Impact of Cumin ( Cuminum cyminum ) Incorporation on the Generation of Heterocyclic Aromatic Amines in Meatballs

: In the current study, the impacts of using cumin (0.5% and 1%, w / w ) in beef meatballs on heterocyclic aromatic amines (HAAs) formation and some quality characteristics when cooked at 150 and 250 ◦ C were investigated. It was found that using of cumin at different ratios in beef meatballs showed a signiﬁcant ( p < 0.01) effect on pH, thiobarbituric acid reactive (TBARS) value, and total HAA. The cooking process signiﬁcantly ( p < 0.01) affected the meatballs’ water content, pH, and TBARS values, while the cooking loss and total HAA content of meatballs were signiﬁcantly ( p < 0.01) affected by cooking temperature. It was found that the cumin usage rate in meatballs increased the pH value, while it was found to decrease the TBARS value. As expected, the cooking process was associated with a reduction in water, while it was found to result in an increase in the pH and TBARS values. On the other hand, both cooking temperature and cumin addition to meatballs led to an increase in their total HAA content. Diverse samples exhibited detectable levels of IQx, MeIQx, MeIQ, and PhIP compounds. The presence of MeIQx compound was found in all samples, except for the control group cooked at 150 ◦ C. Additionally, the use of 0.5% and 1% cumin in meatballs cooked at temperature of 250 ◦ C had an inhibitory effect on MeIQx compound. Our results revealed that the incorporation of cumin in the meatballs resulted in an increase in the total HAA content, likely due to its prooxidant effect. However, it was found that the use of cumin at certain rates could reduce the formation of MeIQx compound with an antioxidant effect. However, in this study found that even if 100 g of meatballs with the highest concentration of total HAA were consumed, the maximum HAA exposure limit value (0–15 µ g/day) was not exceeded.


Introduction
Meat is considered an essential food in human nutrition because of its major components, such as protein and fat, and minor components, such as vitamins and minerals.Meat is usually cooked or processed into various products to be suitable for human consumption.The processes used for this purpose cause some changes in the meat, and the nutritional value, texture, and organoleptic properties of the meat are positively or negatively affected depending on the process used [1].In fact, lipid oxidation, which occurs as a result of contact with air of unsaturated fatty acids during various pretreatments such as mincing, cooking, or transformation into products, has the property of being one of the reactions that have a very important impact on health and quality [2].The use of antioxidant substances to protect foods from oxidative deterioration comes to the fore among the methods applied [3].
On the other hand, due to their carcinogenic and toxic properties, synthetic antioxidants have been replaced by the search for antioxidant substances found in the natural structures of food or medicinal materials [4,5].It is known that Maillard reaction products, phospholipids, sterols, and antioxidant compounds found in the structures of various vegetables, fruits, and seeds have an effect on reducing oxidative damage in many diseases such as cardiovascular disease, cataracts, arthritis, diabetes [5,6].Food phenolics mainly have antioxidant activity as singlet oxygen quenchers, reducing agents and hydrogen donors.Some phenolics have chelating effects on metal ions that catalyze oxidation reactions.Through complex formation with catalytic metal ions, flavonoids exhibit the ability to scavenge and neutralize diverse free radicals (peroxide, hydroxyl, and superoxide radicals).In addition, flavonoids are also known to prevent oxidative rancidity in foods by inhibiting the enzymes lipoxygenase and cyclooxygenase.In this context, herbs and spices are known for their high antioxidant content and have been safely consumed for centuries [6].
Spices, which have an extensive history of utilization and have functions such as preserving foods, prolonging their preservation period, and adding color, taste, and aroma to food items, have been employed for therapeutic purposes in folk medicine for centuries [7][8][9].Many spices are known to have significant anticarcinogenic, antioxidant, and antimutagenic effects [8,10].In this direction, cumin (Cuminum cyminum), which ranks among the most extensively used spices, belongs to the Apiaceae family and contains 2.5-6% essential oil, 10-23% oil, 15-25% protein, in addition to varying proportions of tannin, flavonoids, resin, and gum [9][10][11].It has been reported that antioxidant substances such as cuminal, pinocarveol, γ-terpinene, carotol and linalool 1-methyl-2-(1-methylethyl) benzene isolated from cumin have free radical scavenging and metal chelating effects [6].In addition, studies have shown that the polyphenols found in cumin have antioxidant properties and it has been emphasized that they are good free radical scavengers [4,7].By cooking of meat, some compounds that have been proven to be mutagens and/or carcinogens by epidemiological studies are generated.Heterocyclic aromatic amines (HAAs) occupy an important position among these compounds [12,13].HAAs are chemical substances occurred throughout the cooking of protein-rich foods at temperatures higher than 150 • C [14,15].HAAs, which were initially identified in meat and fish in 1977 [16], are divided into two main chemical classes according to their formation temperatures [13].These are thermal (aminoimidazoazoarenes) and pyrolytic (aminocarbolines) HAAs.Aminoimidazoazoarenes are occurred during reactions in-volving creatine, creatinine, amino acids and reducing sugars (hexoses) at temperatures ranging from 150 • C to 300 • C, whereas aminocarbolines are typically generated through the pyrolysis of amino acids and proteins at temperatures above 300 • C [17][18][19].In the light of epidemiological studies, it has been determined that the majority of HAAs exhibit mutagenic properties and nearly all of them are carcinogenic [20].It is stated that there is a correlation between pancreatic, colorectal and breast cancers and high consumption of fish and meat, and a large part of this positive correlation is due to HAAs [15,21].The cooking conditions (time, equipment, temperature, and method), meat type, pH, lipid oxidation, fats, heat transfer, mass transfer, carbohydrates, water activity, creatine, amino acids, and antioxidant substances are effective on the types and amounts of generation of HAAs [12,22].
Extensive research in the literature has focused on the formation of HAAs [23][24][25][26].Recently, studies have progressed in the direction of the factors influencing the generation of HAAs and the reduction of their generation.In this context, the impacts of several spices on HAA generation have been investigated.The number of studies with various spice additions is quite high in the literature [27][28][29][30].On the other hand, as far as we know, there is just a single study in the literature investigating the impact of cumin on the generation of HAAs [31].In their study, Puangsombat et al. [31] examined the impact of both single concentration and single temperature on HAA formation, and fewer HAA compounds were investigated compared to the current research.Therefore, in the current study, the impact of using cumin in meatballs on the generation of HAA was investigated and it was aimed to consolidate the data obtained with the previous valuable study on this subject and to obtain more data.Meatballs with different amounts of cumin added (0.5% and 1%) were cooked at two temperatures (150 • C and 250 • C) and the amount and type of HAA were investigated.In addition, various quality criteria of meatballs in both raw and cooked forms were determined.

