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Effect of selected local spices marinades on the reduction of heterocyclic amines in grilled beef (satay)
LWT - Food Science and Technology 63 (2015) 919e926
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Effect of selected local spices marinades on the reduction of heterocyclic amines in grilled beef (satay) S. Jinap a, b, *, Shahzad Zafar Iqbal a, Roshny M.P. Selvam a a b
Food Safety Research Centre (FOSREC), Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2015 Received in revised form 16 April 2015 Accepted 19 April 2015 Available online 28 April 2015
The present study focused to investigate the effect of some selected spices (turmeric, torch ginger, lemongrass and curry leaves) on the formation of heterocyclic amines (HCAs, IQx, MeIQ, MeIQx, DiMeIQx, IQ, norharman, harman and AaC) in grilled satay (beef). Satay samples were marinated with 1 e4 g/100 g concentration of turmeric and 2.5e10 g/100 g concentration of torch ginger, lemongrass and curry leaves, at different medium (70 C) and well done (80 C) doneness cooking temperatures. The concentration of HCAs in grilled satay samples were analysed using LC/MS technique. The results have shown that turmeric reduced maximum 82 ng/100 g level of IQ (11.43 ± 0.53 to 2.05 ± 0.18 ng/g) at 4 g/ 100 g concentration at medium doneness and 44.4 ng/100 g level was reduced of IQ, when satay was marinated with 10 g/100 g lemongrass concentration at medium doneness. The highest level of MeIQX was reduced to 83 ng/100 g of satay meat marinated in 10 g/100 g torch ginger at medium doneness. Curry leaves with 10 g/100 g concentration marinated beef meat was reduced the level of IQ 78.5 at medium doneness. The use of local spices in marinating of grilled beef will certainly inhibit/reduce the level of toxic and harmful HCAs. Significance of results for industry: The use of native spices during grilling of meat will minimize the formation of heterocyclic amines and therefore will help to avoid toxic and carcinogenic effects from these toxins. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Local spices Beef meat HCAs
1. Introduction Studies have proved that different kind of harmful components, including various mutagens and carcinogens, may be produced, when meat is heat-treated using traditional procedures such as €gerstad & Skog, 2005). It has frying, barbecuing and smoking (Ja been observed that all types of meat such as beef, pork, goat and lamb, may produce heterocyclic aromatic amines (HCA) when cooked at high temperatures. HCAs are recognized as mutagenic and carcinogenic compounds (Damasius, Venskutonis, Ferracane, & Fogliano, 2011). Currently, more than 25 HCAs have been isolated and recognized in cooked foods, since their discovery (Alaejos & Afonso, 2011; Murkovic, 2007) and divided in two main families:
* Corresponding author. Food Safety Research Centre (FOSREC), Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: þ60 3 8946 8393; fax: þ60 3 8942 3552. E-mail address: [email protected] (S. Jinap). http://dx.doi.org/10.1016/j.lwt.2015.04.047 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
aminoimidazo-azaarenes or thermic HCAs and amino-carbolines or pyrolytic HCAs. These HCAs includes 2-amino-3- methylimidazo [4,5-f]quinoline (IQ), 2-amino-3-methylimidazo [4,5-f] quinoxaline (IQx), 2-amino-3,4-dimethylimidazo [4,5-f]quinolone (MeIQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) (Knize, Dolbeare, Carroll, Moore, & Felton, 1994). Thermic HCAs are produced as a result of complex reaction between creatine/creatinine, free amino acids and sugars through the Maillard reaction at temperatures between 150 and 250 C €gerstad, Skog, Arvidsson, & Solyakov, 1998; Nagao, Honda, & (Ja Seino, 1977). However, the pathway of pyrolytic HCAs production was not so clear but suggested that it may be produced as a result of pyrolysis of proteins or amino acids heated at higher temperatures i.e. > 250 C (Matsumoto, Yoshida, & Tomita, 1981). In Southeast Asia (Malaysia, Indonesia, Thailand, and Singapore), satay is a very popular grilled food similar to shish kebab which is also popular in western and middle-eastern countries. It consists of dice-sized chunks or slices of boneless meat
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(chicken, beef, mutton, pork, fish), on skewers made from the midrib of coconut leaf or bamboo. It is usually grilled over wood or charcoal fires, and then served with various spicy seasonings depending on the satay recipe. Mostly charcoal grilling is the common cooking method for satay prepared in Southeast Asian countries. Compared to other cooking practices such as prolonged heating or frying, this fast grilling method can produce a different mixture of HCAs. The HCAs (IQ,MeIQ, MeIQx and PhIP) are identified as compounds reasonably anticipated to be a human carcinogen. However, the International Agency for Research on Cancer has classified MeIQx, MeIQ and PhIP as reasonably anticipated to be a human carcinogen, while IQ is placed in the list of a probable human carcinogen (IARC, 1993). Epidemiological studies (Knutsen, Binderup, Vikse, & Øvrebø, 2007; Norat et al., 2005) have shown an increased risk of cancer in the intestine, bladder, prostate, breast and pancreas after high consumption of fried, well-done and barbecued meat. Previous studies have revealed that by adding ingredients such as tomatoes to marinades before cooking, is effective to reduce the concentration of HCA (Persson, Graziani, Ferracane, Fogliano, & Skog, 2003), tart cherry tissue (Britt, Gomaa, Gray, & Booren, 1998), and spices (such as onion and garlic) (Shin, Gomaa, Strasburg, & Gray, 2002). The present study is focused to investigate the effect of local Malaysian spices including turmeric, lemongrass, torch ginger and curry leave on the reduction of HCAs in grilled beef (satay) at different doneness cooking.
Massachusetts, USA). MCX cartridges were preconditioned with ethyl acetate (2 mL). The samples (n ¼ 3) of fresh beef (sirloin) 5 kg of local origin were purchased from local markets of Selangor, Malaysia. Visible connective tissues and external fat were removed before storage in freezer at 20 C. The samples of beef were thawed overnight in a cold room (4 ± C). The pH of meat samples was determined prior to analysis. The meat was then cut into big pieces again the grain, in which each piece has weight about 100 ± 0.5g. All the cut meat samples were put into polyethylene containers and stored in the refrigerator (4 ± 1 C). 2.2. Local spices preparation The fresh turmeric, torch ginger flower, curry leaves and lemongrass were obtained from local market, Selangor, Malaysia. The skins of fresh turmeric samples were removed and the curry leaves were stripped off from their stalks. The lemongrass was separated from its stalks and leaves. All the cleaned spices were brought to washing, wiped with paper towel and chopped into small pieces separately. The weight of each spice was noted and kept in polyethylene container. The samples were then kept in freezer (20 C) for overnight and then brought to freeze drying. The samples were freeze-dried for two days, weighed and then grounded using a dry grinder. The homogenized powder samples were kept in polyethylene containers and stored at 20 C. The moisture content of each powdered sample was determined prior to analysis.
2. Materials and methods
2.3. Preparation of marinades formulation
2.1. Chemicals
A range of each individual spice was developed prior to this analysis based on the acceptable maximum level in satay recipes and these levels are optimised. The optimised range for turmeric was 0, 1, 2, 3, and 4 g/100 g, while other spices have ranges 0, 2.5, 5, 7.5 and 10 g/100 g of meat. Each spice concentration was applied (2 replicates) on meat samples, then mixed and left for 24 h. The meat samples having no treatment of spices were control samples. Percentage of marinade uptake for each treatment was determined just after marination (0 h) and after 24 h (Table 1). The weight of sample before marination (W0) and after marination (W1) was recorded. The percentage weight gain was calculated based on formulation.
Standards of HCAs used, 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino- 3,8-dimethyl-imidazo[4,5-f] quinoxaline (MeIQx), 2amino-3,4,8- trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), norharman, harman and AaC were purchased from Toronto Research Chemicals (Toronto, Canada). A stock solution of 100 mg/g in methanol was prepared for each HCAs. Diatomaceous earth (Extrelut 20) was obtained from the International Sorbent Technology (Hengoed Mid Gleam, UK) and Oasis MCX cartridges (3 cm3/60 mg) were purchased from Waters (Milford,
Table 1 Marinate uptake of meat at 0 and 24 h duration. Spices
Concentration of spices (g/100 g)
Marinate uptake at 0 h (g/100 g)
Turmeric
1 2 3 4 2.5 5.0 7.5 10 2.5 5.0 7.5 10 2.5 5.0 7.5 10
0.95 1.82 2.79 3.69 2.15 4.05 6.69 8.22 2.20 2.34 2.97 3.31 2.29 2.42 2.71 2.91
Lemongrass
Torch ginger flowers
Curry leaves
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.17aA 0.12bA 0.05cA 0.13dA 0.19aA 0.27bA 0.28cA 0.35dA 0.02aA 0.06aA 0.12bA 0.16bA 0.03aA 0.03abA 0.06bcA 0.16cA
Marinate uptake after 24 h (g/100 g) 0.38 1.21 2.08 3.00 0.91 2.24 3.77 4.66 1.05 1.35 1.85 2.39 1.32 1.40 1.57 1.69
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.18aA 0.08bB 0.04cB 0.16dA 0.30aB 0.11bB 0.44bcB 0.32cB 0.04aB 0.08abB 0.08abB 0.52bA 0.06Ab 0.04abB 0.00abB 0.09bB
Values with same English lowercase within row of each species are not significant (p 0.05). Values with same English uppercase letter within column of marinate uptake (0 and 24 h) are not significant (p 0.05).
