The Development of Seaweed-Derived Bioactive Compounds for Use As Prebiotics and Nutraceuticals Using Enzyme Technologies

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‘This is the peer reviewed version of the following article: Charoensiddhi, S., Conlon, M. A., Franco, C. M. M., & Zhang, W. (2017). The development of seaweed-derived bioactive compounds for use as prebiotics and nutraceuticals using enzyme technologies. Trends in Food Science & Technology, 70, 20–33. https:// doi.org/10.1016/j.tifs.2017.10.002 which has been published in final form at http://dx.doi.org/10.1016/j.tifs.2017.10.002

The development of seaweed-derived bioactive compounds for use as prebiotics and nutraceuticals using enzyme technologies

Suvimol Charoensiddhi, Michael A. Conlon, Christopher M.M. Franco, Wei Zhang

PII: S0924-2244(17)30294-7 DOI: 10.1016/j.tifs.2017.10.002 Reference: TIFS 2089

To appear in: Trends in Food Science & Technology

Received Date: 19 May 2017 Revised Date: 28 September 2017 Accepted Date: 9 October 2017

Please cite this article as: Charoensiddhi, S., Conlon, M.A., Franco, C.M.M., Zhang, W., The development of seaweed-derived bioactive compounds for use as prebiotics and nutraceuticals using enzyme technologies, Trends in Food Science & Technology (2017), doi: 10.1016/j.tifs.2017.10.002.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 ACCEPTED MANUSCRIPT 1 The development of seaweed-derived bioactive compounds for use as prebiotics and

2 nutraceuticals using enzyme technologies

4 Suvimol Charoensiddhi a,b , Michael A. Conlon c, Christopher M.M. Franco a,b , Wei Zhang a,b,*

6 a Centre for Marine Bioproducts Development, College of Medicine and Public Health,

7 Flinders University, Bedford Park, South Australia 5042, Australia

8 b Medical Biotechnology, College of Medicine and Public Health, Flinders University,

9 Bedford Park, South Australia 5042, Australia

10 e-mail: [email protected]

11 e-mail: [email protected]

12 e-mail: [email protected]

13 14 c CSIRO Health and Biosecurity, Kintore Avenue, MANUSCRIPT Adelaide, South Australia 5000, Australia 15 e-mail: [email protected]

17 * Corresponding author. Tel.: +61 8 72218557; fax: +61 8 7221 8555; e-mail address:

18 [email protected]

20 21 ACCEPTED 22

25 2 ACCEPTED MANUSCRIPT 26 Abstract

27 Background: Seaweeds are a large and diverse group of photosynthetic macro-algae found

28 across the world’s oceans. There is a growing recognition that they are important sources of

29 bioactive compounds with a variety of biological activities that could potentially contribute to

30 functional food and nutraceutical industries.

31 Scope and approach: The complex structure and distinctive components of seaweed cell

32 walls, which differ significantly from terrestrial plants, presents a major challenge for the

33 effective extraction of bioactive compounds from inside the cells. Enzyme technologies have

34 been used to improve the extraction, hydrolysis, and structure modification efficiently with a

35 high degree of environmental sustainability. This review critically analyses the advances,

36 challenges, and future directions in applying enzyme technologies to improve the extraction

37 and processing of bioactive compounds from seaweeds and their potential applications in

38 functional foods and nutraceuticals. 39 Key findings and conclusions: Different enzymatic MANUSCRIPT processes have been demonstrated to (1) 40 assist the extraction by breaking down the seaweed cell walls, and (2) degrade or hydrolyse

41 macromolecules including polysaccharides and proteins. These enzymatic processes improve

42 the yield and recovery of bioactive compounds and enhance their biological properties with

43 regard to prebiotic, antioxidant, ACE inhibitory, anti-inflammatory, and antiviral effects.

44 Seaweed-derived bioactive compounds from these processes present significant new

45 opportunities in developing novel food applications. The current food regulations and safety 46 requirements forACCEPTED seaweeds and their products are addressed for commercial product 47 development.

48 Key words Biological properties; Enzymatic process; Food safety; Functional food;

49 Macroalgae; Prebiotic activity

50 3 ACCEPTED MANUSCRIPT 51 1. Background

52 Marine macroalgae or seaweeds constitute approximately 25,000-30,000 species

53 (Santos et al., 2015), with a great diversity of forms and sizes. They can be categorized into

54 different taxonomic groups reflecting their pigmentation, including red algae

55 (Rhodophyceae), brown algae (Phaeophyceae), and green algae (Chlorophyceae) (Mohamed

56 et al., 2012). Seaweeds have become an appealing source for commercial applications as they

57 have fast growth rates and do not require arable land, fresh water, or even fertilizer when

58 compared with terrestrial plants (Lorbeer et al., 2013). The cultivation of seaweeds has been

59 growing rapidly and is now practiced in about 50 countries, and 28.5 million tonnes of

60 seaweeds and other algae were harvested in 2014 to be used for direct consumption, or as a

61 starting material for the production of food, hydrocolloids, fertilizers, and other purposes

62 (FAO, 2016). Recently, the annual global production of alginate, carrageenan, and agar

63 which are the most important seaweed hydrocolloids for various applications across the food, 64 pharmaceutical, and biotechnology industries has MANUSCRIPTreached 100,000 tonnes and a gross market 65 value just above USD 1.1 billion (Rhein-Knudsen et al., 2015).

66 Seaweeds also contain a great variety of structurally diverse bioactive metabolites not

67 produced by terrestrial plants (Gupta and Abu-Ghannam, 2011). Seaweeds are rich in

68 carbohydrates, proteins, polyunsaturated fatty acids (PUFAs) including omega-3 fatty acids,

69 and minerals as well as polyphenols, pigments (chlorophylls, fucoxanthins, phycobilins), and

70 mycosporine-like amino acids (MAAs). These compounds possess various biological 71 functions includingACCEPTED antioxidant, anti-HIV, anticancer, antidiabetic, antimicrobial, 72 anticoagulant, antivirus, anti-tumor, anti-inflammatory, immunomodulatory, prebiotic and

73 cholesterol lowering effects (Holdt and Kraan, 2011). Although seaweed bioactive

74 compounds are attractive for commercialisation in different functional food and nutraceutical

75 products, the use of seaweed for this purpose is still not extensive. 4 ACCEPTED MANUSCRIPT 76 The industrial use of enzymes to extract natural compounds from terrestrial plants for

77 food and nutraceutical purposes has been developed and reported as a promising technology

78 with a number of benefits such as saving process time and energy and improving the

79 reproducible extraction process at the commercial scale (Puri et al. 2012). However, the

80 efficiency of enzymatic extraction procedures for the retrieval of active compounds from

81 seaweeds may be inhibited by the more complex and heterogeneous structure and

82 composition of seaweed cell walls in comparison to plants, which have a cell wall mostly

83 consisting of cellulose and hemicellulose.

84 The main cell wall and storage polysaccharides of seaweeds vary with taxonomy. The

85 structural polysaccharides of green seaweeds are sulphated polysaccharides, such as ulvans

86 and sulphated galactans, xylans, and mannans, while the main storage polysaccharide is

87 starch. Brown seaweeds contain laminarins as the storage polysaccharide, and the main cell

88 walls are composed of alginic acids, fucoidans, and sargassans. On the other hand, red 89 seaweed cell walls consist of agars, carrageenans, MANUSCRIPT xylans, water-soluble sulphated galactans, 90 and porphyrins (mucopolysaccharides), and the main storage polysaccharide is floridean

91 starch (amylopectin-like glucan) (Mišurcová, 2012; Kraan, 2012). This is further complicated

92 by a tightly-integrated network of biopolymers in seaweed cell walls, mainly

93 polysaccharides, that are associated with proteins, proteoglycans, polymeric phenols, and

94 various bound ions including calcium and potassium (Jeon et al., 2012; Synytsya et al.,

95 2015). An example of the structure model of a brown seaweed cell wall is shown in Fig. 1. In 96 addition, marineACCEPTED algae have adapted to salty environments, unlike land plants. Charoensiddhi 97 et al. (2016b) reported that salt buffers significantly reduced the extraction efficiency of

98 carbohydrates from brown seaweed, compared with pure water. This may be due to the

99 ability of pure water to cause an osmotic shock and the rapid influx of water into seaweed

100 cells, which disrupts the structural integrity of the cell wall and facilitates extraction. The 5 ACCEPTED MANUSCRIPT 101 high salt content in seaweeds may inhibit enzyme-assisted extraction processes, particularly

102 those requiring buffer systems.

