Accepted Manuscript

In vitro modulation of human gut microbiota composition and

metabolites by Bifidobacterium longum BB-46 and a citric pectin

Fernanda Bianchi, Nadja Larsen, Thatiana de Mello Tieghi, Maria

Angela T. Adorno, Susana M.I. Saad, Lene Jespersen, Katia
Sivieri

PII: S0963-9969(18)30895-0
DOI: https://doi.org/10.1016/j.foodres.2018.11.010
Reference: FRIN 8073
To appear in: Food Research International
Received date: 12 September 2018
Revised date: 23 October 2018
Accepted date: 6 November 2018

Please cite this article as: Fernanda Bianchi, Nadja Larsen, Thatiana de Mello Tieghi,
Maria Angela T. Adorno, Susana M.I. Saad, Lene Jespersen, Katia Sivieri , In vitro
modulation of human gut microbiota composition and metabolites by Bifidobacterium
longum BB-46 and a citric pectin. Frin (2018), https://doi.org/10.1016/
j.foodres.2018.11.010

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 ACCEPTED MANUSCRIPT

In vitro modulation of human gut microbiota composition and metabolites by

Bifidobacterium longum BB-46 and a citric pectin

a,b
Fernanda Bianchi , Nadja Larsen b, Thatiana de Mello Tieghi a, Maria Angela T. Adorno c,

Susana M.I. Saad d,e, Lene Jespersen b, Katia Sivieri a*

a
Department of Food and Nutrition, School of Pharmaceutical Sciences, State University of São Paulo (UNESP),

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Araraquara, SP, 14801-902, Brazil

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b
Department of Food Science. Faculty of Science, University of Copenhagen, Frederiksberg C 1958, Denmark
c
Department of Hydraulics and Sanitation, School of Engineering of São Carlos, University of São Paulo (USP),

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São Carlos ,SP, 13563-120 Brazil
d
Department of Biochemical and Pharmaceutical Technology. University of São Paulo (USP), São Paulo, SP,
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05508-000, Brazil
e
Food Research Center. University of São Paulo (USP), São Paulo, SP, 05508-000, Brazil
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* Corresponding author at: Faculty of Pharmaceutical Sciences (UNESP), 1 Km Araraquara–Jaú Highway, 14801-
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902, Araraquara (SP), Brazil. Tel.: +55 16 3301-6931.

E-mail address: katiasiv@hotmail.com (K. Sivieri).

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Abstract

The gut microbiota composition and its metabolites have high impact on human health.
Exploitation of prebiotics and probiotics for modulation of gut microbiota can lead to promising
outcomes. This study aimed to evaluate the effects of the probiotic strain Bifidobacterium
longum BB-46 alone and in combination with a citric pectin from lemon on the gut microbiota
from healthy adults using the Simulator of Human Intestinal Microbial Ecosystem (SHIME®).
Changes in microbiota composition and in metabolic activity were assessed by the 16S rRNA

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gene sequencing and by analyses of short-chain fatty acids (SCFAs) and ammonium ions (NH4+).
An increase in the relative abundances of Firmicutes (especially the members of

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Lachnospiraceae and Lactobacillaceae families) and Bacteroidetes was observed during

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treatment with B. longum BB-46 alone in all compartments of the colon. Treatment with B.
longum BB-46 and pectin stimulated an increase in the proportions of genera Faecalibacterium,
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Eubacterium and Lactobacillus, as well as in the Ruminococcaceae family in the transverse and
descending colons. Concurrently, the butyrate levels increased in these two compartments.
Additionally, the combination of B. longum BB-46 and pectin reduced the abundance of
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proteolytic bacteria Bacteroides, Clostridium, Peptoniphilus, and Streptococcus, along with

decreased NH4+ production. No significant changes could be observed on NH4+ production by
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treatment with B. longum BB-46, nor did it increase the amount of SCFAs. In this study, we
observed that although each treatment was able to modulate the microbiota, the combination of
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B. longum BB-46 and pectin was more efficient in decreasing the intestinal NH4+ levels and in
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increasing butyric acid-producing bacteria. These findings indicate that B. longum BB-46,
especially when combined with the specific citric pectin, might have beneficial impact on human
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health.
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Keywords: human gut microbiota, pectin, Bifidobacterium longum BB-46, metabolites, 16S

rRNA gene sequencing, SHIME®.

