*Article* **On the Health Benefits vs. Risks of Seaweeds and Their Constituents: The Curious Case of the Polymer Paradigm**

**João Cotas 1,2, Diana Pacheco 1,2, Glacio Souza Araujo 3, Ana Valado 2,4, Alan T. Critchley 5,\* and Leonel Pereira 1,2**


**Abstract:** To exploit the nutraceutical and biomedical potential of selected seaweed-derived polymers in an economically viable way, it is necessary to analyze and understand their quality and yield fluctuations throughout the seasons. In this study, the seasonal polysaccharide yield and respective quality were evaluated in three selected seaweeds, namely the agarophyte *Gracilaria gracilis*, the carrageenophyte *Calliblepharis jubata* (both red seaweeds) and the alginophyte *Sargassum muticum* (brown seaweed). It was found that the agar synthesis of *G*. *gracilis* did not significantly differ with the seasons (27.04% seaweed dry weight (DW)). In contrast, the carrageenan content in *C*. *jubata* varied seasonally, being synthesized in higher concentrations during the summer (18.73% DW). Meanwhile, the alginate synthesis of *S*. *muticum* exhibited a higher concentration (36.88% DW) during the winter. Therefore, there is a need to assess the threshold at which seaweed-derived polymers may have positive effects or negative impacts on human nutrition. Furthermore, this study highlights the three polymers, along with their known thresholds, at which they can have positive and/or negative health impacts. Such knowledge is key to recognizing the paradigm governing their successful deployment and related beneficial applications in humans.

**Keywords:** polysaccharides; health benefits; health risks; biomedical; polymer seasonal variation

### **1. Introduction**

The growing demand for seaweed feedstock is noteworthy, particularly since 1990, reaching its peak in 2018, when 31.5 million tons fresh weight (FW) of seaweeds were sustainably cultivated (this is the latest year for which reliable data are available), while around 1 million tons FW of seaweeds were exploited from wild stocks [1]. Seaweeds' rich nutritional profile (including phenolic compounds (e.g., phlorotannins), protein (e.g., phycobiliproteins), carbohydrates (e.g., alginates, fucoidans, ulvans, agars, and carrageenans), lipids (especially, ω-3 fatty acids), vitamins (in particular, A, B, C, D, E, and K and their precursors) and essential minerals (e.g., calcium, iron, iodine, magnesium, and potassium)) has led to their incorporation in the daily diet of several Asian and European countries [2–8]. Furthermore, a significant amount of the total annual seaweeds feedstock is used by the global phycocolloid industry [1]. The main phycocolloids (i.e., algal-derived) or polysaccharides for these industries are agars and carrageenans (i.e., extracted from red seaweeds) and alginates (i.e., extracted from brown seaweeds) [7,9,10]. Polysaccharides (sugars) are highly valuable macronutrients, which are indeed abundant in seaweeds, as they act as structural components of the varied morphologies of the thalli. In fact, these molecules can represent

**Citation:** Cotas, J.; Pacheco, D.; Araujo, G.S.; Valado, A.; Critchley, A.T.; Pereira, L. On the Health Benefits vs. Risks of Seaweeds and Their Constituents: The Curious Case of the Polymer Paradigm. *Mar. Drugs* **2021**, *19*, 164. https://doi.org/ 10.3390/md19030164

Academic Editor: María Lourdes Mourelle

Received: 19 February 2021 Accepted: 16 March 2021 Published: 19 March 2021

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**Copyright:** © 2021 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/).

up to half of thallus dry weight [11–13]. Seaweeds are a polyphyletic group, and across the >12,000 species, a wide range of polysaccharides are synthesized, differing within species (even at the level of alternation of generations (i.e., n vs. 2n) of the same species) and may vary due to biotic and abiotic stimuli [12–16]. Moreover, the selection of the methods of extraction and purification may also directly affect their yield and purity [17].

Such biomolecules are receiving a high degree of economic interest from several industries, including the feed, food, cosmetics and pharmaceuticals industries, due to their rheological (i.e., gelling/thickening) and increasingly wide range of biological activities. In particular, interest has blossomed in the food and pharmaceutical industries, because of many research studies on their bioactive properties, which include: antitumor [18], anticoagulant [19,20], anti-thrombotic [21,22], antiviral [23–25], immunomodulatory [26,27] and anti-fungal [28,29] activities, presented collectively and/or individually by these phycocolloids [30].

Currently, many seaweed polysaccharides are widely used in the food industry as stabilizers or emulsifiers for their gelling properties [31]. In fact, agar (E 406) [32,33], carrageenan (E 407), processed carrageenan (E 407a) [34,35] and alginate (E 401) [36,37] are authorized food additives and are generally recognized as safe (GRAS) for human consumption. Therefore, when food products with these seaweed polysaccharides (as additives) are ingested, they present properties similar to a dietary fiber, not least since humans do not have the required enzymes to break the glycosidic bonds of the long-chain carbohydrates [38]. Indubitably, humans need to metabolize sugars in order to fuel the body and the nervous system [39]. Hence, the inclusion of polysaccharides such as agar, carrageenan and alginate play important roles in human nutrition, since they promote satiety and intestinal function regulation, and enhance intestinal flora, consequently achieving higher nutrient absorption rates [11,16,40–42]. The consumption of algal polysaccharides provides several additional health benefits, such as regulation of glycemic index values and reduction of low-density lipoprotein (LDL) cholesterol [11,41,43,44].

Despite the many health benefits that seaweeds and their compounds provide, there are some concerns hampering some consumers from including them in their daily diet. Among these are iodine, metals, pesticides, antibiotics and or other noxious compounds (e.g., radionuclides), which some seaweeds may accumulate from coastal seawater [45–47]. Thus, the European Food Safety Authority (EFSA) and the competent North American authorities, e.g., the Food and Drug Administration (FDA), have established a threshold for the consumption of certain seaweeds and their components, through several health risk assessment studies [48–50]. It is key to note that only specific seaweeds are on these lists, while other species that are not in those lists are considered "novel food" and require a considerable amount of generational data and clinical trial evidence before being allowed in food supply chains.

The carrageenophyte *Calliblepharis jubata* is widely distributed through the European coastline, while the agarophyte *Gracilaria gracilis* and the alginophyte *Sargassum muticum* are widespread throughout the globe, and all are considered edible seaweeds [2]. All the forementioned species are perennial species in Portugal, meaning that they are present throughout the year [51]. Thus, they are species with potential for industrial exploitation.

Herein, this study aimed to analyze the seasonal variation of the polysaccharide content of three different seaweed species of agarophyte, carrageenophyte and alginophyte. It was further assessed whether the polysaccharide content met the threshold established by the competent authorities, thereby guaranteeing the safety for human consumption of the wild seaweeds. Additionally, the literature was reviewed in order to understand the positive and potentially negative effects of seaweed polysaccharides on human health.

### **2. Results**

Throughout the seasons evaluated, the carrageenophyte *Callibepharis jubata* (Figure 1a) produced the lowest polysaccharide content, as compared to the agarophyte *Gracilaria*

*gracilis* (Figure 1b) and the alginophyte *Sargassum muticum* (Figure 1c). Furthermore, different seasonal patterns were observed with respect to their polysaccharide profiles.

**Figure 1.** Three seaweeds studied at the collection site (Figueira da Foz, Portugal): (**a**) *Calliblepharis jubata* (Rhodophyta—carrageenan-bearing); (**b**) *Gracilaria gracilis* (Rhodophyta—agar bearing); (**c**) *Sargassum muticum* (Phaeophyta—alginate-bearing).

### *2.1. Red Seaweed Polymers*

### 2.1.1. Carrageenan Content and Identification

Regarding the red seaweed *C. jubata* (Figure 2), this species presented the lowest content during the autumn, with a concentration of 10.37 ± 0.416% DW. However, during the summer it was possible to extract 18.73 ± 2.382% DW carrageenan, which was the highest value during the observation period.

