Influence of Fermentation with Different Lactic Acid Bacteria and in

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Influence of fermentation with different lactic acid bacteria and in vitro digestion on the biotransformation of phenolic compounds in fermented pomegranate juices Estefanía Valero-Cases, Nallely Nuncio-Jáuregui, and Maria Jose Frutos J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04854 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 10, 2017

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Journal of Agricultural and Food Chemistry

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Influence of fermentation with different lactic acid bacteria and in vitro digestion on

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the biotransformation of phenolic compounds in fermented pomegranate juices

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Estefanía Valero-Casesa, Nallely Nuncio-Jáureguia, María José Frutosa*

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Research Group on Food Quality and Safety. Food Technology Department, Miguel

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Hernandez University. Ctra. Beniel, km 3.2, 03312-Orihuela, Alicante, Spain

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*Corresponding author: María José Frutos. Tel.: +34966749744; Fax: +34966749677. E-

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mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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This study describes the effect of fermentation and the impact of simulated gastrointestinal

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digestion (SGD) of four fermented pomegranate juices with different lactic acid bacteria

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(LAB) on the biotransformation of phenolic compounds. The changes of the antioxidant

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capacity (AOC) and of LAB growth and survival in different fermented juices were also

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studied. Two new phenolic derivatives (catechin and α-punicalagin) were identified only in

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fermented juices. During SGD, the AOC increased together with the phenolic derivatives

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concentration mainly in the juices fermented with Lactobacillus. These derivatives were

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formed due to the LAB metabolism of the ellagitannins, epicatechin and catechin after

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fermentation and during SGD. The FRAP assay performance might be associated with the

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degradation and biotransformation of catechin. The fermented pomegranate juices with

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these LABs increased the bioaccessibility of phenolic compounds ensuring the survival of

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LAB after SGD, suggesting a possible prebiotic effect of phenolic compounds on LAB.

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Keywords: Lactobacillus, Bifidobacterium, antioxidant capacity, probiotic, bioactive

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compounds.

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INTRODUCTION

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The polyphenols are part of our diet and exert antioxidant properties; the main dietary

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sources are wine, fruit, juices, leguminous and vegetables.1 The bioaccessibility of some

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polyphenols is very low because of their degree of polymerization and glycosylation

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pattern.2 Therefore, their physiological benefits depend on the quantity of phenolic

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compounds that are available (bioavailability) to be absorbed in the intestine.3,

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suggests that a large rate of phenolic compounds reaches the colon and due to the action of

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gut microbiota, these compounds are transformed, leading to the production of metabolites

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that can be absorbed producing a physiological effect.2, 5

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Curiously, everybody has an individual microbiota composition, so that the bioavailability

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of polyphenols for the production of microbial metabolites is subjected to inter-individual

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variability. Therefore, the health effects are not the same for everyone.2, 6, 7

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In recent years, pomegranate juice has been investigated due to its beneficial properties

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because of its elevated concentration of polyphenols.8 Those polyphenols include mainly

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anthocyanins, procyanidins, phenolic acids, flavonol glycosides and hydrolysable tannins

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such as ellagitannins, gallotannins and punicalagins.9 However, humans cannot absorb

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those hydrolysable tannins and therefore, these compounds are hydrolyzed to ellagic acid

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that is also poorly absorbed being metabolized in the colon by the microbiota to produce

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urolithins.10 The urolithins together with other phenolic metabolites are mainly responsible

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for the health properties.9 The capacity of the lactic acid bacteria to metabolize the phenolic

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compounds depends on the species or on the strain.11,

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polyphenols can occur during a food fermentation process by lactic acid bacteria.12-14 Those

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bacteria are the main source of probiotics which are “live microorganisms that, when

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administered in adequate amounts confer health benefits on the host”.15 Although the health

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4

This

The microbial conversion of


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beneficial effects due to Lactobacillus and Bifidobacterium have been shown, their function

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as antioxidants has not been fully investigated.16

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The aim of this study was to investigate the biotransformation of the phenolic compounds

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in pomegranate juices (a) after fermentation by four lactic acid bacteria and (b) during in

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vitro digestion of unfermented and fermented juices. The antioxidant properties and the

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LAB survival were also investigated in different fermented pomegranate juices to study the

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influence of fermentation and in vitro digestion on the polyphenols biotransformations.

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MATERIALS AND METHODS

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Activated bacterial strains and culture preparations

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Lactobacillus acidophilus CECT 903 (LA), Lactobacillus plantarum CECT 220 (LP),

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Bifidobacterium longum subsp. infantis CECT 4551 (BL), Bifidobacterium bifidum CECT

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870 (BB), were purchased from the Spanish Type Culture Collection (CECT, Valencia,

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Spain) in lyophilized form. To obtain the pre-inoculum, each strain was re-suspended in 10

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mL of Man Rogosa Sharpe (MRS) broth (Oxoid; Madrid, Spain) at 37 °C during 24 hours

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under aerobic conditions to Lactobacillus strains and 48 h under anaerobic conditions to

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Bifidobacterium strains. After this time, to obtain an initial biomass of about 8 Log Colony

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Forming Units per mL (CFU/mL), 1 mL of each pre-inoculum was inoculated in 100 mL of

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MRS broth and incubated 24-48 h at 37 ºC. The cultures were separated by centrifugation

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at 2000 × g for 10 min at 4 °C and washed twice with sterile phosphate buffer saline (PBS)

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and stored with glycerol at -80 ºC until used.

