Inhibitory effects of apple peel polyphenol extract on ...

Meat Science 117 (2016) 57–62

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Inhibitory effects of apple peel polyphenol extract on the formation of heterocyclic amines in pan fried beef patties Kebba Sabally a, Lekha Sleno b, Julie-Anne Jauffrit b, Michèle M. Iskandar a, Stan Kubow a,⁎ a b

School of Dietetics and Human Nutrition, McGill University, 21111 Lakeshore, Ste-Anne-de-Bellevue H9X 3V9, QC, Canada Chemistry Department, Université du Québec à Montréal, P.O. Box 8888, Succ. Centre Ville Montreal, QC H3C 3P8, Canada

a r t i c l e

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Article history: Received 26 October 2015 Received in revised form 16 February 2016 Accepted 23 February 2016 Available online 26 February 2016 Keywords: Heterocyclic amines Beef patties Dried apple peel powder Inhibition Liquid chromatography-tandem mass spectrometry

a b s t r a c t The efficacy of polyphenol-rich dried apple peel extract (DAPP) to inhibit the formation of heterocyclic aromatic amines (HCAs) during frying of beef patties was assessed after DAPP was applied at 0.1, 0.15 and 0.3% (w/w) either on the surface of the patties or mixed inside the patty prior to frying. 2-Amino-3,8dimethylimidazo[4,5f]quinoxaline (MeIQx), 2-amino-1-ethyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-dimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) were quantified after frying. HCA concentrations decreased (p b 0.05) upon both surface and mixed applications of DAPP at all of the tested doses. Surface application of 0.3% DAPP showed greater (p b 0.05) inhibitory effects on HCA formation by 68% for MeIQx, 56% for 4,8-DiMeIQx and 83% for PhIP as opposed to 41%, 21% and 60% respectively, for the mixed DAPP application of 0.3%. The present study results indicate that surface application of DAPP in meat preparation prior to panfrying can be a useful approach to minimize the formation of genotoxic HCAs in fried beef patties. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Epidemiological data has indicated that food-derived carcinogens from cooked meat and meat products predispose individuals to gastrointestinal cancer, particularly of the colon, which is the second leading cause of cancer mortality in North America and most industrialized countries (Rohrmann, Hermann, & Linseisen, 2009; Goldman & Shields, 2003). Heterocyclic amines (HCAs) are carcinogenic and/or mutagenic compounds mainly formed in muscle foods, especially meat and fish, via the Maillard reaction with creati(ni)ne, amino acids and sugars as the precursors (Kizil, Oz, & Besler, 2011). The major classes of HCAs include amino-imidazo-quinolines, or amino-imidazo-quinoxalines (called IQ-type compounds). 2-Amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx), 2-amino1-ethyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8dimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) are the most abundant of the HCAs formed in grilled beef, bacon, fish and poultry (Knize, Dolbeare, Carroll, Moore, & Felton, 1994; Skog, Johansson, & Jägerstad, 1998; Sander, Linseisen, & Rohrmann, 2010). Although 2amino-3-methylimidazo[4,5-ƒ]quinoxaline (IQx) is less abundantly formed following frying or grilling, it is considered among the most carcinogenic of the HCAs (Moller et al., 2002). It has been reported that there exists a high risk for colorectal cancer for individuals Abbreviations: DAPP, dried apple peel powder; HCAs, heterocyclic amines. ⁎ Corresponding author. E-mail address: [email protected] (S. Kubow).

http://dx.doi.org/10.1016/j.meatsci.2016.02.040 0309-1740/© 2016 Elsevier Ltd. All rights reserved.