Marinate uptake loss 0e24 h (g/100 g) 60.00a 33.52b 25.45b 18.70b 57.67a 44.69b 43.65b 43.30b 52.27a 42.31b 37.71c 27.79d 42.36a 42.15a 42.07a 41.92a
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Weight gain ðg=100gÞ ¼
W1 W0 100 W0
2.4. Preparation and grilling conditions of satay After marinade, sample from each experiment was cut into cubes by maintain the weight of each sample to 1 g. The cubes were skewed into bamboo sticks in one stick and then the prepared sticks were brought to grilling on electric grill. The grilling method was designed as described by Smith, Ameri, and Gadgil (2008) with some modification in grilling conditions. The satay samples were grilled at 300 C to achieve medium and well doneness of grilling. The surface temperature of griller and internal temperature of each satay was taken after grilling. The weight losses of satay samples were also determined. The hardness and colour of each grilled sample was also analysed. The medium doneness was 70 ± 4 C (3 min) and well done (80 ± 4 C, for 4 min). 2.5. Extraction of heterocyclic amines HCA were extracted from meat samples using the method developed and validated by Gross, Grüter, and Heyland (1992), and modified by Messner and Murkovic (2004). A sub-sample (1 g) of ground meat sample was dissolved in 12 mL of 1 mol/L NaOH. The suspension was homogenised using a shaker (Memmert, Berlin, Germany) at high speed for 3 h. The samples were then mixed with 13 g diatomaceous earth, and the mixtures were poured into an Extrelut 20-ml column. A 50 mL volume of ethyl acetate was used as extraction solvent and a volume of 25 mL collected from the 50 mL sample was passed through the MCX cartridges. The MCX cartridge was then washed with 2 mL of 0.1 mol/L HCl followed by 2 mL methanol. The analytes were then eluted with 2 mL of methanol: concentrated ammonia (19:1, mL:mL) before they were evaporated to dryness under a stream of nitrogen, and the final extract was dissolved in 100 mL methanol. 2.6. Mass spectrometry parameters An agilant 1200 LC modules (Agilent Technologies, USA), consisted of a quaternary pump and an auto sampler (Agilent Technologies, USA) coupled with Agilent 6410 triple quadruple mass spectrometer (Agilent Technologies, USA) with a pneumatically assisted electro spray interface was used. Data acquisition was performed in the positive ion mode. The additional operating conditions were as follows: capillary voltage ¼ 4 kV, nebulizer pressure ¼ 45 kPa, drying gas flow ¼ 9 L/min, dry gas temperature ¼ 300 C and fragmentation voltage was 190 V. For quantification and identification, data acquisitions were operated in the MS/MS mode. The selected reaction monitoring (SRM) analysis was performed as follow: m/z 199 / 184 (collision energy, CE 25 eV) for IQ, m/z 213 / 198 (CE 25 eV) for MeIQ, m/z 200 / 185 (CE 25 eV) for IQx, m/z 214 / 131 (CE 41 eV) for MeIQx, m/z 169 / 115 (CE 37 eV) for Norharman, m/z 228 / 213 (CE 21 eV) for DiMeIQx, m/z 183 / 115 (CE 37 eV) for Harman, m/z 184 / 140 (CE 33 eV) for AaC, and m/z 225 / 210 (CE 25 eV) for PhIP. The dwell time was set at 0.5s. For data acquisition and processing the Agilent Mass Hunter qualitative and quantitative analysis software was used. 2.7. Sensory tests A total of 40 panelists (graduate students) from the Faculty of Food Science and Technology participated in the pre-selection of panelists for the sensory evaluation of beef satay. They were
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selected through series of basic test and triangle test. After tests panelists (n ¼ 30) were selected for further training on the sensory evaluation of beef satay. Information on evaluation techniques used was presented to the panelists before the actual sensory test. The panelists were then performed the threshold test and quantitative descriptive analysis QDA tests at different sessions. The panellists first evaluated beef meat that had not been marinated to determine the preferred (optimal) effect of local spices at different doneness. Sensory scores (7 point hedonic scale) were rated using a 7 cm scale for colour (0e7, very light brown to brunt) texture (0e7, very tender to hard). The panellists have ranked the samples by a mark on the scale. For each spice, one portion was prepared without the addition of spice concentration (as control), while the other were prepared with different concentrations of spices. The satay was prepared in duplicate. Samples were cooked 1 h before sensory evaluation and kept warm in a warmer plate until being evaluated. All products were presented in three-digit codes. A general discussion session was held following the solution tasting to standardize the scores and the definition of the attributes. Fig. 1a and b shows the effect of different concentrations of selected spices marination on colour and texture on meat and satay, respectively. 2.8. Statistical analysis The data of samples (3 2) was given as mean ± standard deviation. The statistical analyses (Levene's test for homogeneity, ShapiroeWilk test for normality) were performed by using the statistical package MINITAB 16. Two way analysis of variance was performed to determine the significant difference among treatments at p 0.05. 3. Results and discussion 3.1. Marination uptake Table 1 shows the marination of spices at 0 and 24 h duration. The results showed that with increase in the concentration of spices the marination uptake was also increased. However after 24 h of marination the uptake of marination decreased subsequently, might be due to the loss of water. 3.2. Colour and tenderness of satay Table 2 shows visual observation on colour intensity of grilled satay marinated with selected spices at different concentrations. Browning of cooked muscle food indicates the presence of complexes formed from maillard reaction. According to Kizil, Oz, and Besler (2011), AIAs or IQ type of HCAs are generally produced from heat induced maillard reaction which involves free amino acids, creatine or creatinine and reducing sugar. Thus, Maillard reaction may results in the formation of HCAs. The longer the cooking time more browning and HCAs formation will occur. Table 2, all medium cooked control, turmeric and lemongrass treated samples had less browning than that of well-cooked samples. Marination process has effect on the reduction of browning of samples. Marinated sample might undergo less browning reaction compared to un-marinated samples (Smith et al., 2008). Thus, marinate uptake of meat and its corresponding weight loss influences the surface browning of meat. It has been observed that turmeric, lemongrass and torch ginger flower marination had good reduction of browning of sample. The results showed that torch ginger and curry leaves marinated samples had decreased browning up to 5 g/100 g of that spices. No differences of browning observed on medium and well-cooked torch ginger and curry
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Fig. 1. a: The effect of different concentrations of selected spices i.e. turmeric, curry leaves, torch ginger and lemon grass on meat. b: The effect of different concentrations of selected spices i.e. turmeric, lemon grass, torch ginger and curry leaves on satay.
leaves treated satay. The reduction of browning may be due to the presence of phenolic compounds in spices. The weight gain of meat obtained by marination and its corresponding weight loss are good determinants of meat tenderness. During marination, marinades slowly penetrate into meat. This results in weakening of the structures due to swelling of the meat, increased proteolysis and conversion of collagen to gelatine. This process results in decrease of mechanical resistance of meat and increased meat tenderness (Cheok, Chin, Yusof, Kamal, & Sazili, 2011). The results have shown that the tenderness of satay
marinated at maximum concentration of all spices was greater than that of minimum concentration. All medium cooked samples except 7.5 g/100 g torch ginger treated sample were tender compared to that of well-cooked samples as shown in Table 3. 3.3. Effect of local spices on HCAs reduction The concentration of HCAs in grilled beef samples were analysed using LC/MS and the range in control sample was 5.39 ± 0.40 to 43.52 ± 0.80 ng/g. The DiMeIQX has found in highest level with
S. Jinap et al. / LWT - Food Science and Technology 63 (2015) 919e926 Table 2 Visual observation of colour intensity of grilled satay marinated with selected spices at different concentrations. Spices
Control Turmeric
Lemongrass
Torch ginger flowers
Curry leaves
Concentration of spices (g/100 g)
colour intensity of satay Medium
Well done
1 2 3 4 2.5 5.0 7.5 10 2.5 5.0 7.5 10 2.5 5.0 7.5 10
þþþþþþ þþþ þþþ þþ þþ þþþþþ þþþ þþþ þþ þþþþþ þþþþ þþþ þþþ þþþþþþþ þþþþþþ þþþþþþ þþþþþþ
þþþþþþþ þþþþþ þþþþ þþþ þþ þþþþþþ þþþþ þþþ þþ þþþþþ þþþþ þþþ þþþ þþþþþþþ þþþþþþ þþþþþþ þþþþþþ
þ very light brown. þþ light brown. þþþ looked brown. þþþþ deep brown. þþþþþ light black. þþþþþþ moderately black. þþþþþþþ indicating black (burnt).