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Fermented pomegranate juices

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Pomegranate juices in aseptic bags of 4.5 Kg were provided by Probelte Biotecnología,

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S.L. (Murcia, Spain). According to the supplier´s certificate of analysis, the pomegranate

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juice had 14.9% of soluble solids content, pH 3.7, an acidity of 0.31 g citric acid/100 mL,


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as well as compliance with the microbiological criteria specified (total plate count, yeast

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and mold, Salmonella sp. and Escherichia coli).

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The pomegranate juices were put into sterile borosilicate glass bottles (250 mL) with

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polypropylene screw caps. Each pomegranate juice bottle (250 mL) was inoculated with

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1% (v/v) of individual LAB previously prepared and incubated at 37 °C for 24 h to obtain

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four different fermented pomegranate juices (FPJ) with an initial concentration of 6 Log

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CFU/mL: FPJ with Bifidobacterium bifidum (FPJBB), FPJ with Lactobacillus plantarum

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(FPJLP), FPJ with Bifidobacterium longum subsp. infantis (FPJBL) and FPJ with

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Lactobacillus acidophilus (FPJLA). The fermented juices were compared with two

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different non-fermented controls: control juices with incubated at 37 ºC (CPJI) or stored

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under refrigerated conditions at 4 ºC (CPJR). The fermented pomegranate juices were

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analyzed after the incubation period together with the unfermented control juices.

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Simulated gastrointestinal digestion

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The in vitro digestion assay was carried out according to Valero-Cases and Frutos17 with

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some modifications. For the assays, 100 mL of each juice was subjected to simulated

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gastrointestinal digestion for 180 min at 37 °C under stirring. The simulated gastric juices

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(SGJ) were prepared with 400 mL of PBS to pH 3 with 1M HCl (Panreac; Barcelona,

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Spain) and pepsin was added to reach a concentration of 3 g/L (Oxoid; Madrid, Spain).

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After the 60 min of gastric digestion, the simulated intestinal juices (SIJ) were prepared

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increasing the pH to7 with 1 M NaHCO3 (Panreac; Barcelona, Spain) and adding 4.5 g/L of

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bile salts and 1 g/L of pancreatin (Sigma; Madrid, Spain) and they were incubated during

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120 min at 37 ºC. Samples were taken in each of the fermented and control juices after

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incubation time and at the different stages of in vitro digestion: after 60 min of SGJ, after

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60 min of SIJ (SIJ1) and after 120 min of SIJ (SIJ2).



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Microbiological analysis

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The growth and survival of LAB after fermentation and during in vitro digestion were

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determined by plate count. Samples (10 mL) of each FPJ were taken after fermentation and

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after different steps of in vitro digestion (SGJ, SIJ1 and SIJ2). Suitable dilutions (0.1 mL)

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were spread in triplicate on MRS agar plates and incubated for 24-48 h at 37 ºC under

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aerobic and anaerobic conditions depending on the LAB conditions. The results were

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expressed as Log CFU/mL of fermented pomegranate juice or SGJ or SIJ.

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Extract preparation

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For the determination of the phenolic compounds and the antioxidant activity after the

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incubation time, the pomegranate juices extracts were prepared as previously described by

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Nuncio-Jáuregui, et al.18 with least modifications: all different juices (5 mL) were mixed

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with 10 mL of MeOH/water (75:25 v/v) with 0.1 % HCl using an Utra-Turrax (T25, IKA,

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China) for 5 min and centrifuged at 15000 x g for 15 min at 4 °C. The control samples

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stored at 4 ºC followed the same extraction process. The supernatants were filtered

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(0.45 µm, Millipore; Spain) and stored at -80 ºC until analysis. Samples from in vitro

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gastrointestinal digestion (10 mL) were taken directly at every digestion step and

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centrifuged and stored following the same procedure described above.

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HPLC-DAD Analysis (Identification and quantification of phenolic compounds)

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The identification and quantification of phenolic compounds was done following the

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Robles-Sánchez, et al.19 method with some adaptations on the binary gradient elution

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system. High performance liquid chromatography studies were performed using an Agilent

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1200 series HPLC system (Agilent Technologies, Waldbronn, Germany) coupled with a

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diode array detector (DAD) equipped with a reversed-phase column C18 Waters Spherisorb

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ODS-1 (250 mm × 4.6 mm, 5 µm particle size, Mediterranea SEA18; Teknokroma S.C.L.,


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Barcelona, Spain). A binary gradient elution system was composed of solvent A: deionized

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water with 1% formic acid and solvent B: acetonitrile with 1% formic acid. The elution

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profile was as follows: 95% (A) at 0 min, 87% (A) at 15 min, 85% (A) at 20 min, 70% (A)

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at 25 min and continued isocratically for 3 min, then changing to 55% (A) at 32 min and

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continued isocratically for 3 min, then changing to 10% (A) at 40 min and continued

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isocratically for 5 min, changing to 95% (A) at 60 min. The flow rate was 1 mL/min with

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an injection volume of 20 µL and the temperature of the column was kept at 30 °C.