103 This review aims to critically analyse the potential role of enzyme technology in

104 assisting with the extraction and digestion of bioactive compounds from seaweeds, and to

105 understand the advantages, limitations, challenges, and future development directions in the

106 application of the seaweed-derived ingredients in functional food products and nutraceuticals.

107

108 2. Seaweed bioactive compounds and their potential as functional foods and

109 nutraceuticals

110 Carbohydrates account for the majority of seaweed biomass. Polysaccharides and

111 oligosaccharides have therefore been the key focus of many studies looking at seaweed-

112 derived compounds. Aside from those, phenolic compounds and proteins from seaweeds have

113 also been widely studied as potential functional ingredients. Therefore, these three classes 114 will be the main focus of this review. MANUSCRIPT 115 2.1. Polysaccharides

116 From an economic perspective, seaweed polysaccharides are the most important products

117 produced from seaweeds (Michalak and Chojnacka, 2015). As the major components in

118 seaweeds, polysaccharides account for up to 76% of the dry weight (DW) (Holdt and Kraan,

119 2011). Seaweeds contain a high total dietary fibre content: 10-75% for brown seaweed, 10-

120 59% for red seaweed, and 29-67% for green seaweed. Seaweeds are particularly rich in 121 soluble dietary ACCEPTEDfibre, which accounts for 26-38%, 9-37%, and 17-24% in brown, red, and 122 green seaweed, respectively (de Jesus Raposo et al., 2016). Most of these polysaccharides can

123 be fermented by gut microbiota, which may provide a health benefit to humans through a

124 prebiotic effect (O’Sullivan et al., 2010), which will be discussed in more detail later.

125 Additionally, sulphated polysaccharides have shown anti-inflammatory, antioxidant, 6 ACCEPTED MANUSCRIPT 126 antibacterial, and immunological activity. These include fucoidans (L-fucose and sulphate

127 ester groups) from brown seaweeds, agars and carrageenans (sulphated galactans) from red

128 seaweeds, and ulvans (sulphated glucuronoxylorhamnans) and other sulphated glycans from

129 green seaweeds (Synytsya et al., 2015).

130 2.2. Phenolic compounds

131 Phlorotannins are the major phenolic compounds found in brown seaweeds, constituting up to

132 14% of dry seaweed biomass, while other phenolic compounds are found at lower levels in

133 some red and green seaweeds. Phlorotannins are highly hydrophilic components formed by

134 the polymerization of phloroglucinol (1,3,5-trihydroxybenzene) monomer units with a wide

135 range of molecular weights between 126 Da and 650 kDa. They can be categorized into four

136 groups based on their linkages which are fuhalols and phlorethols (ether linkage), fucols

137 (phenyl linkage), fucophloroethols (ether and phenyl linkage), and eckols (dibenzodioxin

138 linkage) (Li et al., 2011). Phlorotannins have been explored as functional food ingredients 139 with many biological activities such as antioxidant MANUSCRIPT, anti-inflammatory, antidiabetic, anti- 140 tumor, antihypertensive, and antiallergic activities (Freitas et al., 2015).

141 2.3. Proteins

142 Bioactive proteins and peptides from seaweeds have been demonstrated to have antioxidant,

143 antihypertensive, and anticoagulant activities (Harnedy and FitzGerald, 2011). Generally, a

144 higher content of proteins is found in green and red seaweeds (10-47% of DW) compared to

145 brown seaweeds (3-15% of DW) (Wijesekara and Kim, 2015). Important bioactive proteins 146 from red and greenACCEPTED seaweeds include lectin and phycobiliprotein and bioactive peptides from 147 brown seaweeds have also been reported with angiotensin-I-converting enzyme (ACE-I)

148 inhibitory potential (Fitzgerald et al., 2011). Additionally, most seaweed species are a rich

149 source of essential and acidic amino acids (Freitas et al., 2015).

150 7 ACCEPTED MANUSCRIPT 151 3. Seaweed-derived components as prebiotic sources

152 Interest in prebiotics as functional foods has increased due to their recognised health

153 benefits. The global prebiotics market size was over USD 2.9 billion in 2015, and continued

154 growth is expected due to rising consumer awareness of gut health issues (Grand View

155 Research, 2016). The term ‘prebiotic’ was introduced by Gibson et al. (2004, 2010), and

156 defined as substrates that (1) are resistant to gastrointestinal digestion and absorption, (2) can

157 be fermented by the microbes in the large intestine, and (3) selectively stimulate the growth

158 and/or activity of the intestinal microbes leading to health benefits of the host.

159 Dietary habits influence the composition and metabolic activity of the human gut

160 microbiota (Conlon and Bird, 2015; Flint et al., 2015). Several studies have demonstrated that

161 a higher intake of plant dietary fibre affects the makeup of beneficial intestinal microbiota

162 and metabolites, and inhibits the growth of potential pathogens compared with animal-

163 dominated diets richer in fat and protein (Wu et al., 2011; Claesson et al., 2012; Zimmer et 164 al., 2012; David et al., 2014). de Jesus Raposo MANUSCRIPTet al. (2016) suggested that most seaweed 165 polysaccharides such as alginates, fucoidans, laminarins, porphyrins, ulvans, and

166 carrageenans can be regarded as dietary fibre, as they are resistant to digestion by enzymes

167 present in the human gastrointestinal tract, and selectively stimulate the growth of beneficial

168 gut bacteria. Likewise, ingested polyphenols with complex structures can also reach the large

169 intestine where they can be converted into beneficial bioactive metabolites by microbes

170 (Cardona et al., 2013), as shown in Fig. 2, and this has been shown to occur for phlorotannins 171 from brown seaweedACCEPTED (Corona et al., 2016). The fermentation of seaweed components by 172 beneficial bacteria has also been shown to generate beneficial metabolites such as short chain

173 fatty acids (SCFA), particularly butyrate, acetate, and propionate (de Jesus Raposo et al.,

174 2016). The beneficial effects of SCFA on host health include protection from obesity, chronic

175 respiratory disease or asthma, cancer, and inflammatory bowel, as well as modulation of 8 ACCEPTED MANUSCRIPT 176 immunity, glucose homeostasis, lipid metabolism, and appetite regulation (Bultman, 2016;

177 Dwivedi et al., 2016; Koh et al., 2016; Morrison et al., 2016).

178 3.1. In vitro studies

179 In vitro studies commonly use anaerobic human fecal fermentation for 24 h as a

180 model for gut fermentation processes. Using this approach, Deville et al. (2007) reported that

181 laminarin could stimulate the production of SCFA; especially butyrate and propionate. Low

182 molecular weight (MW) polysaccharides from the red seaweed Gelidium sesquipidale caused

183 a significant increase in populations of Bifidobacterium , as well as an increase in acetate and

184 propionate (Ramnani et al., 2012). Rodrigues et al. (2016) obtained a similar result using an

185 extract from the brown seaweed Osmundea pinnatifida . Charoensiddhi et al. (2016a, 2017)

186 also demonstrated that extracts from the brown seaweed Ecklonia radiata stimulated the

187 production of SCFA and the growth of beneficial microbes such as Bifidobacterium and

188 Lactobacillus . In addition, Kuda et al. (2015) demonstrated that sodium alginate and 189 laminaran from brown seaweeds inhibited theMANUSCRIPT adhesion and invasion of pathogens 190 (Salmonella Typhimurium, Listeria monocytogenes , and Vibrio parahaemolyticus ) in human

191 enterocyte-like HT-29-Luc cells. Also, the effect of intact brown seaweed Ascophyllum

192 nodosum on piglet gut flora was studied by simulating small intestinal and caecal conditions,

193 with antibacterial effects, especially on E.coli , and a decrease in fermentative activity being

194 clearly observed (Dierick et al., 2010).

195 The digestibility of seaweed components can be determined in vitro to verify whether 196 they are decomposedACCEPTED by human digestive enzymes, and thus their likelihood of reaching the 197 large bowel and its resident microbiota. Neoagaro-oligosaccharides and glycerol galactoside

198 from red seaweeds, for instance, have shown that they were not digestible by enzymes

199 typically present in the small intestine (Hu et al., 2006; Muraoka et al., 2008).

200 9 ACCEPTED MANUSCRIPT 201 3.2. In vivo studies

202 Apart from in vitro studies, health benefits attributed to oligosaccharides and

203 polysaccharides derived from seaweeds have been demonstrated by in vivo animal models.

