Ulva Seaweed-Derived Ulvan: A Promising Marine Polysaccharide for Biomaterial Design

1. Introduction

In recent years, seaweed, or macroalgae, has attracted significant attention for its potential as a sustainable source of food and non-food industries, such as food additives, animal feeds, pharmaceuticals, nutraceuticals, cosmetics, textiles, biostimulants, bioplastics, and biofuels [1,2,3,4,5,6]. Seaweed are simple aquatic plants that thrive in diverse environments, ranging from freshwater to highly saline oceans [7]. From an environmental point of view, seaweed is a highly autotrophic plant [8]. Through photosynthesis, seaweeds produce significantly more organic matter and consume CO₂. The photosynthetic efficiency of seaweeds was 3–5 times higher than terrestrial plants [7,9] making it highly effective in converting sunlight and CO₂ into biomass. The carbon uptake rates reach 8–20 tons per hectare/year [4,8,9] contributing to carbon sequestration and helping mitigate climate change. Furthermore, the biomass productivity of seaweed was greater than terrestrial biomass [10]. For instance, the yield of brown seaweed was 225 tons dry weight/Ha/year, 2.3 yield was 2.3 folds higher than sugarcane [9]. In addition, seaweed has lower lignin content than land plants [6,11,12,13] so the process of extracting seaweed components will be easier with lower energy input.

Based on the pigments contained in their thallus, seaweeds are classified into three major groups: red seaweeds (Rhodophyta), green seaweeds (Chlorophyta), and brown seaweeds (Phaeophyta) [4,7]. Currently, red, and brown seaweed are widely known both in terms of the number of species and their commercial production. Suntana et al. reported that the number of red and brown seaweed species reached 4000 and 1500 species respectively, while green seaweed only 900 species [14]. Apart from being obtained from the wild, several types of seaweed can also be cultivated well [15]. Seaweed cultivation offers several advantages over terrestrial plant farming, making it a sustainable and environmentally friendly choice. On seaweed cultivation, there is no need for arable land [15,16] and reduce competition with terrestrial agriculture which ends the need for arable land and reduces competition with terrestrial agriculture [6]. In addition, unlike terrestrial plants, seaweed does not require fresh water or chemical fertilizers [16], conserving valuable resources and minimizing pollution.

Green seaweed, especially Ulva, is one of the promising species in terms of availability, sustainability, and potential applications and uses. Ulva is commonly known as sea lettuce and is seen worldwide [17,18]. Remarkably, Ulva shows adaptability to various salinities, growing well in marine and brackish water and freshwater [19,20]. Some species of Ulva caused green tide bloom. Green tide blooms occur more global seawater eutrophication and temperature rise [21]. The largest Ulva-forming green tide happened in the Yellow Sea of China, covering 1746 km² and producing over 24 million tons of biomass [22]. Ulva thrives in diverse habitats, including tidal pools and rocky shores, and can grow optimally in water temperatures of 20 – 30 °C [17,23,24]. Daily growth rate (DGR) in the open sea was 20.1 ± 1.8 % day⁻¹. Interestingly, it was cultivated in the pond or tanks, the DGR reached 27.9 ± 4.4 % day⁻¹ [18]. Studies have also shown that Ulva achieves higher growth rates than other seaweed massively cultivated such as Porphyra umbilicalis, Chondrus crispus, and Laminaria saccharina [25,26].

Ulva is composed of mainly carbohydrates (47 – 67 %) [27] consisting of starch (4 %), cellulose (9 – 10 %), hemicellulose (14 – 32 %) and Ulvan (13 – 39 %). The protein content of Ulva was (12 – 30 %) and fat (1 – 4 %). Like seaweed, Ulva also has ash (21 – 45 %) [27,28,29,30] consisting of macro and micro minerals. Ulva sp. as a nutraceutical agent can be used as food that shows beneficial physiological functions, improves well-being, and reduces the risk of certain diseases, including inflammatory disorders, cancer, bacterial infections, and viral infections; it also acts as an immunomodulating and hypolipidemic agent [31]. Generally, Ulvan is extracted from seaweed by conventional methods carried out separately. This process will produce waste and environmental problems. Through the concept of integrated biorefinery [6,29,30,32,33,34,35], the production of ulvan with various byproducts will be able to increase the added value of the Ulva seaweed industry. Through the biorefinery approach, process efficiency can be increased, for example increasing from 26% to 39%, as well as cellulose as a by-product increasing from 12% to 39% [28]. Although several papers have comprehensively reviewed ulvan [31,36], to the best of our knowledge, there are few reported concepts of biorefinery concept with green extraction ulvan which focuses on ulvan characteristics for biomaterial purposes. Moreover, there are no studies that have revealed its transition from resource-extraction understanding-based research to biomaterial design application-focused work while addressing the chemical and biological engineering aspects of its exploration. In line with this knowledge gap, this review further explores the comparative advantages of ulvan by comparing its properties in relation to other seaweed-derived polysaccharides and examining the challenges it faces in future applications for biomaterial design across various fields.