who frequently eat well-done grilled meat that potentially contains elevated levels of HCAs (Sinha et al., 1998). The levels of HCAs formed in meats prepared by common household cooking methods are normally small (0.1–50 ng/g) (Santos et al., 2004; Puangsombat, Gadgil, Houser, Hunt, & Smith, 2012); however, the amounts in meats or poultry that are cooked well-done, or the grilled pan scrapings often used for gravy, can be as high as 500 ppb (Skog et al., 1998). In view of the risks associated with consuming HCAs, there is a need to reduce exposure by blocking HCA formation such as adding an ingredient during the cooking of meats to prevent their production (Balogh, Gray, Gomaa, & Booren, 2000). Fortification of foods with food-derived antioxidants such as polyphenols has been under active research since free radicals are thought to be involved in HCA formation through the Maillard reaction (Schwab et al., 2000). A large population study has suggested protection against the development of HCA-induced colon cancer by high dietary intake of flavonoids (Rohrmann et al., 2009), which are found in fruits and vegetables along with teas, chocolate and red wine. Both individual phytochemicals and plant extracts have been reported to inhibit the generation of HCAs from meats, which includes flavonoids from black, white and green tea (Dhawan et al., 2002; Quelhas et al., 2010), the flavonoid chrysin (Turesky, Taylor, Schnackenberg, Freeman, & Holland, 2005), citrus flavonoids (Bear & Teel, 2000) as well as a variety of phenolic-rich fruit and vegetable extracts (Edenharder, Sager, Glatt, Muckel, & Platt, 2002; Cheng, Wu, Zheng, & Peng, 2007). Apple is a rich source of flavonoids and the direct mixing of a single dose of a crude apple extract (0.1%) into ground beef patties was shown to inhibit HCA formation caused by frying, which

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K. Sabally et al. / Meat Science 117 (2016) 57–62

was related to the presence of proanthocyanidins, phloridzin and chlorogenic acid (Cheng et al., 2007). It is possible that the inhibition of HCA formation could be further enhanced using dried apple peel powder (DAPP) as peels have markedly higher levels of total antioxidant capacity than either the flesh plus peel or flesh of all the apple varieties examined (Wolfe, Wu, & Liu, 2003). In the present work, we investigated the use of DAPP in a dose response manner to minimize the production of the three most abundant HCAs, MeIQx, 4,8-MeIQx and PhIP in fried beef patties at a high frying temperature (223 °C). As ingredients have generally been applied upon the surface of meats to prevent HCA production, studies are lacking regarding the direct comparison of the blending of additives within the meat product versus their application on the meat surface in terms of the formation of HCAs after frying. An additional aim was therefore to study the influence of surface versus mixed application of DAPP on HCA formation. 2. Materials and methods 2.1. Materials The standards IQx, MeIQx, 4,8-DiMeIQx and PhIP and deuterated internal standards of 2-amino-3-trideuteromethylimidazo[4,5-f]quinoline (D3-IQx), 2-amino-8-methyl-3-trideuteromethylimidazo[4,5f]quinoxaline (D3-MeIQx) and 2-amino-1-trideuteromethyl-6phenylimidazo[4,5-b]pyridine-D3 (D3-PhIP) were obtained from Toronto Research Chemicals (North York, ON). Ammonium acetate and sodium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO). Deionized water was produced using a Sybron/Branstead PCS unit (Barnstead/Thermolyne, Dubuque, IA). Solid-phase extraction OASIS mixed cation exchange columns (MCX) were purchased from Waters (Bristol, CT). Ethyl acetate, acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ). The DAPP samples were provided by Leahy Orchards Inc. (Franklin, QC). DAPP contains phloridzin quercetin 3-O-glucoside, quercetin 3-Ogalactoside, quercetin 3-O-arabinoside, quercetin 3-O-xyloside and quercetin 3-O-rhamnoside as the major polyphenols (Denis et al., 2013). The total phenolic content of DAPP used in the present study was 2.76 g gallic acid equivalent/100 g dry weight. 2.2. Sample preparation and thermal treatment of ground beef patties Freshly ground beef (advertised as 85% lean beef) was purchased from a local supermarket (Marché Richelieu, Sainte Anne de Bellevue, QC) and used in the frying tests within 1 h of purchase. Samples of 100 g ground beef were used to prepare beef patties with thickness about 1.5 cm and diameter about 10.2 cm using a burger patty maker (Ares, Pointe-Claire, QC). The treatments consisted of 0, 0.1, 0.15 and 0.3% of DAPP (w/w) composed of 100% dried apple peel with a high flavonoid content (Denis et al., 2013). DAPP was either applied to the surface of the ground beef patties or thoroughly mixed and kept at ambient conditions for 30 min prior to frying. Ground beef patties without added DAPP were treated as controls for each experiment. The ground beef patties were pan fried on each side for 10 min at 223 °C using a Cleveland Tilting Skillet Model SET-10 (Burkett Restaurant Equipment and Supplies, OH), preheated to 223 °C (Balogh et al., 2000; Knize et al., 1994). Following frying, the samples were immediately ground using a Robot Coupe Blixer (Robert Coupe, MS). 2.3. Solid phase extraction of heterocyclic amines The extraction of HCAs (Santos et al., 2004) was performed on 1.5 g beef patty samples placed into 50 mL glass extraction tubes into which were added 50 μL of the trideuterated IQ, MeIQx and PhIP internal standard (IS) mixture (D3-PhIP, 0.2 μg/mL; D3-IQ, 0.5 μg/mL; D3-MeIQx, 0.5 μg/mL methanol). There was no commercially available internal

