3.1. Chemical Composition Analysis
Proximate composition of grilled burgers on the 1st and 6th day of storage is shown in
Table 2.
The burgers with olive oil (OO) showed the highest moisture value on day 1 (54.7%) whereas the canola oil group (CO) had the lowest water content value (49.1%) (p < 0.001). On day 6, the control group with reduced fat content had the highest water content (59.25 ± 0.04%) and burgers from the groups with sunflower oil (51.26 ± 0.15%) and linseed oil (51.83 ± 0.30%) had the lowest. Most of the groups experienced statistically significant (p ≤ 0.05) reduction in moisture content during storage. The highest fat content on day1 was present in samples with sunflower oil (15.27 ± 0.26%), the lowest was recorded in samples belonging to CON_LOW FAT (12.16 ± 0.09%), OO (12.23 ± 0.16%) and MF (12.01 ± 0.11%) groups. On day 6, statistically significantly (p ≤ 0.05) the lowest fat content was observed in the reduced fat control group (10.28 ± 0.16%). At the same time, the highest protein content, at 30.37 ± 0.15%, was observed in this group. Burgers with linseed oil contained the lowest proportion of protein in the proximate composition, which was 26.21 ± 0.26%. When analyzing the salt content of the burgers, the highest salt content was found in the control group (1.42 ± 0.04%) and the lowest salt content was found in the burgers with sunflower oil (0.45 ± 0.02%) and canola oil (0.61 ± 0.02%). A statistically significant (p ≤ 0.05) reduction in the percentage salt content of the samples was observed in most groups during storage. The samples with milk fat (MF) contained the lowest level of connective tissue on day 1 and day 6 (day 1 3.60 ± 0.06%; day 6 1.82 ± 0.09%) and the samples from CON group contained the highest level. As for the connective tissue content, its percentage decreased statistically significantly (p ≤ 0.05) during storage in each group.
The proximate composition is a very important parameter because it can directly affect the physical properties of burgers (springiness, chewiness, hardness), fatty acid profile and PAH formation during grilling [
16]. According to studies conducted by Trujillo-Mayol et al. [
17], burger composition also determines the amount of heterocyclic aromatic amines and acrylamide formed during burger frying. A key parameter affecting the amount and profile of PAHs is the total fat content, as confirmed by Wongmaneepratip and Vangnai [
18] in their study. The analysis of water content led to the conclusion that in low-fat burgers and those containing pre-emulsified vegetable oils, moisture retention is attributed to the stabilizing effect of oil in an established emulsion system, as confirmed and reported in the study by Afshari et al. [
8]. The significantly lower protein content in burgers with vegetable oil (CO, LO, OO) was due to the partial substitution of beef tallow. Scientific reports indicate a significant protein content in animal fat and the results obtained are confirmed in the literature [
19,
20]. The diversity between the groups in terms of fat content was related to differences in the fatty acid profile, technological properties and production method of the beef tallow substitutes used. Ingredients available in local stores were used in the experiment. Canola oil and sunflower oil as a partial substitute of beef tallow resulted in increased fat content in the samples. These oils are commonly used in the preparation of heat-treated foods, which is associated with a properly adapted production process to ensure the desired technological properties (high thermal hydrolysis point temperature). Olive oil and linseed oil, used in meals consumed without heat-treatment, are characterized by different properties, which led to a reduced fat content in the samples compared with the control, CON_LOW FAT, SO and CO groups, which could be explained by increased fat leakage during thermal processing.
