Cholesterol photosensitised oxidation of beef meat under standard and modified atmosphere at retail conditions

Meat Science 81 (2009) 224–229

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Cholesterol photosensitised oxidation of beef meat under standard and modified atmosphere at retail conditions Emanuele Boselli a, Maria Teresa Rodriguez-Estrada b, Giorgio Fedrizzi c, Maria Fiorenza Caboni b,* a

Dipartimento SAIFET, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy Dipartimento di Scienze degli Alimenti, Università di Bologna, Viale G. Fanin, 40, 40127 Bologna, Italy c Istituto Zooprofilattico Sper. della Lombardia e dell’Emilia Romagna, Rep. Merceologia degli Alimenti di Origine Animale, Via Fiorini, 5, – 40127 Bologna, Italy b

a r t i c l e

i n f o

Article history: Received 15 June 2008 Received in revised form 19 July 2008 Accepted 22 July 2008

Keywords: Photosensitised oxidation Beef meat Lipids Modified atmosphere Cholesterol oxidation

a b s t r a c t The effect of the fluorescent light exposure and type of packaging (normal atmosphere and oxygen-rich atmosphere) was evaluated on the oxidation parameters (peroxides and cholesterol oxidation products) of raw beef slices placed in packed vessels and refrigerated. The concentration of COPs in meat treated under modified atmosphere ranged from 0.15 to 0.52 mg/100 g meat (average value of 0.27 mg COPs/ 100 g meat), which was twice as much as the average COPs content (0.14 mg/100 g) of meat packed under air (0.04–0.27 mg COPs/100 g meat). The main cholesterol oxide was 7k, which represented about one third of the total cholesterol oxides, followed by 7b-OH (20–25% of total COPs), 7a-OH (about 20%) and b-epoxy (12–18%). In normal atmosphere, photoxidation was a superficial process, since an inverse correlation between meat slice weight and COPs content on a lipid basis was observed, unlike in a high oxygen (32%) atmosphere. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Cholesterol is a monounsaturated lypophilic biomolecule, which is arranged with the 3-hydroxyl group and the A ring exposed to the outer side of the double phospholipid layer in the biological membrane and the side chain located among the alkylic chain of phospholipids (Cercaci, Rodriguez-Estrada, Lercker, & Decker, 2007; Smith, 1987). It is, therefore, prone to both enzymic and chemical oxidation with the formation of compounds potentially harmful to human health, such as cholesterol oxidation products (COPs) (Garcia-Cruset, Carpenter, Codony, & Guardiola, 2002; Osada, 2002; Schroepfer, 2000). COPs are likely to be involved in lipid metabolism, various chronic and degenerative diseases (such as cancer, aging and atherosclerosis) and disturbance of cell functionality (Garcia-Cruset et al., 2002; Leonarduzzi, Sottero, & Poli, 2002; Osada, 2002; Schroepfer, 2000). COPs contain an additional hydroxy, ketone or epoxide group on the sterol nucleus and/or a hydroxyl group on the side chain of their molecules (Lercker & Rodriguez-Estrada, 2002). Cholesterol oxides are present in quite low amount in raw food of animal origin (meat, milk, eggs, sea products), but their concentration increases dramatically in high-temperature treated food, after exposure to light, metals, natural sensitizers and oxygen, as well as in highly processed food products cooked meat (Badiani et al., 2002; Ferioli, Caboni, & Dutta, 2008; Galvin, Lynch, Kerry, * Corresponding author. Tel.: +39 0512096009; fax: +39 0512096017. E-mail address: [email protected] (M.F. Caboni). 0309-1740/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2008.07.023