2. Ulva, Structural Characteristics, and Extraction of Ulvan

2.1. Ulva

As is generally observed in the seaweed, the entire body of Ulva is known as a thallus which is composed of hold fast, stipe, and blade standing for the function of root, stem, and leave in the higher plant such as seagrass. The size of the thallus reaches 100 cm. The color of the thallus ranges from bright green to dark green, depending on the species and environmental conditions. The dominance of chlorophyll pigments causes green color. The shapes of the thallus are thin, flat, and sheet-like resembling lettuce leaves. Some species may appear tubular or filamentous [15]. Examples of species, color and shape are presented in Figure 1.

2.2. Ulvan Chemical Structure characteristic

The chemical structure of ulvan polysaccharide consist of repeating sugar which unique to Ulva species (Figure 2). The major monosaccharide sugars are rhamnose, glucuronic acid, xylose, iduronic acid, and galactose. Sulfate group is attached in all rhamnose and some xylose [37]. Based on thallus morphology, Ulva divides into blade and filamentous. The nomenclature of repeating disaccharide is first initiated by Kidgell et al. [36]. The repeating sugar in ulvan polysaccharide of blade and filamentous Ulva are different, resulting different sugar composition. In their new study, it was found that in the filamentous U. ralfsii and U. prolifera, sulfate also found in glucuronic acid [38]. Besides sulfate, ulvan also have carboxyl group on uronic acid in the form of glucuronic acid and iduronic acid. A lot of hydroxyl group makes ulvan hydrophilic and water-soluble.

2.3. Ulvan Extraction Strategy

In ulvan extraction, the quantity and quality of the extracted ulvan depend on the extraction method, purification process, and biomass sources. The ulvan polysaccharide extraction method is selected based on the properties of the ulvan molecule, its physicochemical characteristics, and its interactions with other components [39]. Broadly, ulvan extraction can be categorized into three approaches: physical, chemical, and enzymatic. The extraction process primarily focuses on ulvan due to its high abundance. However, co-extraction during the process often results in the presence of salts, proteins, pigments, and other polysaccharides, such as cellulose, glucuronan, xyloglucan, and starch. The intended final product plays a critical role in determining the specific steps of ulvan extraction. Figure 3 illustrates the schematic of ulvan extraction from Ulva. In this work, we will unravel the steps of ulvan extraction, emphasizing yield, strategies, and the management of side products in the context of Ulva biorefinery.

In the biorefinery concept, the goal is to optimize the use of biomass resources by maximizing benefits and profitability while minimizing waste [40]. As previously mentioned, Ulva holds significant potential, with ulvan being its most unique and valuable component. Therefore, focusing on ulvan as the primary extraction target, while managing other potentials through sequential extraction, presents a highly attractive approach. In the context of ulvan biorefinery, side products can be categorized into co-products, ulvan itself, and residual products.

The co-products in Ulva include salts, pigments, lipids, and proteins. Extracting co-products helps reduce co-extracted impurities in ulvan, thereby increasing its yield. Since Ulva is harvested from the sea, it contains a high salt content, which can impact the extraction process. Reducing salt content enhances protein yield while decreasing the mineral content. For instance, extracting Ulva with distilled water at 25 °C for 2 hours or at 40 °C for 30 minutes removes 22 % of the salt without significantly affecting ulvan extraction [41]. In addition to salt, starch can be extracted using cold distilled water. Extraction of starch from U. ohnoi with cold distilled water and a homogenizer yields 7.33 % of the dry weight of Ulva [30].

Another valuable co-product is pigment, which can be extracted using ionic liquids or organic solvents [42]. Ethanol is the most commonly used solvent for pigment extraction from Ulva, yielding 3.6 mg/g of dry weight [43]. Cascading extraction, starting with salt removal, increased the ulvan yield from 3.7 % to 8.2 % of Ulva dry weight, while subsequent pigment extraction showed no significant impact on ulvan yield [44]. Besides pigments, ethanol also extracts lipids and proteins from Ulva [44,45]. The compounds extracted with an ethanol-to-water ratio of 70:30 exhibit bioactivity, with an IC50 of 1.2 g/mL of biomass in the solvent [46]. Although pigment removal at the beginning of the process has minimal effect on ulvan yield, pigments themselves have significant applications in fields such as cosmetics, food additives, and pharmaceuticals. Therefore, their early extraction holds considerable value on its own.

The extraction of ulvan requires careful optimization of solvents and conditions, as these not only influence ulvan yield but also affect the co-extraction of other materials. Table 1 summarizes the ulvan yields, molecular properties, and extraction conditions across various Ulva species. Solvents used for ulvan extraction are typically categorized as acidic, alkaline, or neutral. Among these, acidic solvents generally produce higher yields compared to alkaline solvents [45]. Interestingly, even hot distilled water, a neutral solvent, can achieve good ulvan yields with a wide range of molecular weights [36]. Acidic solvents likely enhance ulvan extraction by breaking down the cell wall matrix and disrupting ionic interactions between ulvan and other components, making the polysaccharide more accessible for solubilization. In contrast, alkaline solvents may lead to degradation or selective solubilization of other polysaccharides, reducing ulvan recovery.