standard for 4,8-MeIQx-D3; therefore, we used the most structurally similar compound available, MeIQx-D3, as the internal standard for 4,8-MeIQx. For the calibration standards, 1.5 mg of “blank” beef (cooked at 223 °C for 5 min on each side) was extracted similarly but with the addition of a 100 μL of a mixture of unlabeled standards of 4,8-DiMeIQx, MeIQx (0.00, 0.005, 0.01, 0.02, 0.05, 0.1 and 0.2 μg/mL methanol) and PhIP (0.0, 0.02, 0.05, 0.1 and 0.2 μg/mL) for the external calibration curve. A 6 mL aliquot of 1 M sodium hydroxide was then added to all beef samples and vortexed for 2 min, which was followed by the addition of 8 mL ethyl acetate. After vortexing for another 2 min, the samples were centrifuged at 748 ×g for 5 min. After removal of 6 mL of supernatant, an additional 6 mL ethyl acetate was added and the extraction procedure was repeated as described above. The ethyl acetate supernatants containing the extracted HCAs were combined and evaporated to dryness under nitrogen. The dried samples were re-solubilized in 2 mL methanol prior to cleaning and concentration by solid phase extraction (SPE) using mixed mode 1 cc, 30 mg, OASIS MCX cartridges (Waters, MA). The cartridges were preconditioned with 1 mL of methanol and equilibrated with 1 mL of water and the samples were loaded. The cartridges were first rinsed with 1 mL of water containing 2% formic acid, followed by 1 mL of methanol and then the HCAs were eluted twice with 0.75 mL methanol containing 5% ammonium hydroxide into Eppendorf tubes. Eluted samples were dried and stored at –30 °C prior to liquid chromatography-tandem mass spectrometry (LC–MS/MS). Prior to LC–MS/MS analysis, dried samples were reconstituted in 100 μL methanol and filtered using 0.45 μm polypropylene spin filters into LC vials prior to injection of 20 μL into the LC–MS/MS instrument.

2.4. LC–MS/MS analysis LC–MS/MS analysis was performed on a Shimadzu Nexera UHPLC system (Tokyo, Japan) coupled with a QTRAP 5500 hybrid triple quadrupole-linear ion trap (AB Sciex, Concord, ON, Canada) mass analyzer in positive ion mode. Chromatographic separation was achieved using a Phenomenex Gemini-NX column (100 × 4.6 mm, 5 μm particles) with mobile phases A: 20 mM ammonium acetate, pH 6 and B: acetonitrile. The elution gradient consisted of starting conditions at 5% B held for 1 min, followed by a linear increase to 30% B at 15 min, to 60% B at 18 min and 90% B at 18.5 min and held for an additional 1.5 min at a flow rate of 1 mL/min. Ion source conditions were as follows: ion spray source voltage, 5000 V; curtain gas, 40 psi; temperature, 550 °C, nebulizer/drying gases, 60 psi; declustering potential, 80 V. Each multiple reaction monitoring transition was optimized using standard HCA solutions and the following transitions and optimized parameters were used: IQx m/z 200 → 185 with collision energy (CE) of 36 V and m/z 200 → 158 (CE 46 V); IQ-D3 m/z 202 → 184 (CE 41 V) and m/z 202 → 157 (CE 47 V); MeIQx m/z 214 → 199 (CE 34 V) and m/z 214 → 131 (CE 51 V); MeIQx-D3 m/z 217 → 199 (CE 34 V) and m/z 217 → 131 (CE 51 V); 4,8-DiMeIQx m/z 228 → 213 (CE 32 V) and m/z 228 → 212 (CE 42 V); PhIP m/z 225 → 210 (CE 39 V) and m/z 225 → 140 (CE 61 V); PhIP-D3 m/z 228 → 210 (CE 39 V) and m/z 228 → 140 (CE 61 V). The first transition was used for quantification and the second was used for confirmation. Data were acquired using Analyst 1.5.1 software and processed (peak integration and quantitation) using MultiQuant 2.1 for quantitative HCA analysis. For each analyte, an external calibration curve was produced and the resulting μg/mL values were calculated for each beef extract sample. Three independent experiments were conducted for each study and triplicate samples were used. These values were then transformed into ng HCA/g beef for comparison between control (no treatment) and different samples treated with DAPP. Final results are shown after converting HCA quantities as a function of the amount contained in the controls (percent of control). The precision of the method for the analysis of HCAs is seen in Fig. 1 from the standard curves along with the percent accuracies and r2 values. The precision