3.2. Color Measurement
The CIE L*a*b* color components for raw and grilled burgers on the 1st and 6th days of storage are shown in
Table 3. It was found that 6 days of storage had no statistically significant (
p > 0.05) effect on the color change in burgers, either raw or grilled. Among raw burgers, the value of parameter L* (darkest) was lowest for samples from the control group (day 1 46.65 ± 2.18) and the control group with reduced fat content (day 1 47.66 ± 2.33). These values were statistically significantly (
p ≤ 0.05) lower than in samples where beef tallow was partially replaced with other fats. Among all groups of raw burgers, the highest value of parameter L* (brightest) was recorded for samples with sunflower oil (day 1 52.73 ± 2.92). The parameter a* of raw burgers did not differ statistically significantly, neither between 1 and 6 days of storage, nor between the test groups with different recipes. The lowest values of the parameter b* were found in samples of the control group (day 1 10.46 ± 1.85) and the reduced-fat control group (day 1 13.15 ± 2.12). The parameter b* differed statistically significantly (
p ≤ 0.05) between the control groups, whereas no statistically significant differences were observed between the remaining test groups, of which the highest value of the parameter b* was found for the sample with linseed oil (day 1 16.41 ± 1.64). In Foggiaro et al.’s [
21] study, pork back fat in burgers was replaced by walnut and algal oil mixture hydrogel or pistachio and algal oil mixture hydrogel. The change in recipe resulted, as in the present study, in a significant increase in the values of the L* and b* parameters in the raw burgers [
21]. The increased proportion of the b* component is probably due to natural differences between vegetable fats and milk fat, characterized by their yellow color, and beef tallow, which has a color more similar to white. A higher proportion of the yellow component may also be related to a higher degree of lipid oxidation in samples with vegetable oils [
22,
23].
The highest value of parameter L* of heat-treated burgers occurred in the samples from the control group (day 1 = 35.32 ± 0.87) and samples with olive oil (day 1 = 34.17 ± 1.67). The lowest value of the parameter L* was recorded for the samples with sunflower oil (day 1 = 28.09 ± 2.77) and milk fat (day 1 = 27.64 ± 2.08). The parameter a* reached the lowest values in samples from the control group with sunflower oil and milk fat (7.26–7.30 on day 1), and these values did not differ statistically significantly (
p > 0.05). The SO (day 1 8.51 ± 2.04) and MF (day 1 8.72 ± 1.29) groups had the lowest value of component b*, whereas the CON group had the highest value (11.94 ± 0.62 on day 1 and 11.69 ± 0.56 on day 6). The results showed that the reduction in beef tallow content or its partial replacement with other fat affects the color of raw and grilled burgers. The lower value of L* and b* parameters in grilled burgers in which animal fat was replaced by another ingredient is confirmed by a study by Botella-Martinez et al. [
24] where 50% or 100% of the animal fat in the samples was replaced by a gelled emulsion elaborated with cocoa bean shell flour and walnut oil. Similar conclusions were reached in the study by Wongmaneepratip and Vangnai [
18], where the color of marinated and then grilled chicken breasts was measured. The addition of palm oil and sunflower oil to the marinades resulted in a significant change in color components L* and a* compared with the control group. The results obtained in this study are comparable to the burger color presented by Heck et al. [
25] The authors also confirmed that heat treatment contributes to the reduction in L* and a* color components. A study conducted by Szpicer et al. [
7] confirmed that the addition of canola oil to beef burgers causes a significant decrease in the parameter b* (
p < 0.05). The increase in parameter L* in samples with vegetable oils can be explained by the fact that meat emulsions with such oils had a much smaller oil globules, which reflect more light (have larger surface area) than larger animal fat globules. Similar conclusions were reached in the studies by Youssef and Barbut [
26] and Selani et al. [
27]. A similar relationship was obtained in their study by Rodríguez-Carpena et al. [
28], where they added avocado, sunflower and olive oils to pork burgers. The observed color differences between raw and grilled samples are related to the several changes that occur during thermal processing. These include the Maillard reaction, denaturation of proteins, and loss of fat and water. Grilling led to an increase in the variability of the proportions of color components between the test groups due to the different properties of the beef tallow substitutes used to prepare the burgers [
29].
3.3. Texture Measurement
The springiness, chewiness, and hardness measured in the grilled burgers are presented in
Table 4.
