Inhibitory effects of black cumin on the formation of heterocyclic aromatic amines in meatball.

The impact of black cumin usage on some qualitative properties and formation of heterocyclic aromatic amines (HAAs) in meatball production was investigated. It was found that black cumin usage rate, cooking process and temperature had a significant effect (p<0,01) on the water content, pH, and thiobarbituric acid reactive substances (TBARS) values of meatballs. On the other hand, black cumin usage significantly (p<0,01) reduced the water content and cooking loss. The water content and cooking loss of the meatballs decreased with increases in the usage rate. While IQx, IQ, MeIQ, 7,8-DiMeIQx, 4,8-DiMeIQx, AαC, and MeAαC could not be detected in meatballs, varying amounts of MeIQx (up to 1,53 ng/g) and PhIP (up to 1,22 ng/g) were determined. The total amounts of HAAs ranged between non-detected (nd) to 2,75 ng/g. Both the usage rate and cooking temperature had a very significant effect (p<0,01) on the total contents of HAAs. The total amounts of HAAs were decreased in correlation with the increases in the usage rate; the proportion which is increased when the cooking temperature increased as well. Results of the present study suggested that addition of black cumin may have a substantial role in decreasing the TBARS value, cooking loss, and HAA contents during meatball production. Therefore, using of black cumin in meatball production has been suggested.

Introduction

Foods, complex mixture of the micro compounds such as essential trace elements and vitamins and the macro compounds such as protein, fat, and carbohydrates, play an important role in living organisms with other ingredients such as ditary fibers and antioxidants. On the other hand, various hazards, including microbiological and chemicals can be introduced into the food chain from farm to table. Therefore, nutition not only plays a pivotal role in the prevention of some diseases but also may be considered as a risk factor for some other emerging diseases [1].

Some epidemiological investigations have revealed the role of nutrition in the development of many comon cancers [2,3]. Recently, it has been widely accepted that some mutagenic and/or carcinogenic substances are formed during cooking of foods rich in protein, such as meat [4,5]. The group of heterocyclic aromatic amines (HAAs) is of these substances [6]. In 1977, it was reported that some chemical compounds were formed in products such as meat and fish cooked at high temperatures and these compounds were later defined as HAAs, based on their chemical structures [7].

HAAs have two basic chemical groups; 1) Aminoimidazoarenes, IQ type or thermal HAAs, are formed during cooking of food at temperatures of 150–300 °C. 2) Aminocarbolines, non-IQ type or pyrolitic HAAs, are formed at temperatures above 300 °C [3]. Precursors of HAAs are creatine/creatinine, reducing sugars, amino acids, peptides and proteins, heat and mass transfer, lipids and lipid oxidation [6,8]. Compared to other known food mutagens, HAAs were found to be 100-fold more mutagenic than aflatoxin B1 and 2000-fold more mutagenic than benzo[a]pyrene [3]. On the other hand, according to the International Agency for Research on Cancer (IARC), IQ has been classified as a possible human carcinogen (class 2A) and MelQ, MelQx, PhIP, AαC, MeAαC, Trp-P-1, Trp-P-2, and Glu-P-1 are labelled as probable human carcinogen (class 2B) [9]. Therefore, the presence of HAAs in foods should be prevented and/or reduced.

Since radical reactions had an important role in the formation of HAAs, and it is, therefore, expected that antioxidants can reduce the formation of HAAs in meat and meat products. Indeed, it was reported that antioxidants could inhibit the formation of HAAs and this effect was explained due to the ability of antioxidants to interfere in different stages of the formation reactions of HAAs [10,11]. However, same antioxidants had a prooxidant effect depending on their concentration in food and caused an increase in the formation of HAAs. To reduce the formation of HAAs, synthetic and/or natural antioxidants can be added to meat and meat products. Due to their carcinogenic potential, the use of synthetic antioxidants has been banned from European countries and others, such as Japan and Canada, due to their carcinogenic potential [12,13]. Therefore, during the recent years, food rich in natural antioxidants such as plants or spices have gained much attention [14-18].

Black cumin (Nigella sativa L.) is a spice belonging to the family of Ranunculaceae [19]. Black cumin is grown in many parts of the world [20]. It is reported that the seeds of black cumin contain dietary proteins (26,7%), fats (28,5%), and carbohydrates (40,0%) [20]. In the Traditional Middle Eastern medicine, the seeds have been used as a remedy for treatment of multiple diseases (a natural remedy for asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness and influenza) for more than 2000 years [19,21]. Black cumin seed essential oil contains high concentrations of thymoquinone (37,6%) and its related compounds such as thymosl and dithymoquinone and p-cymene (31,4%) with minor amounts of longifolene, carvacrol and thymohydroquinone [19,20].

Black seed showed the most potent radical scavenging better antioxidant activity compared to synthetic antioxidants (BHA and BHT) [19]. In addition, it was documented that black cumin had antimicrobial, anticarcinogen, antioxidant effects and antitumor and antiinflammatory activity [20,21]. It is known that the major component in black cumin is thymoquinone compound and this compound is considered to be a nutritional component [22] and thymoquinone is also considered as potent antioxidant, antiinflammatory, anticarcinogen, antibacterial, antifungal, and antimutagenic agent [19,21].

