Impact of in Vitro Gastrointestinal Digestion and Transepithelial

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Impact of in Vitro Gastrointestinal Digestion and Transepithelial Transport on Antioxidant and ACE-Inhibitory Activities of Brewer´s Spent Yeast Autolysate Elsa F. Vieira, Jose das Neves, Rui Vitorino, Diana Dias da Silva, Helena Carmo, and Isabel M.P.L.V.O. Ferreira J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02719 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

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Journal of Agricultural and Food Chemistry

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Impact of in Vitro Gastrointestinal Digestion and Transepithelial Transport on

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Antioxidant and ACE-Inhibitory Activities of Brewer´s Spent Yeast Autolysate

3 4

Elsa F. Vieira1, José das Neves2,3,4, Rui Vitorino5,6, Diana Dias da Silva7, Helena

5

Carmo7, Isabel MPLVO. Ferreira1*

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1

8

Bromatologia e Hidrologia, Faculdade de Farmácia, Universidade do Porto, 4050-313

9

Porto, Portugal

LAQV/REQUIMTE, Departamento de Ciências Químicas, Laboratório de

10

2

11

135, Porto, Portugal

12

3

13

Portugal

14

4

15

Saúde & Instituto Universitário de Ciências da Saúde, 4585-116, Gandra, Portugal

16

5

iBiMED – Instituto de Biomedicina da Universidade de Aveiro, Aveiro, Portugal

17

6

Departamento de Fisiologia e Cirurgia Cardiotorácica da Faculdade de Medicina da

18

Universidade do Porto, Portugal

19

7

20

Toxicologia, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo

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Ferreira, 228, 4050-313 Porto, Portugal

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* corresponding author: (Fax: +351226093390; Phone: +351220428642; email:

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[email protected])

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-

INEB – Instituto de Engenharia Biomédica, Universidade do Porto, 4200-135, Porto,

CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da

UCIBIO/REQUIMTE, Departamento de Ciências Biológicas, Laboratório de

ACS Paragon Plus Environment


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Short title: Digestion and Transepithelial Transport of Brewer´s Spent Yeast

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Autolysate

26 27

ABSTRACT

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Brewer’s spent yeast (BSY) autolysates may have potential applications as food

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ingredients or nutraceuticals due to their antioxidant and ACE-inhibitory activities. The

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impact of simulated gastrointestinal (GI) digestion; the interaction with intracellular

31

sources of oxidative stress, the intestinal cell permeability of BSY peptides and the

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antioxidant and ACE-inhibitory activities of BSY permeates were assayed.

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Gastrointestinal digestion of BSY autolysates enhanced antioxidant and ACE-inhibitory

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activities as measured in vitro. No cytotoxic effects were observed on Caco-2 cells after

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exposition to the digested BSY autolysates within a concentration range of 0.5 to 3.0

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mg peptides/mL. A protective role to induced oxidative stress was observed. The

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transepithelial transport assays indicate high apparent permeability coefficient (Papp)

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values for BSY peptides across Caco-2/HT29-MTX cell monolayer (14.5-26.1x10−6

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cm/s) and for Caco-2 cell monolayer model (12.4-20.8x10-6 cm/s), while the antioxidant

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and ACE-inhibitory activities found in fluxes material from the basolateral side suggest

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transepithelial absorption of bioactive compounds.

42 43

Keywords: Brewer’s spent yeast, gastrointestinal digestion, transephitelial transport,

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ACE-inhibitory activity, antioxidant activity


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INTRODUCTION

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Brewer´s spent yeast (BSY), the second major by-product from brewing, is

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currently used as a low-valuable animal feed product.1,2 However, it is assigned GRAS

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(Generally Recognized as Safe) status and can be valorized through the production of

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added value new functional food ingredients, such as β‐glucans from cell wall3 and

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nucleotides.4

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BSY extracts can be obtained by autolysis, plasmolysis and hydrolysis although

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the most frequent is autolysis.5 The production of BSY autolysates usually involves

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incubation of cell suspensions of BSY at temperatures ranging from 45 to 60°C with a

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reaction time between 8 and 72 h.5-7 The autolysis of intracellular content by native

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proteases after removal of BSY cell walls under refrigerated conditions is advantageous

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8

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can be used to obtain peptide rich autolysates. The antioxidant and ACE-I activities of

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BSY autolysates7 foresee potential applications as food or nutraceutical ingredients.