concentration 39.49 ± 0.98 43.52 ± 0.80 ng/g at medium (70 C) and well doneness (80 C) treatments, respectively. The effect on reduction of HCAs using local spices, turmeric, lemongrass, torch ginger and curry leaves with concentration of 1e4 g/100 g for turmeric and 2.5e10 g/100 g for rest of the spices were used to marinades beef samples. Fig. 2a, b, c and d represents the reduction of HCAs in beef marinated at different concentration of local spices at medium and well done doneness, respectively. Fig. 2a shows that turmeric with 4 g/100 g concentration has shown maximum reduction of IQ level (82 ng/100 g) in marinated beef
Table 3 Texture evaluation of grilled satay marinated with selected spices at different concentrations. Spices
Control Turmeric
Lemongrass
Torch ginger flowers
Curry leaves
þ very much tender. þþ very tender. þþþ tender. þþþþ texture is right. þþþþþ not tender not hard. þþþþþþ slightly hard. þþþþþþþ too hard.
Concentration of spices (g/100 g)
Hardness of satay Medium
Well done
e 1 2 3 4 2.5 5.0 7.5 10 2.5 5.0 7.5 10 2.5 5.0 7.5 10
þþþþþ þþþþ þþþþ þþþ þþ þþþþ þþþþ þþþ þþþ þþþþ þþþþ þþþ þþ þþþþ þþþ þþþ þþ
þþþþþþ þþþþ þþþþ þþþþ þþþ þþþþþ þþþþþ þþþþþ þþþþ þþþþþ þþþþ þþþ þþþ þþþþþ þþþþþ þþþþ þþþ
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from 11.43 ± 0.53 to 2.05 ± 0.18 ng/g. However, 44.4 ng/100 g level of IQ was reduced when beef was marinated with 10 g/100 g lemongrass concentration i.e. from 11.43 ± 0.53 to 6.35 ± 0.46 ng/g at medium doneness (Fig. 2b). The level of MeIQX was reduced to 83 ng/100 g (from 12.03 ± 1.14 to 1.99 ± 0.64 ng/g) of beef meat marinated in 10 g/100 g torch ginger flower at medium doneness (Fig. 2c). Curry leaves with 10 g/100 g concentration marinated beef meat was reduced the level of IQ to 78.5 ng/100 g (from 11.43 ± 0.53 to 2.46 ± 0.38 ng/g) at medium doneness (Fig. 2d). The most effective concentration of turmeric was 4 g/100 g which reduced the level of different HCAs ranged from 21 to 82 ng/100 g. The 4 g/ 100 g (turmeric) and 10 g/100 g concentration of lemongrass, torch ginger and curry leaves marinades have shown significantly (p < 0.05) lower formation of HCAs in grilled beef meat. Furthermore, the reduction effect of turmeric, lemongrass, torch ginger and curry leaves marinated beef on Norharman, Harman and AaC are represented in Fig. 3a, b, c and d, respectively. The maximum reduction 60 ng/100 g in AaC level (reduced from 4.58 to 1.82 ng/g) was observed using turmeric marinates (4 g/100 g) beef sample at medium doneness. The lemongrass and torch ginger marinates with 4 g/100 g concentration reduced the level of AaC 54 (reduced from 4.58 to 2.07 ng/g) and 85 ng/100 g (reduced from 4.58 to 0.65 ng/g), respectively at medium doneness. However, curry leaves marinate beef sample with 4 g/100 g concentration reduced 79 ng/100 g (reduced from 7.03 to 1.45 ng/g) of Harman at medium doneness. Different studies (Damasius et al., 2011; Gibis & Weiss, 2012; lu & Üren, 2014; Quelhas et al., 2010) Jinap et al., 2013; Kes¸kekog have been conducted to reduce the level of HCAs in meat. Damasius et al. (2011) have used basil, oregano, marjoram, rosemary, sweet grass, savoury, thyme and coriander extracts for the reduction of HCAs in beef meat samples. The results have shown that the addition of 0.2 g/100 g and 0.5 g/100 g of thyme, savoury and oregano extracts slightly decreased the formation of 1-methyl-6phenyl-1H-imidazo [4,5-b]pyridin-2-amine (PhIP). Oregano and basil extracts have shown the best positive and negative effects on the formation of PhIP. However, in another study green tea marinade was examined in pan-fried beef cooked at 180e200 C for the reduction of HCAs. They have observed that marinating in green tea resulted in a significant decrease (p < 0.05) the levels of PhIP and AaC (Quelhas et al., 2010). Our present study results were supported by Gibis and Weiss (2012), who used oil-based marinades containing grape seed extract formulated in a water/oil emulsion or rosemary extract in oil on the formation of HCAs in fried beef patties. They have shown that after application of marinades and frying, four HCAs MeIQx (2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine), Norharman, and Harman were found at low levels in all fried patties, MeIQx (0.3e1.0 ng/g), and PhIP (0.02e0.3 ng/g). The content of MeIQx and PhIP were significantly reduced by 57 and 90 ng/ 100 g (p < 0.05), respectively. Other studies using different cooking methods and different ingredients in marinades have shown considerable reduction/inhibition of HCAs formation in grilled beef. In our previous study (Jinap et al., 2013) have shown that cooking methods also affect the concentration of HCAs in grilled chicken and beef (satay). The satay samples which were microwaved prior to grilling showed significantly (p < 0.05) lower HCAs concentration as compared to those charcoal grilled. Both medium and well done cooked beef and chicken satay samples that were microwaved and deep fried as an alternative method to grilling were proven to produce significantly lesser HCAs as compared to charcoal-grilled and microwaved prior to grilling. In other study sugar and honey marinades were used in grilled chicken to observe the formation of HCAs and documented a substantial reduction in the concentration of MeIQ, PhIP, DiMeIQx,
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Fig. 2. The effect of different concentrations of selected spices (i.e. turmeric (2a), lemon grass (2b), torch ginger (2c) and curry leaves (2d) on the reduction of HCAs level in satay at different doneness ( ).
Fig. 3. The effect of different concentrations of selected spices i.e. turmeric (3a), lemon grass (3b), torch ginger (3c) and curry leaves (3d) on the reduction of norharman, harman and AaC level in satay at different doneness ( , ).
