Effect of cooking methods on cholesterol, mineral composition and formation of total heterocyclic aromatic amines in Muscovy drake meat 1
1
Omojola, A. B., 1Saheed, H., 2Attoh-Kotoku, V. and 3Wogar, G.S.I
Meat Science Laboratory, Department of Animal Science, University of Ibadan, Ibadan,
Nigeria 2
Animal Science Department, Kwame Nkrumah University of Science and Technology, Kumasi, 3
Ghana
Animal Science Department, University of Calabar, Calabar, Nigeria Abstract
Background: Cholesterol, minerals and heterocyclic aromatic amines (HAA) are of great importance in determining meat quality to humans and therefore of concern to meat consumers because of health reasons. The objective of this study was to evaluate the effects of various cooking methods (grilling, deep frying, pan frying and roasting) on the formation of total HAAs, cholesterol and mineral retention on Muscovy drake breast meat. Methodology/Results: The total HAAs and cholesterol contents were determined by highperformance liquid chromatography (HPLC) while, the minerals were measured by atomic absorption spectroscopy (AAS). Results showed that Muscovy drake breast meat cooked by deep frying contained the highest content of total HAAs (31.83 ng/g) followed by roasted (30.12 ng/g), pan fried (29.23ng/g), and grilled fillets (27.34 ng/g). Cholesterol was higher in deep fried fillets (59.09 mg/g), followed by roasted fillets (58.61 mg/g) while grilling resulted in the least cholesterol value (50.04 mg/g). The mineral content was highest in deep fried samples and least in roasted fillets. Magnesium retention was affected by cooking method and was highest in deep fried samples (110.57%) compared to grilled (97.97 %), pan fried (88.99 %) and roasted fillets (78.46%). Generally, the total HAAs, Cholesterol and the mineral content in cooked Muscovy drake breast meat varies with cooking method. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6687
This article is protected by copyright. All rights reserved
Conclusion: Moderate consumption of deep fried Muscovy drake meat should be encouraged as deep frying increased cholesterol, HAAs levels and mineral content of meat compared to other cooking methods investigated. Key words: Muscovy meat, cooking method, cholesterol, heterocyclic amine, mineral INTRODUCTION Meat as the flesh of animals used for food is a relevant dietary source of proteins, essential amino acids, chemical elements (e.g. iron, zinc) and vitamins (e.g. B12, D). Yet, the healthy perception of people about meat is tarnished by its negative association with cholesterol and other non-nutritional or toxic contaminants activated during cooking. These may include poly aromatic hydrocarbons (PAH) in smoked products and heterocyclic aromatic amines (HAA) in cooked products. Heterocyclic aromatic amines (HAAs) are mutagenic products formed when meat or fish are thermally processed1. They are formed when meats are exposed to temperatures greater than 150°C during preparation and cooking2, 3. Many cooking methods, including frying, roasting, smoking, broiling, and baking have been reported to induce HAA formation4. Fats of animal origin are also considered in general not very healthy because of their high level of saturated fatty acid (SFA) and cholesterol content5. Generally, meat contains 125 mg cholesterol per 100 g on an average. The consumption of animal products has therefore, been associated with chronic illnesses such as obesity and cardiovascular related illnesses6. The implication of meat products as the major cause of cardiovascular related diseases are based on the amount and type of cholesterol in muscle foods. In western societies, coronary heart disease and arteriosclerosis are strongly related to the dietary intake of cholesterol, sodium and saturated fatty acid and are among the important causes of sudden human deaths7. In recent time, consumption of duck meat in Nigeria has decreased probably due to lack of correct and adequate information about the nutritional value of the meat. The ideal cooking This article is protected by copyright. All rights reserved
method of the meat that will not compromise the health of consumers has also not been explored. More importantly, consumers in general are skeptical of duck meat consumption as it is widely believed that its cholesterol level is high. With this perception, it therefore become expedient to investigate the effect of different cooking methods on cholesterol, total HAAs and mineral profile of Muscovy drake breast meat.