In addition to solvent selection, an extraction temperature of at least 80 °C is recommended for optimal results. Assisted extraction strategies, such as microwave, ultrasonic, autoclave, hydrothermal, or enzymatic techniques, can further enhance ulvan yield. Enzymatic assistance using cellulase, for example, is energy-efficient and achieves higher yields compared to non-assisted methods [47]. While physical-assisted methods are highly effective in increasing yield, they tend to consume significantly more energy compared to enzymatic approaches. Physical-assisted methods, such as microwave and ultrasonic techniques, improve extraction by disrupting cell walls through rapid heating or mechanical vibrations [45,48]. Enzymatic approaches target specific bonds, like glycosidic linkages, to release ulvan with minimal structural damage and energy use [49].

The solid fraction generated during ulvan extraction constitutes a significant portion of the residual product. This solid fraction contains cellulose, protein, and minerals, with its composition varying based on the pretreatment and extraction processes used. For instance, the residual solid obtained from hydrothermal extraction combined with supercritical CO₂ and ethanol pretreatment is rich in cellulose and protein but has lower heavy metal and mineral content, making it a promising candidate for food or feed applications [50,51]. The cellulose derived from Ulva exhibits unique characteristics, which make it suitable for a wide range of applications. Separating protein and cellulose from the residual solid can yield more valuable side products, enhancing the overall economic and environmental sustainability of the ulvan biorefinery process.

During the extraction of ulvan, various other components are co-extracted, which are typically considered impurities. To obtain pure ulvan, these impurities must be effectively removed. Ulvan, a water-soluble polysaccharide, is insoluble in alcohol [5]. This property allows for its selective precipitation, while other soluble compounds remain in the liquid phase. Even with extended pretreatment strategies, the precipitation step remains critical for isolating pure ulvan [44]. This step separates ulvan from pigments, lipids, and proteins. Subsequent processes, such as centrifugation, evaporation, and ultrafiltration, are employed to further separate the liquid and solid phases following precipitation.

Alcohol precipitation is a convenient and widely used method; however, its effectiveness is limited by the insolubility of certain impurities, such as salts and starch. Post-treatment using enzymes or strong chemicals, such as peracetic acid, provides an effective means of minimizing co-extracted components [5]. For example, α-amylase can be used to reduce starch content, while proteinase K is effective for protein removal [59]. Dialysis and ultrafiltration are commonly used to remove salts from ulvan extracts. Before dialysis, the precipitated ulvan must be rehydrated using distilled water or an appropriate buffer solution. Dialysis typically employs membrane tubing with a molecular weight cut-off (MWCO) ranging from 12–14 kDa [60]. The choice of downstream processing steps for ulvan purification depends on its intended applications, as ulvan has a wide molecular weight range, spanning from over 400 kDa to less than 3.9 kDa [36]. Molecular weight-based purification is often performed using Size Exclusion Chromatography (SEC), which allows for precise separation based on size. The molecular weight of ulvan plays a crucial role in determining its rheological properties and bioactivity, with higher molecular weights favoring structural applications and lower molecular weights enhancing bioavailability in pharmaceutical uses.

SEC separates ulvan based on particle size, which is determined by the hydrodynamic volume of the molecules in solution. This separation can be used to estimate molecular weight by comparing the elution profile of ulvan to a calibration curve generated with standards of known molecular weight. High-performance SEC is typically performed using distilled water as the mobile phase and carried out on a High-Performance Liquid Chromatography system equipped with an Infrared detector [58]. Further separation may be required if the target product is a rare sugar, such as rhamnose. Rare sugars such as rhamnose are valuable for their applications in pharmaceuticals and as precursors in chemical synthesis, necessitating further refinement to achieve the desired purity and concentration.

3. Biological Properties of Ulvan

Ulvan holds significant potential as a biomaterial, largely due to its exceptional biological properties that are crucial for the functionality of biomaterials. This review highlights ulvan's biological activities, focusing on its role as a biomaterial with key attributes such as excellent biocompatibility, immunomodulatory effects, anticoagulant activity, and antimicrobial properties. These properties are essential for biomaterials, as they enable compatibility with biological systems and enhance therapeutic efficacy, making ulvan a promising candidate for various biomedical applications.

3.1. Biocompatibility Profile

Biomaterials must be compatible with biological tissues, meaning they should not provoke harmful immune responses or toxicity when introduced into the body. This is a critical characteristic for materials used in implants, drug delivery systems, and tissue engineering. The biocompatibility of ulvan has been assessed through various techniques, including cytotoxicity tests, haemolysis assays, and studies of cellular uptake and interactions.

Cytotoxicity testing has been performed both in vitro on cell cultures and in vivo using experimental animals [61,62]. Ulvan’s cytotoxicity has been studied across a range of cell types, including fibroblast cells (e.g., mouse C3H [L929], 3T3) [63,64], macrophage cell lines (e.g., RAW 264.7, peritoneal, and J774A.1) [65,66,67,68], gut cells (e.g., IPEC-1) [69,70,71], myoblast cell ( e.g. mammalian L6 cells) [72,73,74], HaCaT keratinocytes [75], Vero cells [76] and in animal models such as mice and rats [77]. T The most used method for cytotoxicity testing is the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which measures cell viability by detecting the reduction of yellow MTT dye into purple formazan crystals by metabolically active cells, indicating mitochondrial activity.

