Thermal Stability of Total Lipids Extracted from Zlatibor Bacon

Abstract

In this study, non-oxidative and oxidative thermogravimetric analysis was applied as a method for determining the thermal stability of the total lipids extracted from Zlatibor bacon in different phases of production (raw samples, 10th and 30th day of smoking and 30th day of storage). The activation energies of the thermal degradation and oxidation of the total lipids extracted from examined samples were determined. A moderate increase of the activation energy, as well as the other information gained from TG analyses, indicated that there were no significant oxidative changes in Zlatibor bacon lipids during the production and storing.

Keywords:

• Zlatibor bacon
• TG analysis
• Lipids
• Peroxidation

INTRODUCTION

Zlatibor bacon is one of the shelf-stable meat products, traditionaly manufactured in the area of Zlatibor mountain (southwest of Serbia), during the winter months (November–February). The characteristic of the Zlatibor bacon is exceptional organoleptic qualities, one of them being the aroma, which is very pleasant and specific for this type of product. Besides all other quality factors, nutritive and sensory characteristics of meat products, such as Zlatibor bacon, depend a great deal of the stability of their lipids.[1– 4]

Lipid oxidation is one of the most important degradation processes in which derived products cause alteration of the quality attributes of meat and leads to rancidity in meat, which is associated with the development of warmed-over flavor in meat.[5, 6] Besides changes in sensory quality, lipid oxidation causes a reduction of the nutritive value of meat as a result of oxidation of essential fatty acids[7, 8] and vitamins.[9] Susceptibility of meat to lipid oxidation depends on several factors, such as the unsaturated fatty acid content[10, 11] the fatty acid composition of lipid fractions,[12, 13] and the balance between antioxidant/prooxidant factors.[14, 15] Polyunsaturated fatty acids and oxygen in the presence of catalysts, heat, light, high energy radiation, metal ion, or metalloproteins[16, 17] are degradated via formation of free radicals, causing flavor, texture, color, and nutritional deterioration of muscle and fat foods.[18– 21] Lipid oxidation is thought to promote the oxidative damage of proteins through the prooxidant activity of primary (hydroperoxides) and secondary (aldehydes, ketones) lipid oxidation products.[22] Thus, lipid oxidation is the major process by which nutritional and sensory quality traits decline in meat and meat products.

It is well known that smoking of meat and its products is one of the oldest methods for preservation. Smoke has bactericidal and antioxidative effects due to the presence of phenol, volatile acids, carbonyl compounds, and other volatile products, the carriers of the aroma.[23– 26] It is probable that salt- and phenol-type components of smoke, which function as antioxidants, inhibited the oxidation of the lipids with the samples in the smoking phase. A lipophilic character of the phenols should favor their penetration into lipids and consequently their antioxidative effect. The metoxyphenols with an alkenyl or an alkyl side-chain appear to possess both a higher antioxidant activity and a higher lipophilicity compared to the carbonyl-containing methoxyphenols.[27] Most of the polyhidroxyphenols exhibit a chain-bracing effect during radical autooxidation.[28] Ahmad et al.[29] observed that curing, sodium ascorbate, and smoking treatments of buffalo meat was found to be most effective in increasing the shelf life of the controlled meat sample.

Thermogravimetric analysis (TGA) is well established as a routine analytical tool for the study of thermal behavior of different materials. TGA of lipids is an appropriate analytical method commonly used to correlate the kinetic parameters of thermal degradation of lipids and their composition. With TG analysis, it is possible to estimate lipids' resistance to oxidation, measure the mass gain due to oxygen caption of the lipid sample during oxidation, and the initial and final oxidation temperature. By application of these principles, Cross,[30] Hassel,[31] and Coni et al.[32] determined vegetable oil stability and resistance to oxidation. Buzas and Kurucz[33] developed a simple and fast method, suitable for investigating the oxidative stability and oxidative state of edible oils, as well as storage time.

Skala et al.[34] studied the behavior of white meaty hog intramuscular lipids during thermal treatment and storage. Non-isothermal non-oxidative and oxidative TG analysis was applied to investigate the rate of oxidation and the evolution of volatile compounds of experimental samples. The kinetics of the oxidation of intramuscular lipids were determined on the basis of the results obtained by this method during thermal treatment and storage. The effects of heat-induced changes of intramuscular connective tissue and collagen on meat texture properties of beef semitendinosus muscle were investigated in the study by Chang et al.[35] The instrumental texture profile analysis data of heated meat also showed significant differences between two heating modes and studied temperatures.