Morrissey, & Buckley, 2000; Rodriguez-Estrada, Penazzi, Caboni, Bertacco, & Lercker, 1997) irradiated (Ahn, Nam, Du, & Jo, 2001) and dry-cured meat products (Vestergaard, & Parolari, 1999). The extent of meat oxidation is also related to the content of natural antioxidants and the unsaturation degree of the fatty acids (Bou, Grimpa, Baucells, Codony, & Guardiola, 2006; Decker, & Xu, 1998; Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998). Unlike previous studies, the aim of the current work was to evaluate the influence of both fluorescent light exposure and packaging under modified atmosphere, on lipid oxidation of beef slices stored in a bench refrigerator of a retail shop. Markers of the total primary lipid oxidation (peroxide value) and cholesterol oxidation products were used to monitor lipid oxidation. Storage time was chosen according to the typical shelf-life of beef slices packed in a closed vessel: 8 h storage under light exposure was selected as the maximum for the meat slices packed in normal atmosphere, whereas storage lasted 8 days for meat packed in a high oxygen partial pressure. For meat irradiation, a warm-tone lamp emanating red light with low emission in the blue region was used. 2. Materials and methods 2.1. Sampling Meat samples were obtained from the half-carcase of a Garronese breed cow slaughtered at 165 kg weight and stored for 5 days at 3 °C. The inside round was excised from the half-carcase and trimmed from the superficial lipids and connective tissue; the

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meat sample was divided longitudinally and the terminal parts were excluded. The inside round was then cut with an automated cutter and 26 slices were obtained: the meat was 5–6 mm thick and had a weight ranging from 100 to 120 g (thin slices). In addition, 6 meat slices were cut with a thickness of 1 cm with a weight that varied between 135 g and 187 g (heavy slices). A total of 32 slices were, thus, obtained. Of the 32 slices, fourteen thin slices and three heavy slices were packed individually in polyethylene vessel, wrapped with a transparent shrink film (14 lm thickness) with 10445 ml/m2/24 h of oxygen permeability. The packed slices were subjected to the following storage conditions: (a) Two vessels were immediately frozen ( 18 °C) and represented the control (t001 and t002); (b) three vessels were stored in the dark at 4 °C overnight (t01, t02 and t03); (c) three vessels were stored at 4 °C in the dark (wrapped with tin foil) for 8 hours (8hD1, 8hD2 and 8hD3); (d) three vessels were stored at 4 °C under a daylight lamp for 4 hours (4h1, 4h2 and 4h3); (e) three vessels (8h1, 8h2 and 8h3) and the three heavy slices (8hH1, 8hH2 and 8hH3) were stored at 4 °C under a daylight lamp for 8 h. The remaining 12 thin slices and 3 heavy slices were packed in vessels under modified atmosphere (oxygen 32%, nitrogen 30%, carbon dioxide 38%) using a sealing film by means of a Tetra Laval Food Tiromat Compact packaging machine (Pully, Switzerland). The packed slices were subjected to the following storage conditions under modified atmosphere; (f) three vessels were immediately frozen ( 18 °C) and represented the control (MAt01, MAt02 and MAt03); (g) three vessels were stored at 4 °C under a daylight lamp for 4 days (MA4d1, MA4d2 and MA4d3); (h) three vessels were stored at 4 °C in the dark (wrapped with tin foil) for 8 days (MA8dD1, MA8dD2 and MA8dD3); (i) three vessels (MA8d1, MA8d2 and MA8d3) and the three heavy slices were stored at 4 °C under a daylight lamp for 8 days (MA8dH1, MA8dH2 and MA8dH3). The storage at 4 °C was in a bench refrigerator as usually happens in a retail shop. The warm-tone lamps had a colour temperature of 3000 K (low emission in the blue radiation) and a power of 36 W (Osram, Milan, Italy). The lamps were located 1.5 m above the samples, as in a retail shop. 2.2. Reagents and solvents Analytical grade solvents and reagents were utilised. The standards supplied by Sigma Chemical Co. (St. Louis, MO, USA), are listed as follows: 19-hydroxycholesterol (19-OH), cholesterol, dihydrocholesterol, 3b-hydroxycholest-5-en-7-one (7k), 5,6aepoxy-5a-cholestan-3b-ol (a-epoxy), 5,6b-epoxy-5b-cholestan3b-ol (b-epoxy), cholest-5-en-3b,20a-diol (20-OH) and 5a-cholestan-3b,5,6b-triol (triol). The standards cholest-5-en-3b,7a-diol (7a-OH) and cholest-5-en-3b,7b-diol (7b-OH) were purchased from Steraloids (Wilton, NH, USA). 2.3. Extraction procedure The lipids were extracted according to a modified version of the method described by Folch, Lees, & Sloane-Stanley (1957), as reported previously (Boselli et al., 2005). The frozen samples were minced and 60 g were homogenised with 500 ml of a chloroform:methanol solution (1:1, v/v) in a glass bottle with screwcap. The bottle was kept in an oven thermostated at 60 °C for

