When it comes to extracting carrageenan, alkaline treatment is often utilized, and the effectiveness of this procedure is heavily reliant on the type of alkaline solution used. This approach was initially developed for the algae Chondrus crispus; however, it is now applicable to all types of algae. Jönsson et al. (2020) recently reviewed this method and published its schematic protocol. In addition to being known as “refined carrageenan,” the original carrageenan is recovered by extracting carrageenan from cleaned and washed seaweed using hot alkaline solutions at temperatures ranging from 95 to 110°C. When a solution such as sodium hydroxide, calcium hydroxide, or potassium hydroxide is added to the seaweed extract for many hours, depending on the kind of seaweed species and size of extraction scales, an alkaline atmosphere can be created (Mishra et al., 2006). Afterward, the residual seaweed matrix that did not dissolve is removed, resulting in a carrageenan solution that is more concentrated than before. The carrageenan may then be precipitated in one of two methods, depending on how much carrageenan is needed. The first method involves the addition of alcohol (mostly isopropanol), while the second involves the formation of gel in a potassium chloride solution (known as the “gel press process”; Lipnizki, 2010). The alcohol-precipitation method can be used to purify all types of carrageenans; however, the gel extraction method can only be used for κ-carrageenan (FAO, 2014). Squeezing or freeze-thawing are used to remove the remaining water from the gel after being gelled in the gel press procedure. As a result, the gel press product often has higher potassium chloride concentrations than the alcohol precipitated product. After the carrageenans have precipitated, they must be dried, pulverized, and redissolved to obtain a clear solution of purified carrageenan.

The semi-refining process is a relatively simple type of alkali extraction for carrageenan because it does not retrieve carrageenan from the seaweed matrix. Instead, it treats the seaweed matrix with aqueous potassium hydroxide at around 75°C for 2 h to dissolve and eliminate soluble compounds other than carrageenans, such as salts, soluble sugars, and soluble sugars proteins (Heriyanto et al., 2018). The reagent’s hydroxide component enters the seaweed and reduces the amount of sulfate in the carrageenan while simultaneously increasing the amount of 3,6-anhydrogalactose in the seaweed. The reagent’s potassium component reacts with the carrageenan in the seaweed to form a gel, which prevents the carrageenan from melting in the hot solution. Any soluble protein, carbohydrate, and salts are eliminated when the solution is drained from the seaweed. The residue is rinsed several times to remove the alkali and anything else that might dissolve in the water during the rinsing process. The alkali-treated seaweed is dried for about two days in hot areas before being cut and ground into a powder as SRC or seaweed flour. Bono et al. (2014) investigated the influence of process conditions on the viscosity and gel strength of SRC generated from the red seaweed K. alvarezii and determined the optimal conditions for SRC production using alkaline treatment. The optimal alkaline treatment parameters were discovered to be a cooking temperature of 80°C, a cooking time of 30 min, and a potassium hydroxide concentration of 10% w/w, which resulted in a gel viscosity of 1291.84 cP and a gel strength of 94.29 g/cm².

The alkali solution used in both the RC and SRC extraction procedures is intended to improve polysaccharide extraction and accelerate 6-sulfate removal from the monomer form 3,6-anhydro-D-galactose, thereby increasing gel strength and product reactivity on the protein (Corp, 1977). Apart from dissolving the carrageenans, the alkaline solution also transforms the biological precursors υ-carrageenan and μ-carrageenan into usable κ-carrageenan and ι-carrageenan, respectively. A cost comparison of SRC and RC technology reveals that the SRC extraction method is less expensive than the RC extraction process because carrageenan precipitation and solvent recovery costs are avoided in the SRC extraction process. However, when compared to the RC, the extracted primary product of the SRC process is of lower quality and is referred to as seaweed flour or alkali modified flour, with the SRC being unsuitable for human food application but designed for pet food production because of the flour is colored and often has a high bacterial population (Rhein-Knudsen et al., 2015). On the other hand, the RC is of higher quality and is referred to as raw carrageenan (Ortiz-Tena et al., 2017). Despite the benefits of alkali treatment, the procedure invariably results in some polysaccharide degradation due to the stresses of the processing environment (heat, alkalinity; Stanley, 1990). It is also possible that a higher concentration of sodium hydroxide will promote carrageenan depolymerization (Varadarajan et al., 2009).

