Volatile profile and fatty acid composition of kavurma (a cooked uncured meat product) produced with animal fat combinations

ABSTRACT In this research, the effects of animal fat combinations (100% beef intermuscular fat, BIF); 100% beef kidney fat, BKF; 100% sheep tail fat, STF; 50% BIF + 50% BKF; 50% BIF + 50% STF; 35% BIF + 35% BKF + 30% STF) on volatile profile and fatty acid composition as well as selected physicochemical and sensorial properties of kavurma (a traditional Turkish cooked uncured meat product) were investigated. The group containing 50% BIF + 50% BKF had higher scores than other groups in terms of overall apperance, and taste-odor. The use of STF had a negative effect on sensory parameters. The lowest and the highest saturated fatty acid ratio were determined in the group with 100% STF and the group with 100% BKF, respectively. Among volatile compounds identified, aldehydes were the mostly found chemical group and hexanal was determined as major compound in all groups. As a result, the use of sheep tail fat increased unsaturated fatty acid ratio but negatively affected sensory properties of kavurma.


Introduction
Meat is known as one of the most perishable foods and also susceptible to microbial contamination. [1] For this reason, various methods have been used throughout the history to preserve meat. There are a limited number of traditional products in which meat is conserved in fat after being cooked. [2] One of these products is kavurma. Kavurma is a traditional Turkish meat product, made with frying of small meat pieces in fat and again conserving it in fat used for frying. [3] In the production of kavurma, meat obtained from carcasses of beef or sheep after the cutting into small pieces are fried in open or closed cauldrons in animal fats. [4,5] After the cooking process, meat is filled into natural or artificial casing and the fat used while cooking is added over it in a way to fill in the gaps between meat and to cover the surface. [3] In this way, an anaerobic environment is partially created. After the cooling process, kavurma is offered to market as vacuum packed as a whole or after being sliced.
Kidney fat and other internal fats are the most commonly used fat types in production of traditional kavurma. Intermuscular fat can be also used in kavurma production. Kidney fat and other fats depositing surrounding organs (internal fats) are previously melted in the case that it is used in the production, meat fats can be used by melting or without melting. These fats have high melting points and relatively more cheaper than the other types of fat. Meat fat (intermuscular fat) is seldom used in industrial production. [4] Moreover, sheep tail fat may also be used in kavurma production as this type of fat is viewed as improving aroma and taste. [6] It has been stated that sheep tail fat can also be used in the production of this product at certain ratios. [7] The only additive used in kavurma production is salt. Another property of this product is the fact that nitrate and/or nitrite is not used in its production. [3,6,8] Although it was stated that the salt ratio used in production varies between 2 and 5%, [9] salt is used in industrial production today at 2% and even lower ratios.
In the production, temperature varies depending on the type of cauldron (open or closed) used during frying. While the temperature is around 100°C in closed systems, the temperature can reach up to 150-170°C in open cauldron during cooking in hot fat. The cooking time varies also depending on the raw material used, equipment and the temperature applied. [3,9] The animal fats used in kavurma are one of the important factors affecting the general characteristics of the product. [4] Aksu [6] investigated the effects of different fat combinations on fatty acid composition and some physicochemical properties of kavurma. On the other hand, Uzun and Bozkurt [10] investigated the change in volatile compounds during cooking. However, no study in which the effects of different animal fats (meat fat, beef kidney fat, and sheep tail fat) and the combinations of these on the volatile compounds and sensorial properties of the product are investigated, has been encountered in the literature. Determination of the effects of different animal fat combinations (beef intermuscular fat (BIF), beef kidney fat (BKF), sheep tail fat (STF), beef intermuscular fat + beef kidney fat, beef intermuscular fat + sheep tail fat, beef intermuscular fat + beef kidney fat + sheep tail fat) on the volatile compounds, fatty acid profile, sensorial, and some physicochemical properties of kavurma was aimed in the present study.