**Figure 2.** Carrageenan content analyzed seasonally. The extraction yields are expressed as mean ± standard deviation (*n* = 3). a,b The same letters indicate no significant differences at the *p*-value < 0.05 level.

The FTIR-ATR spectrum (Figure 3) of the phycocolloids extracted from *C. jubata* showed bands at approximately 930 and 845 cm−1. However, an additional well-defined feature was visible at around 805 cm−1, indicating the presence of two sulfate ester groups on the anhydro-D-galactose residues, a characteristic band of the iota-carrageenan (Table 1) [52,53].

**Figure 3.** FTIR-ATR spectrum of the carrageenan extracted from *Calliblepharis jubata*.

**Table 1.** FTIR-ATR band identification and characterization of the red seaweed *Calliblepharis jubata* polysaccharides (carrageenan), based on the literature [53,54].


### 2.1.2. Agar Content and Identification

The red seaweed *G. gracilis* produced the highest agar concentration (Figure 4) during the autumn, with 27.04 ± 2.684% DW. However, this did not differ greatly seasonally. Thus, its production was not statistically significantly different over the study period.

**Figure 4.** Agar content analyzed seasonally. The extraction yield results are expressed as mean ± standard deviation (*n* = 3). <sup>a</sup> The letters indicate no significant differences at the *p*-value < 0.05 level.

Agars differ from carrageenans, as they have an L-configuration for the 4-linked galactose residue; nevertheless, they have some structural similarities with carrageenans (Table 2). The characteristic broad band of sulfate esters, generally between 1210 and 1260 cm−<sup>1</sup> (Figure 5), was much stronger in carrageenans than agars [54].


**Table 2.** FTIR-ATR band identification and characterization of the red seaweed *Gracilaria gracilis* polysaccharides (agar), based on [53,54].

**Figure 5.** FTIR-ATR spectrum of the agar extracted from *Gracilaria gracilis.*

### *2.2. Brown Seaweed Polymers*

Alginate Content and Identification

Regarding the non-indigenous seaweed *S. muticum* (Figure 6), the highest alginate concentration was observed during winter (e.g., 36.88 ± 2.953% DW).

The main polysaccharide found in the brown alga (*S. muticum*) was alginic acid, a linear copolymer of mannuronic (M) and guluronic acid (G). Different types of alginic acid present different proportions and/or alternating patterns of different guluronic (G) and mannuronic (M) units. The presence of these acids can be identified from their characteristic bands in the vibrational spectrum (Figure 7). The extracted colloid showed two characteristic bands: 806 cm−1, assigned to M units, and 788 cm−1, assigned to G units, suggesting the presence of similar amounts of mannuronate and guluronate residues (Table 3) [54,55].

**Figure 7.** FTIR-ATR spectrum of the alginate extracted from *Sargassum muticum*.



### **3. Discussion**

Seasonal variation in the concentration of different polysaccharides such as carrageenan, agar and alginate was investigated in order to better understand the impact of the season on the polysaccharide yield and quality, due to their economic value and applications. From an industrial management point of view, it is pivotal to assess the extraction yield and the costs associated with the production of these seaweed polysaccharides. The FTIR-ATR is a known method for characterizing and evaluating the overall quality and composition of the seaweed polymers, mainly on the basis of the concentration or modifications in the sulfate esters groups, which are among those groups that can vary seasonally [56–59]. The results of FTIR-ATR evaluation demonstrated similar spectra between seasons, thus revealing that the quality of the polysaccharide extracted differed only in terms of their yield in the *C. jubata* and *S. muticum*.

With respect to the carrageenophyte *C. jubata*, the optimum season to harvest this seaweed for the highest yield of carrageenan was mid-spring to the beginning of the summer. This red seaweed synthesizes lower carrageenan concentrations during the autumn and winter [59]. This observation was supported by other reports assessing seasonal yield variation in carrageenan on the Normandy (France) and Portuguese coasts [59,60]. It was found that in Normandy, carrageenan yields fluctuated from 15% (in winter) to 45% DW (at the end of the spring/beginning of the summer) [60]. On the Portuguese coast, the lowest carrageenan content was found during winter (i.e., 4% DW), but with a maximum yield during the spring (i.e., 40.4% DW, a 10 fold increase) [59]. Previous research has shown

that *C. jubata* collected during spring 2020, at the same sampling site as the present study, produced a carrageenan yield of 28% DW [61]. The FTIR-ATR analysis of carrageenan from *C. jubata* was in concordance with the analysis of Pereira et al. [53], which detected iota-carrageenan with a low/residual content of kappa-carrageenan.

The increased accumulation of this biological reserve by *C. jubata* could be explained by the fact that the seaweed is typically from cold-temperate waters, so the increase of the surface seawater temperatures (SST) during the summer could be a stressor enhancing carrageenan biosynthesis [60].

The agarophyte *G. gracilis* is an opportunistic seaweed that is present in temperatewarm waters, and is already an important commercial source of agar, of which there is currently a global supply shortage [62–64]. Previous research has highlighted seasonal differences in the yield of agar from this species [65–67]. In agreement with the results of this study, *G. gracilis* collected on the Patagonian coast (Argentina) recorded the highest agar production during the summer and spring seasons (i.e., 41 and 30% DW, respectively) [65,66]. Using materials collected from the Venice Lagoon (Italy), an average agar yield of 25% DW was reported [67]. The lower agar yield during the spring/summer, can be attributed to the increased nutrient concentrations and lower turbidity and planktonic blooms that often characterize these seasons [67]. The FTIR-ATR analysis of the agar from *G. gracilis* demonstrated the presence of typical bonds for agar, with sulfate esters being evident. The observations are similar to the results obtained by Pereira et al. [54] with other agarophyte species (e.g., *Gelidium* spp.).

The alginophyte *S. muticum* is a brown seaweed introduced and well established in European and North American waters [62,68–71]. This seaweed can be used as a feedstock for alginate extraction, e.g., *S. muticum* harvested in Morocco produced a 25.6% DW yield at the beginning of spring [55,72]. The FTIR-ATR spectra of the alginate from *S. muticum* presented alginate peaks, but sulfate esters were also revealed, which could have been derived from sulfated polysaccharides such as fucoidan and laminarin [54].

The FTIR-ATR spectra were very similar between the seasons, demonstrating that the main factor was the quantity and not the quality. These results are in concordance with literature reports [54,59,64].

Seaweed polysaccharides, with a high molecular weight, are generally considered to be good dietary fibers. Specific applications of these are recognized as key players in human health and disease prevention [73]. These benefits are especially enhanced because there is an interplay with the gut microbiome at intestinal, as well as systemic, levels, resulting in homeostasis between the host and the microflora. Food intake can modify the microflora equilibrium positively or negatively, resulting in immunological, physiological, metabolic and even psychological effects. Consequently, the human diet can modulate health status: indubitably, we are what we eat [74–76].

In addition to their biological properties, seaweed polysaccharides also have innate properties that are very important for intestinal health; these include mainly the viscosity and the high potential for water-binding activity, which adjusts the transit time of food through the gut. Such properties are demonstrated to promote satiety and weight loss; additionally, they delay gastric emptying, thereby promoting glycemic control (i.e., reducing the incidence of diabetes). In the intestinal tract, all seaweed-derived polysaccharides are reported to enhance gut transit, maintaining regular stool bulking, and promoting beneficial alterations to the composition of the microbiome. Taken together, these benefits result in improved metabolization of volatile fatty acids (VFAs), which are also considered to be short chain fatty acids (SCFAs) by members of the microflora, promoting positive impacts in the gastrointestinal system, and thus resulting in the improved status of cardiometabolic, immune, bone, and mental health conditions [3,77–80].

