Five marinades were developed using natural antioxidants. Based on a consumer test of the appearance, three marinades were chosen for further investigation: an oregano, an acerola and a Dijon marinade, the Dijon marinade containing acerola, sumac and oregano. A barbecue and a sumac marinade were not included in the further study. In a home use test, consumers barbecued pork chops with the three marinades. Most of the consumers preferred the oregano and the Dijon marinade, while the acerola marinade was less liked. After controlled cooking using both direct and indirect heat to a core temperature of 65°C and 80°C, the content of the heterocyclic aromatic amines (HCA) PhiP, MeIQx, DiMeIQx, Harman and Norharman was analysed. All of the marinades reduced the content of MeIQx and DiMeIQx, although only with indirect heat, while PhiP was reduced using both grilling methods. Surprisingly, in particular the content of Harman—and to a lesser extent Norhaman—was very high in the Dijon-marinated chops. The results demonstrated that it was possible to develop well-liked marinades that can reduce the formation of selected HCAs.
When meat is heated, potentially harmful compounds (heterocyclic aromatic amines (HCA)) can be formed. Two pathways exist: a Maillard-like reaction involving creatine at temperatures ranging between 160˚C and 250˚C [
HCAs have been proposed as one of the mechanisms behind the observed association between meat intake and colorectal cancer [
The formation of HCAs is a redox reaction, and antioxidants have the potential to reduce the formation of the HCAs. Several studies have shown that natural antioxidants in the form of spices, berries, wine or beer can reduce the formation of HCAs in beef [
Most of the studies investigating different natural antioxidants have used either a pure spice or berries or an extract of these in the experiments [
The aim of this investigation was to evaluate the effect of palatable complex marinades based on acerola, oregano and sumac on the inhibition of the formation of heterocyclic amines and to relate the marinades to the consumer responses.
In our study, we focused on marinades based on one herb (oregano) and two berries (acerola and sumac). Oregano (oregano vulgare) has high antioxidant capacities because of its content of polyphenols, flavonoids and different compounds in the essential oil such as carvacrol, thymol, γ-terpinene and linalool [
The common ingredients of the marinades were purchased at a local grocer. Frozen acerola puree (Bloch & Schou Aps, Viborg, Denmark) was freeze-dried before use. Oregano and sumac (ENES Gewürze GmbH, Geesthacht, Germany) were used dried as they were bought. The acerola and oregano marinade was an oil/water emulsion (O/W). The Dijon marinade and sumac marinade was oil based while the barbecue marinade was water-based. The ingredients are given in
Boneless pork loins (m. longissimus dorsi) with approximately 3 mm fat rim, purchased from a commercial Danish slaughterhouse, were used for all experiments. The loins were sliced into 15 chops of 2 cm thickness.
Two different consumer studies were performed: a central location test (CLT) of the appearance of the chops and a home-use test (HUT) of the three selected marinades to assess the liking after barbecuing.
The CLT was carried out in a canteen setting in Taastrup, a suburb of Copenhagen, the capital of Denmark. A total of 109 consumers participated (aged between 20 and 60 years, 61% male and 39% female). The consumers evaluated ten samples: the five marinades either as pre-marinated chops or presented next to the chops in a small bag (see
Seventy-six families, recruited from the area around Copenhagen, participated in the HUT. Up to four persons per family could evaluate the samples, but from most families
Acerola | Dijon | Oregano | Sumac | Barbecue | |
---|---|---|---|---|---|
Water | E1_52.7 | 41.1 | |||
Oil | E_20 | 22.2 | 76.1 | 70.4 | |
Dijon mustard | 30.9 | 1.2 | |||
Acerola, freeze dried | 6.0 | 0.6 | 2.5 | 2.9 | |
Oregano, dried | 5.6 | 4.1 | |||
Sumac | 7.1 | 7.0 | |||
Sugar, brown | 12.3 | 5.2 | |||
Ginger purée | 14.0 | ||||
Garlic purée | 7.4 | 2.1 | |||
Coriander seeds | 7.0 | ||||
Cumin | 7.0 | ||||
Chili powder | 1.1 | ||||
Spice blend2 | 9.3 | ||||
Thyme, dried | 1.7 | ||||
Onion powder | 1.1 | ||||
Pepper, grounded | 0.6 | ||||
Tomato concentrate | 28.9 | ||||
Soy sauce | 18.5 | ||||