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IQ, IQx, and norharman in chicken marinated with honey. A correlation study indicated that adding honey into the recipe retarded the formation of most HCAs, whereas table sugars enhanced the formation of all HCAs except norharman, harman, and AaC (Hasnol, lu and Üren (2014) have Jinap, & Sanny, 2014). Similarly Kes¸kekog used different cooking methods (oven roasting, pan cooking, charcoal-barbecue, and deep-fat frying) for the reduction of six heterocyclic aromatic amines; IQ, MeIQx, 4,8-DiMeIQx, PhIP, norharman, and Harman in beef and chicken meatballs with a 0.5 g/ 100 g pomegranate seed extract. They found that total HCAs formation was reduced by 39 and 46 ng/100 g in beef meatballs cooked by charcoal-barbecue and deep-fat frying, respectively. In the chicken meatballs, the highest inhibitory effects were 75 ng/ 100 g for PhIP, 57 ng/100 g for norharman, 28 ng/100 g for harman, 46 ng/100 g for IQ, and 49 ng/100 g for MeIQx. When the pomegranate seed extract was added to the chicken meatballs cooked by deep-fat frying, the total HCAs formation was inhibited by 49 ng/ 100 g, in contrast the total HCAs contents after oven roasting increased by 70 ng/100 g. Oz and Kaya (2011) have showed that black pepper reduced 100 ng/100 g PhIP in meatball fried. On the perspective of total HCAs amount, black pepper has an inhibitory effect (ranging from about 12 to 100 ng/100 g). However, € and Ozsaraç € Haskaraca, Demirok, Kolsarıcı, Oz, (2014) have found that the addition of green tea extract and microwave pre-cooking did not present any detectable effect on reducing the formation of HCAs in fried chicken drum samples and chicken wing samples produced using a laboratory model under controlled frying conditions. Previously, several studies have been conducted to investigate the effect of spices and herbs on the formation of HCAs. Oz and Kaya (2011) has observed that black pepper has inhibited completely the formation of PhIP and 4,8-DiMeIOx in meatball. In addition, the inhibitory effect of black pepper on the formation of HCAs in meatballs fried at 225 C was 100 g/100 g. Similarly, Balogh, Gray, Gomaa, and Booren (2000) studied the effect of vitamin E on HCA formation in fried beef patties and found that addition of vitamin E (1 and 10 g/100 g based on fat content) to surface of ground beef patties reduced PhIP concentrations in cooked patties by 69 and 72 ng/100 g, respectively. In another study, Salmon, Knize, and Felton (1997) reported that marination of chicken breast meat reduced PhIP concentrations by 92e99 ng/100 g. However, the
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mechanism of reduction in HCA formation following marinating meat samples is not clearly understood. It has been documented that the inhibition of HCAs by incorporation of antioxidants is probably because of inactivation of free radicals. Antioxidants could act as inhibitors along different pathways of the reaction to prevent the mutagens formation, through radical quenchers and free radical scavenger's activity (Vitaglione & Fogliano, 2004). In addition, Johansson and J€ agerstad (1996) examined the effects of prooxidant and antioxidant on HCAs inhibition and suggested that prooxidant or antioxidant effect was dependent on concentration. 3.4. Weight loss for satay samples The percentage weight loss from the cooking of satay samples varied among treatment of local spices as shown in Table 4. The percentage weight loss during cooking increased with degree of doneness. The percentage weight loss for satay samples treated with local spices was significantly (p < 0.05) different. This is probably due to water loss during grilling. The weight loss of present study is comparable to previous study conducted by Jinap et al. (2013), in which they have observed that the percentage weight loss during cooking increased with degree of doneness for chicken (21.0 ± 0.25 to 33.2 ± 0.65 g/100 g) and beef (25.4 ± 0.35 to 39.3 ± 0.31 g/100 g). The weight loss during cooking is probably due to the loss of water suggested by Skog and Solyakov (2002). In their study, they observed that pre-treatment with microwaves before cooking has reduced the formation of heterocyclic amines owing to the loss of precursors by leakage. Felton, Fultz, Dolbeare, and Knize (1994), reported that microwave pre-treatment for 0e3 min before frying at either 200 C or 250 C for 6 min per side of Hamburger patties reduced heterocyclic aromatic amine precursors (creatine, creatinine, amino acids, glucose), water, and fat by up to 30 g/100 g. 4. Conclusions The results have shown that local spices i.e. turmeric, lemongrass, torch ginger and curry leaves have effectively reduced the level of HCAs in grilled beef. The reduction of HCAs from 40 to 85 ng/100 g have been documented using optimum concentrations of selected spices i.e. 4 g/100 g turmeric and 10 g/100 g torch ginger, lemon grass and curry leaves. The use of these spices is applicable
Table 4 Cooking loss (g/100 g) and internal temperature of satay grilled at two different degree of doneness. Spices
Control Turmeric
Lemongrass
Torch ginger flowers
Curry leaves
Concentration of spices (g/100 g) e 1 2 3 4 2.5 5.0 7.5 10 2.5 5.0 7.5 10 2.5 5.0 7.5 10
Medium (3 min per side) Cooking loss (g/100) 39.16 37.86 37.22 35.87 33.84 29.28 38.95 37.38 31.76 36.40 35.93 34.37 31.68 35.29 33.17 31.74 30.47
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
aB
1.40 0.68aB 1.77abB 0.58abB 1.63bB 1.83aB 0.48aB 1.86aB 0.44bB 0.68abB 1.77abB 0.56abB 1.63bB 4.62abB 2.78abB 1.05bB 0.43bB
Well done (4 min per side)
Internal temperature ( C) 73.67 72.33 71.67 72.67 71.00 72.67 71.67 70.33 69.33 72.67 71.67 68.33 68.33 73.33 72.33 69.67 68.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
aB
0.58 0.58aB 1.53aB 1.15aB 1.73aB 1.15abB 1.53abB 1.53abB 1.53bB 1.53aB 1.53aB 0.58bB 0.58bB 1.15aB 0.58abB 1.15bCB 1.53cB
Values with same English lowercase letter within row of each spices are non-significant (p 0.05). Values with same English uppercase letter within column of cooking loss are non-significant (p 0.05). Values with same English lowercase letter within column of internal temperature are non-significant (p 0.05). (e) control.
Cooking loss (g/100 g) 47.29 45.93 45.08 43.64 40.57 47.02 44.47 44.67 41.91 45.62 43.63 42.92 41.66 46.60 44.03 42.53 41.03
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
aA
1.07 0.50abA 0.61abA 0.81bA 1.98cA 1.25aA 2.18abA 0.44bA 0.75cA 1.62abA 2.20abA 0.39abA 2.32bA 1.30abA 1.42bcA 1.31bcA 0.99cA
Internal temperature ( C) 83.33 81.67 81.00 79.67 82.67 83.00 80.33 80.00 78.33 82.67 79.67 78.67 77.33 82.67 81.67 79.67 78.00
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.58aA 1.53aA 1.73aA 1.53aA 1.15aA 1.00aA 0.58bA 1.53bA 0.58bA 1.15aA 1.15bA 1.15bA 0.58bA 1.53aA 1.53abA 0.58bcA 1.00cA
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to various meat systems, the food safety concerns associated with the dietary intake of HCAs from meats could be significantly decreased by adding these spices with recommended concentrations to ground meat. Therefore, the results of the current study would be useful an effective system to minimize HCAs formation in cooked satay, using different cooking methods.
References Alaejos, M. S., & Afonso, A. M. (2011). Factors that affect the content of heterocyclic aromatic amines in foods. Comprehensive Reviews in Food Science and Food Safety, 10, 52e108. Balogh, Z., Gray, J. I., Gomaa, E. A., & Booren, A. M. (2000). Formation and inhibition of heterocyclic aromatic amines in fried ground beef patties. Food and Chemical Toxicology, 38, 395e401. Britt, C., Gomaa, E. A., Gray, J. I., & Booren, A. M. (1998). Influenced by cherry tissue on lipid oxidation and heterocyclic amine formation in ground beef patties. Journal of Agricultural and Food Chemistry, 46(12), 4891e4897. Cheok, C. Y., Chin, N. L., Yusof, Y. A., Kamal, M. S. M., & Sazili, A. Q. (2011). Effect of marinating temperatures on physical changes of traditionally marinated beef satay. Journal of Food Processing and Preservation, 35, 474e482. Damasius, J., Venskutonis, P. R., Ferracane, R., & Fogliano, V. (2011). Assessment of the influence of some spice extracts on the formation of heterocyclic amines in meat. Food Chemistry, 126, 149e156. Felton, J. S., Fultz, E., Dolbeare, F. A., & Knize, M. G. (1994). Effect of microwave pretreatment on heterocyclic aromatic amine mutagens/carcinogens in fried beef patties. Food and Chemical Toxicology, 32(10), 897e903. Gibis, M., & Weiss, J. (2012). Antioxidant capacity and inhibitory effect of grape seed and rosemary extract in marinades on the formation of heterocyclic amines in fried beef patties. Food Chemistry, 134, 766e774. Gross, G. A., Grüter, A., & Heyland, S. (1992). Optimization of the sensitivity of highperformance liquid chromatography in the detection of heterocyclic aromatic amine mutagens. Food and Chemical Toxicology, 30, 491e498. € F., & Ozsaraç, € Haskaraca, G., Demirok, E., Kolsarıcı, N., Oz, N. (2014). Effect of green tea extract and microwave pre-cooking on the formation of heterocyclic aromatic amines in fried chicken meat products. Food Research International, 63, 373e381. Hasnol, N. D. S., Jinap, S., & Sanny, M. (2014). Effect of different types of sugars in a marinating formulation on the formation of heterocyclic amines in grilled chicken. Food Chemistry, 145, 514e521. IARC. (1993). Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 56, 165e195. €gerstad, M., & Skog, K. (2005). Genotoxicity of heat-processed foods. Mutation Ja Research, 574, 156e172. €gerstad, M., Skog, K., Arvidsson, P., & Solyakov, A. (1998). Chemistry, formation Ja and occurrence of genotoxic heterocyclic amines identified in model systems and cooked foods. Zeitschrift für Lebensmittel-Untersuchung und eForschung A, 207, 419e427.