Ulvan has generally been shown to be non-toxic, with several studies reporting high cell viability across various cell lines exposed to ulvan extracts. Fractions of ulvan from several spesies of Ulva, namely U. pertusa [65,66], U. intestinalis [68,78], U. armoricana [69,70], U. lactuca [63,77,79], U. clathrata [80], U. compressa [81], and U. prolifera [67,82], exhibited over 50 % cell viability at concentrations of ≥500 μg/mL. Furthermore, ulvan derived from Ulva sp. has been proven safe for mammalian L6 cells, showing no cytotoxic effects at concentrations under 90 mg/mL. Similarly, it did not show toxicity in 3T3 cells even at concentrations up to 10,000 mg/mL [83]. In human L929 cells, ulvan remained metabolically active after 72 hours of exposure, with no decrease in cell viability [64].

The hemolysis assay, which measures hemoglobin release from red blood cells, is a simple screening method for potential biocompatibility of a material. Ulvan extracted from U. lactuca demonstrated hemolytic activity, with a recorded hemolysis percentage of 12.38 % at a concentration of 100 μg/mL. The negatively charged oxygen groups on ulvan electrostatically interact with the positively charged phosphatidylcholine lipids on the outer surface of red blood cells, contributing to ulvan’s hemolytic activity [84].

3.2. Immunomodulatory effect

Immunomodulatory properties are a key biological activity that can serve as an essential parameter in biomaterial characterization, especially in applications involving interactions with the immune system, such as tissue engineering, wound healing, implants, and drug delivery. The ability of a biomaterial to modulate immune responses in a controlled manner ensures safety, biocompatibility, and effectiveness in these medical applications [85,86]. Immunomodulation refers to the alteration of the immune system's response, either by enhancing immune responses (immunostimulatory) or reducing excessive immune activity (immunosuppressive). This process involves regulating immune cell functions (like macrophages, T cells, or natural killer cells), cytokine production, and overall immune system responsiveness. The primary goal of immunomodulation is to achieve a balanced immune response that supports effective defense against infections or tumours while preventing immune system overactivation, as seen in autoimmune diseases and allergies [85].

The immunomodulatory effects of ulvan have been widely investigated in various macrophage cell types, including RAW 264.7 cells (Rat Adherent White blood cell 264.7 cells), mouse peritoneal macrophages, J77A.1 cells, and fish head kidney cells [86,87,88,89,90,91,92]. Moreover, ulvan’s impact on immune modulation has been explored in several models, such as fish [93,94], porcine intestinal epithelial cells (IPEC-1 cell line), rats [77,97], mice [77,78,95,96], and chickens [97]. A range of probes are commonly used to evaluate ulvan’s effects on inflammation, including signalling molecules (e.g., cytokines like tumor necrosis factor-alpha (TNF-α) , interleukin, IL (IL1, IL2, IL6, IL10, IL12), C-X-C motif chemokine ligand (CXCL1, CXCL12, CXCL14), and C-C motif chemokine ligand), which are various types of interleukins and chemokines that play roles in immune regulation), active metabolites (e.g., Prostaglandin E2), nitric oxide (NO)), immunoglobulins (e.g. immunoglobulin M (IgM), Intercellular Adhesion Molecule, and Vascular Cell Adhesion Molecule-1), enzymes (e.g., cyclooxygenase-2 (COX-2) , inducible nitric oxide synthase-2 (iNOS-2) , Heme Oxygenase-1, and Myeloperoxidase), and transcription-related molecules (e.g., nuclear factor kappa-B (NF-κB) and mRNA) [65,66,67,78,82,94,95,98].

The immunomodulatory effects of ulvans are closely associated with the inflammatory response, which can be divided into four key stages: inducers, sensors, mediators, and target tissues, as shown in Figure 4. Inflammation begins with inducers, which can be classified as either exogenous or endogenous. Exogenous inducers, such as pathogen-associated molecular patterns, are recognized by specific receptors, while endogenous inducers include damage-associated molecular patterns released from damaged host cells. Other endogenous molecules, like advanced glycation end products and oxidized lipoproteins, are often linked to oxidative stress and can also trigger inflammation. Sensors, such as Toll-like receptors (TLRs), Nod-like receptors, and other pattern recognition receptors, identify these inducers and activate downstream signaling pathways like mitogen-activated protein kinase (MAPK), NF-κB, and SIRT1/FOXO1. These signaling pathways lead to the production of various inflammatory mediators, including chemokines, cytokines, vasoactive amines, eicosanoids, matrix metalloproteinases, nitric oxide, and free radicals. These mediators are involved in several processes, such as pain induction, immune modulation, and tissue repair. The target tissues affected by these mediators show responses such as increased vascular permeability, immune cell recruitment, and either tissue damage or repair. If this process is dysregulated, it may lead to chronic inflammation and associated disorders [65].

Ulvans function as immunomodulatory agents through various mechanisms that influence the immune system, including the suppression of pro-inflammatory cytokine production, modulation of the NF-κB pathway, enhancement of anti-inflammatory cytokines, regulation of TLR signaling, and restoration of gut microbiota balance (Figure 4). The immunostimulatory properties of ulvan are largely determined by its structural features, such as monosaccharide composition, sulfate content, and molecular weight, as discussed by Kidgell (2019) [36]. One of the key mechanisms of ulvans is the regulation of cytokine production, particularly pro-inflammatory cytokines that are crucial in immune responses. Cytokines like TNF-α, IL-6, and IL-1β, which are commonly elevated in inflammatory conditions such as inflammatory bowel diseases (IBD) [100], can be modulated by ulvans. Additionally, ulvans regulate intestinal inflammation by inhibiting the NF-κB pathway. Ali et al. (2016) [101] demonstrated that ulvan from U. pertusa COMP reduces the production of pro-inflammatory cytokines (IL-12, p40, IL-6, TNF-α) by preventing the phosphorylation of IκBα, which inhibits its degradation and the nuclear translocation of NF-κB. This action suppresses the expression of inflammatory genes and helps maintain immune homeostasis.