Worchester sauce | 7.2 | ||||
Apple cider vinegar | 2.2 | 1.7 | 2.1 | ||
Salt | E_4.0 | 7.9 | 5.0 | ||
Maizena | E_2.0 | ||||
Sodium caseinate | E_1.0 | ||||
Guargum | E_0.2 | ||||
Xanthan | E_0.1 | ||||
Total | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
two persons participated. A total of 176 consumers aged between 20 and 60 participated (95 males and 81 females). On a Friday, the consumers were given three packages of chops and the three marinades: acerola, Dijon and oregano, packed in small bags. The consumers were instructed to barbecue the chops at home on the same day during the weekend in the way they would normally do so. On a line scale (11.4 cm) ranging from “very bad” to “very good” anchored 1 cm from each end, the consumers assessed liking of the appearance and odour of the barbecued chop, followed by how much they liked the chop when eaten and finally whether they could imagine buying a chop with this marinade another time. The order of the chops was not controlled, but, since the consumers had to fill in the number of the marinades themselves, a random order between families was expected.
Each family member was asked to complete a short questionnaire including age in
10-year intervals, gender, how often they had barbecued chops in the barbecue season (every day, every week, every month, more seldom) and how often they ate marinated meat (every week, every month, more seldom).
On the Monday, the consumers returned the questionnaires together with one barbecued chop for each of the three types of marinade.
To investigate the effect of the marinades on reduction of the development of HCAs, controlled barbecue cooking was carried out indoor at Grilleriet (www.grilleriet.dk) with a controlled room temperature (20˚C) and a constant airflow and humidity. A Weber One-touch® premium barbecue and coconut charcoal (www.kokosbriketter, Videbæk, Denmark) were used. Both direct heat using a distance of 10 cm from the heat (open barbecue) and indirect heat after initially one minute of direct heat, with a distance of 1 cm from the heat on each side (closed barbecue), were used. When direct heat was used, the chops were turned every two minutes, and when indirect heat in the closed barbecue was used, the chops were turned every third minute to avoid having to open the lid too often. The temperature of the barbecue using direct heat was approximately 270˚C, measured with an infrared thermometer (BUHL & BØNSØE A/S, Virum, Denmark), while the temperature of the barbecue when using indirect heat was between 240˚C and 290˚C, measured with the barbecue’s built-in thermometer. Six chops with each marinade were grilled simultaneously on the same barbecue. Half of them were removed from the heat when they had reached a core temperature of 65˚C, while the remaining three were grilled until they had reached a core temperature of 80˚C. This was repeated twice, giving a total of six chops for each treatment (barbecue type and temperature). In addition to the three marinades, pork chops marinated in rapeseed oil and pork chops without any marinade (blank) were also grilled, giving a total of five different types (three marinades, rapeseed oil, blank), two barbecue types, two core temperatures and six replicates. The 30 treatments were arranged in a Latin square design on pairs of two loins.
Multispectral imaging (Videometer A/S, Hørsholm, Denmark) was used to estimate the core temperature. Based on the average surface spectra of the chops from the controlled cooking (open barbecue), three separate models were predicted by partial least squares regression (Unscrambler X10.3, Camo software A/S, Oslo, Norway)―one for each marinade. The core temperature of the HUT grilled pork chops was estimated from these models.
The HCAs were extracted from the meat crust using hollow-fibre (HF) extraction followed by an LC-MS/MS analysis [
Blended crust was homogenized in 0.05 M NaOH, and spiked with internal standards (harman-d3, norharman-d7, PhIP-d3), (100 μl, 2 mg/l). After mixing the homogenate for 1 h, followed by a centrifugation, 2 mL of the supernatant was transferred for extraction using the HF device. The HF was immersed into the sample and the extraction was carried out for 90 min. The sealed end of the HF was cut off and the acceptor phase inside the fibre lumen was transferred to a vial. The sample amount was adjusted to 0.100 g with Milli-Q water.