Several extraction methods have been implemented, with enzyme-based processing techniques being more promising and environmentally friendly than traditional extraction methods (Tarman et al., 2020). Despite the potential for this technology to be used on an industrial scale, there has been relatively little progress in using enzymes to produce carrageenan from seaweed. Regardless, the cellulase enzyme is one of the most widely used enzymes for carrageenan extraction, as demonstrated in K. alvarezii (Varadarajan et al., 2009). In general, the enzyme cellulase must be added to the mixture (which includes ground seaweed and distilled water) before boiling it for 1 h in a water bath with a shaker at 50°C. The suspensions were then centrifuged, and the supernatants were separated and used to generate the supernatant fraction. Approximately one volume of supernatant is poured into two volumes of 2-propanol, causing the polysaccharides to precipitate as long fibers. The clear solution was removed from the samples by centrifugation (12,000 rpm, 4°C, 30 min), and the samples were referred to as the precipitated fraction. The samples were dried using a rotary evaporator before being freeze-dried in a freeze dryer. Because carrageenan is found in the hydrosoluble portion of the cell wall component (Lechat et al., 1997), the seaweed’s cell wall must be shattered for the carrageenan to be released. Carrageenans can be effectively extracted using cellulases because cellulose is the primary component of seaweed cell walls (Masarin et al., 2016). Endo-cellulases, exo-cellulases, and β-glucosidase are the three major types of cellulose enzymes, and a complete hydrolysis of cellulose microfibrils in the cell wall can be performed by combining these three types of enzymes (Jayasekara and Ratnayake, 2019). Enzymatic hydrolysis can be performed on cellulose in most cases since it is soluble and sensitive to hydrolysis. Furthermore, the absence of lignin in seaweed allows for a faster hydrolysis process (Tarman et al., 2020).

Although this extraction method was considered an alternative to standard extraction (Lechat et al., 1997), the high cost of the enzyme cellulase prevents its widespread use on a commercial manufacturing scale because it is not economically feasible. With a significant decrease in enzyme cost for lignocellulose breakdown, enzyme-based methods may become a viable commercialization option. Besides, the enzyme sulfurylases, which naturally catalyze the transformation of 4-linked α-D-galactopyranosyl units into 3,6-anhydro-D-galactopyranoses, have been examined for their ability to convert the precursors into κ- or ι-carrageenan, which would allow the elimination of alkaline conditions (Genicot-Joncour et al., 2009). However, the viscosity of carrageenan extracted using cellulase treatment was found to be lower than that of carrageenan extracted using standard extraction, which could be due to contaminants present in the extraction process, resulting in a lack of pure carrageenan after alcohol precipitation and centrifugation (Varadarajan et al., 2009). After all, it is still necessary to research ways to improve the method for optimal yield on an industrial scale.

Recent efforts to reduce the use of chemicals have resulted in the development of environmentally friendly solvents and approaches that use less solvent and require less energy and extraction time than traditional methods (Ma et al., 2012; Torres et al., 2021). Microwave-assisted extraction (MAE) was used to extract carrageenans from red seaweeds, yielding κ-carrageenan from K. alvarezii and ι-carrageenan from Eucheuma denticulatum (Uy et al., 2005). Most seaweed producers prefer sunlight for drying their products because it is less expensive and readily available. However, the technique has a number of drawbacks, including the need for a large amount of land to complete the drying process, being highly dependent on weather conditions, and allowing contamination from the surrounding environment. Microwave drying offers an alternative in SRC extraction because it overcomes the challenges of sun drying and dries biomass materials with a high water content in a shorter time than traditional drying methods (Sari et al., 2020).