204 Most such studies have investigated prebiotic effects in rats or mice being fed a seaweed-

205 supplemented diet. Results from Liu et al. (2015) showed an increase in the abundance of

206 beneficial gut microbes such as Bifidobacterium breve and a decrease in pathogenic bacteria

207 such as Clostridium septicum and Streptococcus pneumonia in rats supplemented with the red

208 seaweed Chondrus crispus . Furthermore, an increase in SCFA production and colonic growth

209 was observed, as well as an improvement of host immunity modulation through elevation of

210 the plasma immunoglobulin levels. Supplementation of diet with the brown seaweeds

211 Undaria pinnatifida and Laminaria japonica has resulted in suppressed weight gain of rats,

212 influenced the composition of gut microbial communities associated with obesity by

213 reduction in the ratio of Firmicutes to Bacteroidetes , and reduced populations of pathogenic 214 bacteria including Clostridium , Escherichia , and EnterobacterMANUSCRIPT genera (Kim et al., 2016). The 215 oral administration of fucoidan extracts has also been shown to reduce the inflammatory

216 pathology associated with DSS-induced colitis in mice, indicating its potential for treating

217 inflammatory bowel disease (Lean et al., 2015). In addition, Kuda et al. (2005) reported that

218 rats fed a diet containing laminaran and low MW alginate suppressed the production of

219 indole, p-cresol, and sulphide which are the putative risk markers for colon cancer. Neoagaro-

220 oligosaccharides obtained from the hydrolysis of agarose by β-agarase caused an increase in 221 the numbers of LactobacillusACCEPTED and Bifidobacterium in the feces or cecal content of mice, along 222 with a decrease in putrefactive bacteria (Hu et al., 2006).

223

224

225 10 ACCEPTED MANUSCRIPT 226 4. Current enzymatic processing of seaweed-derived bioactive compounds

227 The selection of appropriate enzymatic processes to best suit the desired applications

228 of the final products is a key factor to achieve an effective output. According to the present

229 research in this area, two major enzyme-enhanced processes are normally implemented and

230 designed in order to develop bioactive compounds from seaweeds. They are enzyme-assisted

231 extraction and the enzymatic hydrolysis of macromolecules.

232 4.1 Enzyme-assisted extraction

233 The disintegration of cell wall structures is an elementary step to facilitate the

234 extraction of bioactive components. Various commercial enzymes, not specific to seaweeds,

235 have been used on seaweed cell walls for the extraction of bioactive compounds from

236 seaweeds. For instance, polysaccharides such as fucoidans are tightly associated with

237 cellulose and proteins (Deniaud-Bouët et al., 2014), which limits their extractability from

238 brown seaweeds. Therefore, hydrolysis of the cellulose and protein network by commercially 239 available cellulases and proteases may enable the MANUSCRIPTdisintegration of the cell wall complex, thus 240 releasing the fucoidans (or other target components).

241 Enzymes trialled for this purpose to date include carbohydrate hydrolytic enzymes

242 (AMG, Celluclast, Termamyl, Ultraflo, Viscozyme, etc.) and proteases (Alcalase,

243 Flavourzyme, Kojizyme, Nutrease, Protamex, etc.) (Hardouin et al., 2014a). The results from

244 Hardouin et al. (2014b) suggested that enzyme-assisted extraction may be a promising

245 technique for the recovery of proteins, neutral sugars, uronic acids, and sulphate groups from 246 green (Ulva sp.),ACCEPTED red (Solieria chordalis ), and brown (Sargassum muticum ) seaweeds. The 247 extract obtained from red seaweed using carbohydrase also showed antiviral activity. Their

248 approach appeared to have several advantages over conventional extraction methods,

249 including a high extraction rate, no special solvent requirements, and savings in time and

250 costs. Similar results were observed by Lee et al. (2010). Extracts from the brown seaweed 11 ACCEPTED MANUSCRIPT 251 Ecklonia cava , obtained using enzyme-assisted extraction with the commercial carbohydrase

252 Celluclast, showed potential for use as a therapeutic agent for diabetes to reduce the damage

253 caused by hyperglycaemia-induced oxidative stress, with several potential benefits for

254 commercialisation including its water solubility, multiple biological activities, high extraction

255 efficiency, and non-toxicity.

256 4.2 Enzymatic hydrolysis of macromolecules

257 The principle motives for the structural modification of seaweed components are the

258 enhancement of their biological activities to produce novel bioactive compounds, as well as

259 their structural determination. Specific enzymes are generally employed in order to (1)

260 degrade or hydrolyse high MW polysaccharides and proteins; or (2) act specifically on target

261 molecules to modify the structure of specific functional groups.

262 de Borba Gurpilhares et al. (2016) reported that hydrolases or lyases (alginate lyase,

263 fucoidanase, agarase, carrageenase, etc.) and sulfotransferases or sulfatases are important 264 seaweed-specific enzymes for preparing tailored MANUSCRIPT oligosaccharides and deducing the native 265 structure of sulphated polysaccharides. The study of Sun et al. (2014) showed that a novel κ-

266 carrageenase isolated from Pedobacter hainanensis offered an alternative approach to prepare

267 κ-carrageenan oligosaccharides by cleaving the internal β-1,4 linkage of κ-carrageenan. The

268 carrageenan oligosaccharides produced were reported as potential pharmaceutical products

269 with multiple biological activities including anticoagulant, antioxidant, anti-inflammatory,

270 and antiviral activities. In addition, alginate modifying enzymes (e.g. alginate acetylases, 271 alginate deacetylases,ACCEPTED alginate lyases, and mannuronan C-5-epimerases) have also been used 272 for modification of M- and G-block and quantification purposes, for instance (Ertesvåg,

273 2015).

274 Food grade hydrolytic enzymes, which are capable of catalyzing seaweed-specific

275 structures, remain uncommon in the commercial market at present. The development of more 12 ACCEPTED MANUSCRIPT 276 specific polysaccharide hydrolytic enzymes is required for digesting the parent backbone of

277 seaweeds and to aid in the production of lower MW oligosaccharides with a variety of

278 biological activities (Vavilala and D’Souza, 2015). Most applications of enzymatic

279 modification are currently focused on polysaccharide degradation, but other types of enzymes

280 such as transferase and synthase should be further developed in order to fulfill the application

281 of enzyme technology for this purpose (Li et al., 2016).

282 4.3 The effects of using enzymes for the extraction and hydrolysis of bioactive products

283 from seaweed for prebiotic and nutraceutical applications

284 Recent publications related to the use of enzyme-assisted extraction and enzymatic

285 hydrolysis on seaweed substrates are summarised in Tables 1 and 2. The benefits of using

286 commercially-available enzymes (Table 1) and seaweed-specific enzymes (Table 2) on the

287 processes, as well as on the biological activities of the seaweed extracts or hydrolysates, are

288 also presented in order to evaluate their potential for use as functional and nutraceutical 289 sources. MANUSCRIPT 290 Most of the research thus far has focused on the use of commercially-available

291 carbohydrate hydrolytic enzymes and proteases. Although they might not hold as much

292 promise as the development of seaweed-specific enzymes, they are convenient for use in

293 commercial applications and are cost effective at present for industry. Also, the use of

294 enzymes to hydrolyse cellulose and protein networks in the cell wall, in order to facilitate the

295 liberation of other target components, can avoid excessive structural degradation common 296 with harsh physicalACCEPTED and chemical treatments. High MW polysaccharides, such as fucoidans of 297 ~390-2200 kDa, have shown potential to induce anticancer activity (Yang et al., 2008).

298 On the other hand, commercial enzymes have been used to reduce the MW of target

299 molecules in some instances. Proteases, for example, are commonly used for hydrolysis of

300 proteins extracted from seaweeds. Commercial carbohydrases have also been applied, in a 13 ACCEPTED MANUSCRIPT 301 small number of cases, to specifically digest target polysaccharides, but this is not common

302 due to the fact that commercially available carbohydrases with activity toward seaweed-

303 specific polysaccharides remain rather rare (and expensive). Given the current lack of

304 commercial enzymes with selectivity toward the cell wall polysaccharides of seaweeds,

305 Sánchez-Camargo et al. (2016) suggested that pressurized liquids are currently a more

306 effective option compared with enzymatic treatments for the recovery of phlorotannins from

307 the seaweed Sargassum muticum . Therefore, the isolation, identification, and purification of

308 enzymes from microorganisms that can degrade the constituents specific to seaweeds needs

309 to be investigated, in order to improve hydrolysis efficiency.