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Different pure standards: ellagic acid, α–punicalagin, β-punicalagin, punicalin, catechin,

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epicatechin and galic acid (Sigma; Madrid, Spain) were used to prepare different

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calibration curves. The standards were dissolved in MeOH/water (75:25 v/v) and acidified

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with 1 % HCl. The chromatograms were carried out simultaneously at 260, 280, 320, 360

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or 520 nm. The identification of phenolic compounds was carried out by comparing the

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retention time and UV absorption spectra with those of the standards, and quantified using

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calibration curves of the standards. The microbial metabolites such as ellagic acid

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derivative, ɑ-punicalagin derivative and catechin derivative were identified according to the

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literature20,

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tentatively quantified using the calibration curves of ellagic acid, ɑ-punicalagin and

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catechin. All determinations were made in triplicate and the results were expressed as

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mg/100 mL.

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Antioxidant capacity determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free

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radical scavenging method

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The free radical scavenging activity was determined using the DPPH (radical 2,2-diphenyl-

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1-picrylhydrazyl) method adopted from Brand-Williams, et al.22 Each supernatant (10 µL)

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was mixed with 40 µL of MeOH and added to 950 µL of DPPH solution. The mixture was

21

by comparing the UV absorption spectrum with those of the standardsand



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shaken and kept during 10 min in a dark room. The absorbance decrease was measured at

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515 nm in an UV–Vis Uvikon XS spectrophotometer (Bio-Tek Instruments, Saint Quentin

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Yvelines, France). The calibration curve was of the form y = 0.2443x + 0.0047 (R2 = 0.999)

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and was made using Trolox as standard solution in the range 0.01-5.00 mmol/ L. The

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analyses were run in three replications and the results were expressed as mmol Trolox/ L of

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juice.

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Antioxidant capacity determined by ferric reducing antioxidant power (FRAP)

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method

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The ferric reducing antioxidant power (FRAP) method was employed adopted fromBenzie

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and Strain.23 Briefly, the FRAP reagent was prepared fresh daily by mixing 300 mmol/L

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acetate buffer (pH 3.6), 10 mmol/L TPTZ solution, in 40 mmol/L HCl and 20 mmol/L

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FeCl3·6H2O solution in a volume ratio of 10:1:1, respectively. For the assays, 10 µL of

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each extract was mixed with 990 µL of FRAP and kept in a dark room for 10 min at 37 ºC.

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Then, the absorbance was measured at 593 nm. The calibration curve was of the form y =

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0.4043x + 0.0626 (R2 = 0.998) using Trolox as standard solution in the range 0.01-5.00

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mmol/ L. The analyses were run in three replications and results were expressed as mmol

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Trolox/ L of juice.

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Antioxidant

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ethylbenzothiazoline-6-sulphonic acid) scavenging method

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The

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method was measured adopted from the method developed by Re, et al.24 Briefly, the

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ABTS

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allowed to react for 12-16 h in the dark at room temperature. The solution was then diluted

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with PBS at pH 7.4 to an absorbance of 0.70 + 0.02 at 734 nm. For the assays, 10 µL of

capacity

determined

by

the

ABTS

radical

ABTS[2,2´-azino-bis-(3-ethylbenzothiazoline-6-sulphonic

+

acid)]

2,2´-azino-bis-(3-

radical

cation

was prepared mixing ABTS 7 mM with K2S2O8 2.45 mM and this mixture was


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each extract was mixed with 990 µL of ABTS and kept in a dark room for 10 min. The

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calibration curve was of the form y = 0.2238x + 0.0322 (R2 = 0.991) using Trolox as

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standard solution in the range 0.01-5.00 mmol/ L. The analyses were run in three

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replications and the results were expressed as mmol Trolox/ L of juice.

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Statistical analysis

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All the experiments and analyses were performed in triplicate. The results were expressed

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as mean ± standard deviation. The mean comparison was performed via SPSS v 21.0

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software package (SPSS Inc., Chicago-Illinois-USA) using analysis of variance (ANOVA)

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followed by a Tukey multiple range test to evaluate the significant differences (p < 0.05).

