Open Access

Cooking, storage, and reheating effect on the formation of cholesterol oxidation products in processed meat products

Lipids in Health and Disease201514:89

https://doi.org/10.1186/s12944-015-0091-5

Received: 30 June 2015

Accepted: 3 August 2015

Published: 11 August 2015

Abstract

Background

Cholesterol is an important biological compound; however, its oxidation products have been proven to be harmful to human health. Cooking, storage, and reheating methods significantly affect the safety of meat products, as they contribute to the production of cholesterol oxidation products (COPs).

Methods

Three cooking methods were used to cook sausages, loin ham, bacon, luncheon meat, and pressed ham, in order to investigate the effect of cooking, storage, and reheating on total cholesterol and on the formation of COPs. Cooked samples were stored at 4 °C and reheated after 3 and 6 storage days by the same cooking method or by microwaving. The samples were assessed for total lipids, cholesterol, and cholesterol oxides.

Results

The average cholesterol content in the processed meat varied from 76.0 mg/100 g to 201.70 mg/100 g. Microwaved ham showed the lowest cholesterol content compared to that of other processed meat products. Significant differences were found in cholesterol content and cholesterol oxidation products depending on cooking, storage, and reheating methods. Six cholesterol oxides were found in processed meat, of which 7β-hydroxycholesterol and α-epoxides were detected as the major oxidation products.

Conclusions

Microwaving and oven grilling resulted in higher production of COPs in processed meat as compared with other cooking methods. Refrigerated storage tended to significantly increase the COPs content.

Keywords

Processed meat products Cooking and reheating methods Total cholesterol Cholesterol oxidation products (COPs)

Background

During the last two decades, the relationship between diet and health has been widely studied and consumers were encouraged to improve their dietary habits. However, the intake of fats, namely saturated fats, is still higher than that specified by the American Heart Association recommendations, according to which total fats should not exceed 30 % of the total caloric intake and saturated fats should represent less than 10 % of the total calories. Processed meat products are highly appreciated by a large number of populations; however, they are rich in cholesterol, lipids, and saturated fatty acids. In general, fats of animal origin are not considered healthy because of their high content of saturated fatty acids and cholesterol [1]. Thermal processing of meat and meat products leads to undesirable changes such as lipid oxidation and protein degradation [2]. Excessive oxidation of meat lipids produces potential precursors of highly reactive aldehydes in tissues and foods, becoming a source of oxidative stress [3, 4]. These aldehydes can be major contributing factors in several pathological conditions such as atherosclerosis, inflammation, arthritis, Alzheimer’s, and Parkinson’s disease [5]. Healthy human plasma contains 12.6 mg/L of COPs [6] and ingestion of foods containing COPs increases these levels in plasma and leads to deleterious health effects.

Cholesterol is a compound of biological importance, widely distributed in food of animal origin. Cholesterol contents of meat and meat products varied considerably but general it is less than 70 mg/100 g except for edible offal and it is assumed that one-third of daily intake come from meat and meat products [7]. However, cholesterol oxidation products (COPs) have been proven to be cytotoxic, mutagenic, and carcinogenic [8], and are also considered to be a primary factor responsible for triggering atherosclerosis [9]. COPs are formed when animal-derived foods are subjected to heating and cooking [10], dehydration [11] storage [1], and irradiation [12]. Colorimetric, chromatographic and enzymatic assessment methods are used for assessing cholesterol and its products [13]. Dominguez et al. [14] reported that, among different cooking methods, i.e., roasting, grilling, microwaving, and frying, used for cooking foal meat, microwaved samples showed the highest oxidation products. Lee et al. [12] studied the effects of various cooking and reheating methods on the total cholesterol and on the formation of COPs in beef loin, and observed a significant reduction in cholesterol and an increase in COPs. Hu et al. [15] compared the effects of cooking methods on pork lipid digestibility and on the formation of COPs, and detected a significantly higher COP formation in microwave-treated samples. In a recent study, Freitas et al. [16] reported the reduction in cholesterol content and the increase in COPs, especially 7-ketocholesterol, in fish fillets cooked by different methods.

Hence, this study aimed to determine the effects of cooking methods, storage, and reheating on the amount of total lipids and cholesterol, and on the formation of COPs in processed meat products.

Methods

Reagents and solutions

Cholesterol, linoleic acid, oleic acid, cholesterol oxide standards [7-ketocholesterol (7-keto), 6-ketocholesterol (6-keto), 7α-hydroxycholesterol (7α-OH), 7β-hydroxycholesterol (7β-OH), 5,6α-epoxycholesterol (5,6α-EP), 5,6β-epoxycholesterol (5,6β-EP), 25-hydroxycholesterol (25-OH), 20-hydroxycholesterol (20-OH), and cholestanetriol (triol)], butylated hydroxytoluene (BHT), Xpyridine, and silicic acid (100 mesh) were purchased from Sigma-Aldrich Co., LLC (Seoul, Korea). Bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1 % trimethylchlorosilane (TMCS) was obtained from Supelco (Bellefonte, PA, USA). HPLC-grade hexane, ethyl acetate, acetone, methanol, chloroform, Celite 545, and calcium phosphate (CaHPO4 .2H2O) were purchased from Fisher Scientific Co. (Malvern, PA, USA).