Skala et al.[36] investigated changes in the lipids of muscle and fatty tissue of different games. The authors established the correlation of kinetic parameters of thermal degradation (activation energy) and lipid composition. Milovanovic et al.[37] also used the game tissues for research. The authors determined the influence of gender, age, storage conditions, and maturing on the thermal stability of the total lipids of intramuscular tissue of fallow deer (doe and deer). They concluded that TG is an appropriate method for detecting fine structural changes in lipids. Many researches of Litwinienko et al.[38– 40] concerned applications of thermal analysis in the investigation of autooxidation of fats, edible oils, and lipids. This process is exothermic and methods of thermal analysis are valuable for studying thermostability, thermooxidation, and autooxidation. Differential scanning calorimetry (DSC)[41] was used to obtain specific heat, heat of fusion, and protein denaturation temperatures of raw skipjack tuna. Apreutesei et al.[42] determined the thermal stability of cholesterol and cholesterol esters, respectively, using a simultaneous recording of thermogravimetric, derivative thermogravimetric, and differential thermal analysis in static air.

In view of the fact that lipid oxidation is a major process by which nutritional and sensory quality characteristics decline in meat products, the aim of this study was to examine thermal stability of the total lipids extracted from Zlatibor bacon during different phases of production by applying oxidative and non-oxidative TG analysis. In the interest of better understanding and confirmation of the observed TG effects, the fatty acid composition of the same samples of lipids were also determined.

MATERIALS AND METHODS

Preparation of Sample

Zlatibor bacon was produced in the Čajetina meat industry, in the city of Čajetina. Shaped bars of raw bacon were salted and “coldly” smoked at the temperatures of 8–10°C (max 12°C) during a 30-day period. On finishing the process of smoking, the bacon was stored in a dry room at the temperature of 10°C for the next 30 days. The following samples of Zlatibor bacon were used for thermogravimetric analysis and fatty acids analysis: raw samples (R), 10th and 30th day of smoking—the end of production (10Sm, 30Sm), and 30th day of storage (30St).

Extraction of Lipids

The total lipids were extracted according to the Folch method.[43] The hydrolysis of total lipids was performed in sodium methanoate solution, while the separated fatty acids were extracted by ethyl ether. The fatty acids were converted to their respective methyl esters by reaction transesterification with three methylsulfonium hydroxide according to method SRPS ISO (2007).

Gas Chromatography

Analysis of fatty acid methyl esters (FAME) was performed using a gas chromatograph (Varian 3400, Varian, USA). Nitrogen was used as the carrier gas with a flow rate of 1.33 ml/min and 1:50 split ratio. The temperature of furnace of the column was programed in the range of 125 to 230°C. The total time of analyses was 50.5 min. The injector temperature was 250°C, while the temperature of the detector was 280°C. Identification of FAME was based on retention times of fatty acid standards Supelco 37 Component FAME Mix, 10 mg/ml in CH₂Cl₂ (47885-U; Bellefonte, PA, USA). Fatty acid composition was expressed as percent of total fatty acid methyl esters, using a Spectra-Physics System I integrator (Varian, USA).

Thermal Analysis

Thermogravimetric (TG) analysis of total lipids was performed on a Perkin-Elmer TGS-2 instrument (Perkin Elmer, USA), at heating rates of 2.5°C min⁻¹ and constant gas flow rate (inert atmosphere–nitrogen and oxidative atmosphere–air) of 25 cm³ min⁻¹. The initial sample masses were 10 mg ± 10%. The mass changes of the total lipids were monitored in the temperature interval 30–220°C.

Kinetic Analysis of the Oxidation and Degradation Processes of Total Lipids

The Doyle-Gorbachev method[44, 45] was applied to determine the activation energies of thermal degradation and oxidation of the total lipids of Zlatibor bacon. In order to investigate the non-oxidative thermal degradation of the total lipids, an analogy between oxidation and homogeneous chemical reactions in the gaseous and liquid phase was introduced. Thus, the oxidation rate may be presented by Eq. (1):

(1)

where k is the rate constant of thermal degradation defined by the Arrhenius equation, while ϕ(m) is a function dependent on the concentration of the component susceptible to thermal degradation. The activation energies of thermal degradation and oxidation of the total lipids of the Zlatibor bacon, were determined on the basis of non-isothermal TG curves at dynamic nitrogen and oxygen atmospheres, by the Doyle–Gorbachev integral method.[44, 45] The accuracy of this method depends on the scale of the activation energy investigation and the temperature range. Thus, homogeneous samples, lower sample weights, and a relatively slow heating rate were selected to diminish the effects of the experimental conditions. The kinetic parameters were calculated using Eq. (2), according to the Doyle–Gorbachev method:

(2)

If the term 2RT E ≪ 1, Eq. (2) transforms into:

(3)

The value of the activation energy corresponds to the slope of the linear dependence 1/T versus −ln.