Ulvans enhance the production of anti-inflammatory cytokines, such as IL-10. This cytokine is vital for suppressing inflammatory responses and promoting tissue repair. By increasing IL-10 levels, ulvans counteract the effects of pro-inflammatory cytokines, further contributing to an anti-inflammatory state [102,103]. Ulvans also play a role in regulating TLR signaling pathways, particularly TLR4. By modulating these pathways, ulvans can inhibit the activation of downstream inflammatory responses, including those mediated by NF-κB and MAPK pathways. This regulation is essential for preventing excessive immune activation and inflammation [102].

The immunomodulatory effects of ulvan are partly attributed to its capacity to alter the gut microbiota. The gut microbiome plays a key role in immune system regulation, and ulvan’s prebiotic properties support the growth of beneficial microbes, enhancing immune function and controlling inflammation [103,104,105,106]. Ulvans influence the gut microbiota by promoting beneficial bacteria and regulating immune responses, which is essential for gut health and preventing inflammation-related disorders. By restoring microbiota balance, ulvans help maintain immune homeostasis, promoting beneficial bacteria and suppressing harmful ones, which is important for preventing dysbiosis and reducing inflammation [107,108].

3.3. Anticoagulation Activity

Anticoagulant activity is a vital characteristic of biomaterials used in medical applications, especially those that come into direct contact with blood. This property ensures biocompatibility, functionality, and safety by preventing blood clot formation (thrombosis), minimizing inflammation, and maintaining proper blood flow, which collectively improve patient outcomes and extend the lifespan of medical devices [109]. The anticoagulant potential of a molecule is typically assessed through three primary tests: activated partial thromboplastin time, thrombin time, and prothrombin time. These tests evaluate how the material influences various pathways in the coagulation cascade, providing critical information about its hemocompatibility.

Studies have indicated that ulvan, a polysaccharide derived from various species of the seaweed genus Ulva, exhibits anticoagulant effects. Key species include U. clathrata [110], U. lactuca [111,112], U. prolifera [113], U. fasciata [112,114], U. nematoidea [115], U. conglobata [116], U. linza [117,118], and U. reticulata [112]. The anticoagulant potency of ulvan differs across species and is influenced by environmental and physiological factors. The anticoagulant activity of ulvan depends on the degree of sulfation and molecular weight, both of which are critical to its effectiveness [115,116,117,118]. Higher sulfation enhances its activity, while molecular weights below 200 kDa can diminish it [115].

Regarding its mechanism of action, ulvan primarily affects the intrinsic and/or common pathways of the coagulation cascade. The intrinsic pathway is triggered by the interaction of Factor XII with an anionic surface, while the extrinsic pathway is initiated when Factor VII binds to tissue factor, a receptor released from damaged cells. Both pathways converge at Factor X, activating the common pathway, which leads to the conversion of prothrombin into thrombin. Thrombin then catalyzes the transformation of soluble fibrinogen into insoluble fibrin, forming the structural framework of a clot [119].

3.4. Antimicrobial Activity

Antimicrobial activity is a crucial characteristic of biomaterials, particularly those used in medical applications, as it significantly enhances their safety and functionality. It helps prevent infections by reducing microbial colonization on the material's surface, which is especially vital for implants, wound dressings, and other medical devices. Additionally, antimicrobial properties protect biomaterials from degradation caused by microbial activity, thereby prolonging their lifespan and maintaining their performance [120].

Ulvan has been studied for its ability to combat various harmful microorganisms. Van Tran et al. [121] were the pioneers in demonstrating the antimicrobial properties of ulvan derived from U. reticulata, showing significant inhibitory effects against two pathogenic bacteria linked to skin diseases and inflammation. They reported inhibition zones of 20 mm against Enterobacter cloacae and 18 mm against Escherichia coli. Fournière et al. [122] also documented the biological activity of ulvan extracts from Ulva species against Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes. In addition, Ibrahim et al. [123] emphasized the potent antimicrobial effects of partially purified ulvan from U. lactuca against certain fish and human pathogens, including E. coli ATCC 8739 and C. albicans ATCC 10,231. However, ulvan showed no antimicrobial activity against two Gram-positive bacteria (S. aureus ATCC 25,923 and L. monocytogenes ATCC 35,152), a Gram-negative bacterium (B. pertussis ATCC 8467), or fungal pathogens (A. niger, P. notatum, and F. solani). The antimicrobial effects of ulvan are attributed to its distinctive chemical structure, which is composed mainly of rhamnose, uronic acid, and sulfate groups [121].