It is now clear that various seaweeds have an interesting dietary fiber content, which can have a positive impact on the health status of production and companion animals, as well as on the health status of humans. Furthermore, this source is natural and uniquely different from crop and fruit plants. However, from this study and others, it is patently

clear that not all seaweed polymers are "the same". They have a structural function in the seaweed thalli and can be expected to vary seasonally. Hence, there is considerable need to quantify them, in order to ensure good intake without passing the intake limits. It has been shown that excessive consumption of dietary fiber can lead to negative impacts on human (and animal) health, e.g., recurrent symptoms of soft stools or diarrhea [3,77–79,81,82]. All good things should be taken in moderation. For instance, the study of Calvante et al. [83] demonstrated that a commercial powder of *Crassiphycus birdiae* at a low dosage, which is the recommend dosage intake of seaweed dried biomass supplement daily (5%) can induce reproductive toxicity and cellular damage when ingested with other chemicals. Thus, the full analysis of seaweed is required to fully understand the impact of seaweed compounds in the food industry and, more importantly, in the seaweed for direct intake [84,85], mainly with respect to metals (arsenic, cadmium, mercury, and lead) and other contaminants. In our study, the polymers were seasonally stable and the major differences in *C. jubata* and *S. muticum* were related to the yield.

The diversity of seaweed polysaccharides (and particularly their lower-molecularweight oligomers) needs to be quantified, due to the negative effects that can arise if their cumulative dosage exceeds the limit of 25 g/day [82,86]. In this case, the consumption of wild harvested seaweeds would need to be limited according to their season of harvest (Table 1). Taking just three examples in the present study, *C. jubata* had the highest values in autumn; *G. gracilis* was the seaweed with the most consistent levels across the seasons; and *S. muticum* had the highest polymer levels during the winter. These observations are important in order to maximize the benefits of ingestion of particular types of seaweeds. This is because if the seaweed intake exceeds the recommended levels, the constituent polysaccharides, such as dietary fibers, can de-regulate the intestinal system, inducing bloating, abdominal pain, flatulence, loose stools or diarrhea, etc., as well as a reduction of blood glycemic values, which for diabetic patients, in particular, is a serious health risk [87].

However, due to the considerable diversity of seaweeds and their composition, the recommended daily intake for a generic "seaweed" is normally only 5 g DW/day, due to its high mineral/metal content; this was demonstrated by Milinovic et al. [88] as a result of the iodine content in seaweeds collected at Figueira da Foz, Portugal, which is a limiting factor in the seaweed intake. Due to the advice presented in the Recommended Dietary Allowance [89,90], it is necessary to standardize of the analyses applied to seaweed, especially for applications in the food industry [91]. Considering the examples in this study, the three species represent between 2 and 7% of the recommended daily intake (Table 4) [82,86].


**Table 4.** Thresholds of daily consumption of seaweeds based on their polysaccharide content.

Table 4 demonstrates that, overall, each of the seaweeds analyzed had a good dietary fiber content, which could be exploited commercially. In particular, *G. gracilis* did not exhibit a great deal of seasonal variability. However, *C. jubata* and *S. muticum* showed significant, although different, seasonal variations. From a commercial point of view, for the greatest benefit to the consumer, the seaweed raw materials need to be harvested in specific seasons in order that the level of polysaccharide content does not exceed the threshold of consumption. However, there is the need for a complete biochemical profile if the

specific seaweed is to be consumed whole [90]. In particular, the macro- and trace elements present in the seaweeds need to be known due to their potential accumulation from the surrounding environment [88,92]. However, the effects due to the intake of whole seaweeds appear to be less when compared with the purified seaweed polysaccharide associated with water, milk, or prepared in a juice [93–95]. Consequently, ongoing research in this area is targeting applications of seaweed polysaccharides in novel foods with nutraceutical properties [6,96,97].

Anti-obesity effects have been described as being among the most beneficial attributes of seaweed polysaccharides for human consumption due to their fermentation in the intestinal tract, thereby reducing the microfloral/bile salt hydrolase activity, which is one theory behind this observed effect [96,98–101]. In this case, the microbiome composition was found to change to an augmented state, including populations of *Bifidobacterium*, *Bacteroides*, *Lactobacillus*, *Roseburia*, *Parasutterella*, *Fusicatenibacter*, *Coprococcus*, and *Fecalibacterium* colonies in in vitro experiments [96,98–101]. The nutritional values of the targeted seaweeds demonstrate a general fluctuation on the basis of the location at which the seaweed was collected, as demonstrated by Pacheco et al. [69]. Thus, the harvest site greatly influences the nutritional value, with carbohydrate yield being one of the principal variations (see Table 5). There is a lack of studies regarding the nutritional profile of *C. jubata*; however, there is a study by Araújo et al. [61] that characterizes its carbohydrate and lipidic profile.

**Table 5.** Range of nutritional values of selected seaweeds analyzed around the world (% DW).


However, as a food supplement, the safety of the dietary inclusion of seaweeds also needs to have various biochemical constituents checked before the alga can be made commercially available for regular human consumption [6,106]. Studies thus far have demonstrated that some wild harvested seaweeds, without thorough analysis of their nutrient and metal concentrations, can provoke negative impacts on health. However, there is little information available on this, relative to the more adverse pathologies (i.e., compared to those described above). This important topic is well described in the reviews of Cherry et al. [96] and Weiner [81], as well as Wierner and McKim [107], who demonstrated that within the daily recommend intake, there are indeed relatively low health risks to consumption. Nonetheless, major concerns have been expressed over seaweed polysaccharides present at low molecular weights, and poligeenan in particular. This has also been referred to as "degraded carrageenan", and is not the natural chemical structure of the polysaccharide. Indeed, it can provoke harmful impacts, such as the powerful induction of inflammation. Intake of degraded carrageenan can happen when the legislation regarding polysaccharide preparation and usage in the food industry is not followed. Because of this, seaweed polysaccharide applications in the food industry are regulated, in order to guarantee the safety of the final product [6,32,34,36,81]. Aside from the general considerations regarding the safety of seaweed polysaccharides, there is ongoing debate arising from several in vitro and in vivo assay reports [108–115]. Unfortunately, there is still a lack of standard methods, and there are only a few in vivo assays with fully characterized seaweed polysaccharides [3,96]. This was demonstrated by Kumar and Sharma [116], where several deaths and illnesses that had been attributed to consumption of seaweeds were found to be mainly due to wild harvesting at unsuitable (polluted) sites, unreasonably high consumption, and the noted presence of highly potent secondary metabolites/toxins (some microalgal bloom related).

However, despite these negative reports, which occur only rarely, judicial (i.e., in moderation) consumption of seaweed polysaccharides have overall positive effects on several aspects of human health. These polymers work as nutraceutical compounds, thereby

promoting human welfare and health. Indeed cautious and responsible consumption of seaweeds is no different from that of other terrestrial and marine food sources [117]. Taken alone, isolated seaweed polysaccharides have demonstrated numerous interesting properties for use in pharmaceutical and medical applications. In this regard, several specific seaweeds and their components are already in use commercially, while others are still in the research and development stage [117]. As commercial examples, the use of alginate in wound dressings, carrageenans in antiviral solutions, and agar in encapsulation of pharmacological drugs are all impressive [118–122]. In experimental development, selected seaweed polymers are being targeted mainly for the development of new hydrogel-based models for various human conditions, such as tumor or cardiovascular diseases, in order to provide more comprehensive models with which to understand drug and human cell interactions without using in vivo animal models, thereby providing more accurate/predictable responses [123]. This approach also enhances the development of new hydrogels for tissue engineering, where seaweed polymers have demonstrated good results in the early stage of development [124–126].

Seaweed polysaccharides can be applied as cosmetic ingredients, being used as gelling agents, thickeners, protective colloid emulsifier and stabilizer agents in hand lotions and liquid soap, deodorants, makeup, exfoliant, cleanser, shaving cream, facial moisturizer/lotion or in creams for acne and anti-aging care [127]. Similarly polysaccharide formulations can also be used in skin protection cosmetics to combat dermatitis, psoriasis, eczema, and dryness [128].