310 It should be noted that most publications using seaweed-specific enzymes to produce

311 low MW seaweed fractions, such as oligosaccharides, have reported prebiotic properties. The

312 introduction of oligosaccharides as prebiotic ingredients in functional food has increased, and

313 a number of studies on plant polysaccharides have indicated that low MW or hydrolysed 314 oligosaccharides have improved fermentability by MANUSCRIPT gut microbial communities (Hughes et al., 315 2007, 2008; Mussatto and Mancilha, 2007). Xu et al. (2018) also reported the potential use of

316 β-agarase with high specific activity for extensive applications for human health in food

317 industry. Neoagaroligosaccharide produced by this enzyme could act as non-digestible

318 oligosaccharide prebiotics with more effective in multiple functions such as antioxidant

319 activities and inhibition of tyrosinase activity compared with products produced by acid

320 hydrolysis. 321 The utilisationACCEPTED of enzymes for polysaccharide degradation may be intensified with 322 physical treatments of seaweed materials before or during enzyme processes. As shown in

323 Tables 1, this approach has shown significant improvements in the time and cost of the

324 process, as well as the extraction yield and biological properties of bioactive compounds, as

325 the disaggregation of the cell walls is expected to improve the accessibility of enzymes to 14 ACCEPTED MANUSCRIPT 326 their substrates. Michalak and Chojnacka (2014), Kadam et al. (2015), Roselló-Soto et al.

327 (2015), Poojary et al. (2016), and Zhu et al. (2017) reported several merits of other novel

328 extraction processes such as electrotechnologies, microwave, ultrasound, supercritical fluid,

329 subcritical fluid, ultra-high pressure, pressurized fluid, and autohydrolysis treatments.

330 Therefore, the intensification of enzyme processes with these techniques could potentially

331 yield additional benefits, particularly with regard to process efficiency for the improvement

332 of prebiotic and other nutraceutical functions in the future, but not many studies have focused

333 on this research area to date.

334 Overall, most of the available literature on the use of enzymes for seaweed extraction

335 and digestion suggests that the use of enzymes has a number of advantages for seaweed

336 extraction and digestion, as they are able to yield target compounds with improved

337 selectivity, high yields, and improved biological properties, while using mild conditions and

338 non-toxic chemicals, and with reduced time, energy and process cost. These capabilities may 339 benefit large-scale operations and improve the prosMANUSCRIPTpects for using seaweeds in functional 340 food and nutraceutical industries if some commercial and technical limitations presented on

341 an industry scale, particularly concerning the stability and cost of enzymes (Puri et al., 2012),

342 and the availability of seaweed-specific enzymes, can be overcome.

343 4.4 Purification

344 After the extraction and hydrolysis of bioactive products from seaweeds, the crude

345 extract mixture may take more purification processes to further enrich bioactive compounds 346 of interest if required.ACCEPTED 347 The most commonly used techniques in the purification of seaweed polysaccharides

348 are ion-exchange chromatography (IEC), size-exclusion chromatography (SEC), affinity

349 chromatography, and membrane filtration (Garcia-Vaquero et al., 2016). Graded precipitation

350 using alcohol or ketone is often firstly used to separate polysaccharides from the extracts as it 15 ACCEPTED MANUSCRIPT 351 is more practical method than column chromatography. In this method, the solubility of

352 larger MW polysaccharides in ethanol or acetone is less than that of smaller MW

353 polysaccharides, so the gradual increase in ethanol or acetone concentrations can precipitate

354 different MW polysaccharides (Zha et al., 2012; Shi, 2016). The application of approximately

355 67% (v/v) ethanol is a commonly practiced to precipitate the polysaccharides from the

356 mixture (Charoensiddhi et al., 2017). In order to further purify specific polysaccharides from

357 brown seaweeds, for instance. The separation of alginate was usually carried out using CaCl 2

358 (McHugh, 2003; Sellimi et al., 2015), while fucoidan and laminarin were purified from the

359 extract using membrane filtration or chromatography methods. Dialysis and ultrafiltration

360 with different molecular weight cut-off membranes were used to separate laminarin from

361 other polysaccharides (Rioux et al., 2010; Kadam et al., 2015). However, some problems of

362 the ultrafiltration method were reported in term of low yield, long time-consuming process,

363 and inaccuracy due to non-spherical shape of polysaccharides (Shi, 2016). Anion-exchange 364 chromatography, particularly DEAE-cellulose, MANUSCRIPT Q-Sepharose Fast Flow, and Macro-Prep 365 DEAE column is also one of the most common purification methods used for fucoidan

366 separation based on its high anionic charges and degree of sulphation (Anastyuk et al., 2017;

367 Wang et al., 2012a; Menshova et al., 2015; Cong et al., 2016; Dinesh et al., 2016; Imbs et al.,

368 2016). In addition, the application of SEC such as Sephacryl S-300 and TSK-GEL column

369 allows fractionation of the fucoidan regarding to their MWs (Cong et al., 2016; Anastyuk et

370 al., 2017). 371 Apart fromACCEPTED polysaccharides, the purification of peptides is mainly based on their 372 ionic charges, size, and hydrophobicity (Vijaykrishnaraj and Prabhasankar, 2015). SDS-

373 PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and Gel permeation

374 chromatography (GPC-HPLC) were the primary techniques used to separate biologically

375 active peptides from red seaweed protein hydrolysates (Harnedy and FitzGerald, 2013). 16 ACCEPTED MANUSCRIPT 376 Phlorotannin for human consumption is commonly extracted using ethanol (Wang et al.,

377 2012b) and enzyme hydrolysis (Cho et al., 2012). However, additional purification processes

378 are required to obtain high-purity phlorotannins. Fractionation using ethyl acetate and

379 Sephadex LH-20 column chromatography was applied to further purify phlorotannins (Wang

380 et al., 2012b; Liu and Gu, 2012). In addition, Kim et al. (2014) demonstrated the purification

381 of highly concentrated phlorotannins using macroporous adsorption resins (Diaion HP-20).

382 The high-purity phlorotannins with 92% of recovery rate from their crude extract achieved

383 from this chromatography method could support more application in food and pharmaceutical

384 industries.

385

386 5. Application and legislation of seaweed use in the functional food sector

387 5.1. Seaweed and the functional food industry

388 Increasing consumer awareness regarding the complex relationship between diet and 389 health is resulting in demand for new functional MANUSCRIPT foods that can specifically contribute to 390 health-promotion or disease prevention, beyond providing basic nutrition (Gul et al., 2016).

391 The global nutraceutical market, including functional foods and beverages as well as dietary

392 supplements, was valued at around USD 250 billion in 2014, and with the rapid increase in

393 consumer demand, is expected to reach around USD 385 billion by 2020 (Suleria et al.,

394 2015). Seaweeds are commonly used as general foodstuffs, such as flavorings for noodles,

395 soups, and meals, as well as in snacks, salads, wrap-up vegetable, and pickled side-dishes, in 396 Japan, China, Korea,ACCEPTED and other coastal populations (Lee, 2008). While the recent studies on 397 seaweed-derived functional food ingredients have shown that seaweeds are a rich source of

398 bioactive compounds with a variety of potential health benefits and product applications

399 (Roohinejad et al., 2017), the volume of clinical research in humans into the health-

400 promoting properties of seaweeds in food and nutraceutical applications is rather limited. 17 ACCEPTED MANUSCRIPT 401 Also, the functionality, bioavailability, bioaccessibility, and toxicity of the seaweed-derived

402 bioactive compounds when incorporated into food products still need to be ensured their

403 compliance with standard food and nutraceutical regulations (Barba, 2017).

404 The commercial products and patent activities on seaweed-derived bioactive

405 compounds have increased recently. For example, polysaccharides extracted from brown

406 seaweeds by acid extraction comprising at least 8% by weight of β-glucans, α-fucans, or their

407 mixture have been patented for prebiotic effect and gut health improvement (O'doherty et al.,

408 2009). The Kabushiki Kaisha Yakult Honsha company from Japan has patented

409 oligosaccharides containing fucoidan and rhamnan extracted using hydrochloric acid or

410 trifluoroacetate from brown seaweeds such as Cladosiphon okamuranus , Chordaricles

411 nemacystus , Hydrilla sp., and Fucus sp., and green seaweed Monostroma nitidum for

412 prevention of gastric ulcers caused by Helicobacter pylori (Nagaoka et al., 2003). Another

413 Japanese company Takara Shuzo and Biolsystems company from Korea have patented the 414 isolation of oligosaccharides consisting of 3,6-anh MANUSCRIPTydrogalactose under acidic conditions 415 and/or enzymatic digestion from red seaweeds such as Gelidium, Pteocladia, and Hypnea .