Sample preparation

Five processed meat products (frankfurter sausage, loin ham, bacon, luncheon meat, and pressed ham (Korean-style luncheon pork)) were purchased from a local market. Frankfurter sausage and bacon were used as such, whereas loin ham, luncheon meat, and pressed ham were cut into 1 cm thickness. Each product was divided into four groups: fresh (control), pan roasting (P), oven grilling (O), and microwave heating (M). The fresh samples were analyzed immediately after cooking, whereas the cooked samples were stored at refrigerated temperature (4 °C) and reheated after 3 and 6 days using the same cooking method or microwave heating, to simulate restaurant preparation. The treatment groups were the following: pan roasting-pan roasting (PP), pan roasting-microwave heating (PM), oven grilling-oven grilling (OO), oven grilling-microwave heating (OM), and microwave heating-microwave heating (MM). Samples for pan roasting (P) were roasted on an electric grill pan (Excel 10 Electric Grill, Tefal Sa, France) at 180 °C until the internal temperature of the samples reached 70 °C. After 3 and 6 days of storage, reheating by pan roasting was done at 180 °C for 5 min (PP) whereas microwave heating was performed in a microwave oven (M-M270TC, LG Electronics, Korea) with 700 W power for 2 min (PM). The samples were cooked by oven grilling in a convection oven (GR-643HT, Tong Yang Magic, Korea) at 150 °C until the internal temperature reached 70 °C. After 3 and 6 days of storage, reheating by oven grilling was done at 150 °C for 10 min (OO) using the same oven, whereas microwave heating was performed in a microwave oven with 700 W power for 2 min (OM). For the MM group, the samples were cooked in a microwave oven for 10 min and reheated in a microwave oven for 2 min, after 3 and 6 days of storage.

Determination of total cholesterol

The fat (0.5 g) was diluted in 10 mL of freshly prepared methanolic potassium hydroxide solution (1 M) and 1 g of sea sand was added. The mixture was heated for 25 min and the supernatant was transferred with a pipette into a 25 mL volumetric flask. The residue was boiled with 6 mL of isopropanol under reflux condenser for 5 min, and the solution was collected, cooled, and diluted to the mark with isopropanol. The turbid solutions were filtered through a Whatman No. 1 filter paper (Whatman Inc., Clifton, NJ). The clear aliquot was used for the cholesterol assay following the kit instructions (Cat. No 139050, Boehringer Mannheim, Germany). The blank sample was prepared by mixing 0.4 mL of the extracted sample solution and 5 mL of solution 4 (cholesterol reagent mixture). The sample solution was obtained by mixing 2.5 mL of the extracted sample solution and 0.02 mL of solution 3. The prepared blank and sample solutions were sealed with paraffin film and incubated at 37-40 °C for 60 min. The absorbance values of the blank (A1) and of the sample (A2) were determined using an UV spectrophotometer (UV1601, Shimadzu Co., Japan) at 405 nm. Cholesterol contents (mg/100 g) were calculated using the following equation:
$$ \mathrm{Cholesterol}\ \mathrm{content}\ \left(\mathrm{mg}/100\ \mathrm{g}\right)=\left[0.711\times \left(\mathrm{A}2-\mathrm{A}1\right)/\mathrm{sample}\ \mathrm{weight}\left(\mathrm{g}\right)\right]\times 100\times 25 $$

Cholesterol Oxidation Products (COPs)

Cholesterol oxidation products were analyzed according to the method of Lee et al. [17]. For the separation of COPs, a solid-phase column was prepared [18, 19] by mixing silicic acid, Celite 545, and CaHPO4 .2H2O (10:9:1, wt/wt/wt) with 30 mL chloroform, and the mixture was packed in a glass column (12 mm × 30 cm). The prepared column was repeatedly prewashed with 5 mL of hexane before sample application. The total lipids were extracted following the method of Folch et al. [20]. The lipid sample (0.2 g) was dissolved in 2 mL hexane:ethyl acetate (100:2, v/v) and applied to a prewashed column. The sample container was washed twice with 2 mL hexane:ethyl acetate and the wash solvent was applied to the column. Neutral lipid and cholesterol (phospholipids) were removed by adding 50 mL of solvent I (CHCl3:CH3OH = 2:1, v/v) and 60 mL of solvent II (hexane:ethyl acetate = 4:1, v/v). Solvent III (40 mL, acetone:ethyl acetate:methanol = 10:10:1, v/v/v) was used at a 1 mL/min flow rate to elute the COPs. The collected solutions were dried on a 50 °C hot plate with nitrogen gas flushing. The dried extracts were derivatized by heating at 80 °C for 1 h in the presence of 200 μL pyridine and 100 μL sylon-bis(trimethylsilyl)trifluoroacetamide + 1 % trimethylchlorosilane. The COPs were analyzed using a gas chromatograph (HP 5890 plus) equipped with an on-column capillary injector and a flame ionization detector. Identification of cholesterol and COPs was based on the comparison of the retention times of the samples with those of the standards, on co-chromatography, and on the characteristics of the absorption spectra.

