3.2.1. Thiobarbituric Acid Reacting Substances (TBARS) Test
The TBARS values of cooked samples are presented in
Figure 3. Generally, over time TBARS values increase in all the cases, but the CTRL set showed significantly higher values immediately after cooking (
p < 0.05), as well as a statistically significant increase during the refrigerated storage (
p < 0.05). In all burgers with CSS, TBARS mean values were lower than the acceptance limit of TBARS for rancidity (1.0 mg MDA(Malondialdehyde)/kg) [
45] until 72 h; after 120 h CSS3% showed a TBARS mean value near to the critical content, while this limit was exceeded in all samples without CSS.
The TBARS test is helpful for a first screening of the oxidation rate of a food, but it does not discriminate which kind of oxidation is occurring. We cannot be sure if what is observed with this assay comes from lipid oxidation itself, or if proteins too have taken part in the process and are the principal cause starting the reactions. As is known, chicken meat is poor in lipids that are mainly unsaturated fatty acids which are the elective substrate for the oxidation. Moreover, [
46] have searched for the lipidic profile of CSS and their results show a small content of lipids, which are mainly saturated fatty acids (SFAs). So, one could refer all the defective odours and tastes to the lipidic oxidation complex of reactions. Unfortunately, the oxidation process is very unstable and sometimes unpredictable. Besides lipids, proteins and iron ions boost the process within other factors (rise in temperature, oxygen and light exposure, salt addition, etc.) making TBARS not such an affordable method to establish the lipidic oxidative status of a food [
47,
48].
Anyway, from our results we can imagine that the contribution of CSS to the global oxidation burgers and its lipids increment are almost zero, while its protective effect seems to be promising. This may depend on the high content in phenolic and bioactive species.
3.2.2. Volatile Compounds (VOCs) and Warmed Off-Flavours (WOF) in Chicken Burgers
Among analyses used in this work, GC-MS analysis was used to trace markers of oxidation as a more reliable method than TBARS or any other faster, but less accurate, method. Results show a complex profile of compounds emerging from oxidative phenomena, Maillard reaction occurrence, and by-products addition.
Table 4 contains all the volatile compounds found in CSS-containing samples. Data depicted refer to T
0 and T
72 samples. Chromatograms are shown as
Supplementary Material (Figures S1–S3).
According to Chen et al. [
49], most of the typical odorants from cooked chicken meat are caused by phospholipids oxidation/degradation that led to the formation of long-chain aldehydes such as hexanal, (Z)-2-decenal and (E)-2-decenal. In any case, even if responsible for WOF development, these aldehydes are key aroma compounds of freshly cooked chicken meat. CSS samples showed these classes even if ketones and esters were not found. T
0 1.5% added samples had hydrocarbons as the first class traced, followed by aldehydes, nitrogen containing compounds and alcohols. T
0 3% added samples showed aldehydes at first place followed by Nitrogen containing compounds, alcohols, hydrocarbons, and other compounds. T
72 containing 1.5% of CSS showed nitrogen containing compounds, hydrocarbons, other compounds, aldehydes, alcohols, and ketones, while 3% addition showed aldehydes, hydrocarbons, alcohols, other compounds, and Nitrogen containing compounds.
In general, patties tested in this study, especially CTRL, seem to have a small compounds presence if compared with other articles [
50,
51,
52]. To our knowledge, this is one of the first studies where cooking conditions did not pass 92 °C and were not prolonged for more than four minutes. These conditions were selected to best simulate domestic conditions using an electric device set at medium cooking heat. Most studies on chicken meat burgers have tested grilled or oven-cooked patties. Other references on chicken meat evaluated entire boiled or roasted chicken. This is a fundamental step in explaining, for example, the absence of sulphur containing volatiles. In line with findings of other researchers [
53], these compounds come from the interaction among Maillard reaction compounds and lipid oxidation products. Thus, quick cooking processes, medium/low heating, or their combination seem not to favour this interaction. These settings did not allow the development of traceable Maillard reaction products (desired and undesired). T
72 (CTRL) samples showed an increase in concentration of hexanal, the emergence of heptanal, some alcohols such as 2-Nonen-1-ol, (E)-, ketones as 2,3-Octanedione and 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione, and just one Sulphur containing compound: N-Methyl-taurine. These are all markers of lipid oxidation and muscle damage. The addition of by-products provoked a mitigation of some WOF species, but also gave to patties specific odorants not conducible to meat oxidation and potentially undesirable.
CSS addition reduced the occurrence of aldehydes such as hexanal that reduced from 55.1% (CTRL T
0) to 11.7% (CSS T
0 1.5%) to 0 (CSS T
0 3%). At T
72 CTRL contained 72% and reduced to 0 in both concentrations. Heptanal was found only in CTRL T
72; octanal too, was just found in CTRL samples and not found in samples 1.5 and 3% at both times. Some alcohols such as 2-Nonen-1-ol, (E)- were limited in T
72 samples, but were present in T
0 CSS 3% with other alcohols, probably from the degradation of lignocellulosic precursors. This same pathway is followed by hydrocarbons which are totally absent in CTRL samples while being present in added patties [
54]. According to data here shown, Nitrogen containing compounds present in CSS formulations probably came from the degradation of CSS proteins and from the Maillard complex of reactions which takes place during the roasting process. As for CSS, the significant role of phenols’ interaction with Maillard reaction products to produce specific compounds can be assumed. Unfortunately, CSS developed p-xylene and o-xylene, involved in the rise of WOF and referred to as “cardboard-like” [
55,
56]. No references are available to compare results obtained, especially for CSS properties.