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1. Introduction

It is estimated that the human gut harbours 3 to 4 million microbial genes, which equates to

approximately 150 times more than the number found in the human genome (Ehrlich, 2010;

Lozupone, Stomabaugh, Gordon, Jansson, & Knight, 2012). The gut microorganisms play an

important role in the host’s health, contributing to homoeostasis of the immune system,

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conversion of food into useful nutrients and protection against invasion by pathogenic

microorganisms (Ehrlich, 2010; Lozupone et al., 2012). However, changes in the microbiota

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composition, caused by many factors such as place of residence, environmental influences,

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lifestyle, antibiotic use and diet (Lozupone et al., 2012), have been linked with several diseases

(Alonso & Guarner, 2013). As diet is considered an important factor affecting the gut microbiota
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composition and therefore the human health (Ramakrishna, 2013), interest in food strategies to
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improve the human gut microbial ecosystem has been growing. Among different strategies, the

use of prebiotics and other fibres, as well as probiotics and combinations of pre- and probiotics
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has been highlighted (Alonso & Guarner, 2013).

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The use of probiotic microorganisms has been extensively investigated in gut disorders.
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Probiotics are able to improve the immune system, the epithelial barrier function and to produce

antibacterial factors (Sherman, Ossa, & Johnson-henry, 2009). Several positive effects of
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probiotic Bifidobacterium longum strains, such as anti-allergy effects (Dev et al., 2008),
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stimulation of gut immune system bacteria (Makioka, Tsukahara, Ijichi, & Inoue, 2018), and

improvements in the intestinal environment and defecation frequency (Yaeshima et al., 1997)

have been demonstrated in animal and human intervention studies. Besides

hypocholesterolaemic (Abd El-Gawad et al., 2005) and anti-pathogenic effects (Pavlović, Hardi,

Slačanac, Halt, & Kocevski, 2006; Silva et al., 2004), strains of B. longum BB-46 have also

showed good survival rate alone and combined with different by-products of fruits under in vitro

simulated gastrointestinal test (Bianchi et al., 2018a, Vieira, Badeni, Albuquerque, Biscola, &
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Saad, 2017), being considered as a probiotic strain (Havas, Kun, Perger-Mészáros, Rezessy-

Szabó, & Nguyen, 2015).

Pectins are important water-soluble dietary fibres present in the cell walls of fruits and

vegetables. They have been defined as emerging prebiotics demonstrating ability to modulate the

gut microbiota, increasing Bifidobacterium spp. and butyric acid-producing bacteria, as for

example Faecalibacterium and other members of Ruminococcaceae family (Bianchi et al.,

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2018b; Gómez, Gullón, Yáñez, Schols, & Alonso, 2016; Gullón et al., 2013; Henningsson,

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Margareta, Nyman, & Björck, 2002; Jiang et al., 2016; Tian et al., 2017). Moreover, pectins can

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increase probiotic bacteria survival, slow gastric transit and decrease glycemic index (Larsen,

Cahú, Saad, Blennow, & Jespersen, 2018; Olano-Martin, Gibson, & Rastall, 2002). Pectins can
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be classified as high-methylated (HM) pectin, with degree of methyl esterification (DM) > 50 %,

and low-methylated (LM) pectin, with DM < 50 % (Onumpai, Kolida, Bonnin, & Rastall, 2011;
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Sila et al., 2009). These structural variations can lead to different effects on the colonic

microbiota (Onumpai et al., 2011).

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Dynamic colonic models have been used to study potential beneficial effects of pre- and
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probiotics in the intestinal microbiota (Bianchi et al., 2014; Bianchi et al., 2018b; Pham &
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Mohajeri, 2018). In vitro models, such as the Simulator of Human Intestinal Microbial
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Ecosystem (SHIME®), simulate the human gastrointestinal tract allowing investigation of the

intestinal microbial composition and its metabolites production and functionalities (Molly,
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Woestyne, Smet, & Verstraete, 1994), avoiding the use of both invasive techniques in humans

and animal ethical management (Parvova, Danchev, & Hristov, 2011). The influence of

Bifidobacterium spp. and a few types of pectin on the gut microbial community has been

demonstrated in several in vitro and in vivo preclinical and clinical studies (Ji et al., 2016;

Maldonado-Gómez et al., 2016; Medina, De Palma, Ribes-Koninckx, Calabuig, & Sanz, 2008;

Moya-Pérez, Neef, & Sanz, 2015; Tian et al., 2017). However, as far as we know, changes in the
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gut microbiota of healthy humans promoted by combination between Bifidobacterium longum

and citric pectin have not been described. As such, the aim of this study was to evaluate the

effects of the probiotic Bifidobacterium longum BB-46 combined with a LM pectin from lemon

on the gut microbiota modulation using the SHIME® model.