From the analysis of the results of textural properties, it can be concluded that the modifications introduced in the burger recipe resulted in the greatest changes in parameters such as chewiness and hardness (both during storage and between burger groups). On the other hand, the springiness parameter did not change much and was in the range of 0.66–0.68. The greatest difference in springiness was observed only for the control group with reduced fat content, where this value decreased in a statistically significant manner (
p ≤ 0.05) from 0.66 on day 1 to 0.59 on day 6. Similarly, for chewiness: the samples from the three groups had the lowest hardness on day 1 in a statistically significant (
p ≤ 0.05) manner: LO (34.99 ± 3.23 N), OO (39.87 ± 4.61 N) and MF (45.62 ± 1.78 N). These values were about twice as low as the highest value recorded for the reduced-fat control group (88.64 ± 9.82 N). On day 6, the lowest hardness value was observed in the canola oil group (45.71 ± 5.95 N). The highest in the control groups (CON_LOW FAT 76.72 ± 12.79 N; CON 74.88 ± 7.99 N). Recipe modifications had a positive effect on reducing the hardness of burgers, as confirmed by the study by Szpicer et al. [
7]. The replacement of beef tallow with canola oil resulted in a 15.39% decrease in hardness compared with the control group containing 21.42 ± 0.26% fat. A decrease in hardness due to replacement of animal fat with vegetable oils was also observed in the study by Lee et al. [
30]. The lack of effect of fat substitution on the elasticity parameter was described by de Oliveira Fagundes et al. [
31], where the authors replaced animal fat with canola oil gels. The increased hardness of burgers with vegetable oils, i.e.,: canola oil and sunflower oil may be due to the lower fat globule of vegetable fat when compared with animal fat and the resulting higher protein–protein and protein-lipid interaction [
22]. Since fat imparts flavor, tenderness, and juiciness to food products, reducing it leads to harder products, as confirmed by Selani et al. [
32]. Additionally, Heck et al. [
25] confirmed that the hardness of burgers increases with increasing levels of animal fat substitutes, which were hydrogelated emulsions of chia and linseed oils.
3.4. Lipid Oxidation Analysis (TBARS)
The degree of lipid oxidation in the prepared raw burgers was examined using thiobarbituric acid and malondialdehyde (
Figure 1). On the first day of testing, samples from all groups showed similar levels of lipid oxidation, ranging from 0.12–0.17 mg MDA/kg of sample. On the 6th day of storage, there was a statistically significant (
p ≤ 0.05) increase in the degree of lipid oxidation in each of the analyzed groups. The highest degree of lipid oxidation was observed in canola oil group (0.99 ± 0.06 mg MDA/kg), SO group (0.91 ± 0.03 mg MDA/kg) and LO group (0.95 ± 0.03 mg MDA/kg). The highest stability to oxidation was present in burgers with reduced fat content (0.43 ± 0.02 mg MDA/kg), where oxidation was about 32.8% slower compared with the control group. Modification in the fat used significantly affected the fat oxidation level after 6 days of storage. The use of vegetable oils (SO, CO, LO, OO) increased the degree of lipid oxidation compared with samples containing animal fats (CON, CON_LOW FAT, MF).
The reduction in lipid oxidation due to reduced fat content in burgers is supported by the study of Patinho et al. [
33]. The authors investigated the possibility of replacing some pork fat in beef burgers with
Agaricus bisporus (AB) mushrooms (5%, 10%, 15% AB and 15%, 10% and 5% pork fat, respectively). In this study, TBARS values increased during storage, but the burgers with fat content reduced by replacing it with common mushrooms had significantly lower TBARS values than the control samples. The TBARS values in the group in which half the fat was replaced with common mushrooms were similar to those obtained in the CON_LOW FAT group in this study and were approximately 0.4 mg MDA/kg. The experiment showed that the addition of common mushrooms resulted in higher oxidative stability and moisture retention, lower fat content, and reduced cooking losses. The obtained product was characterized by good technological quality and desirable sensory attributes.
