Effects of α-tocopherol and citric acid on the oxidative stability of alimentary poultry fats during storage at low temperatures

ABSTRACT

The purpose of this study was to investigate the stability of three alimentary poultry fats (goose, duck, and chicken) by natural antioxidants (α-tocopherol and citric acid). This was targeted to extend their shelf life, and to monitor the quality parameters during refrigerated (+4°C) and frozen storage (–20°C). The addition of natural antioxidants in a proportion of 0.1% has extended the shelf life of goose fat stored at +4°C by 90 days; for goose fat stored at –20°C citric acid has prolonged the shelf life by 150 days, while goose fat with α-tocopherol could be stored for more than 480 days at –20°C without spoilage. Polyunsaturated fatty acids and monounsaturated fatty acids content decreased significantly (p < 0.05) after 480 days of chilled storage for fat samples with α-tocopherol. The natural antioxidants provided good protection against oxidation of poultry fats, and these can be used to monitor the oxidation of fats and to predict their shelf life stability.

KEYWORDS:

• α-Tocopherol
• Citric acid
• Poultry fats
• Fatty acid profile, Oxidation

Introduction

The quality of food of animal origin is currently a major concern for everyone. The consumer, policymaker, and producer as well as the specialist in animal husbandry consider that it is of utmost importance to control and assess the quality of animal products. Alimentary animal fats are produced by melting raw material fats at a temperature ranging between 65 and 70°C, followed by filtration to remove solid impurities and water. Animal fats can be used in a wide variety of applications; they appear as part of the diet, as filler in factory produced meat, in fast-food products, and for industrial purposes such as soap making.

Lipid oxidation is responsible for the loss of nutritional value and the change in sensory characteristics of food products. Maintaining the quality of alimentary animal fats depends upon a variety of factors such as storage temperature, permeability of the packaging material to moisture and air, and type of animal feed. Since lipids are highly susceptible to pro-oxidant factors, this process cannot be stopped, thus reducing the shelf-life of food.^[1]

Oxidation of food can be prevented by synthetic antioxidants including t-butyl-4-hydroxyanisole (BHA), 2,6-di-t-butyl-p-hydroxytoluene (BHT), and tertiary butylhydroquinone (TBHQ).^[2] Recent reports have implicated these compounds in many health risks, including cancer and carcinogenesis, and their safety has been questioned.^[3] Despite the high antioxidant activity of TBHQ, its use has been prohibited in some countries, such as Japan and Canada. Based on the safety concerns, the natural antioxidants have gained increased interest because of the belief that natural food ingredients are better and safter than synthetic ones.^[4]

Antioxidants inhibit lipid oxidation by scavenging radicals via hydrogen donation and also by physically stabilizing the micelles in the microenvironments of the reaction sites.^[5] Besides the degree of fatty acid (FA) unsaturation, the antioxidant effectiveness is influenced by other factors, such as diffusion of oxygen, temperature and light, interaction with pro-oxidants, lipid composition-structure-position, physical structures, stability of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and position of FAs on glycerol.^[6] The interest in natural antioxidants is increased by the suggestion that these compounds display antiatherogenic, anticarcinogenic, and antitumor activity; incorporation of such compounds into products may contribute to the stabilization of food products and also to the consumers health benefit.^[7]

In the food industry, the most commonly used natural antioxidants are tocopherols because of their chemical stability, availability, and low cost. Another antioxidant is citric acid, which aids in retarding oxidative deterioration of lipids by chelation of metal ions in citric acid.^[8] Some researchers have studied the effects of natural antioxidants on the oxidative stability of vegetable oils,^[9–11] and on the shelf life of butter, ghee, fish oils, as well as the thermal stability of sheep tallow olein and lard.^[12–19]

However, there is still a lack of systematic studies for evaluating the antioxidative activity of tocopherols and citric acid in animal fats, and there are no studies regarding the effects of natural antioxidants on the oxidative stability of poultry fats under different storage time and temperature conditions and for the improvement of their storage life. The purpose of this research was to investigate the stabilization with natural antioxidants (α-tocopherol and citric acid) of three alimentary poultry fats (goose, duck, and chicken) in order to extend their shelf life, and to monitor the quality parameters during refrigerated (+4°C) and frozen storage (–20°C).

Materials and methods

Materials

The research was conducted on three types of alimentary poultry fats: goose, duck, and chicken fat. Goose fat was collected from goslings, 16 weeks of age, male and female, from the Landaise breed; duck fat was collected from ducklings, 14 weeks of age, male and female, from the Pekin breed, and chicken fat was collected from broilers, 8 weeks of age, male and female, from the Ross 308 breed. All birds were fed with a commercial protein diet, and from the third week they also had access to vegetation. Poultry diets were based on corn and soybean meal, with small amounts of calcium, phosphorus, salt, vitamins, and trace minerals. The birds’ diet was not supplemented with antioxidants or long-chain polyunsaturated FAs (PUFAs). Raw materials (abdominal fats) were collected immediately after slaughtering. An average weight of 1000 g of abdominal fat was cut into small pieces, heated at 65–75°C, centrifuged, and filtered. Natural antioxidants (α-tocopherol and citric acid) were dissolved in the melted fats (with up to 30 min stirring) in the same proportion of 0.1% (0.1 g/100 g of fat). The initial α-tocopherol content was 40 mg/kg in goose fat, 42 mg/kg in duck fat, 40 mg/kg in chicken fat, and citric acid was not detected. In order to carry out fat analyses during storage for 480 days, more samples of 40 g from each fat were packed in closed jars, stored under refrigeration (+4°C) and freezing (–20°C), and at various time intervals were used for chemical analysis. For evaluation of fat stability and monitoring the deterioration during storage, methods included peroxide value (PV), thiobarbituric acid reactive substances (TBARS), iodine value (IV), and FA composition. Three replications were carried out to examine each sample. All chemicals used were of analytical grade and obtained from Merck (Germany); α-tocopherol and citric acid were purchased from Sigma (Sigma Chemicals, Shanghai, China).