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RESULTS AND DISCUSSION

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Biotransformation of phenolic compounds in fermented pomegranate juices after 24

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hours of incubation

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Figure 1 and 2 shows the biotransformation of phenolic compounds by different lactic acid

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bacteria in pomegranate juices in relation with the control juices (CPJR and CPJI). In the

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control juices stored at different temperatures (4 ºC and 37 ºC), 8 different phenolic

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compounds were identified, catechin, α and β punicalagin, punicalin, epicatechin, gallic

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acid, ellagic acid derivative and ellagic acid. To our knowledge, all these phenolic

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compounds have been reported in pomegranate juices in previous studies.21, 25-27 Most of

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the initial phenolic compound values did not show significant differences between the

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control samples stored at different temperatures (4 and 37 ºC, CPJR and CPJI,

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respectively). However, it seems that when the control juices were stored at 37 ºC, the

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catechin content was lower with respect to the initial concentration in control juices stored

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at 4 ºC (130.87 vs 173.42 mg/100 mL, respectively) (Figure 1). At the same time, the

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microbial fermentation had an increase in the levels of the phenolic compounds; in the



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different experimental fermented juices, a total of 9 compounds were identified in this

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study after fermentation (37 ºC during 24 h). Eight of the phenolic compounds were the

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same as the control juices. However, a new compound was found only in fermented

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pomegranate juices. This new phenolic metabolite was a new catechin derivative identified

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after the fermentation (by comparison of the spectral data with the standard) with respect to

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the control juices (Figure 1). Epicatechin was almost completely metabolized (only traces

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were obtained) by BB, LP & LA and in a lesser extent by BL (3.59 mg/100 mL) (Figure 1).

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Catechin was completely degraded by BB and LA (only traces were obtained) and LP and

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BL in a lesser extent (15.30 and 90.28 mg/100 mL were obtained, respectively). However,

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in control juices (CPJR and CPJI), the amount of epicatechin and catechin was higher than

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in the fermented juices (ca. 48 and 130 mg/100 mL, respectively) (Figure 1). Therefore, this

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microbial-derived catechin could be synthesized from the metabolism of other phenolic

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compounds (epicatechin and catechin) during bacterial fermentation, due to the different

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concentrations in relation with the control samples. These results are in agreement with

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Alberto, Gómez-Cordovés and Manca de Nadra20 who reported the identification of

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intermediate metabolites from catechin due to Lactobacillus hilgardii fermentation.

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Otherwise, a decrease of ca. 40% in the ellagic acid derivative in FPJBB, FPJLB and

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FPJLP was observed with respect to the control juices (Figure 2). Nevertheless, in FPJLA

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the decrease was 70% higher with respect to the unfermented juices.

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On the other hand, with respect to the β-punicalagin and α-punicalagin

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concentrations, in the pomegranate juices fermented by Lactobacillus (FPJLP and FPJLA)

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the concentrations of β and α-punicalagin were lower than in the juices fermented by

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Bifidobacterium strains (FPJBB and FPJBL) (Figure 2). However, the punicalin remained

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without significant differences between all fermented juices while the gallic acid


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concentrations in FPJBL were the lowest (12.94 mg/100 mL). The free ellagic acid

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concentrations in control and fermented juices did not change during incubation time and

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hence did not present significant differences between juices (Figure 2). The low

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biotransformation of ellagic acid in this step might be due to its insolubility in aqueous

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media especially at low pH.28,

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fermented only by different Lactobacillus strains also showed the biotransformation of

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phenolic compounds during fermentation.12

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Previous studies with cherry juice and broccoli puree

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At the same time, the number of viable cells was determined (Table 1) to study the

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relationship between the viable cells concentration and the biotransformation of phenolic

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compounds. In spite of pomegranate juices being a hostile ecosystem (pH, buffering

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capacity) and needing a long time for fermentation for the growth of LAB,13 in the present

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study all strains increased from 6 Log CFU/mL to 7.26-7.78 Log CFU/mL without

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significant differences (p > 0.05) among the LAB used. This growth increase could be in

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relation with the metabolism of most of the pomegranate phenolic compounds in a greater

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or lesser extent, depending on the strain as stated before. Previous studies in pomegranate

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juice fermented by LAB showed the same increase after 120 h at 30 ºC (from ca. 7.0 to 7.3

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Log CFU/mL) and a higher one (8 Log CFU/mL) after 72 h at 37 ºC.21-30 Hence, in this

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study a good growth was observed with different LAB and lower fermentation times (24 h

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at 37 ºC).

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Metabolism and biotransformation of phenolic compounds in fermented pomegranate

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juices after gastric digestion

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The phenolic compounds concentrations were measured with the aim of testing their

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stability after 60 minutes in SGJ in the different juices (Figure 1 and 2). The SGJ

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conditions promoted the degradation of the epicatechin in the control juices with an



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increase (ca. 30%) in the catechin concentration in these juices (Figure 1). With respect to

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the fermented juices, the α- and β-punicalagins concentrations increased (ca. 25% and 30%,

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respectively) in the juices fermented by Lactobacillus (FPJLA and FPJLP), and only in

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FPJLA it was found that the gallic acid was fully metabolized by LA after gastric digestion.