Carrageenans are one of the most bioactive polysaccharides from seaweeds; their chemical structure allows the formation of hydrogels, thereby allowing them to be used in anti-viral and anti-bacterial ingredients in various formulae [128,129]. There are compelling reasons for the use of these compounds, given the high levels of safety, efficacy and biocompatibility reported, in addition to their being biodegradable and non-toxic [118,130]. Furthermore, ancient records show that carrageenan has been used as a traditional medicine to ameliorate coughs and the common cold. These "old wives' tales" have been supported more recently by *in vitro* and *in vivo* assays using animal models. This functionality is mainly derived from the actions of carrageenans in inhibiting the aggregation of blood platelets (i.e., anticoagulant activity) [74,131]. Various carrageenans have other demonstrable bioactivities such as anti-tumor, anti-viral and immunomodulation activities [116,132], which are already being exploited commercially. The anti-viral mechanism is based on blocking the entrance of viral particles into the cell. Good results have been demonstrated against the herpes simplex virus type 1 and type 2, HIV-1, and the human rhinovirus [133,134]. These anti-viral activities have mainly been observed in iota-carrageenan (which is the carrageenan type produced by *C. jubata*) [53,59,133]. However, in pharmacodynamics, carrageenans that are harmful for human consumption (specifically, in the form of oligo-carrageenans or poligeenans) are regularly used as a pro-inflammatory factor in diverse *in vitro* and *in vivo* assays, due to the high inherent bioactivity when degraded to a low molecular weight [117]. Oligo-carrageenans can also be used to induce pleurisy, paw edema and ulceration in animal models, and as such, they are used as tools for medical research [135].

In contrast to carrageenans, agars and alginates are not recognized as bioactive molecules—instead they are seen as excellent polymers with reduced bioactivities (i.e., they are biologically inert) that can be inserted and used as a barrier/encapsulation to stabilize active ingredients and develop new biomedical and pharmaceutical methods and techniques [129,136]. Agar is used in pharmaceutical products such as a bulking and suspension ingredients for medical solutions, anti-coagulant agents, and laxatives in capsules and tablets [132,137]. Alginates are perhaps the most used seaweed polysaccharides in medical and pharmaceutical products already on the market, namely in wound and battle dressings, and also in wound-healing products in the form of hydrogels [34]. Alginates, when used in the biomedical and pharmaceutical areas, are linked to cations, such calcium, sodium or magnesium, to produce a biopolymer with no bioactivity and low toxicity that is

easy to manipulate so as to permit the development of hydrogels for tissue regeneration, as well as application in other areas such as in burn or diabetic wound-healing dressings [118].

However, seaweed polysaccharides have been further explored in drug delivery systems, where the polymers have demonstrated features such as natural biocompatibility, variation of viscosity and gelation conditions, low toxicity, low-cost polymers, and biodegradability, with easy adaptation and manipulation for the assembly of polymerderivatives with new physical characteristics [117,118,138]. Seaweed-derived polysaccharides have adaptable swelling properties that respond to temperature modifications, which is important for on-demand and time-dependent modulation of drug release [139]. In the post-rheology, pharmaceutical and medical arenas, seaweed polysaccharides must have a high level of purity in order to reduce the impact of potential inclusion of impurities in the application of polymers in products and solutions, permitting clean application without any associated health risks or hazards [140].

### **4. Materials and Methods**

### *4.1. Reagents*

The reagents used for carrageenan extraction, i.e., methanol, acetone, ethanol and sodium hydroxide, were acquired from the suppliers José Manuel Gomes dos Santos, Lda., Odivelas, Portugal; Ceamed, Lda., Funchal, Portugal; Valente e Ribeiro. Lda., Belas, Portugal and Sigma-Aldrich GmbH, Steinheim, Germany, respectively.

The reagents used for alginate extraction, i.e., sodium carbonate and hydrochloric acid, were purchased from Fisher Chemical, Leicestershire, United Kingdom.

### *4.2. Seaweed Collection*

Seaweeds belonging to the Rhodophyta, i.e., *Gracilaria gracilis* (Stackhouse) Steentoft, L.M. Irvine & Farnham 1995, and *Calliblepharis jubata* (Goodenough & Woodward) Kützing 1843 and the Ochrophyta, i.e., *Sargassum muticum* (Yendo) Fensholt, were collected during low-low tide the intertidal of Buarcos Bay, located in Figueira da Foz, Portugal (40◦10 18.6 N, 8◦53 44.4 W), Portugal. The seasonal sampling was conducted during 2020 (see Table 6) from sites with well-established seaweed populations without epiphytes or degradation visible to the eye. The specimens were collected from three different tidal pools at the same height on the shore. Approximately 100 g FW of *C. jubata,* 300 g FW of *G. gracilis*, 200 g FW of *S. muticum* were collected. After harvesting, each species was kept separately in plastic bags, inside a cool box, and transported to the laboratory (50 min from the harvest location). Firstly, the thalli were washed with filtered seawater to remove sand and other detritus. Thereafter, the samples were washed with distilled water, aiming for the removal of excess salts caused by the previous washing process and then placed on plastic trays and placed into an air-forced oven (Raypa DAF-135, R. Espinar S.L., Barcelona, Spain) at 60 ◦C, for 48 h. The dried algae were finely ground to make uniform (≤1 mm) samples with a commercial mill (Taurus aromatic, Oliana, Spain) and then stored in a box with silica gel to reduce humidity, in the dark, at room temperature (±24 ◦C).


**Table 6.** Seaweed collection data.

### *4.3. Polysaccharide Extraction*

### 4.3.1. Agar

Agar extraction was based on the technique reported by Li et al. [141], with adaptations. The extraction was performed in triplicate, using 20 g of dried seaweed and 600 mL of distilled water. Afterwards, the solution was placed in an electric pressure cooker

(Aigostar 300008IAU, Aigostar, Madrid, Spain) at a temperature of 115 ◦C with an air pressure of 80 Kpa, for 2 h. The solution was hot filtered, under vacuum, in a Buchner funnel, through a cloth filter. The extract was then vacuum filtered with a Goosh funnel (porosity G2). At room temperature, the filtrate was allowed to gel, frozen overnight and thawed. The thawed gel was finally dried (60 ◦C, 48 h) in an air-forced oven (Raypa DAF-135, R. Espinar S.L., Barcelona, Spain).

### 4.3.2. Carrageenan

The extraction of carrageenan was carried out in triplicate, according to the method defined by Pereira and van de Velde [142]. To remove the organic-soluble fraction, 1 g of milled seaweed was pre-treated with an acetone:methanol (1:1) solution at a final concentration of 1% (*m*/*v*) for 16 h, at 4 ◦C. The liquid solution was decanted, and the seaweed residues were collected and dried at 60 ◦C in an air-forced oven (Raypa DAF-135, R. Espinar S.L., Barcelona, Spain).

Dried samples were immersed in 150 mL of NaOH (1 M) in a hot water bath (GFL 1003, GFL, Burgwedel, Germany), at 85–90 ◦C, for 3 h. The solutions were hot filtered, under vacuum, in a Buchner funnel with a cloth filter. The extract was vacuum filtered with a Goosh funnel (porosity G2). Under vacuum, the extract was evaporated (rotary evaporator model: 2600000, Witeg, Germany) to one-third of the initial volume. The carrageenan was precipitated by adding twice the final volume of 96% ethanol. The polysaccharide was washed with 96% ethanol for 48 h at 4 ◦C and dried in an air forced oven (60 ◦C, 48 h).

### 4.3.3. Alginate

The extraction of alginic acid was performed in triplicate, employing the adjusted method of Sivagnanavelmurugan et al. [143]. Milled seaweed was added to a solution of HCl at 1.23% (1:30 v:v) and kept at room temperature for 48 h. The solution was filtered under vacuum with a Goosh funnel (porosity G2). The residue was rinsed with distilled water two to three times. The residue was submitted to an alkali extraction in 2% sodium carbonate for 48 h. The solution was filtered under vacuum through a cloth filter supported in a Goosh funnel (porosity G2), to remove any residues from the alginate solution. This process was followed by the addition of a solution of 37% HCl to the filtrate, producing the alginic acid precipitation (1 mL of 37% of HCl: 30 mL of the final solution). The precipitate was separated by centrifugation (4000 rpm, for 15 min) and then dried in an air forced oven (60 ◦C, 48 h).