Statistical Analysis

In order to determine the effect of different phase production of Zlatibor bacon on a fatty acid composition of total lipids on each phase of study (raw sample [day 0], smoked sample [10 day and 30 day], and stored sample [30 day]), a one-way analysis of variance (ANOVA) was applied. Tukey's b posteriori test was used to determine significant differences (p < 0.05) among the contents of saturated, monounsaturated, and polyunsaturated fatty acids of total lipids in different phases of manufacturing of the Zlatibor bacon.

RESULTS AND DISCUSSION

The effects of the manufacturing process of Zlatibor bacon on the thermal stability of the total lipids analyzed in nitrogen and air atmosphere, at a heating rate of 2.5°C min⁻¹ are shown in Figs. 1 and 2. In the charts, one can observe a small decrease of mass in inert atmosphere compared to samples in an air-stream and it is in the range of 0.4–1.75%. Furthermore, a stable state (insignificant change in mass) was noticed with the samples in the process of smoking in the stream of nitrogen and, in other words, the sample smoked for 30 days (30Sm) and one smoked for 10 days (10Sm) are similar in weight losses. Comparing all the samples, the largest change of mass was detected with the raw sample (R) regardless of the atmosphere in which the analysis was performed. The sample R, since it is raw, was free of preservatives (which originate from the smoke) that would inhibit the reactivity of the oxygen, resulting in the beginning of the oxidation process and thermal degradation happening at a lower temperature.

Figure 2 The oxidative TG curves of the total lipids extracted from the investigated samples^a of Zlatibor bacon (heating rate 2.5°C min⁻¹, gas flow rate 25 cm³ min⁻¹). ^aR (raw sample); 10Sm (10 days of smoking); 30Sm (30 days of smoking); 30St (30 days of storing).

Figure 1 The non-oxidative TG curves of the total lipids extracted from the investigated samples^a of Zlatibor bacon (heating rate 2.5°C min⁻¹, gas flow rate 25 cm³ min⁻¹). ^aR (raw sample); 10Sm (10 days of smoking); 30Sm (30 days of smoking); 30St (30 days of storing).

The activation energies of the non-oxidative and oxidative thermal degradation of the total lipids of Zlatibor bacon are shown in Table 1. These energies were determined in a mass range from 0.25 to 3.75%. Furthermore, activation energy should be observed as a parameter that shows the sensitivity of the examined sample to thermic changes (heating). It means that the higher the activation energy value is, the faster the change in mass under the influence of the temperature is, i.e., the lipids are more susceptible to the process of oxidation.

Table 1 The activation energies (Ea) of non-oxidative and oxidative thermal degradation of total lipids of Zlatibor bacon, kJ/mol

Sample	Ea (N2, 2.5^o/min) kJ/mol	Ea (O2, 2.5^o/min) kJ/mol
R	40.5	42.4
10Sm	42.1	42.7
30Sm	42.7	41.7
30St	43.6	43.9

The values of the activation energies (Ea) of the samples heated in the air-stream are somewhat higher than the measured activation energies of the same samples heated in inert atmosphere. According to the findings of Bastic,[46] activation energies' values of the process of the thermal degradation of the boar's intramuscular lipids are 57.9 and 61.5 kJ/mol and they refer to the samples heated in the nitrogen or oxygen stream. The same author also concluded that in higher temperatures (above 130°C) increased thermal degradation and auto-oxidation of total lipids occurs, which is especially true for oxygen stream when samples are more prone to the process of oxidation. Measured values for activation energies (Ea) of the total lipids samples of Zlatibor bacon heated in nitrogen stream (40.5–43.6 kJ/mol) and for those heated in air stream (42.4–44.9 kJ/mol) were somewhat smaller than the author's findings.[46] Thus, a moderate increase of the activation energy, determined in this research, shows that total lipids of Zlatibor bacon did not suffer significant change during the process of smoking and storage.