416 This compound could prevent and treat diabetes, rheumatism, and cancer (Enoki et al., 2009;

417 Im and Kim, 2016). Apart from seaweed polysaccharides, bioactive peptides derived from

418 seaweeds have been also developed as commercial products with angiotensin I-converting

419 enzyme (ACE) inhibitory. The Riken Vitamin company, Tokyo, Japan has developed

420 Wakame jelly containing peptides with antihypertensive activity. These peptides were 421 extracted from brownACCEPTED seaweed Undaria pinnatifida using enzyme Protease S “Amano” from 422 Bacillus stearothermophilus (Sato et al., 2002; Hayes and Tiwari, 2015). In addition, the

423 antioxidant components comprising phlorotannin from brown seaweed Ascophyllum nodosum

424 extracted using ultrasound-assisted continuous aqueous extraction process have been also

425 patented for use as functional ingredients in food formulations (Sineiro et al., 2016). 18 ACCEPTED MANUSCRIPT 426 Although the reports of functional food products from seaweed-derived bioactive

427 compounds and their related patents using enzymes to assist extraction processes and

428 improve qualities are still not extensive, enzyme-enhanced processing could have potential

429 for developing ingredients with various functionalities and improving the quality of bioactive

430 compounds from seaweeds (Wijesekara and Kim, 2015). These developments could expand

431 the breadth of product applications in the functional food market. For instance, Rhein-

432 Knudsen et al. (2015) reviewed the advantages of using enzyme-assisted extraction to

433 enhance the value and potential applications of commercial seaweed hydrocolloids. Enzymes

434 facilitated selective extraction at mild conditions, leading to better preserved chemical

435 structures and functional properties, and the avoidance of contaminants. Lakmal et al. (2015)

436 also reported that enzyme-assisted extraction is an effective method to isolate sulphated

437 polysaccharides from seaweeds for new nutraceutical applications, as these polysaccharides

438 exhibit many bioactivities, such as antioxidant, anti-inflammatory, anticancer, and 439 anticoagulant effects. In addition, highly soluble oligosaccharides,MANUSCRIPT produced using enzymatic 440 hydrolysis, can be incorporated into food systems more easily and with greater functional

441 properties (Kang et al., 2014). The potential value-enhancement of seaweed-derived food and

442 nutraceutical ingredients using enzyme-enhanced processing is summarised in Fig. 3.

443 5.2. Relevant regulations on seaweed functional food supplements

444 This review summarises the currently available knowledge on the regulations in

445 various countries relevant to seaweed use in food and functional food supplements. 446 In France,ACCEPTED 21 species of macroalgae, including brown seaweeds ( Ascophyllum 447 nodosum , Undaria pinnatifida , Laminaria japonica , etc.), red seaweeds ( Palmaria palmata ,

448 Porphyra umbilicalis , Gracilaria verrucosa , etc.), and green seaweeds ( Ulva and

449 Enteromorpha species), are authorized as vegetables and condiments for food consumption

450 (CEVA, 2014). Some brown seaweed (Laminaria sp. and Nereocystis sp.), red seaweed 19 ACCEPTED MANUSCRIPT 451 (Porphyra sp. and Rhodymenia palmata (L.) Grev.), and the materials derived from these

452 species are also classified for “Generally Recognized As Safe (GRAS) food substances” in

453 U.S. Food and Drug Administration, SCOGS-Report Number 38 (FDA, 1973). These

454 authorizations present an opportunity for the food industry to include seaweeds as raw

455 materials in food formulations and applications.

456 However, of course these seaweeds and their products still must meet the requirement

457 of consumer safety regulations. The maximum allowed levels of heavy metals including

458 arsenic, lead, cadmium, tin, mercury, and iodine have been defined for edible seaweeds in

459 most jurisdictions, but the maximum allowed bacterial levels within dried seaweeds have

460 been defined only in France. The quality criteria and related regulations applied to seaweeds

461 and their products are summarised in Table 3. The high levels of iodine in seaweeds appear to

462 be gaining increasing cautionary interest. The Australian Quarantine and Inspection Service

463 (AQIS) has now included brown seaweeds on the imported food ‘Risk List’ and are 464 monitored at the border to ensure that only product MANUSCRIPTs with safe levels of iodine are imported ( ≤ 465 1000 mg iodine/kg dried weight) (Food Standards Australia New Zealand; FSANZ, 2010).

466 Also, pregnant and breastfeeding women as well as young children are advised to eat no more

467 than one serve a week of brown seaweed (FSANZ, 2011). Bouga et al. (2015) surveyed the

468 iodine content of seaweed and seaweed-containing products in the UK market, with the

469 median content being 110 g/g and 585 g per estimated serving. They identified 26 products

470 that may lead to an iodine intake over the upper level of European tolerable adult at 600 471 g/day. ACCEPTED 472 The safety of seaweed consumption was also recently evaluated in Belgium and

473 Norway. Belgium’s report from the Superior Health Council (2015) conducted a risk

474 assessment of inorganic arsenic through the consumption of edible seaweeds. They

475 recommend that the consumption of Hizikia fusiforme should be of concern due to high levels 20 ACCEPTED MANUSCRIPT 476 of inorganic arsenic, and the consumption of other seaweeds should be limited to 7 g dried

477 material per day. Norway’s report from its National Institute of Nutrition and Seafood

478 Research (NIFES) (2016) suggested that high levels of inorganic arsenic and cadmium which

479 may limit the use of some seaweed species as food ingredients were particularly found in

480 Norwegian brown seaweeds. However, Smith et al. (2010) compared the heavy metal content

481 of edible seaweeds in New Zealand, including commercially available and wild-harvested

482 seaweeds ( Macrocystis pyrifera , Undaria pinnatifida , Porphyra , Ecklonia radiata , Ulva

483 stenophylla , Durvillaea antarctica , and Hormosira banksii ). None of the seaweeds studied

484 showed significant risks with regard to heavy metals, considering the quantities that they are

485 normally consumed. Moreover, NIFES (2016) also mentioned the risks posed by persistent

486 organic pollutants (dioxin and polychlorinated biphenyls, etc.), toxins (pinnatoxin,

487 aplysiatoxin, and kainic acid, etc.), anti-nutrients (polyphenols), radioactivity (radionuclides),

488 microorganisms (bacteria associated with seaweeds and viral contagious agents), and 489 microplastic (particle size <5 mm) contamination MANUSCRIPT in seaweeds. However, a low risk of food 490 safety hazards was observed in all of these parameters.

491 Apart from the aforementioned regulations applying to seaweeds and their products,

492 some other general regulations should be considered. For example, Codex General Standard

493 for Contaminants and Toxins in Food and Feed (Codex, 1995) provides the toxicological

494 guidance for tolerable intake levels of contaminant for humans. The Provisional Tolerable

495 Weekly Intake (PTWI) of inorganic arsenic, cadmium, lead, mercury, and tin are defined at 496 0.015, 0.007, 0.025,ACCEPTED 0.005, and 14 mg/kg body weight, respectively. Meanwhile, the FSANZ 497 standard 1.3.4 (FSANZ, 2012) ensures that food additives (novel food and nutritive

498 substances, etc.) added to food meet appropriate specifications for identity and purity. These

499 substances must not contain lead at >2 mg/kg DW and arsenic, cadmium, and mercury, each

500 at >1 mg/kg DW. In addition, the guideline levels for determining the microbiological quality 21 ACCEPTED MANUSCRIPT 501 of ready-to-eat foods (FSANZ, 2001) may be applied if seaweeds are used as one ingredient

502 in products specified in this food category.

503

504 6. Conclusion and future perspectives

505 Seaweeds are a valuable source of bioactive compounds, especially with regard to their

506 polysaccharides, phenolic compounds, and proteins. Advanced enzymatic processing is a key

507 trend for the efficient extraction and digestion of value-added bioactive compounds from

508 seaweeds. The potential of these compounds for a number of functional food applications is

509 being increasingly recognised, particularly with regard to prebiotic supplements. Despite the

510 opportunities, challenges remain in improving this technique in terms of its productivity and

511 profitability. Enzyme intensification using other physical inputs, and enzymes with activities

512 specific to seaweed structures, should be further investigated. Furthermore, the scaled up

513 industrial process should be designed to support commercial implementation. The adherence 514 of products to safety legislation are also critical andMANUSCRIPT complex subjects requiring attention in 515 order to push forward with the commercial development of seaweed ingredients for

516 functional food and nutraceutical products.

