Determination of creatine, creatinine, free amino acid and heterocyclic aromatic amine contents of plain beef and chicken juices

Abstract

In the present study, creatine, creatinine, free amino acid and heterocyclic aromatic amine (HAA) contents of plain beef and chicken juices were investigated. For this aim, the boned and boneless beef and chicken juices were obtained by boiling for 3 h at 100 °C without any additives in the laboratory. Creatine and creatinine content of the beef juices varied between 1.33–3.16 mg g⁻¹ and 1.29–1.42 mg g⁻¹, respectively, while creatine and creatinine content of the chicken juices varied between 0.98–1.63 mg g⁻¹ and 0.89–1.30 mg g⁻¹, respectively. The total free amino acid content in the beef juices and the chicken juices ranged between 14.61–20.65 mg 100 gdm⁻¹ and 19.66–57.88 mg 100 gdm⁻¹, respectively. None of the heterocyclic aromatic amines analyzed in the present study could be detected in the beef juices and the chicken juices. Therefore, the boned and boneless beef and chicken juice can be considered as safe from the standpoint of these HAAs and are suggested to use in the preparation of meals.

Introduction

Nowadays, determining that most of the health problems are of nutritional origin has caused a detailed examination of all nutritional factors that have positive and negative effects on human health. In this sense, while food are known to play a role in the prevention and/or treatment of some diseases, they are also known to cause some diseases. On the other hand, a positive relationship between meat consumption and cancer was found. The changes occurred during cooking of meat and meat products were identified and a report published by the World Cancer Research Fund (WCRF) in 2011 recommended the weekly consumption of cooked meat should not exceed 500 g (Anonymous 2020).

The habit of consuming of soup (boiling meat with vegetables and spices) is an old ancient method. For example, while the meat juice (or soup) is a valuable food suitable for especially patients because of their positive effects on protein metabolism and digestive system for Egyptian and Greek doctors. However, after the First World War meat juice preparates were prepared to compensate for the lack of basic foods. With the general improvement in life conditions and development of taste, the reputation of these products has changed considerably among consumers (Caponio et al. 2003).

In making homemade meat juice, it is possible to use cattle tibia as a raw material. Meat juice can also be obtained by boiling chopped beef and cattle tibia with vegetables for a long time. Chicken juice is obtained by boiling chicken meat together with vegetables for a long time.

Among the chemical compounds formed as a result of the reactions during the cooking of protein-rich foods, heterocyclic aromatic amines (HAAs) have an important place (Jägerstad et al. 1998). HAAs are formed during cooking of foods rich in protein such as meat, chicken, and fish, usually at temperatures above 150 °C. HAAs were first identified in grilled meat and fish by Japanese scientists (Nagao et al. 1977) in 1977 and epidemiological studies have proved that most of them are mutagenic and almost all are carcinogenic (Skog et al. 1998). Studies have shown a positive relationship between pancreas, breast, colorectal, prostate and ureter cancers and high consumption of meat and fish (Oz and Kaya 2011). To date, more than 25 HAAs have been isolated and identified from heat-treated food (Oz 2019). The International Agency for Research on Cancer (IARC) declared that 2-amino-3,4-dimethylimidazo [4,5-f] quinoline (MeIQ), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), 2-amino-9H-pyrido [2,3-b] indole (AαC), 2-amino-3-methyl-9H-pyrido [2,3-b] indole (MeAαC), pyridoindole type (3-amino-1,4-dimethyl-5H-pyrido [4,3-b] indole (Trp-P-1), 3-amino-1-methyl 5H-pyrido [4,3-b] indole (Trp-P-2), and dipyridoimidazole [2-amino-6-methyl-dipyrido [1,2-a:30,20-d] imidazole (Glu-P-1) have taken place in 2B class as “possible human carcinogens and 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) in 2A class as "probable human carcinogens" (IARC 1993).

HAAs have two main chemical classes. The first one is aminoimidazoazoarenes (AIAs) and the second is aminocarbolines (Oz and Kaya 2011). AIAs (IQ-type compounds, thermal HAAs) are generally formed in reactions between free amino acids, creatine/creatinine and hexose at temperatures between 150 and 300 °C (Jägerstad et al. 1998). The second group of HAAs, aminocarbolines (non-IQ type compounds or pyrolytic HAAs) usually occur at temperatures above 300 °C as a result of pyrolysis of proteins and amino acids (Oz and Kaya 2011). On the other hand, there are studies showing that HAAs can be formed at lower temperatures (< 100 °C) (Oz and Zikirov 2015).