4. Ulvan in Biomaterial Design

Due to its unique structural and functional properties, ulvan exhibits significant potential across fields. The unique structure come from its β-(1,4) backbone is composed of alternating glucuronic acid (GlcA) and rhamnose (Rha) units, which are often modified by varying patterns of sulfation. These sulfate groups are crucial because they enable intermolecular interactions, such as hydrogen bonding and electrostatic forces, which are essential for gelation [124]. Additionally, ulvan contains carboxyl groups that serve as active sites for synthesizing complexes with other molecules, further expanding its functional versatility. The natural origin of ulvan as a renewable and sustainable material further enhances its relevance in environmentally friendly biomaterial design.

Ulvan has the remarkable ability to transform liquids into semi-solid gels by forming network structures. This process occurs through hydrogen bonding, ionic interactions, or hydrophobic interactions. These properties make ulvan highly valuable in the food, cosmetics, and pharmaceutical industries, where it is used to improve the texture, stability, and consistency of products. We will discuss the ulvan in hydrogel, film, emulsion, and nanocomposite (Figure 5).

4.1. Ulvan-Based Hydrogel

Ulvan’s carboxyl and sulfate groups play a pivotal role in its ability to form hydrogels through various crosslinking methods. One such method involves oxidizing ulvan with sodium periodate, which introduces aldehyde groups along the polysaccharide backbone [125]. These aldehyde groups react with gelatine's amine groups through Schiff-base reactions, forming covalent imine bonds. The Schiff-base reaction occurs when the aldehyde groups (−CHO) in ulvan dialdehyde interact with the primary amine groups (−NH2) in lysine residues of gelatine, resulting in stable C=N bonds [126]. Using phosphate-buffered saline (PBS), hydrogels with 80% gelatine and 20% ulvan showed the lowest swelling capacity (300%), while hydrogels with 40% gelatine and 60% ulvan reached the highest swelling capacity (900%). When deionized water was used, swelling capacity increased significantly, with 80% gelatine and 20% ulvan samples showing 1000% swelling, and 40% gelatine and 60% ulvan samples exhibiting an extraordinary swelling capacity of 2400% [50]. These results demonstrate the tunable properties of ulvan-based hydrogels depending on solvent composition and polymer ratios. This interaction not only creates a robust hydrogel network but also enhances its binding capacity for dyes and heavy metal ions, utilizing the free amine groups from gelatine and carboxyl groups in ulvan [45]. The Schiff-base strategy involving gelatin matrices is applicable for tissue engineering application [127] and bone tissue engineering [128].

Another advanced approach uses enzymatic crosslinking with horseradish peroxidase (HRP) and hydroxyphenyl compounds such as tyramine. Hydroxyphenyl compounds are particularly suitable substrates for HRP due to their reactivity in green chemistry processes [129]. Tyramine is the most commonly used hydroxyphenyl compound in enzymatic gelling systems, offering a safe profile and efficient conjugation via its terminal amine group [130]. In this system, ulvan-tetrahydroxyphenyl (UT) conjugates are synthesized by forming amide bonds between the carboxyl group of ulvan and the amine group of tyramine, facilitated by carbodiimide chemistry [131]. The carboxyl groups are first activated by reacting with EDC and sulfo-NHS in a slightly acidic environment, creating reactive sulfo-NHS esters optimized for polymer bonding. In the presence of hydrogen peroxide (H₂O₂), HRP catalyzes the oxidative coupling of hydroxyphenyl compounds through a radical mechanism [130]. Covalent bonds form between carbon atoms in the ortho position to the hydroxyl or oxygen atoms of phenol groups, creating a stable crosslinked network. These enzymatically crosslinked hydrogels achieve swelling degrees of approximately 2000%, classifying them as superabsorbent materials [128]. The enzymatic and H₂O₂ conditions can be adjusted to optimize gelation times for applications such as injectable hydrogels [132].

Ulvan’s gelling potential is also being explored in the development of bioinks for 3D printing applications. A bio-ionic liquid derived from ulvan is formulated by mixing it with choline chloride and subjecting it to ethanol precipitation. This process results in a material with excellent viscoelastic strength, making it an ideal candidate for use in advanced 3D bioink formulations [53]. More details on its biocompatibility and specific cell interaction performance would further solidify its relevance in biomedical applications.

The exploration of ulvan hydrogels extends to food applications. When formulated with alginate, ulvan forms a scaffold suitable for probiotic encapsulation. While alginate is widely used as a scaffold for nutrient encapsulation [133] , it has limitations when applied to systems involving living organisms. In contrast, ulvan demonstrates strong prebiotic activity and supports cell proliferation [134] , effectively addressing alginate's limitations in microbial encapsulation. Ulvan-alginate hydrogel beads for probiotic immobilization have been shown to enhance the survival rate of probiotics in both simulated gastric and intestinal fluids [135] . This system offers significant potential for applications in the food and beverage industry.

4.2. Ulvan-Based Film

Ulvan-based film formulations utilize glycerol as a plasticizer to create hydrophilic films. These films are typically composed of ulvan, glycerol, distilled water, and boric acid. To enhance the material’s characteristics, 10% carnauba wax and Tween-80 can be added, resulting in hydrophobic films ideal for food packaging. These hydrophobic films demonstrate impressive mechanical properties, with an average tensile strength of 1 MPa and an average elongation at break of 80% [56]. Further comparisons with conventional gelling agents could provide insight into ulvan’s superior performance and sustainability.