PV determination

PV was determined using ultraviolet-visible (UV-Vis) T60U spectrophotometer (Bibby Scientific, London, UK): operating temperature 5–45°C; field wavelength 190–1100 nm; wave length accuracy 0.1 nm. This protocol was based on the spectrophotometer determination of ferric ions (Fe³⁺) derived from the oxidation of ferrous ions (Fe²⁺) by hydro peroxides, in the presence of ammonium thiocyanate (NH₄SCN). Thiocyanate ions (SCN⁻) react with Fe³⁺ ions to give a red–violet homogeny that can be determined spectrophotometrically; the absorbance of each solution was read at 500 nm. To quantify PV, a calibration curve (absorbance at 500 nm versus Fe³⁺ expressed in µg) was constructed. PV was expressed as meq O₂/kg fat.^[20]

TBARS test

TBARS determination was carried out as follows: TBA Reagent (0.02 M 2-thiobarbituric acid in 90% glacial acetic acid) was prepared, then 1 g of fat sample was measured into a glass-stoppered test tube and 5 mL of TBA reagent was added. The tube was stoppered and the contents were mixed. Then the tube was immersed in a boiling water bath for 35 min. A distilled water-TBA reagent blank was also prepared and treated like the sample. After heating, the sample was cooled in tap water for 10 min. A portion was transferred to a cuvette, and the optical density of the sample was read against the blank at a wavelength of 538 nm in a UV-Vis T60U model spectrophotometer. The optical density value was converted to the moles of malondialdehyde (MDA) per gram of sample by using a standard curve. A standard curve was prepared by making appropriate dilutions of the 1 × 10⁻³ M 1,1,3,3-tetraethoxypropane standard solution, to give amounts ranging from 1 × 10⁻⁸ to 7 × 10⁻⁸ mol of MDA in 1 mL. These dilutions were reacted with TBA reagent and the optical densities were measured at the wavelength of 538 nm in the spectrophotometer. TBARS value was expressed as milligrams of MDA/kg fat.^[21]

IV determination

IV was determined according to the Hanus method. Approximately 0.5 g sample (dissolved in 15 mL CCl₄) was mixed with 25 mL Hanus solution (IBr) to halogenate the double bonds. After storing the mixture in the dark for 30 min, excess IBr was reduced to free I₂ in the presence of 20 mL of KI (100 g/L) and 100 mL distilled water. Free I₂ was measured by titration with 24.9 g/L Na₂S₂O₃·5H₂O using starch (1.0 g/100 mL) as an indicator. IV was expressed as g I₂/100 g fat.^[22]

FA content

In a round-bottomed flask was introduced a 2-mL sample to which was added 20 mL of sulphuric acid methanol solution and three pieces of porous porcelain; a reflux cooler was fitted to the flask and it was boiled for about 60 min in a water bath. The completion of the reaction was determined by clarifying the solution and noting the absence of fat globules. The content of the flask was cooled to room temperature and then was passed quantitatively in a separatory funnel using 20 mL of water, and the methyl esters were extracted in two stages with 20 mL of heptanes. The extracts were combined into another separatory funnel and washed with 20 mL of water until the sulphuric acid was completely removed, the removal being controlled with methyl orange. The extracts were then dehydrated by the addition of anhydrous sodium sulphate and filtered into a flask, the solvent was distilled off in a water bath under vacuum, and the solvent traces were removed from the sample by blowing with nitrogen. Methyl esters were collected in 1 mL hexane, and 1 μL sample was injected in the gas chromatograph. FA composition was determined using a Shimadzu GC-17 A gas chromatograph (Tokyo, Japan) coupled with a flame ionization detector. The gas chromatography column is Alltech AT-Wax (60 m × 0.32 mm × 0.5 μm), stationary phase (polyethylene); helium was used as a carrier gas at a pressure of 147 kPa, temperature of the injector and detector was set to 260°C, and the oven program was the following: 70°C for 2 min, then the temperature was raised to 150°C with a gradient of 10°C/min, a level of 3 min, and the temperature was raised to 235°C with a gradient of 4°C/min. Identification and quantification of FAs was performed by comparison with standards. Results were expressed as g/100 g total FAs.^[22]

Statistical analysis

The effects of the storage temperature, storage time, and the addition of antioxidants on PV, TBARS, and IV were analyzed via factorial analysis of variance (ANOVA) using the General Linear Model in Minitab 16.1.0 (LEAD Technologies, Inc., Charlotte, NC, USA). Three replications were carried out to examine each sample. The effect of storage temperature on FA content was carried out using the one-way ANOVA. Tukey’s honest significance test was carried out at a 95% confidence level (p < 0.05). The Pearson’s correlation (ɑ = 0.05) with two-tailed probability values was used to estimate the strength of association between chemical parameters.

Results and discussion

In the present research, the effects of natural antioxidants (α-tocopherol and citric acid added in the concentration of 0.1%) on the oxidative stability and deterioration rates of three alimentary poultry fats (goose, duck, and chicken) were determined under different storage conditions. Chemical analyses, such as PV, TBARS, IV, and FA composition, were carried out to monitor fat quality under defined storage conditions. The results of chemical analyses for goose, duck, and chicken fat, and fats with added antioxidants are presented in Tables 1–4, respectively.

Table 1. Changes in quality parameters of goose fat and goose fat with added antioxidants stored at two different temperatures.