Meat contains creatine and creatinine that can react with free amino acids and sugars throughout the cooking process (Felton et al. 1994). There is creatine on the basis of the mutagenic activity of the meat (Jägerstad et al. 1998). During the heat treatment, creatine is transformed into creatinine that forms the imidazo part of AIAs (Jägerstad et al. 1998). High processing temperatures lead to a rapid decrease in the amount of creatine and a rapid increase in the amount of creatinine. It was reported that most of this transformation is reported to occur in the first 40 min of heat treatment (Oz and Kaya 2011).

Although there has been a lot of research including our studies in the literature about the structure, mutagenic activity, carcinogenic activity, the level of formation in different types of food, the effect of different cooking methods and the effect of different substances (natural or synthetic) on the formation of HAAs (Murkovic et al. 1998; Zöchling et al. 2002; Oz and Kaya 2011; Oz and Seyyar 2016; Oz 2019; Elbir and Oz 2020), the number of studies focusing on HAAs in meat juices is rather limited. In these studies, qualitative parameters were not studied in detail and only beef juice is usually used as a material. In addition, there is no study on HAA content of beef juices and chicken juices frequently used in the preparation of various types of food (soup, rice, gravy, etc.). Determination of qualitative parameters and HAA amounts in beef juices and chicken juices is very important to get a database and evaluate the relationship between nutrition and health. The present study was, therefore, undertaken to investigate some qualitative parameters (creatine, creatinine and free amino acid profile) and HAA presence in the boned and boneless beef and chicken juices.

Materials and methods

Material

For the production of beef juice, boned (2.5 kg) and boneless (2.5 kg) round part of two beef carcasses (24 h post-mortem) were obtained from the Erzurum Meat and Milk Institution Meat Combine (January 2018). To prepare chicken juice, 6 fresh chicken carcasses (about 2.5 kg) in total were used in the present study. Carcasses were obtained from the distributor of a nation-wide known company in Erzurum that sells only chicken meat (January 2018). Two of the chicken carcasses (two replications) were used in the analyses of the raw samples. The remaining 4 carcasses were used for the boned and boneless chicken juices (two replications). For the boned chicken juices, leg meat (with skin, 8 pieces) obtained from the chicken carcass was used, while the breast meat (without skin, 8 pieces) obtained from the chicken carcass was used for the boneless chicken juices. Additionally, we paid attention to obtain the chicken carcasses from the same party and size.

Preparation of the beef and chicken juices

A steel cooker was used to prepare of beef and chicken juices in the laboratory, drinking quality water (about 2.5 L) added to the steel cooker to cover the beef and chicken meat, and without the addition of other substances such as additives and/or spices boiling process was carried out. Meat juice preparation was carried out under normal conditions at atmospheric pressure and at a temperature of about 100 °C for 3 h, followed by removal of the meats, the juices were filtered after cooling, and placed into ready-molds and frozen and stored at − 18 °C until analysis. Meat juices were used in the frosted state without thawing in the analyzes.

Chemicals

Chemicals and solvents were of high-performance liquid chromatography (HPLC) or analytical grade. Water was from a Milli-Q water purification system (Millipore, Bedford, Massachusetts, USA). All solutions except for HPLC-grade solvents were passed through a 0.45 μm filter (PTFE, Milex, Massachusetts, USA). The 37 fatty acid methyl ester (FAME) mix standard (CRM47885) and amino acid standards (AAS18) were obtained from Supelco (Taufkirchen, Germany). Creatine standard (C0780) and creatinine standard (C4255) were purchased from Sigma-Aldrich (Taufkirchen, Germany). The following HAA standards [2-amino-3-methylimidazo [4,5-f] quinoxaline (IQx); 2-amino-3-methylimidazo [4,5-f] quinoline (IQ); 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx); 2-amino-3,4-dimethylimidazo [4,5-f] quinoline (MeIQ); 2-amino-3,7,8-trimethylimidazo [4,5-f] quinoxaline (7,8-DiMeIQx); 2-amino-3,4,8-trimethylimidazo [4,5-f] quinoxaline (4,8-DiMeIQx); 2-amino-3,4,7,8-tetramethylimidazo [4,5-f] quinoxaline (4,7,8-TriMeIQx); 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP); 2-amino-9H-pyrido [2,3-b] indole (AαC) and 2-amino-3-methyl-9H-pyrido [2,3-b] indole (MeAαC)] were purchased from Toronto Research Chemicals (Downsview, Ontario, Canada). The stock standard solutions were prepared according to Oz and Seyyar (2016). An Oasis MCX cartridge (3 cm³ 60 mg⁻¹, 30 mm) of Waters (Milford, Massachusetts, USA) was used for the solid phase extraction of HAAs.

