**1. Introduction**

Jellyfish (JF) are mainly available and consumed as food in Asian countries. However, their use in food preparation has recently spread widely worldwide in the form of readyto-use products, also attributed to the availability on internet market channels [1,2]. In recent years, JF food products have also become more popular in Western Countries [3], possibly due to an increase in JF populations related to environmental factors, such as rising temperatures, marine pollution, oxygen depletion, and a reduction of marine predator populations [4].

Jellyfish blooms and the invasive behavior of some species make them a good candidate for potential resources for food and other applications [5–7].

JF for human consumption are generally prepared by separating the umbrella from the oral arms and washing them extensively in order to eliminate mucus, gonads, sand, and superficial microorganisms. Then, these highly perishable JF tissues are treated with mixtures of NaCl and aluminum salts (alum) [1,8] in order to stabilize them, thus extending

**Citation:** Ramires, F.A.; De Domenico, S.; Migoni, D.; Fanizzi, F.P.; Angel, D.L.; Slizyte, R.; Klun, K.; Bleve, G.; Leone, A. Optimization of a Calcium-Based Treatment Method for Jellyfish to Design Food for the Future. *Foods* **2022**, *11*, 2697. https://doi.org/10.3390/ foods11172697

Academic Editor: Maria Lisa Clodoveo

Received: 19 July 2022 Accepted: 1 September 2022 Published: 4 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

their shelf life, reducing any microbial issues, and promoting the expected organoleptic characteristics and texture so highly appreciated by Eastern people. In Asia, although ancient recipes and empirical procedures are still followed [9], new methods still based on the use of alum have been developed [1], since Eastern cuisines pay high attention to product texture and taste. However, the traditionally preserved JF products available on the market contain high levels of aluminum, which is strongly bound to the tissue [10] and cannot be eliminated through the usual washes applied before consumption.

The research on new JF stabilization procedures and treatments for food uses is recently moving to limit the use of alum, due to its toxicity [11,12], and to obtain semi-finished and finished products closer to Western Countries' style and expectations. Pedersen et al. [13] reported the possible substitution of alum with other tanning salts, such as iron salts, with a mechanism similar to a tanning process. In addition, the same authors produced alum-free crisps by soaking the jellyfish in ethanol and drying it afterward.

At present, JF is a novel food in Europe [14] and is limited by several issues, such as the (*i*) very high aluminum content of Asian traditionally preserved JF products and *(ii*) lack of safe stabilization methods for treating and processing JF tissues according to EU safety standards. Consequently, the development of a new, safe, and validated technology for processing JF could encourage regulatory authorities to approve the use and commercialization of edible JF species.

In our previous work, we proposed new parameters for the risk assessment of JF as food in Europe [15]. They were newly identified and applied to JF and JF-derived products not already included in the European regulation on seafood safety [16,17]. More recently, we proposed a new procedure [18] to process JF raw materials using calcium salts, which were selected from the food additives allowed in several Western Countries (the EU, Australia, USA, and New Zealand). It was observed that calcium salts were able to work as firming and stabilizing agents for JF biomasses, thus opening the opportunity to prepare safe semi-finished products suitable for subsequent food applications. Bleve et al. [18] set up the procedure under controlled conditions, thus demonstrating the microbiological safety of the method.

In this paper, the above-mentioned method was further optimized by modifying the JF pre-treatment step and including washes with sterile seawater; furthermore, the whole procedure was validated by using several JF species with different characteristics. Two different strategies are described here in order to substitute the use of sterile seawater for JF washing, with this step being quite challenging and not applicable on an industrial scale. Additionally, JF treated with calcium salts were tested for safety and quality aspects— the treated tissues were analyzed for protein, fatty acids, amino acids, element content, and antioxidant activity. The process efficacy was initially tested on the model species *Rhizostoma pulmo* and successively verified on three other potentially edible JF species (*Cothyloriza tuberculata*, *Phylloriza punctata*, and *Rophilema nomadica*). A comparison of the JF treated with the optimized processing method proposed here with JF prepared with the traditional Asian methods was also carried out.