So far, various studies dealing with the addition of natural and synthetic antioxidants and also food additives to meat and meat products have been published [10, 14–17, 23–27], however, to the best of our knowledge, none of the studies are concerned with the effects of black cumin on the formation of HAAs. Therefore, the purpose of the present study was to investigate the influence of adding of black cumin (0,5 and 1%) in meatball preparation on the formation of HAAs in meatball cooked at different cooking temperatures (150°C, 200°C and 250°C) as well as on the various quality criteria of meatballs.

Materials and method

Materials

Raw materials

Beef muscle (M. Gluteus medius) and intermuscular fat were obtained from a local slaughterhouse (Meat and Milk Institution, Erzurum, Turkey). All subcutaneous fats on meat were removed and used after homogenization. The fat content of meatball dough was adjusted to 15% fat with the intermuscular fat from the same carcass. Black cumin was purchased from a spice store in Erzurum.

HAA standards

The HAA standards were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). 4,7,8-TriMeIQx was used as an internal standard. The stock solutions were prepared according to the method described by Oz and Çakmak [28].

Preparation of meatballs

The meatball dough with 15% fat was divided into three portions; one portion was used as control group (without addition of black cumin), and black cumin was added to other portions at two different rates (0,50 and 1%). All groups were shaped into meatballs (7 x 1 cm) after one night storage at 4°C. No salt or spices were added to meatball dough to avoid any possible interaction.

Cooking conditions

Hot plate heated to 150°C, 200°C and 250°C without any fat or oil was used for the cooking of the meatballs. All samples were turned over after half the cooking time (8 min). The surface temperature was measured by a digital thermocouple with a surface probe (Testo 926, Lenzkirch, Germany).

Water content

Water contents of the meatballs were determined as weight loss of 10 g homogenized samples following dryness at 102°C for 24 h [29].

pH value

pH values were measured in the samples which was homogenized with distilled water (1:10 w/v) using a pH meter (ATI ORION 420, MA 02129, USA). The pH meter was calibrated using buffer solutions (pH 4.0 and pH 7.0) [29].

Cooking loss

Weight of the meatballs was measured before and after cooking [30].

TBARS value

Thiobarbituric acid reactive substances (TBARS) values of the samples were determined by the method of Kılıç and Richards [31]. The absorbance of the samples was measured at a wavelength of 532 nm aganist blank. 1, 1, 3, 3- tetraethoxypropane was used fort he calculation of k value. Results were expressed as mg malondialdehyde (MDA)/kg sample.

Determination of HAA content

The HAAs contnet of the samples was determined according to Messner and Murkovic [32] with minor modifications [1]. Solid phase extraction method (Oasis cartridges, 3 cm3/60 mg Waters, Milford, MA) was used for the analysis. For this aim, 1 g meatball sample was dissolved in 12 ml 1 M NaOH. The suspension was homogenised by using a magnetic stirring for 1 h at 500 rpm at room temperature. The alkaline solution was mixed with 13 g diatomaceous earth (Extrelut NT packaging material, Merck, Darmstadt, Germany) and then poured into empty Extrelut columns. The extractions were made by using 75 ml ethyl acetate and the eluate was passed through coupled Oasis MCX cartridges. The cartridge was washed with 2 ml of 0.1 M HCl and 2 ml MeOH. The analytes were eluted with 2 ml of MeOH-concentrated (25%) ammonia (19/1, v/v). The eluted mixtures were evaporated to dryness at 50°C and the final extracts were dissolved in 100 μl MeOH just before measurement. A mixture of purified water/acetonitrile/methanol/glacial acetic acid (76/14/8/2, v/v/v/v) at pH 5,0 (adjusted with ammonium hydroxide 25%) was used as Solvent A, while solvent B was acetonitrile (100%). Separation process was conducted on AcclaimTM 120 C18 3 μm (4,6 x 150 mm) Tosoh Bioscience GmbH (Stuttgart, Germany) at 35°C. The flow rate was 0,7 ml/min. The gradient programme was as follows: 0% B, 0–10 min; 0–23% B, 11–20 min; 23% B, 21–30 min; 0% B, 31–45 min. The injection volume was 10 ml.

Statistical analyses

The experiment was a completely random block design with two replicates. The data obtained in the current study were subjected to analysis of variance. The differences between means were evaluated by Duncan’s multiple range test.

Results and discussion

Analyses of raw materials

Water content, pH, and TBARS values of the meat, intermuscular fat and raw meatballs with 15% fat were shown in Table 1. The obtained results are in line with those reported by others [33-37].