Material
In this research, the beef round muscle (M.Gluteus medius) and intermuscular fat were acquired from the local butcher (Erzurum, Türkiye), while cumin was obtained from a local market (Erzurum, Türkiye).The muscle and the intramuscular fat were obtained from two different male cattle and used raw material.In the selection of these materials, care was taken to use animal meats of the same race, gender, and age.At 24 h after slaughter, these raw materials were delivered to the laboratory under controlled cold chain.After the connective tissues and visible fat of the supplied meat were removed, they were processed through grinding to produce minced meat, which was then employed in the preparation of meatball dough.Then, meatball dough was prepared at the rate of 15% fat from intermuscular fat obtained from the same carcass as the meat minced by the Babcock method.After dividing the meatball dough into 3 equivalent pieces, a group selected depending on chance was evaluated as a control group, while the remaining 2 groups were mixed by adding 0.5% and 1% cumin.In the current study, in the selection of M. Gluteus medius muscle in meatball production, it was taken into consideration that its chemical composition is suitable for meatballs and that this muscle is generally preferred in studies.In determining the concentrations of cumin, other studies examining the impact of spices and their extracts on HAA generation were taken into consideration [32][33][34][35].
The homogenized meatball doughs were kept at 4 • C for 6 h and then shaped in 7 × 1 cm dimensions by means of a steel mold.In the production of meatballs, no other additives and/or spices were used to prevent any interaction on the analyses.

Method 2.2.1. Cooking Conditions
An electric hot plate (Hot Plate, Test Laboratory Equipment, 460C, ˙Istanbul, Türkiye), was utilized to cook the meatballs at two temperatures (150 • C and 250 • C) for a total of 8 (4 + 4) min.No fats/oils or water were used in the cooking process.Once cooled to room temperature, the meatballs were homogenized by a blender.For the HAA analysis, homogenized meatball samples were stored (−18 • C) [26].

Determination of the Water Content
The percentage of water in meatballs was founded by drying 10 g of sample weighed in the drying cups in an oven (Redline, RE53, Binder, Germany) at 100 • C (approximately 1 night) [36].

Determination of Cooking Loss
The meatballs, which had been weighed prior to cooking, were cooled to approximately 25 • C after cooking on the hot plate and weighed, and the cooking losses were determined by calculating the difference in % [37].

