Use of Saccharomyces cerevisiae To Reduce the Bioaccessibility of

Mar 13, 2017 - This study evaluates the capacity of Saccharomyces cerevisiae to reduce the amount of mercury solubilized after gastrointestinal digest...
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The Use of Saccharomyces cerevisiae to Reduce the Bioaccessibility of Mercury from Food Carlos Jadán-Piedra, Marta Baquedano, Sergi Puig, Dinoraz Velez, and Vicenta Devesa J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00285 • Publication Date (Web): 13 Mar 2017 Downloaded from http://pubs.acs.org on March 15, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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The Use of Saccharomyces cerevisiae to Reduce the Bioaccessibility of Mercury

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from Food

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Carlos Jadán-Piedra, Marta Baquedano, Sergi Puig, Dinoraz Vélez, Vicenta Devesa. *

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Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), C/ Catedrático Agustín Escardino

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7, 46980 - Paterna (Valencia), Spain.

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* To whom correspondence should be addressed (telephone (+34) 963 900 022; fax (+34) 963 636

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301; e-mail: [email protected])

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Running header: Reduction of Hg bioaccessibility by Saccharomyces cerevisiae

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ABSTRACT

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Food is the main pathway of exposure to inorganic mercury [Hg(II)] and methylmercury

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(CH3Hg). Intestinal absorption of these mercury species is influenced by their chemical form,

15

the luminal pH or the diet composition. In this regard, strategies have been proposed for

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reducing mercury absorption using dietary components.

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The present study evaluates the capacity of Saccharomyces cerevisiae to reduce the

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amount of mercury solubilized after gastrointestinal digestion which is available for intestinal

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absorption (bioaccessibility). The results show that S. cerevisiae strains reduce mercury

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bioaccessibility from aqueous solutions of Hg(II) (89 ± 6%) and CH3Hg (83 ± 4%), and from

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mushrooms (19-77%), but not from seafood. The formation of mercury-cysteine or mercury-

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polypeptide complexes in the bioaccessible fraction may contribute to the reduced effect of

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yeasts on mercury bioaccessibility from seafood. Our study indicates that budding yeasts

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could be useful for reducing the intestinal absorption of mercury present in water and some

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food matrices.

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Keywords: Mercury, bioaccessibility, Saccharomyces cerevisiae, seafood, mushrooms.

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1. INTRODUCTION

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Mercury is a metal found in the environment, where it is of natural or anthropogenic

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origin. Food is the main pathway of exposure to inorganic divalent mercury [Hg(II)] and

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methylmercury (CH3Hg). Seafood products, especially large predators, are main dietary

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sources of CH3Hg, the concentrations of which often exceed the maximum limits

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contemplated by current legislation (1 mg/kg).1 The European Food Safety Authority (EFSA)

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reports a maximum CH3Hg intake of 1.57 µg/kg body weight/week in the European adult

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population.2 This value is close to the tolerable weekly intake (TWI) proposed by the Joint

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FAO/WHO Expert Committee on Food Additives (JEFCA) (1.6 µg/kg body weight).3 With

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regard to inorganic mercury, the food groups “fish and other seafood” and “non-alcoholic

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beverages” are among the greatest contributors to the intake of this mercury species in the

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European population.2 However, mushrooms are the food matrices with the highest inorganic

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mercury concentrations, reaching levels in the order of 20 mg/kg dry weight.4 JEFCA reports

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a mean weekly intake of inorganic mercury from food of 2.16 µg/kg body weight for the adult

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population, this value being lower than the TWI proposed by the JEFCA (4 µg/kg body

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weight).3

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Mercury toxicity is dependent upon the chemical form of the element, dose, age of the

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individual, duration of exposure, exposure route and dietary habits. Methylmercury affects

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mainly the nervous system, whereas the main targets of inorganic mercury are the kidneys.2,3

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Furthermore, CH3Hg is considered to be neurotoxic during development,5 and mainly because

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of this the international public health safety authorities have established recommendations

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referred to the consumption of certain fish species in vulnerable population groups.6,7

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EFSA states that in some population groups the intake of CH3Hg is up to 6-fold higher

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than the recommended levels, and that this can result in health problems. On the other hand,

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EFSA also underscores that decreasing the intake of seafood is a strategy that cannot be

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applied without also taking into account that these foods are an important source of nutrients.2

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It is therefore of interest to seek alternatives allowing a decrease in mercury exposure through

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foods. One possibility is to reduce the amount of ingested mercury that is actually absorbed

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and reaches the bloodstream, i.e., to act upon the oral bioavailability of mercury. It is known

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that the absorption of Hg(II) is variable and relatively low, with percentages of between 1-

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38% that moreover depend on the solubility of the mercury salt involved.3 In this respect,

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halides, sulfates and nitrates show high solubility, with moderate absorption levels, while

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mercury sulfide is scantly soluble and thus undergoes minimum absorption.3 In contrast, the

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reported absorption rates for CH3Hg exceed 80%,2 even when consumed through seafood.8

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Some in vivo studies have described modifications in the toxicokinetics of mercury in the

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presence of food components or dietary supplements. An increase in CH3Hg elimination has

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been reported in animals fed different types of dietary fiber, the authors attributing this to

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interruption of the enterohepatic recirculation.9 Orct et al.

