Bifidobacterium lactis HN019: Survival, acid production and impact on sensory acceptance of fermented milk

Abstract

Bifidobacterium lactis HN019 was monitored for its viability and acid production during milk fermentation and cold storage. Milk was fermented for 72 h at 37°C, and the shelf life of the resulting probiotic milk was evaluated at 4°C for 30 d. Additionally, lactic and acetic acids and lactose contents during the fermentation were measured by in situ quantitative proton nuclear magnetic resonance spectroscopy (isq ¹ H NMR), and the sensorial acceptance of the fermented milk was evaluated by 80 panellists. The pH of the milk fermented by B. lactis HN019 reduced from 6.36 to 3.97 after 72 h incubation. The viability of the evaluated strain was maintained above 9.21 up to a decrease to 8.85 log CFU/mL after 48 h fermentation. With respect to metabolism, isq ¹ H NMR revealed that the acetic and lactic acid contents increased from 0.47 ± 0.06 g/L to 16.36 ± 1.86 g/L and 2.59 ± 0.10 g/L to 9.40 ± 0.18 g/L, respectively, indicating high production of acetic acid by HN019 during the 72 h fermentation. On the other hand, lactose content decreased throughout the fermentation period. In fact, strain HN019 showed a high content of acetate throughout fermentation; however, exhibited the stability required in fermented milks with high viable cell counts and low activity of post-acidification during the shelf life. In sensory evaluation, the panellists preferred the probiotic milk fermented for 8 h because after 72 h the acetic flavour was already perceptible.

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Food Research International
journal homepage: www.elsevier.com/locate/foodres
In vitro effects of Bifidobacterium lactis-based synbiotics on human faecal
bacteria
Fernanda C. Henrique-Bana
a,b,⁎
, Xuedan Wang
a
, Giselle N. Costa
c
, Wilma A. Spinosa
b
,
Lucia H.S. Miglioranza
b
, Eleonora Scorletti
d,e,f
, Philip C. Calder
d,e
, Christopher D. Byrne
d,e
,
Glenn R. Gibson
a
a
Department of Food and Nutritional Sciences, The University of Reading, Reading RG6 6AP, UK
b
Department of Food Science and Technology, State University of Londrina, Celso Garcia Cid Road, Km 380, 86051-970 Londrina, PR, Brazil
c
Program of Master in Dairy Science and Technology, University Pitágoras Unopar, Rua Marselha, 591, 86041-140 Londrina, PR, Brazil
d
Nutrition and Metabolism, Human Development and Health, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK
e
National Institute for Health Research Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton National Health
Service (NHS) Foundation Trust, Southampton SO16 6YD, UK
f
Department of Gastroenterology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA
ARTICLE INFO
Keywords:
Probiotic
Prebiotic
Maltodextrin
Faecal microbiota
Batch culture system
Short-chain fatty acids
ABSTRACT
Synbiotic supplements contain pre- and probiotics and are used to modulate gut microbiota composition. This
study aimed to investigate effects of two synbiotic mixtures on human faecal bacteria in vitro. Short chain
fructooligosaccharides (FOS) (1% w/v) combined with either Bifidobacterium lactis Bb12 or Bifidobacterium lactis
HN019 (10
6
colony-forming units (CFU)/mL)] were added to pH-controlled anaerobic batch cultures inoculated
with human faeces. Maltodextrin (1% w/v), FOS (1% w/v) and the probiotic strains were also tested in-
dividually. Effects on bacteria, short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) were
assessed over 48 h. With maltodextrin, FOS and the synbiotic mixtures, there was a significant increase in total
bacteria and bifidobacteria numbers, compared to the negative control or probiotics alone. Increases in
Atopobium cluster and Clostridium coccoides-Eubacterium rectale group occurred with FOS and maltodextrin, re-
spectively. Additionally, maltodextrin, FOS and synbiotics resulted in a greater production of acetate and bu-
tyrate (SCFAs) compared to the negative control and probiotics alone, whereas concentrations of iso-valerate
(BCFA) were lower with these treatments. In conclusion, synbiotic-induced in vitro bacterial changes and changes
in SCFAs concentrations were not different from those observed with FOS alone. These data suggest that me-
tabolic effects of these synbiotics are largely driven by the prebiotic component.
1. Introduction
In recent years, studies have indicated that probiotics, prebiotics
and their combination (synbiotics) may contribute to the maintenance
or improvement of health and prevention of diseases by modifying the
gut microbiota (Hadi, Mohammadi, Miraghajani, & Ghaedi, 2018;
Krumbeck et al., 2018; Markowiak & Śliżewska, 2017). Probiotics are
defined as ‘live microorganisms that, when administered in adequate
amounts, confer a health benefit on the host’(Hill et al., 2014). Pre-
biotics also target gut microbiota with the goal of improving health.
Whereas probiotics are live microorganisms, a prebiotic is ‘a substrate
that is selectively utilized by host microorganisms conferring a health
benefit’(Gibson et al., 2017). Thus, prebiotics serve as nutrients for
beneficial microorganisms harboured by the host, including adminis-
tered probiotic strains and indigenous (resident) microorganisms
(Gibson et al., 2017). However, in the past few years, prebiotics have no
longer seen simply as boosters of bifidobacteria and lactobacilli but are
now recognised for their effects on system-wide metabolic and phy-
siological readouts (Sanders, Merenstein, Reid, Gibson, & Rastall,
2019).
A number of fermentable carbohydrates have been reported to
convey health benefits to the host, but other substances such as poly-
phenols and polyunsaturated fatty acids might also exert prebiotic ef-
fect. In this context, fructooligosaccharides (FOS) and
https://doi.org/10.1016/j.foodres.2019.108776
Received 4 July 2019; Received in revised form 18 October 2019; Accepted 26 October 2019
⁎
Corresponding author at: Department of Food Science and Technology, State University of Londrina, Celso Garcia Cid Road, Km 380, 86051-970 Londrina, PR,
Brazil.
E-mail addresses: ferchenrique@gmail.com,lab.alimentos@uel.br (F.C. Henrique-Bana).
Food Research International 128 (2020) 108776
Available online 08 November 2019
0963-9969/ © 2019 Published by Elsevier Ltd.
T