2. Materials and Methods

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2.1. Bacterial strain and pectin

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The probiotic strain Bifidobacterium longum BB-46 was provided by Chr. Hansen A/S

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(Hoersholm, Denmark) and maintained in de Man Rogosa Sharpe (MRS) broth with 20 % (w/w)
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glycerol at -80 ºC. Before the experiments, the strain was grown in MRS broth supplemented

with L-cysteine (0.05 %) at 37 ºC for 24 hours. Bacterial cells were collected by centrifugation
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(3,000 × g for 10 min), washed and resuspended in saline solution (NaCl 0.85 % (w/v)). The

pectin used in this study was a harsh-extracted LM lemon pectin provided by CP Kelco ApS
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(Lille Skensved, Denmark).

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2.2. The Simulator of Human Intestinal Microbial Ecosystem (SHIME®)

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The SHIME® (registered trade name from Ghent University and ProDigest) is a simulator of

human intestinal microbial ecosystem in which environmental conditions (pH, residence time
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and temperature) are controlled by a software (Molly et al., 1994). It consists of five

double-jacketed vessels simulating the stomach, duodenum and ascending, transverse and

descending colons. The five vessels were continuously stirred with a magnetic stirrer and the

temperature was kept at 37 °C. The system was maintained anaerobically through a daily N2

flushing of 30 min. The pH in the ascending (pH between 5.6–5.9), transverse (pH between 6.1–

6.9) and descending colon (pH between 6.6–6.9) was automatically adjusted by addition of
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NaOH 1 M or HCl 0.1 M (Molly et al., 1994; Possemiers, Verthé, Uyttendaele, & Verstraete,

2004). Each compartment of the colon was filled with the SHIME® feed (carbohydrate-based

medium that allows adaptation of microorganisms to specific environmental conditions of the

colon in terms of pH range, retention time and available carbon sources) in specific volumes as

previously described (Bianchi et al., 2014; Molly et al., 1994). The stomach conditions as well as

the use and preparation of pancreatic juice (composed by Oxgall 6.0 g/L, NaHCO3 12.5 g/L and

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pancreatin 0.9 g/L) were based on the studies conducted by Bianchi et al. (2014) and Bianchi et

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al. (2018a).

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2.3. Experimental protocol using the SHIME® model
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By using the SHIME® model, we evaluated the impact of the probiotic bacterium
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Bifidobacterium longum BB-46 alone (T1) and combined with lemon pectin (T2) on the

intestinal microbiota. Before starting the experiments, the colon vessels (simulating the

ascending, transverse and descending colons) were inoculated with faecal microbiota from 3
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healthy adults (BMI between 18.5 and 25 kg/m2 and waist circumference < 80 cm). The faeces
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samples (≈ 13.5 g from each donor) were collected, diluted in 200 mL of phosphate buffer

containing Na2HPO4 0.05 mol/L, NaH2PO4 0.05 mol/L and Na-thioglicolate 0.1 % (pH 6.5),
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stirred for 10 min in an homogenizer (Stirrer model 130, Norte Científica, São Paulo, BR.) and
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centrifuged (3,000 × g for 15 min). The supernatants were subsequently added (40 mL) to the

three colon vessels (Bianchi et al., 2014; Molly et al., 1994). All donors had no history of

antibiotic treatment (at least 6 months prior to this study) and had not consumed probiotic

products over the past 3 to 6 months.

The experimental protocol included a two-week control period (without intervention) after

inoculation of the stool sample in the three colon vessels. This period allows the microbial

community to adapt to the prevailing nutritional and physicochemical conditions in each region
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of the colon and is necessary for microbiota stabilization (Molly et al., 1994). During the control

period, 200 mL of the SHIME® feed entered through the system two times a day for two weeks.

After this period, the first vessel (stomach) was fed with B. longum BB-46 (final stomach

concentration of 108 CFU/mL) (T1) during one week, followed by one more week of feeding the

stomach with B. longum BB-46 combined with 2 % (w/v) of lemon pectin (T2). Both additions

were carried out along with 200 mL of the SHIME® feed twice a day, i.e. 8 g of pectin per day.

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The stomach contents were later automatically and sequentially transferred to duodenum,

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ascending, transverse and descending colons. After the T2 period, one-week post-treatment (PT)

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was performed (200 mL of the SHIME® feed through the system twice a day).
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2.4. Microbiota composition using 16S rRNA gene sequencing
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Microbiota profiles of each experimental period (control, treatments and post-treatment)

from the three colon vessels of the SHIME® model (two technical replicates per treatment) were

determined using tag-encoded 16S rRNA gene sequencing NextSeq (Illumina, San Diego, USA).
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The microbiota samples from SHIME® (4 mL) were centrifuged (10,000 × g for 5 min) and
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the pellet freeze-dried. Isolation of total bacterial DNA was performed using