The chromatographic separations were performed with the following modifications: using an ACQuity UPLC BEH C18 (2.1 × 50 mm 1.7 µm) column at 40˚C (Waters Corporation, Milford, Mass., USA) and a Krud Katcher UHPLC in-line filter (0.5 µm depth filter x0.004 in ID, Phenomenex, Torrace, CA, USA) on a uHPLCN exera X2 system (Shimadzu, Kyoto, Japan). Separation was achieved with a binary mobile phase at a flow rate of 0.5 mL/min. Solvent A was acetonitrile, and solvent B was 10 mM ammonium formiate at pH 3.0. The gradient elution programme was: 0 - 0.2 min, 95% A, 0.2 - 6.0 10% A, 6.1 - 9.0 min 95% A, return to original condition. The injection volume was 5 µL. The analysis used a hybrid triple quadrupole (QqQ)/linear ion trap mass spectrometer from AB Sciex (QTRAP® 6500, AB SCIEX, Concord, ON, Canada).
The data from the CLT appearance test were analysed using Panel check (version 1.4.0, Nofima, Norway). A PCA analysis was performed using unstandardised data, first on all data followed by the subset of the chops presented with the marinade in a bag.
Liking data from the HUT were analysed using a mixed model analysis of variance with the marinade as fixed effect and the family as random effect (Proc mixed, SAS version 9.4, Cary NC, USA).
The logarithmic content of HCAs in the pork chops after controlled cooking was analysed using an analysis of variance with marinade, temperature, grill method and the interaction between these as fixed effects (Proc mixed, SAS version 9.4, Cary NC, USA). The post-hoc comparison was performed using Bonferroni adjustment for multiple comparisons.
In a preliminary study, the antioxidative of several herbs, berries and other active ingredients was assessed using two assays: oxygen consumption and DPPH. This was compared with literature data on ORAC values. On this basis, oregano, sumac and acerola was chosen for the further experiment. In the development of the marinades, other ingredients were added to get the best gastronomic quality. Some of these ingredients are also known to have antioxidative properties. A list can be seen in
The main difference between the samples in the CLT of appearance was whether they were pre-marinated or packed with the marinade in a bag placed next to them (
A closer examination of the samples with the marinades in the bag (right plot) showed that especially the acerola marinade was perceived as being strange and sticky, while especially oregano and Dijon were perceived as being appetizing, exciting, familiar and natural. Barbecue marinade was more sticky, familiar and natural compared
Oxygen consumption1 μmol TE/100 g2 | DPPH1, %3 | ORAC μmol TE/100g | |
---|---|---|---|
Oregano, dried | 6.2 | 12.0 | 200,1294 |
Sumac, bran | 3.9 | 11.5 | 312,4004 |
Acerola | 59.0 | 8.8 | 70,0005 |
Garlic, raw | 1.7 | 7.1 | 53464 |
Ginger | - | - | 28,8114 |
Thyme | 2.4 | 11.2 | 157,3804 |
Chilli powder | 2.9 | 13.2 | 23,6364 |
Onion | - | - | 8635 |
1from a prestudy. 2μmol TE/100 g (micromol Trolox Equivalent per 100 grams). 3Scavenging activity in 5 mL DM/mL. 4from http://modernsurvivalblog.com/health/high-orac-value-antioxidant-foods-top-100/. 5from http://superfoodly.com/superfoodly-orac-values.pdf.
with sumac, which was perceived as being strange, but also exciting. Based on this, the oregano and Dijon marinades were chosen for the HUT and controlled cooking. However, since acerola has strong antioxidant properties, and since a closer examination of the data showed that a small segment of the consumers actually liked it (data not shown), this marinade was selected as the third in the HUT and controlled cooking.
In the HUT, the consumers assessed liking after barbecuing at home. In general, the consumers liked all three types of marinades, with all of them getting an average score above the middle of the scale (
The surface colour of the chops cooked by the consumers was measured using Videometer Lab and related to the surface colour of the chops from the controlled cooking in order to estimate the core temperature (
The meat was estimated to be cooked to below 65˚C by 42% of the consumers, between 65˚C and 70˚C by 24% and between 70˚C and 80˚C by 34%. The methodology of comparing the cooked surface of the consumer chops with the cooked surface of the chops from the controlled cooking is somewhat imprecise. Nevertheless, it was shown that in general only relatively few consumers (14%) cooked the chops to above 75˚C.