Kavurma production
The shoulder beef meat was used in kavurma production. The beef meat fat, beef kidney fat, and sheep tail fat or their combinations were used in the production. Before using, animal fats were ground through a 3 mm plate and then melted at 60°C in water bath. After filtration, the fats melted were kept at −20°C until use.
In the study, six groups kavurma were produced based on the animal fat combinations (100% BIF; 100% BKF; 100% STF; 50% BIF + 50% BKF; 50% BIF + 50% STF; 35% BIF + 35% BKF + 30% STF). 75% meat, 23% fat, and 2% salt were used in the production. The shoulder beef meat provided for kavurma production was cut into 4 × 5 cm pieces after excessive connective tissues and excessive fats had been removed. The meat pieces were transferred into the steam jacketed cauldron, and salt was added to it. Then, after the meat had been roasted for 30 min at 60°C by adding 20% of the total fat, the remaining fat was added, and the cooking process was maintained for 90 min at around 100°C. After this period, the meat and fat that had been cooled to 60°C were filled in about 3 kg of artificial casings and cooled to 4°C. The production was repeated twice. Moisture content, fat level and pH value were analyzed according to Gökalp et al. [11] Sensory evaluation The sensory assessment of kavurma samples was carried out by 10 experts, 5 women, and 5 men. Panel members were selected from the staff, the faculty members of the Department of Food Engineering. Overall appearance, taste and odor, texture, color of fat, and overall acceptability were evaluated by using hedonic type a scale (1-9 scales: 1: very bad to 9: very good). Kavurma samples after storing for 2 days in refrigerator were sliced around 1 cm thick, and one portion from each sample unit was randomly selected and served to the panelists for evaluation at room temperature. Two replicates per treatment were tested following a completely randomized design with two samples per plate. Water and bread were provided between samples. Each sample was separately assessed in two independent repetition by expert panel consists of 10 members. The 20 individual results were taken into consideration for assessment of each trait.

Fatty acid composition analysis
The extraction of fats was carried out with the method given by Folch et al. [12] The method specified by Metcalfe and Schmitz [13] was used in preparing methyl esters of fatty acids. 1 g of sample was weighed into a tube and 1.5 mL of 2 M methanolic NaOH was added and capped under nitrogen gas. They were left for 1 h at 80°C for saponification After cooling, 2 mL BF3 methanol was added and capped with nitrogen gas again and left for 30 min at 80°C. Samples were cooled and 1 mL of hexane and 1 mL of deionized water was added and vortexed. The tubes were centrifuged for 10 min at 6000 rpm. Upper layer was transferred to new tubes containing sodium sulfate. After addition of 1 mL of hexane, 2 mL of upper layer were transferred to amber vials. Vials were sealed under nitrogen and were maintained until analysis at −18°C. Fatty acid compositions of fats were determined by gas chromatography (GC, Agilent Technologies 6890N) with an FID detector. GC system was equipped with a capillary column (DB23, 60 m × 250 µm × 0.15 µm). The oven temperature was increased from 100°C to 200°C with a rate of 5°C/min and from 200°C to 250°C with a rate of 4°C/min. The injection block and dedector temperatures were 250°C and 280°C, respectively. Helium was used as a carrier gas with a 1.2 mL/min flow rate. A fatty acid methyl ester mix (Supelco, FAME-mix, 4-7801, Bellefonte, PA, USA) was used as standard.

Volatile compounds analysis
The extraction of volatile compounds was done by using Solid Phase Microextraction (SPME) technique with a CAR/PDMS (Supelco 75 μm, CAR/PDMS, USA) fiber and the extraction was carried out according to Kaban. [14] Before the analysis, the fibers were preconditioned in the injection port of the GC (Agilent 6890N) as indicated by the manufacturer. 5 g of sample were placed into a 40 mL SPME vial (Supelco, Bellefonte, PA, USA) sealed with a PTFE-faced silicone septum (Supelco, Bellefonte, PA, USA). The vial was left at 30 ͦ°C in a thermo block (Supelco, Bellefonte, PA, USA) during 1 h to equilibrate its headspace. Then, a SPME fiber was exposed to the headspace while maintaining the sample at 30°C during 2 h. After the extraction, the compounds adsorbed by fiber was desorbed from the injection port of the gas chromatography for 6 min at 250°C, and the compounds were determined by a mass selective detector (Agilent 5973).
The injector port was in the splitless mode. Volatiles were separated in a DB-624 (J&W Scientific, 60 m x 0.25 mm x 1.4 μm) capillary column. Helium was used as carrier gas. The temperature program was started when the fiber was inserted and held at 40°C for 6 min and subsequently programmed from 40°C to 110°C at 3°C/min and at a rate of 4°C/min from 150°C, then, at a rate of 10°C/min from 210°C, where it was held for another 12 min. GC-MS interface was maintained at 280°C. Mass spectra were obtained by electron impact at 70 eV, and data were acquired across the range 30-400 amu. The results were evaluated by comparing with mass spectra from a database developed by NIST and WILEY, or standard molecules (for calculating Kovats indices, Supelco 44585-U, Bellefonte PA USA) and by matching their retention indices with those in the literature. Each sample was analyzed as three replicates.