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However, the challenge before in vivo efficacy studies is to understand the impact of

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simulated gastrointestinal (GI) digestion; intestinal cell permeability and interaction

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with intracellular sources of oxidative stress.

, since mild autolysis conditions with lower temperatures and reduced autolysis time

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The human colorectal adenocarcinoma Caco-2 cell line has been widely used to

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evaluate the cytotoxicity of bioactive compounds at concentrations used to exert the

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desired bioactivity in the body, as well as, to study the potential for inhibiting

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intracellular oxidation.9 Moreover, Caco-2 cell line has been used as in vitro model of

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absorptive enterocytes to evaluate the permeability of drugs and food compounds.9,10,11

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However, only few studies addressed the intestinal transport of complex mixtures of

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peptides.11-13 Furthermore, to the best of our knowledge this is the first study in which



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the permeability of a BSY autolysate is performed. The Caco-2 cell monolayers exhibit

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spontaneous enterocyte-like differentiation under standard culture conditions, showing

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morphological polarity and expression of brush-border hydrolases, transporters,

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microvilli and tight junctions, thus mimicking the main characteristics of the human

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small intestine.9,11,14 Nevertheless, this model presents limitations since besides the

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absorptive enterocytes (80%) the human intestinal epithelial includes other types of

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cells, such as enteroendocrine, goblet and paneth cells. Another disadvantage of the

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Caco-2 cell line is that the tightness of the monolayer resembles that of the colon and

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not the small intestine, therefore paracellular transport can be underestimated.15 To

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overcome these limitations, co-cultures of Caco-2 and mucus-secreting cells (HT29-

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MTX) have been proposed as a more bio-relevant model of the intestinal

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epithelium.10,14,15,16

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The major goals of the present study were to investigate: (i) the effect of in vitro

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GI digestion on antioxidant and ACE-I activity of BSY autolysate and the ability of the

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GI digest to protect Caco-2 cell line regarding viability, mitochondrial integrity and

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oxidative stress, (ii) the resistance to brush-border peptidases and the susceptibility to

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intestinal transepithelial transport of BSY peptides, comparing two cell monolayer

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systems broadly used as models for the small intestine epithelium (Caco-2 and Caco-

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2/HT29-MTX co-culture cell monolayers), and (iii) antioxidant and ACE-inhibitory

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activities of fluxes material from the basolateral side to ascertain transepithelial

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absorption of bioactive compounds from BSY autolysates.

91 92 93 94


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MATERIAL AND METHODS

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Chemicals. Pepsin; pancreatin; commercial angiotensin-I-converting enzyme (ACE, EC

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3.4.15.1, 5.1 U/mg); bovine serum albumin (BSA); trifluoroacetic acid (TFA); 6-

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hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox); dimethyl sulfoxide

100

(DMSO); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the

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Millipore UF membrane 5 kDa were purchased from Sigma-Aldrich (St. Louis, MO).

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The o-aminobenzoylglycyl-p-nitro-phenylalanylproline (o-ABz-Gly-Phe(NO2)-Pro) was

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purchased

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Dulbecco’s Modified Eagle Medium (DMEM), heat-inactivated fetal bovine serum

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(FBS), penicillin/streptomycin, trypsin-EDTA and Hank’s balanced salt solution

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(HBSS, pH 7.0-7.4) were purchased from Invitrogen (Carlsbad, CA). Falcon®

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translucent polyethylene terephthalate (PET) cell culture inserts (3.0 µm pore size, 24

108

mm diameter inserts, 4.2 cm2 effective growth area) were acquired from BD

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Biosciences (Franklin Lakes, NJ, USA) and 6-well and 96-well microplates were

110

purchased from Corning Costar (Sigma-Aldrich, St. Louis, MO).

from

Bachem

Feinchemikalien

(Bubendorf,

Switzerland).

GIBCO

111 112

Cells. Human colon carcinoma (Caco-2) cell line was obtained from the American Type

113

Culture Collection (ATCC) and mucus producing HT29-MTX cell line was kindly

114

provided by Dr. T. Lesuffleur (INSERM U178, Villejuif, France).