Determination of HAAs
After combining the meatball sample (1 g) with 12 mL of NaOH (1 M) and stirring for 1 h, the Extrelut NT packaging material (10 g) was placed on it and mixed thoroughly and packaging was done with an Oasis MCX cartridge.Afterwards, the extraction was done out with ethyl acetate (75 mL), methanol (2 mL), and HCl (0.1 N, 2 mL), and in the last step, the extract was eluted with 2 mL of methanol:ammonia (NH 3 , 32%) (19:1).The obtained extract was stored at −18 • C until High Performance Liquid Chromatography (HPLC) analysis.Prior to the analysis, the extract was placed in an oven at 45 • C overnight to eliminate methanol:NH 3 , and subsequently, methanol (100 µL) containing the internal standard (4,7,8-TriMeIQx) was added for analysis using the HPLC-Diode Array Detector system [39].The incorporation of an internal standard (4,7,8-TriMeIQx, 1 ng/mL) was intended to increase the precision and accuracy of the obtained results and to understand whether there was a shift in the other HAAs' peaks or not.In HPLC analysis, the injection volume was determined as 10 µL.In this study, the levels of HAAs present in the samples were measured by conducting standards at different concentrations.The identification of HAAs involved a comparison of their retention times in the samples and the retention times of HAAs whose UV spectra are known as standard.For the quantitative determination of HAAs, the external calibration curve method was employed.The basis for determining the recovery was the standard addition method.For this purpose, a mix stock solution at concentrations of 1, 2.5, 5 and 7.5 ng/g was added to the raw sample before extraction.The peak area against the amount of analyte added was drawn as curve.The recoveries were calculated based on the ratio between the slopes of the linear regression lines for the standard addition and the HAA standard solutions.HPLC conditions (Table 1) for the HAA analysis were referenced by Oz et al. [40].25), and the differences between the outcomes were investigated through the application of the Duncan multiple comparison test.

Water Content, pH, and TBARS Results of Meat Muscle, Intermuscular Fat, and Meatball Dough
The quality of meat and meat products relies on essential parameters such as water percentage, pH value, and lipid oxidation (TBARS value), all of which can be impacted by the cooking process.In addition, they can be related for the formation of HAAs [41,42].The results of water percentage, pH and TBARS values of meat muscle, intermuscular fat, and meatball dough, were presented in Table 2.As can be presented in Table 2, water, pH, and TBARS values were determined as 74.70%, 5.46, and 0.31 mg MDA/kg in beef round meat; 13.89%, 6.47, and 0.38 mg MDA/kg in (intermuscular) meat fat; and 65.62%, 5.63, and 0.34 mg MDA/kg in raw meatballs, respectively (Table 2).There are similar results in the literature [32,35].

Water Percentage, pH, TBARS, Cooking Loss, and HAA Results of All Meatball Samples
In the present study, the average water percentage, pH, TBARS, cooking loss, and total HAA results of all meatball samples based on the cumin usage rate (0, 0.5, and 1%), cooking process (raw and cooked) and cooking temperature (150 and 200 • C) were given in Table 3.The usage rate effect covers the cooking process (raw and cooked meatballs, n = 2), cooking temperatures (150 and 200 • C, n = 2) and replication (n = 2).Therefore, the number of samples was 8.The cooking process effect covers the cumin usage rate (0%, 0.5% and 1%, n = 3), cooking temperatures (150 and 200 • C, n = 2) and replication (n = 2).Therefore, the number of samples was 12. On a different note, the influence of cooking temperature influence covers the cumin usage rate (n = 3), cooking process (raw and cooked meatballs, n = 2) and replication (n = 2).Therefore, the number of samples was 12.

Water Percentage in Meatballs
The percentage of water in raw and cooked meatball samples are presented in Table 3.Our results revealed that the cooking process exhibited a statistically significant impact (p < 0.01) on the water percentage beef meatballs, whereas the incorporation of cumin at various ratios and the cooking level (temperature) did not show statistically significant effect (p > 0.05).It was determined that the water content of raw meatballs decreased as expected with the cooking process.It is known that the water content of the meat decreases with the cooking process due to the evaporation of the free water content of the meat and the changes in the connective tissue and proteins [43].Similar changes in water content due to the effect of cooking have been demonstrated by other researchers [28,44].