Statistical analysis
The results of analyses which depend on the animal fat combination were analyzed according to a completely randomized design. The data were tested by variance analysis, and differences between means were evaluated by Duncan's multiple range test using SPSS 13 statistics software (2004). Moreover, principal components analysis (PCA) was carried out to study the structure of dependence and correlation among the variables in the groups by using Unscrambler software (Version 10.01, Camo Process AS., Oslo, Norway).

Moisture, fat, and pH values
The pH and moisture values and fat contents of kavurma groups that were produced by using animal fat combinations were given in Table 1. Although there were some differences, pH was found to be over 6.00 due to the protein denaturation during cooking in all groups. Similar results were also determined by Vural and Öztan, [3] Aksu. [6] Significant (p < 0.05) differences were also observed in terms of moisture content between kavurma groups. The lowest moisture value was determined in the group produced by using 100% BIF, and the highest average moisture value was determined in the group produced by using 100% STF. The average fat content was determined to be less in kavurma group produced by using 100% STF compared to other groups. According to these results, the fat used is an important factor for cooking. Different animal fat combinations in kavurma production cause different moisture and fat values under same cooking conditions.

Sensorial properties of kavurma
The appearance of cooked meat can be influenced by many factors such as pH, meat source, packaging conditions, fat content, added ingredients, and preservation treatments. [15] Fat content, one of them, is an important factor for flavor and palatability of meat. Therefore, the fat used in production is an important factor in the development of sensorial properties in kavurma. The sensorial analysis results of kavurma groups produced by using animal fat combinations were given in Table 2. Kavurma samples produced using 50% BIF +50% BKF showed high values in terms of overall appearance and taste-odor. However, the use of 100% STF significantly decreased the overall appearance, taste-odor, color of fat, and general acceptability values of kavurma. The lowest value for fat color was determined in 100% STF group. In addition, the ratio of STF had a negative effect on texture and the lowest value was observed in group containing 50% BIF +50% STF. On the other hand, the group with 100% STF showed lower texture value compared to 100% BIF group. While the group containing 100% BIF had the highest texture value, the group containing 100% BKF had the highest general acceptability value. The group containing 100% BKF and the group containing 50% BIF + 50% BKF showed higher values in terms of color of fat compared to other groups. According to these results, sensorial properties are negatively affected in kavurma depending on the increase in the ratio of sheep tail fat.

Fatty acid composition of kavurma
The results concerning the fatty acid composition of kavurma samples were given in Table 3. It was determined that different fat and fat combination had a very significant (p < 0.01) effect on saturated fatty acids (excluding arachidic acid, C 20:0 ). Palmitic acid (C 16:0 ) and stearic acid (C 18:0 ) fatty acids were determined as major saturated fatty acids in kavurma groups. Similar results were also reported by Aksu. [6] Among groups, the group produced only with sheep tail fat (100% STF) showed the lowest saturated fatty acid ratio (42.31%±1.63). The highest value was determined as 58.51% ±0.23 in the group in which only kidney fat (100% BKF). Unsaturated fatty acid oleic acid (C 18:1n9 ) was determined as the major unsaturated fatty acid in all kavurma groups. In addition to this, the groups containing 100% BIF (46.81%±2.93), 100% STF (45.35% ±0.91), and 50% BIF + 50% STF (45.54%±3. 21) showed higher values in terms of oleic acid (C 18:1n9 ) compared to other groups. Regarding the unsaturated fatty acid, the highest value (57.70%±1.61) was determined in the group produced using 100% sheep tail fat, and the lowest value was determined as 41.30%±0.50 in the group with 100% BKF. Similarly, the polyunsaturated fatty acid ratio of the group containing 100% STF was also determined to be higher compared to other groups. According to these results, the use of sheep tail fat in kavurma production significantly increases the unsaturated fatty acid ratio.