Determination of creatine content

The analyses of creatine content of the samples were performed according to Polaket al. (2009). Briefly, the sample (0.25 g) weighed into the beaker, 100 mL of trichloroacetic acid was added, homogenized for 5 min; filtered through filter paper to separate the precipitated protein. Subsequently, a total of 20 mL was removed from the extract and deoiled with 10 mL of diethyl ether. After separation of the phases (10 min), 4 mL of the purified extract was mixed with 2 mL of 1-naphthol and 2 mL of diacetyl in sodium hydroxide solution. At the end of the mixing, the mixture was heated at 40 °C for 5 min and the creatine content was determined spectrophotometrically at 520 nm.

Determination of creatinine content

The creatinine content of the samples was analyzed according to Polak et al. (2009). 1 g sample weighed into a beaker; 20 mL of trichloroacetic acid was added and homogenized for 5 min and filtered through filter paper to remove the precipitated protein. Subsequently, a total of 8 mL was removed from the extract and deoiled with 4 mL of diethyl ether. After separation of the phases (10 min), 4 mL of the purified extract was mixed with 1.5 mL of picric acid in sodium hydroxide solution. After the mixing procedure, the mixture was heated at 40 °C for 10 min and the creatine content was measured spectrophotometrically at 500 nm.

Determination of free amino acid content

The determination of free amino acid contents of the samples was performed according to Ceylan and Aksu (2011). According to the method; 4 g of sample was weighed and 40 mL of cold 0.1 N HCl was added and homogenized for 45 s. After 12 h in the refrigerator, it was centrifugated at 12,000 rpm and 4 °C for 50 min. After taking 20 mL of the supernatant and filtering through a 0.22 µm filter, it was analyzed with high pressure liquid chromatography (HPLC). In the HPLC analysis, Zorbax Eclipse AAA Column (4.6 × 150 mm, 3.5 µm) was used as column and 40 °C was selected as column temperature, NaH₂PO₄ (pH 7.8) was used as Solvent A, and ACN:MeOH:H₂O (45:45:10) was used as Solvent B.

Determination of HAA content

The extraction of HAA was performed as described by the Oz and Seyyar (2016) method. 1 g of sample was weighed, dissolved in a beaker in 12 mL of 1 M NaOH, and then magnetically stirred for 1 h at 500 rpm. After stirring, 10 g of Extrelute NT packaging material (refill material, Merck, Darmstadt, Germany) was added and thoroughly stirred via a spatula. Oasis MCX cartridges (3 cm³ 60 mg⁻¹, Waters, Milford, Massachusetts, USA) were connected to the vacuum manifold system (Supelco, Visiprep) and pre-conditioned with ethyl-acetate and then connected to the extraction cartridges and packed in the reservoir. Then cartridges were washed with ethyl acetate, hydrochloric acid, methanol, and the HAAs in the samples were eluted with a solution of methanol/ammonia (95:5). The samples were kept at − 18 °C until the HPLC analysis and dried in an oven at 50 °C 1 day before the analysis. HPLC analysis started by adding 100 μL of methanol to the vial.

The content of HAAs was detected with an HPLC (Thermo Ultimate 3000, Thermo Scientific, USA) containing a diode array detector (DAD-3000). Separation process was performed at 35 °C at a flow rate of 0.7 mL min⁻¹, on a reversed phase analytical column [Acclaim 120 C18 3 mm (4.6 × 150 mm), Tosoh Bioscience GmbH (Stuttgart, Germany)], using solvent A (methanol:acetonitrile:water:acetic acid, 8:14:76:2, adjusted to pH 5.0 with ammonium hydroxide 25%) and solvent B (acetonitrile) solutions (Oz and Seyyar 2016). Gradient program was as follows; 0% B, 0–10. min; 0–23% B, 11–19. min; 100% B, 20–22. min; 0% B, 23–33. min. 10 μL injection was made from the samples. Quantitative determination was performed using the external calibration curve method (Oz and Seyyar 2016).

Statistical analysis

The present study is completely randomized design in nature and was carried out with two replications. The results were analyzed by using an SPSS package program, and Duncan’s multiple range test was used in evaluating the differences between average values considered as significant.

Results and discussion

Creatine content of the juices

Creatine contents of the boned and boneless beef used to produce beef juices were determined as 3.92 ± 0.58 mg g⁻¹ and 4.45 ± 0.17 mg g⁻¹, respectively. Similar results for beef were also obtained in different studies (Polak et al. 2007; Mora et al. 2010). Polak et al. (2007) found that the creatine content of 14 days matured raw beef (Longissimus dorsi muscle) meat as 5.86 mg g⁻¹, 28 days matured raw beef as 5.66 mg g⁻¹. Puangsombat et al. (2012) found the creatine content of minced raw beef as 2.53 mg g⁻¹. Purchas et al. (2004) reported that creatine contents of different muscles of raw lamb meat varied between 4.64 and 18.66 mg g⁻¹, while creatine contents of different muscles of lamb cooked at 70 °C for 90 min varied between 2.65 and 11.88 mg g⁻¹. Pais et al. (1999) reported that the creatine content of raw beef as 6.33 mg g⁻¹. On the other hand, there are studies indicating that beef contains much higher amounts of creatine.