Jinap, S., Mohd-Mokhtar, M. S., Farhadian, A., Hasnol, N. D. S., Jaafar, S. N., & Hajeb, P. (2013). Effects of varying degrees of doneness on the formation of Heterocyclic Aromatic Amines in chicken and beef satay. Meat Science, 94, 202e207. €gerstad, M. (1996). Influence of pro- and antioxidants on the Johansson, M. A. E., & Ja formation of mutagenic-carcinogenic heterocyclic amines in a model system. Food Chemistry, 56(1), 69e75. lu, H., & Üren, A. (2014). Inhibitory effects of pomegranate seed extract on Kes¸kekog the formation of heterocyclic aromatic amines in beef and chicken meatballs after cooking by four different methods. Meat Science, 96, 1446e1451. Kizil, M., Oz, F., & Besler, H. T. (2011). A review on the formation of Carcinogenic/ Mutagenic heterocyclic aromatic amines. Journal of Food Processing and Technology, 2(5), 1000120. http://dx.doi.org/10.4172/2157-7110.1000120. Knize, M. G., Dolbeare, F. A., Carroll, K. L., Moore, D. H., & Felton, J. S. (1994). Effects of cooking time and temperature on the heterocyclic amine content of fried beef patties. Food and Chemical Toxicology, 32(7), 595e603. Knutsen, H., Binderup, M. L., Vikse, R., & Øvrebø, S. (2007). Vurdering af helserisiko ved konsum av grillet mat. Norges Vitenskapskomiteen for mattrygghet (VKM). Norwegian, English summary www.vkm.no. Matsumoto, T., Yoshida, D., & Tomita, H. (1981). Determination of mutagens, aminoa-carbolines in grilled foods and cigarette smoke condensate. Cancer Letters, 12, 105e110. Messner, C., & Murkovic, M. (2004). Evaluation of a new model system for studying the formation of heterocyclic amines. Journal of Chromatography B, 802(1), 19e26. Murkovic, M. (2007). Analysis of heterocyclic aromatic amines. Analytical and Bio Analytical Chemistry, 389, 139e146. Nagao, M., Honda, M., & Seino, Y. (1977). Mutagenicities of smoke condensates and the charred surface of fish and meat. Cancer Letters, 2, 221e226. Norat, T., Bingham, S., Ferrari, P., Slimani, N., Jenab, M., Mazuir, M., et al. (2005). Meat, fish, and colorectal cancer risk: the European prospective Investigation into cancer and nutrition. Journal of the National Cancer Institute, 97, 906e916. Oz, F., & Kaya, M. (2011). The inhibitory effect of black pepper on formation of heterocyclic aromatic amines in high-fat meatball. Food Control, 22, 596e600. Persson, E., Graziani, G., Ferracane, R., Fogliano, V., & Skog, K. (2003). Influence of antioxidants in virgin olive oil on the formation of heterocyclic amines in fried beef burgers. Food and Chemical Toxicology, 41(11), 1587e1597. Quelhas, I., Petisca, C., Viegas, O., Melo, A., Pinho, O., & Ferreira, I. M. P. L. V. O. (2010). Effect of green tea marinades on the formation of heterocyclic aromatic amines and sensory quality of pan-fried beef. Food Chemistry, 122, 98e104. Salmon, C. P., Knize, M. G., & Felton, J. S. (1997). Effects of marinating on heterocyclic amine carcinogen formation in grilled chicken. Food and Chemical Toxicology, 35, 433e441. Shin, W. J., Gomaa, E. A., Strasburg, G. M., & Gray, J. I. (2002). Inhibition of heterocyclic aromatic amine formation in fried ground beef patties by garlic and selected garlic-related sulfur compounds. Journal of Food Protection, 65, 21e24. Skog, K., & Solyakov, A. (2002). Heterocyclic amines in poultry products: a literature review. Food and Chemical Toxicology, 40, 1213e1221. Smith, J. S.., Ameri, F., & Gadgil, P. (2008). Effect of marinades on the formation of heterocyclic amines in grilled beef steaks. Journal of Food Science, 73, 100e105. Vitaglione, P., & Fogliano, V. (2004). Use of antioxidants to minimize the human health risk associated to mutagenic/carcinogenic heterocyclic amines in food. Journal of Chromatography B, 802, 189e199.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Effect of selected local spices marinades on the reduction of heterocyclic amines in grilled beef (satay) S. Jinap a, b, *, Shahzad Zafar Iqbal a, Roshny M.P. Selvam a a b
Food Safety Research Centre (FOSREC), Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2015 Received in revised form 16 April 2015 Accepted 19 April 2015 Available online 28 April 2015
The present study focused to investigate the effect of some selected spices (turmeric, torch ginger, lemongrass and curry leaves) on the formation of heterocyclic amines (HCAs, IQx, MeIQ, MeIQx, DiMeIQx, IQ, norharman, harman and AaC) in grilled satay (beef). Satay samples were marinated with 1 e4 g/100 g concentration of turmeric and 2.5e10 g/100 g concentration of torch ginger, lemongrass and curry leaves, at different medium (70 C) and well done (80 C) doneness cooking temperatures. The concentration of HCAs in grilled satay samples were analysed using LC/MS technique. The results have shown that turmeric reduced maximum 82 ng/100 g level of IQ (11.43 ± 0.53 to 2.05 ± 0.18 ng/g) at 4 g/ 100 g concentration at medium doneness and 44.4 ng/100 g level was reduced of IQ, when satay was marinated with 10 g/100 g lemongrass concentration at medium doneness. The highest level of MeIQX was reduced to 83 ng/100 g of satay meat marinated in 10 g/100 g torch ginger at medium doneness. Curry leaves with 10 g/100 g concentration marinated beef meat was reduced the level of IQ 78.5 at medium doneness. The use of local spices in marinating of grilled beef will certainly inhibit/reduce the level of toxic and harmful HCAs. Significance of results for industry: The use of native spices during grilling of meat will minimize the formation of heterocyclic amines and therefore will help to avoid toxic and carcinogenic effects from these toxins. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Local spices Beef meat HCAs
1. Introduction Studies have proved that different kind of harmful components, including various mutagens and carcinogens, may be produced, when meat is heat-treated using traditional procedures such as €gerstad & Skog, 2005). It has frying, barbecuing and smoking (Ja been observed that all types of meat such as beef, pork, goat and lamb, may produce heterocyclic aromatic amines (HCA) when cooked at high temperatures. HCAs are recognized as mutagenic and carcinogenic compounds (Damasius, Venskutonis, Ferracane, & Fogliano, 2011). Currently, more than 25 HCAs have been isolated and recognized in cooked foods, since their discovery (Alaejos & Afonso, 2011; Murkovic, 2007) and divided in two main families:
* Corresponding author. Food Safety Research Centre (FOSREC), Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: þ60 3 8946 8393; fax: þ60 3 8942 3552. E-mail address: [email protected] (S. Jinap). http://dx.doi.org/10.1016/j.lwt.2015.04.047 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
aminoimidazo-azaarenes or thermic HCAs and amino-carbolines or pyrolytic HCAs. These HCAs includes 2-amino-3- methylimidazo [4,5-f]quinoline (IQ), 2-amino-3-methylimidazo [4,5-f] quinoxaline (IQx), 2-amino-3,4-dimethylimidazo [4,5-f]quinolone (MeIQ), 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) (Knize, Dolbeare, Carroll, Moore, & Felton, 1994). Thermic HCAs are produced as a result of complex reaction between creatine/creatinine, free amino acids and sugars through the Maillard reaction at temperatures between 150 and 250 C €gerstad, Skog, Arvidsson, & Solyakov, 1998; Nagao, Honda, & (Ja Seino, 1977). However, the pathway of pyrolytic HCAs production was not so clear but suggested that it may be produced as a result of pyrolysis of proteins or amino acids heated at higher temperatures i.e. > 250 C (Matsumoto, Yoshida, & Tomita, 1981). In Southeast Asia (Malaysia, Indonesia, Thailand, and Singapore), satay is a very popular grilled food similar to shish kebab which is also popular in western and middle-eastern countries. It consists of dice-sized chunks or slices of boneless meat
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(chicken, beef, mutton, pork, fish), on skewers made from the midrib of coconut leaf or bamboo. It is usually grilled over wood or charcoal fires, and then served with various spicy seasonings depending on the satay recipe. Mostly charcoal grilling is the common cooking method for satay prepared in Southeast Asian countries. Compared to other cooking practices such as prolonged heating or frying, this fast grilling method can produce a different mixture of HCAs. The HCAs (IQ,MeIQ, MeIQx and PhIP) are identified as compounds reasonably anticipated to be a human carcinogen. However, the International Agency for Research on Cancer has classified MeIQx, MeIQ and PhIP as reasonably anticipated to be a human carcinogen, while IQ is placed in the list of a probable human carcinogen (IARC, 1993). Epidemiological studies (Knutsen, Binderup, Vikse, & Øvrebø, 2007; Norat et al., 2005) have shown an increased risk of cancer in the intestine, bladder, prostate, breast and pancreas after high consumption of fried, well-done and barbecued meat. Previous studies have revealed that by adding ingredients such as tomatoes to marinades before cooking, is effective to reduce the concentration of HCA (Persson, Graziani, Ferracane, Fogliano, & Skog, 2003), tart cherry tissue (Britt, Gomaa, Gray, & Booren, 1998), and spices (such as onion and garlic) (Shin, Gomaa, Strasburg, & Gray, 2002). The present study is focused to investigate the effect of local Malaysian spices including turmeric, lemongrass, torch ginger and curry leave on the reduction of HCAs in grilled beef (satay) at different doneness cooking.
