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Bioactive Constituents, Metabolites, and Functions

Gastrointestinal bioaccessibility and colonic fermentation of fucoxanthin from the extract of the microalga Nitzschia laevis Bingbing Guo, teresa oliviero, Vincenzo Fogliano, Yuwei Ma, Feng Chen, and Edoardo Capuano J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02496 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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Journal of Agricultural and Food Chemistry

Gastrointestinal bioaccessibility and colonic fermentation of fucoxanthin from the extract of the microalga Nitzschia laevis

Bingbing Guo1, 2, Teresa Oliviero2, Vincenzo Fogliano2, Yuwei Ma2, Feng Chen1,3*, Edoardo Capuano2* 1. Institute for Food and Bioresource Engineering, College of Engineering, Peking University, Beijing, 100871, China. 2. Food Quality and Design Group, Department of Agrotechnology and Food Sciences, Wageningen University, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands 3. Institute for Advanced Study, Shenzhen University, Nanshan District, Shenzhen, Guangdong 518060, China

*Corresponding authors: Edoardo Capuano, [email protected], +31 317485690 Feng Chen, [email protected], +86 6276664

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ACS Paragon Plus Environment


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Abstract

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The extract of microalga Nitzschia laevis (NLE) is considered a source of dietary fucoxanthin,

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a carotenoid possessing a variety of health benefits. In the present study, the bioaccessibility

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and deacetylation of fucoxanthin was studied by simulated in vitro gastrointestinal digestion

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and colonic batch fermentation. In the gastric phase, higher fucoxanthin loss was observed at

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pH 3 compared to pH 4 and 5. Lipases are crucial for the deacetylation of fucoxanthin into

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fucoxanthinol. Fucoxanthinol production decreased significantly in the order: Pure

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Fucoxanthin (25.3%) > NLE (21.3%) > Fucoxanthin-Containing-Emulsion (11.74%). More

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than 32.7% of fucoxanthin and fucoxanthinol was bioaccessible after gastrointestinal

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digestion of NLE. During colon fermentation of NLE, a higher loss of fucoxanthin and

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changes of short chain fatty acids production were observed but no fucoxanthinol was

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detected. Altogether, we provided novel insights on fucoxanthin fate along the human

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digestion tract and showed the potential of NLE as a promising source of fucoxanthin.

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Keywords: Fucoxanthin; Microalgae; Digestion; Fermentation

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Introduction

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Fucoxanthin, accounting for 10% of total carotenoids in nature,1 is an algal carotenoid. It

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possesses a variety of health-beneficial bioactivities: anti-oxidant, anti-diabetes, anti-

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carcinogenic, anti-allergic, anti-inflammatory, anti-osteoporotic activities, among the others.2

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Particularly, both mice and human studies have proved fucoxanthin was effective in reducing

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body weight.3-5 Therefore, fucoxanthin-containing products are now widely available on the

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market as functional food supplements, predominantly indicated for weight loss.

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Fucoxanthin is mainly found in brown algae (seaweeds) and diatoms (microalgae). Most of

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fucoxanthin-containing products that are available are brown algae extracts supplemented

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with oil and/or polyphenols from tea or other sources. However, fucoxanthin content in brown

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algae is much lower than that of microalgae.6 Moreover, environmental issues like land-

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occupation, extraction technology, by-products waste and contamination are to be considered

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when dealing with production of fucoxanthin from brown algae.7-9 Fucoxanthin can be

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effectively produced by microalgae: Fucovital®, a patented, all-natural 3% fucoxanthin

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oleoresin produced and extracted from Phaeodactylum tricornutum, was granted New Dietary

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Ingredient Notification (NDIN, #1048, 2017) by FDA. Microalgae, diatoms, specifically,

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have been also screened to produce fucoxanthin.6 Among them, notably, Nitzschia laevis is a

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very productive microalga which was also successfully cultured heterotrophically on

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glucose.10 The biomass concentration of Nitzschia laevis in the fermenter could reach

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17.25g/L and the fucoxanthin productivity of mixed-cultured Nitzschia laevis could be as

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high as 16.5 mg/(L·day), which is the highest reported for diatoms up to now.11 Moreover,

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other functional ingredients including carotenoids (e.g. diadinoxanthin, diatoxanthin, β-

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carotene and lycopene), polyphenols, lipids (especially PUFAs) are usually present in the

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extract of Nitzschia laevis.12 All these make this extract a potential sustainable source of

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fucoxanthin.

