# The Potential of _Phaeodactylum_ as a Natural Source of Antioxidants for Fish Oil Stabilization
## 3 Results and Discussion
### _Phaeodactylum_ Total Lipid Extract-Fish Oil Mixtures (Part II)
#### 3.2.3 Secondary Oxidation  
The clear separation regarding the course of primary oxidation between 'Pure _Phaeodactylum_ total lipid extract', 'Mixture 79/21' and 'Mixture 49/51', on the one hand, and 'Mixture 6/94', 'Mixture 1/99' and 'Pure Fish oil' on the other hand, was also visible for the secondary oxidation products, as can be seen in Figure 5 showing the progression of (E,E)-2,4-Heptadienal, (E)-2-Pentenal and (Z)-2-Butenal as secondary n-3 LC-PUFA oxidation indicators. For all three compounds, the 'Mixture 6/94', 'Mixture 1/99', and 'Pure Fish oil'  
Figure 4: Primary oxidation of six different _’Phaeodactylum_ total lipid extract–fish oil’ systems during storage at 37 \({}^{\circ}\)C. ‘Pure _Phaeodactylum_ total lipid extract’, 'Mixture 79/21’ and 'Mixture 49/51’ (respectively possessing 100.0%, 78.6% and 49.2% (\(w/w\)) of _Phaeodactylum_ total lipid extract) were stored for 10 weeks (**a**). 'Mixture 6/94’, 'Mixture 1/99’, and ‘Pure Fish oil’ (respectively possessing 6.4%, 0.7% and 0.0% (\(w/w\)) of _Phaeodactylum_ total lipid extract) were stored for 4 weeks (**b**). The peroxide values (mean \(\pm\) sd, \(n=3\)) are expressed as delta values, representing the relative increase or decrease compared to day zero. Information on statistical significance can be found in Supplementary Materials (Table S3).  
systems showed a clear, significant increase (of respectively ca. 0.02 ug/mg, ca. 0.3 ug/mg and ca. 0.15 ug/mg) already after 4 days of storage. Other than in Part I, this significant increase in secondary oxidation products did not appear later than that of the primary oxidation products. Again, the relatively higher amount of oxygen as compared with the amount of bulk lipids could have led to accelerated oxidation, making the differentiation between the onset of primary and secondary oxidation less clear. The fact that also for the secondary oxidation products a clear, significant increase was already observed after the first time point indicated thorough lipid oxidation for all three systems. The 'Pure _Phaedactylum_ total lipid extract', 'Mixture 79/21', and 'Mixture 49/51' systems, on the other hand, showed a flat course throughout 10 weeks of storage for all three compounds. This indicated the absence of clear secondary lipid oxidation, which is in line with the _Phaedactylum_ hexane-isopropanol (3:2) extract in the research of [PERSON] et al. [16], and which also corresponds with the above-described small increase in primary oxidation products for these three systems. However, other than for the primary oxidation results, [PERSON] analysis showed that for the (E,E)-2,4-heptadienal and (E)-2-pentenal content, a small but significant decrease (of ca. 0.002 ug/mg) occurred after the first time point. Similarly, as described for _Nannochloropsis_ biomass by [PERSON] et al. [18], a reason for this reduction could be that volatile organic compounds (VOCs), previously formed in vivo in _Phaedactylum_ and co-extracted in the total lipid extract, were already present at the start of the storage experiment and decomposed during storage. Although in the literature, (E,E)-2,4-heptadienal and (E)-2-pentenal are not specifically mentioned as known _Phaedactylum_ VOCs [43], [PERSON] and [PERSON] [44] described the in vivo formation of lipid oxidation derived unsaturated aldehydes (encompassing (E,E)-2,4-heptadienal and (E)-2-pentenal) as a defense mechanism for diatoms, which would explain their presence. Anyhow, it is clear that this phenomenon (although significant) was only minor compared to the clear secondary lipid oxidation in the 'Mixture 6/94', 'Mixture 1/99', and 'Pure Fish oil' systems.