517

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1000

1001

1002 42 ACCEPTED MANUSCRIPT 1003 Figure

1004 Fig. 1 Structure model of the brown seaweed cell wall; some sulphated fucans are tightly

1005 associated with cellulose microfibrils (flat ribbon-like shape), and they are embedded within

1006 the alginate network. Hemicellulose components (short chain form) link with the cellulose by

1007 hydrophobic interactions and connect with the sulphated fucans. Alginates and phenolic

1008 compounds are associated and can form high molecular weight complexes. Proteins are

1009 linked with sulphated fucans and covalently attached to phenolics. (adapted from Kloareg et

1010 al., 1988; Michel et al., 2010; Deniaud-Bouët et al., 2014)

1011

1012 Fig. 2 Seaweed components and their potential health benefits through prebiotic effects

1013

1014 Fig. 3 Potential of using enzyme-enhanced processes to improve the value and uses of

1015 seaweed-derived food and nutraceutical ingredients 1016 MANUSCRIPT 1017 Table

1018 Table 1: Studies reporting the use of commercially-available enzymes in the development of

1019 seaweed bioactive compounds; B, R, and G denote brown, red, and green seaweed,

1020 respectively. ↑ and ↓ means increase and decrease, respectively, compared to the control.

1021

1022 Table 2 Studies reporting the use of seaweed-specific enzymes in the development of seaweed

1023 bioactive compounds; B, R, and G denote brown, red, and green seaweed, respectively. ↑ and

1024 ↓ means increaseACCEPTED and decrease compared to control.

1025

1026 Table 3 Quality criteria and regulations applied to seaweeds and their products ACCEPTED MANUSCRIPT

1 Table 1: Studies reporting the use of commercially-available enzymes in the development of seaweed bioactive compounds; B, R, and G denote

2 brown, red, and green seaweed, respectively. ↑ and ↓ means increase and decrease, respectively, compared to the control.

Active components Substrates Enzymes Processes Advantages Reference or activities Enzyme-assisted extraction Ecklonia radiata Celluclast E/S ratio 100 µL: 1 g, Prebiotic Improved biological properties Charoensiddhi (B) (Novozyme) water, pH 4.5, 50 °C, ↑ Total SCFA and butyric acid production et al. (2016a) 24 h ↑ Bifidobacterium and Lactobacillus (In vitro human fecal fermentation 24 h) Ulva - Endo-protease E/S ratio 60 mg: 1 g, Antiviral High yield, improved biological properties Hardouin et al. armoricana (G) - Multiple-mix of water pH 5.9-6.2, Antioxidant ↑ Yield; Endo-protease (2-fold) (2016) glycosyl-hydrolases 50 °C, 3 h ↑ Organic matter, neutral sugar, and protein; - Exo-β-1,3(4)- All enzymatic extracts (up to 2-fold, 2.7-fold, glucanase and 1.75-fold, respectively) (Novozymes) ↑ Activities against Herpes simplex virus type-1 MANUSCRIPT - At EC 50 ; Multiple-mix of glycosyl-hydrolases (373 g/mL) and Exo-β-1,3(4)-glucanase (321 g/mL), Control (>500 g/mL) ↑ Free radical scavenging capacity - At IC 50 ; Endo-protease (1.8 and 12.5 mg/mL) and Multiple-mix of glycosyl-hydrolases (6 mg/mL), Control (>500 g/mL) Lessonia α-amylase E/S ratio 100 mg: 1 g, ACE inhibitor High yield, improved biological properties Olivares- nigrescens (Sigma) 0.2 N phosphate ↑ Yield (2.3-fold) Molina and (B) buffer, pH 6, 60 °C, ↑ ACE inhibition (1.04-fold) Fernández

17 h ↓ IC 50 (4.8-fold) (2016)

Osmundea Viscozyme E/S ratio 50 mg: 1 g, Prebiotic Improved biological properties Rodrigues pinnatifida (Sigma-Aldrich) water, pHACCEPTED 4.5, 50 °C, ↑ Total SCFA production et al. (2016) (R) 24 h ↑ Bifidobacterium spp. after 6 h fermentation (In vitro human fecal fermentation 24 h)

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Active components Substrates Enzymes Processes Advantages Reference or activities Chondrus - Cellulase Enzyme 0.5%, water, Antiviral High yield, improved biological properties Kulshreshtha crispus (R) - β-glucanase 50 °C, 3 h ↑ Yield (2-3-fold) et al. (2015) Codium fragile - Ultraflo ↑ Recovery of protein, neutral sugars, uronic (G) - Neutrase acids, and sulphates; C. crispus (up to 3-4-fold) (Novozyme) ↑ Activity against Herpes simplex virus Type 1 - EC 50 at MOI 0.001 and 0.01 ID 50 /cells; C. fragile (1.4-folds and 2.3-fold, respectively) Osmundea - Alcalase E/S ratio 50 mg: 1 g, Antioxidant High yield, improved biological properties Rodrigues pinnatifida - Flavourzyme water, pH 8 (Alcalase), Prebiotic ↑ Yield; C. tomentosum (up to 1.4-fold) et al. (2015) (R) - Cellulase pH 7 (Flavourzyme), α-Glucosidase ↑ Antioxidant (ABTS, DPPH, Hydroxyl radical Sargassum - Viscozyme L and pH 4.5 (Cellulase, Inhibitor Superoxide radical); S. muticum muticum (Sigma-Aldrich) Viscozyme), 50 °C, ↑ Lactobacillus acidophilus La-5 and (B) 24 h Bifidobacterium animalis BB-12; Codium S. muticum and C. tomentosum tomentosum (In vitro enumeration of viable cells) (G) ↑ % Inhibition of α-glucosidase; MANUSCRIPTC. tomentosum (1.4-fold) Palmaria Xylanase E/S ratio 17.8 g/kg, R-phycoerythrin High yield and selectivity Dumay et al. palmata (R) (Sigma-Aldrich) 50 mM acetate buffer ↑ Yield 62-fold (2013) pH 5, 24 °C, 320 min ↑ Purity index up to 16-fold Enteromorpha - Viscozyme L E/S ratio 20 mg: 1 g, Antioxidant Reported biological properties Ahn et al. prolifera - Promozyme Optimum pH and Anti- - High antioxidant activity (DPPH, H 2O2 (2012) (G) - Flavourzyme temperature, 8 h acetylcholinesterase scavenging, ferrous ion chelating, and reducing 500 MG (AChE) power); Viscozyme and Protamex extracts - Protamex Anti-inflammatory - High AChE inhibitory activities at 1 mg/mL; (Novozyme) Promozyme extract (93.6%) and Flavourzyme extract (89.9%) Sargassum Neutrase 0.8 L E/S ratio 0.1 g: 1 g, Anticancer Reported biological properties Ko et al. coreanum (Novozyme) 0.2 M phosphateACCEPTED - DNA fragmentation, apoptoic body and cells; (2012) (B) buffer, pH 6, 50 °C, Crude polysaccharide >30 kDa fraction 12 h