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mercury levels in rats co-administrated with HgCl2 (6.45 µmol kg−1 body weight) and

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Na2SeO3 (19.4 µmol kg−1 body weight). In addition to food components, studies have also

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examined the possible role of probiotics in reducing mercury entry into the bloodstream. A

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recent study has reported a decrease in blood mercury levels in pregnant women treated with

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Lactobacillus rhamnosus GR-1.11 Kinoshita et al.12 described the capacity of lactobacilli to

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bind mercury in vitro, this possibly being responsible for the decrease in bioavailability

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observed in the population based study. Likewise, certain strains of Saccharomyces cerevisiae

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have been shown to be useful in reducing Hg(II) present in solution,13 and therefore might

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exert effects similar to those described for lactobacilli in human populations.

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recorded a decrease in plasma

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The present study seeks to identify strains of S. cerevisiae capable of reducing the amount

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of mercury contained in food that can be absorbed in the gastrointestinal tract. For this

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purpose an in vitro study has been made to evaluate the capacity of different strains of S.

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cerevisiae to bind to Hg(II) and CH3Hg and their effect upon the bioaccessibility (i.e., the

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soluble amount obtained after gastrointestinal digestion that is available for absorption) of

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mercury present in water and food.

81 82

2. MATERIALS AND METHODS

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2.1. Culture conditions of Saccharomyces cerevisiae. The strains of S. cerevisiae used in the

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present study are described in Table 1. Yeasts were maintained on solid YPD medium [1%

85

(w/v) of yeast extract, 2% (w/v) of bacteriological peptone, 2% (w/v) of glucose (Sigma,

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Spain), and 1.5% (w/v) of bacteriological agar]. For each assay, independent yeast colonies

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were incubated overnight in liquid synthetic complete medium [0.17% (w/v) of yeast nitrogen

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base without amino acids and without (NH4)2SO4, 0.5% (w/v) of (NH4)2SO4, 0.2% (w/v)

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synthetic complete drop-out Kaiser mixture (Formedium, Norfolk, United Kingdom), and 2%

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(w/v) of glucose] at 30ºC and 190 rpm. Next, yeast cells were collected by centrifugation at

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4,000 rpm for 2 min, washed twice with phosphate buffered saline (PBS, Hyclone, Fisher,

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Spain), and then used for the assayed described below.

93 94

2.2. Mercury binding assays. Laboratory yeast strain BY4741 (strain #1) was incubated

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under agitation with 10 mL of the standard solutions of Hg(II) and CH3Hg (1 mg/L) prepared

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in PBS from commercial standards of Hg(NO3)2 (Merck, Spain) and CH3HgCl (Alfa Aesar,

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Spain) respectively. Incubation was performed at an approximate concentration of 5 × 107

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cells/mL [4 optical densities (OD)/mL], at different temperatures (30ºC and 37ºC), and at

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37ºC for different times (30, 60 and 120 min). After the incubation period, the samples were

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centrifuged at 4,000 rpm for 2 min. The mercury content in the supernatant and in the cell

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pellet was analyzed according to the protocol described in section 2.5.

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2.3. Mercury bioaccessibility assays. Bioaccessibility was evaluated using the in vitro

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gastrointestinal digestion model described by Jadán-Piedra et al.,14 with minor modifications.

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Two different assays were performed:

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a) Study of the mercury biosorption capacity of S. cerevisiae during the gastrointestinal

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digestion of standard solutions of Hg(II) and CH3Hg.

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b) Study of the mercury biosorption capacity of S. cerevisiae during the digestion of

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swordfish (Xiphias gladius), yellow fin tuna (Thunnus albacares) and dehydrated mushrooms

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(Boletus edulis and Amanita caesarea).

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For this study we chose S. cerevisiae strain BY4741, which was added at a concentration

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of 4 OD/mL to standard solutions of mercury (1 mg/L) or food (5 g of seafood or 0.5 g of

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mushrooms), and the combination was then subjected to digestion. In both cases, the volume

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of the gastrointestinal digestion process was 50 mL. The pH was adjusted to 2.0 with 6 M

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HCl (Merck), and a sufficient volume of pepsin solution (0.1 g of pepsin/mL prepared in 0.1

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M HCl) was added to provide 0.2 mg of pepsin/mL of digestion solution. The mixture was

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incubated at 37°C during 2 h under constant agitation (120 rpm). After gastric digestion, the

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pH was increased to 6.5 with 1 M NH3 (Panreac, Spain), and a solution of pancreatin and bile

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extract [0.004 g/mL of pancreatin and 0.025 g/mL of bile extract in 0.1 M NH4HCO3

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(Merck)] was added to yield a final concentration of 0.025 mg of pancreatin/mL of solution

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and 0.3 mg of bile extract/mL of solution. The mixture was incubated at 37°C during 2 h

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under constant agitation (120 rpm).

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The pH was then adjusted to 7.2 with NH3 and the samples were centrifuged at 10,000 rpm

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during 30 min at 4ºC. The concentration of mercury in the soluble fraction obtained after

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centrifugation (bioaccessible fraction) was analyzed according to the protocol described in

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section 2.5. Bioaccessibility was calculated as a percentage using the following equation:

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Bioaccessibility (%) = [A/B] × 100

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Journal of Agricultural and Food Chemistry

where A is the concentration of mercury in the bioaccessible fraction and B is the concentration of mercury in the standard solution or in the food before digestion.

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The enzymes and bile salts used during digestion were acquired from Sigma-Aldrich,

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Spain: porcine pepsin (activity 944 U/mg of protein), porcine pancreatin (activity equivalent

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to the specifications of 4×US Pharmacopeia/mg of pancreatin) and bile extract (glycine,

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conjugates of taurine and other bile salts).