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20 min before adding 250 ml chloroform. After 3 min of homogenisation, the contents of the bottle were filtered. The filtrate was mixed thoroughly with a 1 M KCl solution and left overnight at 4 °C in order to obtain phase separation. The lower phase containing the lipids was collected and dried with a rotary evaporator. The fat content was determined gravimetrically. 2.4. Determination of total cholesterol and cholesterol oxidation products (COPs) A 250 mg lipid subfraction of the Folch extract was treated with a known amount of the internal standard solutions (12.5 lg of 19OH and 5 mg dihydrocholesterol, for the determination of COPs and total cholesterol, respectively). Subsequently, the sample was dried under nitrogen and treated with 10 ml of 1 N KOH solution in methanol, for saponification at room temperature for 18 h (Sander, Addis, Park, & Smith, 1989). For the extraction of the unsaponifiable matter, 10 ml of water and 10 ml of diethyl ether were added to the samples, which were vigorously shaken. The diethyl ether fraction was then separated; the extraction with 10 ml of diethyl ether was repeated twice. The three portions of diethyl ether were pooled, treated with 5 ml of a 0.5 N KOH solution and extracted. The resulting ether extract was washed twice with 5 ml of water. The ether solution was finally evaporated in a rotary evaporator, after elimination of excess water by addition of anhydrous sodium sulphate. One tenth of the unsaponifiable matter was used for the determination of total cholesterol and the remainder for COPs analysis. The determination of total cholesterol (sum of free and esterified) was achieved by means of capillary gas chromatography (CGC) after silylation (Sweeley, Bentley, Makita, & Wells, 1963). The gas chromatograph (HRGC Mega2 Series, Fisons, Rodano, Italy) was equipped with a split/splitless injector and a flame ionisation detector. A fused-silica capillary column (30 m  0.32 mm i.d.  0.25 lm film thickness) coated with 100% dimethyl-polysiloxane (DB-1, J&W Scientific, Folsom, CA, USA), was used. The oven temperature was programmed from 250 to 325 °C at 3 °C/min; the injector and detector temperatures were both set at 325 °C. Helium was used as carrier gas at a flow rate of 2 ml/min; the split ratio was 1:15. COPs purification was performed with nine tenths of the unsaponifiable matter by solid phase extraction (SPE) using a NH2 cartridge, according to Rose-Sallin, Hugget, Bosset, Tabacchi, and Fay (1995). After silylation, the derivatised COPs were injected into a CGC under the same conditions as reported for the determination of total cholesterol. The identification of COPs was confirmed by comparison with the retention time and mass spectra of the COP standards. The mass spectra were obtained using the same column mounted in a 3400 GC from Varian (Palo Alto, CA, USA) coupled to an ion trap mass spectrometer (Magnum, Finnigan, San José, CA, USA). 2.5. Determination of peroxide value (PV) The peroxide value was determined in 50 mg of lipid extract, according to Takagi, Mitsuno, and Masumura (1978), a iodometric procedure suitable for small lipid samples. The peroxides present in the meat lipids oxidised iodide to iodine and, after 5 min, the excess of iodide ion was immediately converted to a cadmium complex under a nitrogen atmosphere. The iodine was measured at 358 nm with a double beam UV/visible spectrophotometer Jasco model UVIDEC-430 (Tokyo, Japan). The PV was calculated from the absorbance. For the quantitative determination of PV, a calibration curve was prepared by adding solutions of potassium dichromate at different concentrations to the KI solution. The same procedure was used for the measurement of the released iodine absorbance (358 nm), which was plotted against the active oxygen content.