Statistical analysis

Statistical analysis was performed by one-way analysis of variation (ANOVA) using SAS software (SAS, Release 8.01, SAS Institute Inc., Cary, NC), and Duncan’s multiple range test was employed to differentiate the significance among mean values.

Results and discussion

Total fat and cholesterol content

Total fat and cholesterol contents of processed meat products cooked and reheated by different methods after 3 and 6 days of storage at 4 °C are shown in Tables 1 and 2. The highest fat content was found in fresh bacon (48.5 %), followed by sausages (24.7 %), luncheon meats (23.6 %), and pressed ham (16.3 %), and the lowest fat was found in fresh loin meat (8.3 %). The fat content of meat products varied significantly as a result of cooking and reheating as well as during storage. Bacon showed non-significant changes upon cooking and reheating; however, a considerable reduction in fat content was observed after storing the product for 6 days. The cholesterol content of fresh samples did not vary significantly during storage at 4 °C for 6 days (Table 2). Cooking and reheating of meat products greatly affect their cholesterol content, and maximum reduction in cholesterol was observed for oven grilling and microwave cooking and reheating. Among the tested samples, the highest cholesterol content (201.70 mg/100 g) was observed in bacon, and the lowest (76.00 mg/100 g) in pressed ham. In the present study, the wide range of cholesterol content from pressed ham to bacon is probably due to a high variability in the original samples. Rodriguez-Estrada et al. [2] reported the presence of higher amounts of cholesterol in fresh samples compared with the cooked samples, and attributed these differences to the loss of cholesterol during the cooking process. Cooking and reheating resulted in the oxidation of cholesterol and production of COPs. These findings are in agreement with the results obtained by Freitas et al. [16], who reported a decrease in the cholesterol content of fish fillets and an increase in COP formation upon cooking. Badiani et al. [21] reported that cooking induced a remarkable decrease in moisture content, which varies depending on the different cooking methods, leading to various cholesterol levels due to the differences in cooking and reheating techniques. The results obtained in the current study also confirm the findings of Baggio and Bragagnolo [22], who observed that storage time did not alter the cholesterol content of processed meat samples.
Table 1

Changes of the crude fat content (%) of processed meat products in fresh, cooked, and re-heated at day 3 and 6 of storage

Sample

Cooking method

Re-heating (day)