	Goose fat			Goose fat with 0.1% α-tocopherol			Goose fat with 0.1% citric acid
Days of storage	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)
at +4°C
0	1.10^k ± 0.01	0.60^m ± 0.06	84.7^a ± 0.3	1.11^ij ± 0.03	0.62^j ± 0.05	84.5^a ± 0.2	1.10^j ± 0.02	0.59^j ± 0.03	84.2^a ± 0.5
60	2.03^j ± 0.03	1.76^l ± 0.01	83.4^ab ± 0.4	1.73^i ± 0.03	0.92^ij ± 0.01	84.1^a ± 0.5	1.83^i ± 0.04	1.07^i ± 0.07	83.8^a ± 0.6
120	3.80^hi ± 0.02	3.24^jk ± 0.02	81.8^bc ± 0.1	2.81^h ± 0.05	1.77^i ± 0.04	83.1^b ± 0.4	2.97^h ± 0.03	1.98^h ± 0.06	83.3^ab ± 0.4
180	7.33^g ± 0.05	4.87^hi ± 0.04	80.7^de ± 0.3	4.02^g ± 0.07	2.90^h ± 0.07	82.3^bc ± 0.2	4.25^fg ± 0.06	3.01^g ± 0.01	82.0^bc ± 0.1
240	9.51^ef ± 0.04	7.25^fg ± 0.03	78.5^f ± 0.2	5.70^f ± 0.01	4.37^gh ± 0.06	80.4^c ± 0.3	5.92^ef ± 0.05	4.52^fg ± 0.04	79.8^c ± 0.2
300	11.14^d ± 0.06	9.02^de ± 0.05	76.2^g ± 0.4	7.79^e ± 0.02	6.02^f ± 0.03	78.3^d ± 0.2	7.81^e ± 0.01	6.23^e ± 0.02	77.5^de ± 0.3
360	12.70^c ± 0.01	10.84^d ± 0.07	74.3^h ± 0.5	9.78^cd ± 0.04	7.88^de ± 0.02	76.7^e ± 0.1	9.92^cd ± 0.07	7.96^de ± 0.05	75.8^ef ± 0.2
420	14.04^bc ± 0.03	12.75^c ± 0.01	71.7^i ± 0.3	11.75^c ± 0.03	10.05^c ± 0.05	75.6^ef ± 0.1	11.94^b ± 0.04	10.12^c ± 0.07	74.4^g ± 0.2
450	14.74^b ± 0.05	13.62^b ± 0.06	71.1^ij ± 0.4	12.68^b ± 0.05	11.14^b ± 0.07	74.8^g ± 0.4	13.01^ab ± 0.05	11.39^b ± 0.04	73.1^h ± 0.4
480	15.11^a ± 0.01	14.59^a ± 0.03	70.7^jk ± 0.1	13.46^a ± 0.02	12.25^a ± 0.04	74.1^gh ± 0.3	13.73^a ± 0.01	12.51^a ± 0.03	72.2^hi ± 0.5
at –20°C
0	1.10^k ± 0.01	0.60^m ± 0.06	84.7^a ± 0.3	1.11^ij ± 0.03	0.62^j ± 0.05	84.5^a ± 0.2	1.10^j ± 0.02	0.59^j ± 0.03	84.2^a ± 0.5
60	1.51^kj ± 0.02	1.32^l ± 0.03	83.9^ab ± 0.2	1.23^ij ± 0.03	0.75^ij ± 0.02	84.2^a ± 0.4	1.44^i ± 0.04	0.97^hi ± 0.01	84.0^a ± 0.3
120	2.40^j ± 0.04	2.43^k ± 0.05	82.8^b ± 0.5	1.91^hi ± 0.01	1.31^i ± 0.05	83.7^ab ± 0.2	2.13^hi ± 0.02	1.55^h ± 0.07	83.5^ab ± 0.5
180	3.91^hi ± 0.06	3.81^j ± 0.02	81.3^c ± 0.4	2.75^gh ± 0.06	2.05^hi ± 0.04	82.8^b ± 0.5	2.95^h ± 0.07	2.31^gh ± 0.02	82.4^bc ± 0.1
240	6.58^gh ± 0.03	5.58^gh ± 0.04	79.2^de ± 0.1	3.71^g ± 0.05	3.09^h ± 0.07	81.0^bc ± 0.3	4.02^g ± 0.06	3.48^g ± 0.06	80.2^c ± 0.2
300	9.67^ef ± 0.07	7.52^ef ± 0.01	77.3^fg ± 0.1	5.60^f ± 0.07	4.15^gh ± 0.06	79.3^c ± 0.4	5.88^ef ± 0.05	4.69^fg ± 0.04	78.3^d ± 0.3
360	11.29^e ± 0.02	9.46^de ± 0.05	75.8^gh ± 0.2	7.10^e ± 0.04	5.37^f ± 0.01	78.1^d ± 0.2	7.59^e ± 0.03	5.99^e ± 0.03	76.9^de ± 0.5
420	12.51^cd ± 0.01	11.34^d± 0.07	74.6^h ± 0.4	8.40^d ± 0.03	6.67^ef ± 0.04	77.1^de ± 0.3	8.96^d ± 0.04	7.28^de ± 0.05	75.6^ef ± 0.4
450	13.24^c ± 0.03	12.22^c ± 0.04	73.6^hi ± 0.4	9.13^cd ± 0.01	7.26^de ± 0.05	76.6^e ± 0.3	9.65^cd ± 0.07	7.91^de ± 0.01	74.8^g ± 0.3
480	14.02^b ± 0.05	13.31^b ± 0.01	72.4^j ± 0.3	9.72^cd ± 0.02	7.89^de ± 0.03	76.0^ef ± 0.1	10.31^bc ± 0.01	8.64^d ± 0.04	74.1^gh ± 0.2

^aPV: peroxide value; TBARS: thiobarbituric acid reactive substances; IV: iodine value.

^bValues are expressed as mean ± standard deviation of three replicates for each parameter.

^cDifferent letters in the same column indicate statistically significant differences at p < 0.05 (Tukey’s test).

Table 2. Changes in quality parameters of duck fat and duck fat with added antioxidants stored at two different temperatures.