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Active components Substrates Enzymes Processes Advantages Reference or activities Ecklonia cava Celluclast E/S ratio 100 µL: 1 g, Anti-diabetic Reported biological properties Lee et al. (B) (Novozyme) water, pH 4.5, 50 °C, - IC 50 : α-glucosidase 0.62 and α-amylase 0.59 (2012b) 24 h mg/mL, Acarbose (0.68 and 0.71 mg/mL, respectively) Ecklonia cava Celluclast E/S ratio 10 µL: 1 g, Fucoidan High yield, reported biological properties Lee et al. (B) (Novozyme) water, 50 °C, 24 h Anti-inflammatory ↑ Yield (1.3-fold) (2012a) ↑ Total carbohydrate (1.4-fold) ↑ Sulphate content (1.7-fold) - Inhibit nitric oxide production from macrophages (RAW 264.7) Caulerpa Pepsin E/S ratio 100 mg: 1 g, Angiotensin I- Reported biological properties Lin et al. microphysa (Sigma) 0.2 M phosphate converting enzyme - ACE Inhibitory activity IC 50 0.2 mg/mL (2012) (G) buffer, pH 3, 37 °C, (ACE) inhibitor - Inhibit the growth of transplanted 12 h Anti-tumor myelomonocytic leukaemia (WEHI-3) and human promyelocytic leukaemia (HL-60) cell lines MANUSCRIPT- Increase DNA damage (concentration >100 g/mL) Laminaria - AMG 300L E/S ratio 20 mg: 1 g, Antioxidant Reported biological properties Sevevirathne japonica - Celluclast 1.5L FG Optimum pH and Anti-Alzheimer’s - High total phenolic; Alcalase extract et al. (2012) (B) - Dextrozyme temperature, 8 h Anti-inflammatory (14.4 mg Gallic acid equivalent/g) - Alcalase 2.4L FG - High total flavonoid; Celluclast extract - Flavourzyme (5.1 mg Quercetin equivalent/g) 500 MG - High antioxidant activity (DPPH, H 2O2 - Protamex scavenging, metal chelating, and reducing (Novozyme) power); Flavourzyme and Dextrozyme extracts - High AChE inhibitory activity at 0.25 mg/mL; Flavourzyme extract (90%) and Celluclast extract (60%) ACCEPTED - High activity in nitrite scavenging; Protamex and AMG extracts

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Active components Substrates Enzymes Processes Advantages Reference or activities Ecklonia cava Kojizyme E/S ratio 100 g: 1 g, Immune regulation Reported biological properties Ahn et al. (B) (Novozyme) water, pH 6, 40 °C, Increase the production of IL-2 through the (2011) 12 h activation of NF-κB, and induce the proliferation of lymphocytes with the coordinated stimulation of IL-2 Ecklonia cava AMG 300 L E/S ratio 10 L: 1 g, Anticoagulant Improved biological properties Wijesinghe (B) (Novozyme) Optimum pH and Sulphated ↑ Prolong bleeding time at dosage of 300 µg/kg et al. (2011) temperature, 12 h polysaccharides (>1800 s), Control (900 s) Porphyra tenera - Viscozyme L E/S ratio 20 mg: 1 g, Antioxidant Reported biological properties Senevirathne (R) - Maltogenase Optimum pH and Anti-Alzheimer’s - High antioxidant activity (DPPH, H 2O2 et al. (2010) - Alcalase 2.4L FG temperature, 8 h Anti-inflammatory scavenging, ferrous ion chelating, and reducing - Flavourzyme power); Alcalase and Maltogenase extracts 500 MG - High AChE inhibitory activities at 1 mg/mL; (Novozyme) Flavourzyme extract (99.3%) and Viscozyme extract (82.7%) Palmaria Umamizyme E/S ratio 50 mg: 1 g, Antioxidant High yield, improved biological properties Wang et al. palmata (R) (Amano water, pH 7, 50 °C, MANUSCRIPT↑ Yield (2-fold) (2010) Enzyme Inc.) 24 h ↑ Total phenolic (3-fold) ↑ Antiradical power (3.5-fold) ↑ ORAC value (4-fold) Enzyme-assisted extraction intensified with other processes Grateloupia Mixture enzyme: Ultrasound-assisted Carbohydrates High yield Le Guillard turuturu Sumizyme TG (Shin enzymatic extraction Amino acids ↑ Liquefaction (1.7-fold) et al. (2016) (R) Nihon Chemical), E/S ratio 10 mg: 1 g, ↑ Carbohydrates (3.3-fold) Sumizyme MC water, pH 5.5, 40 °C, ↑ Amino acids (2.1-fold) (Takabio), 6 h ↑ Nitrogen (1.8-fold) ® Multifect CX 15L Sonitube 35 kHz, ↑ Carbon (2.2-fold) (DuPont), 400 W Ultraflo XL ACCEPTED (Novozymes)

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Active components Substrates Enzymes Processes Advantages Reference or activities Ecklonia radiata Viscozyme L Microwave-assisted Phlorotannins Improved extraction time, cost-savings, and Charoensiddhi (B) (Novozyme) enzymatic extraction Antioxidant extraction efficiency et al. (2015) E/S ratio 100 µL: 1 g, ↑ Phlorotannins (1.3-fold) 0.1 M sodium acetate ↑ Antioxidant FRAP and ORAC (1.4-fold) buffer, pH 4.5, 50 °C, - Short extraction time 5-30 min 30 min under open- vessel microwave intensification Gracilaria Proteolytic enzyme Ultrasound-assisted Anticoagulant High yield, improved biological properties Fidelis et al. birdiae (R) enzymatic extraction Antioxidant ↑ Total yield (15.9-fold) (2014) 2 step: 0.1 M sodium Sulphated ↑ Sulphated polysaccharide: sugar/sulphate (5.1), hydroxide, 22°C; Polysaccharides Control (8.5) Sonication: 60°C, 30 ↑ Anticoagulant and antioxidant (~2-fold) min, 60 W Enzyme: pH 8.0, 60°C, 12 h - Palmaria Pancreatin, Ultrasound-assisted Iodine (minerals) MANUSCRIPT High yield Romaris- palmate (Sigma-Aldrich) enzymatic extraction ↑ Large amount of extracted iodine Hortas et al. - Porphyra E/S ratio 0.2 g: 1 g, 76-96% of total iodine. (2013) umbilicalis 0.2 M dihydrogen (R) phosphate/sodium - Ulva rigida hydroxide buffer, pH (G) 8.0 under ultrasound - Laminaria irradiation 45 kHz, ochroleuca 50 °C, 12 h - Undaria pinnatifida (B)

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Active components Substrates Enzymes Processes Advantages Reference or activities Enzymatic hydrolysis of target molecules Saccharina Trypsin E/S ratio 50 mg: 1 g, Antibacterial Selectivity, improved biological properties Beaulieu longicruris (Sigma-Aldrich) 20 mM phosphate - Degree of hydrolysis (DH); Fraction >10 kDa et al. (2015) (B) buffer, pH 7, 30 °C, (45.4% w/w) (Protein 24 h ↑ Antibacterial activity against Staphylococcus extracts) aureus ↓ Maximum specific growth rate at 2.5 mg/mL from 0.19 to 0.02 max /h Pyropia - Alkaline protease Simple hydrolysis (A) ACE inhibitor Selectivity, improved biological properties Cian et al. columbina (Danisco) Alkaline protease: Antioxidant - Peptide A (MW 2.3 kDa) (2015) (R) - Flavourzyme E/S ratio 4 mg: 1 g, Antiplatelet Peptide AF (MW 2.3 kDa and 287 Da) (Protein (Sigma) water, pH 9.5, 55 °C, aggregation Control (MW >175 kDa and 52.1 kDa) extracts) 2 h ↑ ACE inhibition IC 50 ; Peptide A (4.5-fold) Sequential hydrolysis ↑ Antioxidant ABTS and DPPH radical (AF) Inhibition IC 50 ; Peptide AF (3.7-fold and 2.1- Start with Alkaline fold, respectively) protease hydrolysis 2 MANUSCRIPT↑ Copper-chelating activity and Antiplatelet h, following to aggregation; Peptide AF (4.2-fold and 3-fold, Flavourzyme respectively) hydrolysis E/S ratio 5 mg: 1 g, water, pH 7, 55 °C, 4 h (total 6 h) Pterocladia Viscozyme L Enzyme 30 units, Phenolics Selectivity, high yield, improved biological Fleita et al. capillacea (Sigma-Aldrich) 0.1 M sodium acetate Antioxidant properties (2015) (R) buffer, pH 4.5, 37 °C, Antibacterial ↑ >50% of phenolic content and DPPH (Sulphated 30 min scavenging polysaccharide ↑ Anti-bacterial activity, Control = antibiotics extracts) (tetracycline and cefuroxime) ACCEPTED

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Active components Substrates Enzymes Processes Advantages Reference or activities Agarose Celluclast 1.5L E/S ratio 20 mL: 1 g, Antioxidant Selectivity, reported biological properties Kang et al. (Sigma) (Novozyme) 0.05 M acetate buffer, - Hydrolysis yield 20.5% and low viscosity (2014) pH 5, 50 °C, 24 h - High solubility up to 86.8% - High water and oil adsorption capacities (3.0 and 4.7 g/g, respectively) - High antioxidant activity (DPPH 24.6%, ABTS 83.8%, and FRAP 1.6) Porphyra - Flavourzyme Subsequent hydrolysis ACE inhibitor Selectivity, Improved functional properties Cian et al. columbina (Sigma) Protease: Antioxidant ↓ MW of peptides (2013) (R) - Fungal protease E/S ratio 50 mg: 1 g, (Asp, Glu, Ala, Leu) (Residue cake) (Genencor) pH 4.3, 55°C, 3 h ↑ Protein solubility Flavourzyme: - ACE inhibition ~45%