A study conducted by Szpicer et al. [
7], where the effect of replacing 40% of beef tallow with canola oil on the TBARS value in beef burgers was analyzed, confirmed, similar to this study, that replacing animal fat with vegetable oil can cause an increase in oxidative processes both on the first day of testing and during storage. The increase in TBARS values due to replacement of animal fat with vegetable fat was also confirmed in the study by Heck et al. [
25], where beef burgers with 40% replacement of pork fat by a hydrogel emulsion of chia oil and linseed oil had higher lipid oxidation from 0.27 to 0.53 mg MDA/kg.
A study by Trujillo-Mayol et al. [
17] showed that an effective method to reduce the degree of lipid oxidation in beef burgers stored for 10 days can be the addition of avocado peel extract to the burger recipe. This is because plant extracts, which are rich in phenolic compounds, have the ability to limit the oxidative reactions in meat products. According to Lu et al. [
34], the susceptibility of acids to oxidation increases in proportion to the number of unsaturated bonds in each fatty chain. Oils with high linolenic and linoleic acid content, i.e., linseed oil and sunflower oil, have the highest susceptibility to oxidation. The oxidative stability of olive oil, similarly to canola oil, is much higher due to its high oleic acid content. In conclusion, the use of beef tallow substitutes characterized by high unsaturated fatty acid content leads to increased lipid oxidation and thus to higher MDA content in the samples during storage. This is confirmed in the scientific literature in studies by Botella-Martínez et al. [
29], Heck et al. [
22], Lucas-Gonzalez [
35] and Moghtadaei [
36], among others.
3.5. Fatty Acids Profile Analysis
The fatty acid profile of the samples grilled on the 1st and 6th day of storage was analyzed. The mean proportions of each fatty acid, the content of total saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA), and the PUFA/SFA and n6/n3 ratio are shown in
Table 5 and
Table 6.
The saturated fatty acid content was highest on both day 1 and day 6 in the group, in which beef tallow was replaced by milk fat (day 1 48.76%; day 6 49.77%). The lowest SFA value on day 1 was observed in samples with canola oil (26.53%), similar to day 6 (27.48%). Similar SFA values were also obtained for groups with sunflower oil, linseed oil and olive oil. Analyzing the individual saturated fatty acids, the statistically significantly (p ≤ 0.05) highest content of C12:0, C14:0, C15:0 and C:16:0 fatty acids was recorded in the group with milk fat (MF on day 1 and day 6). The highest C20:0 eicosanoic acid content was observed in the MF group (day 1 = day 6 = 0.67) and the lowest in the LO group (day 1 = 0.32; day 6 = 0.34). In most cases, no statistically significant differences were observed between day 1 and day 6 except for C14:0 fatty acid in the CON, CON_LOW FAT, SO and OO groups and C16:0 fatty acid in the OO group, where values on day 1 were significantly (p ≤ 0.05) higher than on day 6.
The lowest statistically significant (p ≤ 0.05) content of total monounsaturated fatty acids on day 1 and day 6 was observed in the linseed oil burger group (day 1 33.66%; day 6 34.02%). These burgers had the lowest content of all monounsaturated fatty acids (MUFA) on day 1 and day 6. The only exception to this was C18:1 trans acid, which was at its lowest level of 0.02% in the burgers from the LO group. The MUFA found to have the highest amount in the burgers was C18:1 cis acid, and its highest value was present in the burgers with olive oil (58.83%). No C24:1 fatty acid was detected in any study group except the CO group (day 1 = day 6 = 0.04%).
The group with linseed oil added had the highest (day 1 38.52%, day 6 37.09%), statistically significant (p ≤ 0.5), polyunsaturated fatty acid (PUFA) content. The lowest PUFA contents on day 1 were observed in the CON_LOW FAT and MF groups. Of the polyunsaturated fatty acids, linoleic acid (C18:2 9.12 cis) was of the highest amount, with the highest content on days 1 and 6 present in the linseed oil group (day 1 34.37%, day 6 33.22%). By analyzing the PUFA to SFA ratio, it was observed that PUFA dominated in the LO (1.37) samples on day 1 and SO (1.03) and LO (1.28) samples on day 6. The significantly statistically highest ratio of n6 to n3 fatty acids was observed in the sunflower oil group (day 1 57.8 and day 6 92.76). The other study groups had similar n6/n3 ratio values, ranging from 3.79 to 10.75 on day 1 and from 3.92 to 10.99 on day 6. Analyzing the differences between the KT profile on day 1 and day 6, it was observed that SFAs had the highest stability during storage, whereas PUFAs had the lowest stability (they oxidize the fastest).