### *4.4. Carbohydrate Characterization*

### Polysaccharide Analysis

For Fourier-Transform Infrared Spectroscopy–Attenuated Total Reflection (FTIR-ATR) analyses, the dried polysaccharides were powdered using a commercial mill, and then subjected to direct analysis. FTIR-ATR spectra were recorded on a Perkin Elmer Spectrum 400 spectrometer (Waltham, MA, USA), with no need for sample preparation, since these assays only require dried samples [52–54]. All spectra presented are the average of two independent measurements from 650–1500 cm−<sup>1</sup> with 128 scans, each at a resolution of 2 cm−1. The FTIR-ATR spectra in the manuscript were performed with the polymers extracted in the autumn.

### *4.5. Statistical Analyses*

The statistical analyses were performed using Sigma Plot v.14. This included an ANOVA analysis to assess statistically differences between the extraction yields. Holms-Sidak multiple comparison analysis was used after the rejection of the ANOVA null hypothesis, to discriminate any differences. The analyses were considered statistically different when *p*-value < 0.05. Error bars are the standard deviation of the mean.

### **5. Conclusions**

The seaweeds analyzed in this study demonstrated that wild harvested materials can indeed vary in terms of polysaccharide yield. Such variance could significantly change the nutritional value/properties on a seasonal basis. Therefore, the direct intake of seaweeds should be carefully analyzed.

Seaweed-derived polymers (polysaccharides) as food sources/ingredients are compared to dietary fiber due to their high molecular weight and because algal polysaccharides are not digestible compounds, being an important nutraceutical for good gastrointestinal functioning. However, if the polysaccharide is degraded (by over hydrolysis), the low-molecular-weight fractions can have negative impacts on human health. Likewise, over-dosage/consumption may be an issue, and thus moderation in all things is a key.

However, for the commodity food sector, there is a need to guarantee similar nutritional values in all supplies, independent of the season. In this context, seaweed cultivation can present a solution for controlling seaweed food safety; this is something in which Asian countries are already well practiced [144–148], and which Western countries need to learn and adapt to.

In the future, long-term assays should be conducted in different years to understand any fluctuations that may occur. Thus, seaweed cultivation (on land or in the sea) may provide more homogenous raw materials. In the nutraceutical/biomedical field, there is a need to understand the digestive part of the polymers in order to provide greater security for the consumption of seaweed polymers. As demonstrated, there is a need to understand the ecological factors affecting seaweed biomass in order to obtain safe and high-quality polymers to support their many applications in the food industry.

**Author Contributions:** Conceptualization, J.C., D.P., G.S.A., A.V., A.T.C. and L.P.; Seaweed laboratory work, J.C., D.P., G.S.A.; writing—original draft preparation, J.C., D.P., G.S.A.; writing—review and editing, J.C., D.P., A.V., A.T.C. and L.P.; supervision, A.V., A.T.C. and L.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data available from authors.

**Acknowledgments:** This work was financed by national funds through FCT (Foundation for Science and Technology), I.P., within the scope of the projects UIDB/04292/2020 (MARE, Marine and Environmental Sciences Centre). Diana Pacheco thanks to PTDC/BIA-CBI/31144/2017-POCI-01 project -0145-FEDER-031144-MARINE INVADERS, co-financed by the ERDF through POCI (Operational Program Competitiveness and Internationalization) and by the Foundation for Science and Technology (FCT, IP). João Cotas thanks to the European Regional Development Fund through the Interreg Atlantic Area Program, under the project NASPA (EAPA\_451/2016).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


## *Review* **Microalgal Peloids for Cosmetic and Wellness Uses**

**M. Lourdes Mourelle \*, Carmen P. Gómez and José L. Legido**

FA2 Research Group, Applied Physics Department, University of Vigo, 36310 Vigo, Spain; carmengomez@uvigo.es (C.P.G.); xllegido@uvigo.es (J.L.L.) **\*** Correspondence: lmourelle@uvigo.es; Tel.: +34-696413531

**Abstract:** Peloids have been used for therapeutic purposes since time immemorial, mainly in the treatment of locomotor system pathologies and dermatology. Their effects are attributed to their components, i.e., to the properties and action of mineral waters, clays, and their biological fraction, which may be made up of microalgae, cyanobacteria, and other organisms present in water and clays. There are many studies on the therapeutic use of peloids made with microalgae/cyanobacteria, but very little research has been done on dermocosmetic applications. Such research demonstrates their potential as soothing, regenerating, antioxidant, anti-inflammatory, and antimicrobial agents. In this work, a method for the manufacture of a dermocosmetic peloid is presented based on the experience of the authors and existing publications, with indications for its characterization and study of its efficacy.

**Keywords:** peloids; microalgae; cyanobacteria; cosmetics; dermocosmetics; mineral water; seawater

### **1. Introduction**

Peloids are therapeutic agents used in spas and thalassotherapy centers since time immemorial, mainly for treatment of osteo-articular and dermatological disorders, sports injuries, and generally in rehabilitation programs. Their use in cosmetics also dates back a long time, especially the ones made from clays, which are used in wellness programs and thermal spa centres nowadays [1].

Peloids are comprised of a solid fraction that includes various sediments, clays and peat, and a liquid fraction that can be either mineral-medicinal water (mineral water), seawater, or salt/brackish lake water. A biological fraction, consisting of microbiota present in mineral-medicinal water, clays, peat or sediments, and the microorganisms that thrive in the mixture during the peloid maturation processes, can also be present [2]. It is precisely during this maturation process (prolonged contact between solid substrate and liquid) that the different biological action compounds, partly responsible for the therapeutic actions, are formed [3].

Peloids either form "in situ" through contact between the mineral-medicinal water and the sediments surrounding it or are prepared artificially by mixing the above components [4]. When preparing peloids artificially, the biological fraction (microalgae and cyanobacteria) is usually from the natural mineral-medicinal water, while in the case of marine silt peloids, it is from cultivated microalgae; maturation times vary from 1–18 months but usually do not exceed 3 months [5]. According to Gomes et al. (2013), peloids can be classified regarding their origin, composition, and applications into "natural peloid" or "peloid *sensu strictu*"; "inorganic," "organic," or "mixed peloids"; and "medical" or "cosmetic" peloids (Figure 1) [4]. During the 3rd Symposium on Thermal Mud, held in 2004 in Dax, it was agreed to distinguish between the two main types of peloids: (i) muds or clays that are just mixed with mineral water with no maturation process—the extemporaneous or prepared *ad hoc* peloids—and (ii) muds or clays mixed with mineral water, including naturally or artificially matured peloids. Figures 2–5 show two different types of natural-maturation and artificial-maturation peloids [4].

**Citation:** Mourelle, M.L.; Gómez, C.P.; Legido, J.L. Microalgal Peloids for Cosmetic and Wellness Uses. *Mar. Drugs* **2021**, *19*, 666. https://doi.org/ 10.3390/md19120666

Academic Editor: Orazio Taglialatela-Scafati

Received: 21 October 2021 Accepted: 24 November 2021 Published: 26 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

In order to evaluate peloid suitability for therapeutic and cosmetic purposes, the thermal properties of the mixtures like density, specific heat, thermal conductivity, and retention capacity are studied, as well as other properties related to applicability such as viscosity and pH [6].

Applicability, spreadability, user, and efficacy tests should be performed when used in dermocosmetics and/or wellness programs in thermal spas and thalassotherapy centres. Marketing of cosmetic peloids must comply with national legislation, which usually includes safety reports, user and efficacy tests, etc.

Thermal spas and wellness centers seldom use microalgae peloids in dermocosmetics, which is why this study reviewed such peloids and proposed a method for their manufacture. Therefore, the intention was to encourage spas to manufacture their own products for use in cosmetic and wellness applications through the required research and experience.