The MAE extraction method begins with an alkaline treatment, followed by microwave-assisted extraction using a microwave vessel system at full power and a frequency of 2,450 MHz (Vázquez-Delfín et al., 2014). In brief, 1 g of materials was hydrated for 12 h in 50 ml distilled water (aqueous treatment) and 3% potassium hydroxide (alkaline treatment), and then placed in a closed-vessel system designed to minimize solvent and analyte loss during operation. Extraction can be carried out at high temperatures in closed containers, which accelerates the mass transfer of target carrageenan compounds from K. alvarezii samples (Bouanati et al., 2020). Temperature and pressure sensors were used to monitor internal temperature and pressure conditions and to regulate extraction conditions, with a maximum pressure of 159 kPa during the extraction and vessels designed to dissipate excess pressure. Following the completion of the vessel system, the extracted carrageenan was purified and recovered as described above. Nonetheless, Vázquez-Delfín et al. (2014) also reported a decrease in carrageenan yield after alkali treatment in the MAE method, which could be attributed to unavoidable polysaccharide breakdown and loss under rigorous hot alkaline or elevated temperature processing conditions.

The ultrasound-assisted extraction (UAE) method has been used for decades to extract a wide range of natural raw materials, including from red algae (Cikoš et al., 2018; Bordoloi and Goosen, 2020). This method has recently gained popularity because it requires less equipment and procedures (Tiwari and Troy, 2015), and it is considered an environmentally friendly technology (Talmaciu et al., 2015; Hanjabam et al., 2019). In comparison with other green techniques, ultrasonic technology is significantly easier, takes less time, and is less reliant on the algae biochemical composition as required for enzyme-assisted extraction (Hahn et al., 2012; Zhu et al., 2017b). Youssouf et al. (2017) demonstrated that using the UAE method to extract carrageenans from K. alvarezii resulted in higher yields in less time than traditional extraction while not altering the chemical structure of the carrageenan.

The three essential steps of carrageenan extraction using the UAE method are algae pretreatment, carrageenan extraction, and carrageenan purification (Youssouf et al., 2017). The pretreatment process was started by incubating dried seaweed (K. alvarezii) in an 80% ethanol solution at room temperature overnight. The pre-treated seaweed was then filtered before being subjected to an ultrasound at 150 W for 15 min to extract the carrageenan. The movement of ultrasonic waves mediates the extraction process, which results in the transformation of ultrasonic waves into mechanical energy. This mechanical energy then ruptures the cell wall, reducing particle size as a result of the rupture and releasing the carrageenan from the cell wall (Nigam et al., 2022). Following the extraction process, the carrageenan was available in the form of a solution, and any remaining algal residues were removed using a hot filtering process. It was necessary to place the filter at 4°C for several hours to obtain carrageenan extract in the form of a gel, and gel carrageenan was frozen and lyophilized to obtain the final carrageenan powder (Youssouf et al., 2017). Ultrasounds allowed for the extraction of 50–55% of carrageenans from red seaweeds such as K. alvarezii and E. denticulatum in 15 min, and a longer ultrasonic treatment (30 min) may not result in a higher extraction yield (Youssouf et al., 2017; Azis, 2019).

Temperature, light intensity, and nutrition were among the environmental parameters influencing Kappaphycus development, with temperature appearing to be the most critical factors determining carrageenan output (Glenn and Doty, 1992). As the temperature rises, so does the yield of carrageenan until it reaches the optimal temperature, at which point the production rate begins to fall rapidly (Bulboa et al., 2008). Temperature variations have also been found to affect the quantity and quality of carrageenan in K. alvarezii (Heriyanto et al., 2018). According to Orbita (2013), the maximum carrageenan yield was obtained from K. alvarezii when the water temperature was between 28°C and 31°C, but the growth and carrageenan content were very low when the temperature exceeded the optimal range, and the seaweed also died from “ice–ice” disease. Kumar et al. (2020) discovered that seaweeds incubated above 32°C produced significantly lower carrageenan yields than seaweeds incubated at 28°C and that temperatures above the optimal range reduced gel strength and viscosity. Temperatures above 32°C also resulted in lower pigment content and, as a result, lower photosynthetic efficiency, poor growth rates, lower carrageenan yield and quality, and “ice–ice” symptom and biomass loss to thallus fragmentation. Overall, for most K. alvarezii, even minor temperature variations (above the ideal temperature) can harm the seaweed’s health, which will undoubtedly impact the production of carrageenan yield.