PowerLyzer@PowerSoil DNA Isolation Kit (Qiagen, Valencia, USA) according to its manual.
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The DNA library for amplicon sequencing was prepared as described by Williams et al. (2017).
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Briefly, the V3 region (≈190 bp) of the 16S rRNA gene was PCR amplified using primers

compatible with the Nextera Index Kit (Illumina) (Primers NXt_388 and NXt_518). The PCR I

was performed using 12 μL of AccuPrime SuperMix II (Life Technologies, Camarillo, USA),

5 μL of genomic DNA (≈20 ng/μL), 0.5 μL of each primer (10 μM) and nuclease-free water to a

total volume of 20 μL. The DNA was amplified using the following setup: initial denaturation at

95 °C for 2 min, 33 cycles of denaturation at 95 °C for 15 s, annealing of primer at 55 °C for 15 s

and elongation at 68 °C for 30 s, followed by an extension at 68 °C for 4 min and final cooling to
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4 °C. To incorporate primers with adapters and indexes, a second PCR was performed (PCR II)

using 12 μL of Phusion High-Fidelity PCR Master Mix (Thermo Fisher Scientific,Tewksbury,

USA) and 2 μL of primers P5 and P7 (Nextera Index Kit). PCR reactions contained 2 μL PCR I

product and nuclease-free water for a total volume of 25 μL. The DNA was amplified using the

following setup: initial denaturation at 98 °C for 1 min, 13 cycles of denaturation at 98 °C for

10 s, annealing of primer at 55 °C for 20 s and elongation at 72 °C for 20 s, followed by an

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extension at 72 °C for 5 min and final cooling to 4 °C. After PCR II, the amplified fragments

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with adapters and tags were purified using AMPure XP beads (Beckman Coulter Genomic,

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Indianapolis, USA), providing a size selection step and removing short library fragments

(Williams et al., 2017).

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The sequencing was performed on the Illumina NextSeq instrument as a part of a flowcell

using a 2 × 150 cycles MID output kit V2 (Illumina, San Diego, USA). The raw dataset
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containing pair-ended reads with corresponding quality scores were merged and trimmed using

settings as described by Williams et al. (2017). Quantitative Insight Into Microbial Ecology
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(QIIME) open source software package (1.7.0 and 1.8.0) was used for subsequent analysis steps
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(Caporaso et al., 2011). The UPARSE pipeline was employed to purge the dataset of chimeric
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reads and to construct de novo Operational Taxonomic Units (OTU). As a reference database,
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the Green Genes 16S rRNA gene collection was used (McDonald et al., 2012). Aiming to

normalize different depths of sequencing samples, the matrices’ abundance of taxonomic units,
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of each sample, was divided by the total number of pairings. By means of the alpha rarefaction

workflow, the alpha diversity measures, expressed as observed species (sequence similarity of

97 % OTUs) values, were computed for rarefied OTU tables (23,000 reads/sample) (Williams et

al., 2017).
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2.5. Metabolite production: short-chain fatty acids (SCFAs) and ammonium ions (NH4+)

Samples (50 mL) were collected weekly from each compartment (ascending, transverse and

descending colons) during the control, treatments T1 and T2 and post-treatment period for

SCFAs and NH4+ analyses. The analyses were carried out in triplicates.

The determination of SCFAs was previously described by Adorno, Hirasawa, & Varesche

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(2014). For this purpose, we used a gas chromatograph equipped with a flame-ionization gas

detector, a capillary split/splitless injector and an HP-INNOWAX column (30 m × 0.25 mm ×

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0.25 μm) (Agilent Technologies, La Jolla, USA). Hydrogen was the carrier gas at a flow rate of

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1.45 mL/min. The temperature of both the detector and injector was 240 °C (Adorno et al.,

2014).
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NH4+ levels were determined by a selective ion meter (HI 4101 model, Hanna Instruments,
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Leighton Buzzard, UK) coupled with an ammonium ion-selective electrode (Orion 95–12). The

samples (10 mL) collected from each simulated region of the colon were added to 0.2 mL of

ammonia pH-adjusting ionic strength adjuster (ISA) solution (Orion, Thermo Fisher,
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Millersburg, USA) and then the NH4+ levels (mmol/L) were computed.
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2.6. Statistical analysis

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One-way ANOVA followed by a Tukey post-hoc test (p < 0.05) was applied to test
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significant differences between results (composition of microbiota, production of SCFAs and

NH4+) using Biostat 5.0 software (IBM, Belém, BR) (Ayres, Ayres, Ayres, & Santos, 2007). A

simple correspondence analysis was used to observe correlation between the different treatments

and the microbiota composition using Minitab Software (State College, USA) (Minitab, 2010).