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Fig. 1. Calibration curves of heterocyclic amines standards MeIQx (A), 4,8-DiMeIQx (B), and PhIP (C) with corresponding percent accuracies and r2.

ranges from 87–107% accuracy for MeIQx, 95–107% for 4,8-DiMeIQx and 84–110% for PhIP. 2.5. Statistical analysis Data are expressed as mean ± SEM. Data were analyzed by two-way ANOVA with treatment method (surface, mixed) and treatment dose (0, 0.1, 0.15 and 0.3%) as factors followed by Tukey's post hoc multiple comparison test to determine statistically significant differences between groups. For PhIP, the reciprocal transformation was used to obtain a normal distribution and the transformed data was analyzed. The backtransformed means and standard errors are reported for these two variables. A p-value less than or equal to 0.05 was considered significant. Statistical analyses were performed using Sigma Plot version 12.0 (Systat Software, Inc.).

(n = 3) (Fig. 1). MeIQx and 4,8-DiMeIQx had dynamic ranges of 0.005–0.2 and 0.005–0.1 μg/mL, respectively, whereas PhIP was quantifiable from 0.02–0.2 μg/mL. The lower limit of quantification values were 0.33 ng/g for MeIQx and 4,8-DiMeIQx, and 1.33 ng/g for PhIP. The method was suitable for the analysis and quantification of MeIQx, 4,8-DiMeIQx and PhIP in the fried beef patty samples as shown by the chromatograms in Fig. 2a-c. IQx was the least sensitive of the analytes although it was quantifiable from 0.005–0.1 μg/mL of spiked standards; however, no peaks were detected at the proper retention time for IQx in the fried beef samples and so IQx could not be quantified in those samples. The method was highly sensitive as there was baseline separation of the three commercially available internal standards D3-IQx, D3-MeIQx and D3-PhIP as well as for the standards IQx, MeIQx, 4,8-DiMeIQx and PhIP in the tested beef samples. 3.2. Concentrations of HCAs in fried beef patty samples

3. Results and discussion 3.1. LC/MS/MS for quantification of HCAs in beef samples The standard addition calibration curves were within the linearity range of the method. The method was precise for the quantitative analysis of HCAs (MeIQx, 4,8-DiMeIQx and PhIP) as shown by the percent accuracies ranging from 90.9% to 110.5% with an average r2 of 0.99

The HCAs detected after the beef patty frying were PhIP (10.41 ± 0.91 ng/g), MeIQx (16.02 ± 0.70 ng/g) and 4,8-DiMeIQx (4.38 ± 0.43 ng/g) with a total HCA concentration of 30.83 ± 1.92 ng/g (Table 1). The total measurable HCA (PhIP, MeIQx and 4,8-DiMeIQx) content is similar to previous observations as 33.4 ng/g was detected in meatballs fried at 225 °C for 30 min (Shin, Park, & Park, 2003) and 25.65 ng/g was seen following frying of beef for 20 min at 180 °C