The creatine contents of the boned chicken meat and the boneless chicken meat used were determined as 3.80 ± 0.08 mg g⁻¹ and 4.04 ± 0.26 mg g⁻¹, respectively. The creatine contents of the chicken meat were found to be similar to the data in the literature. As a matter of fact, creatine content was determined as 2.21 mg g⁻¹ in raw chicken breast meat and as 2.51 mg g⁻¹ in raw chicken thigh meat by Puangsombat et al. (2012) and as 3.54 mg g⁻¹ in raw chicken breast meat and as 4.44 mg g⁻¹ in raw chicken thigh meat by Pais et al. (1999).

The creatine contents of the boned and boneless beef and chicken juices were given in Table 1. The use of boned and boneless beef to produce beef juices had a significant effect (P < 0.05) on the creatine contents of the beef juices, while the use of the boned and boneless chicken meat to produce chicken juices had no significant effect (P > 0.05) on the creatine contents of the chicken juices. The creatine content (3.16 mg g⁻¹) of the boneless beef juice was found to be significantly (P < 0.05) higher than that of (1.33 mg g⁻¹) the boned beef juice due to the muscle difference used in the preparation of juice (Mora et al. 2010). This result proved that creatine, naturally found in meat, passes into juice because it is soluble in water. Indeed, it is known that creatine and creatinine are non-protein nitrogenous compounds of meat and are soluble in water (Girard 1992).

Table 1.

Creatine and creatinine contents of the boned and boneless beef juices and chicken juices

Sample	n	Creatine (mg g−1) ± SD	Creatinine (mg g−1) ± SD
Beef juice
Boned beef juice	2	1.33 ± 0.58b	1.29 ± 0.04b
Boneless beef juice	2	3.16 ± 0.16a	1.42 ± 0.01a
Sign		*	*
Chicken juice
Boned chicken juice	2	0.98 ± 0.25	0.89 ± 0.07b
Boneless chicken juice	2	1.63 ± 0.33	1.30 ± 0.07a
Sign		Ns	*

Sign.: significance, ns: not significant (P > 0.05), *: P < 0.05,

a–b: means with different letters in the same column are significantly different (P < 0.05)

It is very difficult to compare the creatine content of the beef used in the present study with the data in the literature because beef is generally used as a material and the number of studies related to juice or meat extract is rather limited. On the other hand, no detailed information was given about the samples in these studies, and the analyses were made with different methods and the creatine content was expressed with different units. For example, Arvidsson et al. (1999) found that creatine content of model roast beef juice was 358 μmol g⁻¹, while Borgen et al. (2001) found creatine content in meat juice obtained from roast beef as 340 μmol gdm⁻¹. In another study on meat juice, creatine content of meat juice prepared by adding 1:2 salt (7.5 g L⁻¹) at room temperature was determined as 5959 μmol L⁻¹, while creatine contents of juices, obtained at 55–95 °C for 60 min, were determined to range between 6112 and 6600 μmol L⁻¹ (Cambero et al. 2000). Researchers stated that the cooking process caused an increase in the creatine content, but reported that the creatine content decreased when the temperature was increased from 85 to 95 °C. Differences in the creatine content between the present study and the aforementioned studies are thought to be caused by differences such as animal race, age, ration, muscle difference, selected conditions (temperature, duration, method, foodstuff and additive usage and usage rate) used to prepare meat juice.

The creatine content (0.98 mg g⁻¹) of the boned chicken meat juice was found to be lower than that of (1.63 mg g⁻¹) the boneless chicken meat juice, however, this was not statistically significant (P > 0.05). Borgen et al. (2001) determined the creatine content as 152 μmol g⁻¹ in juice obtained from chicken breast. It is thought that the difference is caused by differences such as meat type, muscle difference, production method, used additives and processing time.

Creatinine content of the juices

The creatinine contents of the boned and boneless beef used to produce the beef juices were determined as 0.31 ± 0.03 mg g⁻¹ and 0.39 ± 0.04 mg g⁻¹, respectively. The creatinine contents of the beef were found to be similar to the data in the literature. In fact, Polak et al. (2007) determined creatinine content of 14 and 28 days matured raw beef Longissimus dorsi muscle as 0.24 and 0.26 mg g⁻¹, respectively.