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2.4. Study of the factors influencing the effect of S. cerevisiae upon the bioaccessibility of

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mercury in food. An evaluation was made of different factors that might influence the effect

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of the yeasts upon the bioaccessibility of mercury from food samples. BY4741 yeast strain

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was used in all assays, except for the study D. The factors analyzed were:

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A. Effect of yeast concentration. Three different yeast concentrations (4, 7 and 10

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OD/mL) were added to samples of swordfish and mushrooms (Amanita cesarea). The

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resulting combination was subjected to the gastrointestinal digestion process described

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in section 2.3.

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B. Effect of the presence of cysteine (Cys) and bovine serum albumin (BSA). This assay

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was carried out to determine whether the binding of mercury to these compounds could

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affect its adsorption/uptake by yeast cells. The solutions of Hg(II) and CH3Hg (1 mg/L)

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added with Cys (5 mg/L; Sigma) and BSA (5 mg/L; Biowest, Labclinic, Spain) were

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subjected to gastrointestinal digestion in the presence of S. cerevisiae (4 OD/mL),

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following the protocol described in section 2.3.

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C. Effect of the presence of Ca(II), Cu(II) and Fe(II). In this assay we determined whether

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the presence of divalent cations (ions with characteristics similar to those of mercury)

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could affect mercurial species retention by the yeasts through competitive mechanisms.

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Solutions of Hg(II) or CH3Hg (1 mg/L) prepared in PBS were independently added

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with CaCl2 (100 mg/L; Panreac), CuSO4 (40 mg/L; Panreac) or FeSO4 (40 mg/L;

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Sigma), these being concentrations commonly found in food. The mixtures were

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incubated (2 h, 37ºC) with S. cerevisiae (4 OD/mL) and then centrifuged at 4,000 rpm

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during 2 min. The cell pellet and supernatant were recovered to determine mercury

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according to the protocol described in section 2.5.

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D. Effect of the S. cerevisiae strain. In addition to BY4741 strain, we studied the mercury

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biosorption capacity of 7 strains of S. cerevisiae from different sources (Table 1) by,

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incubating them at 4 OD/mL with solutions of Hg(II) and CH3Hg (1 mg/L) prepared in

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PBS, as described in section 2.2.

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Gastrointestinal digestions were then performed using three yeast strains (#3, #4 and

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#5) found to be most effective in reducing the solubility of mercury in standard

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solutions at a concentration of 4 OD/mL. These yeast strains were added to the samples

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of swordfish and mushrooms (Tricholoma georgii), and the combination was subjected

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to gastrointestinal digestion (section 2.3).

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2.5. Determination of mercury. Microwave oven-assisted digestion (MARS, CEM, Vertex,

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Spain), with subsequent quantification by cold vapor atomic fluorescence spectrometry (CV-

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AFS; Millenium Merlin PSA 10.025, PS Analytical, Microbeam, Spain), was used for the

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determination of mercury in food samples, bioaccessible fractions, yeast cells and incubation

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media. For digestion, the samples were placed in a Teflon reactor, with the addition of 4 mL

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of 14 M HNO3 (Merck) and 1 mL of H2O2 (30% v/v, Panreac). The reactor was irradiated in

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the microwave oven at a power setting of 800 W (180ºC/15 min). After the digestion process,

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the digests were left to stand overnight to eliminate the nitrous vapors. Finally, the solution

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was made up to a final volume with HCl 0.6 M.

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The quantification of mercury was carried out against a Hg(II) calibration curve in the

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concentration range of 0.05-2 ng/mL. Quality control was based on the analysis of the sample

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of RTC QCI1014 mercury certified water (certified value: 40.8 ± 1.19 µg/L, LGC Standards,

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Spain).

181 182

2.6. Statistical analysis. The statistical analysis of the results was based on the Student t-test

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for paired data or single-factor analysis of variance (ANOVA) with post hoc multiple

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comparisons using the Tukey HSD test (SigmaPlot, version 13.5). Statistical significance was

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considered for p ≤ 0.05.

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3. RESULTS

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3.1. Studies on the mercury biosorption capacity of S. cerevisiae

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3.1.1. Influence of temperature. The effect of S. cerevisiae upon uptake/adsorption of the

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different mercury species was initially evaluated at 30ºC, this being the optimum temperature

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for growth of the yeast. The results showed laboratory yeast strain BY4741 (#1 in Table 1) to

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be able to accumulate a large percentage of added Hg(II) (59 ± 2%) and CH3Hg (76 ± 4%)

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(Figure 1). However, since the purpose of our study was to evaluate the effect of S. cerevisiae

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during gastrointestinal digestion, and that the latter takes place at 37ºC, we considered

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advisable to determine whether this difference in temperature affected the mercury

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biosorption capacity. The results obtained indicate that the temperature increment did not

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reduce the efficiency of the mercury uptake/adsorption process [Figure 1, Hg(II): 73 ± 3%;

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CH3Hg: 82 ± 2%].

199 200

3.1.2. Influence of time. The kinetics of mercury binding by the laboratory yeast strain at 37ºC

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was analyzed using incubation times of 30, 60 and 120 min (Figure 2). The biosorption of

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Hg(II) increased significantly with the duration of exposure, from 35 ± 1% after 30 min to 73

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± 3% after 120 min. In contrast, the retention of CH3Hg did not show significant variations

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over time (88 ± 3% after 30 min and 82 ± 2% after 120 min), and was already found to be

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high after 30 min of exposure.