MATERIALS AND METHODS
Experimental animals A total of eighteen (18) matured Muscovy drakes weighing between 2.0 and 2.5 kg were used for the study. The drakes were purchased from a reputable duck farm in Ibadan, Nigeria. The drakes were kept for a period of three weeks prior to slaughtering where they had access to feed and water. Slaughtering and processing The drakes were fasted for 16 h before slaughtering but with access to fresh cool water. The breasts were excised within one hour post-mortem, skinned and divided into symmetric halves. Each half was dissected into 5 fillets (a total of 10 fillets per breast). The fillets were trimmed of any visible fat, ligaments and bones and kept frozen for 24 hours and the breasts were properly thawed before cooking. Meat sample preparation Cooking Before processing, the drake breasts were trimmed to obtain a uniform size and shape of each. After cleaning, a total of one hundred and eighty fillets (weighing about 125.35 ± 4.42 g) were obtained and distributed randomly to four cooking conditions of: grilling, deepfrying, pan-frying, and roasting in a completely randomized design. Each treatment was This article is protected by copyright. All rights reserved
replicated four times with each replicate consisting of nine (9) fillets. Raw fillets were analyzed and served as the standard. Cooking methods For pan-frying, the breasts were fried for 5 min per side without fat or oil in a Teflon-coated pan, which was preheated and the surface temperature was measured as 180 0C. For the deep-frying, fresh soybean oil was used. The Muscovy drake breasts were fried for 10 min in a commercial stainless steel deep-fat fryer, when the temperature of oil reached 180 0
C. For the gas grilling, the drake breasts were grilled for 10 min per side, total cooking time
was 20 min, the distance between samples and burner was about 15 cm. The surface temperature of the samples was about 200 0C. For roasting, the breasts were placed in an oven for 20 min at 200 0C. No condiment, spice or salt were applied to the fillets before and after cooking. The temperature of cooking was monitored by the thin chromium-aluminum thermocouples and cooking terminated when the core temperature of the meat reached the degree of well done (800C) in each of the cooking procedure. All experiments were replicated four times. After all processing methods, the meat samples were allowed to cool down at room temperature. The cooked meat samples were homogenized using commercial blender to obtain a uniform sample and kept frozen at -2 0C in plastic containers until analyzed.
Instrumentation Total Heterocyclic Aromatic Amines (THAAs) THAAs were analyzed after solid-phase extraction using HPLC with UV and fluorescence detectors, as described by Gross and Gruter8 with some minor modifications.
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HPLC analysis was performed on a Waters 600 HPLC system (Milford, Massachusetts, USA) equipped with a photodiode array detector (Waters 2996, Photo Diode Array). The analytical column used was a reversed phased column TSK-gel ODS 80-TM (4.6 mm id×25.0 cm L, 5 μm) from Tosoh Bioscience Gm BH (Stuttgart, Germany).
Cholesterol Total cholesterol was extracted from lyophilized meat after saponification with saturated methanolic KOH, according to the procedure of Naeem et al.9 except that three extractions with cyclohexane were used10. Cholesterol was separated and quantified by normal phase HPLC (Column Zorbax Rx- Sil, 4.6 mm i.d. x 250 mm, 5mm particle size, Chrompack, Bridgewater, N.J.) with an HPLC system (HP 1000 series, Hewlett-Packard, Palo Alto, CA) equipped with an auto sampler and diode array detector adjusted at 206 nm, a solvent (3 % iso propanol in n-hexane) flow rate of 1mL/min. and injection volume of 30 FL. Total cholesterol content of each meat sample was calculated in triplicate based on the external standard technique from a standard curve area vs. concentration.
Minerals Calcium, potassium, magnesium and sodium determination was carried out using Jenway Digital Flame Photometer (PFP7 Model) using the filter corresponding to each mineral element. The concentration of each of the element was calculated using the formula:
%( Mineral element) = Meter Reading (MR) x Slope x Dilution Factor 10000
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Phosphorus was determined by the Spectro-photometric method. The percentage phosphorus was calculated from the formula: %Phosphorus = Absorbance x Slope x Dilution Factor 10000
Statistical analysis The data was subjected to analysis of variance using the SAS procedure11. Where statistical significance was observed the means were compared using Duncan’s multiple-range test12 at the significance level of P=0.05.