E/S ratio 20 mg: 1 g, - IC 50 : ABTS 1.01 and DPPH 0.91 g/L pH 7, 55°C, 4 h - High copper chelating activity ~97.5% Palmaria - Corolase PP E/S ratio 10 L or mg: Anti-diabetic Selectivity, improved biological properties Harnedy and palmata (R) (AB Enzymes) 1 g, water, pH 7, 50 °C, Antioxidant ↑ Amino acid concentration; Alcalase (3-fold) FitzGerald (Protein - Alcalase 2.4L 4 h ACE inhibitor MANUSCRIPT and Corolase (5-fold), Low MW peptide (2013) extracts) (Novozyme) ↑ ACE and DPP IV inhibitory activity: IC 50 ; Alcalase (0.2 and 2.5 mg/mL, respectively) and Corolase (0.3 and 1.7 mg/mL, respectively), Control (>2 and >5 mg/mL, respectively) ↑ ~50% Renin inhibition; Corolase ↑ Antioxidant FRAP and ORAC; Alcalase (8-9-fold) and Corolase (10-13-fold) Enzymatic hydrolysis of target molecules, intensified with other processes Undaria Tunicase Ultra high pressure Anticoagulant Selectivity, Improved biological properties Park et al. pinnatifida (β-glucanase) (UHP)-assisted ↓ Average MW 687 kDa, Control 877 kDa (2012) sporophylls (Daiwakasei) enzymatic extraction ↑ Anticoagulant activity, APTT (1.3-fold) and (B) Enzyme ACCEPTED60 mg: 1g, pH TT (1.6-fold) (Crude 8, 40 °C, 24 h in UHP fucoidan) unit 100 MPa

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Active components Substrates Enzymes Processes Advantages Reference or activities Pyropia AMG Microwave-assisted Antioxidant Selectivity, improved biological properties Lee et al. yezoensis (Novozyme) enzymatic extraction ↑ Degree of hydrolysis (DH 25%) (2016)

(R) E/S ratio 1:10 ↑ Antioxidant IC 50 ; Alkyl radical (196 g/mL) ° pH 4.5, 60 C, 2 h and H2O2 (96 g/mL), Control (554 and >180 under microwave g/mL, respectively) intensification power 400 W 3 *E/S: Enzyme and substrate ratio; EC 50 : Half-maximal effective concentration; IC 50 : Half-maximal inhibitory concentration; MOI: Multiplicity

4 of infection ID 50 /cells, ratio (virus titer)/(cell number/mL); ABTS: 2,2’-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt;

5 DPPH: 2,2-diphenyl-1-picrylhydrazyl; FRAP: Ferric reducing antioxidant power; DPP: Dipeptidyl peptidase; ORAC: Oxygen radical

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1 Table 2 Studies reporting the use of seaweed-specific enzymes in the development of seaweed bioactive compounds; B, R, and G denote brown,

2 red, and green seaweed, respectively. ↑ and ↓ means increase and decrease compared to control.

Active Substrates Enzymes Processes components or Advantages Reference activities Enzymatic hydrolysis of target molecules Ulva sp. β-lyase 0.01 mg/mL of crude Monosaccharides Selectivity Coste et al. (2015) (G) (Alteromonas enzyme mix, pH 6-8, Oligosaccharides ↓ Ulvan MW to 5 kDa, Control 674 kDa (Ulvan extract) species) 20-35 °C, 0-50 h Gracilaria sp. (R) Agarase AS-II Appropriate amount of Prebiotic Selectivity, improved biological properties Wu et al. (2012) Monostroma nitidum (Aeromonas agarase, water, 40 °C, 2 h Oligosaccharides ↑ >3 log cfu/mL Bifidobacterium (G) salmonicida Antiviral pseudolongum BCRC 14673; Gracilaria (Polysaccharide MAEF 108) oligosaccharide extract) - Exhibit the decrease of Japanese encephalitis virus (JEV, Beijing-1 strain) infection Gelidium sp. Agarases E/S ratio 250 or 500 AU: PrebioticMANUSCRIPT Selectivity, improved biological properties Wu et al. (2007) Gracilaria sp. (Aeromonas 0.5 g, buffer solution (0.5 Oligosaccharides ↑ E. faecalis BCRC13076 and L. rhamnosus Porphyra dentate salmonicida M tris-HCl buffer (pH BCRC14068 (R) MAEF108 and 6.2), 1.0 M NaCl, 0.1 M (In vitro enumeration of viable cells) Monostroma nitidum Pseudomonas CaCl 2), 40 °C, 24 h (G) vesicularis (Polysaccharide MA103) extract) Agarose Agarase Appropriate amount of Prebiotic Selectivity, improved biological properties Hu et al. (2006) (Biowest) (E. coli BL21 agarase, water, 37 °C, 12 h Neoagaro- ↑ Bifidobacteria and Lactobacilli (DE3)) oligosaccharides (In vitro enumeration of viable cells) (NAOS) - Resistant to enzymes of the upper gastrointestinal tract ACCEPTED ↑ Bifidobacteria and Lactobacilli (In vivo mice model) ↓ Putrefactive microorganisms ↑ Degrees of polymerization ↑ Prebiotic activity ACCEPTED MANUSCRIPT

Active Substrates Enzymes Processes components or Advantages Reference activities Alginate Alginate lyase E/S ratio 10 U: 0.5 g, Prebiotic Selectivity, improved biological properties Wang et al. (Qingdao Yijia (Vibrio sp. water, 37 °C, 6 h Alginate ↑ Bifidobacterium bifidum ATCC 29521 and (2006) Huayi Import and QY102) oligosaccharides Bifidobacterium longum SMU 27001 Export) (AOS) (In vitro enumeration of viable cells) - Resistant to enzymes of the upper gastrointestinal tract ↑ Bifidobacteria; 13-fold for control and 4.7-fold for FOS, Lactobacilli; 5-fold for control (In vivo mice model) ↓ Enterobacteriaceae and Enterococci Enzyme-assisted extraction intensified with other processes Undaria pinnatifida Alginate lyase Enzyme-assisted Fucoxanthin High yield, cost-saving Billakanti (B) (Sigma-Aldrich) supercritical fluid Lipids ( ω-3 and ↑ Fucoxanthin 1.1-fold et al. (2013) extraction ω-6) ↑ Lipids rich in PUFAs 1.3-fold Enzyme pre-treatment: polyunsaturated Enzyme 0.05% (w/w of fatty acidsMANUSCRIPT dried seaweed), (PUFAs) water pH 6.2, 37°C, 2 h Supercritical fluid extraction: Dimethyl ether extraction with ethanol co-solvent, 60 °C, 40 bar 3

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Upper limit Risk items Australia France USA EU Toxic minerals (mg/kg DW, ppm)

- Inorganic arsenic ≤ 1.0 ≤ 3.0 ≤ 3.0 - Lead ≤ 5.0 ≤ 10 ≤ 3.0 - Cadmium ≤ 0.5 ≤ 3.0 - Tin ≤ 5.0 - Mercury ≤ 0.1 ≤ 0.1 - Iodine ≤ 1000 ≤ 2000 ≤ 5000 - Heavy metals ≤ 40 Bacteria *Dried seaweeds only (cfu/g)

≤ 100 - Aerobes ≤ 10 - Fecal coliforms ≤ 1 - Clostridium perfringens ≤ 100 - Anaerobes - FSANZ (2013) - CEVA (2014) Mabeau and Fleurence - Holdt and Kraan (2011) References - FSANZ (2010) - Mabeau and Fleurence (1993) - EU (2008) (1993) 2

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ACCEPTED ACCEPTED MANUSCRIPT Highlights

• Seaweeds are important source of bioactive compounds for prebiotic and nutraceutical.

• Enzyme extraction and hydrolysis are valuable to produce seaweed bioactive compounds.

• Enzyme processes improve the value of seaweeds and broaden their food applications.

• Enzymes with activities specific to seaweed improve hydrolysis and functionality.

• Food safety focus of seaweed consumption on heavy metals and iodine.

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