Cooking Loss Results of the Meatballs
Cooking loss in meat and meat products is a consequential alteration arising from the cooking process and holds significant economic importance.The color, flavor, juiciness, tenderness, vitamin and mineral levels of the meat are correlated with cooking time and cooking losses [45].The cooking loss results of raw and cooked meatball samples are presented in Table 3.The cooking temperature had a statistically very significant impact (p < 0.01) on the cooking loss of meatballs, while the use of cumin at different ratios did not show a statistically significant impact (p > 0.05).It has been reported that most of the mass loss caused by cooking of meat is in the form of water loss as a result of denaturation and shrinkage, which take places in proteins as a consequence of temperature elevation.As a matter of fact, cooking conditions such as time and temperature along with the specific attributes of the meat such as meat component, size, and pH value, contribute to the variation in cooking loss [45,46].Gerber et al. [47] reported that water removed from meat by the cooking process may also contain other water-soluble components such as proteins, collagen, lipids, polyphosphates, salt, and flavor compounds.While no significant difference was observed in meatball samples with cumin added at different rates compared to the control group, an increment in cooking losses was determined in meatball samples prepared at various temperatures.In this regard, the increase in cooking temperature has an increasing impact on the values of cooking loss.There are studies in the literature with similar results regarding cooking loss in various meat products cooked by different methods [23,46,48,49].

pH Values of the Meatballs
In the current study, the pH values of raw and cooked meatball samples are given in Table 3.While it was determined that the use of cumin in different rates and the cooking process had a very significant effect (p < 0.01) on the pH value of the meatballs, no statistically significant effect (p > 0.05) was observed in terms of the cooking temperature.A statistically significant increase in the pH values was detected of meatball samples to which different amounts of cumin were added compared to the control group.This increase is thought to be related to the pH value of cumin (mean 5.93 ± 0.03).The cooking process caused an increase in the pH value of the meatballs, as expected.Elbir et al. [26] explained the pH increase in meat after cooking with the release of bonds containing imidazole, hydroxyl, and sulfhydryl groups in meat with cooking.It was detected that the cooking temperature did not cause a significant change in the pH value.This result can be explained by the fact that the bonds containing sulfhydryl, imidazole, and hydroxyl groups in meat become sufficiently free even at 150 • C. Similar results on changes in pH have been put forward by other researchers [1,50].

TBARS Values of the Meatballs
TBARS analysis is the common a method used to measure secondary oxidation products that cause oxidative rancidity [2].Oxidation reactions cause the formation of harmful compounds as well as affecting the quality criteria such as shelf life and nutritional value of the products [51].For this reason, TBARS analysis is among the important quality parameters in meat and meat products.The TBARS values of raw and cooked meatball samples are given in Table 3.It was found that the use of cumin had a very significant effect (p < 0.01) on the TBARS value and there was a decrease in the TBARS values independent of the cumin ratio.It is thought that antioxidant substances such as γ-terpinene, linalool, pinocarveol, carotol, and 1-methyl-2-(1-methylethyl) benzene found in cumin are effective in reducing the TBARS value due to their free radical scavenging and metal chelating effects [6].Heat treatment has been reported to disrupt heat-sensitive phenolic compounds and changes their molecular structure in spice [52].Gunasena and Rajapakse [53] reported that temperature and duration have positive effects on antioxidant activity in spices to some extent; however, there are losses in antioxidant activity as the duration is extended.It is considered that the changes in TBARS values depending on the cumin ratio are influenced by the temperature degradation of the antioxidant compounds contained in cumin.It has been reported that phenolic compounds prohibit the formation and the spread of free radical reactions in various ways, and the products formed in the first steps of the Maillard reaction, which occurs in meat at cooking temperatures above 90 • C, show antioxidant properties with free radical scavenging activity [54,55].It was observed that the cooking process had a very significant effect (p < 0.01) on the TBARS value and the cooking process leaded to a significant increase in the TBARS value of the samples.It has been shown that oxidation reactions are affected by various factors (light, temperature, oxygen, metals, radiation, and the presence of antioxidant substances) [56].However, it is also stated that heat treatment is more important than other factors in increasing the lipid oxidation of the samples.The increase in TBARS value seen with the cooking process is due to the increase in the level of malondialdehyde due to the effect of temperature, which is one of the important factors that accelerate oxidation [57].In addition, the increase in TBARS value after cooking could be due to the release of polyunsaturated fatty acids and iron from myoglobin and hemoglobin compounds [35].It was determined that cooking of the meatballs at different temperatures (150 • C and 250 • C) did not lead to significant change (p > 0.05) in the TBARS value.Lu et al. [28] reported that there may be an increase or decrease in TBARS values depending on the type of spice used in meatballs.Researchers attributed the decrease in TBARS value to the antioxidant substances found in the spices used and their activity.As a matter of fact, although there are studies showing that the TBARS value increased with the effect of cooking [49,50], there are also studies showing that it decreased [46].On the other hand, it is stated that the acceptable TBARS value for meat products is a maximum of 3 mg MDA/kg, while values below this threshold do not pose a risk in terms of oxidative changes [57].In this context, it was founded that the TBARS values detected in the control group and the meatball samples with cumin at different rates in the current study were below this limit.