RESULTS AND DISCUSSION The deep fried meat has higher HAA compared to the rest of the cooking methods (Deep frying> Roasting >Pan Frying > Grilling > Raw). The raw meat sample contained no detectable HAAs (Table1). This observation is in line with the report by NTP13. The result could suggest that HAAs are formed during cooking similar to earlier suggestion14. The total HAAs score for processed meat in this study ranged from 27.34 to 31.83bng/g and was
higher than the estimated dietary exposure of 1-17 ng/kg body weight per day15. The
value obtained for pan-fried (29.23 ng/g) in this study was lower than that obtained by Liao et al.16 who showed that total HAAs in pan-fried duck breast meat was 53.3ng/g. Deep fried samples have a value of 31.83ng/g. The high level of total HAAs in deep fried fillets was probably due to the fact that HAAs could migrate via the fats to reach the interior part of the fillets however; this value was found to be higher than the values obtained by17, 16. The value obtained for grilling was 27.3ng/g and was contrary to 32ng/g obtained for charcoal grilling of duck breast meat by earlier authors18,
16
. The difference in values obtained could be
attributed the type of combustible material used. Charcoal was used in their own study while
This article is protected by copyright. All rights reserved
in the present study the fillets were grilled using liquefied gas. Lower levels of total HAAs (6.8ng/g) were reported for roasted duck breast meat18, 16 in contrast to (30.12ng/g) obtained in this study. The varying level of HAAs in the differently processed duck meat in this study was also previously explained by Liao et al
16
that poultry can be prepared under different conditions
and will contain variable levels of HAAs. Total HAAs concentration in cooked meat generally ranges from less than 1 to about 500ng/g but usually less than 100ng/g17. Many studies have shown that cooking conditions are crucial in the formation of HAAs18, 19, 20, 21
According to Solyakov and Skog18 a variety of heterocyclic amines in heat-treated
poultry products can be formed with different cooking methods and cooking conditions such as duration and temperature of cooking as well as the composition of the food22. The temperature and duration of cooking appear to have a much greater influence than the lipid content and animal species from which the muscle was taken. However, grilling and barbecuing, the most common methods for the preparation of fatty meat, usually require higher temperatures (above 200 °C) that already promote HAA formation. Probably the different cooking methods of food and the temperatures influence the amount of HAAs formed23. It is supposed that HAA might originate in Maillard reactions between hexoses and free amino acids (especially creatine and creatinine), however, the exact mechanism has not been established24.
Residual levels of HAAs found in some foods constitute a risk of mutagenicity and carcinogenetic for the consumer. The International Agency for Research on Cancer (IARC) has classified several HAAs as probable (2a) or possible (2b) carcinogens and recommended reducing human exposure to these compounds25. However, there are no EU guidelines and Minimum Recommended Levels (MRLs) of HAs in cooked foods and foodstuffs and it is This article is protected by copyright. All rights reserved
therefore not possible to evaluate the real exposure risk to these cooking toxic products. For instance, reported estimated daily intake of HAAs per caput was 50 ng in China26, 72 ng in Japan27, 160 ng in Sweden28, 330 ng in Switzerland23, 455 ng in US29, 606 ng in Spain30,31. However, there were no documented MLRs for African countries. The quantity of cholesterol in raw and cooked Muscovy drake breast fillets is shown in Table 1. The cholesterol was relatively higher in cooked samples irrespective of the cooking method employed than that in raw meat. The result of this study revealed that cholesterol content was highest (P0.05)
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Table III. Mineral retention of differently processed Muscovy drake meat (%) Cooking Methods Parameters
Grilling
Deep Frying
Pan Frying
Roasting SEM
F-Val
P-Val
Potassium
74.17
69.01
69.64
67.98
1.41
0.93
0.434
Sodium
75.19
69.97
66.26
64.63
1.99
1.42
0.249
Calcium
74.76
73.30
70.42
66.87
1.71
1.04
0.383
Magnesium
97.97ab
110.57a
88.99bc
78.46c
3.18
6.08
0.001
Phosphorus
82.63
78.41
77.35
81.43
1.72
0.50
0.682
abc