### **2. Materials and Methods**

### *2.1. Sample Collection and Pre-Treatment*

*Rhizostoma pulmo*, Macrì 1778 (Cnidaria, Scyphozoa, Rhizostomatidiae) specimens were hand-collected from a motorboat in the Ionian Sea (Ginosa Marina, Italy 40◦2437.5 N 16◦5304.2 E) using a nylon landing net (3.5 cm mesh size) during samplings in the 2019– 2020 summer period and processed by either of the two procedures described below.

In the absence of specific slaughter guidelines for cnidarians [19], the traditional method used in Asian Countries to kill the jellyfish [1] was applied to *R. pulmo* specimens by cutting the oral arms from the umbrella and removing the gastric content. For *Cotylorhiza tuberculata*, *Phyllorhiza punctata*, and *Rhopilema nomadica* samples, also sampled during the 2019–2020 summer period, whole JF were immediately frozen at −40 ◦C. At least 5 different specimens from each species were used; for *Rhizostoma pulmo*, randomized sampling was conducted as in Leone et al. [20].

All the procedures followed in this study are summarized in the scheme reported in Figure 1.

**Figure 1.** Diagram illustrating the procedure of the JF treatment procedures.

*Procedure 1 (JF-B)*. JF oral arms were separated from the umbrellas, immersed in refrigerated seawater, and transported to the laboratory (max 3–6 h). At the laboratory, jellyfish parts were extensively washed for 3–5 min in drinking water.

*Procedure 2 (JF-DW)*. Whole JF were immersed in refrigerated seawater immediately after capture and transported to the laboratory (max 3–6 h). At the laboratory, the jellyfish were immersed in drinking water, the umbrellas were separated from the oral arms, and these parts were extensively washed for 3–5 min.

Following both procedures, the umbrellas and oral arms were placed in sterile foodgrade plastic bags and stored at −80 ◦C or immediately treated in the newly formulated brines with a firming agent.

Procedure 2 (JF-DW) was also applied to *Cotylorhiza tuberculata* samples harvested from the Gulf of Trieste (northern Adriatic Sea, Slovenia) and the Ionian Sea (Italy), and to *Rhopilema nomadica* and *Phyllorhiza punctata* harvested from the Eastern Mediterranean (Israeli coastal waters), frozen at −40 ◦C, and shipped on dry ice to Italy. Frozen material was thawed overnight on ice and then extensively washed with drinking water.

Aliquots of all untreated and treated JF samples (see below) were freeze-dried in order to analyze the elemental, lipid, and amino acid compositions or immediately frozen to evaluate the protein content and antioxidant activity. A commercial product, jellyfish stored in brine, from Japan (Salt-Alum Jp) was also analyzed and used for comparison.

### *2.2. Microbiological Analyses of Pre-Treated Jellyfish*

Ten grams from each JF sample were added to 90 mL of buffered peptone water (Biolife Italiana, Milano, Italy) as a diluent (1:10). For total bacterial counts (TBCs), samples were diluted and plated by the pour plate technique on plate count agar (PCA) (Biolife Italiana, Milano, Italy) at pH 7.0 and incubated at 30 ◦C for 72 h; the enumeration of yeas<sup>t</sup> and molds was performed by incubation at 25 ◦C for 5 days on dichloran Rose–Bengal chloramphenicol agar (DRBC, Thermo Fisher Scientific, Monza, Italy). The presence of Enterobacteriaceae, *Escherichia coli*, *Salmonella enteritidis*, Coagulase-positive staphylococci, *Staphylococcus aureus*, *Vibrio* spp. (*V. cholerae*, *fluvialis*, *parahemolyticus*, and *vulnificus*), *Bacillus* spp. (*B. cereus*, *turigensis*, *megaterium*, and *subtilis*), *Shewanella putrefacens*, *Aereomonas hydrophila*, and *Pseudomonas fluorescens* was assessed following the procedure described by Bleve et al. [18].

For the determination of halophilic microorganisms, JF samples were homogenized with a sterilized blender, and 25 g of each sample was added to peptone seawater 0.1% (*w*/*v* peptone) and artificial seawater. All samples and their respective serial dilutions were plated in different media dissolved in artificial seawater as described by Bleve et al. [15,18]. For each plate, the number of colony-forming units (CFU) per gram of JF was determined.