The partial replacement of beef fat in the burgers contributed to a decrease in saturated fatty acids and an increase in MUFAs and PUFAs, which is consistent with previous studies by Szpicer et al. [
37]. The study then showed that replacing beef fat with canola oil and oat beta-glucan concentrate improved the fatty acid profile. Another method of changing the fatty acid profile of burgers was used by Carvalho et al. [
38], where the fat content of the base composition was reduced by adding wheat fiber at 1.25, 2.50, 3.75, and 5.00 g per 80 g serving of burger (the fiber was hydrated before being added to the rest of the ingredients). The pork fat was reduced so that the 4:1 meat-to-fat ratio was maintained. Only the addition of the greatest amount of fiber negatively affected consumer acceptance of the burgers. The other test groups were evaluated positively and showed no or statistically insignificant changes in process-specific and sensory characteristics compared with the control group. As in the study conducted by Afshari et al. [
8], the applied lipid modification improved the fatty acid profile by reducing the percentage of SFA and lowering the n6/n3 acid ratio in CON_LOW FAT, CO and MF groups. The addition of fiber (a mixture of 3.1% inulin and 2.2% β-glucan), canola oil and olive oil as substitutes for beef fat in the study of Afshari et al. [
8] reduced SFA levels from 48% (control group) to about 19–24% and reduced the n6/n3 ratio from 8.6 to about 3. Our study confirmed that replacing animal fat with canola oil, sunflower oil, and linseed oil increases PUFA concentrations due to the presence of linoleic acid (C18:2 n-6) and linolenic acid (C18:3n-3) as the major fatty acids of sunflower oil, canola oil, and linseed oil, respectively. According to the study by Selani et al. [
32], the addition of canola oil to the formulation may contribute to the reduction in fatty acids such as myristic, palmitic and stearic acids compared with other vegetable fats.
3.6. The Effect of Fat Replacement on the Formation of PAHs
According to Wongmaneepratip and Vangnai [
18], the specific mechanism of PAH formation is not well known, but some researchers have suggested that these compounds may be formed in free-radical reactions, intramolecular addition reactions or polymerization of small organic molecules.
Samples of burgers grilled on the 1st and 6th day were analyzed to find the profile of polycyclic aromatic hydrocarbons. The content of individual PAHs, the sum of 12 PAHs, the sum of the light compounds, and the sum of heavy compounds are given as arithmetic means in mg/kg for a sample (
Table 7). As anticipated, it was statistically significant (
p ≤ 0.05) that the lowest level of Σ12 PAHs was present for low-fat samples (CON_LOW FAT), where on day 1, the total of 12 PAHs was at 48.46 mg/kg and in day 6 47.68 mg/kg. The highest statistically significant level (
p ≤ 0.05) of PAH was observed in samples with canola oil (day 1 105.5 mg/kg: day 6 102.69 mg/kg). The analysis of the content of the individual light PAHs showed that the group with canola oil showed the highest levels of fluorene (day 1 0.94 ± 0.12 mg/kg), anthracene (day 1 1.49 ± 0.25 mg/kg), chrysene (day 1 22.11 ± 0.56 mg/kg) and benzo[b]fluorine (18.86 ± 0.43 mg/kg) on day one.