The creatinine contents of the boned and boneless chicken meat used to produce chicken juices were determined as 0.19 ± 0.02 mg g⁻¹ and 0.15 ± 0.01 mg g⁻¹, respectively.

The creatinine contents of the boned and boneless beef and chicken juices were given in Table 1. The use of boned and boneless meat to produce meat juice had a significant effect (P < 0.05) on the creatine contents of both the beef and the chicken juices. The creatinine content (1.29 mg g⁻¹) of the boned beef juice was found to be lower than that (1.42 mg g⁻¹) of boneless beef juice. This result is believed to be affected by the pH values of the juices. In the present study, pH value of the boned beef juice (6.96) was found to be significantly higher (P < 0.05) than that (6.73) of the boneless beef juice. As a matter of fact, it was found that pH of the medium was effective in the transformation of creatine into creatinine, and transformation in more acidic conditions is faster (Mora et al. 2008).

Creatinine content was determined as 4.2 μmol g⁻¹ (Arvidsson et al. 1999) in meat juice obtained from roasted veal in a model juice system, and as 2.4 μmol gdm⁻¹ (Borgen et al. 2001) in meat juice obtained from roasted veal. In another study on meat juice, creatinine content of juice prepared at 1:2 ratio by adding salt (7.5 g L⁻¹) at room temperature was determined as 537 μmol L⁻¹, while creatinine content of juice obtained at 55–95 °C for 60 min were found to vary between 625 and 818 μmol L⁻¹ (Cambero et al. 2000). The difference between the present study and the mentioned studies is thought to be due to the effect of factors such as the race, age, muscle type of the animal, cooking temperature, time and additive used in the production of juice.

The creatinine content (0.89 mg g⁻¹) of the boned chicken juice was found to be lower than that (1.30 mg g⁻¹) of the boneless chicken juice. This result was also thought to be influenced by the pH value of the juice. In the present study, the pH value (6.86) of the boned chicken juice was found to be significantly higher (P < 0.05) than that (6.49) of boneless chicken juice. In fact, the transformation of creatine into creatinine is known to be affected by the pH of the environment and the transformation is more rapid in more acidic conditions (Mora et al. 2008). Borgen et al. (2001) determined the creatinine content as 0.5 μmol g⁻¹ in meat juice obtained from chicken breast meat. As in beef, having higher levels of creatinine in chicken juice compared to chicken meat was associated with the transformation of creatine in the meat to creatinine with temperature during the preparation of juice.

Creatinine, like creatine, is a non-protein nitrogenous compound and can be dissolved in water. On the other hand, higher levels of creatinine in juice than in meat was attributed to the transformation of creatine into the creatinine by the effect of temperature during the preparation of the juice. The conversion of creatine to creatinine is indicated to be due to initial concentration, pH, temperature and time (Mora et al. 2010). In addition, many investigators reported that by cooking process, the creatine content in the meat decreases while the creatinine content is increased (Purchas et al. 2006; Mora et al. 2010).

Free amino acid profile of the juices

In the present study, the samples were analyzed in terms of 17 amino acid contents (aspartic acid, glutamic acid, serine histidine, glycine, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline). The limit of detection (LOD) and the limit of quantification (LOQ) values of the amino acid mix stock solution were given in Table 2.

Table 2.

Limit of detection (LOD) and limit of quantification (LOQ) values of free amino acids and heterocyclic aromatic amines (ng g⁻¹)

	LOD	LOQ
Free amino acids
Aspartic acid	0.013	0.039
Glutamic acid	0.012	0.036
Serine	0.012	0.036
Histidine	0.016	0.049
Glycine	0.057	0.172
Threonine	0.051	0.156
Arginine	0.029	0.089
Alanine	0.019	0.057
Tyrosine	0.011	0.034
Cystine	0.047	0.142
Valine	0.024	0.072
Methionine	0.014	0.043
Phenylalanine	0.009	0.027
Isoleucine	0.039	0.118
Leucine	0.013	0.039
Lysine	0.024	0.074
Proline	0.005	0.014
Heterocyclic aromatic amines
IQx	0.004	0.013
IQ	0.009	0.029
MeIQx	0.024	0.081
MeIQ	0.014	0.047
7,8-DiMeIQx	0.005	0.018
4,8-DiMeIOx	0.008	0.025
PhIP	0.025	0.085
AαC	0.012	0.039
MeAαC	0.010	0.035

It is difficult to compare the results obtained in the present study with the data in the literature. Because the factors such as the materials used, and the way of preparation were quite different. On the other hand, in the present study, after the meat juice was prepared, the meat was removed and only the juice was used. Nevertheless, in the present study, it was found that the obtained results of amino acid could be compared with the data in the literature. In the literature review, only two studies that investigated the amino acid content in broth (Arvidsson et al. 1999; Borgen et al. 2001) were found, and in these studies, the lyophilized broth was used as material. For this reason, the amino acid content of beef broth was compared with these studies.