0

3

6

Sausage

Fresh

24.7 ± 0.09AB

24.5 ± 0.66AB

24.1 ± 0.53A

PP

24.6 ± 0.0Ba

23.6 ± 0.01BCb

23.5 ± 0.10Bb

PM

24.6 ± 0.0Ba

23.0 ± 0.98Cb

22.8 ± 0.13Cb

OO

25.2 ± 0.78Aa

24.7 ± 0.50Aa

23.8 ± 0.01ABb

OM

25.2 ± 0.78Aa

24.0 ± 0.11ABCb

23.7 ± 0.10ABb

MM

23.3 ± 0.06Ca

23.0 ± 0.11Cb

23.0 ± 0.12Cb

Loin ham

Fresh

8.3 ± 0.09A

8.1 ± 0.52A

8.1 ± 0.56A

PP

5.7 ± 0.01Ba

5.5 ± 0.01Bb

5.3 ± 0.13Bc

PM

5.7 ± 0.01Ba

5.4 ± 0.01Bb

4.8 ± 0.19Bc

OO

6.8 ± 0.01Ba

5.8 ± 0.03Bb

4.9 ± 0.04Bc

OM

6.8 ± 0.01Ba

5.4 ± 0.10Bb

5.0 ± 012Bc

MM

5.5 ± 0.01Bb

5.8 ± 0.11Ba

5.1 ± 0.08Bc

Bacon

Fresh

48.5 ± 0.51

48.7 ± 0.84

48.6 ± 1.00

PP

49.0 ± 0.57a

47.7 ± 0.01b

46.6 ± 0.09c

PM

49.0 ± 0.57a

47.5 ± 0.71b

44.6 ± 0.56c

OO

47.0 ± 0.0a

45.9 ± 0.29b

45.7 ± 0.09b

OM

47.0 ± 0.01b

48.6 ± 0.38a

43.8 ± 0.10c

MM

48.8 ± 0.62a

45.8 ± 0.02b

42.9 ± 0.10c

Luncheon meat

Fresh

23.6 ± 1.10AB

23.2 ± 0.71A

23.2 ± 0.69BC

PP

24.0 ± 0.07Ab

27.1 ± 0.11Aa

27.2 ± 0.14Aa

PM

24.0 ± 0.07Ab

23.6 ± 0.08Ac

25.9 ± 0.08Aba

OO

24.0 ± 0.11Ac

26.8 ± 0.10Aa

24.6 ± 0.21ABb

OM

24.0 ± 0.11Ba

20.3 ± 0.10Bb

19.2 ± 0.10Cc

MM

23.4 ± 0.09AB

24.3 ± 0.08A

23.5 ± 0.12ABC

Press Ham

Fresh

16.3 ± 0.10B

16.4 ± 0.31B

16.2 ± 0.26B

PP

18.2 ± 0.09Ac

21.4 ± 0.01Aa

19.6 ± 0.08Ab

PM

18.2 ± 0.09Ac

18.8 ± 0.10Ab

19.8 ± 0.13Aa

OO

20.1 ± 0.08Aa

18.4 ± 0.16Ab

19.9 ± 0.55Aa

OM

20.1 ± 0.08Ab

16.6 ± 0.07Ac

21.2 ± 0.01Aa

MM

20.3 ± 0.07Ac

21.4 ± 0.12Aa

20.6 ± 0.08Ab

Means ± SE with different superscript in the same row (a-c) and column (A-C) differ significantly (p < 0.05)

Pan roasting (cooking) + pan roasting (re-heating), PP; pan roasting (cooking) + microwaving (re-heating), PM; oven grilling (cooking) + oven grilling (re-heating), OO; oven grilling (cooking) + microwaving (re-heating), OM; microwaving (cooking) + microwaving (re-heating), MM

Table 2

Changes of cholesterol content (mg/100 g) of meat products in fresh, cooked, and re-heated at day 3 and 6 of storage

Sample

Cooking method

Re-heating (day)

0

3

6

Sausage

Fresh

96.2 ± 11.52

95.3 ± 11.24

94.7 ± 10.99C

PP

103.3 ± 6.57b

86.0 ± 4.63c

137.9 ± 4.78Aa

PM

103.3 ± 6.57b

84.6 ± 5.11c

119.5 ± 3.64Ba

OO

93.0 ± 4.22

88.3 ± 11.88

91.5 ± 1.14C

OM

93.0 ± 4.22a

74.3 ± 6.25b

93.0 ± 3.02Ca

MM

93.4 ± 7.07

90.1 ± 4.07

91.2 ± 4.05C

Loin ham

Fresh

91.7 ± 7.93B

91.9 ± 7.41A

91.9 ± 7.65A

PP

54.6 ± 1.61Cb

47.8 ± 3.20Cc

89.8 ± 2.82Aa

PM

54.6 ± 1.61Cc

71.8 ± 2.54Ba

64.3 ± 3.79Cb

OO

101.4 ± 6.03Aa

52.5 ± 1.54Cc

85.6 ± 2.86ABb

OM

101.4 ± 6.03Aa

55.6 ± 3.04Cc

79.8 ± 0.91Bb

MM

45.1 ± 1.83Da

49.4 ± 4.62Ca

29.5 ± 0.37Db

Bacon

Fresh

201.70 ± 7.95A

201.2 ± 8.68BC

200.8 ± 7.56B

PP

155.4 ± 8.95Bc

205.7 ± 7.61ABb

238.0 ± 9.32Aa

PM

155.4 ± 8.95Bb

232.4 ± 18.58Aa

218.1 ± 18.81Aba

OO

140.8 ± 23.0Bb

158.8 ± 27.23Dab

198.3 ± 9.19Ba

OM

140.8 ± 23.0B

173.9 ± 18.08CD

168.1 ± 10.21C

MM

151.6 ± 4.84B

174.0 ± 6.48CD

163.5 ± 12.63C

Luncheon meat

Fresh

106.2 ± 5.06A

106.1 ± 4.16A

106.2 ± 4.83BC

PP

92.8 ± 9.80B

94.2 ± 14.45AB

81.8 ± 5.29C

PM

92.8 ± 9.80B

98.3 ± 18.34AB

125.3 ± 15.61B

OO

91.8 ± 2.15Ba

77.1 ± 3.94Bb

80.1 ± 2.27Ca

OM

91.8 ± 2.15B

91.7 ± 6.07AB

121.8 ± 31.64B

MM

79.7 ± 6.93Bc

111.1 ± 13.55Ab

214.4 ± 6.50Aa

Press ham

Fresh

76.0 ± 7.30

75.5 ± 6.85B

75.4 ± 6.61B

PP

81.0 ± 4.38a

70.6 ± 3.84Bb

53.2 ± 4.35BCc

PM

81.0 ± 4.38

92.3 ± 11.04A

52.7 ± 43.23BC

OO

67.9 ± 9.10

65.4 ± 9.43B

69.6 ± 3.99B

OM

67.9 ± 9.10a

59.4 ± 11.87Ba

31.1 ± 1.71Cb

MM

67.8 ± 12.47b

62.2 ± 6.01Bb

191.5 ± 9.40Aa

Means ± SE with different superscript in the same row (a-c) and column (A-C) differ significantly (p < 0.05)

Pan roasting (cooking) + pan roasting (re-heating), PP; pan roasting (cooking) + microwaving (re-heating), PM; oven grilling (cooking) + oven grilling (re-heating), OO; oven grilling (cooking) + microwaving (re-heating), OM; microwaving (cooking) + microwaving (re-heating), MM

