## 3 Results
### Conjugated Diene Content and DPPH Antioxidant Activity of Lipids Extracted from Wafers  
Lipid peroxidation is a very complex process leading to the formation of a wide variety of products. At the beginning of the process, hydroperoxides are formed from the polyunsaturated fatty acids. This process is accompanied by the rearrangement of double bonds in order to stabilize the radical state resulting in the formation of conjugated structures [6; 30]. Therefore, dienes are also the primary lipid oxidation products and the content of these is a good indicator of progress in the oxidation process. The increase in conjugated compounds is proportional to the formation of hydroperoxides [31]. Figure 3 shows the results of determining conjugated dienes (CD) in samples (LWS, LWE) during 13 months of storage. Differences between the LWS and LWE samples in terms of CD concentration are clearly visible, as the concentration of CD was always higher in the LWS sample than the LWE up until the 12 th month of storage, which indicates the protective effect of adding the extract. The content of dienes increased from 0.25% to 2.08% for the LWS samples and from 0.38% to 2.05% for the LWE during the whole storage period. There were no significant differences (\(p>0.05\)) between the LWS and LWE samples at the beginning of storage (0 month) and at the end of storage (13 th month). At the beginning of the oxidation process, i.e., in the initiation stage, a lag phase is normally observed. During that period, accumulation of lipid oxidation products is slow. This is mainly due to the slow formation of free radicals prior to hydroperoxides accumulation and that the free radicals preferentially oxidize the antioxidants present in products, which protects lipids at the earliest stages of oxidation [31]. Thus, on the 0 month of storage the CD concentration is similar in LWS and LWE samples. When the oxidation process has proceeded the content of conjugated structure increases in the sample without antioxidant treatment (LWS) in comparison to sample with antioxidant (LWE). Starting from the 3 rd month of storage, LWS showed 1.5-times higher CDs (0.82%) than LWE samples (0.49%) A similar trend between both samples was observed until the 12 th month. However, similarly to the peroxide value measurement, CD content is only effective at the beginning of the oxidation process. At low oxidation stages hydroperoxides undergo low decomposition but at the end of the process other products of lipid oxidation were formed thus the level of CD decreased [31]. Figure 3: Conjugated diene concentration (CD) of standard wafers (LWS) and wafers with extract (LWE) during 13 months of storage. Vertical bars indicate 0.95 confidence intervals, the same letters indicate a lack of significant differences between data (\(\alpha=0.05\)). Thus, on the 13 moth of storage the level of CD decreased in both samples (LWE and LWS) and no significant differences are observed. Summing up, it can be stated that the most pronounced changes of CD content took place during first six months of storage. The CD content doubled in the control wafers between 0 and 3 rd month and between 3 rd and 6 th month of storage. The trend was similar to results noted by [PERSON] et al. [3] where the oxidation of lipid fraction of wafers expressed as peroxide value doubled during the first three months of storage from 1.2 to 2.5 mg \(\mathrm{O_{2}/kg}\)[3]. The dienes and trienes content in stored cookies was also investigated by [PERSON] et al. [28]. They stated very small changes in fat extracted from stored cookies containing green tea extract, for which the lowest content of both dienes and trienes was detected after 3 months; 2.35 and 0.37, respectively. However, the question may arise as to whether the extract added to the LWE sample protects the lipids against formation of conjugated dienes. Therefore, the antioxidant activity of both samples (LWS, LWE) was also compared by using the DPPH\({}^{\bullet}\) radical scavenging method. In Figure 4, the results of the DPPH assay are presented.