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2.6. Statistical analysis The Principal Component Analysis (PCA) was performed to recognize differences or groups among the 32 meat samples by means of seven chemical variables (7a-hydroxy, 7b-hydroxy, b-epoxy, aepoxy, 20-hydroxy, 7k and peroxide value). PCA was performed using Unscrambler v 7.6 (Camo Inc., Corvallis, OR, USA). The cluster analysis of the same samples was performed using XLStat software (Addinsoft, New York, USA). 3. Results and discussion The use of modified atmosphere (MA) for the packaging of vessels for beef patties is common in the meat market. An increased percentage of oxygen (up to 80%) stabilises the intense red colour of meat, thus increasing consumer acceptability of the product (Zakrys, Hogan, O’Sullivan, Allen, & Kerry, 2008). However, the lipid oxidation rate is dramatically increased in the presence of high oxygen concentrations and by light exposure in the shop refrigerator; both factors can affect the technological and nutritional quality of the meat (Lund, Hviid, & Skibsted, 2007). The experimental design was aimed to evaluate the influence of both packaging and light exposure on cholesterol oxidation and peroxide value, which were subdivided as follows: (a) the effect of fluorescent light exposure (warm-tone lamps) compared to a dark environment in a normal atmosphere and (b) the effect of a modified atmosphere on similar dark/light storage as in (a). Only the storage time was different, which was chosen according to the shelf-life requirements and commercial distribution needs (Fig. 1). Table 1 reports the mean values (±std. dev.) of the results. The COPs concentration in meat treated under modified atmosphere ranged from 0.15 to 0.52 mg/100 g meat (average value of

Fig. 1. Schematic experimental plan and abbreviations used.

0.27 mg COPs/100 g meat), which was twice as much as the average COP content (0.14 mg/100 g) of meat packed under air (0.04– 0.27 mg COPs/100 g meat). These values are higher than those found in earlier studies on samples kept in unmodified atmosphere, such as those reported by Zubillaga and Maerker (1991) and Hwang and Maerker (1993) (0.0009–0.13 mg/100 g meat), and by Nam, Du, Jo, and Ahn (2001) (39.4 lg COPs/g lipids). These differences might be due to the fact that the meat samples analysed in the cited studies had higher lipid contents (the meat was not as trimmed), thus contained a higher ratio of triacylglycerols to membrane lipids. This resulted in a negative correlation between the cholesterol present in the meat lipid fraction and its total lipid content, as shown in Fig. 2. Moreover, it must be pointed out that Italian slaughtering and processing practices imply a holding period of a few days at 3– 6 °C, which is aimed to improve tenderness and promote the formation of aroma compounds or precursors that develop during cooking. Despite this, the final COP concentration in the raw meat, expressed on a fresh weight, was similar to that reported by Thurner, Razzazi-Fazeli, Wagner, Elmadfa, and Luf (2007), who found 3.4 ± 0.2 mg/kg COPs in raw beef. The cholesterol oxidation ratio (calculated as % COPs/cholesterol) varied between 0.02% and 0.3%. The main cholesterol oxide was 7k, which represented about one third of the total cholesterol oxides in all samples. The second most concentrated oxide was 7b-OH (20–25% of total COPs), followed by 7a-OH (about 20%) and b-epoxy (12–18%). a-epoxy was only seen in a few samples (65% of total COPs, with an average value of 2.8%), as for 20-OH, were its content was very variable and could reach 15%.

Fig. 2. Cholesterol content (expressed on lipid basis) vs fat content of meat samples subjected to different light exposure and packaging conditions.

Table 1 Average content of single and total COPS on lipid basis (lg/g lipids), on fresh weight (lg/g beef) and peroxide value (lg O2/g lipids) in meat samples (±standard deviation) Samples

7a OH

7b OH

b epoxy

a epoxy

20-OH

7k

Total COPs

Peroxide value

Total COPs lg/g beef)