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3.2. Studies on the capacity of S. cerevisiae to reduce bioaccessibility during

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gastrointestinal digestion

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3.2.1. Assays in standard solutions. Figure 3 shows the amount of mercury retained by S.

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cerevisiae strain BY4741 after the gastrointestinal digestion of standard solutions of Hg(II)

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and CH3Hg. Mercury retention by yeast was very high for both mercurial species, with

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percentages corresponding to Hg(II) (89 ± 6%) and CH3Hg (83 ± 4%) similar to those

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obtained in the assays made without digestion (section 3.1). These results indicate that the

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digestion conditions used (pH, enzymes and salt concentrations) did not reduce the mercurial

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species uptake/adsorption capacity of S. cerevisiae.

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3.2.2. Assays in food. Figure 4 shows the bioaccessible mercury contents following

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gastrointestinal digestion of food samples in the absence and presence of yeast strain BY4741

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(4 OD/mL). The results indicate that yeast addition does not result in important changes in the

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amount of mercury present in the soluble fraction of swordfish and tuna. Importantly, a

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statistically significant decrease in bioaccessible content in mushrooms was recorded (19-

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33%). Therefore, these data indicate that budding yeast reduces mercury bioaccessibility from

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mushrooms but not from swordfish or tuna.

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3.3. Factors influencing the reduction of mercury bioaccessibility from food

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3.3.1. Yeast concentration. In samples of swordfish, the addition of yeast at higher optical

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densities (7-10 OD/mL) was not associated to increased mercury retention during digestion

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(data not shown) compared with that recorded at an optical density of 4 OD/mL (section

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3.2.2). In the case of mushrooms, an increase in optical density from 4 to 7 OD/mL was

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associated to a nonsignificant further decrease in mercury bioaccessibility (4 OD/mL: 22%; 7

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OD/mL: 28%). We concluded that, within the studied range, optical densities higher than 4

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OD/mL were therefore not found to be a determining factor in mercury binding in food

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samples.

233 234

3.3.2. Presence of cysteine and albumin. A possible explanation for the inefficacy of yeast in

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reducing the bioaccessibility of mercury in the seafood samples could be that the mercury

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solubilized from the food matrix is present in a chemical form that cannot interact with yeast

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cells. In this regard, some studies have described the formation of complexes of mercury with

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Cys or with polypeptides or proteins, 15,16 which might be unable to interact with yeast. Figure

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5 shows mercury uptake/adsorption by yeast BY4741 in the presence of Cys or BSA during

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the digestive process. Both compounds were associated to significant reductions in the

241

amount of mercury retained by yeast cells compared with those assays in which Cys or BSA

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was not added to the medium. The effect of Cys was particularly notorious, with reductions of

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over 95%. These data suggest that the formation of complexes of Hg(II) and CH3Hg with

244

sulfated amino acids or proteins could prevent the interaction of mercury with S. cerevisiae,

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thereby resulting in no reduction in mercury bioaccessibility.

246 247

3.3.3. Presence of divalent cations. Another possible explanation for the lesser effect of yeast

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upon the bioaccessibility of mercury in food would be the interaction of yeast with other

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matrix components for which they exhibit greater affinity, thereby preventing interaction with

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mercury. We explored the above possibility using divalent cations with characteristics similar

251

to those of the mercurial species studied, and which could compete for the same binding sites

252

or for the same transport mechanisms. Therefore, we studied mercury binding to yeast in the

253

absence of such cations and following the individual addition of Ca(II), Cu(II) and Fe(II). As

254

shown in Figure 6, the presence of these divalent cations exerted no effect upon CH3Hg,

255

though significant reductions were observed in the uptake/adsorption of Hg(II) with Ca(II)

256

(18 ± 3%) and particularly with Cu(II) (36 ± 2%).

257 258

3.3.4. Saccharomyces cerevisiae strain. To distinguish whether the effect in mercury retention

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exerted by the laboratory yeast BY4741 was strain specific or a feature of S. cerevisiae cells,

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we determined the amount of soluble mercury after incubating Hg(II) and CH3Hg standards

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during two hours at 37ºC with seven additional S. cerevisiae strains from different sources

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including laboratory, wine, dietetic, wild and sake yeast strains (Table 1). As shown in Figure

263

7, all the tested strains significantly reduced the mercury contents in solution. Following

264

incubation, the initial amount of added Hg(II) (9200 ng) was found to have decreased to

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between 404 and 2241 ng (Figure 7a). In the case of CH3Hg, with an initially added amount

266

of 8432 ng, the soluble content following exposure to the yeasts ranged between 324 and

267

3028 ng (Figure 7b). These results demonstrate that mercury retention is an intrinsic

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characteristic of S. cerevisiae, but they also suggest that there are differences among yeast

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strains that significantly affect mercury bioaccessibility.

270

From these studies standard solutions, we selected wine strain VRB (strain #4), wine strain

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T73 (strain #3) and dietetic strain Ultralevura (strain #5) that exhibited a strong effect in

272

decreasing Hg(II) and CH3Hg solubility, for assessing their effects upon the bioaccessibility

273

of mercury present in swordfish and mushrooms. As previously shown for laboratory strain

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BY4741 (section 3.2.2), none of these strains modified the bioaccessibility of mercury in

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swordfish (data not shown). The effect in mushrooms was different (Figure 8), with reduction

276

in the bioaccessible mercury contents superior to 50% for the 3 strains (strain #3: 55 ± 3%;

277

strain #4: 74 ± 4%; strain #5: 61 ± 8%). These results strongly suggest that the reduction in

278

mercury bioaccessibility obtained from mushroom samples is a general characteristic of S.

