Fucoidan is a polysaccharide mainly composed of fucose, with a high sulphate content (more than 20%). This carbohydrate moiety is found in marine resources, generally in brown algae, sea cucumbers, and sea urchin eggs (as indicated in Table 1). The chemical structure of fucoidans consists of a backbone of α-(1→3)-L-fucopyranose residues, or alternating α-(1→3)-linked and α-(1→4)-linked L-fucopyranose or α-(1→2)-L-fucopyranose residues (Figure 1), with different substitutions, such as fucose, sulphate, acetate, and uronic acid ^[1][2][1,2]. Fucoidan presents a wide range of pharmaceutical activities, including antioxidant, anticancer, immunomodulatory, anticoagulant, and antimicrobial activities ^[3][4][3,4].

In fact, the preparation and structural characterisation of fucoidan seem to be well elucidated. However, haemorrhagic risk, high molecular weight (MW), high viscosity of fucoidan, and a poor dissolution rate in solution may limit their successful application in functional foods and pharmaceuticals ^[9][10][9,10]. Fucoidans often differ in terms of structural diversity and complexity; therefore, standardisation and quality assurance of fucoidans are challenging. Therefore, there has been a recent increase in interest in degrading fucoidan into fuco-oligosaccharides (FOSs) or low-MW fucoidan (LMWF) to broaden potential applications.

Oligosaccharides consist of a mixture of 2–20 monosaccharides, with a variable extent of polymerisation. They have attracted increasing interest owing to their nutritional and biological properties [11]. Many functional oligosaccharides, such as fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, manno-oligosaccharides, and isomalto-oligosaccharides, have been widely used as prebiotics in food application ^[12][13][14][12,13,14]. Commercial prebiotics are derived from terrestrial plant oligosaccharides. However, research is underway to explore new prebiotics from natural resources, especially from renewable sources. Marine algae have unparalleled advantages as easily renewable and extremely abundant resources, as they grow in seawater and avoid the demand for freshwater. FOSs, also called LMWF, is an oligomer of fucoidan, which is normally obtained from brown algae. FOSs have a degree of polymerisation (DP) < 20 and an average MW of less than 10 kDa. FOSs also show a prebiotic ability and can be fermented by the gut microbiota, exert a range of physiological effects on body functions and improve human health [15]. Therefore, FOSs and other marine algae oligosaccharides are considered promising prebiotics.

2. Preparation of Fucoidan and/or Raw Materials for Fuco-Oligosaccharide Production

The advantages of FOSs include high water solubility, low viscosity, and good biological activity. However, FOSs are seldom found in natural resources. Therefore, the degradation of fucodain is necessary for the production of FOSs. Figure 2 shows a singular diagram of the process of preparing fucoidan from brown algae. The extraction processes are based on aqueous (water, acidic, or alkaline solution) extraction; the polysaccharides are precipitated by ethanol; proteins are removed by the Sevag or enzymatic method, and the low-molecular-weight impurity is discarded by diafiltration [16]. The Sevag method is widely used to discard protein. It was first exploited by Sevag et al., as they used an organic solvent, including chloroform and n-butanol, to precipitate protein from sample solution [17]. An acid or calcium chloride solution added to the polysaccharide mixture has been commonly used to separate alginic acid from fucoidan. Béress et al. precipitated the alginic acid by adding glacial acetic acid, and the process was completed after 12 h at 4 °C [18]. Furthermore, column chromatography, such as ion-exchange and size-exclusion methods, can be used to obtain fucoidan [19]. Currently, three major processes exist to obtain FOSs from fucoidan: degradation by mild acids, enzymes, and radical methods. The mild acids and radical degradation methods are considered to be random and non-specific in degrading fucosidic linkages, while enzymatic method recognizes the specific substrate to cleave fucosidic linkages.

3. Preparation of Fuco-Oligosaccharides

3.1. Mild Acid Hydrolysis

Mild acid hydrolysis usually uses dilute acid solutions, such as sulphuric acid, hydrochloric acid, and trifluoroacetic acid, at suitable heating temperatures and times. FOSs mixtures are obtained after hydrolysis and subsequent neutralisation. Mild acid hydrolysis seems to offer a simple, low-cost production, and an easy operation, especially to fulfil requirements in the laboratory. Therefore, the mild acid hydrolysis method is always adopted by laboratories to screen for potential biological activities of FOSs. An aliquot of 0.05 mol/L sulphuric acid solution was used to degrade Nemacystus decipiens fucoidan at 80 °C for 2 h to obtain FOSs with a DP ranging from 2 to 9, and the FOSs showed antithrombotic activity [20]. A high-MW fucoidan, derived from S. hemiphyllum, was degraded by 0.01 mol/L hydrochloric acid at 85 °C for 15 min to obtain FOSs with a MW of 800 Da. The result showed skin-protective effects against ultraviolet B damage [21]. However, the degradation method is non-specific and may produce a series of FOSs, which increases the challenge of purifying FOSs. Moreover, the sulphated group in fucoidan is acid-labile and easily hydrolysed during acid hydrolysis. Mild acid hydrolysis (parameters used 0.005–0.01 mol/L trifluoroacetic acid at 60–100 °C for 2–4 h) was conducted on fucan sulphate derived from S. herrmanni, to obtain a number of FOSs. They contained two series of FOSs: 2-desulphated and 2-sulphated residues at the reducing ends of FOSs [22]. Pomin et al. found that in the dilute acid hydrolysis of fucoidan, the 2-sulphate ester group is initially cleaved, subsequently degrading the glycosidic bonds between the non-sulphated residue and the 4-sulphate group [23]. The kinetic constant of mild acid hydrolysis on sulphated fucan derived from three sea cucumber species, I. badionotus, H. floridana, and L. variegatus, with core α (1→3)-linked fucose, degraded the sulphate group substitution at the 2-O and 4-O-positions. The mild acid hydrolysis progress was under 0.05 mol/L sulphuric acid at 60 °C for 9 h, and selective 2-desulphation in sulphated fucan and tetrasaccharides FOSs were obtained [24]. Mild acid hydrolysis is suitable for a large-scale production of FOSs. Future studies on the kinetics and mechanism of mild acid hydrolysis should focus on providing fundamental data for industrial application in the large-scale production of FOSs.