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The FPJBB presented the lowest concentrations of these compounds (56.00 and 60.92

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mg/100 mL respectively). A decrease in the concentration of ellagic acid (ca. 30%) in the

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juices fermented by Bifidobacterium (FPJBB and FPJBL) was also observed (Figure 2). At

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the same time, the survival in Bifidobacterium FPJs was higher than in the juices fermented

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by Lactobacillus, with concentrations of 7.40 and 7.18 Log CFU/mL for BB and BL,

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respectively (Table 1). These survival differences could be due to a higher metabolism of

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phenolic compounds by Bifidobacterium strains during SGJ (Figure 1 and 2). In other

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studies with apples and blackberries phenolic compounds such as flavonoids, lignans and

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phenolic acids were stable to the gastric conditions.31,

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dried figs and pomegranate extracts, a slight decrease in such compounds was observed.5, 33

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It has to be pointed out that none of the previous studies was performed with fermented

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food matrices.

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Metabolism and biotransformation of phenolic compounds in fermented pomegranate

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juices during and after intestinal digestion

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The different phenolic compounds were measured in all juices after 60 min (SIJ1) and after

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120 min of intestinal digestion (SIJ2) to study and compare their metabolism and stability

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with AF and SGJ (Figure 1 and 2). After SIJ1 an increase of ellagic acid in all juices was

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observed, with amounts three times higher than those found after gastric digestion (Figure

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2). The SIJ conditions (neutral pH, presence of pancreatic enzymes and bile salts) could

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promote the transformation of ɑ- and β-punicalagins into ellagic acid.34 Consequently, a

32

However, in other studies with


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decrease was observed in those phenolic compounds (ɑ- and β-punicalagin), with a higher

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decrease (ca. 40%) of ɑ-punicalagin content in the fermented juices with respect to the

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control juices (ca. 14%) (Figure 2). This decrease could be related to the generation of a

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new ɑ-punicalagin derivative that was only detected in fermented juices as a possible

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metabolite of the microbial transformations. The concentrations in fermented juices with

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BB, LP y LA were between 30-31 mg/100 mL, while the FPJBL presented lower

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concentrations (25.07 mg/100 mL).

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On the other hand, after SIJ1 a decrease of catechin and epicatechin in the control

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juices was observed (ca. 45% and 64%, respectively) (Figure 1). The decrease in control

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juices could be due to the instability of the catechin at neutral pH.35-37 In this digestion step,

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these compounds (catechin and epicatechin) were not detected in fermented juices because

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of a previous biotransformation of these compounds due to the LAB metabolism. The

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catechin derivative was only detected in fermented juices with respect to the control juices,

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and it was present in higher concentrations (4.37 mg/100 mL) in FPJLA, while the other

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fermented juices only presented trace amounts of these compounds after SIJ1 (Figure 1).

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In different studies with green and black tea, an important decrease was also observed in

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catechin after intestinal digestion.35, 37

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After 120 min of SIJ, following the same pattern as in SIJ1, the phenolic compound

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metabolism observed in fermented juices was higher than in the control juices, probably

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because of the longer time in intestinal juices with the microorganisms. The catechin

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derivative (detected only in fermented juices), increased significantly in FPJLP, FPJBL and

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FPJLA (from trace amounts to 19.22, 6.47 and 9.45 mg/100 mL, respectively) while in

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FPJBB only trace amounts were detected (Figure 1). However, the ɑ-punicalagin

312

derivative concentration in SPJ1 was stable during this same period (Figure 2). This fact



313

may suggest that the bacterial metabolism of ellagitannins, catechin and epicatechin occurs

314

during all the steps of the in vitro digestion, predominantly during the intestinal step.

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Generally, the decrease in phenolic compounds for this period was higher for the fermented

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juices with Bifidobacterium (FPJBB and FPJBL). At the same time, when the cell

317

concentration was compared between the LAB strains, it can be observed that the

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concentrations of the Bifidobacterium strains were higher than the Lactobacillus ones. The

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FPJBL presented the highest cell concentration at the end of in vitro digestion. This fact

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could be related to the higher metabolism of most of the phenolic compounds in these

321

fermented juices with respect to the other fermented ones (Table 1). Regarding the cell

322

concentration, our results showed that although the LAB survival in SIJ2 was lower than in

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the other digestion steps, the cell concentrations were high (>106 CFU/mL) after all the in

324

vitro digestion period. However, the lowest cell survival was observed for FPJLP and the

325

phenolic compound concentration in these juices was higher than in the other FPJs (Table

326

1). The relationship observed in fermented juices between the phenolic compounds and cell

327

concentrations suggests the possible prebiotic effect of phenolic compounds on the LAB.

328

The metabolites excreted by the LABs could produce health benefits through

329

bioaccessibility or bioactivity even though they are not absorbed in the gut.3 This is a

330

preliminary study, thus it could be considered as a basis for future studies to reinforce this

331

hypothesis.

332

Effect of fermentation and in vitro digestion on the antioxidant capacity of

333

pomegranate juices

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During the fermentation and gastrointestinal digestion, different transformations

335

(epimerization, degradation, oxidation and hydrolysis) may occur to the phenolic

336

constituents of the pomegranate juices that could change the pattern (structure and levels)


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of their metabolites.21, 35 Therefore, it is necessary to use different methods for providing an

338

estimate of the in vitro antioxidant capacity. In this study, three methods were used to

339

evaluate the changes in the antioxidant capacity after fermentation and during in vitro

340

digestion. After fermentation, it was observed that the antioxidant capacity was higher in

341

the fermented samples for the three methods, depending on the AOC method used and on

342

the bacterial strain (Table 2). Therefore, the DPPH scavenging activity of fermented juices

343

with BL and LA was higher (p < 0.05) than the control samples, while the ABTS

344

scavenging activity of fermented samples did not change with respect to the control juices.