Means with the same superscript along the same row are not significantly different (P>0.05)
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1
Omojola, A. B., 1Saheed, H., 2Attoh-Kotoku, V. and 3Wogar, G.S.I
Meat Science Laboratory, Department of Animal Science, University of Ibadan, Ibadan,
Nigeria 2
Animal Science Department, Kwame Nkrumah University of Science and Technology, Kumasi, 3
Ghana
Animal Science Department, University of Calabar, Calabar, Nigeria Abstract
Background: Cholesterol, minerals and heterocyclic aromatic amines (HAA) are of great importance in determining meat quality to humans and therefore of concern to meat consumers because of health reasons. The objective of this study was to evaluate the effects of various cooking methods (grilling, deep frying, pan frying and roasting) on the formation of total HAAs, cholesterol and mineral retention on Muscovy drake breast meat. Methodology/Results: The total HAAs and cholesterol contents were determined by highperformance liquid chromatography (HPLC) while, the minerals were measured by atomic absorption spectroscopy (AAS). Results showed that Muscovy drake breast meat cooked by deep frying contained the highest content of total HAAs (31.83 ng/g) followed by roasted (30.12 ng/g), pan fried (29.23ng/g), and grilled fillets (27.34 ng/g). Cholesterol was higher in deep fried fillets (59.09 mg/g), followed by roasted fillets (58.61 mg/g) while grilling resulted in the least cholesterol value (50.04 mg/g). The mineral content was highest in deep fried samples and least in roasted fillets. Magnesium retention was affected by cooking method and was highest in deep fried samples (110.57%) compared to grilled (97.97 %), pan fried (88.99 %) and roasted fillets (78.46%). Generally, the total HAAs, Cholesterol and the mineral content in cooked Muscovy drake breast meat varies with cooking method. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6687
This article is protected by copyright. All rights reserved
Conclusion: Moderate consumption of deep fried Muscovy drake meat should be encouraged as deep frying increased cholesterol, HAAs levels and mineral content of meat compared to other cooking methods investigated. Key words: Muscovy meat, cooking method, cholesterol, heterocyclic amine, mineral INTRODUCTION Meat as the flesh of animals used for food is a relevant dietary source of proteins, essential amino acids, chemical elements (e.g. iron, zinc) and vitamins (e.g. B12, D). Yet, the healthy perception of people about meat is tarnished by its negative association with cholesterol and other non-nutritional or toxic contaminants activated during cooking. These may include poly aromatic hydrocarbons (PAH) in smoked products and heterocyclic aromatic amines (HAA) in cooked products. Heterocyclic aromatic amines (HAAs) are mutagenic products formed when meat or fish are thermally processed1. They are formed when meats are exposed to temperatures greater than 150°C during preparation and cooking2, 3. Many cooking methods, including frying, roasting, smoking, broiling, and baking have been reported to induce HAA formation4. Fats of animal origin are also considered in general not very healthy because of their high level of saturated fatty acid (SFA) and cholesterol content5. Generally, meat contains 125 mg cholesterol per 100 g on an average. The consumption of animal products has therefore, been associated with chronic illnesses such as obesity and cardiovascular related illnesses6. The implication of meat products as the major cause of cardiovascular related diseases are based on the amount and type of cholesterol in muscle foods. In western societies, coronary heart disease and arteriosclerosis are strongly related to the dietary intake of cholesterol, sodium and saturated fatty acid and are among the important causes of sudden human deaths7. In recent time, consumption of duck meat in Nigeria has decreased probably due to lack of correct and adequate information about the nutritional value of the meat. The ideal cooking This article is protected by copyright. All rights reserved
method of the meat that will not compromise the health of consumers has also not been explored. More importantly, consumers in general are skeptical of duck meat consumption as it is widely believed that its cholesterol level is high. With this perception, it therefore become expedient to investigate the effect of different cooking methods on cholesterol, total HAAs and mineral profile of Muscovy drake breast meat.