Cooking methods and COP formation

The data expressed in Table 3 shows the impact of the cooking methods on the formation of COPs in fresh meat products. 7β-OH was observed in sausages and bacon for all cooking methods, whereas only microwave cooking produces 7β-OH in loin ham. However, 7β-OH was not detected in luncheon meat and pressed ham after cooking by all methods. The highest level of 7β-OH (360.54 μg/100 g) was observed in sausages cooked by oven grilling. A low level of 25-OH was found in fresh as well as in cooked loin ham and in oven grilled luncheon meat. A high 25-OH level was observed in sausages cooked by microwave. Cholestanetriol was detected in microwaved sausage (171.6 μg/100 g), pan-roasted bacon (184.2 μg/100 g), and fresh (153.8 μg/100 g) and oven grilled luncheon meat (256.9 μg/100 g). Low levels of α-epoxide were found in fresh, pan roasted, and microwaved sausages, and in oven grilled loin ham and luncheon meat. 20α-OH and 7-keto were not detected in any of the meat products for all the tested cooking methods.
Table 3

The amount of cholesterol oxidation products (μg/100 g) of meat products with different cooking methods

Sample

Cooking method

Cholesterol Oxidation Products

COPs/cholesterol (%)

7β-OH

20α-OH

25-OH

Triol

α-epoxide

7-keto

Sausage

Fresh

n.d.

n.d.

n.d.

n.d.

20.2 ± 34.94

n.d.

0.02 ± 0.04

P

58.3 ± 100.98

n.d.

n.d.

n.d.

29.9 ± 51.69

n.d.

0.08 ± 0.14

O

360.5 ± 624.48

n.d.

n.d.

n.d.

-.

n.d.

0.36 ± 0.63

M

204.3 ± 228.85

n.d.

359.8 ± 598.90

171.6 ± 297.14

17.4 ± 30.08

n.d.

0.73 ± 0.71

Loin ham

Fresh

n.d.

n.d.

43.0 ± 74.39

n.d.

n.d.

n.d.

0.05 ± 0.08

P

n.d.

n.d.

5.5 ± 9.49

n.d.

n.d.

n.d.

0.01 ± 0.01

O

n.d.

n.d.

22.0 ± 38.01

n.d.

11.6 ± 20.12

n.d.

0.04 ± 0.07

M

24.6 ± 33.33

n.d.

127.3 ± 171.25

n.d.

n.d.

n.d.

0.16 ± 0.21

Bacon

Fresh

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

P

134.2 ± 26.97

n.d.

n.d.

184.2 ± 319.09

n.d.

n.d.

0.16 ± 0.15

O

110.6 ± 191.50

n.d.

n.d.

n.d.

n.d.

n.d.

0.05 ± 0.09

M

304.4 ± 527.25

n.d.

n.d.

n.d.

n.d.

n.d.

0.16 ± 0.27

Luncheon meat

Fresh

n.d.

n.d.

n.d.

153.8 ± 266.35

n.d.

n.d.

0.15 ± 0.26

P

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

O

n.d.

n.d.

30.9 ± 53.49

256.9 ± 445.03

50.7 ± 87.73

n.d.

0.32 ± 0.48

M

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

Press ham

Fresh

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

P

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

O

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

M

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

Pan roasting, P; oven grilling, O; microwaving, M; 7β-hydroxycholesterol, 7β-OH; 20α-hydroxycholesterol, 20α-OH; 25-hydroxycholesterol, 25-OH; cholestane-3β,5α,6β-triol, triol; cholesterol-5α,6α-epoxide, α-epoxide; 7-ketocholesterol, 7-keto

The ratio between the total amount of COPs and cholesterol in the different meat products cooked by different methods was found to be very low (0–0.73 %). The highest value was observed in sausages cooked by microwave (0.73 %), whereas bacon contained the highest cholesterol content (Table 2) and a low total COPs (0.16 %) (Table 3). The presence of a higher amount of 7β-OH in cooked meat products is in agreement with the previous finding of Du et al. [23], who observed a higher level of 7α-OH and 7β-OH compared with other COPs in cooked turkey meat. Broncano et al. [24] investigated different cooking methods and their role in lipid oxidation and in the formation of COPs, and reported the presence of higher levels of 7α-OH and 7β-OH than other COPs. The absence or lower level of triol, α-epoxide, 20α-OH, and 7-keto than 7β-OH is in accordance with the findings of Grau et al. [25] and Ghiretti et al. [26]. The higher COP formation owing to microwaving confirms the results of Dominguez et al. [14], who, upon cooking foal meat using roasting, grilling, microwaving, and frying, detected the highest lipid oxidation in microwaved samples. Serra et al. [27] reported 7β-OH and 7-keto as the primary products of cholesterol oxidation, and α- and β-epoxides as the secondary oxidation products.