**Figure 1.** Peloid classification with regard to origin, composition, and applications (from Gomes et al. 2013).

**Figure 2.** Natural maturation peloid (saline mud from Seˇcovlje salt pans, Seˇcovlje Nature Park, Slovenia).

**Figure 3.** Cyanobacteria cultivation for peloid preparation (Dax, France).

**Figure 4.** Tanks for artificial peloid maturation (Dax, France).

**Figure 5.** Thermal mud maturation (Dax, France).

### **2. Peloids for Dermocosmetics and Wellness**

For this review, SciFinder, Pubmed, Web of Science, and Scopus databases were reviewed up to September 2021. Search terms included "pelotherapy," "mud therapy," "peloids and skin," "thermal mud," "microalgae and thermal water," "cyanobacteria and thermal water," mud and cosmetics," "mud and dermocosmetics," "mineral water and skin," and "seawater and skin".

Although frequently used empirically, peloids have important cosmetic actions, which are linked to the improvement of skin hydration, the removal of flaking cells, and the prevention of aging [7–9]. Traditionally, the types of peloids most used in cosmetics are volcanic, sulphurous, and chlorinated bromo-iodics, but this also includes peat due to its content of fulvic and ulmic acids [7].

There is also evidence of their action in treating dermatological diseases such as psoriasis and other skin disorders, an example being that of Dead Sea peloids [10], in which it has been observed to reduce all skin symptoms of this disease (PASI index) [11] when combined with Dead Sea water and phototherapy. This water is furthermore observed to have antimicrobial action [12], an aspect of interest in the treatment of related dermatological alterations such as dermatitis. Other studies have shown that these muds can improve wound healing [13]. The effects of biogleas in thermal muds from Guardia Piemontese-Acquappesa were studied and found to significantly reduce desquamation, erythema, and itching in psoriasis [14].

Similar muds used in skin disorder applications are the Peruíbe muds [15] for psoriasis, dermatitis, acne, and seborrhoea. Peloids from Balaruc-les-Bains have been recently used for their anti-inflammatory, antioxidant, and healing properties [16]. Additionally, Spilioti et al. (2017) investigated the anti-inflammatory properties of 13 mud samples from Greek spa resorts by assessing their effect on the expression of the adhesion molecules ICAM-1 and VCAM-1 by endothelial cells as well as their effects on monocyte adhesion to activated endothelial cells. Most of the mud extracts used in the study inhibited TNF-ainduced expression of VCAM-1 by endothelial cells but showed little alteration on ICAM1 expression. Interestingly, the majority of the examined mud extracts markedly reduced

monocyte adhesion to activated endothelial cells indicating a potent anti-inflammatory activity [17].

In terms of peloid composition for cosmetic and welfare purposes, many studies attribute their curative properties to their clay (less frequently peat or sapropels), mineral and trace elements content, and to the presence of microalgae and cyanobacteria.

### *2.1. Clays and Dermocosmetic Peloids*

The use of clays in the preparation of peloids and dermocosmetic products have been studied by a great number of authors [1,3,18–27]. The main phyllosilicates present in most peloids are smectites, kaolinite, illite, illite–smectite mixed layers, and chlorite in different proportions [21].

Although there are fewer studies published on the composition of sapropels from Lake Techirghiol in Romania [28] and from lakes in Latvia, there are some studies that evaluate their potential medicinal and cosmetic use [29].

A comparative physico-chemical composition study of muds from different areas in the Homogeneous Euganean Hills Hydromineral Basin (B.I.O.C.E.) (Italy) reported the composition of peloids as "clayey-silt" (65.42% silt and 24.62% clay) and "silty-clay" (64.37% clay and 34.41% silt). Their heavy metal content was studied by comparison with commercial cosmetic mud and was found to be higher than in commercial mud; however, no allergic reactions were detected. A proposal to establish a protocol for effective control of these types of natural products has been put forward [30].

### *2.2. Minerals and Trace Elements in Dermocosmetic Peloids*

Peloids for dermocosmetic and wellness applications are characterised by their varied composition in minerals and trace elements. The moisturising, soothing, and regenerating properties of the Dead Sea mud are attributed to a high magnesium content [31], which is well known for its anti-inflammatory and antiphlogistic effects and for its capacity to inhibit the polyamines involved in psoriasis pathogenesis [7]. Dead Sea mud also exhibits antimicrobial action, which is attributed to the high salt and sulphide concentrations plus its low pH, and it is therefore used in the treatment of acne [31].

In the case of the above-mentioned Peruíbe peloids, Da Silva-Cardoso et al. (2015) noted that the mud is enriched with Br, Cr, Sb, SE, and Zn ions during the maturation process and that these may be responsible for their anti-inflammatory properties [15,32].

### *2.3. Microalgae and Cyanobacteria in Dermocosmetic Peloids*

One of the most outstanding and studied characteristics of peloids is their content in microalgae and cyanobacteria, which seem to exert a great influence on their cosmetic properties, since they have been proven to generate biologically active substances (especially during the maturation process), which in turn are responsible for the beneficial effects and actions on the skin [33].

There is abundant recent scientific literature on the biological fraction of peloids, and worth highlighting among them are studies on Euganea basin muds in the Spa area of Abano Terme (Italy). Thus, Ceschi-Berrini et al. in 2004 [34] described the presence of the genus *Phormidium* in thermal waters of the Euganea basin and subsequently identified the presence of acylglycerolipids produced by the aforementioned cyanobacteria, which appeared to confer therapeutic and cosmetic properties to the mud [35]. In a study of microbial diversity in the same area, Moro et al. (2007) [36] described a new species of Cyanoprokaryote called *Cyanobacterium aponinum* in the microbial mats of Euganean thermal springs. Subsequently, Poli et al. (2009) [37] described a thermophilic bacterium in the mud from this thermal basin that they called *Anoxybacillus thermarum*, which provides an idea of the special characteristics of the biological composition of these muds. Additional studies by Moro et al. (2010) expanded the biodiversity of these muds to species of the genus *Leptolyngbya* and *Spirulina* (now *Arthrospira*), suggesting that the cyanobacterial composition of phototrophic mats in the rather unusual environment of the Euganean Thermal District is variable, depending on the physico-chemical features of the different thermal spa waters. In fact, surveys carried out on 90 thermal spas suggest that the cyanobacterial diversity might be related to thermal mud processing in the different maturation tanks with thermal waters at different temperatures [38].

Research on the biological composition and organic matter present in the different maturation stages of Abano muds showed the presence of saturated and unsaturated fatty acids, hydroxyl acids, dicarboxylic acids, ketoacids, and alcohols and an increase in the lipid profile during the maturation process that peaked at six months. The presence of diatoms from clays was observed at the start of maturation; however, cyanobacteria belonging to the Oscillatoriales subsection progressively colonized the mud throughout maturation [39].

Centini et al. [40] recently analyzed the composition and antioxidant capacity of biogleas present in the Satunia Terme mud and confirmed earlier findings on the increased lipid profile during the maturation process and analyzed the hydrophilic fraction. Studies on antioxidant power revealed that bottom mud extracts are more active than surface extracts and that hydrophilic extracts are more active than lipid extracts.

A comprehensive study using more than 650 mud samples from 29 places in the Abano area compared mineralogical and geological parameter variations with chlorophyll A in sludge during the mud maturation and recycling process. The conclusion was that chlorophyll A is converted into its derivatives and generates molecules that pass to the matured mud. Such a decrease in the chlorophyll A amount warrants maturation to take place in open tanks in order to maintain the photosynthetic process and to ensure that the amount of chlorophyll A and its derivatives continue to be sensitive to the supply of fresh mud [41]. Subsequent research by Gris et al. (2020) on the same muds (Euganean Thermal Muds) confirmed that the predominant species is *Phormidium* sp. and that diversity is greater when the temperature is 37–47 ◦C. At lower and higher temperatures, populations lose stability, thus exhibiting a significant change in species composition, low biodiversity, and low cyanobacterial abundance [42]. Zampieri et al. (2020) likewise noted the antiinflammatory activity of exopolyssacharides from *Phormidium* sp present in the Abano muds [43].