Method Validation of HAAs
The standard addition method was preferred to calculate the recoveries of the HAAs examined in the study, and HAA mixture stock solutions at concentrations of 7.5, 5, 2.5 and 1 ng/g were added to the raw meatball samples before extraction.After the extraction and analysis performed for this purpose, the recoveries were determined.The LOD and LOQ values calculated according to the Signal/Noise (S/N) ratios from the HAA concentrations at certain concentrations are given in Table 4.The detected recoveries (81.52-95.64%) of the HAA compounds examined in this study are in agreement with the literature [39].In addition, the chromatograms of the meatballs with the highest total HAA content and HAA mix stock solution are given in Figure 1.

Total HAAs of the Meatballs
Total HAA content of cooked meatball samples are shown in Table 3.It was observed that cumin usage rate and cooking temperature had a statistically very significant effect (p < 0.01).While the total HAA content was detected as 0.33 ng/g in the control group, it was detected as 1.20 ng/g and 1.11 ng/g in the meatballs containing 0.5% and 1% cumin, respectively.The total HAA content was determined as 0.30 ng/g in the meatballs cooked at 150 °C and 1.46 ng/g in the meatballs cooked at 250 °C.It was determined that the total HAA content of the meatballs cooked at 150 °C and added cumin at the rate of 0.5% and 1% consisted of MeIQx and MeIQ compounds, while the total HAA content of the meatballs in the control group cooked at 250 °C consisted entirely of MeIQx compound.The total HAA content all of the meatballs increased in direct proportion to the temperature.It was observed that the use of cumin in meatballs increased the total HAA content compared to the control group, regardless of the rates added.It is thought that cumin, which was used in the meatball preparation in the present research, increased the content of many individual HAA and total HAA due to its pro-oxidant effect under the conditions in the research.Although it is known that the use of products with antioxidant effects has a reducing effect on HAA formation, there are also studies showing a prooxidant effect on the level of HAA formation.Johansson and Jägerstad [58] reported in their study that various antioxidants exhibited prooxidant effects on HAAs formation in model system.It was determined that HAA levels decreased in meats treated with various spice extracts (basil, coriander, thyme, marjoram, rosemary, savory, sweet herb, and thyme extracts) depending on cooking conditions and type of extract, but increased (PhIP) occurred in some samples depending on the type of HAA.In addition, it was emphasized that heating conditions were more effective on HAAs than antioxidants in extracts [59].Cumin contains antioxidant compounds such as γ-terpinene, linalool, quercetin, pinocarveol, carotol, 1-methyl-2-(1-methylethyl)benzene, ferulic acid, gallic