Chromatograms, in all cases, showed a great reduction of WOF or general active odorants. As a demonstration of this, CTRL T0 and T72 images had a resolution with an order of magnitude of 1010, while CSS had values of 108. From pictures, the peak of hexanal that eluted at around 6.48 min is always visible and it is clear how much it decreases in respect of additions of by-products. In all samples at around minutes 13.44 and 16.49, long chain aldehydes were eluted (i.e., octanal, decenal). At around minutes 19.35–37 2-Nonen-1-ol, (E)- was eluted in almost all samples. After minute 19.40, the main compounds traced were siloxanes and low matched compounds.
Of course, a better characterization of the aromatic profile of these molecules is fundamental to understand if and how volatiles from these substrates can have a negative impact on the final flavour of meat products.
3.2.3. Sensory Analysis
Panellists involved in this analysis were asked to try samples T0 and T72 of all formulations on separate days without knowing what they were eating, with the objective of evaluating the presence and the intensity of WOF markers and possible perceptions of extraneous flavours in cooked burgers after refrigerated storage. Descriptors were carefully explained, especially those difficult to associate with food, such as “cardboard-like” or “paint”.
In
Table 5 are reported all the average values for burger samples tasted immediately after cooking (T
0) and after 72 h of refrigerated storage (T
72).
T0 samples of all formulations did not show significative differences, and panellists were not able to trace significant variability from control. Statistical analysis did not show significance between samples even if, looking at the average values, some considerations can be made. The score reported for the bitter descriptor rose to 2.25 for the 3% CSS formulation, while CTRL was 1.25 and CSS 1.5% was 1.87. CSS addition did not influence the average score for all descriptors, and, in most cases, they were the same as CTRL or very near to it. CSS seemed to influence the perception of cooked meat odour rising from 1.75 in CTRL, to 2.87 in 1.5% addition, to 2.75 in 3% addition. This is one of the few situations in which 1.5% received higher scores than 3%. Astringency also registered an increase from CTRL to the 1.5% CSS addition of 3%: from 1.62 to 1.75 to 2.37, respectively.
Generally, all the descriptors for all formulations received increased scores, but some significant differences were traced via the statistical analysis or can be noted from the direct comparison among the average scores. Only two descriptors for CSS formulations were significant (p < 0.05): cooked meat odour and bitter. Cooked meat odour was mitigated, mainly by CSS addition. For this descriptor, the score decreased from 4 (CTRL) to 2.75 (1.5%) and 2.62 (3%). This limitation can be seen neither as negative nor positive. If the cooked meat odour can be directly linked with positive sensations, we do not know the considerations of each panellist regarding cooked meat. Nevertheless, from the explanation of each descriptor and the training, these lower scores do not directly show a positive thing. For better discrimination, a comparison with the roasted descriptor can be made; although not significant, it received lower scores than cooked meat.
The presence of this descriptor was not casual, because it helped in separating what flavours, odours, and aromas can come from a fresh grilled or oven/pan-cooked burger, instead of an already cooked and reheated burger. Panelists were not aware of the cooking process. The ‘roasted’ adjective generally includes those positive flavours coming from the Maillard complex of the reaction, while cooked meat is mainly linked with sensations of staling. In these terms, the mitigation obtained from by-products can be positive, while increasing the stability of the product. Bitterness perceived in CSS formulated patties can be a result of the reheating of meat. The data show an increase from CTRL at 1.37, to 1.62 (CSS 1.5%), and to 2.35 (CSS 3%). Heat can make the condensation or splitting of phenolic species easier. During the roasting process, chlorogenic acids degrade to active taste lactones which give desirable sourness and bitterness [
57]. When exposed to further heating, these species undergo greater degradation which leads to the splitting of quinic acid which, in successive steps will give metallic, lingering bitter phenyl-indanes which are undesirable for coffee taste. Caffeine and, in general, methylxanthines-alkaloids give a bitter and astringent note. Probably, the double exposure to heating, even if at lower temperatures, can determine higher bitterness.
Refrigerated storage can also favour the condensation of flavonoids to tannins or bigger phenolic species, but no sources are available at this time. A general positive comment is that none of the descriptors directly linked with the development of WOF (paint, cardboard-like, vegetable oil-like and sulphur/rubber) was increased. Even if not significative, average scores of the CSS added sample were lowered in respect of CTRL. Control had 2, while CSS fell to 1.75 for 1.5% addition, and to 1.5 for 3% addition. Acidity and even metallic sensations were not increased. To better investigate the significance of cooked meat flavour and bitterness, different factors were considered. Time, Formulation, and Time x Formulation were selected as factors. For cooked meat flavour, the time factor, i.e., the effect of time, was significant for
p < 0.01 while time x formulation factor had a
p < 0.05 (
Table 6). Formulation alone did not influence the results. Conversely, bitterness was influenced only by the formulation with a
p < 0.05. Neither time nor time x formulation factors made an effect. Overall, T
0 samples did not show any significant difference for the cooked meat odour descriptor. After 72 h of refrigerated storage, the time effect greatly influences (
p < 0.01) this characteristic.