Storage, reheating, and COP formation

The cooked meat products stored at 4 °C and reheated after 3 and 6 storage days were assessed for the formation of COPs (Tables 4 and 5). It is evident from the data presented in Table 4 that the COPs observed in cooked products increased significantly upon reheating after 3 storage days. However, 20α-OH was not found in any of the samples reheated by different methods, and 7-keto was detected only in pan roasted luncheon meat reheated by microwave. 7β-OH, 25-OH, and α-epoxide were present in greater amounts than other COPs and were found namely in sausages, loin ham, and bacon. 7β-OH was detected in all samples of sausages, loin ham, and bacon reheated after 3 days by different methods. Similarly, 25-OH and α-epoxide were present in all reheated samples of loin ham. Pressed ham samples also showed cholesterol oxides after 3 days of storage, whereas no oxides were detected after cooking. 7β-OH was found to be present in the highest quantity (497 μg/100 g) pressed ham cooked by oven grilling and reheated with the same method after 3 days of storage. The ratio total amount of COPs/cholesterol in meat products increased relatively after 3 days of storage (0–0.86 %). The data presented in Table 4 shows that the highest ratio (0.86 %) was observed in cooked sausage reheated by microwave.
Table 4

The amount of cholesterol oxidation products (μg/100 g) of meat products cooked with different methods and re-heated by microwaving at day 3 of storage at 4 °C

Sample

Cooking method

Cholesterol Oxidation Products

COPs/cholesterol (%)

7β-OH

20α-OH

25-OH

Triol

α-epoxide

7-keto

Sausage

Fresh

12.7 ± 14.14

n.d.

n.d.

n.d.

n.d.

n.d.

0.01 ± 0.01

PP

155.1 ± 268.60

n.d.

n.d.

283.0 ± 49.01

n.d.

n.d.

0.42 ± 0.42

PM

42.6 ± 73.81

n.d.

396.1 ± 686.09

n.d.

15.7 ± 27.24B

n.d.

0.46 ± 0.69

OO

300.3 ± 520.14

n.d.

n.d.

n.d.

n.d.

n.d.

0.34 ± 0.59

OM

147.3 ± 140.60

n.d.

278.2 ± 481.78

87.8 ± 152.00

n.d.

n.d.

0.57 ± 0.49

MM

55.2 ± 95.63

n.d.

381.2 ± 660.22

n.d.

315.0 ± 320.37B

n.d.

0.86 ± 1.07

Loin ham

Fresh

n.d.

n.d.

43.5 ± 75.38

n.d.

26.0 ± 45.07B

n.d.

0.07 ± 0.13

PP

22.9 ± 29.78

n.d.

196.5 ± 153.41

n.d.

125.2 ± 216.91B

n.d.

0.64 ± 0.63

PM

9.9 ± 17.08

n.d.

13.5 ± 23.38

26.4 ± 45.70

19.4 ± 23.69B

n.d.

0.13 ± 0.10

OO

8.9 ± 8.05

n.d.

58.7 ± 101.69

54.6 ± 94.51

77.1 ± 124.63B

n.d.

0.20 ± 0.20

OM

8.6 ± 14.85

n.d.

25.0 ± 43.34

16.4 ± 28.44

79.7 ± 138.02B

n.d.

0.13 ± 0.16

MM

56.6 ± 98.02

n.d.

241.6 ± 213.09

n.d.

34.2 ± 59.23B

n.d.

0.73 ± 0.64

Bacon

Fresh

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

PP

899.1 ± 1557.30

n.d.

n.d.

245.6 ± 425.41

n.d.

n.d.

0.71 ± 0.84

PM

120.7 ± 209.00

n.d.

n.d.

109.7 ± 189.93

n.d.

n.d.

0.14 ± 0.13

OO

94.7 ± 164.09

n.d.

247.1 ± 427.91

n.d.

751.2 ± 909.52A

n.d.

0.81 ± 0.95

OM

111.6 ± 193.21

n.d.

n.d.

212.5 ± 368.04

n.d.

n.d.

0.25 ± 0.24

MM

589.4 ± 1020.48

n.d.

667.2 ± 1155.64

n.d.

n.d.

n.d.

0.84 ± 0.73

Luncheon meat

Fresh

n.d.

n.d.

n.d.

156.6 ± 271.45

n.d.

n.d.

0.15 ± 0.27

PP

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00

PM

150.5 ± 260.58

n.d.

52.6 ± 91.14

205.8 ± 356.42

37.5 ± 37.68B

26.3 ± 45.51

0.48 ± 0.44

OO

n.d.

n.d.

10.7 ± 18.51

107.1 ± 185.42

n.d.

n.d.

0.13 ± 0.22

OM

87.7 ± 151.86

n.d.

95.1 ± 164.77

n.d.

n.d.

n.d.

0.20 ± 0.17

MM

231.2 ± 440.37

n.d.

n.d.

n.d.

n.d.

n.d.

0.32 ± 0.55

Press ham

Fresh

n.d.

n.d.

48.1 ± 76.50

n.d.

n.d.

n.d.

0.07 ± 0.11

PP

n.d.

n.d.

n.d.

168.4 ± 162.53

n.d.

n.d.

0.22 ± 0.21

PM

n.d.

n.d.