	Duck fat			Duck fat with 0.1% α-tocopherol			Duck fat with 0.1% citric acid
Days of storage	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)
at +4°C
0	0.82^j ± 0.02	0.51^l ± 0.05	79.5^a ± 0.4	0.78^ij ± 0.01	0.48^j ± 0.03	79.9^a ± 0.6	0.80^ij ± 0.04	0.45^j ± 0.01	79.7^a ± 0.2
60	1.84^i ± 0.05	1.65^jk ± 0.03	78.6^ab ± 0.5	1.61^i ± 0.02	0.77^i ± 0.02	79.1^a ± 0.1	1.72^i ± 0.06	0.89^ij ± 0.03	79.2^a ± 0.1
120	3.55^h ± 0.07	3.12^hi ± 0.04	77.3^bc ± 0.3	2.63^hi ± 0.04	1.51^hi ± 0.05	78.2^ab ± 0.5	2.76^ghi ± 0.01	1.63^hi ± 0.07	77.6^bc ± 0.3
180	7.13^f ± 0.06	4.96^g ± 0.01	75.5^cd ± 0.6	3.85^gh ± 0.01	2.59^h ± 0.04	76.7^c ± 0.3	3.96^fg ± 0.07	2.80^h ± 0.05	76.1^c ± 0.1
240	9.27^e ± 0.03	6.86^ef ± 0.07	73.2^d ± 0.6	5.46^fg ± 0.06	4.88^g ± 0.07	75.0^cd ± 0.6	5.68^ef ± 0.04	4.25^gh ± 0.06	74.5^d ± 0.2
300	10.86^d ± 0.05	8.87^de ± 0.06	70.6^ef ± 0.2	7.57^def ± 0.02	6.38^f ± 0.01	73.1^d ± 0.4	7.74^de ± 0.05	5.89^fg ± 0.04	72.6^de ± 0.5
360	12.41^c ± 0.04	10.92^c ± 0.05	68.3^f ± 0.1	9.42^cd ± 0.05	7.57^d ± 0.06	71.7^e ± 0.2	9.88^cd ± 0.01	7.89^cd ± 0.01	71.1^e ± 0.4
420	13.74^b ± 0.01	12.05^b ± 0.04	66.1^g ± 0.5	11.53^bc ± 0.01	9.77^c ± 0.07	68.9^f ± 0.4	11.76^bc ± 0.02	10.02^bc ± 0.02	68.2^f ± 0.1
450	14.39^ab ± 0.03	12.66^ab ± 0.02	65.2^h ± 0.4	12.45^ab ± 0.04	10.76^b ± 0.07	67.8^fg ± 0.3	12.69^ab ± 0.07	10.84^b ± 0.04	67.5^fg ± 0.1
480	14.86^a ± 0.08	13.37^a ± 0.01	64.1^i ± 0.2	13.24^a ± 0.03	11.88^a ± 0.03	66.7^g ± 0.1	13.51^a ± 0.04	11.71^a ± 0.05	66.9^g ± 0.3
at –20°C
0	0.82^j ± 0.02	0.51^l ± 0.05	79.5^a ± 0.4	0.78^ij ± 0.01	0.48^j ± 0.03	79.9^a ± 0.6	0.80^ij ± 0.04	0.45^j ± 0.01	79.7^a ± 0.2
60	1.22^ij ± 0.03	1.21^jkl ± 0.04	78.8^ab ± 0.6	1.02^ij ± 0.01	0.59^ij ± 0.06	79.3^a ± 0.2	1.20^hij ± 0.05	0.67^ij ± 0.05	79.1^a ± 0.4
120	2.13^hi ± 0.01	2.11^hij ± 0.01	77.6^bc ± 0.1	1.65^hij ± 0.02	1.04^hij ± 0.07	78.9^ab ± 0.6	1.94^hi ± 0.01	1.19^hij ± 0.01	78.2^ab ± 0.3
180	3.64^h ± 0.05	3.23^gh ± 0.03	76.0^c ± 0.2	2.43^hi ± 0.06	1.71^hi ± 0.05	77.1^bc ± 0.5	2.72^ghi ± 0.04	1.89^hi ± 0.07	76.5^c ± 0.1
240	6.25^fg ± 0.07	4.71^g ± 0.07	73.9^d ± 0.5	3.42^gh ± 0.08	2.75^h ± 0.04	76.1^c ± 0.3	3.81^fg ± 0.06	2.78^h ± 0.05	75.6^cd ± 0.2
300	9.36^e ± 0.06	6.80^ef ± 0.04	71.8^e ± 0.6	5.31^fg ± 0.04	3.89^gh ± 0.03	73.9^d ± 0.4	5.65^ef ± 0.07	3.99^gh ± 0.06	73.1^d ± 0.5
360	10.99^d ± 0.04	8.91^de ± 0.06	70.3^ef ± 0.4	6.85^efg ± 0.05	5.16^fg ± 0.02	72.2^de ± 0.3	7.17^def ± 0.02	5.03^fgh ± 0.01	71.6^e ± 0.4
420	12.21^cd ± 0.08	11.12^c ± 0.05	68.6^f ± 0.1	8.21^ef ± 0.07	6.46^ef ± 0.01	69.4^ef ± 0.1	8.32^cdef ± 0.01	6.36^ef ± 0.03	68.9^f ± 0.1
450	12.97^c ± 0.04	12.14^b ± 0.01	67.4^fg ± 0.2	8.97^cde ± 0.03	7.06^de ± 0.08	68.3^f ± 0.1	9.06^cde ± 0.04	6.97^de ± 0.04	67.8^fg ± 0.1
480	13.82^b ± 0.01	13.03^ab ± 0.03	66.3^g ± 0.1	9.42^cd ± 0.01	7.78^d ± 0.04	67.4^fg ± 0.2	9.88^cd ± 0.03	7.69^cd ± 0.02	67.1^fg ± 0.3

^aPV: peroxide value; TBARS: thiobarbituric acid reactive substances; IV: iodine value.

^bValues are expressed as mean ± standard deviation of three replicates for each parameter.

^cDifferent letters in the same column indicate statistically significant differences at p < 0.05 (Tukey’s test).

Table 3. Changes in quality parameters of chicken fat and chicken fat with added antioxidants stored at two different temperatures.