Correlations between short-chain fatty acids, NH4+ production and specific bacterial genera were

determined by Spearman correlation test (p < 0.05) using the open-source RStudio software

program (RStudio, 2017).

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3. Results

3.1. Richness and diversity of the gut microbiota

A total of more than 1.7 million high-quality reads (> 200 bp) were obtained from

24 microbiota samples collected during control periods, treatments with Bifidobacterium longum

BB-46 alone and in combination with the pectin and post-treatments using the Nextseq Illumina

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sequencing. After normalizing the data, 552,000 sequences were produced, resulting in 23,000

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sequences per sample. For each sample, a rarefaction curve was constructed to evaluate

sequencing depth and species richness (Fig. S1). The curves showed that the sequencing depth

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was enough to cover most of the bacteria in the SHIME® samples.
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Supplementary Fig. S2 shows alpha diversity within microbiota samples expressed by

indices Chao1 (species richness) and Shannon (diversity). The treatment with B. longum BB-46
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(T1) showed low richness in all colon vessels (average Chao1 of 242) but high diversity (average

Shannon of 5.50) compared to controls (averages of 298 and 4.8 for Chao1 and Shannon,
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respectively). Treatment with B. longum BB-46 with pectin (T2) showed relatively low richness
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and diversity (averages of 216 and 3.92).

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3.2. Changes in the gut microbiota during the treatments with B. longum BB-46 and

B. longum BB-46 combined with citric pectin using 16S rRNA gene sequencing
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Fig. 1 shows the relative abundance of the main bacterial families in different regions of the

SHIME® model: ascending (V3), transverse (V4) and descending (V5) colon vessels. High

relative abundance of Bifidobacteriaceae (V3 = 35 %, V4 = 38 % and V5 = 32 %) and

Coreobacteraceae families (V3 = 14 %, V4 = 7 % and V5 = 5 %), both belonging to

Actinobacteria phylum, as well as Burkholderiaceae (V3 = 10 %, V4 = 17 % and V5 = 17 %)

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and Enterobacteraceae families (V3 = 17 %, V4 = 11 % and V5 = 10 %), both belonging to

Proteobacteria phylum, were found during the control period.

An increase in the abundance of Bacteroidaceae (Bacteroidetes phylum), Lachnospiraceae

(Firmicutes phylum), Enterobacteriaceae (Proteobacteria phylum) and Lactobacillaceae

(Firmicutes phylum) families, as well as a reduction in the abundance of Bifidobacteriaceae

were found in all colon vessels during the treatment with B. longum BB-46 (T1) (Fig.1). An

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increase in the relative abundance of Lactobacillaceae (from 0.28 % to 37.51 %) and

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Enterobacteriaceae (from 16 % to 33 %) was observed during the treatment using

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B. longum BB-46 with pectin (T2) in V3, while a high increase of Ruminococcaceae (from 3 %

to 73 % in V4 and from 10 % to 43 % in V5) and a reduction of Bacteroidaceae and

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Enterobacteriaceae families was observed in the same experimental period (T2) in V4 and V5.

The one-week post-treatment (PT) revealed similar results to treatment T2 in V3 and V5,
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indicating that the modifications obtained during T2 were maintained (Fig. 1).

A simple correspondence analysis was performed to investigate the association between the
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treatments and the microbiota composition (Fig. 2). The correspondence analysis was sufficient
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to interpret the results as seen from the explained variance (Component 1 = 36.3 % and
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Component 2 = 29.8 %). The control period of ascending (V3), transverse (V4) and
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descending (V5) colons were clustered according to the high abundance of Bifidobacteriaceae,

Burkholderiaceae, Streptococcaceae, Erysipelotrichaceae and Coreobacteriaceae families. The

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high proportion of Lachnospiraceae, Bacteroidaceae and Clostridiaceae families found after

treatment T1 accounted for the clustering of samples during this treatment. Grouping of the

treatment T2 and PT from V3 was due to the high abundance of Enterobacteriaceae and

Lactobacillaceae families. The Ruminococcaceae, Rikenellaceae and Eubacteriaceae families

were associated with treatment T2 and PT from V4 and V5.