MATERIALS AND METHODS
Experimental animals A total of eighteen (18) matured Muscovy drakes weighing between 2.0 and 2.5 kg were used for the study. The drakes were purchased from a reputable duck farm in Ibadan, Nigeria. The drakes were kept for a period of three weeks prior to slaughtering where they had access to feed and water. Slaughtering and processing The drakes were fasted for 16 h before slaughtering but with access to fresh cool water. The breasts were excised within one hour post-mortem, skinned and divided into symmetric halves. Each half was dissected into 5 fillets (a total of 10 fillets per breast). The fillets were trimmed of any visible fat, ligaments and bones and kept frozen for 24 hours and the breasts were properly thawed before cooking. Meat sample preparation Cooking Before processing, the drake breasts were trimmed to obtain a uniform size and shape of each. After cleaning, a total of one hundred and eighty fillets (weighing about 125.35 ± 4.42 g) were obtained and distributed randomly to four cooking conditions of: grilling, deepfrying, pan-frying, and roasting in a completely randomized design. Each treatment was This article is protected by copyright. All rights reserved
replicated four times with each replicate consisting of nine (9) fillets. Raw fillets were analyzed and served as the standard. Cooking methods For pan-frying, the breasts were fried for 5 min per side without fat or oil in a Teflon-coated pan, which was preheated and the surface temperature was measured as 180 0C. For the deep-frying, fresh soybean oil was used. The Muscovy drake breasts were fried for 10 min in a commercial stainless steel deep-fat fryer, when the temperature of oil reached 180 0
C. For the gas grilling, the drake breasts were grilled for 10 min per side, total cooking time
was 20 min, the distance between samples and burner was about 15 cm. The surface temperature of the samples was about 200 0C. For roasting, the breasts were placed in an oven for 20 min at 200 0C. No condiment, spice or salt were applied to the fillets before and after cooking. The temperature of cooking was monitored by the thin chromium-aluminum thermocouples and cooking terminated when the core temperature of the meat reached the degree of well done (800C) in each of the cooking procedure. All experiments were replicated four times. After all processing methods, the meat samples were allowed to cool down at room temperature. The cooked meat samples were homogenized using commercial blender to obtain a uniform sample and kept frozen at -2 0C in plastic containers until analyzed.
Instrumentation Total Heterocyclic Aromatic Amines (THAAs) THAAs were analyzed after solid-phase extraction using HPLC with UV and fluorescence detectors, as described by Gross and Gruter8 with some minor modifications.
This article is protected by copyright. All rights reserved
HPLC analysis was performed on a Waters 600 HPLC system (Milford, Massachusetts, USA) equipped with a photodiode array detector (Waters 2996, Photo Diode Array). The analytical column used was a reversed phased column TSK-gel ODS 80-TM (4.6 mm id×25.0 cm L, 5 μm) from Tosoh Bioscience Gm BH (Stuttgart, Germany).
Cholesterol Total cholesterol was extracted from lyophilized meat after saponification with saturated methanolic KOH, according to the procedure of Naeem et al.9 except that three extractions with cyclohexane were used10. Cholesterol was separated and quantified by normal phase HPLC (Column Zorbax Rx- Sil, 4.6 mm i.d. x 250 mm, 5mm particle size, Chrompack, Bridgewater, N.J.) with an HPLC system (HP 1000 series, Hewlett-Packard, Palo Alto, CA) equipped with an auto sampler and diode array detector adjusted at 206 nm, a solvent (3 % iso propanol in n-hexane) flow rate of 1mL/min. and injection volume of 30 FL. Total cholesterol content of each meat sample was calculated in triplicate based on the external standard technique from a standard curve area vs. concentration.
Minerals Calcium, potassium, magnesium and sodium determination was carried out using Jenway Digital Flame Photometer (PFP7 Model) using the filter corresponding to each mineral element. The concentration of each of the element was calculated using the formula:
%( Mineral element) = Meter Reading (MR) x Slope x Dilution Factor 10000
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Phosphorus was determined by the Spectro-photometric method. The percentage phosphorus was calculated from the formula: %Phosphorus = Absorbance x Slope x Dilution Factor 10000
Statistical analysis The data was subjected to analysis of variance using the SAS procedure11. Where statistical significance was observed the means were compared using Duncan’s multiple-range test12 at the significance level of P=0.05.