	Chicken fat			Chicken fat with 0.1% α-tocopherol			Chicken fat with 0.1% citric acid
Days of storage	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)	PV (meq O2/kg)	TBARS (mg MDA/kg)	IV (g I2/100 g)
at +4°C
0	2.30^n ± 0.02	1.08^m ± 0.05	96.8^a ± 0.2	2.28^j ± 0.04	1.07^k ± 0.06	96.6^a ± 0.3	2.26^kl ± 0.01	1.05^l ± 0.04	96.5^a ± 0.3
60	3.80^lm ± 0.06	2.76^l ± 0.01	95.1^b ± 0.4	2.83^ij ± 0.05	1.82^jk ± 0.02	96.4^a ± 0.2	2.97^k ± 0.02	1.97^k ± 0.06	95.9^b ± 0.3
120	6.35^jkl ± 0.07	4.66^jk ± 0.03	93.0^d ± 0.3	4.05^hi ± 0.04	2.94^j ± 0.04	94.3^c ± 0.1	4.34^j ± 0.01	2.84^j ± 0.05	93.8^d ± 0.4
180	9.46^hi ± 0.04	7.38^hi ± 0.04	90.6^f ± 0.1	5.46^gh ± 0.07	4.25^i ± 0.05	91.3^e ± 0.5	5.72^ij ± 0.05	4.15^i ± 0.07	91.1^f ± 0.2
240	12.61^g ± 0.03	10.2^fg ± 0.02	88.5^gh ± 0.2	7.29^ef ± 0.06	5.74^h ± 0.07	89.6^fg ± 0.4	7.51^gh ± 0.07	5.64^h ± 0.03	88.7^g ± 0.2
300	14.81^e ± 0.06	11.87^e ± 0.07	85.6^ij ± 0.2	9.61^d ± 0.03	7.56^fg ± 0.04	86.5^hi ± 0.3	9.88^ef ± 0.06	7.79^fg ± 0.04	85.9^i ± 0.1
360	16.82^d ± 0.01	13.39^d ± 0.06	83.4^jk ± 0.5	12.11^c ± 0.02	9.46^de ± 0.03	84.3^ij ± 0.3	12.38^d ± 0.04	9.68^de ± 0.02	83.8^j ± 0.1
420	18.01^bc ± 0.02	14.48^c ± 0.01	81.7^kl ± 0.4	13.41^b ± 0.04	11.47^c ± 0.02	82.6^j ± 0.2	13.74^b ± 0.03	11.73^c ± 0.01	82.2^k ± 0.4
450	18.61^b ± 0.07	15.26^b ± 0.04	80.3^lm ± 0.3	14.12^ab ± 0.01	12.58^b ± 0.01	81.4^k ± 0.1	14.46^ab ± 0.02	12.41^b ± 0.05	81.1^l ± 0.2
480	19.11^a ± 0.04	16.15^a ± 0.03	77.1^n ± 0.2	14.61^a ± 0.05	13.77^a ± 0.06	80.2^l ± 0.4	14.92^a ± 0.01	13.92^a ± 0.04	78.3^m ± 0.3
at –20°C
0	2.30^n ± 0.02	1.08^m ± 0.05	96.8^a ± 0.2	2.28^j ± 0.04	1.07^k ± 0.06	96.6^a ± 0.3	2.26^kl ± 0.01	1.05^l ± 0.04	96.5^a ± 0.3
60	3.21^m ± 0.01	2.04^lm ± 0.07	95.5^b ± 0.1	2.64^ij ± 0.03	1.53^jk ± 0.04	96.3^a ± 0.1	2.84^k ± 0.01	1.72^k ± 0.03	96.2^a ± 0.4
120	4.72^kl ± 0.03	3.56^kl ± 0.04	93.8^cd ± 0.4	3.54^hij ± 0.06	2.24^ijk ± 0.01	95.4^b ± 0.4	3.92^jk ± 0.04	2.45^j ± 0.02	95.1^b ± 0.3
180	6.81^jk ± 0.05	5.58^hi ± 0.06	91.2^ef ± 0.5	4.85^ghi ± 0.05	3.26^ij ± 0.02	93.5^d ± 0.5	5.24^ijk ± 0.02	3.16^ij ± 0.04	92.7^de ± 0.1
240	9.32^hij ± 0.07	7.79^gh ± 0.01	89.1^fg ± 0.3	6.24^fgh ± 0.07	4.43^i ± 0.03	91.1^e ± 0.3	6.47^hi ± 0.03	4.23^i ± 0.02	90.9^fg ± 0.2
300	12.43^g ± 0.04	10.18^fg ± 0.02	86.7^hi ± 0.2	7.75^def ± 0.02	5.62^h ± 0.05	88.7^gh ± 0.2	7.81^gh ± 0.06	5.78^h ± 0.01	88.3^g ± 0.4
360	14.52^ef ± 0.06	12.29^de ± 0.04	84.5^ij ± 0.3	8.86^de ± 0.01	6.82^fgh ± 0.07	86.4^hi ± 0.3	9.02^efg ± 0.06	6.95^fgh ± 0.03	85.1^i ± 0.2
420	16.11^de ± 0.06	13.98^cd ± 0.03	82.8^jk ± 0.4	9.78^d ± 0.03	7.81^efg ± 0.06	84.1^ij ± 0.4	9.87^ef ± 0.05	7.99^fg ± 0.06	83.6^j ± 0.2
450	16.01^de ± 0.05	14.66^bc ± 0.05	81.5^kl ± 0.1	10.45^cd ± 0.04	8.36^ef ± 0.06	82.9^j ± 0.1	10.65^def ± 0.01	8.59^f ± 0.05	82.1^k ± 0.3
480	17.93^cd ± 0.03	15.54^ab ± 0.07	80.1^lm ± 0.1	11.03^cd ± 0.02	9.17^e ± 0.02	81.7^k ± 0.1	11.34^de ± 0.07	9.42^ef ± 0.07	80.9^l ± 0.4

^aPV: peroxide value; TBARS: thiobarbituric acid reactive substances; IV: iodine value.

^bValues are expressed as mean ± standard deviation of three replicates for each parameter.

^cDifferent letters in the same column indicate statistically significant differences at p < 0.05 (Tukey’s test).

Table 4. Variations in fatty acid composition (% of total fatty acids) of fresh poultry fats and poultry fats with α-tocopherol stored at two different temperatures