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Changes in abundances of various bacterial taxa after treatments T1 and T2 were

significantly different (p < 0.05) from controls (Fig. 3). The treatment with B. longum BB-46

(T1) stimulated proliferation of genera Lactobacillus and Dorea (Lachnospiraceae family),

Bacteroides, Enterobacter, as well as an unclassified genus of Lachnospiraceae and a reduction

in Streptococcus in all three colon vessels. Genus Eubacterium was also stimulated during T1

but only in V5. The treatment with B. longum BB-46 combined with pectin (T2) significantly

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stimulated the genera Lactobacillus, Veilonella, Enterobacter, Klebsiella and Erwinia in V3, and

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significantly increased Faecalibacterium, Eubacterium and unclassified genus of

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Ruminococcaceae in V4 and V5. Furthermore, a depletion of Enterobacter, Klebsiella and

Erwinia, as well as Bacteroides, Peptinophilus and Streptococcus was found during treatment T2
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in V4 and V5, whereas genus Lactobacillus was stimulated in V5.
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3.3. Metabolic activity

Levels of acetic and butyric acids had a significant decrease (p < 0.05) during the treatments
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with B. longum BB-46 (T1) and B. longum BB-46 with pectin (T2) in the ascending colon,
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whereas no visible changes were noted for propionic acid (Fig. 4). On the other hand, a

significant increase (p < 0.05) in butyric acid was observed during the treatment T2 in the
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transverse and descending colons. There were no significant visible changes of propionic and
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acetic acids in the transverse and descending colons during treatments T1 and T2, except for a

reduction of acetic acid in the transverse colon during treatment T1.

A significant decrease in NH4+ production was observed during treatment T2 in all colon

vessels compared to the other periods of study (Table 1). Apparently, treatment T1 did not affect

the NH4+ production. The intestinal levels of NH4+ during PT were higher (p < 0.05) than those

found during treatment T2, but lower than those found in the control period (p < 0.05), thus

showing some residual effects of treatment T2 (Table 1).

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3.4. Correlation between metabolite production and bacterial genera during the treatments

We assessed the correlation between the relative abundance of bacterial genera, SCFAs and

NH4+ to identify the genera that might contribute to the production of SCFA and NH4+ (Fig. 5).

Genera Eubacterium, Dorea and Faecalibacterium positively correlated (r-value = 0.60 ‒ 0.80, p

< 0.05) with butyric acid levels, while an unclassified genus of Ruminococcaceae had a positive

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correlation (p < 0.05) with acetic (r-value = 0.66) and butyric acids (r-value = 0.95) production.

The relative abundance of genera Klebsiella, Enterobacter, Erwinea and an unclassified genus of

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Enterobacteriaceae showed negative correlation (r-value = -0.52 ‒ -0.95, p < 0.05) with acetic,

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propionic and butyric acids. Numbers of Streptococcus, Bacteroides, Clostridium and

Peptoniphilus positively correlated with NH4+ production (r-value between 0.60 and 0.73, p <
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0.05) (Fig. 5).
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4. Discussion
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The effects of B. longum BB-46 in combination with lemon pectin (T2) on microbiota
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composition and activity were evaluated using a gut microbiome model (SHIME®). We also
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evaluated the effects of B. longum BB-46 alone (T1) on the intestinal microbiota. The treatment
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T1 stimulated genera of Firmicutes phylum, such as Lactobacillus (Lactobacillaceae family), an

unclassified genus of the Lachnospiraceae and Dorea (Lachnospiraceae family). The increased
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abundance of Lachnospiraceae members during T1 can most likely be explained by the

production of lactate and/or acetate by the B. longum BB-46, which can be used by

Lachnospiraceae members, stimulating their proliferation (Belenguer et al., 2006; Falony,

Vlachou, Verbrugghe, & De Vuyst, 2006; Flint, Duncan, Scott, & Louis, 2015). During the same

treatment (T1), an increase in the abundance of genus Bacteroides was observed. Bifidobacteria

have been reported to modulate the microbiota in favour of saccharolytic bacteria, such as

Bacteroides, possibly by cross-feeding (Turroni et al., 2016), which could probably explain our
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result. Increased abundances of Bacteroides in this study can be also due to production of

exopolysaccharides by some strains of B. longum, which can be consumed by Bacteroides

species, increasing their abundance (Ríos-Covián et al., 2016a).

B. longum BB-46 combined with pectin (T2) greatly stimulated an unclassified genus of the

Ruminococcaceae family in the transverse and descending colons. The correlation analysis

revealed positive correlation between the unclassified genus of the Ruminococcaceae family and

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levels of butyric acid (Fig. 5). These results are in accordance with previous findings showing

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that family Ruminoccocaeae includes the major butyrate-producing species, having capacity to

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degrade pectin (Lopez-Siles et al., 2012; Louis, Young, Holtrop, & Flint, 2010). Tian et al.

(2017), Gómez et al. (2016) and Jiang et al. (2016) studied the effects of different pectins on the
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gut microbiota and also demonstrated an increase in the abundance of Ruminococcacea

members, such as Faecalibacterium and, consequently, in butyric acid production.