RESULTS AND DISCUSSION The deep fried meat has higher HAA compared to the rest of the cooking methods (Deep frying> Roasting >Pan Frying > Grilling > Raw). The raw meat sample contained no detectable HAAs (Table1). This observation is in line with the report by NTP13. The result could suggest that HAAs are formed during cooking similar to earlier suggestion14. The total HAAs score for processed meat in this study ranged from 27.34 to 31.83bng/g and was
higher than the estimated dietary exposure of 1-17 ng/kg body weight per day15. The
value obtained for pan-fried (29.23 ng/g) in this study was lower than that obtained by Liao et al.16 who showed that total HAAs in pan-fried duck breast meat was 53.3ng/g. Deep fried samples have a value of 31.83ng/g. The high level of total HAAs in deep fried fillets was probably due to the fact that HAAs could migrate via the fats to reach the interior part of the fillets however; this value was found to be higher than the values obtained by17, 16. The value obtained for grilling was 27.3ng/g and was contrary to 32ng/g obtained for charcoal grilling of duck breast meat by earlier authors18,
16
. The difference in values obtained could be
attributed the type of combustible material used. Charcoal was used in their own study while
This article is protected by copyright. All rights reserved
in the present study the fillets were grilled using liquefied gas. Lower levels of total HAAs (6.8ng/g) were reported for roasted duck breast meat18, 16 in contrast to (30.12ng/g) obtained in this study. The varying level of HAAs in the differently processed duck meat in this study was also previously explained by Liao et al
16
that poultry can be prepared under different conditions
and will contain variable levels of HAAs. Total HAAs concentration in cooked meat generally ranges from less than 1 to about 500ng/g but usually less than 100ng/g17. Many studies have shown that cooking conditions are crucial in the formation of HAAs18, 19, 20, 21
According to Solyakov and Skog18 a variety of heterocyclic amines in heat-treated
poultry products can be formed with different cooking methods and cooking conditions such as duration and temperature of cooking as well as the composition of the food22. The temperature and duration of cooking appear to have a much greater influence than the lipid content and animal species from which the muscle was taken. However, grilling and barbecuing, the most common methods for the preparation of fatty meat, usually require higher temperatures (above 200 °C) that already promote HAA formation. Probably the different cooking methods of food and the temperatures influence the amount of HAAs formed23. It is supposed that HAA might originate in Maillard reactions between hexoses and free amino acids (especially creatine and creatinine), however, the exact mechanism has not been established24.
Residual levels of HAAs found in some foods constitute a risk of mutagenicity and carcinogenetic for the consumer. The International Agency for Research on Cancer (IARC) has classified several HAAs as probable (2a) or possible (2b) carcinogens and recommended reducing human exposure to these compounds25. However, there are no EU guidelines and Minimum Recommended Levels (MRLs) of HAs in cooked foods and foodstuffs and it is This article is protected by copyright. All rights reserved
therefore not possible to evaluate the real exposure risk to these cooking toxic products. For instance, reported estimated daily intake of HAAs per caput was 50 ng in China26, 72 ng in Japan27, 160 ng in Sweden28, 330 ng in Switzerland23, 455 ng in US29, 606 ng in Spain30,31. However, there were no documented MLRs for African countries. The quantity of cholesterol in raw and cooked Muscovy drake breast fillets is shown in Table 1. The cholesterol was relatively higher in cooked samples irrespective of the cooking method employed than that in raw meat. The result of this study revealed that cholesterol content was highest (P0.05)
This article is protected by copyright. All rights reserved
Table III. Mineral retention of differently processed Muscovy drake meat (%) Cooking Methods Parameters
Grilling
Deep Frying
Pan Frying
Roasting SEM
F-Val
P-Val
Potassium
74.17
69.01
69.64
67.98
1.41
0.93
0.434
Sodium
75.19
69.97
66.26
64.63
1.99
1.42
0.249
Calcium
74.76
73.30
70.42
66.87
1.71
1.04
0.383
Magnesium
97.97ab
110.57a
88.99bc
78.46c
3.18
6.08
0.001
Phosphorus
82.63
78.41
77.35
81.43
1.72
0.50
0.682
abc
Means with the same superscript along the same row are not significantly different (P>0.05)
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