Three heterocyclic aromatic amines with mutagenic properties, MeIQx, DiMeIQx and PhiP, were analysed (
How much do you like… | Acerola | Dijon | Oregano | p-value |
---|---|---|---|---|
the appearance of the grilled chop? | 7.1b ± 2.3 | 8.1a ± 2.1 | 8.2a ± 2.2 | <0.001 |
the odour of the grilled chop? | 6.5b ± 2.3 | 7.9a ± 2.1 | 8.0a ± 2.2 | <0.001 |
the chop when you eat it? | 6.5b ± 2.4 | 7.9a ± 2.3 | 8.0a ± 2.4 | <0.001 |
Would you eat it again? | 5.7b ± 3.0 | 7.6a ± 2.9 | 8.0a ± 3.0 | <0.001 |
MeIQx | T | Acerola | Dijon | Oregano | Rapeseed Oil | Blank |
---|---|---|---|---|---|---|
Indirect | 65 | 0.08ab (0.10) | 0.14a (0.10) | 0.02b (0.02) | 0.11ab (0.10) | 0.29a (0.14) |
80 | 0.13ab (0.06) | 0.10ab (0.03) | 0.06b (0.07) | 0.29a (0.06) | 0.65a (0.25) | |
Direct | 65 | 0.44a (0.08) | 0.32a (0.13) | 0.47a (0.20) | 0.51a (0.20) | 0.53a (0.24) |
80 | 0.73a (0.47) | 0.95a (0.31) | 1.03a (0.74) | 0.60a (0.08) | 1.11a (0.40) | |
DiMeIQx | Acerola | Dijon | Oregano | Rapeseed Oil | Blank | |
Indirect | 65 | 0.00c1 | 0.03b (0.04) | 0.00c1 | 0.09ab (0.07) | 0.09a (0.09) |
80 | 0.09b (0.04) | 0.00c1 | 0.02c (0.03) | 0.11ab (0.03) | 1.55a (2.28) | |
Direct | 65 | 0.06a (0.07) | 0.05a (0.07) | 0.11a (0.08) | 0.27a (0.10) | 0.19a (0.07) |
80 | 0.49a (0.49) | 0.13a (0.03) | 0.28a (0.18) | 0.13a (0.04) | 1.54a (1.61) | |
PhiP | Acerola | Dijon | Oregano | Rapeseed Oil | Blank | |
Indirect | 65 | 0.25b (0.15) | 0.20b (0.11) | 0.23b (0.19) | 2.17a (0.10) | 4.50a (1.02) |
80 | 0.73bc (0.23) | 0.04d (0.04) | 0.23c (0.16) | 1.57ab (0.06) | 5.22a (1.57) | |
Direct | 65 | 0.66b (0.27) | 0.42b (0.26) | 1.37ab (0.89) | 3.69a (0.20) | 5.43a (2.69) |
80 | 3.11b (5.36) | 0.72b (0.19) | 1.52b (1.08) | 3.23ab (0.08) | 11.35a (3.41) | |
Harman | Acerola | Dijon | Oregano | Rapeseed Oil | Blank | |
Indirect | 65 | 1.92b (0.43) | 32.01a (8.67) | 2.47b (0.20) | 2.97b (1.11) | 2.70b (0.92) |
80 | 4.43b (0.66) | 27.57a (5.47) | 2.43b (0.40) | 2.34b (0.41) | 3.35b (1.39) | |
Direct | 65 | 2.74b (0.88) | 49.58a (15.21) | 4.30b (2.73) | 0.98c (0.20) | 0.93c (0.48) |
80 | 3.02b (1.16) | 66.57a (14.77) | 4.59b (2.55) | 1.09b (0.22) | 3.55b (1.66) | |
Norharman | Acerola | Dijon | Oregano | Rapeseed Oil | Blank | |
Indirect | 65 | 4.02b (1.26) | 11.39a (2.83) | 3.35b (0.37) | 5.76ab (2.49) | 3.44b (1.14) |
80 | 9.42ab (1.63) | 10.67a (2.49) | 4.52b (0.70) | 6.60ab (1.61) | 8.46ab (8.16) | |
Direct | 65 | 2.41b (0.46) | 10.38a (3.23) | 3.52b (1.90) | 1.97b (0.19) | 1.51b (0.43) |
80 | 3.17c (1.76) | 11.62a (1.71) | 3.96bc (1.51) | 2.43c (0.58) | 5.66b (2.50) |
1Values < LOQ.