Massachusetts, USA). MCX cartridges were preconditioned with ethyl acetate (2 mL). The samples (n ¼ 3) of fresh beef (sirloin) 5 kg of local origin were purchased from local markets of Selangor, Malaysia. Visible connective tissues and external fat were removed before storage in freezer at 20 C. The samples of beef were thawed overnight in a cold room (4 ± C). The pH of meat samples was determined prior to analysis. The meat was then cut into big pieces again the grain, in which each piece has weight about 100 ± 0.5g. All the cut meat samples were put into polyethylene containers and stored in the refrigerator (4 ± 1 C). 2.2. Local spices preparation The fresh turmeric, torch ginger flower, curry leaves and lemongrass were obtained from local market, Selangor, Malaysia. The skins of fresh turmeric samples were removed and the curry leaves were stripped off from their stalks. The lemongrass was separated from its stalks and leaves. All the cleaned spices were brought to washing, wiped with paper towel and chopped into small pieces separately. The weight of each spice was noted and kept in polyethylene container. The samples were then kept in freezer (20 C) for overnight and then brought to freeze drying. The samples were freeze-dried for two days, weighed and then grounded using a dry grinder. The homogenized powder samples were kept in polyethylene containers and stored at 20 C. The moisture content of each powdered sample was determined prior to analysis.
2. Materials and methods
2.3. Preparation of marinades formulation
2.1. Chemicals
A range of each individual spice was developed prior to this analysis based on the acceptable maximum level in satay recipes and these levels are optimised. The optimised range for turmeric was 0, 1, 2, 3, and 4 g/100 g, while other spices have ranges 0, 2.5, 5, 7.5 and 10 g/100 g of meat. Each spice concentration was applied (2 replicates) on meat samples, then mixed and left for 24 h. The meat samples having no treatment of spices were control samples. Percentage of marinade uptake for each treatment was determined just after marination (0 h) and after 24 h (Table 1). The weight of sample before marination (W0) and after marination (W1) was recorded. The percentage weight gain was calculated based on formulation.
Standards of HCAs used, 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino- 3,8-dimethyl-imidazo[4,5-f] quinoxaline (MeIQx), 2amino-3,4,8- trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), norharman, harman and AaC were purchased from Toronto Research Chemicals (Toronto, Canada). A stock solution of 100 mg/g in methanol was prepared for each HCAs. Diatomaceous earth (Extrelut 20) was obtained from the International Sorbent Technology (Hengoed Mid Gleam, UK) and Oasis MCX cartridges (3 cm3/60 mg) were purchased from Waters (Milford,
Table 1 Marinate uptake of meat at 0 and 24 h duration. Spices
Concentration of spices (g/100 g)
Marinate uptake at 0 h (g/100 g)
Turmeric
1 2 3 4 2.5 5.0 7.5 10 2.5 5.0 7.5 10 2.5 5.0 7.5 10
0.95 1.82 2.79 3.69 2.15 4.05 6.69 8.22 2.20 2.34 2.97 3.31 2.29 2.42 2.71 2.91
Lemongrass
Torch ginger flowers
Curry leaves
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.17aA 0.12bA 0.05cA 0.13dA 0.19aA 0.27bA 0.28cA 0.35dA 0.02aA 0.06aA 0.12bA 0.16bA 0.03aA 0.03abA 0.06bcA 0.16cA
Marinate uptake after 24 h (g/100 g) 0.38 1.21 2.08 3.00 0.91 2.24 3.77 4.66 1.05 1.35 1.85 2.39 1.32 1.40 1.57 1.69
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.18aA 0.08bB 0.04cB 0.16dA 0.30aB 0.11bB 0.44bcB 0.32cB 0.04aB 0.08abB 0.08abB 0.52bA 0.06Ab 0.04abB 0.00abB 0.09bB
Values with same English lowercase within row of each species are not significant (p 0.05). Values with same English uppercase letter within column of marinate uptake (0 and 24 h) are not significant (p 0.05).
Marinate uptake loss 0e24 h (g/100 g) 60.00a 33.52b 25.45b 18.70b 57.67a 44.69b 43.65b 43.30b 52.27a 42.31b 37.71c 27.79d 42.36a 42.15a 42.07a 41.92a
S. Jinap et al. / LWT - Food Science and Technology 63 (2015) 919e926
Weight gain ðg=100gÞ ¼
W1 W0 100 W0
2.4. Preparation and grilling conditions of satay After marinade, sample from each experiment was cut into cubes by maintain the weight of each sample to 1 g. The cubes were skewed into bamboo sticks in one stick and then the prepared sticks were brought to grilling on electric grill. The grilling method was designed as described by Smith, Ameri, and Gadgil (2008) with some modification in grilling conditions. The satay samples were grilled at 300 C to achieve medium and well doneness of grilling. The surface temperature of griller and internal temperature of each satay was taken after grilling. The weight losses of satay samples were also determined. The hardness and colour of each grilled sample was also analysed. The medium doneness was 70 ± 4 C (3 min) and well done (80 ± 4 C, for 4 min). 2.5. Extraction of heterocyclic amines HCA were extracted from meat samples using the method developed and validated by Gross, Grüter, and Heyland (1992), and modified by Messner and Murkovic (2004). A sub-sample (1 g) of ground meat sample was dissolved in 12 mL of 1 mol/L NaOH. The suspension was homogenised using a shaker (Memmert, Berlin, Germany) at high speed for 3 h. The samples were then mixed with 13 g diatomaceous earth, and the mixtures were poured into an Extrelut 20-ml column. A 50 mL volume of ethyl acetate was used as extraction solvent and a volume of 25 mL collected from the 50 mL sample was passed through the MCX cartridges. The MCX cartridge was then washed with 2 mL of 0.1 mol/L HCl followed by 2 mL methanol. The analytes were then eluted with 2 mL of methanol: concentrated ammonia (19:1, mL:mL) before they were evaporated to dryness under a stream of nitrogen, and the final extract was dissolved in 100 mL methanol. 2.6. Mass spectrometry parameters An agilant 1200 LC modules (Agilent Technologies, USA), consisted of a quaternary pump and an auto sampler (Agilent Technologies, USA) coupled with Agilent 6410 triple quadruple mass spectrometer (Agilent Technologies, USA) with a pneumatically assisted electro spray interface was used. Data acquisition was performed in the positive ion mode. The additional operating conditions were as follows: capillary voltage ¼ 4 kV, nebulizer pressure ¼ 45 kPa, drying gas flow ¼ 9 L/min, dry gas temperature ¼ 300 C and fragmentation voltage was 190 V. For quantification and identification, data acquisitions were operated in the MS/MS mode. The selected reaction monitoring (SRM) analysis was performed as follow: m/z 199 / 184 (collision energy, CE 25 eV) for IQ, m/z 213 / 198 (CE 25 eV) for MeIQ, m/z 200 / 185 (CE 25 eV) for IQx, m/z 214 / 131 (CE 41 eV) for MeIQx, m/z 169 / 115 (CE 37 eV) for Norharman, m/z 228 / 213 (CE 21 eV) for DiMeIQx, m/z 183 / 115 (CE 37 eV) for Harman, m/z 184 / 140 (CE 33 eV) for AaC, and m/z 225 / 210 (CE 25 eV) for PhIP. The dwell time was set at 0.5s. For data acquisition and processing the Agilent Mass Hunter qualitative and quantitative analysis software was used. 2.7. Sensory tests A total of 40 panelists (graduate students) from the Faculty of Food Science and Technology participated in the pre-selection of panelists for the sensory evaluation of beef satay. They were
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selected through series of basic test and triangle test. After tests panelists (n ¼ 30) were selected for further training on the sensory evaluation of beef satay. Information on evaluation techniques used was presented to the panelists before the actual sensory test. The panelists were then performed the threshold test and quantitative descriptive analysis QDA tests at different sessions. The panellists first evaluated beef meat that had not been marinated to determine the preferred (optimal) effect of local spices at different doneness. Sensory scores (7 point hedonic scale) were rated using a 7 cm scale for colour (0e7, very light brown to brunt) texture (0e7, very tender to hard). The panellists have ranked the samples by a mark on the scale. For each spice, one portion was prepared without the addition of spice concentration (as control), while the other were prepared with different concentrations of spices. The satay was prepared in duplicate. Samples were cooked 1 h before sensory evaluation and kept warm in a warmer plate until being evaluated. All products were presented in three-digit codes. A general discussion session was held following the solution tasting to standardize the scores and the definition of the attributes. Fig. 1a and b shows the effect of different concentrations of selected spices marination on colour and texture on meat and satay, respectively. 2.8. Statistical analysis The data of samples (3 2) was given as mean ± standard deviation. The statistical analyses (Levene's test for homogeneity, ShapiroeWilk test for normality) were performed by using the statistical package MINITAB 16. Two way analysis of variance was performed to determine the significant difference among treatments at p 0.05. 3. Results and discussion 3.1. Marination uptake Table 1 shows the marination of spices at 0 and 24 h duration. The results showed that with increase in the concentration of spices the marination uptake was also increased. However after 24 h of marination the uptake of marination decreased subsequently, might be due to the loss of water. 3.2. Colour and tenderness of satay Table 2 shows visual observation on colour intensity of grilled satay marinated with selected spices at different concentrations. Browning of cooked muscle food indicates the presence of complexes formed from maillard reaction. According to Kizil, Oz, and Besler (2011), AIAs or IQ type of HCAs are generally produced from heat induced maillard reaction which involves free amino acids, creatine or creatinine and reducing sugar. Thus, Maillard reaction may results in the formation of HCAs. The longer the cooking time more browning and HCAs formation will occur. Table 2, all medium cooked control, turmeric and lemongrass treated samples had less browning than that of well-cooked samples. Marination process has effect on the reduction of browning of samples. Marinated sample might undergo less browning reaction compared to un-marinated samples (Smith et al., 2008). Thus, marinate uptake of meat and its corresponding weight loss influences the surface browning of meat. It has been observed that turmeric, lemongrass and torch ginger flower marination had good reduction of browning of sample. The results showed that torch ginger and curry leaves marinated samples had decreased browning up to 5 g/100 g of that spices. No differences of browning observed on medium and well-cooked torch ginger and curry
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Fig. 1. a: The effect of different concentrations of selected spices i.e. turmeric, curry leaves, torch ginger and lemon grass on meat. b: The effect of different concentrations of selected spices i.e. turmeric, lemon grass, torch ginger and curry leaves on satay.