Table 1

Water content, pH and TBARS values of the raw materials (mean ± SD).

	n	Water (%)	pH	TBARS (mg MDA/kg)
Meat	2	71,33±0,52 a	5,64±0,09 b	0,804±0,107 a
Intermuscular fat	2	13,97±0,86 c	6,54±0,29 a	0,290±0,082 b
Meatball	2	64,18±0,77 b	5,70±0,16 b	0,656±0,097 a
Sign.		**	**	**

**p< 0,01,

SD: Standard Deviation,

Different letters (a-c) in the same column denote significant differences (p<0,05)

Water contents of the meatballs

The water contents of the meatballs were given in Table 2. There was a very significant effect (p<0,01) of usage rate of black cumin, cooking process and cooking temperature on the water contents of the meatballs. The addition of black cumin to meatball dough significantly reduced the water content compared to the control group samples (p<0,05). As the usage rate of black cumin increased, the water content of the samples decreased. The decrease in the water content of meatballs with adding the black cumin is attributable to the high dry matter content of the black cumin used in the present study (95,70%). Cooking, as expected, caused a decrease in the water content of the samples; the finding which is consistent with others [28,38]. In addition, it was also determined that the water content of the samples decreased as the cooking temperature increased. del Pulgar et al. [39] reported the three main reasons for water loss in meat during cooking process. First, as temperature increases so does the process of evaporation. Second, heating caused denaturation of myosin and shrinkage of myofibrils, which in turn lead to a reduction in the myofibril’s ability to hold water. Finally, a contraction of the perimysial connective tissue causes a compression of the muscle fiber bundles; in accordance this could enhance water release from the meat cut.

Table 2

The average water content, pH and TBARS values, cooking loss and total HAA contents of the samples (mean ± SD).

	nx	Water(%)	pH	TBARS(mg MDA/kg)	Cooking Loss(%)	Total HAA(ng/g)
Usage rate (UR, %)
0	12	61,72 ± 3,95a	5,98 ± 0,14c	1,228 ± 0,425a	31,56 ± 4,74a	1,25 ± 1,22a
0,5	12	60,78 ± 4,84b	6,02 ± 0,14b	1,088 ± 0,283b	30,02 ± 4,98b	0,81 ± 0,79b
1	12	59,66 ± 5,33c	6,03 ± 0,12a	1,023 ± 0,234c	29,07 ± 4,51c	0,65 ± 0,62c
Sign.		**	**	**	**	**
Cooking Process (CP)
Raw	18	64,95 ± 0,38a	5,89 ± 0,03b	0,876 ± 0,032b
Cooked	18	56,49 ± 2,67b	6,13 ± 0,06a	1,349 ± 0,316a
Sign		**	**	**
Cooking Temperature (CT, °C)
150	12	62,25 ± 2,96a	5,97 ± 0,09c	0,959 ± 0,100c	24,38 ± 1,19c	nd
200	12	60,13 ± 5,16b	6,02 ± 0,14b	1,102 ± 0,259b	31,65 ± 1,25b	0,58 ± 0,26b
250	12	59,80 ± 5,53c	6,04 ± 0,15a	1,277 ± 0,455a	34,62 ± 1,30a	2,03 ± 0,58a
Sign.		**	**	**	**	**
Interactions
UR x CP		**	**	**
UR x CT		ns	*	**	ns	**
CP x CT		**	**	**
UR x CP x CT		ns	*	**

** p<0,01,

*p<0,05,

ns: Not Significant (p>0,05),

xn = 6 for cooking loss values

SD: Standard Deviation, nd: Not detected,

Different letters (a-c) in the same column denote significant differences (p<0,05)

pH values of the meatballs

pH values of the meatballs were also given in Table 2. There was a very significant effect (p<0,01) of usage rate of black cumin, cooking process and cooking temperature on the pH values of the meatballs. Black cumin usage in the meatball preparation significantly increased the pH values compared to the control group samples (p<0,05). The pH of the samples was increased with the increased usage rate of black cumin. The increase in pH value of meatballs with adding the black cumin could be attributed to the high pH value of the black cumin used in the present study (6,22). Cooking, as expected, caused an increase in pH values of the samples and this finding is in accordance with that reported by others [18,37,40]. In addition, in the present study, it was also determined that the pH values of the samples increased as the cooking temperature increased. Oz et al. [40] found pH value of 6,09 and 6,17 in raw and cooked meatballs, respectively. The reason behind the increase in pH value of meat owing to cooking might be attributed to the cleavage of bonds involving imidazole, sulfhydryl and hydroxyl groups as reported by Girard [41].

TBARS values of the meatballs

TBARS values of the meatballs were also given in Table 2. There was a very significant effect (p<0,01) of usage rate of black cumin, cooking process and cooking temperature on the TBARS values of the meatballs. Adding of black cumin usage in the meatball preparation significantly reduced TBARS values compared to the control group samples (p<0,05). As the usage rate of black cumin increased, the TBARS values of the samples decreased. The antioxidant effect of black cumin on TBARS value was believed to be due to its phenolic compound. It is known that antioxidant activity of phenolic compounds is linked to their high redox potentials that would allow them to act as reducing agents, hydrogen donors [19]. In addition, it was determined that cooking caused an increase in TBARS values of the samples and the cooking temperature increased, TBARS values of the samples increased. Due to the fact that cooking destroys the cellular structure and inactivates enzymes enabling oxygen to become free from oxymyoglobin, cooking is one of the main reasons of lipid oxidation in meat and meat products [42].

The obtained TBARS value herein with remained below the acceptance threshold of 2 mg MDA/kg [43,44].