Salinity is another abiotic factor that may influence the carrageenan yields of K. alvarezii because it affects the seaweed’s osmotic equilibrium and nutrient absorption (Aris et al., 2021). Since carrageenan is essential for maintaining the ionic equilibrium of the cell, it is well recognized that salinity impacts the osmoregulation mechanism, affecting carrageenan yield and quality (Percival, 1979). However, Setyawidati et al. (2017) reported that the salinity ranges differing between high (33.8–34.8 psu) and low (33.0–34.0 psu) carrageenan production were only minor, implying that salinity amplitude may be insufficient to explain the variability in carrageenan yield. In contrast, Aris et al. (2021) investigated the effect of different salinity levels (30–34 ppt) on the weight of K. alvarezii explants and discovered that the varied salinities affected the growth rate of explants, with the best explant growth occurring at a salinity of 31 ppt and the lowest growth occurring at a salinity of 34 ppt, respectively.

Another factor influencing seaweed carrageenan content is the amount and quality of sunlight that reaches the seaweed. Seaweeds have evolved mechanisms to regulate their photosynthetic activity and adapt to changing light regimes, including the production of pigments that complement the spectral quality of the incident light (Thien et al., 2021). Asni (2021) examined the relationship between seaweed carrageenan yield and season, discovering that the carrageenan content in K. alvarezii was higher during the rainy season than during the dry season. However, the study also stated that an increase in sunlight would cause an increase in photosynthetic rates in seaweed and an increase in carrageenan amount in cultured marine algae (Campo et al., 2009). Seaweed cells typically absorb nutrients (carbon, hydrogen, oxygen, sulfate, and nitrate) and convert them into polysaccharides in the form of carrageenan, which can subsequently be deposited on the cell walls via the photosynthesis process (Hayashi et al., 2007). As a result, if the photosynthetic activity is disrupted, seaweed development may be hampered, and carrageenan levels may become undesirable (Hurtado et al., 2009).

Some seaweed farms in the southern Philippines, such as Tawi-Tawi, that were once very productive, are now producing only moderate seaweed yields. This phenomenon could be attributed to nutrient depletion caused by poor quality planting material or overstocking of seaweed in farms (Santhanaraju Vairappan, 2021). According to Kumar et al. (2015), nutritional availability is one of the factors that may cause seasonal fluctuations in carrageenan content. Farming practices must be improved to address this issue, such as seedling enrichment with biostimulants to increase growth rate and carrageenan yield (Luhan et al., 2014; Hurtado and Critchley, 2018). The essential macronutrients for seaweed development are carbon, nitrogen, and phosphorus, and it is crucial to managing nutrient availability in seaweed aquaculture or farming to achieve a better outcome (Harrison and Hurd, 2001; Msuya and Porter, 2014). Phosphorus is a significant nutrient that, when combined with nitrogen, plays a vital role in the growth of algae and carrageenan production (Chopin et al., 2004; Sarri et al., 2022). A recent study also discovered substantial seasonal and regional variation in farmed K. alvarezii production and product quality in Indonesia, with optimal nutrient availability producing the highest growth and carrageenan yield and quality (Simatupang et al., 2021).

Biotic variables have also been shown to impact carrageenan yield in seaweeds. One of the challenges in seaweed production is regulating epiphytes in farms, which can be detrimental to the overall production. Due to the open nature of the water, other algae have numerous opportunities to travel with the current and adhere themselves as competitors to seaweed that has grown. Epiphytes and diseases are two major environmental factors that can influence the development, output, and carrageenan content of K. alvarezii (Syamsuddin et al., 2019). Suboptimal culture conditions, such as low light intensity, low salinity (below 20 ppt), and high temperature (above 35°C), can lead to the development of “ice–ice” disease, which is frequently associated with the growth of epiphytes on the seaweed and can result in lower production or crop failure (Valderrama et al., 2013).