115 116

Equipments. The RP-HPLC-UV analyses were carried out using an analytical HPLC

117

system (Jasco, Tokyo, Japan) equipped with a quaternary low pressure gradient HPLC

118

pump (Jasco PU-1580), a degasification unit (Jasco DG-1580-53 3-line degasser), an

119

autosampler (Jasco AS-2057-PLUS), a multiwavelengh detector (Jasco MD-910) and a



120

7125 Rheodyne injector valve (CA, USA). The data acquisition was accomplished using

121

Borwin Controller software, version 1.50 (JMBS Developments, Le Fontanil, France).

122

Mass Spectrometry (MS) data were acquired using a matrix-assisted laser

123

desorption/ionisation time of-flight/time-of-flight - 4800 MALDI-TOF/TOF (Applied

124

Biosystems, Darmstadt, Germany) mass spectrometer in the m/z range of 1000-12000.

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The MS data was processed using Data Explorer 4.8 Software (Applied Biosystems).

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Spectrophotometric analyses were carried out using a SPECTROstar Nano-microplate,

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cuvette UV/Vis absorbance reader (BMG Labtech GmbH, Offenburg, Germany).

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Fluorimetric analyses were carried out using a fluorescence microplate reader

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(FLUOstar Optima, BMG Labtech GmbH,).

130 131

Preparation of autolysates from inner content of Brewer’s spent yeast and in vitro

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gastrointestinal digestion. BSY samples (Saccharomyces pastorianus) used to prepare

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Lager beer of Pilsener type were supplied by Unicer brewing (Leça do Balio, Portugal).

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Disruption of cell wall to extract the inner yeast content was performed according to

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Vieira et al.8 BSY autolysates from the inner cell content, presenting 10 mg/mL of

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proteins determined by the Bradford method 17 and pH 6.0, were submitted to autolysis

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at 36°C, 6 h and filtered using a 5 kDa molecular weight cut-off membrane. To mimic

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digestion in the stomach and upper intestine, in vitro pepsin-pancreatin digestion of

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BSY autolysate from the inner cell content was performed according to the method

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described by Samaranayaka et al.18 The digested BSY autolysate was used for screening

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in vitro antioxidant and ACE-I activity and for RP-HPLC-UV analysis.

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In vitro antioxidant activity. Determination of Ferric ion reducing ability (FRAP) was

144

based on the reduction of the ferric-tripyridyltriazine complex to the ferrous form,


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19

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formation of a blue color measured at 593 nm (kinetic method).

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standard at 10-500 µM to produce the calibration curve. Results were expressed as mean

147

values ± standard deviations of µM of Trolox equivalent per mL (µM TE/mL). Assays

148

were performed in triplicate.

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ACE-inhibitory activity. Measurement was performed in triplicate using the

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fluorimetric assay of Sentandreu & Toldrá 20, with the modifications reported by Quiros

151

et al.21 ACE-I percentage (I) was calculated using the equation:

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I=

153

where B is the fluorescence of the ACE solution without an inhibitor (BSY autolysate);

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A is the fluorescence of the tested sample of BSY autolysate; and C is the fluorescence

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of experimental blank (o-ABz-Gly-Phe(NO2)-Pro dissolved in 150 mM Tris-base buffer

156

(pH 8.3), containing 1.125 M NaCl). IC50 values were measured as the concentration

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required to decrease the ACE activity by 50%.21

X 100

Trolox was used as

(1)

158 159

Cell-based assays

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Cell culture maintenance and generation of cell monolayers. Caco-2 and HT29-

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MTX cell lines at passage 36 and 47, respectively, were used. The two cell lines were

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maintained in DMEM supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100

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µg/mL streptomycin, at 37ºC under 5% CO2, water saturated atmosphere. The medium

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was changed every other day. For cell permeability studies, 90% confluent Caco-2 and

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HT29-MTX cells were harvested using trypsin-EDTA and seeded onto cell culture

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inserts mounted in 6-well plates. Seeding density was 2.8x105 cells/mL for Caco-2 cell

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monolayers and Caco-2/HT29-MTX co-culture model (cell ratio of 90:10), as described

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by Antunes et al.10 The culture medium was allowed to differentiate for 21 days before

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permeability experiments. The integrity of the cell monolayers was checked before and



170

after permeability assays by the Transepithelial Electrical Resistance (TEER) using an

171

epithelial voltammeter (EVOM, World Precision Instrument, Sarasota, FL, USA).