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Other butyrate-producing bacteria, such as Eubacterium and Faecalibacterium (Louis et al.,

2010; Moens, Verce, & De Vuyst, 2017), were similarly enriched during treatment T2 in the
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transverse and descending colons, and positively correlated with butyric acid. The increase in
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butyric acid during treatment T2 can also be attributed, at least to a certain extent, to the
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cross-feeding interactions between the probiotic strain B. longum BB-46 and other bacteria, such
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as Faecalibacterium and Eubacterium, stimulated by the presence of pectin. This assumption is

based on previous studies showing that the formation of butyrate by Faecalibacterium

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prausnitzii is enhanced in the presence of bifidobacteria, demonstrating the cross-feeding

between Bifidobacterium and F. prausnitzii (Ríos-Covián et al., 2016b). Moreover, Moens et al.

(2017) demonstrated that some bacterial strains, such as Eubacterium, are able to consume

lactate generated by other bacteria, such as Bifidobacterium spp., converting the lactate into

butyric acid. These findings suggest an indirect interaction between the citric pectin and

B. longum BB-46, resulting in stimulation of different bacterial genera and increase of butyric
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acid. High production of butyrate is commonly considered as beneficial since this metabolite can

have a positive effect on the human health, such as the ability to increase the intestinal barrier

function (Brahe, Astrup, & Larsen, 2016) and protect against colon carcinoma (Hijova &

Chmelarova, 2007).

Production of propionic and acetic acids in the transverse and descending colons seemed to

be unaffected by treatments T1 and T2 in this study, except for a reduction in acetic acid levels

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in the transverse colon during treatment T1. Although a positive correlation was found between

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acetic acid production and the unclassified genus of Ruminococcaceae, which was increased

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during treatment T2, no significant increase in acetic acid was observed during this treatment.

Consistent levels of propionic and acetic acids can be related to bacterial cross-feeding and
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complex interactions among gut microorganisms (Duncan, Louis, & Flint, 2004; Ríos-Covián et

al., 2016b). Duncan et al. (2004), for example, evidenced an in vitro conversion of acetate to
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butyrate by Faecalibacterium spp., which can probably explain the increase in butyric acid and

no alteration in acetic acid found in our study.

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Reduction of acetic and butyric acids in the ascending colon during treatments T1 and T2
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was associated with the high levels of Enterobacteriaceae in this region, which correlated
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negatively with butyric and acetic acids. Likewise, Van Der Wielen, Biesterveld, Hofstra,
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Urlings, & Van Knapen (2000) found a negative correlation between SCFAs and numbers of

Enterobacteriaceae, which can be explained by the competition between the members of

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Enterobacteriaceae and butyrate/acetate-producing bacteria (Van Der Wielen et al., 2000).

Curiously, the highest amount of Klebsiella and other bacterial genera belonging to

Enterobacteriaceae family was found in the ascending colon during treatment T2, while a

significant reduction in these genera was observed in the transverse and descending colons.

These results indicate that the ascending colon conditions (pH between 5.6 ‒ 5.9 and available

carbohydrates from the pectin and the feed medium) were preferable for growth of
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Enterobacteriaceae members, such as Klebsiella. Specifically, the pH of the ascending colon,

corresponding to the optimum pH of polygalacturonase (a pectin-degrading enzyme found in

some species of Klebsiella), might favour pectin utilization and consequent growth of Klebsiella,

usually considered as pathogenic bacteria (Yuan et al., 2012).

The reduction of NH4+ during treatment T2 could be attributed to the decrease in the relative

abundance of Streptococcus, Peptoniphilus, Bacteroides and Clostridium spp., which are

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classified as proteolytic bacteria (Dai, Wu, & Zhu, 2011). Proteolytic bacteria use amino acids as

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sources of nitrogen and carbon, generating NH4+ as one of the intermediate or final metabolites

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(Macfarlane & Cummings, 1991). The simple addition of a carbohydrate (pectin) during the

intervention could also have contributed to the decrease in NH4+, once inhibition of amino acids
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fermentation in favour of carbohydrate fermentation can occur due to the preference of gut

microorganisms to carbohydrates (Ito, Kimura, Deguchi, Yajima, & Kan, 1993). Although an
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increase in Bacteroides and Clostridium spp. was observed in the transverse and descending

colons during treatment T1, no significant visible changes on NH4+ production were found. This
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result can be explained by the balance between an increase in some proteolytic bacteria and a
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decrease in others, such as Streptococcus, or the use of NH4+ by other groups of bacteria. The
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decrease in NH4+ levels in the colon is considered beneficial to the host’s health since high
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amounts of NH4+ can promote colon carcinogenesis by increasing DNA synthesis, culminating in

changes in the morphology and intermediary metabolism of intestinal cells (Davila et al., 2013;
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Ichikawa & Sakata, 1998).