In the interest of confirmation of the observed TG effects, the fatty acid composition of the same samples of lipids were also determined. The results shown in Table 2 were obtained by gas chromatography of the total lipids of fatty tissue of Zlatibor bacon. Based on the corresponding standards, the presence of saturated fatty acids (SFA) (C14:0, C16:0, and C18:0) and unsaturated (C16:1, C18:1, C18:2, C18:3, C20:1, C20:2, C20:3, C20:4) fatty acids was confirmed. The SFA consisted mainly of palmitic (65.3–65.8% of ΣSFA) and stearic acid (˜31% of ΣSFA), whereas the MUFA was mostly composed of oleic acid (88.5–88.8% of ΣMUFA) and PUFA of linoleic acid (81.5–83.1% of ΣPUFA). Within the four groups of the sample (R, 10Sm, 30Sm, 30St), the contents of saturated fatty acids (SFA) were different than those of MUFA, while their PUFA contents varied between 11.6 and 12.6% of total fatty acids. Statistical analysis of contents SFA, MUFA, and PUFA showed that statistical significant difference (p < 0.05, t-test), exist in contents of SFA and PUFA between raw sample (R) and the other samples, as well as in contents of MUFA between the smoked sample (10Sm) and the other samples.

Table 2 Fatty acid content of the total lipids of the Zlatibor bacon in different manufacturing phase, mean, and standard deviation, % = % of total fatty acids. ^a

	Row sample	Smoked samples		Storage samples
Fatty acid	0 days	10 days	30 days	30 days
C14:0 Tetradecanoate	1.38 ± 0.05	1.21 ± 0.05	1.19 ± 0.05	1.19 ± 0.03
C16:0 Hexadecanoic	24.85 ± 0.07	24.57 ± 0.10	24.48 ± 0.20	24.46 ± 0.11
C16:1 cis-9-Hexadecenoic	2.18 ± 0.11	2.14 ± 0.13	2.14 ± 0.11	2.15 ± 0.11
C18:0 Octadecanoic	11.84 ± 0.08	11.59 ± 0.15	11.56 ± 0.10	11.59 ± 0.16
C18:1 cis-9-Octadecenoic	44.67 ± 0.11	44.16 ± 0.13	44.71 ± 0.11	44.71 ± 0.07
C18:1 cis-7-Octadecenoic	2.69 ± 0.10	2.56 ± 0.17	2.65 ± 0.10	2.69 ± 0.05
C18:2 n-6 Linoleic	9.61 ± 0.07	10.33 ± 0.06	10.21 ± 0.14	10.14 ± 0.09
C18:3 n-3 Linolenic	0.268 ± 0.03	0.301 ± 0.03	0.283 ± 0.031	0.282 ± 0.026
C20:1 Eicosenoic	0.91 ± 0.05	0.882 ± 0.092	0.902 ± 0.044	0.798 ± 0.057
C20:2 Eicosadienoic	0.588 ± 0.024	0.785 ± 0.060	0.763 ± 0.066	0.733 ± 0.058
C20:3 n-6 Eicosatrienoic	1.002 ± 0.116	1.078 ± 0.077	1.193 ± 0.114	1.16 ± 0.08
C20:4 Arachidonic	0.100±0.005	0.103± 0.010	0.103± 0.010	0.127± 0.022
ΣSFA	38.05±0.14 b	37.36±0.09 a	37.10±0.25 a	37.26±0.06 a
ΣMUFA	50.45±0.19 a	49.72±0.24 b	50.40±0.10 a	50.35±0.04 a
ΣPUFA	11.57±0.13 b	12.57±0.14 a	12.55±0.12 a	12.44±0.08 a
^aValues in the same row bearing different letters are significantly different (p ≤ 0.05).
SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

Results of fatty-acid contents of the Zlatibor bacon total lipids (Table 2), in which there were no significant differences between final product (30Sm) and stored samples (30St, 10°C), showed that thermal stability of lipids exist, which are in accordance with results of TG analysis, and confirmed these results. The reason of lipids thermal stability lies in the fact that “cold” smoke was used (≈10°C), which means that the sample was not exposed to high temperatures in the process of smoking, which was more a process of drying. Furthermore, the bacon was kept in a dry and cold room, without light, at the end of the smoking process (namely at the end of production process), which means that the possibility for lipids oxidation was minimized.

CONCLUSIONS

TG analysis assisted successfully in thermal stability of lipids and quality evaluation of meat products. This applicability was confirmed by examination of the total lipids samples of Zlatibor bacon taken after different phases of treatment. The non-isothermal TG analysis of the total lipids was performed in nitrogen and air in a selected temperature range. The registered mass losses of the samples in nitrogen were somewhat smaller than the one in the air atmosphere. A moderate difference between Ea samples heated in inert atmosphere (40.5–43.6 kJ/mol) and in the air stream (42.4–44.9 kJ/mol) confirm the stability of lipids. Small differences between Ea values (heated in the air stream), particularly between smoked (final product) and stored samples (30 days, 10°C), as well as between contents of SFA, MUFA, and PUFA in the noted samples, also predict the stability of lipids, namely, long shelf life and, therefore, the quality of Zlatibor bacon if kept in adequate conditions.