Total HAAs of the Meatballs
Total HAA content of cooked meatball samples are shown in Table 3.It was observed that cumin usage rate and cooking temperature had a statistically very significant effect (p < 0.01).While the total HAA content was detected as 0.33 ng/g in the control group, it was detected as 1.20 ng/g and 1.11 ng/g in the meatballs containing 0.5% and 1% cumin, respectively.The total HAA content was determined as 0.30 ng/g in the meatballs cooked at 150 • C and 1.46 ng/g in the meatballs cooked at 250 • C. It was determined that the total HAA content of the meatballs cooked at 150 • C and added cumin at the rate of 0.5% and 1% consisted of MeIQx and MeIQ compounds, while the total HAA content of the meatballs in the control group cooked at 250 • C consisted entirely of MeIQx compound.The total HAA content all of the meatballs increased in direct proportion to the temperature.It was observed that the use of cumin in meatballs increased the total HAA content compared to the control group, regardless of the rates added.It is thought that cumin, which was used in the meatball preparation in the present research, increased the content of many individual HAA and total HAA due to its pro-oxidant effect under the conditions in the research.Although it is known that the use of products with antioxidant effects has a reducing effect on HAA formation, there are also studies showing a prooxidant effect on the level of HAA formation.Johansson and Jägerstad [58] reported in their study that various antioxidants exhibited prooxidant effects on HAAs formation in model system.It was determined that HAA levels decreased in meats treated with various spice extracts (basil, coriander, thyme, marjoram, rosemary, savory, sweet herb, and thyme extracts) depending on cooking conditions and type of extract, but increased (PhIP) occurred in some samples depending on the type of HAA.In addition, it was emphasized that heating conditions were more effective on HAAs than antioxidants in extracts [59].Cumin contains antioxidant compounds such as γ-terpinene, linalool, quercetin, pinocarveol, carotol, 1-methyl-2-(1-methylethyl)benzene, ferulic acid, gallic acid, kaempferol, ellagic protocatechuic acid, caffeic acid and chlorogenic acid.Chlorogenic acid, one of the antioxidant compounds, can be converted into lactones of coumaroyl-caffeoyyl and feruloylquinic acids by heat treatment, as well as into various aromatic compounds consisting of proteins, carbohydrates, fats and aromatic acids [26,60].Many factors are effective on types and amounts of HAA compounds, and compounds are formed by different mechanisms.While creatinine and alkylpyridine free radicals are effective in the formation of MeIQ and IQ compounds, dialkylprazine free radicals, as well as creatinine, are effective in the formation of 4,8-DiMeIQx and MeIQx compounds.It is also added that amino acids and Maillard and Strecker reactions are involved in MeIQx formation [56].As a matter of fact, it has been reported that glycine, lysine, alanine, threonine, and serine play a role in the formation of MeIQx, and lysine, aspartic acid, isoleucine, and phenylalanine play a role in the formation of PhIP [61].In this respect, the high carbohydrate (44.2%) and protein (17.8%) content of cumin provides the appropriate substrate conditions for the formation of the Maillard reaction during cooking [26,62].Puangsombat et al. [31] cooked meatballs to which 0.2% cumin was added at 204 • C and examined the amounts of IQ, IQx, MeIQ, MeIQx and PhIP compounds from HAAs.The amount of MeIQx was determined as 6.94 ng/g, the amount of PhIP was determined as 6.14 ng/g, and the total HAA was determined as 13.08 ng/g.The researchers reported that inhibition of HAAs was dependent on the scavenging activity and total phenolic content of spices.As a matter of fact, there are studies examining the impacts of some spices, extracts, or fruits on the inhibition of HAAs [31,32,63].Zhu et al. [64] emphasized that the inhibitory effect of antioxidant substances on HAAs is provided by the mechanism of clearance of intermediate products such as formaldehyde, such as Strecker aldehydes involved in the formation of MeIQx and IQ, and acetaldehyde involved in the formation of 4,8-DiMeIQx and MeIQ, rather than their free radical scavenging activities.Nuray and Oz [50] reported that antioxidant or prooxidant activities of antioxidant compounds can change depending on various parameters such as concentration, structure, substrate, and test method.