279

cerevisiae species.

280 281

4. DISCUSSION

282

Reducing mercury exposure through the diet is not easy. Extraction of the metal from food

283

before marketing17,18 results in important changes in the physical and nutritional properties of

284

the food due to the application of relatively long treatment processes or the need for food

285

matrix transformation. Another option for reducing exposure is to lower the amount of

286

mercury that reaches the systemic circulation after intake. This can be done in two

287

complementary ways: reducing the amount of mercury rendered soluble after digestion and/or

288

reducing absorption through direct action upon transport across the intestinal barrier. Jadán-

289

Piedra et al.14 suggest the use of food components (tannins, lignin, pectin and some

290

celluloses) in order to lower the bioaccessibility of mercury from seafood. The addition of

291

these components to food significantly reduces (≤ 98%) the amount of toxic element available

292

for absorption after digestion. The present study has continued the search for dietary strategies

293

aimed at lowering the oral bioavailability of mercury, focusing on the use of yeast strains

294

belonging to the species S. cerevisiae.

295

Since ancient times, yeasts belonging to the genus Saccharomyces, particularly S.

296

cerevisiae, have been used in many food-related fermentation processes. Recently, certain

297

strains of S. cerevisiae have been adopted as food supplements due to their high contents in

298

vitamins, minerals, fiber and proteins,19 and have been used as probiotics in the treatment of

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chronic, recurrent or acute diarrhea.20,21 Interest in the use of this yeast as bioabsorbing agents

300

for eliminating metals and metalloids has increased in recent years, since they are safe and

301

easy to produce on a large scale, they are a subproduct of the food industry, and they are easy

302

to study due to their genetic characteristics.22 The capacity of this yeast to retain or bind

303

metals such as lead, cadmium, copper, aluminum, chromium, nickel, zinc and mercury has

304

been demonstrated in aqueous solutions.13,23-25 They are also effective in eliminating metals

305

from waste waters, acting as chelating and flocculating agents.26,27 In this regard, although the

306

application of such yeasts has been limited to environmental samples, their chelating capacity

307

could also be applicable to other areas, including health. Probiotics of the genus Lactobacillus

308

have recently been used in a population-based intervention designed to reduce the absorption

309

of metals ingested via the oral route.11 These authors demonstrated a slight decrease in blood

310

mercury levels in pregnant women. Some strains of S. cerevisiae could also be used in

311

interventions of this kind. Their application moreover would afford other benefits related to

312

their antioxidant and, in some cases, anti-inflammatory effects,28 thereby countering two of

313

the toxic effects associated to continued mercury exposure.

314

The above considerations led us to use S. cerevisiae in the present study. The most relevant

315

results are summarized in table S1 and S2 of supplementary data. Saccharomyces cerevisiae

316

is able to retain mercury under the conditions of gastrointestinal digestion, with reductions in

317

aqueous solutions of the soluble fraction of Hg(II) (89%) and CH3Hg (83%). This yeast was

318

also effective in lowering the bioaccessibility of mercury present in mushrooms (up to 77%),

319

though no such decrease was noted in seafood. This lesser efficacy is possibly attributable to

320

the way in which mercury is released from seafood during digestion, or to the formation of

321

complexes of mercury with amino acids, polypeptides or proteins solubilized during the

322

digestive process. Likewise, certain components of the food matrix itself, such as divalent

323

cations, might exert a negative effect in this regard.

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The application of simulated digestion of mercury standards in the presence of Cys showed

325

this amino acid to significantly reduce the capacity of the yeast to retain Hg(II) and CH3Hg

326

during digestion. Cabañero et al.29 found Hg/Cys complexes to be present in the bioaccessible

327

fraction obtained after the digestion of samples of swordfish, which would explain the lack of

328

efficiency of yeast in reducing the bioaccessibility of mercury from this type of fish. The fact

329

that yeast is more effective reducing mercury solubilized from mushrooms may be due to the

330

fact that these food matrices are deficient in sulfated amino acids,30,31 and therefore the

331

mercury released from the matrix might not form complexes with Cys.

332

The presence of copper also significantly reduced the retention of Hg(II) by

333

Saccharomyces (38%). This decrease could be a result of competition with mercury for the

334

same binding sites and/or same internalization mechanisms. In fact, studies on the biosorption

335

of Cu(II) by S. cerevisiae have revealed important retention of this element.32 An X-ray

336

absorption spectroscopic study with intact S. cerevisiae cells, found accumulated copper to be

337

exclusively linked to sulfide groups.33 The binding of Hg(II) and CH3Hg to thiol groups has

338

also been widely documented, and is even considered to constitute one of the main ways in

339

which mercury accumulates and exerts its toxic activity in other eukaryotic cells.34

340

Furthermore, synchrotron-based X-ray spectroscopic studies of bacteria exposed to Hg(II)

341

have identified sulfhydryl groups as the dominant mercury binding groups in the micromolar

342

and submicromolar range.35 Further studies are necessary to decipher how copper interferes

343

with this type of interaction.