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Fig. 2. Chromatograms of (A) blank beef spiked only with mixed Internal Standards (IS), (B) Blank beef spiked with standard mix of 0.1 μg/ml and IS and (C) grilled control beef spiked only with IS mixed

(Murkovic, Steinberger, & Pfannhauser, 1998). Prior studies have shown that the amounts of HCAs produced are directly proportional to both frying temperature and duration (Chen & Chiu, 1998; Knize et al., 1994). For example, Knize et al. (1994) showed that after frying at 230 °C for 6 and 10 min, they detected PhIP concentrations of 7.8 and 32 ng/g and MeIQx values of 5.6 and 7.3 ng/g, respectively. Likewise, meatballs fried at 275 °C for 5, 10 and 15 min showed corresponding cooking duration related increases in MeIQx concentrations of 2.7 ng/g, 4.2 ng/g and 12.3 ng/g, respectively (Turesky, Bur, Huynh-Ba, Aeschbacher, & Milon, 1988). PhIP was present in similar concentrations to previous work performed by Felton et al. (1986) who detected 15 ng/g PhIP in ground beef fried for 11 min at 300 °C. The presence of detectable HCAs among studies may also depend on the varying thickness of the meat samples, meat cooking method and temperature as other studies have shown non-detectable or much lower concentrations of PhIP and MeIQx following beef patty frying. Abdulkarim and

Scott Smith (1998) were unable to detect PhIP following the frying of 15% fat hamburgers at 230 °C for 15 min while Puangsombat et al. (2012) reported that PhIP and MeIQx were detected in fried beef patties (204 °C for 5 min) at 2.35 ng/g and 3.1 ng/g, respectively. In terms of 4,8-DiMeIQx, it has been detected under more prolonged cooking times as 4,8-DiMeIQx appeared at a concentration of 3.9 ng/g after meatballs were fried for 15 min at 275 °C (Turesky et al., 1988), which matches well with the concentrations observed here. On the other hand, studies involving shorter frying times have not detected this compound such as in hamburgers grilled for 10 min at 250–270 °C (Gross et al., 1993) or in meatballs fried at 275 °C for 5 or 10 min (Turesky et al., 1988). In the present study, IQx was not detectable. Similarly, no IQx was detected following frying for 20 min at 190 °C of hamburgers with a 15% fat composition (Knize et al., 1994) or in grilled pork chops (Zimmerli, Rhyn, Zoller, & Schlatter, 2001). On the other hand, 1.1 ng/g IQx was detected in fried 15% fat hamburgers at 170 °C

Table 1 Effect of DAPP on HCA production in pan-fried beef patties. MeIQx

4,8-DiMeIQx

PhIP

Total HCA

ng/mL

% of control

ng/mL

% of control

ng/mL

% of control

ng/mL

% of control

16.02 ± 0.70a

100

4.38 ± 0.43

100

10.41 ± 0.91

100

30.83 ± 1.92

100

Mixed DAPP application 0.1% 11.42 ± 0.23 0.15% 9.22 ± 0.81 0.3% 9.44 ± 0.28

71.25 ± 1.41 57.56 ± 5.07 58.92 ± 1.76

3.50 ± 0.13 2.88 ± 0.41 3.24 ± 0.12

79.83 ± 2.90 65.58 ± 9.36 73.87 ± 2.78

5.92 ± 0.30 4.18 ± 0.48 4.18 ± 0.13

56.80 ± 2.82 40.15 ± 4.58 40.13 ± 1.21

20.84 ± 0.59 16.28 ± 1.67 16.86 ± 0.45

67.59 ± 1.91 52.82 ± 5.5 54.70 ± 1.45

Surface DAPP application 0.1% 8.75 ± 0.52 0.15% 8.97 ± 0.67 0.3% 5.15 ± 0.11

54.59 ± 3.23 55.96 ± 4.16 32.12 ± 0.66

2.51 ± 0.14 2.36 ± 0.25 1.92 ± 0.04

57.14 ± 3.16 53.72 ± 5.79 43.68 ± 0.93

3.45 ± 0.21 3.33 ± 0.28 1.78 ± 0.02

33.12 ± 2.01 32.01 ± 2.64 17.08 ± 0.14

14.70 ± 0.86 14.66 ± 1.19 8.84 ± 0.11

47.70 ± 2.79 47.55 ± 3.86 28.69 ± 0.37

Control

a

Results are expressed in ng/g.