Cooking loss values of the cooked meatballs

Cooking loss values of the meatballs were also given in Table 2. There was a very significant effect (p<0,01) of usage rate of black cumin and cooking temperature on cooking loss values of meatballs. Black cumin usage in meatball preparation significantly reduced cooking loss values compared to control group (p<0,05). As the usage rate of black cumin increased, the cooking loss values of the samples decreased. This could be attributed to the dry matter content (95,70%) of the black cumin used in the current study. It was found that cooking loss values of the samples increased as the cooking temperature increased.

Rodriguez-Estrada et al. [45] reported that there is a decrease in the weights of meat products as a result of the cooking process; it is assumed that water is removed. Cooking not only removes meat juice from the meat but also some water-soluble compounds therein. Indeed, according to Gerber et al. [46], Laroche declared that meat juices removed during cooking contain some compounds such as myofibrillar or sarcoplasmic proteins, collagen, lipids, salt, polyphosphates, and aroma compounds.

Limit of detection and limit of quantification values and recoveries of HAAs

Limit of detection (LOD) and limit of quantification (LOQ) for HAAs were calculated based on the signal to- noise ratios of 3 and 10, respectively. Recovery rates for the nine HAAs in the samples were determined by the standard addition method. The recovery values of the nine HAAs were ranged between 28,94 and 82,15%. The LOD values were in the range of 0.004 and 0.025 ng/g, while the LOQ values were in between 0,013 and 0,085 ng/g. These values were comparable to those reported in the literature [26,32].

HAA content of the meatballs

HAA content of the meatballs were shown in Table 3. While IQx, IQ, MeIQ, 7,8-DiMeIQx, 4,8-DiMeIQx, AαC and MeAαC compounds were not detected in any of the analyzed samples, varying levels of MeIQx (up to 1,53 ng/g) and PhIP (up to 1,22 ng/g) were determined.

Table 3

The HAA content of the samples (ng/g).

Cooking Temperature (°C)	Usage Rate (%)	IQx	IQ	MeIQx	MeIQ	7,8-DiMeIQx	4,8-DiMeIQx	PhIP	AαC	MeAαC	Total HAA
150	0	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd
	0,5	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd
	1	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd
200	0	nd	nd	0,90	nd	nd	nd	nq	nd	nd	0,90
	0,5	nd	nd	0,50	nd	nd	nd	nd	nd	nd	0,50
	1	nd	nd	0,35	nd	nd	nd	nd	nd	nd	0,35
250	0	nd	nd	1,53	nd	nd	nd	1,22	nd	nd	2,75
	0,5	nd	nd	1,00	nd	nd	nd	0,85	nd	nd	1,85
	1	nd	nd	0,86	nd	nd	nd	0,65	nd	nd	1,51

nd: Not detected

The amount of IQx was below the LOD; the finding which is consistent with others [28,35,47-52]. On the other hand, IQx was determined up to 0,13 ng/g in beef barbecued at up to 300°C for up to 20 min by Turesky et al. [53], up to 0,39 ng/g in beef fried at up to 300°C for up to 24 min by Turesky et al. [53], up to 0,61 ng/g in beef fried at 200°C for up to 6 min by Oz et al. [54], as 1,5 ng/g in grilled beef by Fay et al. [55] and up to 3,65 ng/g in barbecued beef for up to 6 min by Oz et al. [54].

The amount of IQ was below the LOD; the finding which is consistent with others [28,35,47,48]. On the other hand, IQ was determined as 0,02 ng/g in beef fried at 200 °C for 12 min by Felton et al. [56], as 0,5 ng/g in grilled beef by Yamaizumi et al. [57], as 7 ng/g in barbecued beef at up to 500°C for 15 min by Rivera et al. [58] and as 10,2 ng/g in beef fried at 180°C for 20 min by Murkovic et al. [26].

MeIQx was determined in all of the meatballs cooked at 200°C and 250°C, however, the compound could not be detected in the meatballs cooked at 150°C. As the cooking temperature increased, MeIQx content of the samples increased. In addition, as the usage rate of black cumin increased, MeIQx content of the samples decreased. Although MeIQx was not detected in various meat and meat products [18,35,59-63], MeIQx was determined as 0,64 ng/g in fried beef by Wakabayashi et al. [64], as 1 ng/g in beef fried at 250°C for 12 min by Felton et al. [56], as 2,11 ng/g in broiled beef by Wakabayashi et al. [64], up to 8,3 ng/g in beef fried at 190°C for up to 13 min by Gross [65] and as 16,4 ng/g in beef fried at 277°C for 12 min by Thiébaud et al. [66]. The maximum MeIQx content was recorded in cooked beef at a concentration level of 80 ng/g [3].

The amount of MeIQ was below the LOD; the finding which is consistent with others [35,36,40,48,60]. On the other hand, MeIQ was determined up to 0,38 ng/g in beef barbecued at up to 240°C for up to 12 min by Abdulkarim and Smith [59], as 2,46 ng/g in beef fried at 180°C for 20 min by Murkovic et al. [26], and as 8 ng/g in barbecued beef at up to 500°C for 15 min by Rivera et al. [58].