345

The FRAP values of FPJLA and FPJBB were higher (p < 0.05) than the control samples

346

values. However, a continuous increase in the antioxidant capacity was observed in

347

fermented samples during the gastric digestion period, only for the DPPH and ABTS

348

assays. This increase in AOC for these assays could be due to the gastric conditions, with

349

low pH and pepsin activity that led to an improvement in the release of bioactive

350

compounds such as epicatechin, catechin and ɑ-punicalagin (Figure 1 and 2) increasing

351

their bioaccessibility, and thus the interaction with LABs. Huang, et al.,38 found the same

352

results after chemical extraction and in vitro digestion in Chinese bayberry. These results

353

are in agreement with those of Gullon, Pintado, Fernández-López, Pérez-Álvarez and

354

Viuda-Martos21 where the gastric digestion increased the inhibition values of the DPPH and

355

of the ABTS in the pomegranate peel.

356

In the intestinal digestion, the increase observed in the AOC during the SGJ for the

357

ABTS and DPPH methods, remained without significant changes during the two hours of

358

intestinal digestion for all FPJs (Table 2). At the end of the in vitro digestion, the

359

antioxidant capacity for the DPPH and ABTS method was in most of the FPJs, higher than

360

in the control ones. Several authors have reported that the in vitro digestion has a high



361

impact on the AOC measured with these methods, as can be demonstrated in the studies

362

performed in 33 fruit types, where the inhibition of DPPH radical increased after in vitro

363

digestion.39 The results are also in agreement with those reported by Chandrasekara and

364

Shahidi4 and Wootton-Beard, et al.40 who found an increase in the ABTS values after in

365

vitro digestion for millet grain and for 23 vegetable juices, respectively.

366

Nevertheless, during in vitro digestion, the FRAP assay values, in contrast to the

367

DPPH and ABTS assay, showed a decrease in AOC for all juices after the gastrointestinal

368

step. However, this initial decrease in fermented samples was higher (p ≥ 0.05) after 60 min

369

under intestinal conditions. These values obtained after SIJ1 remained stable at the end of

370

SIJ2 with values between 10.7 and 8.4 mmol Trolox/ L. According to a previous study,41

371

the FRAP assay values of the control , fermented

372

conditions can be enhanced by the electron transfer reaction under acid pH conditions.

373

Previous results obtained for catechin instability at neutral pH in control juices and by the

374

metabolism of the LAB in fermented juices (Figure 1 and 2), could be of some relation

375

with the results of the FRAP assay after SIJ1 (Table 2). A possible explanation could be

376

related to the metal-chelating properties of catechin and epicatechin with an important

377

contribution to the antioxidant activity of the juices.42 Therefore, the degradation of

378

catechin and epicatechin in control juices and the formation of the catechin microbial

379

metabolite through LAB metabolism in fermented juices, could be associated with the

380

reduction of the chelating activity resulting in a lower antioxidant activity in the FRAP

381

assay.

and digested juices under gastric

382

In conclusion, the results of the present study showed that the fermentation of

383

pomegranate juices improves the antioxidant capacity evaluated by the DPPH and ABTS

384

assays and modifies the type and amount of phenolic compounds with respect to the


Page 16 of 28

Page 17 of 28


385

unfermented ones. Through the biotransformation of these compounds by lactic acid

386

bacteria, two new phenolic derivatives were obtained (catechin and α-punicalagin). The in

387

vitro digestion improved the antioxidant capacity for the ABTS and DPPH assays.

388

However, the FRAP assay was significantly influenced by the degradation and

389

biotransformation of the catechin and epicatechin. At the same time, the pomegranate juices

390

were a good food matrix to ensure a high viability (≥106 CFU/mL) of all lactic acid bacteria

391

used in this study after in vitro digestion. Therefore, the lactic acid bacteria used in this

392

study can transform the phenolic compounds present in pomegranate juices, suggesting a

393

possible prebiotic effect of phenolic compounds.

394

Microbial metabolites derived from the fermentation of pomegranate juices and the high

395

viability of microorganisms reaching the colon, may contribute to the maintenance of gut

396

health. Further research is needed to understand the mechanisms of action of phenolic

397

microbial metabolites in humans. Acknowledgments

398

The authors acknowledge Mr. Graham Arnold for his assistance during the preparation of

399

the manuscript and Probelte Biotecnología, S.L. (Murcia, Spain) for the donation of the

400

pomegranate juices

401

Supporting information

402

Additional figures. Representative HPLC chromatograms of fermented pomegranate juices

403

and fermented pomegranate juices after gastrointestinal in vitro digestion.