3.2. Enzymatic Hydrolysis

Several hydrolytic enzymes that catalyse the hydrolysis of polysaccharides have been characterised. Normally, the breakdown of fucoidans involves the degradation of glycosidic linkages by fucoidanases (EC 3.2.1.44) and α-L-fucosidases (EC 3.2.1.51, EC 3.2.1.63, EC 3.2.1.111, and EC 3.2.1.127), which converts fucoidans into FOSs [25]. The enzymes that produce FOSs are produced by marine microorganisms and include endo- and exo-enzymes [26]. The well characterized endo-enzymes for fucoidan hydrolysis include endo-fucanases FcnA, FFA1, FFA2, Fp273, Fp277, Fp279, FWf1, FWf2, Fhf1, Fhf2 of the GH107 family and endo-fucanase FunA of the GH168 family, while the α-L-fucosidase is exo-enzymes and described for the families of glycoside hydrolases (GH) 29, 95, and 141 ^[25][27][28][25,27,28]. Enzymatic depolymerisation is highly selective for specific substrates and efficient for producing the desired end-product under selective method conditions. Therefore, with the enzymatic method, unlike the mild acid hydrolysis method, it is possible to achieve hydrolysate of crude fucoidan yielding FOSs. The extent of enzymatic action is strongly dependent on operating conditions, including temperature and pH. Fucoidan with a MW of approximately 400 kDa from F. distichus was incubated together with endo-α-(1,4)-fucoidanase from the marine bacterium Formosa haliotis (Fhf1Δ470), in Tris-HCl buffer (pH 8) at 37 °C for 1 d, yielding FOSs with a MW of 2 kDa [29]. The study demonstrated the use of the enzymatic technique to screen out the tailored fucoidan functional group (FOSs) to specifically improve bone regeneration [29]. Enzymatic depolymerisation of L. japonica fucoidan by fucoidanase—which is produced by Flavobacteriaceae RC2-3—yielded FOSs with a MW < 2 kDa (96.3%) after incubation at 37 °C for 10 h. The FOSs products showed an excellent radical scavenging activity and inhibition of tyrosinase activity, rendering them suitable for application as cosmetic skin-whitening additives [30]. Another advantage of the enzymatic method is that the sulphated groups in FOSs may be preserved during the depolymerisation process. A fucoidanase obtained from the digestive glands of mollusc Lambis sp., specifically, degraded (1→3) and (1→4)-α-L-fucans derived from F. evanescens and F. vesiculosus, yielding sulphated FOSs, without any effect on the sulphate group [31]. To overcome problems of using the enzyme, including those owing to its high cost and poor stability, the immobilised enzyme technique is preferred. The immobilised enzyme technique has the advantages of maintaining enzymatic activity, ease of recovery after coating monolithic substrates, and easy scaling up of processes for industrial application [32]. The enzymatic method is considered a good choice for producing FOSs for applications in the food, cosmetic, and pharmaceutical industries. Therefore, screening for new enzymes that can degrade fucoidan has become an increasingly important focus of scientists and industrial engineers; in particular, searching for new endo-fucoidanases and their subsequent kinetic analysis, molecular cloning, and immobilisation techniques to provide valuable fundamental knowledge to produce highly active FOSs.