5. Conclusions

This study provided novel insights on the synbiotic potential of Bifidobacterium longum

BB-46 with lemon pectin (T2) to modulate the gut microbiota. We observed that each treatment

had a different effect on gut microbiota. Treatment with B. longum BB-46 alone (T1) mainly
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stimulated members of Lachnospiraceae and Bacteroidaceae families, whereas the combination

of B. longum BB-46 with pectin stimulated specific bacterial groups able to degrade pectin and

to increase the production of butyric acid. Using the intestinal model SHIME ®, this study

indicated that the combination (T2) could positively modulate the composition and metabolic

activity of the gut microbiota. Although in this study we could deduce that the increase of butyric

acid as well as of specific bacterial genera, such as Faecalibacterium, occurred partly due to an

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interaction between the probiotic B. longum BB-46 and the pectin, new studies using the B.

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longum BB-46 and pectin alone as well as different types of pectins are welcome to confirm the

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beneficial effects of B. longum BB-46 combined or not with citric pectins on human health.
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Acknowledgements
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We would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo and

DCSR for financial support and fellowships. The authors also wish to thank Karin Meyer Hansen
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(CP Kelco, Denmark) and Thomas Lesser (Chr. Hansen, Denmark) for providing, respectively,
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the pectin from lemon and the Bifidobacterium longum BB-46, and for their valuable discussions
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of the results. This study was supported by Fundação de Amparo à Pesquisa do Estado de São

Paulo (FAPESP) (Projects 2013/50506-8, 2015/13965-0, 2015/08228-6 and 2016/20336-1),

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de

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Desenvolvimento Científico e Tecnológico (CNPq) and by the Danish Council for Strategic

Research (project BioSyn, no 3050-00005B).

Conflict of interest

The authors declare no conflict of interest.

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Figure legends

Fig. 1. Relative abundance of bacterial families in ascending (V3), transverse (V4) and
descending (V5) colon vessels of SHIME®. C= control period; T1= treatment with
Bifidobacterium longum BB-46; T2= treatment with BB-46 and pectin; PT= post-treatment.

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Fig. 2. Simple correspondence analysis showing relationship between the treatments and
bacterial families in the three vessels of SHIME® model. C= control period; T1= treatment

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with Bifidobacterium longum BB-46; T2= treatment with BB-46 and pectin; PT= post-
treatment; V3= ascending colon; V4= transverse colon; V5= descending colon.

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Fig. 3. Cluster analysis of the relative abundance of bacterial genera significantly changed
during treatments in SHIME® colon vessels. Significant increase (p < 0.05) compared to
control is represented by “●” and significant reduction (p < 0.05) compared to control is
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represented by “○” (One-way ANOVA and Tukey post-hoc test). C= control period; T1=
treatment with Bifidobacterium longum BB-46; T2= treatment with BB-46 and pectin; PT=
post-treatment. V3= ascending colon; V4= transverse colon; V5= descending colon.
Unclassified genera are represented by “g_“.
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Fig. 4. Production of butyric, acetic and propionic acids by the microbiota in SHIME® colon
vessels. Different letters denote statistical differences (p < 0.05) between the treatments for the
same vessel (One-way ANOVA and Tukey post-hoc test). C= control period; T1= treatment with
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Bifidobacterium longum BB-46; T2= treatment with BB-46 and pectin; PT= post-treatment; V3=
ascending colon; V4= transverse colon; V5= descending colon.
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Fig. 5. Correlation between SCFA production, ammonium ions, and bacterial genera. Positive
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correlations are represented by blue color and negative correlations by red color. Color intensity
are proportional to the correlation coefficients (Spearman correlation). Significant correlations
are indicated by *(p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001). Unclassified
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genera are represented by “g_ “.

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Table 1
NH4+ production (mmol/L) by the microbiota in SHIME® colon vessels.a

Treatment Treatment with Post-

Control with BB-46 BB-46 and pectin treatment
Ascending colon 16.63±0.11A 15.90±0.16A 4.23±0.27C 7.84±0.19B
Transverse colon 20.51±0.03A 20.87±0.38A 6.31±0.11C 17.21±0.15B
Descending colon 22.73±0.38A 21.20±0.54A 8.58±0.85C 17.37±0.07B

a
Different letters represent statistical difference (p < 0.05) between treatments for the same vessel (One-

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way ANOVA and Tukey post-hoc test).

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Highlights

● Citric pectin combined with probiotic BB-46 modulated the human gut microbiota

● Probiotic BB-46 modified the gut microbiota differently from BB-46 combined with pectin

● Citric pectin combined with BB-46 increased intestinal butyric acid production

● Citric pectin combined with BB-46 decreased intestinal ammonium ions production

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.

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Figure 1
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