404 405 406 407 408 409 410 411

ABBREVIATIONS AOC Antioxidant capacity LAB Lactic acid bacteria CFU Colony Forming Units PBS Phosphate Buffer Saline CPJI Control Pomegranate Juices with Incubation CPJR Control Pomegranate Juices with Refrigeration FPJBB Fermented Pomegranate Juice with Bifidobacterium bifidum



412 413 414 415 416 417 418 419 420 421 422 423

FPJLP FPJBL FPJLA SGD SGJ SIJ SIJ1 SIJ2 AI

Fermented Pomegranate Juice with Lactobacillus plantarum Fermented Pomegranate Juice with Bifidobacterium longum subsp. Infantis Fermented Pomegranate Juice with Lactobacillus acidophilus Simulated Gastrointestinal Human Digestion Simulated Gastric Juice Simulated Intestinal Juices Simulated Intestinal Juices after 60 min Simulated Intestinal Juices after 120 min After Incubation

424

1. Lewandowska, U.; Szewczyk, K.; Hrabec, E.; Janecka, A.; Gorlach, S. Overview of

425

metabolism and bioavailability enhancement of polyphenols. J. Agric. Food Chem. 2013,

426

61, 12183-99.

427

2. Etxeberria, U.; Fernandez-Quintela, A.; Milagro, F. I.; Aguirre, L.; Martinez, J. A.;

428

Portillo, M. P. Impact of polyphenols and polyphenol-rich dietary sources on gut

429

microbiota composition. J. Agric. Food Chem. 2013, 61, 9517-33.

430

3. Fernandez-Garcia, E.; Carvajal-Lerida, I.; Perez-Galvez, A. In vitro bioaccessibility

431

assessment as a prediction tool of nutritional efficiency. Nutr. Res. 2009, 29, 751-60.

432

4. Chandrasekara, A.; Shahidi, F. Bioaccessibility and antioxidant potential of millet grain

433

phenolics as affected by simulated in vitro digestion and microbial fermentation. J. Funct.

434

Foods 2012, 4, 226-237.

435

5. Mosele, J. I.; Macià, A.; Romero, M.-P.; Motilva, M.-J.; Rubió, L. Application of in vitro

436

gastrointestinal digestion and colonic fermentation models to pomegranate products (juice,

437

pulp and peel extract) to study the stability and catabolism of phenolic compounds. J.

438

Funct. Foods 2015, 14, 529-540.

439

6. Moco, S.; Martin, F. P.; Rezzi, S. Metabolomics view on gut microbiome modulation by

440

polyphenol-rich foods. J. Proteome Res. 2012, 11, 4781-90.

441

7. Pandareesh, M. D.; Mythri, R. B.; Srinivas Bharath, M. M. Bioavailability of dietary

442

polyphenols: Factors contributing to their clinical application in CNS diseases. Neurochem.

443

Int. 2015, 89, 198-208.

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Page 23 of 28


Table 1. Growth of different lactic acid bacteria in pomegranate juices after incubation period and their survival during the different steps of the in vitro digestion.

Period AI SGJ SIJ1 SIJ2

FPJBB 7.78 ±0.05D a 7.40±0.01Cc 7.09±0.01Bc 6.79±0.15Ab

FPJBL Log CFU/mL 7.33±0.02Ca 7.67±0.02Da Ba 6.83±0.04 7.18±0.01Cb ABa 6.73±0.02 7.11±0.01Bc Aa 6.71±0.02 6.99±0.02Ac

FPJLP

FPJLA 7.26±0.07Ba 6.93±0.02Aa 6.88±0.01Ab 6.82±0.01Ab

Values are the mean of 3 replications (± standard error). Capital letters refer to the evolution of each period: After Incubation (AI), Simulated Gastric Juice (SGJ), Simulated Intestinal Juices after 60 min (SIJ1) and Simulated Intestinal Juices after 120 min (SIJ2). Lowercase letters are the comparison among Fermented Pomegranate

Juices

with:

Bifidobacterium

bifidum (FPJBB),

Lactobacillus plantarum

(FPJLP),

Bifidobacterium longum subsp. Infantis (FPJBL) and Lactobacillus acidophilus (FPJLA). Values followed by the same letter within the same column or row were not statistically different according to Tukey’s multiple range test (p < 0.05).



Page 24 of 28

Table 2. Antioxidant Capacity of pomegranate juice after incubation period and during the different steps of the in vitro digestion.