leaves treated satay. The reduction of browning may be due to the presence of phenolic compounds in spices. The weight gain of meat obtained by marination and its corresponding weight loss are good determinants of meat tenderness. During marination, marinades slowly penetrate into meat. This results in weakening of the structures due to swelling of the meat, increased proteolysis and conversion of collagen to gelatine. This process results in decrease of mechanical resistance of meat and increased meat tenderness (Cheok, Chin, Yusof, Kamal, & Sazili, 2011). The results have shown that the tenderness of satay
marinated at maximum concentration of all spices was greater than that of minimum concentration. All medium cooked samples except 7.5 g/100 g torch ginger treated sample were tender compared to that of well-cooked samples as shown in Table 3. 3.3. Effect of local spices on HCAs reduction The concentration of HCAs in grilled beef samples were analysed using LC/MS and the range in control sample was 5.39 ± 0.40 to 43.52 ± 0.80 ng/g. The DiMeIQX has found in highest level with
S. Jinap et al. / LWT - Food Science and Technology 63 (2015) 919e926 Table 2 Visual observation of colour intensity of grilled satay marinated with selected spices at different concentrations. Spices
Control Turmeric
Lemongrass
Torch ginger flowers
Curry leaves
Concentration of spices (g/100 g)
colour intensity of satay Medium
Well done
1 2 3 4 2.5 5.0 7.5 10 2.5 5.0 7.5 10 2.5 5.0 7.5 10
þþþþþþ þþþ þþþ þþ þþ þþþþþ þþþ þþþ þþ þþþþþ þþþþ þþþ þþþ þþþþþþþ þþþþþþ þþþþþþ þþþþþþ
þþþþþþþ þþþþþ þþþþ þþþ þþ þþþþþþ þþþþ þþþ þþ þþþþþ þþþþ þþþ þþþ þþþþþþþ þþþþþþ þþþþþþ þþþþþþ
þ very light brown. þþ light brown. þþþ looked brown. þþþþ deep brown. þþþþþ light black. þþþþþþ moderately black. þþþþþþþ indicating black (burnt).
concentration 39.49 ± 0.98 43.52 ± 0.80 ng/g at medium (70 C) and well doneness (80 C) treatments, respectively. The effect on reduction of HCAs using local spices, turmeric, lemongrass, torch ginger and curry leaves with concentration of 1e4 g/100 g for turmeric and 2.5e10 g/100 g for rest of the spices were used to marinades beef samples. Fig. 2a, b, c and d represents the reduction of HCAs in beef marinated at different concentration of local spices at medium and well done doneness, respectively. Fig. 2a shows that turmeric with 4 g/100 g concentration has shown maximum reduction of IQ level (82 ng/100 g) in marinated beef
Table 3 Texture evaluation of grilled satay marinated with selected spices at different concentrations. Spices
Control Turmeric
Lemongrass
Torch ginger flowers
Curry leaves
þ very much tender. þþ very tender. þþþ tender. þþþþ texture is right. þþþþþ not tender not hard. þþþþþþ slightly hard. þþþþþþþ too hard.
Concentration of spices (g/100 g)
Hardness of satay Medium
Well done
e 1 2 3 4 2.5 5.0 7.5 10 2.5 5.0 7.5 10 2.5 5.0 7.5 10
þþþþþ þþþþ þþþþ þþþ þþ þþþþ þþþþ þþþ þþþ þþþþ þþþþ þþþ þþ þþþþ þþþ þþþ þþ
þþþþþþ þþþþ þþþþ þþþþ þþþ þþþþþ þþþþþ þþþþþ þþþþ þþþþþ þþþþ þþþ þþþ þþþþþ þþþþþ þþþþ þþþ
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from 11.43 ± 0.53 to 2.05 ± 0.18 ng/g. However, 44.4 ng/100 g level of IQ was reduced when beef was marinated with 10 g/100 g lemongrass concentration i.e. from 11.43 ± 0.53 to 6.35 ± 0.46 ng/g at medium doneness (Fig. 2b). The level of MeIQX was reduced to 83 ng/100 g (from 12.03 ± 1.14 to 1.99 ± 0.64 ng/g) of beef meat marinated in 10 g/100 g torch ginger flower at medium doneness (Fig. 2c). Curry leaves with 10 g/100 g concentration marinated beef meat was reduced the level of IQ to 78.5 ng/100 g (from 11.43 ± 0.53 to 2.46 ± 0.38 ng/g) at medium doneness (Fig. 2d). The most effective concentration of turmeric was 4 g/100 g which reduced the level of different HCAs ranged from 21 to 82 ng/100 g. The 4 g/ 100 g (turmeric) and 10 g/100 g concentration of lemongrass, torch ginger and curry leaves marinades have shown significantly (p < 0.05) lower formation of HCAs in grilled beef meat. Furthermore, the reduction effect of turmeric, lemongrass, torch ginger and curry leaves marinated beef on Norharman, Harman and AaC are represented in Fig. 3a, b, c and d, respectively. The maximum reduction 60 ng/100 g in AaC level (reduced from 4.58 to 1.82 ng/g) was observed using turmeric marinates (4 g/100 g) beef sample at medium doneness. The lemongrass and torch ginger marinates with 4 g/100 g concentration reduced the level of AaC 54 (reduced from 4.58 to 2.07 ng/g) and 85 ng/100 g (reduced from 4.58 to 0.65 ng/g), respectively at medium doneness. However, curry leaves marinate beef sample with 4 g/100 g concentration reduced 79 ng/100 g (reduced from 7.03 to 1.45 ng/g) of Harman at medium doneness. Different studies (Damasius et al., 2011; Gibis & Weiss, 2012; lu & Üren, 2014; Quelhas et al., 2010) Jinap et al., 2013; Kes¸kekog have been conducted to reduce the level of HCAs in meat. Damasius et al. (2011) have used basil, oregano, marjoram, rosemary, sweet grass, savoury, thyme and coriander extracts for the reduction of HCAs in beef meat samples. The results have shown that the addition of 0.2 g/100 g and 0.5 g/100 g of thyme, savoury and oregano extracts slightly decreased the formation of 1-methyl-6phenyl-1H-imidazo [4,5-b]pyridin-2-amine (PhIP). Oregano and basil extracts have shown the best positive and negative effects on the formation of PhIP. However, in another study green tea marinade was examined in pan-fried beef cooked at 180e200 C for the reduction of HCAs. They have observed that marinating in green tea resulted in a significant decrease (p < 0.05) the levels of PhIP and AaC (Quelhas et al., 2010). Our present study results were supported by Gibis and Weiss (2012), who used oil-based marinades containing grape seed extract formulated in a water/oil emulsion or rosemary extract in oil on the formation of HCAs in fried beef patties. They have shown that after application of marinades and frying, four HCAs MeIQx (2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine), Norharman, and Harman were found at low levels in all fried patties, MeIQx (0.3e1.0 ng/g), and PhIP (0.02e0.3 ng/g). The content of MeIQx and PhIP were significantly reduced by 57 and 90 ng/ 100 g (p < 0.05), respectively. Other studies using different cooking methods and different ingredients in marinades have shown considerable reduction/inhibition of HCAs formation in grilled beef. In our previous study (Jinap et al., 2013) have shown that cooking methods also affect the concentration of HCAs in grilled chicken and beef (satay). The satay samples which were microwaved prior to grilling showed significantly (p < 0.05) lower HCAs concentration as compared to those charcoal grilled. Both medium and well done cooked beef and chicken satay samples that were microwaved and deep fried as an alternative method to grilling were proven to produce significantly lesser HCAs as compared to charcoal-grilled and microwaved prior to grilling. In other study sugar and honey marinades were used in grilled chicken to observe the formation of HCAs and documented a substantial reduction in the concentration of MeIQ, PhIP, DiMeIQx,
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Fig. 2. The effect of different concentrations of selected spices (i.e. turmeric (2a), lemon grass (2b), torch ginger (2c) and curry leaves (2d) on the reduction of HCAs level in satay at different doneness ( ).