t00

12.3 ± 4.3

15.2 ± 6.0

10.4 ± 1.7

2.9 ± 1.3

3.8 ± 1.4

33.2 ± 13.6

77.8 ± 28.0

27.1 ± 9.1

0.11 ± 0.04

Unmodified atmosphere t0 4h 8h 8hD 8hH

11.6 ± 2.8 23.2 ± 4.4 22.1 ± 15 16.9 ± 8.9 9.1 ± 5.0

13.2 ± 3.2 28.1 ± 6.7 26.0 ± 17.0 19.4 ± 10.0 10.5 ± 6.2

9.6 ± 2.0 16.0 ± 3.4 16.4 ± 7.3 11.8 ± 6.2 7.4 ± 4.0

0.8 ± 1.4 3.8 ± 0.8 3.7 ± 1.5 2.7 ± 2.0 0.8 ± 1.0

1.1 ± 1.9 2.9 ± 0.6 2.4 ± 0.7 2.7 ± 0.3 3.0 ± 0.3

21.4 ± 5.6 39.3 ± 7.6 33.3 ± 22.6 28.3 ± 13.0 18.1 ± 9.3

57.7 ± 15.0 113.0 ± 22.0 104 ± 63.0 81.7 ± 40.0 48.8 ± 26.0

24.8 ± 3.5 30.1 ± 4.9 34.8 ± 26 22.4 ± 6.8 13.8 ± 0.4

0.10 ± 0.04 0.22 ± 0.06 0.18 ± 0.07 0.16 ± 0.1 0.09 ± 0.04

Modified atmosphere MAt0 MA4d MA8d MA8dD MA8dH

20.9 ± 7.0 25.1 ± 2.7 31.1 ± 13 29.3 ± 4.6 30.8 ± 4.3

25.3 ± 12 30.3 ± 2.7 40.0 ± 16.0 37.9 ± 5.6 41.5 ± 7.5

15.8 ± 6.6 16.9 ± 2.4 24.9 ± 8.4 20.4 ± 3.0 19.7 ± 2.3

2.8 ± 3.0 4.3 ± 0.8 5.6 ± 1.8 5.0 ± 0.8 4.0 ± 0.7

17.7 ± 4.3 2.6 ± 0.2 0.9 ± 1.6 0.9 ± 1.5 2.8 ± 0.3

41.0 ± 20.2 40.9 ± 5.1 54.4 ± 23.0 49.4 ± 8.6 57.6 ± 8.1

124.0 ± 53.0 120.0 ± 13.0 157.0 ± 61.0 143.0 ± 21.0 156.0 ± 23.0

32.8 ± 13.0 27.0 ± 6.6 15.7 ± 3.1 25.6 ± 5.2 16.9 ± 7.5

0.22 ± 0.1 0.26 ± 0.07 0.33 ± 0.2 0.31 ± 0.08 0.24 ± 0.01

Control

Abbreviations: MA, modified atmosphere; d, day; D, dark environment; H, heavy meat slice; 4 h, 4 h; 8 h, 8 h; t, control samples; 20-OH, 20-hydroxycholesterol; b-epoxy, b-epoxycholesterol; a-epoxy, a-epoxycholesterol; 7a-OH, 7a-hydroxycholesterol; 7b JY, 7b-hydroxycholesterol.

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3.1. Storage under unmodified atmosphere Previous works carried out on raw meat have shown the relationship between PV and the COP contents. In turkey patties (Boselli et al., 2005), as well as pork slices (Cardenia, Rodriguez-Estrada, Cumella, Massimini, & Lercker, 2008), COPs formation was delayed with respect to peroxides. In the present study, a high positive linear correlation between PV and each COP was observed (0.69 < r2 < 0.72), except for 20-OH, where no correlation was found. The samples stored for one night at 4 °C (t0) did not have a significantly higher content of total COPs with respect to the meat that had been immediately frozen (t00) (Table 1). After 4 h of light exposure, a marked increase in both PV (+20%) and COP contents (+100%) was registered. Prolonged light exposure (up to 8 h) resulted in a further increase of PV, but COPs remained practically constant, due to higher rates of evolution of these products with respect to their formation. The average cholesterol oxidation after 8 h light exposure (104 lg COP/g of lipids), was about 25% higher than in the meat samples stored for the same time in darkness (82 lg COP/g of lipids). Comparison between ‘thin’ and ‘thick’ (heavy) 8 h illuminated slices, showed average PV’s of 35 and 13 mg O2/kg fat, respectively, whereas their COP contents were 104 and 49 lg/g of lipids.