Period

DPPH

ABTS

FRAP

AI SGJ SIJ1 SIJ2 AI SGJ SIJ1 SIJ2 AI SGJ SIJ1 SIJ2

CPJR 20.41 ±0.59Cab 14.40±1.27Ba 7.17±0.26Aa 7.50±0.33Aa 13.83±1.16Ac 14.66±0.49Abc 14.47±0.36Abc 13.87±0.27Aa 13.94±0.56Ba 10.80±0.51Aa 10.15±0.43Ab 10.68±0.53Ab

CPJI

FPJBB

18.85±1.40Ca 13.07±1.33Ba 9.79±2.60ABa 6.95±0.74Aa 12.52±0.15Aabc 14.03±0.85Bd 13.84±0.40Babc 14.02±0.60Ba 13.32±0.39Ca 12.27±0.24Ba 10.16±0.43Ab 10.01±0.04Ab

FPJLP (mmol Trolox/ L) 20.40±0.95Aab 22.22±1.05Abc Bb 27.27±0.25 27.99±0.60Bb Bb 27.22±0.46 26.50±0.18Bb Bb 27.26±0.37 27.05±1.13Bb Aab 11.97±0.23 12.87±0.92Abc Bd 15.41±0.27 15.74±0.44Bd Bbc 15.35±0.50 14.56±0.20Bc Bb 15.39±0.40 15.16±0.68Bb Cb 15.99±1.46 13.51±0.58Ca Ba 12.05±0.72 11.98±0.99Ba Aab 9.10±0.69 8.58±0.21Aa Ab 8.42±0.18 8.41±0.24Ab

FPJBL 24.04±0.75Ac 27.32±0.19ABb 27.08±0.47Bb 26.39±0.42ABb 11.88±0.82Aab 13.23±0.21Bab 13.52±0.31Bab 14.44±0.45Bab 14.68±0.91Cab 11.31±1.41Ba 8.63±0.35Aa 8.71±0.13Ab

FPJLA 22.93±0.23Ac 27.90±0.45Cb 26.32±0.37Bb 26.56±0.49Bb 10.67±0.25Aa 12.08±0.40ABa 12.48±1.14Ba 14.63±0.53Cab 16.06±1.35Cb 12.41±0.16Ba 8.99±0.08Aa 8.84±0.32Ab

Values are the mean of 3 replications (± standard error). Capital letters are the evolution of period: After Incubation (AI)*, Simulated Gastric Juice (SGJ), Simulated Intestinal Juices after 60 min (SIJ1) and Simulated Intestinal Juices after 120 min (SIJ2). Lowercase letters are the comparison among Pomegranate Juices [Unfermented Control Pomegranate Juice Refrigerated (CPJR), Unfermented Control Pomegranate Juice Incubated (CPJI); and Fermented Pomegranate Juice with: Bifidobacterium bifidum (FPJBB), Lactobacillus plantarum (FPJLP), Bifidobacterium longum subsp. Infantis (FPJBL) and Lactobacillus acidophilus (FPJLA)]. Values followed by the same letter within the same column or row were not statistically different according to Tukey’s multiple range test (p < 0.05). *AI refers to the results after incubation period for CPJI, FPJBB, FPJLP, FPJBL and FPJLA compared to CPJR that were not incubated and remained at 4 ºC as refrigerated control juices.


Page 25 of 28


Figure 1. Evolution of flavan-3-ol after incubation period and during the different steps of the in vitro digestion in different fermented pomegranate juices. The bars are the mean of 3 replications (± standard error). Capital letters refer to the flavan-3-ol evolution: After Incubation period (AI)*, Simulated Gastric Juice (SGJ), Simulated Intestinal Juices after 60 min (SIJ1) and Simulated Intestinal Juices after 120 min (SIJ2) for the same type of juice. Lowercase letters refer to the comparison among pomegranate juices for the same step: Control Pomegranate Juice Refrigerated (CPJR), Control Pomegranate Juice Incubated (CPJI), Fermented Pomegranate

Juice

with:

Bifidobacterium

bifidum

(FPJBB),

Lactobacillus

plantarum

(FPJLP),

Bifidobacterium longum subsp. infantis (FPJBL) and Lactobacillus acidophilus (FPJLA). *AI refers to the results after incubation period for CPJI, FPJBB, FPJLP, FPJBL and FPJLA at 37 º C compared to CPJR that was not incubated and remained refrigerated at 4 ºC.




Page 26 of 28

Page 27 of 28


Figure 2. Ellagitannins evolution after incubation period and during in vitro digestion in different fermented pomegranate juices. The bars are the mean of 3 replications (± standard error). Capital letters refer to the ellagitannins evolution: After Incubation period (AI)*, Simulated Gastric Juice (SGJ), Simulated Intestinal Juices after 60 min (SIJ1) and Simulated Intestinal Juices after 120 min (SIJ2) for the same type of juice. Lowercase letters refer to the comparison among pomegranate juices for the same step: Control Pomegranate Juice Refrigerated (CPJR), Control Pomegranate Juice Incubated (CPJI), Fermented Pomegranate Juice with: Bifidobacterium bifidum (FPJBB), Lactobacillus plantarum (FPJLP), Bifidobacterium longum subsp. infantis (FPJBL) and Lactobacillus acidophilus (FPJLA). *AI refers to the results after incubation period for CPJI, FPJBB, FPJLP, FPJBL and FPJLA at 37 º C compared to CPJR that was not incubated and remained refrigerated at 4 ºC.



TOC GRAPHIC 4 different fermented juices

Bioconversion of phenolic compounds?


Page 28 of 28

Influence of Fermentation with Different Lactic Acid Bacteria and in

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