The amount of 7,8-DiMeIQx was below the LOD; the finding which is consistent with others [50,52,62]. On the other hand, 7,8-DiMeIQx was determined as 0,2 ng/g in grilled beef by Fay et al. [55], and up to 1,75 ng/g in beef fried at up to 275°C for up to 15 min by Klassen et al. [67].

The amount of 4,8-DiMeIQx was below the LOD; the finding which is consistent with others [60,62]. On the other hand, 4,8-DiMeIQx was determined up to 1,2 ng/g in beef fried at up to 250°C for 12 min by Felton et al. [61] and as 4,5 ng/g in beef fried at 277°C for 12 min by Thiébaud et al. [66]. The maximum 4,8-DiMeIQx content was recorded in cooked beef at a concentration level of 15 ng/g [3].

PhIP was not detected in all of the meatballs cooked at 150°C and in the meatballs with black cumin and cooked at 200°C. In the control group meatballs cooked at 200°C, the compound was detected but its amount could not be determined (not quantified). On the other hand, PhIP was determined in all of the meatballs cooked at 250°C. In the control group meatballs, as the cooking temperature increased, PhIP content increased. The use of black cumin in the preparation of meatball caused a reduction in PhIP content of the samples cooked at 200°C and 250°C. There are studies in the literature showing that PhIP was not detected in various meat and meat products [25,35,36,40,62]. On the other hand, PhIP was determined as 0,56 ng/g in fried beef by Wakabayashi et al. [64], up to 13,3 ng/g in beef fried at up to 250°C for 12 min by Felton et al. [61] and as 15,7 ng/g in broiled beef by Wakabayashi et al. [64]. The maximum PhIP content was recorded in cooked beef at a concentration level of 182 ng/g [3].

The amount of AαC was below the LOD; the finding which is consistent with others [47,68,69]. On the other hand, AαC was determined as 1,20 ng/g in broiled beef by Wakabayashi et al. [64], up to 3,32 ng/g in beef fried at up to 300°C for up to 24 min by Turesky et al. [53], up to 7,75 ng/g in beef barbecued at up to 300°C for 20 min by Turesky et al. [53] and as 21 ng/g in beef fried at 277°C for 12 min by Thiébaud et al. [66].

The amount of MeAαC was below the LOD; the finding which is consistent with others [36,47,52,68,69]. On the other hand, MeAαC was determined up to 0,14 ng/g in beef fried at up to 300°C for up to 24 min by Turesky et al. [53] and up to 0,29 ng/g in beef barbecued at up to 300°C for 20 min by Turesky et al. [53].

Total HAA content of the meatballs ranged from 0,5–2,75 ng/g, whereas in the case of meatballs cooked at 150°C HAAs were not detected. It was determined that increases in the cooking temperature would increase the total HAAs of both control and meatballs supplemented with black cumin. While the total HAA content of the control group meatballs cooked at 150°C was not detectable, the total HAA contents of the control group meatballs cooked at 200°C and 250°C were 0,90 ng/g and 2,75 ng/g, respectively. On the other hand, the total HAA content of the meatballs including black cumin at 0,5% and cooked at 150°C was not detectable, whereas the total HAA contents of those meatballs cooked at 200°C and 250°C were 0,50 ng/g and 1,85 ng/g, respectively. While the total HAA content of the meatballs including black cumin at 1% and cooked at 150°C was not detectable, the total HAA contents of those meatballs cooked at 200°C and 250°C were 0,35 ng/g and 1,51 ng/g, respectively. It was also found that the use of black cumin in the preparation of meatball caused a reduction in total HAA content of the samples and as the use rate of black cumin increased, the total HAA content of the meatballs decreased. While the total HAA content of the meatballs including black cumin cooked at 150°C was not detectable, the use of black cumin at 0,5% in meatball production caused a reduction on the total HAA content of the meatballs cooked at 200°C (44,44%) and at 250°C (32,73%). Similarly, the use of black cumin at 1% in meatball production also caused a reduction on the total HAA content of the meatballs cooked at 200°C (32,73%) and at 250°C (45,09%). On the other hand, the total HAA contents of the meatballs cooked at 200°C were belonging to MeIQx compound. While the total HAA contents of the control group meatballs cooked at 250°C were determined to be consisted of MeIQx (55,64%) and PhIP (44,36%), the total HAA contents of the meatballs containing different amounts of black cumin and cooked at the same temperature were consisted of MeIQx (54,05–56,95%) and PhIP (43,05–45,95%).

Oz and Kaya [16] investigated the effect of black pepper on the formation of HAA and found that the total HAA (IQ, MeIQ, MeIQx, 4,8-DiMeIQx, and PhIP) content ranged between 1,40–37,81 ng/g in their control group meatballs cooked at 175–250°C. The researchers declared that use of black pepper at 1% inhibited total HAA content up to 100%.