Studies carried out on mud from Pausilya Therme di Donn'Anna (Italy) revealed antimicrobial capacity and identified seven taxa of green algae, two taxa of cyanobacteria, and even diatom taxa. In terms of the microalgae community, mud samples ripened for 6 months (6-month mud) presented a higher biodiversity compared to mud allowed to ripen for 1 month (1-month mud). The most abundant benthic microalgae taxa, identified in both samples and isolated exclusively from ripened mud, are *Chlorella* sp., *Coccomyxa* sp., *Scenedesmus* sp., *Leptolyngbya* sp., *Anabaena* sp., *Cocconeis placentula*, *Rhoicosphenia abbreviata* and *Navicula cincta*. *Nostoc* sp., *Scenedesmus* sp., *Chlamydomonas* sp., *Pseudococcomyxa simplex*, *Monodus* sp., *Gomphonema acuminatum*, *Amphora ovalis*, and *Nitzschia palea* [44].

In a like manner, the microbiological diversity of waters and muds from Sirmione Terme was characterised (using next-generation sequencing technology) by studying the different mud maturation stages: young (2-month old), intermediate (4-month old), and mature (6-month old). The results showed that three genera predominate: *Pelobacter*, *Desulfomonile*, and *Thiobacillus* and that *Pelobacter* levels increase during maturation while those of *Desulfomonile* and *Thiobacillus* decrease. The increase in phospholipid and sulpho- glycolipid fraction of mature muds reported by other authors [45] was attributed to *Pelobacter* by these authors.

Muds from Balaruc-les-Bains (France) have also been analyzed to study the molecules responsible for their antioxidant, anti-inflammatory, and healing properties. Nine strains were analyzed and although no antioxidant activity was detected, a strong anti-inflammatory potential was observed for *Planktothricoides raciborskii*, *Nostoc* sp., and *Pseudo-chroococcus couteii*, and a slight wound-healing function was detected in extracts from *Aliinostoc* sp. [46], which is an activity of great interest in dermocosmetic and well-being treatments. Recent studies using morphological, ultrastructural, and molecular methods clearly identified the

nine cyanobacterial isolates from the Thermes de Balaruc-Les-Bains muds as belonging to the orders Chroococcales: *Pseudochroococcus coutei*; Synechococcales: *Leptolyngbya boryana*; Oscillatoriales: *Planktothricoides raciborskii*, *Laspinema* sp., *Microcoleus vaginatus*, and *Lyngbya martensiana*; and Nostocales: *Nostoc* sp., *Aliinostoc* sp., and *Dulcicalothrix* sp. [47,48].

Dead Sea muds are well known for their use in the treatment of psoriasis [49]. They are high-salinity muds in which nine extremely halotolerant Bacillus species have been identified, one of them being *B. Paralicheniformis*, which confer a high antimicrobial action [50]. Subsequent studies have confirmed the antimicrobial property of *Bacillus persicusi* against different Gram+ and Gram− pathogens [51].

Organic fractions of mud from other environments have also been studied. Dolmaa et al. (2017) studied silty mud containing sulphide from Noggon Lake (Mongolia) and found that soluble organic matter contains a high percentage of hydrocarbons and their derivatives (33.68%) and that the lipid group contains fat-soluble vitamins including vitamins A, D, E, and their derivatives, plus steroids, which the authors relate to therapeutic and cosmetic properties [52].

Bigovic et al. (2019) examined the organic composition of Igalo Bay peloids (Montenegro), and they found them to mostly contain (saturated and unsaturated) fatty acids as well as essential amino acids, many of which have significant physiological, medical, and pharmaceutical properties [53].

Research carried out on the mineral peloids from Mariánské Lázne (Czech Republic) reported a new species of the genus *Aquitalea* (previously identified in humic lakes and peat marshes), which they called *Aquitalea pelogenes* ("derived or generated from mud"). They also found a profile of quinones and fatty acids upon analyzing the dry biomass. The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids, and one unidentified aminophospholipid, to which the authors attributed the therapeutic properties [54].

Other studies reported changes in the microbial community composition of the peloid throughout maturation, wherein main changes take place in the early stages, with there being hardly any change between 3 and 6 months [55].

### *2.4. Safety of Peloids for Application in Dermocosmetics*

Given that peloids are applied topically and in many cases on skin with dermatological disorders, their safety must be monitored for the possible presence of trace toxic metals and pathogenic microorganisms.

Ma'or et al. (2015) studied the safety of Dead Sea muds used in cosmetics, by evaluating traces of nickel and chrome, and concluded that nickel and chrome concentrations measured in the mud are safe for human health insofar as systemic toxicity is concerned. They also observed that skin exposure to nickel and chrome is much lower since both metals mainly attach to the clay components in mud and are not easily released into the aquatic solution. The use of Dead Sea mud is not recommended for Ni−- or Cr−-sensitive persons [56].

Recently, Pavlovska et al. (2017) recommended testing in natural peloids (to be used as a raw material for pharmaceutical applications) not only heavy metals but also pesticides such as chlororganics, which are widely used as effective help to combat unwanted plant pests and pathogens and which have bioaccumulation and bioconcentration capabilities [29].

To ensure the quality and safety of the peloids, some properties should be determined; the most common are granulometry, plasticity, CEC and exchangeable cations, water content, pH, specific surface area, swelling power and swelling index, abrasiveness, density, rheological properties (viscosity), and thermal properties such as: specific heat capacity, thermal conductivity, thermal diffusivity, and thermal retentivity. For cosmetic uses could be also of interest to determine the parameters of hardness, springiness, adhesiveness, and cohesiveness, which are related to their visco-elastic properties [3]. From the microbiological quality and hygiene perspective, microbiological analyses such as total viable

count (TVC), total coliforms, *E. coli*, enterococci, *S. aureus, P. aeruginosa*, and sulfite-reducing clostridia and dermatophytes fungi, must also be carried out [57].

### **3. Proposal for a Procedure to Manufacture Microalgae Peloids**

This work puts forward a method that uses clays, microalgae/cyanobacteria, and mineral-medicinal water or seawater to manufacture peloids for use in cosmetics and in health and wellness programs at wellness centers.

Such peloids can be manufactured in the thermal spa itself for use with patients on the premises. Some examples of use in Europe are Abano Terme and Montecatini Terme (Italy), Dax Thermes, Eugenie-les-Bains or Barèges (France), Bad Bayersoien (Germany), and the thermal spas of Archena, Bohí, and El Raposo in Spain, these being mainly used to treat rheumatology and locomotor system disorders. Worth highlighting in Spain are the spas at Isla de la Toja and Balneario de Compostela that manufacture their own peloids for use in dermatology and dermocosmetics, with interesting results in psoriasis and dermatitis [58,59]. In such cases, the peloid is considered as a spa product derived from mineral-medicinal water and is governed by the spa legislation of each country. An example is shown in Figure 6.

However, if the product is destined for marketing as a cosmetic product, it is governed by cosmetic regulations. REGULATION (EC) No 1223/2009 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 30 November 2009 on cosmetic products (https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:02009R1223-20190813 accessed on 21 October 2021) defines the stages involved in the manufacture and marketing of a cosmetic product in Europe, including the lifecycle of a cosmetic product, from its conception in R&D laboratories to the monitoring of its effects and effectiveness after marketing.

**Figure 6.** Manufactured peloid; application for psoriasis and dermatological conditions (La Toja thermal spa, Pontevedra, Spain).

### *3.1. Composition of a Peloid*

A peloid is comprised of a solid fraction or substrate made of clays, sediments, or peat and a liquid fraction made of mineral-medicinal water, seawater, or brackish/salt-lake water, and it may contain a biological fraction from the water or the solid substrate [2,8]. When manufacturing peloids for dermocosmetic purposes, one should use high-quality clays to guarantee safety and effectiveness on skins, which in many cases are damaged. Their composition is shown in Figure 7.