The presence of epiphytes in seaweed production is a common occurrence, but they can become a significant issue that reduces seaweed productivity (Lüning and Pang, 2003; Vairappan, 2006; Hurtado et al., 2007). Sargassum polycystum is one of the most common epiphytes attached to seaweed thalli, exacerbating biomass loss in coastal farms (Aslan et al., 2020). Several other epiphytes isolated from the K. alvarezii seaweed included Entheromorpha intestinalis, Ceramium sp., Neosiphonia apiculata, Chaetomorpha crassa, Hypnea sp., and Gracilaria sp. (Mulyaningrum et al., 2019). It has been demonstrated that epiphytes cover the surface of seaweed thalli, considerably reducing nutrient absorption (Wakibia et al., 2006; Vairappan et al., 2007). According to Aeni et al. (2019), the combined effects of “ice–ice” disease and epiphyte development decreased the growth rate during K. alvarezii cultivation in southeast Sulawesi. Mulyaningrum et al. (2019) compared healthy seaweed to infected seaweed and discovered that healthy seaweed had a higher carrageenan yield and gel strength than infected seaweed. In many cases, the epiphyte was more prevalent and occurred more frequently in conjunction with “ice–ice” disease (Vairappan et al., 2007, 2010; Eranza et al., 2017).

While epiphytes attach to the seaweed thalli, “ice–ice” disease-causing bacteria commonly infect the thalli tips. The bacteria are considered to act as secondary causative agents, but the mechanism by which they do so is currently unknown. Certain studies have shown that the pathogenic bacterium Vibrio sp. can quickly adhere to and dominate seaweed tissue as a first stage in the infection process, based on its motile capabilities (Largo et al., 1995, 1998). When exposed to harsh conditions, the bacteria can use carrageenan in seaweed thalli as a carbon source by reaching the medullary layer of diseased branches (Tahiluddin and Terzi, 2021). A similar ability of Pseudoalteromonas carrageenovora to produce the specific κ-carrageenase enzyme and degrade κ-carrageenan in K. alvarezii suggested that this bacterium might cause “ice–ice” disease symptoms such as thalli whitening and scaling (Riyaz et al., 2019).

The antibacterial properties of seaweeds have opened up a wide range of potential applications in food and pharmaceutical research (Fayaz et al., 2005). Kappaphycus alvarezii contains various nutritional compounds, including carrageenan, that can be used as a nutritional supplement. The polymer has shown significant antibacterial activity against a wide range of human pathogens, including Bacillus subtilis, Staphylococcus aureus, Lactobacillus acidophilus, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Vibrio cholerae (Pushparaj et al., 2014). According to a study, gram-positive bacteria are more vulnerable to seaweed extracts than gram-negative bacteria, and this vulnerability to algal extracts is attributable to the differences in the structure and content of their cell walls (Čagalj et al., 2022). Additionally, K. alvarezii carrageenan has shown excellent suppression of Mycobacterium tuberculosis (Mayakrishnan et al., 2015). Molecular docking with specific software and visualization tools was used to investigate the mechanism of action of K. alvarezii against M. tuberculosis. In the study, κ-carrageenan was discovered to be able to inhibit the InhA enzyme, which was found to exist in both strains of M. tuberculosis and thus controls the activity of the pathogenic bacteria. Besides, oxidized κ-carrageenans prepared with hydrogen peroxide were capable of damaging the bacterial cell wall and cytoplasmic membrane, effectively suppressing bacterial growth, and potentially acting as a new antibacterial agent against a variety of food-borne pathogens (Zhu et al., 2017a).When determining antimicrobial activity in polysaccharides derived from seaweeds, criteria such as molecular weight, charge density, sulfated content in the case of sulfated polysaccharides, and structural and conformation features are considered (Pérez et al., 2016). The antimicrobial mechanism induced by macroalgal compounds, including carrageenan oligosaccharides, is generally based on the modification of bacterial cell porousness, which results in the loss of macromolecules, disruption of membrane function, and ultimately the destruction of pathogen cells (Ismail et al., 2020).