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Bioavailability studies reported that most fucoxanthin is absorbed as fucoxanthinol.13 A study

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on mice reported that fucoxanthin was converted into fucoxanthinol in the gastrointestinal

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tract through deacetylation, possibly by lipase or cholesterol esterase13 and fucoxanthinol was

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then converted into amarouciaxanthin by de-epoxidation in the liver in mice.14 Fucoxanthinol

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was detected in the liver, the lung, the spleen and the heart, while amarouciaxanthin was

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accumulated in the adipose tissue.14 Human studies have been conducted with fucoxanthin

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from brown algae and both fucoxanthin and fucoxanthinol have been detected in plasma.15

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However, very little is known about the behaviour of fucoxanthin in the human digestion tract

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and the factors affecting its bioaccessibility and conversion into fucoxanthinol from

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fucoxanthin-containing products. In particular, fucoxanthin is easily oxidised in presence of

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oxygen and is unstable in acidic condition,16 indicating a possibility that fucoxanthin could be

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lost during the gastric or small intestinal digestion. Also, different bioaccessibility of

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fucoxanthin was observed for fucoxanthin-containing capsules and milk powder

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supplemented with different oils.17-18 Therefore, the inherent stability of fucoxanthin and the

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interaction with other compounds (e.g. lipids) during digestion needs to be considered.

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It is widely recognized that a substantial, if not predominant, part of dietary carotenoids

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escape absorption in the small intestine and ends up in the colon where they can shape gut

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microbiota19 or gut metagenome.20 Specifically, the ability of fucoxanthin-containing extract

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of Nitzschia laevis to change the gut microbiota composition when administered to mice for

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two months was reported.12 In addition, gut microbiota affected carotenoids bioavailability

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by altering the carotenoids absorption or degradation patterns.21 A previous study reported

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that gut microbiota may contribute to 11% of variance to the serum xanthophyll 4


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concentration.22 However, how the fucoxanthin escaping absorption in the small intestine

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would interact with gut microbiota, e.g. whether it is metabolized or not, needs to be clarified.

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In the present study, the fucoxanthin deacetylation and its bioaccessibility from the extract of

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Nitzschia laevis were investigated using simulated in vitro digestion models. The effect of

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gastric pH, the kinetics of deacetylation and bioaccessibility in the small intestine were

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investigated. Moreover, fucoxanthin recovery and the effect on SCFAs production were, for

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the first time, analysed through in vitro colonic fermentation with human faeces.

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Materials and Methods

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Chemicals and reagents

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Pure Fucoxanthin (PF, #16337), porcine pepsin (P6887), porcine pancreatin (P1750, 4×USP),

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porcine bile salt preparation (B8631), Orlistat (#04139) and Butylated hydroxytoluene (BHT)

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were purchased from Sigma Aldrich (Merck KGaA, Germany). Olive oil (extra virgin,

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Carapelli, USA) was bought from local market in the Netherlands. KCl, KH2PO4, NaCl,

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MgCl2× (H2O)6 and CaCl2× (H2O)2 and pure ethanol were purchased from VWR International

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B.V. (Netherlands). KH2PO4, NaCl , (NH4)2CO3, NaOH, HCl and Tween-80 were purchased

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from Sigma Aldrich Chemie B.V. (Netherlands), as well as the yeast extract, peptone, mucine

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and L-Cysteine HCl. Acetonitrile and chloroform were bought from LPS B.V. (Oss, the

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Netherlands). All the chemicals used are of analytical and chromatographic grade.

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Preparation of Nitzschia laevis extract (NLE)

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The NLE was obtained according to the method described in our previous paper.6 Briefly,

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about 100 mg of lyophilized powder was grounded with liquid nitrogen. Immediately, 10 mL

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ethanol was added into the ground cells for extraction, followed by end-to-end shaking for 1

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hour. Supernatants were collected by centrifugation (2500g × 5 min, 4 °C). The extraction

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was conducted twice and the supernatants were combined. NLE was obtained by evaporating

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with nitrogen at 22 °C. All the procedures were carried out under red light. The composition

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of NLE was investigated in our previous study.12 Fucoxanthin content was about 5.1% and no

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fucoxanthinol is detected in the freshly extracted NLE. Notably, there are more than 7% lipids

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in NLE.

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Preparation of Fucoxanthin-Containing-Emulsion

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The fucoxanthin-Containing-Emulsions (FCE) were prepared as previously described 23 by

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mixing [13500 rpm, 10 min (UltraTurrax, IKA-Werke, Staufen, Germany)] pure fucoxanthin

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and olive oil (5%, w/w) with an aqueous solution containing Tween-80 (1%, w/w). The

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obtained FCE were stored at -20 °C in the dark and used within 3 days.