Volatile compounds of kavurma samples
A total of 44 volatile compounds included in nine different chemical groups as aldehydes, ketones, aromatic hydrocarbons, aliphatic hydrocarbons, alcohols, esters, furans, acids, and terpenes were defined in kavurma samples. Results regarding the volatile compounds of kavurma groups were given in Table 4. The use of different fat or fat combination had significant or very significant effect on only a few compounds (benzaldehyde, nonanal, 3-methyl-2-butanone, 2,5-octadiene, undecane, and ethanol). The reactions such as carbohydrate fermentation, lipolysis, proteolysis, lipid oxidation, and amino acid catabolism are important sources in the formation of volatile compounds. In addition, changes in the fatty acid composition of lipids can be accepted an indirect measure of susceptibility to lipid oxidation. [16] The free fatty acids formed as a result of lipolysis carried out by lipase and phospholipase enzymes can be precursors of lipid oxidation. The compounds such as aldehydes, alcohols, and ketones formed by autooxidation have volatile character and play an important role in the development of the product's taste and odor. [17] However, aldehydes present in trace concentrations may affect the aroma due to their low-odor thresholds. [18] Among the aldehydes, hexanal is the most common volatile compound sourced from cooked meat, and it is accepted as an index of meat flavor deterioration during early storage stages of cooked meat. [19,20] In this study, hexanal was also the major compound in all groups. However, the highest hexanal value was found in the group containing 100% BKF. This result may be explained by the highest linoleic acid content of 100% BKF. Medina et al. [21] and Zhang et al. [22] also suggested that hexanal may source from linoleic acid. While benzaldehyde was determined to be at the lower level in STF group compared to other groups, nonanal was not determined in 100% BKF group (Table 4). Four different alcohols were detected in kavurma groups. However, only ethanol showed significant differences between kavurma groups (Table 4).
Ketones are another group of volatile compounds that may influence the flavor properties of kavurma. 3-methyl-2-butanone founded statistically significant showed the highest value in 100% BKF. On the other hand, 2,5-octadiene showed the highest value in mixture of fats containing STF. In this study, 10 aliphatic hydrocarbons were identified in the kavurma groups. The use of different animal fat had no significant effect on aliphatic hydrocarbons except undecane. Although aliphatic hydrocarbons do not contribute to meat aroma, they are a part of meat aroma and formed as a result of the oxidation of fatty acids. [18] When volatile compounds are evaluated, in general, it can be concluded that the use of sheep tail fat in kavurma production, contrary to expectations, does not make a significant contribution to the volatile profile. In addition, as it can be conferred from the results of the sensory analysis, increasing STF rate has a negative effect on sensory properties. However, no significant changes occur in case of keeping STF rate at 30%.

PCA analysis
PCA was applied to assess the relationships between animal fat combinations and the unsaturation level of the fatty acid composition in kavurma samples (Fig. 1). Two principal components explained 100% of the total variance (97% and 3% for PC1 and PC2, respectively). The biplot obtained from PCA indicated that SFA and BIF+BKF showed a negative correlation with PC1 and PC2. In contrast, PUFA, USFA, MUFA, STF, BIF, and BIF+STF placed on the positive axis of PC1. STF showed positively high correlated with PUFA in PC1 and PC2 (Fig.1). SFA was related to BKF and the groups containing BKF. The relationship of PCA results between sensorial properties and the unsaturation level of the fatty acid composition in kavurma samples was given in Fig. 2. Principal component analysis revealed that SFA is negatively related to sensorial properties in PC1, whereas PUFA, USFA, and MUFA were separated from the sensorial properties (Fig. 2).

Conclusion
The use of BIF, BKF, STF, or their combinations in kavurma production significantly affects the fatty acid composition and sensorial parameters. BIF, BKF, or mix of these two types fat in equal proportions gives similar results in terms of sensory properties. The use of STF increases the unsaturated fatty acid content but causes significant decreases in sensorial parameters. This decrease is a consequence of STF having a high degree of unsaturation and low melting temperature. Because of this, STF must not be used at over a proportion of 30%. Moreover, STF used in kavurma production with intent to enrich the aroma does not cause significant differences in volatile composition of the product.

Funding
This study has been supported by the Research Council of Atatürk University (BAP 2010/59). The financial support of Atatürk University is gratefully acknowledged.