# Valorization Potential of Oilseed Cakes by Subcritical Water Extraction
## 4 Discussion
### Total Lipid and Protein Content  
For all three samples the highest extractability of lipid compounds was achieved when adding 0.05 mol/L HCl as a modifier (48.55 \(\pm\) 0.23, 50.75 \(\pm\) 0.21 and 32.87 \(\pm\) 0.18 g/100 g dry extract for pumpkin, hemp and flax dried seed cakes, respectively), followed by extraction in a carbon dioxide atmosphere (38.87 \(\pm\) 0.22, 32.04 \(\pm\) 0.18 and 23.49 \(\pm\) 0.17 g/100 g dry extract, respectively). Since both extraction systems are, in fact, more reactive mediums in comparison to pure water, the results indicated strong bonds of lipid compounds with matrix constituents. Adding hydrochloric acid potentiated breakage of bonds between lipid compounds and matrix constituents. A similar effect was observed for the dissolution of carbon dioxide, forming carbonic acid, which further dissociates to hydrogen ion and bicarbonate. Since lipid components were extracted even with pure water, it can be speculated that in the extraction of hydrophobic compounds from these plant matrices, the limiting step is dissociation of hydrophobic molecules from complexes with matrix constituents. Another fact supporting this assumption, are the good lipid yields attained in the carbon dioxide atmosphere, since the reactivity of subcritical water in a carbon dioxide atmosphere is improved, potentiating hydrolytical reactions due to formation of carbonic acid [17]. The contents of lipids obtained by pure water were not negligible, being 28.54 \(\pm\) 0.18, 10.1 \(\pm\) 0.21 and 6.97 \(\pm\) 0.10 g/100 g dry extract for pumpkin, hemp and flax seed cakes, respectively. Thus, water polarity at 160 \({}^{\circ}\)C was such to be able to dissolve hydrophobic compounds from the studied samples. The protein contents determined in the extracts reflect the content of protein degradation products, i.e., the content of free amino acids and oligopeptides. For the tested samples, the hydrolysis of proteins was more pronounced in carbon dioxide atmosphere, which was reflected in the highest protein contents in the obtained extracts; 4.87 \(\pm\) 0.08, 6.83 \(\pm\) 0.9 and 2.84 \(\pm\) 0.03 g/100 g dry extract for pumpkin, hemp and flax dried seed cakes, respectively. As expected, the content of proteins was the lowest (\(p\) < 0.05) in extracts obtained in a nitrogen atmosphere with pure water (1.94 \(\pm\) 0.04, 4.83 \(\pm\) 0.06 and 1.30 \(\pm\) 0.01 g/100 g dry extract for pumpkin, hemp and flax dried seed cakes, respectively). The protein content was comparable in carbon dioxide atmosphere and with the addition of HCl modifier. Close protein contents were observed under these treatment conditions, especially for flax and hemp seed cakes. The highest content of proteins (6.83 \(\pm\) 0.9 g/100 g dry extract) was observed in hemp extracts treated in a carbon dioxide atmosphere, whereas the highest content of lipids (50.75 \(\pm\) 0.21 g/100 g dry extract) was observed in hemp extracts obtained with the addition of HCl modifier. Flax extracts obtained in nitrogen atmosphere had the lowest contents of both proteins (1.30 \(\pm\) 0.01 g/100 g dry extract) and lipids (6.97 \(\pm\) 0.10 g/100 g dry extract). It can reasonably be expected that matrix protein hydrolysis would increase with the increase in temperature, time or catalyst/modifier concentration. This should be taken into consideration if the goal of oilseed valorization is protein hydrolysis. For obtaining protein hydrolysates from different sources enzymatic hydrolysis using different enzyme mixtures is the commonly applied approach [18]. [PERSON] and [PERSON] [19] achieved a protein yield of 10.7% by treating defatted biomass of black soldier fly larvae (_Hermetia illicens_) using bromelain. [PERSON] et al. [20] compared trypsin hydrolysis with subcritical water hydrolysis of three model proteins: haemoglobin, bovine serum albumin, and \(\beta\)-casein. The authors confirmed high protein sequence coverage (>80%) for subcritical water hydrolysates of all three model proteins, at relatively mild conditions (160 \({}^{\circ}\)C). [PERSON] at al.