**1. Introduction**

One of the challenges for coming decades in nutraceutical research is to find new potential resources that will provide an easy and low-cost approach to food necessities for the coming centuries. In this sense, jellyfish is a compartment in the marine food web (the greatest biome on earth) that often achieves high increases of biomass [1]. However, only recently has this wide group of organisms been starting to be seen from a more positive point of view [2], contrasting with the deleterious impact that it has on several economic human activities such as fisheries [3], clogging of power plants [4] and tourism [5]. This dark side of jellyfish blooms has been the only vision that has prevailed in the general human perception for many decades [6]. One example of the new positive point of view of the rapid increment of biomass in jellyfish is the cannonball, *Stomolophus meleagris*, in the Gulf of California, where a small Mexican community obtains benefits from switching for one month from fishing fish to fishing jellyfish, processes them, and sells them to the Asiatic market [7]. Another example of the explosive increment of jellyfish is the case of *Cotylorhiza tuberculata* in the Mar Menor lagoon in the

Mediterranean Sea, where during summer, fishermen are paid by regional authorities to catch jellyfish and bury them. Some summers they have caught more than five thousand tons of jellyfish [8,9].

The potential of jellyfish for human use is not only as a food resource [2,10–13], which is traditionally in Asian cuisine [14,15], but also recently as potential cosmeceutical and pharmacological applications [16]. Also, recently, jellyfish collagen has been studied for several biomedical applications [17], including antioxidant [18–20] and melanogenesis-inhibitory activity [18], skin photo-protection from Ultraviolet radiation [21,22], immunostimulatory effects [23,24], and antihypertensive effects [25].

The case we present here is unknown by the scientific community, and regards the potential use of the organic compounds and marine products from jellyfish. The jellyfish in the present work is *Rhizostoma luteum* Quoy and Gaimard, 1827 (Cnidaria: Scyphozoa: Rhizostomeae), which went scientifically unnoticed for more than 60 years until it was rediscovered and genetically characterized in 2012 [26]. Later, it was proved to be widely spread along the east coast of the Atlantic Ocean (from Portugal to South Africa) and in the Alboran Sea (Mediterranean Sea) and is much more frequent than previously thought, but was long misidentified for some of its congeners from the same geographical zones [27].

The purpose of the work is to describe the organic compounds for *Rhizostoma luteum* for the first time, with the hypothesis that it can be a candidate for the isolation and production of bioactive compounds in several fields of food, cosmetics, and biomedical industries. This hypothesis is based on three facts inherent to this jellyfish species: (1) it is from the family of the *Rhizostomaes*, which is the family of jellyfish preferable for consumption in Asiatic cuisine [28]; (2) The natural adult size of this jellyfish is very large, reaching more than 60 cm in umbrella size, weighing 13 kg and having 3 m tentacles [26,27,29], making one of the largest species of this family and therefore converting each individual into considerable amount of biomass; and (3) its life cycle has been recently described and closed entirely in captivity [29], which is a very difficult achievement since it is an open-ocean species. These three facts sugges<sup>t</sup> that, in the case the potential use of *Rhizostoma luteum*, the exploitation of this species' biomass can be obtained both from the natural environment and from controlled culture in captivity. However, before using it, it is mandatory to characterize the different organic compounds and other properties inherent to this jellyfish.

The main aim of this work is the identification of the primary metabolites (fatty acids) with antioxidant activity of the jellyfish *Rhizostoma luteum*. This goal was achieved by combining the original data on biochemical composition, protein, and phenol contents with their antioxidant activity, and the analysis of lipid content (identifying each of the presented fatty acids). The results presented here sugges<sup>t</sup> this jellyfish to be a promising sustainable source for the production of several natural products. *Rhizostoma luteum* appears all year around on the coasts of the east Atlantic Ocean and Alboran Sea [29], providing a highly unexploited biomass. Now a positive association of this giant jellyfish species and an isolation of its bioactive compounds can be established.

### **2. Results and Discussion**

### *2.1. Jellyfish Biomass Characterization*

Biometric and average biomass data of individuals of *Rhizostoma luteum* are shown in Table 1.

The specimens used are young medusae, with an age range from about 23–140 days (Table 1), with a proportionally increasing biomass with jellyfish age. The relation between fresh weight and diameter in *Rhizostoma luteum* has been described by [29], while the relationship between fresh weight (FW) and dry weight (DW) is documented for the first time for this species. The variability of the biometric measures, including the FW, the FW:diameter ratio and DW percentage values, is representative of the different growth stages of the jellyfish [30]. After lyophilization, the DW of *R. luteum* ranges from 3.9–31% of the FW, displaying a high variability in tissue consistency. The highest values correspond with the older jellyfish employed in this study.



FW, fresh weight; DW, dry weight; \* data are expressed as range and/or means standard deviation (5 < *n* < 61).

The first parameter that should be assessed for evaluation in its potential use as biomass is the total energy or gross energy value, which is directly related to DW. The DW of *R. luteum* is similar to the measured range of the FW of *Cotylorhiza tuberculata* (3.9–32.4%), which is higher than *Aurelia* sp. 1 (2.2–3%) and *Rhizostoma pulmo* (4.1–6.8%) [16]. These values provide important information for the potential exploitation of *R. luteum* jellyfish biomass. It has recently been documented using different techniques that jellyfish may practically represent the total diet of several organisms, such as leatherback sea turtles and several fish [31–34].