HAA Content of Meatballs
The HAA contents of cooked meatball samples are given in Table 5.In the present study, IQ, 7,8-DiMeIQx, 4,8-DiMeIQx, AαC and MeAαC could not be detected in any sample, while IQx, MeIQx, MeIQ, and PhIP were determined at varying levels in different samples.In the meatball, IQx compound was detected only in the meatball samples with 1% cumin and cooked at 250 • C (0.08 ng/g).IQx compound, one of the less studied compounds in the literature, was detected up to 0.39 ng/g in pan-fried meat samples [65], up to 3.48 ng/g in a pan-and oven-cooked beef samples [32], and up to 3.06 ng/g in commercial beef samples [55].It was stated that this compound could not be founded in meat samples [66], in meatball samples [67], and in burger samples [68].Puangsombat et al. [31] stated that IQx compound was not detected in meatball samples they cooked for 10 min at 204 • C by adding cumin at a rate of 0.2%.
In this study, the amount of IQ compound in the meatballs was determined below the LOD value.It is stated that IQ compound was not founded in pan-fried beef and lamb chops [69]; in pan-fried beef meatballs [34]; and in meatball samples cooked on a hot plate [35].IQ compound was not defined in meatball samples cooked (204 • C/10 min) by adding cumin at a rate of 0.2% [31].
MeIQx was founded in all meatball samples except the control group, which was cooked at 150 • C.There are studies in which the MeIQx compound has been detected [34,56] in various meat products and has not been detected [50].It was determined that the addition of cumin increased in the MeIQx compound in meatballs cooked at 150 • C compared to the control group.However, increasing the cumin content from 0.5% to 1% in meatballs showed a reducing effect on the formation of MeIQx.It was determined that the increase in the cumin ratio in the meatball samples cooked at 250 • C compared to the control groups exhibited an inhibitory effect on MeIQx.In this context, it was determined that 0.5% cumin addition provided 16.48% inhibition, while 1% cumin addition provided 42.86% inhibition in the meatballs cooked at 250 • C. It has been stated that the addition of garlic to meatballs by Shin et al. [70], the addition of hibiscus extract to meatballs by Gibis and Weiss [71], the addition of red pepper to beef by Oz and Kaya [72], the addition of dried apple peel powder to meatballs by Sabally et al. [63], and the addition of black cumin to meatballs by Oz [49] exhibited inhibitory effects on MeIQx.Puangsombat et al. [31] stated that MeIQx compound was detected as 6.94 ng/g in meatball samples cooked at 204 • C by adding cumin at a rate of 0.2%, and a decrease in the amount of MeIQx compound was observed compared to the control group.
It was observed that the use of cumin increased MeIQx formation in the meatballs cooked at 150 • C compared to control group meatballs, and decreased MeIQx formation in the meatballs cooked at 250 • C. It is thought that this situation may be due to the fact that cumin shows both an antioxidant and a pro-oxidant effect depending on the cooking temperatures applied.Because, it is stated in the literature that antioxidant substances can have antioxidant and pro-oxidant effects depending on the ambient temperature [59,60].
MeIQ compound was below the LOD value in the control group meatballs cooked at 150 • C and 250 • C, while it was found between 0.06-0.23 ng/g in the meatballs with 0.5% and 1% cumin.In the meatballs cooked at 150 • C and 250 • C, the addition of cumin at a rate of 1% had an inhibitory effect on MeIQ compound compared to the addition of cumin at a rate of 0.5%.It has been reported that MeIQ compound was detected 0.1-3.5 ng/g in beef patties fried (175-225 • C and 12-20 min) by Balogh et al. [73]; 2.02-6.35ng/g in beef cooked (150-250 • C and 10 min) by Tengilimo glu-Metin and Kızıl [32]; 3.03-18.09ng/g in fried meatball samples to which different spices were added by Lu et al. [28].There are also studies in the literature in which MeIQ compound could not be founded [34,74].Puangsombat et al. [31] stated that MeIQ compound was not detected in meatball samples cooked for 10 min at 204 • C by adding cumin at a rate of 0.2%.
PhIP compound could not be founded in the meatballs cooked at 150 • C and the control group meatballs cooked at 250 • C, while it was detected as 0.82 ng/g and 1.21 ng/g in the meatballs with cumin added at a rate of 0.5% and 1% and cooked at 250 • C, respectively.While PhIP compound was detected in meat samples up to 15.2 ng/g [65], the compound was determined in meatball samples up to 0.06 ng/g [71], up to 13.85 ng/g [28], up to 2.08 ng/g [74], and up to 3.1 ng/g [34].Korkmaz and Oz [44] and Uzun and Oz [35] could not detect the PhIP compound in the meatball samples.PhIP compound was observed as 6.914 ng/g in meatball samples cooked at 204 • C for 10 min by adding cumin at a rate of 0.2%, and there was a decrease in the PhIP compound compared to the control group [31].It has been revealed that PhIP compound is occurred as a result of the Maillard reaction between glucose, phenylalanine, and creatine.In the current study, increasing the cumin ratio in the meatballs cooked at 250 • C increased the amount of PhIP compound.It is thought that the fact that cumin contains 44.2% carbohydrates and 4.23% phenylalanine contributes to the formation of PhIP [60,62].
MeAαC and AαC compounds could not be founded in any of the meatball samples.Tengilimoglu-Metin and Kizil [32] stated that AαC compound ranged between nd-8.48 ng/g in the control group and meat samples to which artichoke extract was added; Khan et al. [74] reported that it ranged between 0.31-0.65 ng/g in control group goat meatballs and goat meatballs to which Chrysanthemum morifolium extract was added.Tengilimoglu-Metin et al. [23] stated that MeAαC compound changed between nd-0.48 ng/g in meat samples to which they added hawthorn extract and cooked at different temperatures.There are studies in the literature in which AαC and MeAαC compounds were not detected.It was stated that AαC and MeAαC compounds could not be determined in beef burgers [68], in meatball samples added with different onion extracts [50], in meatball samples in which black cumin was added at different ratios [49], and in commercially beef samples [55].