**Figure 7.** Composition of a peloid (MM: mineral-medicinal).

### 3.1.1. Solid Substrate: Clays

As indicated earlier, the solid component of a peloid can be diverse. In order to achieve good thermo-physical characteristics and applicability, we propose the use of clays containing smectite (bentonite) and kaolinite, since the former have very good plastic properties [21] and the latter help regulate skin secretions and the final pH of the mixtures [8].

### 3.1.2. Solid Substrate: Mineral-Medicinal Water and Seawater

Each mineral-medicinal water is unique and thus the first step is gaining knowledge of its chemical composition, including the majority and trace elements, as well as physicochemical characteristics such as pH, electrical conductivity, and the possible presence of dissolved gases.

All mineral-medicinal waters must be analyzed periodically to guarantee quality before and during their application in spas, as provided for in the legislation of the different countries. This is why all of them are analyzed and quality is guaranteed. However, given that often only the major elements are analyzed, it is of utmost importance to analyze the trace elements when developing peloids for cosmetic us since their dermocosmetic potential lies in them [60]. Table 1 summarizes the principal majority and trace elements in mineral-medicinal waters of interest for the manufacture of cosmetic products.

**Table 1.** Majority and trace elements in mineral-medicinal waters that have an effect on the skin (Mourelle & Gómez, 2015).



**Table 1.** *Cont.*

### 3.1.3. Microalgae and Cyanobacteria

They are one of the differential components in a peloid; and given that each mineralmedicinal water is unique, one needs to study the type of microalgae/cyanobacteria present therein. The plankton composition in seawater differs through latitudes unlike the composition of seawater, which is similar at all latitudes, and hence one needs to study the type of microalgae present in a particular environment.

We therefore propose that microalgae/cyanobacteria cultures be sourced from the mineral-medicinal waters or seawater, by means of a process adapted to the characteristics of each species or genus predominant therein. The culture process includes growth in an appropriate medium (mineral-medicinal or seawater) with the necessary nutrients and light stimulation depending on the type of species (Figure 8).

**Figure 8.** Photobioreactor with microalgae (cultivation at FA2 lab; Applied Physics Department; University of Vigo).

### *3.2. Preparation of a Dermocosmetic Peloid with Microalgae*

The process of preparing a peloid with microalgae or cyanobacteria involves a few prior stages in which raw materials are first studied before carrying out tests on the mixtures. The stages are summarised in Figure 9 and in the following subsections: (i) selection of raw materials (clays, thermal waters, and microalgae cyanobacteria); (ii) characterization of raw materials (different test and determinations to asess its suitability and optimal properties); (iii) preparation and testing mixtures (using different proportions of the raw materials); (iv) characterization of the peloid sample (including maturation process if necessary); and (v) use and effectiveness test.

**Figure 9.** Peloid manufacture: procedure and test.

### 3.2.1. Selection of Raw Materials

Raw materials or initial materials (clays, microalgae, and waters) are selected for the intended purposes. Given that the peloid is intended for dermocosmetic and/or welfare uses, the clays selected must be of a high quality and have an affinity for the skin (kaolinites, bentonites, etc.). The water used is the one present at the spa: mineral-medicinal water (or seawater), and the microalgae/cyanobacteria can either be those present at the thermal spa or the thalassotherapy center, but others acquired lyophilized or frozen can also be used.

### 3.2.2. Characterization of Raw Materials

All raw materials must be properly characterised. The most frequent tests performed on clays are mineralogical analysis; chemical composition; granulometry; SEM study; swelling; cation exchange capacity and exchangeable cations; percentage of water, solids, and ash; and differential thermal analysis and thermogravimetry [6,21,61–63].

The spa water or seawater must also be analyzed to study the majority and trace elements, in addition to other physico-chemical analyses. The most important parameters are temperature, electrical conductivity, dry residue, turbidity, cations and anions, dissolved gases, radioactivity, hardness, and alkalinity. One also needs to study properties such as density, thermal conductivity, specific heat, viscosity, and thermal diffusivity [63–65].

It is furthermore important to characterize microalgae or cyanobacteria and undertake studies to isolate and obtain a mono-specific and clonal culture. The sample is characterized through a chemical analysis, determination of crystalline phases, and by studying its composition (proteins, lipids, carbohydrates, vitamins, etc.) [66].

### 3.2.3. Preparation and Testing of Mixtures

Mixtures are prepared using different proportions of the three raw materials and tested for texture, spreadability, ease of application, etc.

The mixtures are then selected, characterized, and subject to use and efficacy tests.

### 3.2.4. Characterization of the Peloid Sample

The most common analyses carried out on the sample of the selected peloid or mixture are density, thermal conductivity, specific heat, viscosity, rheological behavior, and thermal diffusivity [62,65,67,68]. For a peloid to be suitable for pelotherapy uses, it should have several properties, such as a low cooling rate, a high absorption capacity, a high cationic exchange capacity, good adhesiveness, handling easiness, and a pleasant feeling when applied to the skin. Among all the above properties, the cooling rate is one of the most critical ones, since the heat contributed by the peloid also plays a role as a therapeutic agent. In many therapeutic applications, therefore, the peloid must be kept at a higher temperature than that of the patient's body during application [6].

If peloids need maturing, then one must also establish the temperature, light, agitation, etc. conditions. In any case, the characterization analyses are the same, and samples need to be taken after 15 days, 1 month, 2 months, etc. until the maturation process is complete and no further changes in the physico-chemical parameters are observed [39,55,61,69,70].

### 3.2.5. Use and Effectiveness Tests

Different analyses and tests are carried out on volunteers to evaluate user acceptance of the peloid and its effectiveness. Inclusion and exclusion criteria for both tests must be established, taking into account that these preliminary studies are carried out on healthy persons. Additional controlled clinical trials must be done if the peloid is finally destined to treat skin conditions such as psoriasis, dermatitis, etc.

The use test consists of a set of questions related to texture, ease of application, sensations during and after application, skin condition after product removal, etc. In Figure 10, an example of a microalgal peloid is shown.

**Figure 10.** Application of microalgal peloid (Talaso Atlántico, Baiona, Pontevedra, Spain).

Efficacy studies are usually objective determinations done through skin biometrology techniques, such as hydration (by corneometry), grade of sebum (with sebumeter), skin elasticity (cutometry or elastometry), and, sometimes, transepidermal water loss [71–73].

### **4. Conclusions**

Peloids have been used for therapeutic purposes since time immemorial, mainly in the treatment of locomotor-system pathologies and dermatology. Their effects are attributed to their components, i.e., to the properties and action of mineral waters, clays, and their biological fraction, which may be made up of microalgae, cyanobacteria, and other organisms present in water and clays. Different studies show that the biological fraction and the maturation process (in which components remain in contact for a certain length of time) contribute to the formation of biologically active compounds.

Even though there are many studies on the therapeutic use of peloids made with microalgae/cyanobacteria, very little research has been done on dermocosmetic applications. Such research demonstrates their potential as soothing, regenerating, antioxidant, anti-inflammatory, and antimicrobial agents. Their effect is related to the presence of unsaturated fatty acids, acylglycerolipids, sulfoglucolipids, vitamins, alcohols, phenols, etc., as well as sulphur derivatives, minerals (Ca, Mg, etc.), and trace elements (Zn, Se, Si, etc.).

Each thermal spa has a unique natural mineral water with specific physico-chemical characteristics, which are the basis of their therapeutic actions (along with other mechanisms related to the application technique). Moreover, specific microbiota consisting mainly of microalgae and/or cyanobacteria are often found in it. This is why thermal spas, thalassotherapy centres, and wellness centres in general should progress towards making their own dermocosmetic products using their natural mineral water or seawater; a solid substrate, preferably clay; and the microalgae/cyanobacteria. Hence, a method for the manufacture of a dermocosmetic peloid was presented based on the experience of the authors and existing publications, with indications for its characterization and efficacy study.

**Author Contributions:** Writing—original draft preparation, M.L.M.; writing—review and editing, M.L.M.; C.P.G., and J.L.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

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