4.3. Ulvan-Based Nanocomposite

Hybrid hydrogels that incorporate ulvan exhibit enhanced mechanical and functional properties. For instance, ulvan crosslinked with citric acid and hybridized with sodium carboxymethyl cellulose (Na-CMC) achieves a swelling degree of up to 387.5%, highlighting its improved mechanical stability. This hybrid hydrogel is synthesized using a single-pot heating method, where a fixed citric acid concentration of 20 wt% is used to crosslink ulvan and Na-CMC. The simplicity of this process enhances its applicability in scalable production methods. Comparatively, ulvan hydrogels without Na-CMC show a swelling capacity of 248.7%, demonstrating that hybridization significantly improves performance. [54]

Comprehensive utilization of Ulva is further demonstrated by Mariia et al. [136]. Ulvan is reacted with chitosan to form polyelectrolyte complexes involving anionic and cationic side chain interactions. The sulfate groups in ulvan carry a negative charge (anionic), while the amine groups in chitosan are positively charged (cationic). Combining chitosan/ulvan with cellulose nanocrystals (CNCs), extracted from the solid residue of ulvan extraction, results in nano-bio composite hydrogels designed for wound healing applications. Chitosan hydrogels incorporating 20% CNC show enhanced tensile strength of 1.2 MPa and a significant increase in swelling capacity, indicating improved mechanical properties. Furthermore, these nanocomposites exhibit excellent cell proliferation, remarkable biocompatibility, and non-toxicity. The sustained release of epidermal growth factors from these hydrogels accelerates wound healing, demonstrating their advanced therapeutic potential.

Further advancements in ulvan research include ulvan hybrid with carrageenan and polyvinyl alcohol (PVA). For ulvan hybrid carrageenan, the synthesis of ulvan-amide derivatives through carbodiimide chemistry. These derivatives enable the creation of ulvan-kappa-carrabiose hybrid polysaccharides, expanding the scope of ulvan-based materials [137]. For ulvan nanofiber fabrication with PVA, ulvan-polyvinyl alcohol (PVA) nanofibers, produced via electrospinning with a 1:2 ulvan/PVA ratio, demonstrate promising potential for advanced material applications, including biomedical and environmental uses [125]. Specific examples of these applications, such as filtration membranes or antimicrobial coatings, would add depth.

4.4. Ulvan-Based Emulsion

A detailed evaluation of ulvan highlights its significant potential in various industries, especially within food and cosmetic formulations. Ulvan’s capacity to hold water and oil, quantified at 3.17 g water/g ulvan and 2.66 g oil/g ulvan respectively [45], makes it particularly useful for stabilizing emulsified products. This capability helps prevent liquid separation (syneresis) while enhancing viscosity and texture, ensuring consistency in complex formulations [138].

In emulsified systems, increasing ulvan concentrations from 1 % to 3 % enhances both emulsion activity and stability. At its peak concentration of 3 %, ulvan achieves an emulsion activity index of 69.66 m²/g and a stability of 72.39 % [45]. While polysaccharides are typically recognized for their stabilizing rather than emulsifying roles, ulvan stands out due to its surface-active functional groups, such as carboxyl, sulfate, and hydroxyl, as well as hydrophobic protein-like elements [139]. The molecular structure of ulvan, which includes a high molecular weight and glucuronic acid content, significantly bolsters its ability to form stable emulsions by increasing solution viscosity and strengthening interfacial layers [140].

Ulvan also exhibits excellent foaming capabilities, achieving a foaming capacity of 75 % and a stability of 54 % at 3 % concentration [45]. These properties are crucial for developing consistent and appealing textures in food and cosmetic products [140]. As ulvan concentrations increase, its ability to stabilize foam improves, largely due to its high molecular weight. This allows it to create robust networks at the air-water interface, effectively delaying the merging of gas bubbles and prolonging foam integrity. Such characteristics are highly beneficial for applications in whipped desserts, aerated beverages, and cosmetic mousses, where stable foam structures are essential [139,140].

5. Comparative Advantages of Ulvan

One of the key advantages of ulvan is its potentially high yield due to the high and robust biomass of Ulva species, which can outperform other seaweed sources such as agar, carrageenan, alginate, laminaran, fucoidan, porphyran, floridean, and xylofucoglycan [141]. As mentioned in the previous section, ulvan extracted from Ulva is derived from an undesired seaweed, which can be produced 7 – 10 times faster and in higher quantities than other commercially available seaweed biomass in the current seaweed industry. This characteristic ensures a sustainable production pipeline, addressing scalability issues that often limit other polysaccharides.

The extraction of ulvan is also reported to be relatively easy and simple, making it a more accessible and cost-effective option compared to other marine polysaccharides. Compared to other marine polysaccharides, ulvan demonstrates superior yield potential, with up to ~41 % extraction efficiency from dry Ulva biomass [56,142]. This is higher than the yields typically reported for agar (10 – 15 %) or fucoidan (5 – 10 %). The ability of Ulva species to proliferate in eutrophic environments further enhances its suitability for large-scale production, reducing reliance on pristine ecosystems required for agarophytes or carrageenophytes. Chemically, with ulvan’s unique properties, stands as an equally competitive seaweed polysaccharide alongside others such as agar, carrageenan, alginate, laminaran, fucoidan, porphyran, floridean starch, and xylofucoglycan, offering distinct advantages in yield, functionality, and bioactivity.