Correlation Analysis
The correlation analysis of all parameters examined in meatball samples is given in Figure 2. A strong and very significant positive correlation (r = 0.708; p < 0.01) was determined between total HAA content and cooking loss, while a strong and very significant negative correlation (r = −0.857;p < 0.01) was found between pH and TBARS values.The water percentage of the meat is important in transporting the precursors of the HAAs to the meat surface.During cooking, the substances that are removed with the water come to the outer surface of the meat and contribute to the formation of HAA [24].Indeed, the negative correlation between water content and cooking loss confirms this situation.Therefore, increased cooking loss results in higher HAA content.In this study, the use of cumin in meatball samples increased the pH value.On the other hand, the use of cumin in meatballs decreased the TBARS value with its antioxidant effect.In this context, the negative correlation between TBARS and pH value is explained by the use of cumin.MeAαC and AαC compounds could not be founded in any of the meatball samples.Tengilimoglu-Metin and Kizil [32] stated that AαC compound ranged between nd-8.48 ng/g in the control group and meat samples to which artichoke extract was added; Khan et al. [74] reported that it ranged between 0.31-0.65 ng/g in control group goat meatballs and goat meatballs to which Chrysanthemum morifolium extract was added.Tengilimoglu-Metin et al. [23] stated that MeAαC compound changed between nd-0.48 ng/g in meat samples to which they added hawthorn extract and cooked at different temperatures.There are studies in the literature in which AαC and MeAαC compounds were not detected.It was stated that AαC and MeAαC compounds could not be determined in beef burgers [68], in meatball samples added with different onion extracts [50], in meatball samples in which black cumin was added at different ratios [49], and in commercially beef samples [55].

Correlation Analysis
The correlation analysis of all parameters examined in meatball samples is given in Figure 2. A strong and very significant positive correlation (r = 0.708; p < 0.01) was determined between total HAA content and cooking loss, while a strong and very significant negative correlation (r = −0.857;p < 0.01) was found between pH and TBARS values.The water percentage of the meat is important in transporting the precursors of the HAAs to the meat surface.During cooking, the substances that are removed with the water come to the outer surface of the meat and contribute to the formation of HAA [24].Indeed, the negative correlation between water content and cooking loss confirms this situation.Therefore, increased cooking loss results in higher HAA content.In this study, the use of cumin in meatball samples increased the pH value.On the other hand, the use of cumin in meatballs decreased the TBARS value with its antioxidant effect.In this context, the negative correlation between TBARS and pH value is explained by the use of cumin.

Conclusions
In the present study, the effect of cumin addition to meatballs cooked at two different temperatures (150 and 250 °C) on HAA formation was investigated.The results showed that the addition of cumin increased the formation of total HAA content in the meatballs compared to the control group meatballs.On the other hand, it has been reported that the maximum acceptable intake of heterocyclic aromatic amines (HAAs) is 0-15 µg/day [75], and even if 100 g of the meatballs with the highest total HAA content (samples with 1% cumin and cooked at 250 °C) were consumed in the current study, 0.201 µg of HAAs would be exposed.The inhibitory effect of cumin use in meatball production on HAAs was determined in MeIQx compound, and the highest inhibition (42.86%) belongs to meatballs cooked at 250 °C and added cumin at a rate of 1%.The phenolic compounds in the structure of cumin are thought to inhibit HAA formation due to their antioxidant and radical scavenging effects.On the other hand, model system studies are needed to explain the clear mechanisms in this regard.

Conclusions
In the present study, the effect of cumin addition to meatballs cooked at two different temperatures (150 and 250 • C) on HAA formation was investigated.The results showed that the addition of cumin increased the formation of total HAA content in the meatballs compared to the control group meatballs.On the other hand, it has been reported that the maximum acceptable intake of heterocyclic aromatic amines (HAAs) is 0-15 µg/day [75], and even if 100 g of the meatballs with the highest total HAA content (samples with 1% cumin and cooked at 250 • C) were consumed in the current study, 0.201 µg of HAAs would be exposed.The inhibitory effect of cumin use in meatball production on HAAs was determined in MeIQx compound, and the highest inhibition (42.86%) belongs to meatballs cooked at 250 • C and added cumin at a rate of 1%.The phenolic compounds in

Figure 1 .
Figure 1.The chromatograms of the meatballs with the highest total HAA content and the HAA mix stock solution.

Figure 1 .
Figure 1.The chromatograms of the meatballs with the highest total HAA content and the HAA mix stock solution.

Table 1 .
HPLC conditions for the HAA analysis.

Table 2 .
Water percentage, pH and TBARS values of beef, intermuscular fat and meatball dough with 15% fat.

Table 3 .
The average water percentage, pH, TBARS, cooking loss values and total HAA contents of the meatball.

Table 5 .
The individual HAA content of the meatball samples (ng/g).