Fatty acids	Goose fat			Duck fat			Chicken fat
	Control (fresh fat)	Goose fat with 0.1% α-tocopherol after 480 days of storage at +4°C	Goose fat with 0.1% α-tocopherol after 480 days of storage at –20°C	Control (fresh fat)	Duck fat with 0.1% α-tocopherol after 480 days of storage at +4°C	Duck fat with 0.1% α-tocopherol after 480 days of storage at –20°C	Control (fresh fat)	Chicken fat with 0.1% α-tocopherol after 480 days of storage at +4°C	Chicken fat with 0.1% α-tocopherol after 480 days of storage at –20°C
Myristic (14:0)	1.89 ± 0.03	2.18 ± 0.02	1.98 ± 0.03	2.18 ± 0.03	2.46 ± 0.02	2.29 ± 0.05	0.73 ± 0.03	0.87 ± 0.04	0.57 ± 0.04
Myristoleic (14:1)	1.47 ± 0.05	1.59 ± 0.06	1.49 ± 0.01	1.79 ± 0.05	1.92 ± 0.05	1.86 ± 0.04	0.50 ± 0.07	0.74 ± 0.09	0.63 ± 0.07
Pentadecanoic (C15:0)	2.65 ± 0.02	3.00 ± 0.05	2.85 ± 0.07	2.89 ± 0.02	3.11 ± 0.01	2.92 ± 0.02	1.63 ± 0.09	1.80 ± 0.06	1.66 ± 0.05
Palmitic (C16:0)	10.99 ± 0.01	11.54 ± 0.06	11.41 ± 0.02	11.41 ± 0.01	12.05 ± 0.06	11.34 ± 0.03	9.72 ± 0.08	10.12 ± 0.05	9.87 ± 0.01
Palmitoleic (C16:1)	11.13 ± 0.04	10.98 ± 0.01	11.08 ± 0.06	10.80 ± 0.04	10.68 ± 0.04	10.72 ± 0.06	8.09 ± 0.04	8.21 ± 0.03	8.13 ± 0.04
Hexadecadienoic (C16:2)	4.08 ± 0.05	3.77 ± 0.06	3.90 ± 0.06	3.78 ± 0.05	3.56 ± 0.06	3.75 ± 0.04	5.66 ± 0.09	5.60 ± 0.08	5.62 ± 0.02
Hexadecatrienoic (C16:3)	3.65 ± 0.02	3.44 ± 0.04	3.56 ± 0.01	3.25 ± 0.02	3.07 ± 0.03	3.22 ± 0.02	4.82 ± 0.05	4.76 ± 0.04	4.80 ± 0.06
Hexadecatetranoic (C16:4)	2.59 ± 0.04	2.28 ± 0.03	2.48 ± 0.06	2.17 ± 0.01	2.05 ± 0.01	2.10 ± 0.05	2.87 ± 0.01	2.81 ± 0.07	2.84 ± 0.08
Heptadecanoic (C17:0)	0.11 ± 0.05	0.32 ± 0.01	0.21 ± 0.05	0.09 ± 0.06	0.27 ± 0.05	0.22 ± 0.01	0.13 ± 0.04	0.38 ± 0.01	0.20 ± 0.03
Stearic (C18:0)	8.51 ± 0.01	8.93 ± 0.06	8.50 ± 0.06	8.93 ± 0.07	9.24 ± 0.03	9.06 ± 0.03	7.18 ± 0.06	7.68 ± 0.05	8.25 ± 0.09
Oleic (C18:1)	29.64 ± 0.03	28.75 ± 0.02	29.26 ± 0.01	29.30 ± 0.03	29.10 ± 0.01	29.60 ± 0.04	34.03 ± 0.01	33.91 ± 0.04	33.96 ± 0.05
Linoleic (C18:2)	19.82 ± 0.06	19.58 ± 0.04	19.69 ± 0.06	19.41 ± 0.02	19.08 ± 0.04	19.35 ± 0.02	20.80 ± 0.03	19.64 ± 0.02	19.89 ± 0.08
Linolenic (C18:3)	1.19 ± 0.02	1.14 ± 0.02	1.15 ± 0.05	1.25 ± 0.04	1.11 ± 0.05	1.20 ± 0.06	1.11 ± 0.08	0.95 ± 0.01	0.98 ± 0.04
Stearidonic acid (C18:4)	1.62 ± 0.05	1.50 ± 0.03	1.58 ± 0.03	1.41 ± 0.01	1.30 ± 0.03	1.37 ± 0.07	1.73 ± 0.06	1.53 ± 0.07	1.60 ± 0.01
Total SFA	24.15 ± 0.05	26.03 ± 0.06	24.95 ± 0.02	25.50 ± 0.04	27.13± 0.03	25.83 ± 0.01	19.39 ± 0.04	20.85 ± 0.06	20.55 ± 0.04
Total MUFA	41.90 ± 0.04	41.26 ± 0.03	41.59 ± 0.06	42.23 ± 0.02	41.70 ± 0.05	42.18 ± 0.06	42.62 ± 0.06	42.86 ± 0.01	42.72 ± 0.07
Total PUFA	32.95 ± 0.06	31.71 ± 0.02	32.46 ± 0.03	31.27 ± 0.01	30.17 ± 0.02	30.99 ± 0.03	36.99 ± 0.03	35.29 ± 0.08	35.73 ± 0.08
PUFA/SFA	1.36 ± 0.02	1.21 ± 0.05	1.30 ± 0.03	1.22 ± 0.06	1.11 ± 0.04	1.19 ± 0.05	1.90 ± 0.06	1.69 ± 0.05	1.83 ± 0.08

^aSFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

^bValues are expressed as mean ± standard deviation of three replicates for each parameter.

PV and TBARS test measured in regular time intervals allowed monitoring the oxidative stability of fats. TBARS test measured the formation of secondary oxidation products such as aldehydes and carbonyl compounds, which contribute to the off flavor characteristic of oxidized products.^[23] It was observed that PV and TBARS values gradually increased in all fat samples while IV decreased during storage at both temperatures. The decrease in IV values indicates a reduction of fat unsaturation that was considerably lower at –20°C than at +4°C during storage.

The changes in PV, TBARS, and IV values of goose fat during storage at +4°C and –20°C, and after the addition of 0.1% α-tocopherol and 0.1% citric acid are presented in Table 1. There are limits for quality and acceptability of fats for human consumption: for PV, the limit of acceptability is 10 meq O₂/kg of fat, and for TBARS value, this limit is 8 mg MDA/kg of fat.^[19] PV of goose fat with 0.1% α-tocopherol and 0.1% citric acid reached the acceptability limit for human consumption in 360 days during storage at +4°C, while during storage at –20°C the acceptability limit was reached in 450 days for goose fat with citric acid. Goose fat with α-tocopherol stored at –20°C did not exceed the acceptability limit within 480 days. TBARS values and peroxide index values in goose fat were significantly correlated with the storage time (r = 0.84 and r = 0.87, respectively; p < 0.001). PV and TBARS test increased with time, and the differences in PV and TBARS values were also increased with storage temperature as storage time advanced.

The addition of natural antioxidants in proportion of 0.1% extended the shelf life of goose fat stored at +4°C by 90 days; for goose fat stored at –20°C, citric acid prolonged the shelf life by 150 days, while goose fat with α-tocopherol could be stored for more than 480 days at –20°C without spoilage. The PV, TBARS, and IV values were significantly influenced (p < 0.001) by the storage temperature and time. Antioxidant application had a statistically significant (p < 0.01) effect on the PV, TBARS, and IV of the goose fat samples.