112.2 ± 194.33

n.d.

n.d.

n.d.

0.15 ± 0.26

OO

497.0 ± 860.89

n.d.

n.d.

n.d.

n.d.

n.d.

0.63 ± 1.10

OM

n.d.

n.d.

91.8 ± 159.05

n.d.

n.d.

n.d.

0.15 ± 0.26

MM

n.d.

n.d.

106.5 ± 184.50

n.d.

n.d.

n.d.

0.13 ± 0.22

Means ± SE with different superscript in the same column (A-C) differ significantly

Pan roasting (cooking) + pan roasting (re-heating), PP; pan roasting (cooking) + microwaving (re-heating), PM; oven grilling (cooking) + oven grilling (re-heating), OO; oven grilling (cooking) + microwaving (re-heating), OM; microwaving (cooking) + microwaving (re-heating), MM; 7β-hydroxycholesterol, 7β-OH; 20α-hydroxycholesterol, 20α-OH; 25-hydroxycholesterol, 25-OH; cholestane-3β,5α,6β-triol, triol; cholesterol-5α,6α-epoxide, α-epoxide; 7-ketocholesterol, 7-keto

Table 5

The amount of cholesterol oxidation products (μg/100 g) of meat products cooked with various methods and re-heated by microwaving at day 6 of storage at 4 °C

Sample

Cooking method

Cholesterol Oxidation Products

COPs/cholesterol (%)

7β-OH

20α-OH

25-OH

Triol

α-epoxide

7-keto

Sausage

Fresh

35.3 ± 0.49

n.d.

13.3 ± 14.56

41.8 ± 68.60

35.1 ± 47.79

n.d.

0.13 ± 0.02C

PP

127.3 ± 141.26

n.d.

56.6 ± 97.99

43.8 ± 75.89

20.2 ± 34.94

n.d.

0.24 ± 0.12C

PM

51.1 ± 88.42

n.d.

303.9 ± 526.40

72.9 ± 126.26

n.d.

n.d.

0.39 ± 0.50C

OO

68.0 ± 117.73

n.d.

36.9 ± 63.91

125.9 ± 218.12

n.d.

n.d.

0.25 ± 0.15C

OM

194.3 ± 242.03

n.d.

29.9 ± 51.78

913.8 ± 1582.68

130.0 ± 225.16

n.d.

1.32 ± 1.81BC

MM

339.5 ± 437.60

n.d.

52.0 ± 89.99

120.6 ± 187.71

n.d.

n.d.

0.54 ± 0.26C

Loin ham

Fresh

n.d.

n.d.

43.7 ± 75.79

n.d.

25.8 ± 44.74

n.d.

0.07 ± 0.13C

PP

14.1 ± 24.33

n.d.

147.2 ± 232.12

36.0 ± 62.31

119.7 ± 167.93

n.d.

0.57 ± 0.70C

PM

9.3 ± 16.11

n.d.

n.d.

n.d.

36.7 ± 63.53

n.d.

0.09 ± 0.11C

OO

15.6 ± 26.95

n.d.

114.9 ± 156.50

79.7 ± 138.07

191.0 ± 204.85

n.d.

0.39 ± 0.30C

OM

17.2 ± 29.86

n.d.

243.7 ± 388.99

125.2 ± 216.89

188.8 ± 178.94

37.7 ± 65.30

0.59 ± 0.48C

MM

23.5 ± 40.75

n.d.

131.33 ± 187.97

-

50.8 ± 87.99

n.d.

0.45 ± 0.44C

Bacon

Fresh

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00C

PP

141.4 ± 244.96

n.d.

n.d.

164.2 ± 284.39

n.d.

n.d.

0.19 ± 0.17C

PM

98.1 ± 069.85

n.d.

n.d.

106.9 ± 105.64

238.0 ± 412.15

n.d.

0.28 ± 0.30C

OO

98.7 ± 100.05

n.d.

n.d.

228.2 ± 395.25

n.d.

n.d.

0.25 ± 0.24C

OM

75.9 ± 131.39

n.d.

1718.6 ± 2976.73

2646.3 ± 4583.49

358.7 ± 470.27

n.d.

3.30 ± 2.29A

MM

91.1 ± 157.75

52.8 ± 91.46

n.d.

n.d.

347.6 ± 602.05

n.d.

0.32 ± 0.31C

Luncheon meat

Fresh

n.d.

n.d.

n.d.

154.4 ± 267.47

69.2 ± 77.20

n.d.

0.22 ± 0.26C

PP

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.00 ± 0.00C

PM

122.8 ± 212.66

47.9 ± 82.89

n.d.

n.d.

n.d.

n.d.

0.21 ± 0.36C

OO

n.d.

n.d.

82.1 ± 142.08

158.8 ± 274.96

n.d.

n.d.

0.26 ± 0.44C

OM

888.6 ± 1539.04

22.4 ± 38.75

84.4 ± 146.13

n.d.

102.0 ± 176.73

n.d.

1.20 ± 1.56BC

MM

142.2 ± 246.24

27.2 ± 47.47

n.d.

n.d.

n.d.

n.d.