The JF samples were also submitted to an accredited external laboratory for independent analyses (Laboratori Artas Società Cooperativa, Poggiardo, Lecce, Italy). Ten grams of each JF sample were added to 90 mL of buffered peptone water (Biolife Italiana, Milano, Italy) as a diluent (1:10) and homogenized for 2 min in a Stomacher in accordance with specific standard methods for the total bacterial count (UNI EN ISO 4833-1:2013), coliforms (ISO 4832:2006), β-glucuronidase-positive *Escherichia coli* (ISO 16649-2:2001), coagulasepositive staphylococci (UNI EN ISO 6888-2:1999), and yeas<sup>t</sup> and molds (ISO 21527-1:2008, ISO 21527-2:2008). For the detection of the pathogenic bacteria *Salmonella* spp. (UNI EN ISO 6579-1:2017) and *Listeria monocytogenes* (ISO 11290-1:2017), 25 g of jellyfish samples were suspended in 225 mL of buffered peptone water (Biolife Italiana, Milano, Italy) and Fraser broth at half concentration (Biomerieux, Marcy l'Etoile, France), respectively, as diluents.

### *2.3. Jellyfish Treatment in Brine*

Solutions of calcium citrate and calcium lactate were prepared using 0.1 M calciumcitrate solution or 0.1 M calcium–lactate hydrate solution and adjusted to pH 5.0 by using the corresponding alpha organic acids, 1 M citric acid or 1 M lactic acid 85% (*v*/*v*), all from Sigma-Aldrich (Darmstadt, Germany). These concentrations were arbitrarily chosen and tested for JF stabilization treatment, as already previously described by Bleve et al. [18].

In order to determine these treatment conditions, JF tissue samples were soaked in calcium salt brines at different pH values ranging from 3 to 6 for 10 days. A starting brine pH value of 5 was determined as the best compromise, obtained after the evaluation of the effects produced by calcium salts on JF tissue's traits, such as texture and appearance, and safety aspects. More acidic pH values (<5) exerted undesirable effects on JF consistency by damaging (attacking and corroding) the tissue (data not shown).

Pre-treated jellyfish (umbrella and oral arms washed for 3–5 min in drinking water, JF-B, or JF-DW) were immersed in brines at a 1:1 ratio (*v*/*<sup>v</sup>*, JF tissue:brine) in food-grade glass or plastic containers. These technological phases were arbitrarily transferred from the

conditions generally used for vegetable stabilization treatment and directly tested for the first time for their possible adaptation to JF tissues. The containers were stored at 4 ◦C for 5 days and then JF tissues (umbrella and oral arms) were removed, washed with drinking water to eliminate excess salts, sealed in food-grade plastic bags, and stored at −80 ◦C for further tests. Aliquots of each sample were also freeze-dried and stored.

### *2.4. JF Treatment with NaCl and Aluminum Salt*

*R. pulmo* specimens were also processed by the traditional Asian method [21,22] using salt and alum (Salt–Alum JF-DW). Briefly, *R. pulmo* umbrellas were separated from the oral arms and extensively washed for 3–5 min with drinking water. The washed umbrellas were covered with a salt mix containing 90% (*w*/*w*) NaCl and 10% KAl(SO4)2·12H2O (alum) (*w*/*w*) (Cruciani Prodotti Crual, Roma, Italy) using about 100 g of salt–alum mix per 1 kg of JF biomass and incubated at 4 ◦C in a food-grade glass container. After 4 days, brines released from the JF tissues were removed and the umbrellas were covered with a salt mixture containing 92.5% (*w*/*w*) NaCl and 7.5% alum (*w*/*w*). After 4 days, the same procedure was repeated, but the percentage of alum in the salt mix was reduced to 5% (*w*/*w*) and finally to 2.5% (*w*/*w*). At the end of the process, the salted jellyfish samples were left to dry on a draining rack at room temperature for 4 days, inverting them several times to drain and remove excess water. The entire process took 20 days. Aliquots of each sample were also lyophilized.