Elhamirad and Zamanipoor^[12] investigated the thermal stability of phenolic antioxidants in tallow olein at 120, 140, 160, and 180°C. The researchers suggested that quercetin and ellagic acid had the highest thermal stability, while gallic acid and caffeic acid exhibited the least thermal stability. Gramza et al.^[13] examined the antioxidative activity of water and ethanol extracts of green and black tea leaves against the oxidation of lard and heated sunflower oil. The authors reported that the highest antioxidant activity, measured as an induction period, with 1000 ppm green tea ethanol extract, was comparable to α-tocopherol activity in sunflower oil, and in lard, the longest induction period was measured with 500 and 1000 ppm of green tea ethanol extract. The study also showed an influence of epicatechin gallate, epicatechin, and catechin contents in the tea extracts on the antioxidant activity in lipids. The effect of temperature on the antioxidant activity of α- and δ-tocopherol in pork lard using the Oxipres apparatus was also studied.^[15] The research showed that the activity of α-tocopherol was constant in the temperature range from 80 to 110°C and decreased with increasing temperature; both tocopherols were ineffective at 150°C; the δ-tocopherol activity was two times higher than the α-tocopherol activity at 80°C and their activities were the same at 130°C.

TBARS values increased significantly with storage time and temperature in duck fat (Table 2). In a similar study,^[14] TBARS values were also reduced in butter stored at –20°C, by the addition of BHA, BHT, and α-tocopherol. The lowest TBARS values of fats treated with antioxidants were determined for duck fat, and those values were considerably lower at –20°C. TBARS values and peroxide index values in duck fat were significantly correlated with the storage time (r = 0.73 and r = 0.79, respectively; p < 0.001). During the storage, the TBARS and PV values of the fat samples increased at both storage temperatures; the biggest increase occurred in fats stored at +4°C compared to –20°C, and the rate of increase was lower during frozen storage. TBARS values of duck fat with α-tocopherol and citric acid reached the acceptability limit for human consumption in 360 days during storage at +4°C, while for duck fat with both antioxidants stored at –20°C did not exceed the acceptability limit within 480 days. The PV, TBARS, and IV values were significantly influenced (p < 0.001) by the storage temperature and time.

The addition of α-tocopherol and citric acid in proportion of 0.1% extended the shelf life of duck fat stored at +4°C by 90 days, while duck fat with both antioxidants could be stored for more than 480 days at –20°C without spoilage. PV and TBARS analysis revealed that the oxidation was initially low and increased rapidly in the control samples during storage; however, a much slower rate of increase in oxidation was observed in samples treated with a natural antioxidant.

Ozturk and Cakmakci^[14] have compared the effects of natural and synthetic antioxidants on the oxidative stability of butter. The study reported that butter samples with 50 ppm antioxidant could be stored for more than 180 days at 4°C, and both synthetic and natural antioxidants were capable of protecting the butter samples against oxidation during storage at +4°C and –20°C. The ability of some phenolic compounds to inhibit butter oxidation was evaluated.^[16] The researchers have monitored the PVs and TBARS during storage of butter at 50°C and at 110°C. They found that gallic acid, caffeic acid and catechin, each at 80 mg/L, inhibited butter oxidation at 50°C to a degree equal to that of butylated hydroxyanisole at 200 mg/L and gallic acid, at 80 mg/L, was more effective than butylated hydroxyanisole, at 200 mg/L, in inhibiting butter oxidation at 110°C. Asha et al.^[17] evaluated the antioxidant activities of butylatedhydroxyanisole and orange peel powder extract in ghee (butter oil) stored at different temperatures (6; 32; 60°C) during a period of 21 days. The study showed that ghee incorporated with orange peel extract showed stronger activity in quenching radicals and least development of PV, TBARS, and free FAs than ghee incorporated with butylatedhydroxyanisole and control.

The changes in PV, TBARS, and IV values of chicken fat during storage at +4°C and –20°C, and after the addition of 0.1% α-tocopherol and 0.1% citric acid are presented in Table 3. Iodine index values of chicken fat were significantly influenced by storage temperature and time. The highest levels of IV were found in chicken fat, followed by goose and duck fat. The highest level of PV was observed in chichen fat stored at +4°C. Regardless of the type of fat and storage temperature, the highest PV level was found at 480 days of storage. TBARS values and peroxide index values in chicken fat were significantly correlated with the storage time (r = 0.93 and r = 0.98, respectively; p < 0.001). PV of chicken fat with added antioxidants reached the acceptability limit for human consumption in 300 days during storage at +4°C, while during storage at –20°C the acceptability limit was reached in 420 days. The PV and TBARS values of the antioxidant-containing samples were significantly lower (p < 0.01) than those of the control group. The highest PV was observed in the control samples, followed by 0.1% citric acid and 0.1% α-tocopherol for all poultry fats and the changes were more pronounced at +4°C than at –20°C. Antioxidant application had a statistically significant (p < 0.01) effect on the chemical parameters of the chicken fat samples.

The addition of natural antioxidants has extended the shelf life of chicken fat stored at +4°C by 120 days, and of chicken fat stored at –20°C by 180 days. Among the fat samples with added antioxidants it was observed that the largest increase in PV and TBARS values occurred in chicken fat for both storage temperatures. In general, the TBARS and PV levels of the samples treated with natural antioxidants were lower than the control, especially for the research at –20°C. During frozen storage, the increases in PV and TBARS of the samples treated with antioxidants were not significantly different from each other. With respect to storage time, the oxidative rancidity of the fat samples increased during the storage period. α-Tocopherol and citric acid significantly inhibited lipid oxidation in poultry fats as indicated by PV and TBARS.