344

We can conclude that S. cerevisiae is able to retain in vitro the main mercurial species in

345

water and foods. The binding capacity of this yeast is maintained under the conditions of the

346

gastrointestinal digestion process employed in our study, resulting in a decrease in the

347

bioaccessibility of the mercury present in aqueous solutions or in mushrooms. However,

348

effective biosorption was not observed in swordfish or tuna. This may be attributable to the

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formation of mercury-cysteine or mercury-polypeptide complexes in the soluble fraction,

350

which are not retained by yeast, or to the presence of matrix components that interact with

351

yeast cells in the same way as mercury. These effects of S. cerevisiae could be modified in

352

vivo due to the composition and interactions found in the lumen, which are more complex

353

than those simulated by the in vitro digestion process used in our study. In vivo studies are

354

therefore needed to confirm the results obtained here. It also would be interesting to

355

characterize the mechanisms involved in mercury retention, with a view to designing

356

adequate strategies for reducing oral exposure to mercury.

357 358

Acknowledgements. We are grateful to Drs. Amparo Querol and Gianni Liti for yeast strains

359

used in this study.

360 361

Funding sources. This work was supported by the Spanish Ministry of Economy and

362

Competitiveness grants AGL2012-33461, AGL2015-68920 and BIO2014-56298-P, for which

363

the authors are deeply indebted. Carlos Jadán-Piedra received a Personnel Training Grant

364

from SENESCYT (Ecuadorian Ministry of Higher Education, Science, Technology and

365

Innovation) to carry out this study.

366

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REFERENCES

368

1. European Union. Commission Regulation (EU) No 420/2011 of 29 April 2011 amending

369

Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in

370

foodstuffs. OJ 2011, L 111/3-111/6.

371

2. European Food Safety Authority (EFSA). Scientific Opinion on the risk for public health

372

related to the presence of mercury and methylmercury in food. EFSA J. 2012, 10, pp. 2985.

373

3. Joint FAO/WHO Expert Committee on Food Additives (JECFA). Safety evaluation of

374

certain food additives and contaminants. In Seventy-second meeting of JEFCA, WHO Food

375

Additives Series No. 63. World Health Organization, Geneva, 2011.

376

4. Kalač, P.; Svoboda, L. A review of trace element concentrations in edible mushrooms.

377

Food Chem. 2000, 69, 273–281.

378

5. Grandjean, P.; Weihe, P.; White, R. F.; Debes, F.; Araki, S.; Yokoyama, K.; Murata, K.;

379

Sørensen, N.; Dahl, R.; Jørgensen, P. J. Cognitive deficit in 7-year-old children with prenatal

380

exposure to methylmercury. Neurotoxicol. Teratol. 1979, 19, 417–428.

381

6. United States Environmental Protection Agency/Food and Drug Administration

382

(EPA/FDA). Advisory: Mercury in fish and shellfish and federal consumption advice

383

(EPA/FDA). 2004. URL (https://www.epa.gov/choose-fish-and-shellfish-wisely/what-you-

384

need-know-about-mercury-fish-and-shellfish) (accessed 7.06.16).

385

7. Agencia española de Consumo, Seguridad Alimentaria y Nutrición (AECOSAN).

386

Recomendaciones de consumo de pescado para poblaciones sensibles debido a la presencia de

387

mercurio.

388

(http://aesan.msssi.gob.es/AESAN/web/rincon_consumidor/subseccion/mercurio_pescado.sht

389

ml) (accessed 10.06.16).

390

8. Yannai, S.; Sachs, K. M. Absorption and accumulation of cadmium, lead and mercury from

391

foods by rats. Food Chem. Toxicol. 1993, 31, 351–355.

2011.

URL

17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 32

392

9. Rowland, I. R.; Mallett, A. K.; Flynn, J.; Hargreaves, R. J. The effect of various dietary

393

fibers on tissue concentration and chemical form of mercury after methylmercury exposure in

394

mice. Arch. Toxicol. 1986, 59, 94–98.

395

10. Orct, T.; Lazarus, M.; Jurasović, J.; Blanusa, M.; Piasek, M.; Kostial, K. Influence of

396

selenium dose on mercury distribution and retention in suckling rats. J. Appl. Toxicol. 2009,

397

29, 585–589.

398

11. Bisanz, J. E.; Enos, M. K.; Mwanga, J. R.; Changalucha, J.; Burton, J. P.; Gloor, G. B.;

399

Reid, G. Randomized open-label pilot study of the influence of probiotics and the gut

400

microbiome on toxic metal levels in Tanzanian pregnant women and school children. mBio,

401

2014, 5, e01580–14.

402

12. Kinoshita, H.; Sohma, Y.; Ohtake, F.; Ishida, M.; Kawai, Y.; Kitazawa, H.; Saito, T.;

403

Kimura, K. Biosorption of heavy metals by lactic acid bacteria and identification of mercury

404

binding protein. Res. Microbiol. 2013, 164, 701–709.

405

13. Infante, J. C.; De Arco, R. D.; Angulo, M. E. Removal of lead, mercury and nickel using

406

the yeast Saccharomyces cerevisiae. Rev. MVZ Córdoba, 2014, 19, 4141–4149.

407

14. Jadán-Piedra, C.; Sánchez, V.; Vélez, D.; Devesa, V. Reduction of mercury

408

bioaccessibility using dietary strategies. LWT– Food Sci. Technol. 2016, 71, 10–16.

409

15. Yasutake, A.; Hirayama, K.; Inoue, M. Interaction of methylmercury compounds with

410

albumin. Arch. Toxicol. 1990, 64, 639–643.