The free amino acid profiles of the boned and boneless beef and chicken juices were given in Table 3. The use of the boned beef and the boneless beef used to produce the beef juices had only a significant effect on (P < 0.05) on aspartic acid and had no significant effect (P > 0.05) on other free amino acids analyzed in the present study and total amino acid content, while the use of the boned chicken and the boneless chicken used to produce the chicken juices had only a significant effect on (P < 0.05) on total amino acid content and had no significant effect (P > 0.05) on individual free amino acids analyzed in the present study.

Table 3.

The free amino acid profile of the boned and boneless beef juices and chicken juices (mg 100gdm⁻¹)

Sample	n	Aspartic acid	Glutamic acid	Serine	Histidine	Glycine	Threonine	Arginine	Alanine
Beef juice
Boned beef juice	2	0.33 ± 0.04a	2.97 ± 0.87	0.89 ± 0.30	1.23 ± 0.44	1.94 ± 0.57	0.29 ± 0.11	0.29 ± 0.04	7.90 ± 2.23
Boneless beef juice	2	0.06 ± 0.04b	1.82 ± 0.10	0.63 ± 0.11	0.36 ± 0.01	1.17 ± 0.07	0.20 ± 0.04	0.25 ± 0.11	6.77 ± 0.18
Sign		*	Ns	Ns	Ns	Ns	Ns	Ns	Ns
Chicken juice
Boned chicken juice	2	1.08 ± 0.28	4.30 ± 5.41	1.40 ± 1.52	0.42 ± 0.45	0.51 ± 0.24	2.19 ± 1.09	2.93 ± 3.76	1.26 ± 0.65
Boneless chicken juice	2	1.95 ± 1.90	6.83 ± 6.48	4.06 ± 3.96	0.71 ± 0.51	5.18 ± 6.78	2.89 ± 1.54	2.51 ± 2.04	17.19 ± 15.63
Sign		Ns	Ns	Ns	Ns	Ns	Ns	Ns	Ns

Sample	n	Tyrosine	Cystine	Valine	Methionine	Phenylalanine	Isoleucine	Leucine	Lysine	Proline	Total
Beef juice
Boned beef juice	2	0.82 ± 0.01	0.22 ± 0.18	0.43 ± 0.15	0.14 ± 0.02	0.50 ± 0.13	0.35 ± 0.01	0.61 ± 0.18	1.24 ± 0.23	0.55 ± 0.30	20.65 ± 5.40
Boneless beef juice	2	0.68 ± 0.57	0.20 ± 0.07	0.15 ± 0.10	0.22 ± 0.13	0.34 ± 0.03	0.28 ± 0.04	0.29 ± 0.04	0.83 ± 0.11	0.40 ± 0.27	14.61 ± 1.48
Sign		Ns	Ns	Ns	Ns	Ns	Ns	Ns	Ns	Ns	Ns
Chicken juice
Boned chicken juice	2	1.50 ± 1.29	1.06 ± 0.93	0.68 ± 0.77	0.15 ± 0.08	0.62 ± 0.13	0.22 ± 0.11	0.40 ± 0.26	0.87 ± 0.45	0.11 ± 0.07	19.66 ± 15.97 b
Boneless chicken juice	2	3.03 ± 2.57	0.21 ± 0.00	0.58 ± 0.42	0.74 ± 0.47	1.41 ± 0.73	0.93 ± 0.57	1.04 ± 0.57	2.56 ± 0.33	6.09 ± 5.75	57.88 ± 46.32 a
Sign		Ns	Ns	Ns	Ns	Ns	Ns	Ns	Ns	Ns	*

Sign.: significance, Ns: not significant (P > 0.05), *P < 0.05, **P < 0.01,

a–b: means with different letters in the same column are significantly different (P < 0.05)

Free amino acid compositions of the beef juices and chicken juices obtained in the present study were comparable with the data in the literature. Arvidsson et al. (1999) found aspartic acid as 4.03 μmol g⁻¹, glutamic acid as 6.26 μmol g⁻¹, serine as 4.25 μmol g⁻¹, histidine as 1.84 μmol g⁻¹, glycine as 8.80 μmol g⁻¹, threonine as 2.74 μmol g⁻¹, arginine as 3.47 μmol g⁻¹, alanine as 35.40 μmol g⁻¹, tyrosine as 2.03 μmol g⁻¹,valine as 4.23 μmol g⁻¹, methionine as 1.11 μmol g⁻¹, phenylalanine as 1.85 μmol g⁻¹, isoleucine as μmol g⁻¹, leucine as μmol g⁻¹, lysine as μmol g⁻¹, proline as μmol g⁻¹ in meat juice model system.