172

According to the literature only monolayers presenting TEER values higher than 200 Ω

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cm2 should be used for Caco-2/HT29-MTX co-culture model 16 whereas for Caco-2 cell

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monolayers over 250 Ω cm2 is considered as a threshold value.22

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Cell viability determination. Caco-2 cell viability was determined using the MTT

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assay.23 Caco-2 cells were seeded onto 96-well plates at a density of 8x104 cells/well.

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After overnight incubation Caco-2 cells were exposed during 24 h (37°C, 5% CO2) to

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the digested BSY samples at six different concentrations: 0.5; 1.0; 2.0; 3.0; 4.0 and 6.0

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mg peptides/mL, which were prepared from freeze-dried digested BSY extracts. A

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negative control (NEGc, cells treated with medium only) and a positive control (POSc,

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cells treated with medium and 0.1% Triton X-100) were also included. The period of 24

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h exposition was selected because although the contact between xenobiotics and

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enterocytes usually ranges between 2–4h, several phenomena may extend the

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permanence of these substances in the gastrointestinal tract (e.g., enterohepatic

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circulation, impaired bowel movements, gastrointestinal pathology, etc.). Therefore, the

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selected time-point represents a conservative worst case approach for evaluating

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toxicity of digested BSY samples. After cell treatment with the test samples, the culture

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medium was aspirated and the attached cells were rinsed with 200 µL HBSS, followed

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by the addition of fresh culture medium containing 0.25 mg/L MTT. After 30 min of

191

incubation (37°C, 5% CO2), the formed intracellular crystals of formazan were

192

dissolved using 100 µL of DMSO and determined by measuring the absorbance at 570

193

nm. Data were obtained from three independent experiments, with each plate containing


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six replicates for each test sample. Cell viability (%) was calculated relative to the

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maximum viability of NEGc, as follows:

196

Cell viability % =

197

where Absorbance of NEGc was used as a measure of the formazan formed in negative

198

control cells and Absorbance of sample as the measure of the formazan formed after

199

sample test exposure.

!"#

$ × 100

(2)

200 201

Mitochondrial integrity determination. Assessment of mitochondrial integrity was

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performed by measuring the tetramethylrhodamine ethyl ester perchlorate (TMRE)

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inclusion, according to Dias da Silva et al. 24 Caco-2 cells at the density of 105 cells/well

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were seeded onto 96-well plates. After 24 h, the media was gently aspirated and the

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cells were incubated with digested BSY autolysate at six different peptide

206

concentrations for 24 h (37°C, 5% CO2). Then, cells were rinsed twice with HBSS and

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incubated at 37ºC with 100 µL of 2 mM TMRE for 30 min. Then, the media was gently

208

aspirated and replaced by 0.2% BSA in HBSS. Fluorescence was measured at 37ºC set

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to 544 nm excitation and 590 nm emission. TMRE mitochondrial inclusion (%) was

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calculated relative to the maximum levels of the NEGc. Data were obtained from three

211

independent experiments, with each plate containing six replicates of each test sample.

212 213

ROS levels production. The intracellular reactive oxygen species (ROS) production

214

was monitored by means of the 2`,7`-dichlorodihydrofluorescein diacetate (DCFH-DA)

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assay, according to Dias da Silva et al.24 DCFH-DA penetrates into cells and is

216

hydrolyzed to DCFH by intracellular esterases; the presence of ROS can oxidize DCFH

217

to form DCF, a fluorescent product.

218

density 105 cells/well were seeded onto 96-well plates and allowed to attach for 24 h.

24

For this determination, Caco-2 cells at the



219

On the day of the experiment, the differentiated cells were rinsed with HBSS and

220

incubated with 200 µL per well of digested BSY autolysate at six different peptide

221

concentrations for 24 h (37°C, 5% CO2). A negative control (NEGc, cells treated with

222

medium only) was also included. After the treatment period, cells were rinsed twice

223

with HBSS and incubated with 10 mM DCFH-DA for 30 min (37°C, 5% CO2). After

224

removal of the DCFH-DA and further washing with HBSS, the formation of 2`,7`-

225

dichlorodihydrofluorescein (DCF), due to the oxidation of DCFH-DA in the presence of

226

intracellular ROS, was measured at an excitation wavelength of 485 nm and an

227

emission wavelength of 530 nm. Cell ROS production (%) was calculated relative to the

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maximum ROS levels of NEGc. Data were obtained from three independent

229

experiments, with each plate containing six replicates of each test sample.