Shin et al. [14] found that the addition of 20 g of minced garlic cloves to ground beef patties would reduce the total HAAs (MeIQx, DiMeIQx and PhIP) by 68%. In another study conducted the same group, it was reported that 24.6 ng/g total HAA (MeIQx, DiMeIQx, and PhIP) was determined in control group fried at 225°C for 20 min [15]. The authors also found that the addition of diallyl disulfide and dipropyl disulfide to ground beef patties inhibited total HAA formation by up to 78% and 70%, respectively.

The use of spices with antioxidant properties in meat preparation could interfere with different stages of HAA formation. The exact mechanism of inhibiting the HAA formation by antioxidants is still not fully understood. However, it has been generally accepted that antioxidants inactivate free radicals. Due to their antiradical activity, antioxidants could act as inhibitors in the mutagens formation [10]. On the other hand, it was justified that prooxidant or antioxidant effect of antioxidants was highly dependent upon the concentration [25]. In this context, Oz et al. [38] investigated the effect of direct addition of conjugated linoleic acid (CLA) to beef chops cooked at 150°C, 200°C and 250°C on formation of HAAs and found that while direct addition of 0.05% CLA to beef chops increased (3.85–68.75%) total HAA amount at all cooking temperatures, direct addition of 0.1% CLA to the beef chops decreased (18,75–31,54%) the total HAA amount at all cooking temperatures. Similarly, Oz and Çakmak [28] investigated the effects of CLA usage in meatball production on the formation of HAA in meatballs and found that the use of CLA displayed an inhibitory as well as a stimulatory on total HAA content of meatballs depending on the usage rate and cooking temperatures.

In terms of total HAAs content, it is somehow difficult to compare the results of the present study with the others. This is due to the fact that the current study is the first study on the effect of black cumin usage in the preparation of meatball on the formation of HAAs. On the other hand, the amount of total HAAs in the present study was lower than other studies. The little differences might be arise from the type of the meat used, animal feeding conditions, the various cooking conditions, extraction methods, and type of chromatographic detection systems. On the other hand, according to Knize et al. [70], who cited data by Pearson and others, the total amounts of IQ, MeIQx, and DiMeIQx were 7300 ng/g in beef fried at 215°C; the value which is considerably higher than both results of the present study and others [14–16, 28, 38].

In the present study, it is seen that even if 100 g of the control group meatballs at 250°C whose total amount of HAA content is the highest, is eaten, the intake amount is quite far below the maximum acceptable daily consumption (15 μg/day) stated by Skog [5].

Conclusion

As a conclusion, the results showed that adding black cumin to meatball would reduce the levels of both individual HAAs (MeIQx and PhIP) and total HAAs. The inhibitory effect of black cumin on HAAs could be attributed to the antioxidant effect of its phenolic compounds.

31 Jul 2019

PONE-D-19-18609

The effect of the use of black cumin in meatball preparation on the formation of heterocyclic aromatic amines

PLOS ONE

Dear Dr. Oz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please revise this paper.

We would appreciate receiving your revised manuscript by Sep 14 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Aneta Agnieszka Koronowicz, PhD

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Editor,

The article is deal with the inhibitory effect of black seed on the formation of heterocyclic amines in meatball. The topic is interest and the article is good and easy to follow. It can be published in your journal after done necessary corrections. My specific comments and questions about the article are below.

- The author should use black cumin or Nigella sativa throughout the article.

- Title: The title of the article should be “Inhibitory effects of black cumin on the formation of heterocyclic aromatic amines in meatball”.

- Page 2, line 31: significantly reduced? Give P values in statistical analysis.

- Page 4, line 98-99: No need to this sentence, repetition for the above stated sentence (line 83-84).

- Page 6, line 143: blind or blank?

- Page 7, line 151: distilled water or purified water?

- Page 15, line 349-350: What do you mean? This sentence is not clear.

- Page 16, line 377: Give the references.

Reviewer #2: The paper is very interesting. From chemical point of view, there is nothing new, just another scenario of Maillard reaction. The data are brief and clear. The paper is well written. And it includes the relevant literature. It seems from the data presented that the black cumin can prevent the formation of genotoxic mutagenic amines to some extent. Taking into account nutritional aspects, it is a valuable finding. Therefore, I recommend the present study to be published as it is.

Reviewer #3: In my opinion this manuscript may be published in PLOS ONE only after a major revision. Generally, English must be improved. In this study, the Authors estimated the effect of use of black cumin in meatball preparation on formation of HAAs. In my opinion, it would improve a paper a lot if the Authors added (e.g., in introduction) any information about black cumin as a source of antioxidants. Beside essentiale oil containing mainly carvone and limonen, black cumin seeds may contain also phenolic acids (e.g., caffeic acid), flavonoids, leuco-anthocyanins that may have a crucial role in scavenging of free radicals. The last ones may be responsible for formation of HHAs.

The remaining comments to the Authors are given below.