It was determined that 1.7 μmol gdm⁻¹ aspartic acid, 2.6 μmol gdm⁻¹ glutamic acid, 3.5 μmol gdm⁻¹ serine, 1.3 μmol gdm⁻¹ histidine, 9 μmol gdm⁻¹ glycine, 2.3 μmol gdm⁻¹ threonine, 2.8 μmol gdm⁻¹ arginine, 28 μmol gdm⁻¹ alanine, 1.5 μmol gdm⁻¹ tyrosine, 3.2 μmol gdm⁻¹ valine, 0.7 μmol gdm⁻¹ methionine, 1.2 μmol gdm⁻¹ phenylalanine, 1.7 μmol gdm⁻¹ isoleucine, 3.80 μmol gdm⁻¹ leucine, μmol gdm⁻¹ lysine, 1.90 μmol gdm⁻¹ proline were determined in juice obtained from veal roast by Borgen et al. (2001). On the other hand, the researchers could not detect cystine in their samples.

In the present study, the total free amino acid content of the boned beef juices and the boneless beef juices was determined as 20.65 and 14.61 mg 100 gdm⁻¹, respectively. Borgen et al. (2001) reported that total free amino acid content of juice obtained from beef was found as 110 μmol gdm⁻¹, while Arvidsson et al. (1999) found total free amino acid content in juice obtained from veal roast as 163 μmol g⁻¹.

In a study carried out in chicken juice model system, Borgen et al. (2001) found aspartic acid as 9.4 μmol gdm⁻¹, glutamic acid as 26.3 μmol gdm⁻¹, serine as 16.1 μmol gdm⁻¹, histidine as 7.2 μmol gdm⁻¹, glycine as 12.8 μmol gdm⁻¹, threonine as 11.5 μmol gdm⁻¹, arginine as 9.6 μmol gdm⁻¹, alanine as 23.7 μmol gdm⁻¹, tyrosine as 2.7 μmol gdm⁻¹, valine as 9.1 μmol gdm⁻¹, methionine as 3.7 μmol gdm⁻¹, phenylalanine as 4.5 μmol gdm⁻¹, isoleucine as 7.7 μmol gdm⁻¹, leucine as 27.9 μmol gdm⁻¹, lysine as 11 μmol gdm⁻¹, proline as 8 μmol gdm⁻¹. On the other hand, the researchers could not detect cystine in their samples.

In the present study, the total free amino acid contents of the boned chicken juices and the boneless chicken juices were determined as 19.66 and 57.88 mg 100 gdm⁻¹, respectively. Borgen et al. (2001) found the total amount of free amino acid in a chicken meat juice obtained from chicken breast meat as 222 μmol g⁻¹.

Heterocyclic aromatic amine contents of the meat juices.

In the present study, the standard addition method was used to determine the recoveries of HAAs and HAA mix stock solution at known concentrations (10 ng g⁻¹, 7.5 ng g⁻¹, 5 ng g⁻¹, 2.5 ng g⁻¹, 1 ng g⁻¹, 0.5 ng g⁻¹) that were added to the samples. LOD and LOQ values based on Signal/Noise (S/N) ratios of HAAs studied in the present study are also given in Table 2. The recoveries (28.94–82.15%) were in accordance with existing studies (Felton et al. 1994; Knize et al. 1994; Murkovic et al. 1998).

In the present study, none of the HAAs analyzed could be detected in the boned and boneless beef and chicken juices. This could be due to the unavailability of sugar during preparation of the meat juices and the low cooking temperature in boiling method. It is known that sugar is one of the precursors of the HAAs formed in meat. However, the studies show that the effect of sugar on the formation of HAAs depends on the amount and type of sugar (Oz and Kaya 2011). As known, the precursors of the HAAs such as sugar are water-soluble. Therefore, sugar naturally present in meat may dissolve into water with the effect of boiling temperature without participating in HAA formation reactions. In addition, the meat juices produced in the laboratory were the plain meat juices and in their preparation no additives, vegetables and/or spices was used. In the studies conducted with commercial beef extract, different results were obtained regarding HAAs. In some studies, HAAs could not be detected in beef extracts analyzed, but in other studies, it was determined at very high levels. These differences are thought to be influenced by factors such as the preparation of beef extract, extraction method used in the analysis, chromatographic method and detector difference.

In some studies (Holder et al. 1996; Mardones et al. 1998; Toribio et al. 2000a), IQ could not be determined in beef meat extracts, while it was determined as 0.2 ng g⁻¹ Pais et al. (1997a, 1997b). On the other hand, there are studies showing that higher levels of IQ were determined in meat extracts. Indeed, Jägerstad et al. (1998) up to 15 ng g⁻¹, Toribio et al. (2000b) up to 37.5 ng g⁻¹ and Toribio et al. (1999) up to 43 ng g⁻¹.