Selmi et al.^[18] studied the stability of sardine oil stored at +4°C and +35°C for 28 days with or without the addition of α-tocopherol (50 and 100 ppm). The study reported that PVs and TBARS of control sardine oil significantly increased to 29.9 meq O₂/kg oil and 46.48 mg MDA/kg during storage at +35°C, but the increase was considerably less (4.36 meq O₂/kg oil and 13.21 mg MDA/kg oil, respectively) in oil stored at +4°C. The authors concluded that storage at +4°C combined with the addition of α-tocopherol in concentration of 100 ppm had a beneficial effect on sardine oil stability. Zuta et al.^[19] investigated the effect of α-tocopherol added in concentrations of 50, 100, 250, and 500 ppm on the oxidation of unrefined mackerel oil sored at +4 and –40°C over a period of 66 days. The researchers concluded that the higher concentrations of α-tocopherol, were less effective in controlling oxidation in the oils than lower α-tocopherol levels; the treatment of mackerel oil with 50 or 100 ppm α-tocopherol at –40°C produced the lowest oxidation over the 66 day period and was considered as adequate levels of incorporation in crude mackerel oil to minimize oxidation.

Delles et al.^[24] demonstrated that dietary antioxidant supplementation can minimize the negative impact of oxidized oil on the quality of broiler meat packaged in different atmospheric environments. Botsoglou et al.^[25] showed that dietary olive leaves were more effective than oregano at inhibiting lipid oxidation of turkey breast fillets during refrigerated storage, but were inferior to dietary supplementation of α-tocopheryl acetate.

In this study, it was found that chicken fat was the most sensitive to deterioration and showed the lowest oxidative stability among the examined fat samples. Samples of fats with 0.1% α-tocopherol stored at +4°C preserved acceptable characteristics up to 360 days for goose and duck fat, 300 days for chicken fat, while during frozen storage the acceptability tolerance was found to be 420 days for chicken fat; goose and duck fat could be stored for more than 480 days without spoilage. Samples of fats with 0.1% citric acid stored at +4 °C preserved acceptable characteristics up to 360 days for goose and duck fat, and 300 days for chicken fat, while during storage at –20°C the acceptability tolerance was found to be 420 days for chicken fat and 450 days for goose fat; duck fat could be stored for more than 480 days without spoilage. The addition of antioxidants had a statistically significant (p < 0.01) effect on the PV, TBARS, and IV values of the poultry fat samples.

The total lipids of the fresh fat samples showed a predominance of monounsaturated FAs (MUFAs) and PUFAs. The highest content of oleic (18:1) and linoleic (18:2) acids was determined in chicken fat, followed by the goose and duck fat (Table 4). The highest level of PUFAs was found in chicken fat followed by the goose and duck fat, and the highest level of MUFAs was noticed in chicken fat, followed by the duck and goose fat. Saturated fatty acids (SFAs) content increased during both storage temperatures for poultry fats treated with α-tocopherol by 480 days of storage. Storage time showed to be most correlated with MUFA for chicken (r = 0.94), followed by the duck (r = 0.88) and goose fat (r = 0.71).

In all fat samples, the PUFA/SFA ratio was within the recommendations (>0.4) and the decrease of PUFA, in contrast to SFA, led to no significant decrease (p ≥ 0.05) in this ratio during the process of frozen storage. The total PUFA was significantly affected by storage temperature in poultry fats treated with antioxidants (to a greater extent at +4°C than at –20°C). Taking into account that in chicken fat stored at +4°C was found the highest level of PV, the decrease of total PUFA during storage may be due to the oxidation of PUFAs. Linoleic acid (C18:2) significantly decreased in chicken fat with α-tocopherol stored under refrigeration (with 1.16 units) and freezing (with 0.91 units). Linolenic acid (C18:3), also decreased significantly in chicken fat with α-tocopherol stored under refrigeration (with 0.16 units) and freezing (with 0.13 units).

Analysis revealed an effect of the storage temperature on the overall FA composition of fats treated with α-tocopherol by 480 days of storage compared with fresh fats. A significant difference (p < 0.05) in total FAs was registered by 480 days of storage at +4°C, and no significant differences (p ≥ 0.05) were registered by 480 days of storage at –20°C compared with control. Variation in FA composition of different animal fats could be related to storage temperature, frozen storage preserving their acceptability properties. Zeb and Murkovic^[26] reported that PUFA autoxidation rate depends not only on their unsaturation degree, but also on the position of the FAs in the triacylglycerols. It has been reported that stability of triacylglycerols was compromised when EPA was highly concentrated within rather than between the triacylglycerol molecules.^[27] The researchers concluded that a higher oxidative stability was achieved when unsaturated FAs were at sn-2 position, demonstrating that the type of the unsaturated FAs and substrate also influence the oxidation. Jankowski et al.^[28] determined the effect of diets with a different n-6/n-3 PUFA ratio (7.31, 4.43, and 0.99), resulting from the addition of different dietary oils: soybean, rapeseed, and linseed on the FA profile, oxidative status and sensory properties of turkey breast meat. The researchers showed that breast meat of turkeys fed with linseed oil was characterized by higher concentrations of total PUFA, a significantly lower n-6/n-3 PUFA ratio and a higher TBARS content, and after 4 months of deep-freeze storage the n-6/n-3 PUFA ratio did not deteriorate. The natural antioxidants (α-tocopherol and citric acid) were effective antioxidants and provided good protection against oxidation of poultry fats; they can be used to monitor the oxidation of fats and to predict their shelf life stability. The results also showed that the storage temperature had important effects on the storage stability of poultry fats.

Conclusions

Lipid oxidation in poultry fats varied with the temperature and the duration of storage; lower temperature had a greater retardation effect on rancidity than that occurring at higher temperatures, especially during longer storage periods. The PV and TBARS values of the antioxidant-containing samples were significantly lower (p < 0.01) than those of the control group. Sample of fats with 0.1% α-tocopherol stored at +4°C preserved acceptable characteristics up to 360 days for goose and duck fat, 300 days for chicken fat, while during frozen storage the acceptability tolerance was found to be 420 days for chicken fat; goose and duck fat could be stored for more than 480 days without spoilage. Sample of fats with 0.1% citric acid stored at +4°C preserved acceptable characteristics up to 360 days for goose and duck fat, and 300 days for chicken fat, while during storage at –20°C the acceptability tolerance was found to be 420 days for chicken fat and 450 days for goose fat; duck fat could be stored for more than 480 days without spoilage. Statistical analysis of the data revealed that the development of rancidity in poultry fats was significantly (p < 0.01) reduced by the addition of α-tocopherol and citric acid in concentration of 0.1%, but α-tocopherol resulted in a better protection than citric acid.