The highest content of heavy PAHs was recorded in canola oil samples (day 1 60.75 mg/kg, day 6 59.56 mg/kg). This group showed statistically significantly (p ≤ 0.05) highest contents of 4 heavy PAHs (BbFL 2.47 ± 0.23 mg/kg, BkF 4.86 ± 0.31 mg/kg, DBahA 46.97 ± 1.06 mg/kg, BghiP 3.15 ± 0.30 mg/kg) on both test days. The lowest benzo[a]pyrene content in a statistically significant manner (p ≤ 0.05) was found in burgers from the reduced fat group (1.23 ± 0.21 mg/kg). Indeno[1,2,3-cd]pyrene was not detected in any of the study groups. Analysis of changes in PAH content between day 1 and day 6 showed that statistically significant changes (p ≤ 0.05) were least frequent in the reduced-fat group and most frequent in the OO and CO groups.
The PAH results presented in this study are comparable to the results obtained in Onopiuk et al. [
39], where levels of individual PAHs as well as their total are presented for charcoal-grilled pork. Slightly higher BaA, BaP, BaF values for grilled pork tenderloin were obtained in by Cordeiro et al. [
40], where they studied the effect of using elderberry vinegar, white and red wine vinegar, cider vinegar, and fruit vinegar with raspberry juice, as marinades, on PAH formation during high-temperature heat treatment. There is great difficulty in discussing and directly comparing PAH results between studies. This is mainly due to the large number of factors affecting the formation of PAHs such as: type of meat, fat content, presence of other ingredients, heat treatment conditions and many others [
2]. The mechanism of PAH formation, which is not fully understood, is also an important difficulty, whereas studies confirm a strong relationship between fat content and type of fat and PAH levels in food products. In a study by Lu et al. [
34], 40% of pork fat was replaced with sunflower oil, olive oil or grape seed oil. The researchers demonstrated that oil type has an effect on PAH content, and the direction of these changes exhibits differences that depend on the heat treatment temperature used. The use of olive oil reduced the PAH content by more than 50% compared with the control group for a temperature of 220 °C. In the present study, a reduction in PAH levels due to the use of olive oil of about 30% was achieved. In the study by Hu et al. [
41], beef was injected with the following fats: colza oil, soybean oil, canola oil, sunflower oil, butter, and pork fat. The research findings showed that the PAH content increased significantly due to the use of canola oil, which is consistent with the results obtained in this study. Hu et al. [
41] also showed that the total PAH content of the samples with pork fat was significantly higher than in the samples with butter, similar to the MF burgers in the present study. The use of fat replacements for heat-treated meat products can be an effective way to reduce the levels of cyclic organic benzene derivatives. Natural vegetable oils such as olive oil and linseed oil contain fatty acids that play a role in the fragmentation of hydrocarbons as well as the cyclization of aromatic compounds. The lowest PAH content in the CON_LOW FAT group may be due to the lowest fat and highest water content. Research by Kafouris et al. [
14] confirm a strong positive correlation between fat content and PAH formation in meat products. The highest PAH levels in the canola oil burger group can be linked to the fastest lipid oxidation process in this group. According to Lu et al. [
42], lipid oxidation and protein oxidation are associated with the development of HCA and PAHs through the interaction of radicals generated by lipid oxidation, lipid pyrolysis and Maillard reaction during the heat treatment.
The correlation coefficients between the profiles of selected fatty acids of raw burgers and PAHs of grilled burgers are presented in
Table 8 (selected highest correlation coefficients are shown in the table). Based on the results, high correlation was found between fluorene, benz(a)anthracene, benzo[b]fluoranthene and benzo[k]fluoranthene content and fatty acids such as γ-linolenic acid GLA (C18:3, 6, 9, 12), eicosenoic acid (C20:1), erucic acid (C22:1) and nervonic acid (C24:1). Benzo[a]pyrene showed a positive correlation with ∑PUFA (r = 0.758) and PUFA/SFA (r = 0.779). Unsaturated fatty acids have a significant impact on the formation of PAHs in heat-treated food, as confirmed by a study by Lee et al. [
43]. The PAH that had the highest correlation with fatty acids was benzo[g,h,i]perylene BghiP.
There was no statistically significant correlation between the basic composition (content of: water, fat, protein, connective tissue and salt) and the fatty acid profile. Similar results were obtained when analyzing the correlation of TBARS value and fatty acids.