for 16 min (Britt, Gomaa, Gray, & Booren, 1998) and 1.4 ng/g IQx was noted in meatballs fried at 175 °C for 15 min (Oz & Kaya, 2011a). Frying temperature and duration appear to affect IQx formation, which could have led to the lack of its detection in the present work. For example, 4.85 ng/g IQx was detected in meatballs fried for 15 min at 200 °C, but was not measurable in meatballs fried for the same duration at 225 °C (Oz & Kaya, 2011a), similar to the frying temperature and duration used in our study. 3.3. Effect of DAPP dose and type of application on HCA formation When DAPP was applied to the surface of beef patties at 0.1%, 0.15% and 0.3% prior to frying at 223 °C, total HCA formation was inhibited by 52%, 55% and 71%, respectively. The mixed application of DAPP at concentrations of at 0.1%, 0.15% and 0.3% within the beef patties was associated with inhibition by 32%, 45% and 45%, respectively. Hence, greater inhibition of total HCA formation was noted by surface versus mixed application of DAPP. Inhibition of the formation of individual HCAs was shown by the application of DAPP prior to frying regardless whether DAPP was applied on the surface or mixed within the patty (Table 1). A decrease in MeIQx formation was observed with both mixed and surface application, with all treatment doses leading to a statistically significant decrease (Table 1). In terms of mixed application of DAPP, significantly (p b 0.05) greater inhibition was seen with 0.15% and 0.3% versus 0.1% while surface DAPP application at 0.3% showed greater inhibition of MeIQx than was observed at 0.10% and 0.15% (Table 1). The above findings coincide with observations of concentration-dependent effects of antioxidants on HCA formation (Johansson & Jägerstad, 1996). Surface application, however, led to a more efficient suppression of MeIQx formation (up to 68% at the 0.3% dose) than mixed application (up to 41% at the 0.3% dose) and the effect of surface versus mixed application was statistically significant (p b 0.05) at the doses of 0.1 and 0.3% of DAPP (Table 1). Similar results were observed for 4,8-DiMeIQx as surface application led to 43–57% decrease whereas a 21–35% decline was seen with the mixed application with all doses corresponding to a significantly greater (p b 0.05) effect with surface versus mixed application (Table 1). On the other hand, no difference was observed in 4,8-DiMeIQx formation between different concentrations of surface and mixed application. The greatest inhibition of HCA formation was observed with PhIP with a maximum inhibition of 83% observed with the 0.3% dose after surface application and a 60% maximum inhibition of PhIP formation associated with the mixed application (Table 1). Mixed application of DAPP showed significantly (p b 0.05) greater inhibition of PhIP at 0.15% and 0.3% versus 0.1% whereas surface DAPP application at 0.3% showed greater inhibition than 0.15% and 0.1% (Table 1). The effect of surface versus mixed application was statistically significant (p b 0.05) at all treatment doses except for 0.15% for MeIQx and 4,8-DMeIQx (Table 1). The above findings demonstrate that surface versus mixed application of DAPP generally leads to a more robust decrease in total HCA formation in pan-fried beef patties by approximately 22–27%. The demonstrated relationship between increased temperature and increased HCA concentration (Chen & Chiu, 1998; Knize et al., 1994) suggests that the exterior portions of meat most exposed to direct cooking generate higher amounts of HCAs, which can explain the better efficacy shown by DAPP surface application. Regardless, integration of DAPP within the meat patty was efficacious in limiting HCA formation, which suggests that mixing of polyphenol-rich extracts within meat patties could still serve as a useful alternate approach. The inhibition of HCA formation by DAPP pretreatment is explainable by the antioxidant properties previously demonstrated for DAPP (Denis et al., 2013) since antioxidants are considered to play an important role in decreasing HCA formation in fried meats (Gibis & Weiss, 2012; Rahman, Sahar, Khan, & Nadeem, 2014). Support for this contention has been noted by single antioxidant application in terms of vitamin E, which when added at either at 1% and 10% of total fat content