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Simulated in vitro gastrointestinal digestion

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All samples were digested using a static in vitro digestion system consisting of simulated oral

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phase, gastric phase and intestinal phase with modifications.24-25 The composition (%, w/w) of

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the Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF, pH 3.0 ± 0.05) and

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Simulated Intestinal Fluid (SIF, pH 7.0 ± 0.05) were as reported.24

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For the oral phase, for 5 mL samples (fucoxanthin content 100 µg), 3.5 mL of SSF stock

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solution (37 °C) and 25 µL of CaCl2 (0.3 M) and 1.475 mL of Milli-Q water (VEOLIA water,

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veolia water solutions and technologies Netherlands B.V.) were added and mixed. For the

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experiment of oxidation prevention, 0.02% and 0.04% BHT were added.

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Subsequently, to start the gastric phase, 5 mL of oral bolus was mixed with 3.75 mL SGF

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(37 °C). Then 0.8 mL porcine pepsin stock solutions of 25000 U/mL (2000 U/mL in final 6


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chyme) in SGF (37 °C) was added, followed by 2.5 µL of CaCl2 (0.3 M). Last, about 0.1 mL

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HCl (1 M) was added to reach pH 3. Finally, 0.349 mL Milli-Q water was added to get the

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final volume (10 mL). Then the chyme was shaken at 37 °C for 2 h without light. All reactors

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were filled with nitrogen when closed before starting the digestion. In the experiments at

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different gastric pH, pH was adjusted to 4 and 5 before starting the gastric phase with HCl.

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At the end of the gastric digestion step, about 0.075 mL NaOH (1 M) was added to reach pH

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7.0, inhibiting the gastric enzyme activity. Thereafter, to 10 mL of gastric chyme, 5.5 mL SIF

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(37 °C), 2.5 mL of porcine pancreatin (trypsin activity 800 U/mL and the lipase activity was

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around 280U/mg) in SIF (37 °C), 1.25 mL fresh bile stock solutions (10 mM in the final

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intestinal chyme), 20 µL of CaCl2 (0.3 M) and 0.655 mL Milli-Q water were added. Finally,

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the chyme was incubated in a shaking water bath for 2 h at 37 °C and the digestion was

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stopped by cooling down immediately in an ice bath. To study the kinetics of fucoxanthin

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micellarization, samples were collected at 0, 10, 20, 30, 60 and 120 min, respectively. For the

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inhibition of lipase activity, 10 μg/mL Orlistat was added into pancreatin dissolved in SSF

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and the mixture was incubated at 37 °C for 30 minutes before undertaking the simulated

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pancreatic digestion.26 In this experiment, samples were only collected after 120 min. Also,

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for the experiment with BHT, samples were collected at the end of intestinal digestion. All

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intestinal digests were centrifuged for 10 min at 20,000g (4 °C) and the supernatants were

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collected and filtered through a 0.22 μm membrane filter (Phenomenex, Netherlands).

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Fucoxanthin in the filtered supernatants was supposed to have been incorporated into the

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micellar phase. All tubes used here were filled with nitrogen before starting the digestion

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procedure and all procedures were conducted under red light.

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In vitro colonic fermentation

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Fresh faecal samples were donated by three healthy adults (Two Chinese and one Dutch,

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Aged 23-28, BMI 18.5-23.9) who declared no smoking and no antibiotic consumption for 6

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months before the beginning of the study. The faecal samples were prepared according to

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protocols previously reported with modifications.27-28 Briefly, about 20.0 g faeces were

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diluted by 100 mL anaerobic phosphate buffer before homogenization in a stomacher bag.

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The resulting faecal suspension was filtered and considered as the faecal microbiota inocula.

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The basal medium, consisting of 5.22 g/L K2HPO4, 16.32 g/L KH2PO4, 2.0 g/L NaHCO3, 2.0

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g/L Yeast extract, 2.0 g/L Peptone, 1.0 g/L Mucine, 0.5 g/L L-Cysteine HCl and 2.0 mL/L

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Tween-80, was used after a 30-minutes flushed with nitrogen before autoclaving. Fucoxanthin

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(100 µg) from all fucoxanthin-containing products dissolved in SIF was added, together with

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a 10% inoculation. The fermentation was then started at 37 °C with continuous shaking (60

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rpm) under anaerobic conditions without light. In parallel, two different controls were

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conducted under the applied conditions: (i) the fucoxanthin-containing fractions were

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fermented without inoculation to determine possible chemical reactions; (ii) the faecal

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suspension was fermented without fucoxanthin-containing products to be served as a negative

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control. All incubations were performed in triplicate and sampled at 0, 2, 10, 24, 48, 72h,

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respectively. All the samples were centrifuged immediately when sampled and the

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supernatants were stored at -20 °C until use.