### *2.2. Jellyfish Carbon, Nitrogen and Sulfur Content*

The carbon, nitrogen and sulfur content of the jellyfish *Rhizostoma luteum* were 5.9 ± 0.3%, 1.6 ± 0.1% and 1.9 ± 0.2% of DW, respectively (mean ± standard deviation, *n* = 6 (Batch numbers: 3, 5–9)). The C:N molar ratio was 3.7 ± 0.1%. Similar values of carbon and nitrogen content have been reported in various of jellyfish such as *Aurelia aurita*, *Chrysaora fuscenses* and *Cyanea capillata* [35] and to the symbiotic jellyfish *Cassiopea xamachana* [36,37]. The C:N molar ratio of *R. luteum* is in the same range as overall medusae and ctenophores [38], and as the symbiotic adults of *Cotylorhiza tuberculata* [39] and *Cassiopea xamachana* [36,37].

### *2.3. Jellyfish Protein Content*

The total freeze-dried tissue of 8 different batches were subjected to phosphate-buffered saline (PBS) solvent extraction treatment. The whole tissues of *Rhizostoma luteum* contained proteins soluble in polar solvents, specifically in aqueous solution with a mean value of 13.7 ± 3.4 mg of proteins of per g of dry weight (mean ± standard deviation, *n* = 8 (Batch numbers: 1, 2, 4–7, 9, 10)). The values ranged between 8.4–18.7 mg of proteins/g of DW. Compared to other species, such as *Aurelia* sp. 1, *Cotylorhiza tuberculata* and *Rhizostoma pulmo*, with 22, 35 and 37 mg of proteins/g of DW, respectively [16], *R. luteum* contained less soluble protein but in the same order of magnitude. The content of protein in jellyfish is consistently the most abundant organic fraction [38] and has been studied in other rhizostomae jellyfish such as *Rhopilema asamushi* [40] and from *Cyanea nozakii* [41] for collagen isolation and characterization, which has been suggested for multipurpose uses in cosmetics and nutraceutical sectors [42].

### *2.4. Phenolic Compound Content in Jellyfish Aqueous Soluble Extract*

The total phenolic content of the jellyfish *Rhizostoma luteum* was 1964.9 ± 386.4 μg GAE (Gallic Acid Equivalent) per gram of DW in the PBS extract (mean ± standard deviation, *n* = 8 (Batch numbers: 1, 2, 4–7, 9, 10)). The values ranged between 1289.6–2597.1 μg GAE/g DW. The phenolic content in jellyfish is not well documented. Compared to *Aurelia* sp. 1 with 116 μg GAE/g DW in the PBS extract [16], *R. luteum* is in a position between *C. tuberculata* and *R. pulmo* reaching 1818 and 2079 μg GAE/g DW, respectively [16]. Also, phenolic compounds were detected in the podocyst and adults of *Chrysaora quinquecirrha* [43,44] and in *Cyanea capillata* and *C. tuberculata* [30,44].

The biostability and biochemical properties of collagen-based tissues may be enhanced by the polyphenols through modulation of mechanisms of collagen fiber cross-linking at different levels (molecular, inter-molecular and inter-microfibrillar) [45,46]. The high phenolic content of *R. luteum* (this study), *C. tuberculata* and *R. pulmo* [16], all of them large rhizostomae jellyfish, may be the reason for the robust and hardened mesoglea, compared to the flexible and soft *Aurelia* spp.

### *2.5. Antioxidant Activity in Jellyfish Aqueous Soluble Extract*

The total antioxidant activity of the jellyfish *Rhizostoma luteum* was 32,598 ± 9015 nmol of TE (Trolox Equivalent) per gram of DW in the PBS extract (means ± standard deviation, *n* = 8 (Batch numbers: 1, 2, 4–7, 9, 10)). The values ranged between 15,468–43,501 nmol of TE/g DW. These values of *R. luteum* are the highest values measured in a jellyfish so far, but in the same magnitude as other rhizostomae jellies such as *R. pulmo* and *C. tuberculata*, with antioxidant activity of 22,520 and 25,621 nmol of TE/g DW, respectively [16]. Meanwhile, *Aurelia* sp. 1 antioxidant activity was much lower, with measured values of 7651 nmol of TE/g DW [16].

The high values of *R. pulmo* and *C. tuberculata* compared to *Aurelia* sp. 1 were presumably attributed to the protein and phenols contents, although other unidentified compounds could be included [16]. Similarly, high values of antioxidant activity present in *R. luteum* in the present study could also be related to the protein and phenol content. The antioxidant activity referring to protein content, both in PBS extract, for *R. luteum* is 2379 nmol of TE/mg of proteins, showing evidence that this high antioxidant activity could be ascribed to the inherent protein properties of this jellyfish. Values of this ratio are double those reported by *R. pulmo* and *C. tuberculata* [16].