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decreased PhIP formation by 69% and 72%, respectively (Balogh et al., 2000). Polyphenols have been indicated to inhibit HCA production via the trapping of free radicals generated from the Maillard reaction (Vitaglione & Fogliano, 2004). Several studies have shown reduction of HCA formation in fried meat products following application of a variety of polyphenol-containing products including wine, tea, cherries, beer as well as extracts from hibiscus, rosemary, grape seed and various fruits (Britt et al., 1998; Cheng et al., 2007; Gibis & Weiss, 2010, 2012; Melo, Viegas, Petisca, Pinho, & Ferreira, 2008; Persson, Graziani, Ferracane, Fogliano, & Skog, 2003). Flavonoid compounds have previously been demonstrated to inhibit PhIP formation (Weisburger, Nagao, Wakabayashi, & Oguri, 1994). We observed that both surface and mixed application of DAPP provided greater inhibitory effects on PhIP formation, despite the relatively greater concentrations of MeIQx. This observation is consistent with several findings showing similar effects following treatment of ground beef patties with spice powders, essential oils and plant extracts including apple peel extract (Rounds, Havens, Feinstein, Friedman, & Ravishankar, 2012). The high degree of inhibition of PhIP formation by DAPP is significant in view of evidence that the presence of this compound has been particularly linked with the mutagenic and carcinogenic properties of cooked meat (Sugimura, Wakabayashi, Nakagama, & Nagao, 2004). The degree of HCA reduction appears to depend on the potency and concentration of the applied plant extract as well as the concentration of the HCAs in the control meat samples. In a study of beef chops fried at 175°C for 15 min with a total HCA content of 2.88 ng/g, surface application of red pepper at 1% showed a reduction of MeIQx and PhIP formation by 88% and 100%, respectively (Oz & Kaya, 2011b). In relation to the present findings, the greater efficacy of red pepper could be due to a combination of the high dose of red pepper used and relatively low HCA formation in the control samples. Cheng et al. (2007) evaluated the inhibitory effects on HCA formation following incorporation of 0.1% of four fruit extracts (apple, elderberry, grape seed, and pineapple) within meat patties, which were fried on each side for 6 min at 210 °C. They showed that apple extracts inhibited the formation of PhIP, MeIQx and 4,8-DiMeIQx by 69%, 59% and 62%, respectively, whereas our findings show that surface application of 0.3% DAPP resulted in a relatively greater inhibition of PhIP of 83% together with similar decreases of MeIQx and 4,8DiMeIQx of 68% and 57%, respectively. Direct comparison between the two studies is complicated by the lower frying temperatures and shorter frying time in the Cheng et al. (2007) study that resulted in much lower mean concentrations of 4,8-DiMeIQx (0.95), MeIQx (2.96) and PhIP (10.10) ng/g than detected in the present study. Previous work has indicated that potency of antioxidant food ingredients to inhibit HCA production can vary according to the cooking temperature used. For example, surface application of 1% red pepper on beef chops fried at 175 °C showed 100% inhibition of total HCAs whereas only a 75% decrease was observed at 225 °C (Oz & Kaya, 2011b). Rounds et al. (2012) have shown that mixed application into ground beef patties of a more than 10-fold higher dose of apple skin extract (5%) than used in the present work resulted in a similar reduction of PhIP (82.1%) and MeIQx (76.1%). In another study, mixed application of 1% apple extract into ground beef patties exerted no effect on HCA formation while 3% apple extract reduced PhIP and MeIQx by 50.9% and 65.2%, respectively (Rounds, Havens, Feinstein, Friedman, & Ravishankar, 2013). The major dose-related differences in HCA inhibition using apple extracts seen previously in comparison to the present findings could be related to wide variations in proanthocyanidin content noted among apple varieties due to differences in genetic capacity for synthesis of secondary metabolites (USDA, 2004). Proanthocyanidin content can also be affected by season to season climatic variations, farming practices and post-harvest handling (USDA, 2004). Different extraction techniques used for the preparation of apple extracts could also play a role (Rounds et al., 2012). In conclusion, the present study highlights the use of DAPP in meat preparation as a means to reduce formation of genotoxic HCAs during

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