Page 3 line 58 „aminoimidazoaazoarenes” shouild be changed into „aminoimidazoarenes”

Page 3 line 63 It is not clear if antioxidants are precursors of HAAs ort hey may play a role of inhibitors of HAAs or any others contaminants

Page 3 line 64 „ HAAs were found to be” should be changed as „HAAs content (or level) were found to be 100-fold higher”

Page 3 line 75 According to the Authors „some antioxidants had a prooxidant effect”, however,

the same antioxidants could possess prooxidant or antioxidant activity depending on their concentration in food”

Page 4 line 80 „natural antioxidants” should be changed as „food rich in natural antioxidants such as”, line 83 „protein, fat and carbohydrate” should be changed into „proteins, fats and harbohydrates”

Page 4 line 90 „carvacrola” should be changed into „carvacrol”, line 95 „ antiinflamatory” should be changed as „antiinflamatory activity (or properties)”

Page 4 lines 96&98 „thymokinone” should be changed as „thymoquinone”

Page 4 lines 98&99 The last sentence is the exact repetition what was written before (lines 83&84)

Page 5 line 101 „food ingredients” should be changed as „food additives”

Page 6 line 150 It is not clear in which way the mentioned solvents were used during the solid phase extraction methods, so a more detailed description of the SPE extraction is needed

Page 7 line 153 Please, give a information that the gradient method was used during chromatographic analysis, moreover, 3 mm should be changed into 3µm (in case of particles diameter)

Page 7 line 161 Please, use the same designation in the description of Table 1 as in Table 1 (eg. „meat” in Table 1 and „beef muscle” in line 161

Page 8 line 199 „groups by Girard” should be changed as „ groups as reported by Girard”

Page 13 line 314 „ranged between nd – 2,75 ng/g” should be changed into „ ranged from 0,5 – 2,75 ng/g, whereas in the case of meatballs cooked at 150°C HAAs were not detected”

Page 15 line 355 „radical quenchers and free radical scavenging activity are designation of the same phenomenon – antiradical activity

Page 16 line 373 „the chromatographic detector” should be changed as „ type of chromatographic detection”

Page 16 lines 382&385 The last sentence is not so adequate to the conclusion of this manuscript

Table 2 The Authors have given the values of determinated parameters in first line named as Usage rate (0-1) but nothin is known about temperature of cooking (in case of first line), so detailed information could be helpful for the readers (under the table or in the text),

With respect to the values of parameters given in line No 3 named as cooking temperature, did the Authors give the average values for various usage rate (0–1 %), please to explain

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: report.pdf

Click here for additional data file.

1 Aug 2019

Plos One

PONE-D-19-18609

“The effect of the use of black cumin in meatball preparation on the formation of heterocyclic aromatic amines”

Dear Editor,

Thank you for your useful comments. I have modified the manuscript accordingly. The corrections according to reviewers comment were given in yellow in the text. The detailed corrections are also listed below point by point:

Response to the reviewers:

Review Comments to the Author

Reviewer #1:

Dear Editor,

The article is deal with the inhibitory effect of black seed on the formation of heterocyclic amines in meatball. The topic is interest and the article is good and easy to follow. It can be published in your journal after done necessary corrections. My specific comments and questions about the article are below.

- The author should use black cumin or Nigella sativa throughout the article.

“Black cumin” was used throughout the article.

- Title: The title of the article should be “Inhibitory effects of black cumin on the formation of heterocyclic aromatic amines in meatball”.

I would like to thank for the reviewer comment. The title of the article has been changed as “Inhibitory effects of black cumin on the formation of heterocyclic aromatic amines in meatball”.

- Page 2, line 31: significantly reduced? Give P values in statistical analysis.

P value has been added to the sentence.

- Page 4, line 98-99: No need to this sentence, repetition for the above stated sentence (line 83-84).

Thank you for the reviewer’s attention. The second sentence has been discarded form the text.

- Page 6, line 143: blind or blank?

“Blind” has been changed as “blank”.

- Page 7, line 151: distilled water or purified water?

“Water” has been changed as “purified water”

- Page 15, line 349-350: What do you mean? This sentence is not clear.

Thank you. The sentence has been modified.

- Page 16, line 377: Give the references.

The references have been added to the text.

Reviewer #2: The paper is very interesting. From chemical point of view, there is nothing new, just another scenario of Maillard reaction. The data are brief and clear. The paper is well written. And it includes the relevant literature. It seems from the data presented that the black cumin can prevent the formation of genotoxic mutagenic amines to some extent. Taking into account nutritional aspects, it is a valuable finding. Therefore, I recommend the present study to be published as it is.

I would like to thank for the reviewer comment.

Reviewer #3: In my opinion this manuscript may be published in PLOS ONE only after a major revision. Generally, English must be improved. In this study, the Authors estimated the effect of use of black cumin in meatball preparation on formation of HAAs. In my opinion, it would improve a paper a lot if the Authors added (e.g., in introduction) any information about black cumin as a source of antioxidants. Beside essentiale oil containing mainly carvone and limonen, black cumin seeds may contain also phenolic acids (e.g., caffeic acid), flavonoids, leuco-anthocyanins that may have a crucial role in scavenging of free radicals. The last ones may be responsible for formation of HHAs.

I would like to thank you for the reviewer’s useful comment. A native speaker has checked the article and the necessary corrections have been done on it. About the antioxidant activity of the black cumin, black cumin seeds contain fixed oil and volatile oil including thymoquinone and monoterpenes such as r-cymene and a-piene. However, it is reported that thymoquinone is major component of black cumin and it is considered as potent antioxidant, anticarcinogenic and antimutagenic. In the present study, usage of black cumin in meatball production inhibited the formation of HAAs (not promoted) and its inhibitory effect could be attributed to its thymoquinone content. The sentence about the antioxidative properties of black cumin has been modified.