411

16. Krupp, E. M.; Milne, B. F.; Mestrot, A.; Meharg, A. A.; Feldmann, J. Investigation into

412

mercury bound to biothiols: structural identification using ESI-ion-trap MS and introduction

413

of a method for their HPLC separation with simultaneous detection by ICP-MS and ESI-MS.

414

Anal. Bioanal. Chem. 2008, 390, 1753–1764.

415

17. Hajeb, P.; Jinap, S. Effects of washing pre-treatment on mercury concentration in fish

416

tissue. Food Addit. Contam. 2009, 26, 1354–1361.

18

ACS Paragon Plus Environment

Page 19 of 32

Journal of Agricultural and Food Chemistry

417

18. Hajeb, P.; Jinap, S. Reduction of mercury from mackerel fillet using combined solution of

418

cysteine, EDTA, and sodium chloride. J. Agric. Food Chem. 2012, 60, 6069–6076.

419

19. Ferreira, I. M. P.; Pinho, O.; Vieira, E.; Tavarela, J. G. Brewer's Saccharomyces yeast

420

biomass: characteristics and potential applications. Trends Food Sci. Technol. 2010, 21, 77–

421

84.

422

20. Czerucka, D.; Piche, T.; Rampal, P. Review article: yeast as probiotics – Saccharomyces

423

boulardii. Aliment. Pharmacol. Ther. 2007, 26, 767–778.

424

21. McFarland, L.V. Systematic review and meta-analysis of Saccharomyces boulardii in

425

adult patients. World J. Gastroenterol. 2010, 16, 2202–2222.

426

22. Wang, J.; Chen, C. Biosorption of heavy metals by Saccharomyces cerevisiae: a

427

review. Biotechnol. Adv. 2006, 24, 427–451.

428

23. Bakkaloglu, I.; Butter, T. J.; Evison, L. M.; Holland, F. S.; Hancock, I. C. Screening of

429

various types biomass for removal and recovery of heavy metals (Zn, Cu, Ni) by biosorption,

430

sedimentation and desorption. Water Sci. Technol. 1998, 38, 269–277.

431

24. Özer, A.; Özer, D. Comparative study of the biosorption of Pb(II), Ni(II) and Cr(VI) ions

432

onto S. cerevisiae: determination of biosorption heats. J. Hazard Mater. 2003, 100, 219–229.

433

25. Park, J. K.; Lee, J. W.; Jung, J. Y. Cadmium uptake capacity of two strains of

434

Saccharomyces cerevisiae cells. Enzyme Microb. Technol. 2003, 33, 371–378.

435

26. Machado, M. D.; Santos, M. S. F.; Gouveia, C.; Soares, H. M. M.; Soares, E. V. Removal

436

of heavy metals using a brewer’s yeast strain of Saccharomyces cerevisiae: the flocculation as

437

a separation process. Bioresour. Technol. 2008, 99, 2107–2115.

438

27. Machado, M. D.; Janssens, S.; Soares, H.; Soares, E. V. Removal of heavy metals using a

439

brewer’s yeast strain of Saccharomyces cerevisiae: advantages of using dead biomass. J.

440

Appl. Microbiol. 2009, 106, 1792–1804.

19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 32

441

28. Pothoulakis, C. Review article: anti-inflammatory mechanisms of action of

442

Saccharomyces boulardii. Aliment. Pharmacol. Ther. 2009, 30, 826–833.

443

29. Cabañero, A. I.; Madrid, Y.; Cámara, C. Mercury–selenium species ratio in representative

444

fish samples and their bioaccessibility by an in vitro digestion method. Biol. Trace Elem. Res.

445

2007, 119, 195–211.

446

30. Oyetayo, F. L.; Akindahunsi, A. A.; Oyetayo, V. O. Chemical profile and amino acids

447

composition of edible mushrooms Pleurotus sajor-caju. Nutr. Health 2007, 18, 383–389.

448

31. Al-Enazi, M. M.; El-Bahrawy, A. Z.; El-Khateeb, M. A. In vivo evaluation of the proteins

449

in the cultivated mushrooms. J. Nutr. Food Sci. 2012, 2, 176–180.

450

32. Dutta, A.; Zhou, L.; Castillo-Araiza, C.; De Herdt, E. Cadmium(II), lead(II), and

451

copper(II) biosorption on baker’s yeast (Saccharomyces cerevisiae). J. Environ. Eng. 2015,

452

142, 10.1061.

453

33. Desideri, A.; Hartmann, H. J.; Morante, S.; Weser, U. An EXAFS study of the copper

454

accumulated by yeast cells. Biol. Met. 1990, 3, 45–47.

455

34. Clarkson, T. W.; Magos, L. The toxicology of mercury and its chemical compounds. Crit.

456

Rev. Toxicol. 2006, 36, 609–662.

457

35. Myneni, S. C. B. Soft X-ray spectroscopy and spectromicroscopy studies of organic

458

molecules in the environment. Rev. Mineral. Geochem. 2002, 49, 485–579.

459

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FIGURE CAPTIONS

461

Figure 1. Effect of temperature upon the retention of Hg(II) and CH3Hg (1 mg/L) by S.

462

cerevisiae laboratory strain BY4741 (4 OD/mL) following an incubation period of 120 min.

463

Values expressed as ng of mercury (mean ± SD, n=3). Asterisks indicate statistically

464

significant differences in yeast retention at 37ºC with respect to 30ºC (p ≤ 0.05).

465

Figure 2. Effect of incubation time upon the retention of Hg(II) and CH3Hg (1 mg/L) by S.