Fig. 3. The effect of different concentrations of selected spices i.e. turmeric (3a), lemon grass (3b), torch ginger (3c) and curry leaves (3d) on the reduction of norharman, harman and AaC level in satay at different doneness ( , ).
S. Jinap et al. / LWT - Food Science and Technology 63 (2015) 919e926
IQ, IQx, and norharman in chicken marinated with honey. A correlation study indicated that adding honey into the recipe retarded the formation of most HCAs, whereas table sugars enhanced the formation of all HCAs except norharman, harman, and AaC (Hasnol, lu and Üren (2014) have Jinap, & Sanny, 2014). Similarly Kes¸kekog used different cooking methods (oven roasting, pan cooking, charcoal-barbecue, and deep-fat frying) for the reduction of six heterocyclic aromatic amines; IQ, MeIQx, 4,8-DiMeIQx, PhIP, norharman, and Harman in beef and chicken meatballs with a 0.5 g/ 100 g pomegranate seed extract. They found that total HCAs formation was reduced by 39 and 46 ng/100 g in beef meatballs cooked by charcoal-barbecue and deep-fat frying, respectively. In the chicken meatballs, the highest inhibitory effects were 75 ng/ 100 g for PhIP, 57 ng/100 g for norharman, 28 ng/100 g for harman, 46 ng/100 g for IQ, and 49 ng/100 g for MeIQx. When the pomegranate seed extract was added to the chicken meatballs cooked by deep-fat frying, the total HCAs formation was inhibited by 49 ng/ 100 g, in contrast the total HCAs contents after oven roasting increased by 70 ng/100 g. Oz and Kaya (2011) have showed that black pepper reduced 100 ng/100 g PhIP in meatball fried. On the perspective of total HCAs amount, black pepper has an inhibitory effect (ranging from about 12 to 100 ng/100 g). However, € and Ozsaraç € Haskaraca, Demirok, Kolsarıcı, Oz, (2014) have found that the addition of green tea extract and microwave pre-cooking did not present any detectable effect on reducing the formation of HCAs in fried chicken drum samples and chicken wing samples produced using a laboratory model under controlled frying conditions. Previously, several studies have been conducted to investigate the effect of spices and herbs on the formation of HCAs. Oz and Kaya (2011) has observed that black pepper has inhibited completely the formation of PhIP and 4,8-DiMeIOx in meatball. In addition, the inhibitory effect of black pepper on the formation of HCAs in meatballs fried at 225 C was 100 g/100 g. Similarly, Balogh, Gray, Gomaa, and Booren (2000) studied the effect of vitamin E on HCA formation in fried beef patties and found that addition of vitamin E (1 and 10 g/100 g based on fat content) to surface of ground beef patties reduced PhIP concentrations in cooked patties by 69 and 72 ng/100 g, respectively. In another study, Salmon, Knize, and Felton (1997) reported that marination of chicken breast meat reduced PhIP concentrations by 92e99 ng/100 g. However, the
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mechanism of reduction in HCA formation following marinating meat samples is not clearly understood. It has been documented that the inhibition of HCAs by incorporation of antioxidants is probably because of inactivation of free radicals. Antioxidants could act as inhibitors along different pathways of the reaction to prevent the mutagens formation, through radical quenchers and free radical scavenger's activity (Vitaglione & Fogliano, 2004). In addition, Johansson and J€ agerstad (1996) examined the effects of prooxidant and antioxidant on HCAs inhibition and suggested that prooxidant or antioxidant effect was dependent on concentration. 3.4. Weight loss for satay samples The percentage weight loss from the cooking of satay samples varied among treatment of local spices as shown in Table 4. The percentage weight loss during cooking increased with degree of doneness. The percentage weight loss for satay samples treated with local spices was significantly (p < 0.05) different. This is probably due to water loss during grilling. The weight loss of present study is comparable to previous study conducted by Jinap et al. (2013), in which they have observed that the percentage weight loss during cooking increased with degree of doneness for chicken (21.0 ± 0.25 to 33.2 ± 0.65 g/100 g) and beef (25.4 ± 0.35 to 39.3 ± 0.31 g/100 g). The weight loss during cooking is probably due to the loss of water suggested by Skog and Solyakov (2002). In their study, they observed that pre-treatment with microwaves before cooking has reduced the formation of heterocyclic amines owing to the loss of precursors by leakage. Felton, Fultz, Dolbeare, and Knize (1994), reported that microwave pre-treatment for 0e3 min before frying at either 200 C or 250 C for 6 min per side of Hamburger patties reduced heterocyclic aromatic amine precursors (creatine, creatinine, amino acids, glucose), water, and fat by up to 30 g/100 g. 4. Conclusions The results have shown that local spices i.e. turmeric, lemongrass, torch ginger and curry leaves have effectively reduced the level of HCAs in grilled beef. The reduction of HCAs from 40 to 85 ng/100 g have been documented using optimum concentrations of selected spices i.e. 4 g/100 g turmeric and 10 g/100 g torch ginger, lemon grass and curry leaves. The use of these spices is applicable
Table 4 Cooking loss (g/100 g) and internal temperature of satay grilled at two different degree of doneness. Spices
Control Turmeric
Lemongrass
Torch ginger flowers
Curry leaves
Concentration of spices (g/100 g) e 1 2 3 4 2.5 5.0 7.5 10 2.5 5.0 7.5 10 2.5 5.0 7.5 10
Medium (3 min per side) Cooking loss (g/100) 39.16 37.86 37.22 35.87 33.84 29.28 38.95 37.38 31.76 36.40 35.93 34.37 31.68 35.29 33.17 31.74 30.47
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
aB
1.40 0.68aB 1.77abB 0.58abB 1.63bB 1.83aB 0.48aB 1.86aB 0.44bB 0.68abB 1.77abB 0.56abB 1.63bB 4.62abB 2.78abB 1.05bB 0.43bB
Well done (4 min per side)
Internal temperature ( C) 73.67 72.33 71.67 72.67 71.00 72.67 71.67 70.33 69.33 72.67 71.67 68.33 68.33 73.33 72.33 69.67 68.67
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
aB
0.58 0.58aB 1.53aB 1.15aB 1.73aB 1.15abB 1.53abB 1.53abB 1.53bB 1.53aB 1.53aB 0.58bB 0.58bB 1.15aB 0.58abB 1.15bCB 1.53cB
Values with same English lowercase letter within row of each spices are non-significant (p 0.05). Values with same English uppercase letter within column of cooking loss are non-significant (p 0.05). Values with same English lowercase letter within column of internal temperature are non-significant (p 0.05). (e) control.
Cooking loss (g/100 g) 47.29 45.93 45.08 43.64 40.57 47.02 44.47 44.67 41.91 45.62 43.63 42.92 41.66 46.60 44.03 42.53 41.03
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
aA
1.07 0.50abA 0.61abA 0.81bA 1.98cA 1.25aA 2.18abA 0.44bA 0.75cA 1.62abA 2.20abA 0.39abA 2.32bA 1.30abA 1.42bcA 1.31bcA 0.99cA
Internal temperature ( C) 83.33 81.67 81.00 79.67 82.67 83.00 80.33 80.00 78.33 82.67 79.67 78.67 77.33 82.67 81.67 79.67 78.00
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.58aA 1.53aA 1.73aA 1.53aA 1.15aA 1.00aA 0.58bA 1.53bA 0.58bA 1.15aA 1.15bA 1.15bA 0.58bA 1.53aA 1.53abA 0.58bcA 1.00cA
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to various meat systems, the food safety concerns associated with the dietary intake of HCAs from meats could be significantly decreased by adding these spices with recommended concentrations to ground meat. Therefore, the results of the current study would be useful an effective system to minimize HCAs formation in cooked satay, using different cooking methods.
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