3.3. Radical Depolymerisation

Radical depolymerisation is a process that uses radicals to attack glyosidic linkages of polysaccharides to form oligosaccharides. The radicals, including HOO⁻•, •O₂⁻, and HO•, can be generated by hydrogen peroxide (H₂O₂) and a catalytic metal system. Depolymerisation by H₂O₂ involves radicals (HOO•, •O₂⁻, and HO•) attacking the glycosidic linkages [33]. This method has the advantages of good reproducibility and controllability. The main parameters that influence the efficacy of depolymerisation include the temperature, concentration of H₂O₂, reaction time, and substrate concentration. Radical depolymerisation of fucan sulphate from S. herrmanni was performed by adding H₂O₂ and copper (II) acetate monohydrate. On average, the MW of fucan sulfate was degraded from 790.8 kDa to 8.32–22.11 kDa. The resulting products exhibited good anticoagulant activity [34]. Moreover, a combination of ascorbic acid and H₂O₂ was used to degrade fucoidan at 25 °C for 16 h, to obtain a fraction with the MW of approximately 3 kDa, which exhibited anti-lung-cancer activity in vitro [35]. Qi et al. reported that radical depolymerisation can maintain the sulphate content or other functional units in the degradation process [36]. Qi et al. degraded U. pinnatifida fucoidan using a photocatalytic method, which processes the reaction in H₂O₂ and TiO₂ under xenon light for 3 h. While the MW of 190 kDa was degraded to 3 kDa, the resulting product exhibited anticoagulant activity [36]. Moreover, photoirradiation techniques for the degradation of polysaccharides have been attracting increasing attention. These techniques provide efficient degradation methods for the production of FOSs from fucoidan, because photoirradiation can be achieved at room temperature and under normal pressure. Ultraviolet and gamma radiation are typically used to degrade fucoidan. They are an energetic form of electromagnetic radiation, with a short wavelength and high energy. Water radiolysis is the major effect of gamma irradiation of solutions. Higher gamma irradiation doses may result in a lower MW of fucoidan: the MW of F. vesiculosus fucoidan degraded from 210 kDa to 85 kDa and 7 kDa following irradiation doses of 8 kGy and 100 kGy, respectively, while the latter degraded product showed the highest antioxidant activity [37]. In addition, photoirradiation combined with radical depolymerisation increased the efficiency of FOSs production. The average MW of fucoidan decreased to 6.7 kDa at an irradiation dose of 20 kGy, while the degradation efficiency increased by 5% when a 10% H₂O₂ concentration was used in combination with gamma radiation. The hydroxyl radical scavenging abilities of degraded product of FOSs were more effective than fucoidan [38]. Park and Choi also used a combination of gamma radiation at a dose of 100 kGy and H₂O₂ to degrade U. pinnatifida fucoidan from MW 378 kDa to 6 kDa. Park and Choi found that degraded fucoidan has a higher inhibitory activity against tyrosinase and antioxidant activity than fucoidan [39].

4. Purification of Fuco-Oligosaccharides

The mixtures of FOSs produced by degradation methods include a variety of compounds, i.e., monosaccharides, FOSs ranges of different DPs, and large MW fucoidan. To obtain high-purity FOSs (for use in studies of the structure–function relationship and mechanism, and for applications in functional food or pharmaceutical areas), some suitable purification steps should be performed. Figure 3 shows a schematic diagram of the production and purification of FOSs. Step-gradient ethanol precipitation is a convenient and time-saving method for the large-scale production of FOSs. Different MWs can be prepared by adding an ethanol gradient at a final concentration of 10–80%. However, this method yields a broad MW distribution of FOSs, and it is difficult to obtain high-purity FOSs. For example, Fernando et al. performed a step-gradient ethanol precipitation to exclude high-MW fucoidan, and a broad MW distribution of FOSs, ranging from 8 to 25 kDa, was achieved [40]. Thus, ethanol precipitation is useful as a preliminary step for discarding high-MW fucoidans or proteins. Membrane filtration is another green technique for the purification of FOSs, and its performances are quite similar to gradient ethanol precipitation, which obtained a broad MW distribution of FOSs. Zhao et al. purified a degraded fucoidan mixture, using an ultrafiltration membrane (10 kDa and 2.5 kDa MW cut-off) to obtain the FOSs fraction, and the fraction range 2.5–10.0 kDa was collected [9]. The degraded fucoidan samples passed through a 30-kDa-MW cut-off membrane and subsequently through a 1-kDa membrane, obtaining an FOSs fraction MW range of 1–30 kDa [41]. Dialysis bags with different MW cut-off membranes have been used to obtain MW values of lower than 10 kDa fraction [42]. Column chromatography, including ion-exchange chromatography and size-exclusion chromatography, is widely used for fractionation oligosaccharides during purification. Size-exclusion chromatography has been used to fractionate oligosaccharides according to their MW, and this is suitable for the purification of FOSs and achieving high purity. Commercially available size-exclusion chromatography resins have been used to purify FOSs. The purified FOSs fractions are suitable for structural identification and pharmaceutical activity investigation. After mild acid hydrolysis the fucoidan hydrolysate sample was purified using a Bio-Gel P-2 column, and a high-purity fucotrioligosaccharide was obtained [43]. A mixture of oligosaccharides, produced from acid hydrolysis, was further purified using Bio-Gel P-4 and P-2, to obtain a high-purity yield of 6–9% of monosulphated fucobiose and monosulphated fucotriose [44]. Ion-exchange chromatography for FOSs purification proceeds according to the sulphate content of the FOSs, which leads to different ionic interactions with the resins. The FOSs mixture produced from fucoidan was degraded by the enzymatic method and further purified by ion-exchange chromatography on Q-Sepharose, to yield DP 4–10 FOSs with different sulphate contents [45].