3.2. Storage under modified atmosphere The samples packed under modified atmosphere were characterised by a faster increase of both primary and secondary oxidation parameters, with respect to the samples stored under normal conditions. After 12 h at 4 °C in the dark, PV slightly increased, as compared to that of t00. The COP content was twice as much as reported for unmodified atmospheres. In addition, PV was not related to the COP contents (r2 = 0.05). The light exposure led to a decrease of hydroperoxides, whereas oxysterols increased slightly. In fact, average cholesterol oxidation after 8 h light exposure (157 lg COP/g of lipids), was about 10% higher than in the meat samples stored for the same time in darkness (143 lg COP/g of lipids). It should be noted that the differences in the oxidation processes between ‘thick’ and ‘thin’ slices stored in modified atmosphere were not marked. 3.3. Effect of the mass/surface ratio on lipid oxidation Since the mass/surface ratio could affect the extent of oxidation, this effect was assessed by calculating the correlation between the oxidation parameters (expressed on a lipid basis) and the corresponding meat weight in samples packed under different atmospheres. No correlations were found between PV and meat weight for both packaging systems. An inverse, linear correlation was only found between COP content and meat weight in samples packed in unmodified atmospheres (Fig. 3). It is evident that, in the latter case, the oxidation was superficial, due to the low partial oxygen pressure. On the contrary, the high partial oxygen pressure of the modified atmosphere vessels led to a brilliant red colour, but also gave rise to oxidation of inner lipids, regardless of slice thickness (Fig. 4). 3.4. Multivariate statistical analysis The present experimental model is suitable for a multivariate statistical analysis, which is a powerful tool to demonstrate sam-

Fig. 3. COP contents (expressed on lipid basis) vs meat weight in meat stored under unmodified atmosphere.

Fig. 4. COP contents (expressed on lipid basis) vs meat sample weight in meat stored in modified atmosphere.

ple groupings and differences, as well as positive or negative relationships among all variables. The PCA of the seven variables across the 32 meat samples resulted in the model depicted in Fig. 5, where the first two principal components explained 87% of the total variance. The figure reports the scores of the samples according to these first two components. The loadings of the variables are also reported. From Fig. 5, it is clear that the extent of the primary oxidation (hydroperoxides) can be distinguished from the secondary oxidation of cholesterol (located at high values of PC1 and PC2 about 0). In fact, the less oxidised samples are those located at negative values of PC1 and 2 (left bottom of the plot). On the contrary, the samples packed in modified atmosphere (MAt01, MAt02, MAt03) at time 0 already had higher PV’s than the controls packed in normal atmospheres (the top one of the arbitrary circles in Fig. 5). The samples stored in modified atmosphere increased their COP contents in a time-dependent way; in fact, the trend is towards the bottom right corner of the plot (the bottom circle groups most samples kept under modified atmosphere for 4 or 8 days). As aforementioned, the effect of light was not as significant as the effect of atmosphere composition on the samples; in fact, no different groupings between the dark and lit environments were noted in both the normal and MA stored samples. Fig. 6 shows the dendrogram resulting from a cluster analysis obtained using Ward’s method (Euclidean distance). The dendrogram confirmed the results of the PCA and shows four statistically different clusters: the first group corresponded to the control samples in modified atmosphere (MAt01, MAt02, MAt03), the second group contained all the other samples stored in MA and in normal atmosphere for 4 h (high peroxides, high oxysterols), the third group was formed by three outliers, and the fourth group corresponded to both the less oxidised samples (t0) and the 8 h samples, where peroxides were markedly decomposed into secondary oxidation products.

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Fig. 5. Principal Component Analysis of meat samples subjected to different light exposure and packaging conditions: factor 1 versus factor 2 score plot (see Table 1 for abbreviations).

Fig. 6. Dendrogram obtained from the cluster analysis of meat samples subjected to different light exposure and packaging conditions (see Table 1 for abbreviations).

4. Conclusions Fluorescent light exposure (with low emission in the blue radiation) and packaging conditions play different roles on lipid oxidation in beef slices stored in a refrigerator. When samples were stored in a normal atmosphere, photosentised oxidation was mainly a superficial process, since the ‘thick’ meat slices reached a lower relative oxidation level due to the lower surface/volume ratio. The effect of the partial pressure of oxygen on lipid and cholesterol oxidation in beef meat was greater than that of exposure to a warm-tone fluorescent light; in fact, the high oxygen packaging led to a marked increase of the oxidation parameters (both peroxides and COPs), resulting in the disappearance of the inverse correlation between meat weight and COP content on a lipid basis. The decreasing trend of the peroxide value in high oxygen atmospheres is a consequence of the formation of secondary oxidation products, such as COPs, whose content, in fact, increased. The present data suggest that using high oxygen atmospheres for raw beef is a questionable packaging solution under retail conditions, as this type of packaging has a significant negative impact

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