In some studies (Mardones et al. 1998), MeIQx could not be determined in beef meat extracts, while it was determined as 1 ng g⁻¹ by Pais et al. (1997b) and 1.1 ng g⁻¹ by Pais et al. (1997a). On the other hand, there are also studies showing that higher levels of IQ were determined in meat extracts. Indeed, Holder et al. (1996) determined MeIQx up to 30 ng g⁻¹, Toribio et al. (2000a) up to 33.4 ng g⁻¹, Toribio et al. (1999) up to 41 ng g⁻¹, Toribio et al. (2000b) up to 46.1 ng g⁻¹ and Jägerstad et al. (1998) up to 80 ng g⁻¹.

In some studies (Holder et al. 1996; Toribio et al. 2000b), MeIQ could not be determined in beef meat extracts, while it was determined as 0.2 ng g⁻¹ by Pais et al. (1997b), 0.3 ng g⁻¹ by Pais et al. (1997a), up to 6 ng g⁻¹ by Jägerstad et al. (1998) and 17.3 ng g⁻¹ by Toribio et al. (2000a).

While 4,8-DiMeIQx in beef extracts could not be determined in some studies (Holder et al. 1996; Mardones et al. 1998), it was determined as 0.2 ng g⁻¹ by Pais et al. (1997a), 1.4 ng g⁻¹ by Pais et al. (1997b), up to 9 ng g⁻¹ by Jägerstad et al. (1998), 12.4 ng g⁻¹ by Toribio et al. (2000a) up to 13.3 ng g⁻¹ by Toribio et al. (2000b) and up to 14 ng g⁻¹ by Toribio et al. (1999). On the other hand, 7,8-DiMeIQx in beef meat extracts could not be determined in the study by Toribio et al. (2000b).

In beef extracts, PhIP was determined as 0.2 ng g⁻¹ by Holder et al. (1996), 0.3 ng g⁻¹ by Pais et al. (1997a), 0.4 ng g⁻¹ by Pais et al. (1997b), 7.42 ng g⁻¹ by Martncalero et al. (2007) and 10 ng g⁻¹ by Jägerstad et al. (1998). On the other hand, there are also studies showing that higher levels of PhIP were determined in meat extracts. Indeed, Toribio et al. (2000a) determined PhIP as 28.8 ng g⁻¹, Toribio et al. (2000b) up to 31.3 ng g⁻¹ and Toribio et al. (1999) up to 38 ng g⁻¹.

In some studies (Martncalero et al. 2007), AαC could not be determined in beef meat extracts, while Toribio et al. (2000b) determined but not quantified, Pais et al. (1997a) determined as 0.2 ng g⁻¹, Pais et al. (1997b) as 0.3 ng g⁻¹, Holder et al. (1996) as 0.8 ng g⁻¹, Galceran et al. (1996) as 2 ng g⁻¹, Jägerstad et al. (1998) up to 3 ng g⁻¹, Toribio et al. (2000a) as 6.4 ng g⁻¹ and Skog et al. (1998) up to 8.1 ng g⁻¹. On the other hand, some studies (Toribio et al. 2000a; Martncalero et al. 2007) were not identified MeAαC in beef meat extracts, whereas Toribio et al. (2000b) detected the compound but did not determine the amount, Pais et al. (1997a) determined as 0.2 ng g⁻¹, Pais et al. (1997b) as 0.4 ng g⁻¹ and Skog et al. (1998) up to 4 ng g⁻¹.

In another study conducted with meat extract, in the microwave oven meat extract, 5.9 ng g⁻¹ IQx, 46 ng g⁻¹ MeIQx, 6.2 ng g⁻¹ 4,8-DiMeIQx and 7.5 ng g⁻¹ PhIP were determined, while 7,8-DiMeIQx was below the detectable limit (Fay et al. 1997). In the same study, it was reported that PhIP and 7,8-DiMeIQx could not be detected in the vacuum dried meat extract while 2.1 ng g⁻¹ IQx, 29 ng g⁻¹ MeIQx, 4.8 ng g⁻¹ 4,8-DiMeIQx were determined (Fay et al. 1997).

Conclusion

As a result, it was determined that beef juice and chicken juice produced in the laboratory contain some dry matter including the precursors of HAAs. On the other hand, the amounts of HAAs analyzed in these meat juices were below the detectable limit (< LOD). Therefore, the boned and boneless beef and chicken juices can be used in the preparation of meals due to the fact that these meat juices are considered as safe from the standpoint of HAAs analyzed in the current study.