The remaining comments to the Authors are given below.

Page 3 line 58 „aminoimidazoaazoarenes” shouild be changed into „aminoimidazoarenes”

Thank you for the reviewer’s attention. “aminoimidazoaazoarenes” has been changed as “aminoimidazoarenes”.

Page 3 line 63 It is not clear if antioxidants are precursors of HAAs ort hey may play a role of inhibitors of HAAs or any others contaminants

The reviewer is right. Therefore, the word of “antioxidants” has been discarded from the text.

Page 3 line 64 „ HAAs were found to be” should be changed as „HAAs content (or level) were found to be 100-fold higher”

In here, I compared the mutagenicities of HAAs and the other known food mutagens such as aflatoxin B1 and benzo(a)pyrene. However, the sentence has been modified.

Page 3 line 75 According to the Authors „some antioxidants had a prooxidant effect”, however,

the same antioxidants could possess prooxidant or antioxidant activity depending on their concentration in food”

The sentence has been modified.

Page 4 line 80 „natural antioxidants” should be changed as „food rich in natural antioxidants such as”, line 83 „protein, fat and carbohydrate” should be changed into „proteins, fats and harbohydrates”

The corrections have been done. Thank you.

Page 4 line 90 „carvacrola” should be changed into „carvacrol”, line 95 „ antiinflamatory” should be changed as „antiinflamatory activity (or properties)”

Page 4 lines 96&98 „thymokinone” should be changed as „thymoquinone”

The corrections have been done. Thank you.

Page 4 lines 98&99 The last sentence is the exact repetition what was written before (lines 83&84)

Thank you for the reviewer’s attention. The sentence has been discarded form the text.

Page 5 line 101 „food ingredients” should be changed as „food additives”

“food ingredients” has been changed as “food additives”.

Page 6 line 150 It is not clear in which way the mentioned solvents were used during the solid phase extraction methods, so a more detailed description of the SPE extraction is needed

Thank you. The part of SPE has been modified.

Page 7 line 153 Please, give a information that the gradient method was used during chromatographic analysis, moreover, 3 mm should be changed into 3µm (in case of particles diameter)

Gradient programme has been added to the text and 3 mm has been changed as 3 µm. Thank you.

Page 7 line 161 Please, use the same designation in the description of Table 1 as in Table 1 (eg. „meat” in Table 1 and „beef muscle” in line 161

“Beef muscle” has been changed as “meat”.

Page 8 line 199 „groups by Girard” should be changed as „ groups as reported by Girard”

Thank you. The correction has been done.

Page 13 line 314 „ranged between nd – 2,75 ng/g” should be changed into „ ranged from 0,5 – 2,75 ng/g, whereas in the case of meatballs cooked at 150°C HAAs were not detected”

Thank you. The correction has been done in the text.

Page 15 line 355 „radical quenchers and free radical scavenging activity are designation of the same phenomenon – antiradical activity

This part has been corrected.

Page 16 line 373 „the chromatographic detector” should be changed as „ type of chromatographic detection”

Thank you. The correction has been done in the text.

Page 16 lines 382&385 The last sentence is not so adequate to the conclusion of this manuscript

The reviewer is right. Therefore, the sentence has been moved to results and discussion section. Thank you.

Table 2 The Authors have given the values of determinated parameters in first line named as Usage rate (0-1) but nothin is known about temperature of cooking (in case of first line), so detailed information could be helpful for the readers (under the table or in the text),

With respect to the values of parameters given in line No 3 named as cooking temperature, did the Authors give the average values for various usage rate (0–1 %), please to explain

Thank you for your comment. Table 2 shows the average values of the results. Therefore, n (number of sample) was added to the Table 2. I think n explains the situation.

The revised manuscript has been resubmitted to your journal. I look forward to your positive response as soon as possible. Is there anything I can do for that, please don’t hesitate to contact me.

Sincerely yours,

Dr. Oz

Submitted filename: Response to reviewers.doc

Click here for additional data file.

14 Aug 2019

Inhibitory effects of black cumin on the formation of heterocyclic aromatic amines in meatball

PONE-D-19-18609R1

Dear Dr. Oz,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Aneta Agnieszka Koronowicz, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Editor,

The article is deal with the inhibitory effect of black seed on the formation of heterocyclic amines in meatball. The topic is interest and the article is good and easy to follow. In addition, the author has modified the article according to the reviewers comments. It can be published in your journal with this form.

Sincerely yours,

Reviewer #3: In my opinion the manuscript entitled "Inhibitory effects of black cumin on the formation of heterocyclic aromatic amines in meatball" may be published in PLOS ONE

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

20 Aug 2019

PONE-D-19-18609R1

Inhibitory effects of black cumin on the formation of heterocyclic aromatic amines in meatball

Dear Dr. Oz:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Aneta Agnieszka Koronowicz

Academic Editor

PLOS ONE