Carrageenan derived from red algae has been shown to possess antiviral properties against a wide range of enveloped and non-enveloped viruses (Ahmadi et al., 2015; Talarico and Damonte, 2016). Since the early 1900s, marine algal sulfated carrageenan has been extensively studied for its antiviral activity, which includes inhibiting viral attachment to the host cell surface, viral internalization and uncoating, and viral transcription and replication (Shi et al., 2017; Álvarez-Viñas et al., 2021). According to Talarico and Damonte (2016), the ability of sulfated polysaccharides to prevent viral entry into the host is the most well-studied biological feature responsible for antiviral action. Due to having the highest sulfate content among the sulfated carrageenans, λ-carrageenan has been shown to have better antiviral activity than κ- and ι-carrageenans (Hans et al., 2021). The chlorosulfonic acid-pyridine approach, sulfuric acid (using a mixture of concentrated sulfuric acid and butanol), and sulfur trioxide-pyridine procedures are commonly used for sulfating polysaccharides to improve their antiviral activities (Ahmad, 2021). The presence of an adequate sulfation degree can be deduced from the fact that sulfation of κ- and ι-carrageenan increased their activity to levels comparable to those of λ-carrageenan; however, no increase was observed after sulfation of λ-carrageenan (Álvarez-Viñas et al., 2021). Yamada et al. (2000) expanded on the significance of this feature by showing that desulfation of the original carrageenans reduced their anti-Human Immunodeficiency Virus (HIV) activity. However, previous research discovered that λ-carrageenan and ι-carrageenan had significantly different anti-HIV potencies despite having comparable sulfate content (Yamada et al., 1997). This finding suggests that antiviral activities are not entirely dependent on sulfate content; instead, the positions and densities of individual sulfate groups on sugar chains appear to be significant considerations (Besednova et al., 2019). Carrageenans are also notable for their ability to inhibit the Human Papillomavirus (HPV) in vitro, exerting their inhibitory effects at an early stage of viral infection (Buck et al., 2006). The effectiveness of carrageenan against a variety of sexually transmitted viruses, including the Herpes Simplex Virus (HSV), was discovered to be due to a stiff interaction between the virus and the carrageenan, which causes the viruses to lose their ability to infect cells, effectively reducing virus multiplication and increasing mortality (Carlucci et al., 2002; Bai and Tuvikene, 2021). Shi et al. (2017) described the mechanism of macroalgal polysaccharides against viral diseases during the initial viral attachment phase, where an immune response is triggered when the polysaccharide attaches to virions or the associated protein receptor immunomodulators, activating natural killer cells or inducing an immunological response as part of the process.

The ability of polysaccharides to successfully inhibit the proliferation and development of cancer cell colonies in vitro, and their ability to effectively suppress the growth and spread of malignancies in experimental animals when administered in vivo, is still being investigated (Lin et al., 2020). Carrageenans have been shown to suppress tumor cell proliferation and adhesion to diverse substrates by preventing connections between cancer cells and the basement membrane (Pangestuti and Kim, 2014). They also exhibit anticancer activity by improving immunity, which may be related to the activation of the immunocompetence of the body, and specifically targeting key apoptotic molecules, making them potential chemotherapeutic or chemopreventive agents (Yuan et al., 2011; Liu et al., 2019). Calvo et al. (2019) discovered that both κ- and ι-carrageenans, and carrageenan oligosaccharides such as disaccharides (carrabioses) and disaccharide-alditols (carrabiitols) have cytotoxic effects on LM2 cancer cells, killing them via an apoptotic mechanism. Furthermore, previous research has shown that carrageenan can arrest the cell cycle at specific stages, such as the G2 or S phases, indicating a mechanism affecting the tumor cell cycle (Prasedya et al., 2016). These activities have piqued the interest of researchers in recent years because cancer is regarded as one of the most severe diseases, posing a threat to human health and life. Unfortunately, most cancer treatments are currently hazardous, frequently affecting not only tumor cells but also normal healthy cells throughout the body. As a result, contemporary pharmacology and biotechnology are charged with the critical mission of discovering novel, effective, and non-toxic anticancer compounds derived from natural sources. The Joint FAO/WHO Expert Committee on Food Additives (JECFA; 2001) concluded that carrageenans are safe for human consumption and thus a safe alternative to chemotherapy based on numerous toxicological studies conducted on animal models (EFSA Panel on Food Additives and Nutrient Sources Added to Food (ANS) et al., 2018).