*Non-detected
Data are expressed as the mean ± SD. The lower-case letters show the statistical difference between cell disruption methods (P< 0.05).
According to the results of fatty acids, there were not significant differences in the cell disruption groups compared to the control groups. Among the total crude lipid methods, Bligh and Dyer showed better results in terms of fatty acids. It was thought that the temperature applied in Soxhlet damaged the lipid material. Lower fatty acid amounts were obtained in all Soxhlet applied groups. It also appears that Soxhlet method damaged to DHA. In the study by Guckert et al. (1988) on lipid solvent system for the analysis of lipid classes inChlorella , Bligh and Dyer provided the most quantitative and reproducible recovery of all Chlorella lipid classes, also, degradation of the polyunsaturated fatty acids was observed during the Soxhlet procedure, similar with our study. Tang et al. (2011) in their study on supercritical CO2 and Soxhlet extraction of lipids and enrichment of DHA from oil-rich microalgaeSchizochytrium limacinum , DHA content was obtained in these groups as 27.5% and 15.4%, respectively. It was concluded supercritical CO2 extraction exhibits many advantages over the Soxhlet extraction for the DHA enrichment and purity. In our study, cell disruption methods, particularly osmotic shock, enzyme and HCl resulted in high yields of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids in the extracted oil. The highest DHA was detected in the BDE group. The enzymatic hydrolysis was shown to be an effective and nontoxic procedure for improving of extraction efficiency of intracellular compounds (Zhang et al., 2019). Gomes et al. (2020) reported that the microbial cell disruption using enzymes is a promising and highly energy-efficient technology. Furthermore, enzymatic lysis process is considered to be more environmentally friendly (Lee et al., 2017). Taher et al. (2014) in the study on extracting lipid with enzyme (lysozyme and cellulase) and acid treatment from Scenedesmus sp., reported C16:0 as the major saturated fatty acid which was 16.4 ± 0.08%, 11.9 ± 0.3% and 15.1 ± 0.2% in the enzyme lysozyme and cellulase, and acid treatments, respectively. Total fatty acids were 84.3%, 76.9%, 79.7%, 82.2% and 79.0%, in the groups of untreated freeze-dried algae, acid treatment, lysozyme and cellulase enzyme, respectively. Treatments decreased the total fatty acids compared with untreated algae, especially in the acid applied group. Yu et al. (2015) in their study investigating the effect of autoclaving, bead-beating, microwaving, sonication, acid (HCl) digestion, and Soxhlet extraction to lipid extraction fromChlorella sorokiniana , declared that in Soxhlet method saturated fatty acids 16:0 and 18:0 were affected, and lower results were obtained compared with other methods. In autoclaving and microwaving methods, 16:1, 18:0, 18:2 and 18:3 were not obtained when these fatty acids were relatively high in HCl digestion, especially for 18:2 with 28.9 ± 1.4%. In our study, most of fatty acids were obtained with HCl digestion. Although HCl that was reported to cause harm in the literature, did not cause any loss in fatty acids in our study. But Taher et al. (2014) reported that using acids requires special materials of construction, which is not economic for large-scale applications. Safety issues, and wastewater treatment are also essential for strong acid treatment (Lee et al., 2017) and it is thought that the method will not be safe in applications such as feed, food and cosmetics. In our study, effective results were obtained in terms of fatty acids in the OS and UH groups with the BD method. In the study by Prabakaran and Ravindran (2011) on cell disruption methods (autoclaving, bead beating, microwave, sonication, 10% NaCl solution) for lipid extraction fromChlorella sp., Nostoc sp. and Tolypothrix sp. sonication was found as the most effective method. Osmotic shock has advantages such as; lower energy consumption, easier scale-up, faster extraction- suitable for all cell types, however it has disadvantages like generation of waste salt water, salt’s high cost (Lee et al., 2017) time consuming. Sonication also has disadvantages such as damaging chemical structure of molecules (Byreddy et al., 2015).
In the present study, palmitic acid and DHA constitute most of the total fatty acids. It is an expected result that palmitic is high in aquatic plants, and the high amount of DHA, as a valuable fatty acid in terms of human health, is an important criterion. High DHA adds value to our product. It is thought that the evaluation of this algae for use in areas such as food and feed will make an important contribution. Ju et al. (2020) in their study the resulting microalgal lipids are mainly docosahexaenoic acid (DHA) and palmitic acid, also they reported palmitic acid is a saturated fatty acid (SFA) that is used as an emollient and diluent in the cosmetics industry. Fedorova-Dahms et al. (2011) reported that Schizochytrium sp. oil contains 40-45% DHA and up to 10% EPA. Leaño et al. (2003) found the amount of lipid in the range of 13.0-39.1%, in their studies of growth and fatty acid production of Schizochytrium sp. at different salinity and temperatures. In the study by Li et al. (2009) on was usedSchizochytrium as supplements in Ictalurus punctatus feed, the highest PUFA in dry algae was determined as DHA with 31.39% and Ʃn-3 PUFA was 37.08%. It was concluded that addition of 2% dried algae in the diet markedly improves the levels of 22:6 n-3 and Ʃn-3 LC-PUFAs in the edible tissue of fish. DHA and palmitic acid were high in % total fatty acids similar with our study. In our study; ƩSFA, ƩMUFA and ƩPUFA were obtained in the range of 67.08-85.82%, 7.38-10.53%, and 4.91-20.87%, respectively. In the study of Byreddy et al. (2015) saturated, monounsaturated and polyunsaturated fatty acids were obtained in the range of 49%-57%, 31%-35% and 2%-18%, respectively forSchizochytrium sp.. ƩPUFA obtained in our study higher than that reported by Byreddy et al. (2015). n-3 FAs have beneficial effects against chronic metabolic diseases, such as obesity, diabetes, and cardiovascular diseases, different cancers, asthma, inflammatory bowel disease, rheumatoid arthritis, psoriasis and osteoporosis (Prato et al., 2019). Because of their health benefits of long-chain n-3 polyunsaturated fatty acids, especially DHA, consumer preference increased for the products fortified with these desirable nutrients (Liu et al., 2020). n-3, n-6 and n-3/n-6, DHA/EPA, PUFA/SFA, unsaturated (UNS)/SFA ratios are widely used to evaluate the nutritional value of lipid (Prato et al., 2019). In our present study Ʃn-3 and Ʃn-6 were detected between 4.58 ± 2.44-19.53 ± 0.09% and 0.33 ± 0.07-1.79 ± 0.00%, respectively. n-6/n-3 ratio was detected in the range of 0.06 ± 0.00-0.09 ± 0.09. Gonçalves et al. (2021) reported that n-6/n-3 ratio below 4.0 in a diet indicates desirable quantities for human health. In our study, this ratio was below the limits that Gonçalves et al. (2021) reported, in our study. Increasing consumption of fish and fish products rich in n-3 PUFA (polyunsaturated fatty acids), which is important for human health, and poor in n-6 PUFA, should be encouraged (Metin et al., 2021). PUFA/SFA ratio was obtained between 0.06 ± 0.03-0.31 ± 0.00 values. Due to the high content of palmitic acid, this ratio was obtained at low levels. However, the values were close to the minimum limit value of 0.45 specified by Gonçalves et al. (2021) and Liu et al. (2020) except for the SC group.
As a result; as Zhang et al. (2019) reported; compared with conventional methods, the use of emerging techniques allowed the recovery bio-molecules avoiding toxic solvent, high temperature and treatment time.