466

cerevisiae strain BY4741 (4 OD/mL) at 37ºC. Values expressed as ng of mercury (mean ±

467

SD, n=3).

468

Figure 3. Yeast mercury retention after subjecting the aqueous standard solutions of Hg(II)

469

and CH3Hg (1 mg/L) with S. cerevisiae strain BY4741 (4 OD/mL) to gastrointestinal

470

digestion. Values expressed as ng of mercury (mean ± SD, n=3).

471

Figure 4. Bioaccessible mercury content after subjecting samples of swordfish (Xiphias

472

gladius), mushrooms (Amanita cesarea and Boletus edulis) and tuna (Thunnus albacares) to

473

gastrointestinal digestion in the absence or presence of S. cerevisiae strain BY4741 (4

474

OD/mL). Values expressed as ng of mercury (mean ± SD, n=3). Asterisks indicate

475

statistically significant differences with respect to digestion without yeast (p ≤ 0.05).

476

Figure 5. Mercury retention by S. cerevisiae strain BY4741 (4 OD/mL) during the digestion

477

of standard solutions of Hg(II) and CH3Hg (1 mg/L) in the absence or presence of cysteine

478

(Cys, 5 mg/L) or albumin (BSA, 5 mg/L). Values expressed as ng of mercury (mean ± SD,

479

n=3). Asterisks indicate statistically significant differences with respect to digestion without

480

BSA and Cys (p ≤ 0.05).

481

Figure 6. Mercury retention by S. cerevisiae strain BY4741 (4 OD/mL) during the incubation

482

of standard solutions of Hg(II) and CH3Hg (1 mg/L) in the absence or presence of Ca(II) (100

483

mg/L), Fe(II) (40 mg/L) and Cu(II) (40 mg/L). Values expressed as ng of mercury (mean ± 21

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484

SD, n=3). Asterisks statistically significant differences with respect to incubation without

485

cations (p ≤ 0.05).

486

Figure 7. Soluble mercury contents after two hours of incubation at 37ºC of different strains

487

of S. cerevisiae (4 OD/mL) with a 1 mg/L solution of Hg(II) (Figure 7a) or CH3Hg (Figure

488

7b). Values expressed as ng of mercury (mean ± SD, n=3). Asterisks indicate statistically

489

significant differences with respect to incubation without yeast (p ≤ 0.05). See Table 1 for a

490

description of the yeast strains used.

491

Figure 8. Bioaccessible mercury content after subjecting the mushroom samples (Tricholoma

492

georgii) to gastrointestinal digestion in the absence or presence of different strains of S.

493

cerevisiae (4 OD/mL). Values expressed as ng of mercury/g of sample (mean ± SD, n=3).

494

Asterisks indicate statistically significant differences with respect to digestion without yeast

495

(p



0.05).

See

Table

1

for

a

description

of

the

yeast

strains

used.

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Table 1. Saccharomyces cerevisiae strains used in the study.

Strain number

Name

Characteristics

1

BY4741

Haploid laboratory

2

BY4743

Diploid laboratory

3

T73

Wine

4

VRB

Wine

5

Ultralevura

Dietetic

6

YPS128

American wild

7

UWOPS03-461.4

Malaysian wild

8

Kyokai 6

Sake

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Figure 1.

12000

Hg standard Yeast retention at 30ºC Yeast retention at 37ºC

Hg contents (ng)

10000

*

8000

*

6000

4000

2000

0 Hg(II)

CH3Hg

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Figure 2.

9000

Retention of Hg by yeast (ng)

8000

7000

6000 Hg (II) CH3Hg

5000

4000

3000 30

60

120

Time (min)

25

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Figure 3.

25000

Hg standard Yeast retention after digestion

Hg contents (ng)

20000

15000

10000

5000

0

Hg(II)

CH3Hg

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Figure 4.

w/o yeast w/ yeast

Bioaccessible Hg contents (ng)

500

400

300

200

* 100

*

0 s ladiu ias g h ip X

ares dulis area alb ac tus e a ces s it u n n a Bole Am Thun

27

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Figure 5.

100 w/o BSA or Cys BSA Cys

Yeast retention (ng)

80

60

* 40

20

*

* *

0

Hg(II)

CH3Hg

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Figure 6.

120 w/o cations Ca(II) Fe(II) Cu(II)

Yeast retention (ng)

100

80

* 60

*

40

20

0 Hg(II)

CH3Hg

29

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Figure 7.

10000

7a

Contents of soluble Hg (ng)

8000

6000

4000

* 2000

*

*

*

* *

*

* 8 ra in

7 St

St

ra in

ra in

6

5 St

St

ra in

ra in

4

3 St

St

St

ra in

ra in

2

1 ra in St

w/ o

ye as t

0

10000

7b

Contents of soluble Hg (ng)

8000

6000

4000

* *

2000

*

* *

* *

*

0 w/o

t 8 7 6 5 4 3 2 1 as ain ain ain ain ain ain ain ain ye St r St r St r St r St r St r St r St r

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Figure 8.

Bioaccessible Hg contents (ng/g)

500

400

300

*

*

200

* 100

0 w/o yeast

Strain 3

Strain 4

Strain 5

31

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Reduction of Hg

500

Food samples

bioaccessibility B ioacc essib le Hg contents (ng/g )

Hg

Page 32 of 32

+ S. cerevisiae Gastrointestinal digestion

400

300

200

100

0

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w/o yeast w/o yeast

Strain 3

Strain 4 w/ yeast

Strain 5