Notably, unlike agar or carrageenan, which require heat for gel formation, ulvan can form gels under non-thermal conditions through ionic cross-linking with CaCl₂ and H₃BO₃ [143]. This characteristic could position it as a potential alternative to alginate, a widely used marine polysaccharide This property is advantageous for applications requiring cold processing, such as in the pharmaceutical and food industries. For instance, ulvan-based hydrogels can encapsulate heat-sensitive bioactives, a limitation for thermally reliant gelling agents. Furthermore, the high sulfate content of ulvan has garnered interest in the biomedical field, as it may confer anticoagulant properties, similar to heparin, leading to it being referred to as the "vegan heparin" [144]. Compared to other seaweed polysaccharides, ulvan has also demonstrated versatility in its bioactive compounds, exhibiting a range of beneficial properties, such as antioxidant, immunomodulatory, anticancer, and antimicrobial activities [141,145]. The richness of sulfated moieties in ulvan’s structure also favors its potential for engineering and modification, opening up opportunities for the development of innovative polymeric materials and applications [146]. The comprehensive comparison between ulvan and other marines’ polysaccharide shows in Table 2.

Despite potential of ulvan discussed in the previous chapter, it faces certain challenges that could hinder its broader adoption in commercial and industrial needed. Key limitations include:

Extraction Complexity and Standardization. The extraction process for ulvan remains less standardized compared to traditional polysaccharides like agar or carrageenan. Variability in the chemical composition of Ulva biomass due to environmental factors affects the consistency and quality of ulvan. Green solvent and enzyme-assisted methods show promise for improving efficiency but require further optimization for industrial scalability.

Seasonal Yield Variability. Currently, most Ulva cultivation occurs in wild or semi-wild conditions, where environmental factors such as temperature, nutrient availability, and seasonal variations significantly affect the yield and quality of ulvan. This lack of controlled cultivation introduces variability and limits the scalability of ulvan production.

Functional Limitations. Although ulvan exhibits excellent gelling and bioactive properties, its mechanical strength in hydrogels is often lower compared to alginate or carrageenan. This limitation restricts its use in applications demanding high structural integrity, such as wound dressings or load-bearing scaffolds in tissue engineering.

Processing Costs. Specialized extraction methods, including green solvents and enzymatic approaches, increase production costs. The absence of well-established infrastructure for ulvan processing further compounds this issue, making it less competitive than agar or alginate in certain markets.

Future Directions. To overcome these limitations, research efforts should focus on:

• Enhanced Extraction Techniques: Developing cost-effective, standardized methods for high-purity ulvan production.
• Cross-Linking Strategies: Enhancing mechanical properties of ulvan-based materials through innovative cross-linking and composite formation.
• Sustainability Metrics: Incorporating life cycle assessments to establish ulvan as a green alternative to synthetic and traditional polysaccharides.
• Controlled Cultivation: Advancing aquaculture techniques for Ulva species to ensure stable and high-quality ulvan yields independent of seasonal and environmental variations.
• Establishing high-quality ulvan standards depends on its intended use, whether as feed, a food source, or a bioactive compound in the biomedical field.

By addressing these challenges, ulvan has the potential to surpass its current limitations and establish itself as a cornerstone versatile material in biomaterial science and industrial applications.

Lastly, the evaluation of Ulvan as a biomaterial emphasizes its potential across three key strategies: Bioactive Ingredient, Scaffold-Backbone Design and Dual Function Synergetic (Figure 6). While substantial progress has been made, critical gaps remain in integrated functionality, mechanical property optimization, and encapsulation stability. Addressing these challenges could unlock transformative applications for Ulvan in biomaterial design, particularly in biomedical and industrial fields.

6. Conclusion

Ulva provide the potent as the versatile new generation of seaweed that more robust and sustainable. The cultivation of Ulva offers environmental benefits, including high carbon sequestration, rapid growth, and low resource requirements. Integrated green biorefinery of Ulva approaches enhance its polysaccharide extraction efficiency of ulvan as the main sugar. It enables the recovery of valuable co-products such as pigments, proteins, and cellulose, boosting sustainability. Ulvan's superior yield and scalability compared to other marine polysaccharides position it as an economically viable alternative.

Ulvan, a sulfated polysaccharide derived from Ulva seaweed, is a promising biomaterial due to its unique structure and exceptional bioactive properties, including immunomodulation, anticoagulant activity, antimicrobial effects, and biocompatibility. Its ability to form hydrogels, films, scaffolds, and nanocomposites makes it valuable for biomedical, food, and environmental applications. Unlike traditional polysaccharides, ulvan’s non-thermal gelation and high sulfate content provide advantages for preserving heat-sensitive bioactives and mimicking heparin-like anticoagulant properties.

Despite its potential, challenges in extraction optimization and impurity management remain. Advancing green extraction methods and exploring innovative applications will unlock ulvan’s full potential. With its sustainability, versatility, and scalability, ulvan is poised to play a vital role in advancing biomaterial design and addressing global challenges in healthcare, food security, and environmental sustainability.