*2.6. Lipid Content*

Total lipid content of the jellyfish *Rhizostoma luteum* was around 0.94 g per 100 g of DW (standard deviation = 0.03, *n* = 6 (Batch numbers: 3, 5–8, 10)). This value was one order of magnitude lower than both *Aurelia* sp.1 and *R. pulmo* (4 g/100 g DW) and two orders of magnitude lower than *C. tuberculata* (12.3 g/100 g DW; [16]). The low values of *R. luteum* are probably due to the age of the specimens examined. In the case of symbiotic jellyfish such as *C. tuberculata*, the high lipid content could be related to the photosynthetic membranes of the zooxanthellae [30,47].

The fatty acid (FA) quantitative composition (as percentage values) of *R. luteum* showed that polyunsaturated fatty acids (PUFA) accounted for half of the total FA (49%), followed by saturated fatty acids (SFA), representing one-third of the total FA (about 30%), and finally monounsaturated fatty acids (MUFA), representing around 21% of the total FA (Table 2). In the case of *Aurelia* sp. 1, *C. tuberculata* and *R. pulmo*, the proportions were different to *R. luteum*, with SFA being the most abundant FA (two-thirds of the total FA), followed by PUFA (two-thirds of the total FA) and a small amount of MUFA (4–15%) [16].

Polyunsaturated fatty acids of *R. luteum* consisted mostly of <sup>ω</sup>-*6* arachidonic (C20:4), <sup>ω</sup>-*3* linoleic (C18:3) and the essential <sup>ω</sup>-*6* linoleic (C18:2) acids. Saturated fatty acids consisted mostly of stearic (C18:0) and palmitic (C16:0) acids. Among MUFA, oleic acid (C18:1) was the prevalent FA (Table 2). This composition of fatty acids shows remarkable differences to the other three species studied previously (see Table 4 in [16]). The proportion of PUFA of both <sup>ω</sup>-*6* arachidonic (C20:4) and <sup>ω</sup>-*3* linoleic (C18:3) in *R. luteum* are the highest documented for jellyfish and the essential <sup>ω</sup>-*6* linoleic (C18:2) acid is only topped by the symbiotic jellyfish *C. tuberculata* [16], giving a peculiar composition of PUFA in *R. luteum*. In the case of MUFA oleic acid, (C18:1) is in the same order of magnitude with *C. tuberculata* and higher than *Aurelia* sp.1 and *R. pulmo* [16]).


**Table 2.** Fatty acid composition of *Rhizostoma luteum* expressed as percentage of the total fatty acid ± standard deviation (SD).

RT: retention time; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; \*: essential fatty acids. *n* = 6.

Both <sup>ω</sup>-*6* and <sup>ω</sup>-*3* PUFAs were abundant in the jellyfish *Rhizostoma luteum*, with the ratio of <sup>ω</sup>-*6* to <sup>ω</sup>-*3* of 2.14. The Σ <sup>ω</sup>-*6* was 33.4% while the Σ <sup>ω</sup>-*3* was 15.6%. The total <sup>ω</sup>-*3* PUFAs in *R. luteum* is similar to *Aurelia* sp. 1, *C. tuberculata* and *R. pulmo* [16], but the total <sup>ω</sup>-*6* PUFAs in *R. luteum* is double the other three documented jellyfish. Therefore, the ratio <sup>ω</sup>-*6* to <sup>ω</sup>-*3* of the other jellyfish is lower (between 0.4 to 0.8) [16] than in *R. luteum*. The role of <sup>ω</sup>-*3* PUFAs in a diverse biological process is well known, including development, growth, tissue, and cell homeostasis [48]. Regarding the numerous human health benefits of <sup>ω</sup>-*3* PUFAs, these include antiarthritis, anti-inflammatory, antioxidant, antihypertensive, anticancer, hypo-trigleceridemic, antiaging and antidepressive effects [49]. A high ratio of <sup>ω</sup>-*6* to <sup>ω</sup>-*3* in the Western diet in humans appears to be related to pathogenesis of chronic disease due to its proinflammatory effects [50]. The fatty acid composition of *R. luteum* discovered in this study should be considered for new applications due to its nutraceutical value.

### **3. Materials and Methods**

### *3.1. Materials and Chemicals*

Methanol and ethanol were purchased from Merck (Darmstadt, Germany); potassium persulfate (dipotassium peroxodisulfate), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), gallic acid, PBS, Folin-Ciocalteu's phenol reagent, Bradford reagent, fatty acid methyl esters (FAME) Supelco 37 Component Mix were all purchased from Sigma-Aldrich Química (Madrid, Spain). All other reagents were of analytical grade.

### *3.2. Sample Collection and Preparation*

Specimens of *Rhizostoma luteum* jellyfish were reared in the laboratory (ICMAN-CSIC) from polyps. The origin of this living collection is from planulae gathered directly from the gonad of a free-swimming female medusa offshore of the coast of the Alboran Sea (Spain) in October 2015 (details in Kienberger et al. [29]). For the present study, a total of 303 specimens were used. After the biometric measurements (weight and diameter) were taken, and due to their small size, specimens were grouped to reach enough biomass to be analyzed and lyophilized. Samples were freeze-dried for 4 days at −55 ◦C using a chamber pressure of 0.110 mbar in a freeze dryer (LyoAlfa 15, Telstar Life Science Solutions, Madrid, Spain). Lyophilized samples were weighed to annotate the dry weight and stored at −20 ◦C until use.