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Fucoxanthin analysis

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For the in vitro gastrointestinal digestion samples, the fucoxanthin and its metabolites were

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extracted as previously described17 with tiny modifications. A total of 10 mL digest was

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mixed with 5 mL chloroform, as well as 0.5 g NaCl, followed by end-to-end shaking for 30

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min. Then the chloroform layer was collected through centrifugation (4700rpm × 15 min, 8


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4 °C). The extraction was repeated twice and both the chloroform fractions were combined.

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Chloroform was then evaporated with nitrogen and the samples were re-dissolved in ethanol

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before analysis with high performance liquid chromatography (HPLC). Same method was

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applied to the supernatants in the batch fermentation experiments (see previous section).

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Fucoxanthin was determined and quantified using a HPLC-DAD system (Ultimate 3000,

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Thermo scientific, Netherlands) equipped with an Onyx Monolithic C18 column (100 × 4.6

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mm, pore size 130 Å, Phenomenex, Netherlands). The column temperature was kept at 30 °C.

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The mobile phase was a mixture of acetonitrile and water (85:15) and the flow rate was 1.5

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mL/min. The injection volume was 10 µL. Fucoxanthin and fucoxanthinol were identified at

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450 nm and quantified using external calibration curves.

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The bioaccessibility/recovery of fucoxanthin was defined as the sum of fucoxanthin and

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fucoxanthinol content that are detected in the supernatants, divided by total fucoxanthin

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initially present in the samples before digestion. Specific calculation was as follows:

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Bioaccessibility/Recovery (%) =

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Cm represents the fucoxanthin or fucoxanthinol amounts in the micellar phase where Cfucoxanthin

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represents the total fucoxanthin content (µg) in the samples.

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SCFAs analysis

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Fecal fermented samples (1 mL) were thawed and centrifuged at 9000g × 5 min at 4 °C. The

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resulting supernatants were then filtered using 0. 2 μm RC filter. A total of 250 μL of internal

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standard (0.45 mg/mL 2-ethylbutyic acid in 0.3M HC and 0.9M oxalic acid) was added to 500

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μL sample. Finally, the solutions were mixed and centrifuged at 20,000g for 4 minutes before

𝐶𝑚fucoxanthin + 𝐶𝑚fucoxanthinol 𝐶fucoxanthin

× 100

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injection to gas chromatography (GC-2014, Shimadzu, Hertogenbosch, Netherlands),

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equipped with a capillary fatty acid-free Stabilwax®-DA column (1 μm × 0.33 mm × 30m;

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Restek, America), a flame ionization detector, and a split injector. The injection volume was

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1.0 µL. The carrier gas was nitrogen, and the temperature of the injector and detector was

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100 °C and 250 °C, respectively. The temperature profile starts with 100 °C, then increased at

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10.8 °C/min to 180 °C and kept for 2 minutes. Then it increased at 50.00 °C/min until 240 °C

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and kept for 2 minutes. Standard solutions of acetic acid, propionic acid, butyric acid, valeric

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acid, isovaleric acid and isobutyric acid in concentrations of 0.01-0.45 mg/mL were prepared

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and used for identification and quantification.

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Statistical analysis

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All the data was expressed as mean ± SD. Shapiro-Wilks test was used for the evaluation of

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normality of distribution and Leven test was applied for the equality of variance. One-way

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ANOVA was performed using SPSS followed with Tukey test to evaluate differences

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between groups. All figures were drawn with Origin 8.5 (OriginLab Corporation,

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Massachusetts, USA). Statistical significance was set at P ≤0.05. All statistical analyses were

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performed with SPSS 23.0 (SPSS, Chicago, IL, USA).

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Results and Discussion

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Effect of gastric pH on the recovery of fucoxanthin

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The effect of gastric pH on fucoxanthin recovery was tested and the results were shown in

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Figure 1. No significant difference for fucoxanthin bioaccessibility was observed when PF

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and FCE were digested at pH 4 and 5 compared to pH 3. However, gastric bioaccessibility of

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fucoxanthin from NLE was significantly lower at pH 3 compared with that of pH 4 and 5.

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This last result agrees with previous results showing that fucoxanthin exhibits the lowest 10


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stability at pH

Gastrointestinal Bioaccessibility and Colonic Fermentation of

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