0.23 ± 0.40

Press ham

Fresh

n.d.

n.d.

48.1 ± 76.50

n.d.

73.9 ± 111.53

n.d.

0.16 ± 0.11

PP

n.d.

19.7 ± 34.07

96.0 ± 138.77

n.d.

26.4 ± 45.66

18.3 ± 31.77

0.20 ± 0.18

PM

n.d.

n.d.

n.d.

449.8 ± 779.10

164.8 ± 285.46

n.d.

0.81 ± 1.40C

OO

1017.2 ± 1761.78

n.d.

n.d.

n.d.

n.d.

n.d.

1.63 ± 2.83ABC

OM

1175.2 ± 2012.57

n.d.

n.d.

461.2 ± 798.81

164.6 ± 285.09

n.d.

2.87 ± 2.78AB

MM

n.d.

37.7 ± 65.31

n.d.

n.d.

n.d.

177.7 ± 307.76

0.36 ± 0.46C

Means ± SE with different superscript in the same column (A-C) differ significantly

Pan roasting (cooking) + pan roasting (re-heating), PP; pan roasting (cooking) + microwaving (re-heating), PM; oven grilling (cooking) + oven grilling (re-heating), OO; oven grilling (cooking) + microwaving (re-heating), OM; microwaving (cooking) + microwaving (re-heating), MM; 7β-hydroxycholesterol, 7β-OH; 20α-hydroxycholesterol, 20α-OH; 25-hydroxycholesterol, 25-OH; cholestane-3β,5α,6β-triol, triol; cholesterol-5α,6α-epoxide, α-epoxide; 7-ketocholesterol, 7-keto

Generally, the amount of COPs in samples reheated after 6 days of storage at 4 °C (Table 5) was higher than that in samples reheated after 3 days of storage, suggesting that increasing the storage time after cooking increased the COP content in processed meat products. 20α-OH was not detected in any of the cooked samples nor in those reheated after 3 days; however, it appeared in samples reheated after 6 days of storage. A similar trend was also observed for 7-keto. The ratio between the total amount of COPs and cholesterol in meat products also increased after 6 days of storage (0.00-3.30 %), with the highest ratio in bacon cooked by oven grilling and reheated by microwave. The concentration of COPs in pressed ham was not detected after cooking and increased significantly upon reheating; the highest total amount of COPs/cholesterol ratios were observed in OM (2.87 %) and OO (1.63 %). Our findings are in line with previous investigations, indicating that storage of cooked meat results in an increase in COP concentration upon reheating. Monahan et al. [28] reported that the rate of cholesterol oxidation in pork products significantly increased during storage after cooking. A higher level of lipid oxidation was found by El-Alim et al. [29] in microwave cooked meat patties after 7 storage days at 4 °C. Hu et al. [15] also observed significantly higher COP levels in microwave cooked samples compared with the COP content in samples cooked by other methods. Processed meat initially free of COPs showed a minor increase in COP content during storage at −4 °C for 4 weeks and a reduction of 15 to 24 % in free cholesterol [30]. Juarez et al. [31] investigated the impact of different cooking methods on buffalo meat composition. Greater oxidative changes were observed in boiled and grilled samples, based on higher TBARS (thiobarbituric acid reactive substances) values. Ferioli et al. [32] evaluated lipid and cholesterol oxidation in fresh and cooked minced beef stored at 4 °C; the COP content increased by 6 times after 2 weeks of storage. They also revealed that the increase in the COP content was higher in cooked meat than in fresh samples. Our investigation confirms these results, as a significant increase in the COP content was detected after refrigerated storage of the cooked meat samples.

Conclusions

Cooking of processed meat products resulted in a decrease of lipids and total cholesterol and in an increase in COP formation. Microwaving and oven grilling led to the production of higher amounts of COPs as compared with other cooking methods. The refrigerated storage of cooked products and subsequent reheating remarkably increased the amount of COPs in processed meat products. However, this increase was found to be lower in fresh products than in cooked products.

Abbreviations

COPs: 

Cholesterol oxidation products

BHT: 

Butylated hydroxyl toluene

GC: 

Gas chromatography

SAS: 

Statistical analysis system

7β-OH: 

7β-hydroxycholesterol

20α-OH: 

7α-hydroxy-cholesterol

25-OH: 

25-hydroxycholesterol

Triol: 

Cholestanetriol

α-epoxide: 

5,6α-epoxycholesterol

7-keto: 

7-ketocholesterol

Declarations

Acknowledgements

This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development (Project No. 011617), Rural Development Administration and Research Institute of Eco-friendly Livestock Science, Institute of Green Bio Science and Technology, Seoul National University, Korea.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Agricultural Biotechnology, Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Science, Seoul National University
(2)
National Institute of Food Science and Technology, University of Agriculture
(3)
CJ Food Research Center
(4)
Department of Health Administration and Food Hygiene, Jinju Health College
(5)
National Institute of Animal Science
(6)
College of Agriculture and Life Science, Arsi University

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Copyright

© Khan et al. 2015

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