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In parallel, the cellular protection of digested BSY autloysate against a cytotoxic agent

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was induced by addition of 1 mM hydrogen peroxide. For this purpose, cells were also

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submitted to the 24 h treatment period, but in this case, 1 mM hydrogen peroxide was

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added 6 h before ROS measurement. Cell ROS production (%) was calculated relative

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to the maximum ROS levels of positive control (POSc, cells treated with hydrogen

235

peroxide).

236 237

Permeability experiments. Cell monolayers were pre-equilibrated with fresh HBSS

238

solution, at 37°C, for 30 min. The apical (donor) compartment was filled with 1.5 mL of

239

digested BSY autolysate in HBSS to a final concentration of 3.0 mg peptides/mL and

240

basolateral (receptor) compartment was filled with 2.5 mL of HBSS. Cell monolayers

241

were allowed to incubate at 37°C under 5% CO2 and 95% relative humidity. Samples

242

(1.0 mL) were collected from the basolateral side at 15, 30, 60, 120 and 180 minutes to

243

analyze the peptides transported across cell monolayers. Fresh HBSS was added in


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order to complete the initial volume at the basolateral compartment. TEER values were

245

also determined at the end of the experiment after monolayer washing with HBSS. A

246

control sample, containing only HBSS and no sample solutions was included in the

247

experimental setup. After the permeability experiments, the action of cell proteases on

248

collected permeates was immediately stopped by cold at -90ºC and freeze-dried. In

249

order to perform the antioxidant and in vitro ACE-I assays, freeze-dried permeates were

250

dissolved in HBSS (20% the original volume added to apical side). For

251

chromatographic analysis, freeze-dried permeates were dissolved to the original volume

252

(added to apical side) in solvent A (0.1% TFA in water). The efficiency of peptide

253

transport, expressed as percentage of permeability for each peak (P%), was calculated

254

according to Cinq-Mars et al.13 as follows:

255

P% = .( X 100

256

where Ab and Aa correspond, respectively, to the RP-HPLC peak areas of the peptide

257

fraction detected in the basolateral and in the apical side and V (mL) is the volume of

258

sample loaded in the apical side. In addition to P(%), the apparent permeation

259

coefficient (Papp), expressed in cm/s, was calculated according to Ferraro et al.25 from

260

the following equation:

261

)app = ..,

262

where Q is the total amount of permeated peptides during the 180 minutes of the

263

transport experiment (mg), A is the diffusion area (4.2 cm2), C is the apical

264

compartment concentration at time zero (mg/mL), and t is the time of the experiment(s).

+

(3)

(4)

265 266

Reverse-phase HPLC-UV and MALDI-TOF/TOF. The RP-HPLC-UV analysis of

267

digested BSY autolysate (0 min) and respective permeates (15, 30, 60, 120 and 180



268

min) from Caco-2 and Caco-2/HT29-MTX co-culture cell monolayers permeability

269

assays was performed according to the method described by Ferreira el al.26

270

Digested BSY autolysate and five-fold concentrated permeates from Caco-2/HT29-

271

MTX cell monolayers were analyzed using matrix-assisted laser desorption/ionisation

272

time of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry to determine the

273

molecular mass of the peptide mixture. Samples were cleaned with ZipTip C18

274

(Millipore) using the manufacturer’ instructions. Then, the eluted samples were

275

premixed with matrix (3 mg/mL alpha-cyano-4-hydroxycinnamic acid (CHCA) in 50%

276

(v/v) aqueous acetonitrile, 0.1% TFA), spotted onto a target plate, and dried at room

277

temperature. The 4800 MALDI-TOF/TOF was calibrated using horse myoglobine (m/z

278

16952.56 (+1); m/z 8476.78 (+2); m/z 5651.85 (+3)) and cytochrome c (m/z 12349.72

279

(+1); m/z 6177.94 (+2)).

280 281

Statistical analysis. The student t-test was performed to compare the permeability

282

profile between the two cell models; one-way analysis of variance (ANOVA) was

283

carried out for comparing multiple samples, followed by Duncan’s post-hoc test.

284

Differences with p

Impact of in Vitro Gastrointestinal Digestion and Transepithelial

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