while the reduction was only significant for MeIQx in the oregano marinade, in which the formation of HCAs in general was almost totally inhibited. No effect was seen when cooking over direct heat for DiMeIQx and MeIQx. In contrast, PhiP was inhibited independently of the cooking method and temperature and was only insignificant for the oregano marinade when cooking over direct heat to a core temperature of 65˚C.
Harman and Norharman have comutagenic activity, meaning that they themselves have no mutagenic effects but can increase the mutagenicity of other HCAs (Jägerstad, Skog, Arvidsson, & Solyakov, 1998). Unfortunately, a consistent and very high concentration of, in particular, Harman, but also Norharman, was seen after marinating with the Dijon marinade. The concentration was much higher than in the control sample. No clear reducing effect of the other marinades was seen on Harman and Norharman compared with the blank sample.
Cold pressed rapeseed oil used for the marinades has an antioxidant effect on its own, and it was therefore of interest also to investigate the effect of the pure oil. No significant reduction of pure rapeseed oil was seen for any of the investigated HCAs except Norharman cooked over direct heat to 80˚C. However, a numerical decrease was seen, indicating that the oil might have some properties that inhibit the formation of HCAs.
Barbecuing is one of the cooking methods that results in the highest content of HCAs [
Based on the test of the appearance, three marinades were chosen for further study― Dijon and oregano, which were clearly appreciated, and acerola, due to its promising antioxidant activity, even though only a few of the consumers liked the appearance. This lower liking was also found during the HUT, with the acerola marinade getting lower scores for all attributes, especially the question “would you eat it again?”, compared with the Dijon and oregano marinades.
When the controlled grilled samples were used to estimate the core temperature of the consumer samples, it was seen that only approximately one third of the consumers cooked the meat to above 70˚C. This is in contrast to a previous study in Denmark, in which more than half of the estimated core temperatures in consumer-cooked pork chops were 75˚C or more [
In the controlled barbecuing, coconut charcoal was used because they are said to give a higher and more stable temperature (Grilleriet®, personal communication), which is crucial to keeping the conditions as controlled as possible during the cooking process. It has been shown that the concentration of PhiP is lower in meat cooked on this type of charcoal compared with wood charcoal [
PhiP was the most abundant HCA, which confirms several other studies [
The acerola marinade was neither well-liked nor as effective as the oregano marinade in reducing the HCAs, especially at 65˚C, even though acerola is known to be an effective antioxidant [
This study clearly demonstrates that it is possible to tailor-make marinades that are effective in inhibiting the formation of heterocylic aromatic amines based on knowledge about the antioxidant capacity of herbs and berries and shows that the herbs and berries were effective as ingredients in a complex marinade. However, it was also shown that knowledge about the antioxidant capacity was in itself insufficient to predict the effectiveness of the marinade, since the content of Harman and Norharman was surprisingly high in the Dijon marinade. Using consumer studies is an effective and powerful way to investigate liking of marinades.
This study was funded by the Pig Levy fund, to whom we are very grateful. A special thanks to master’s student Lone Green Ensig, who developed the marinades and performed the consumer studies, and to Jane Skou Olsen, Tine Frogne and Pia Krall Fauschou, who performed the chemical analysis of the HCAs. Claus Borggaard is thanked for doing the data analysis on the multispectral imaging data.
Aaslyng, M.D., Lund, B.W. and Jensen, K. (2016) Inhibition of Heterocyclic Aromatic Amines in Pork Chops Using Complex Marinades with Natural Antioxidants. Food and Nutrition Sciences, 7, 1315-1329. http://dx.doi.org/10.4236/fns.2016.714120