galactooligosaccharides (GOS) currently dominate the prebiotic cate-
gory (Gibson et al., 2017). These oligosaccharides selectively promote
beneficial microorganisms within the gut microbiota and increase
production of short-chain fatty acids (SCFAs), among many other
benefits (Salazar et al., 2015; Simpson & Campbell, 2015). The SCFAs
are crucial for intestinal health and can also influence sites distant to
the gut, since they may supply energy to colon enterocytes and function
as signaling molecules, influencing intestinal permeability. SCFA re-
ceptors are not only found in the gut, but are also expressed in immune
cells and human white adipose tissue (Choque Delgado & Tamashiro,
2018; Gibson et al., 2017).
In a general sense, probiotic and prebiotic interventions serve to
increase the community of beneficial microorganisms and products of
their growth and metabolism in the host (Sanders et al., 2019). In this
manner, it is known that microbiota composition can also be success-
fully modulated by certain synbiotic treatments. In vivo and in vitro
studies have shown the positive effects of specific synbiotics (Freitas
et al., 2018; Kim, Keogh, & Clifton, 2018; Krumbeck, Walter, & Hutkins,
2018; Panigrahi et al., 2017); however, the development of effective
combinations remains a challenging issue. It is currently unknown if it
is possible for a probiotic strain to benefit from the presence of a pre-
biotic substrate in the competitive environment of the human gut
(Krumbeck et al., 2018).
Bifidobacterium lactis Bb12 and B. lactis HN019 are widely used bi-
fidobacterial strains in fermented dairy production that may convey
health-promoting properties in human hosts (Jungersen et al., 2014;
Meng et al., 2016). One approach to enrich for bifidobacteria, increase
the number of responders, and enhance their metabolic activity would
be to administer a prebiotic together with a select strain or that use the
prebiotic as a growth substrate.According to ecological theory, the
provision of resources in a microbial community leads to a relaxation of
competition and therefore could enhance colonization success of pro-
biotic strains (Walter, Maldonado-Gómez, & Martínez, 2018). Despite
the proven bifidogenic effect of FOS, bifidobacteria ability to use dif-
ferent carbohydrates varies among strains (Milani et al., 2015).
The synbiotic combination of B. lactis Bb12 and FOS, was used in the
INvestigation of SYbiotic TrEatment (INSYTE) study (www.clinicaltrials.
gov registration number NCT01680640); a unique proof-of-concept
double blind, randomised, placebo-controlled trial with a ~12 month
intervention in patients with non-alcoholic fatty liver disease (NAFLD)
(Scorletti et al., 2018). The aim of the current study was to investigate
whether: (a) Bifidobacterium lactis Bb12 and FOS and (b) B. lactis HN019
and FOS, were more effective than maltodextrin, FOS or either pro-
biotic alone, using an in vitro faecal bacterial culture system. The po-
tential fermentation properties of the two synbiotics were investigated
using pH-controlled anaerobic faecal batch cultures. Levels of selected
microbial groups and concentrations of SCFAs and BCFAs were then
quantified in order to assess relationships between changes in organic
acids and variations in microbial populations.
2. Materials and methods
2.1. Substrates
Maltodextrin and FOS with a degree of polymerization < 10 were
supplied by Chr. Hansen, Hørsholm, Denmark. Freeze-dried
Bifidobacterium animalis subsp. lactis HN019 (DuPont-Danisco, Madison,
USA) and Bifidobacterium animalis subsp. lactis Bb12 (Chr. Hansen,
Hørsholm, Denmark) were stored at −80 °C. Plates of de Man-Rogosa-
Sharpe (MRS) agar (Oxoid Ltd, Basingstoke, UK) were inoculated with
the bifidobacterial strains and incubated at 37 °C in an anaerobic
chamber (10% CO
2
, 10% H
2
and 80% N
2
, Don Whitley Scientific Ltd,
Shipley, UK) for 48 h. After incubation, bottles containing 9 mL of MRS
broth were then inoculated with one colony from each plate, and in-
cubated for 24 h under the same conditions, as mentioned above.
2.2. Faecal sample preparation
Faecal samples were collected from three healthy individuals (2
women and 1 man aged between 25 and 35 years) who had not taken
antibiotics, probiotics or prebiotics for at least six months before the
faecal collection, and who had no history of gastrointestinal disorders.
The number of volunteers selected in this study was based on previously
batch cultures studies (Monteagudo-Mera et al., 2018; Ramnani,
Costabile, Bustillo, & Gibson, 2015; Wang et al., 2019). Faecal samples
were collected, on site, on the day of the experiment and placed in an
anaerobic jar (AnaeroJar™2.5L, Oxoid Ltd), including a gas-generating
kit (AnaeroGen™, Oxoid). Samples were prepared as reported by
Guergoletto, Costabile, Flores, Garcia, and Gibson (2016) and 15 mL of
the resulting faecal slurries were immediately used to inoculate the
batch-culture systems.
2.3. In vitro batch culture fermentation
Sterile stirred batch culture fermentation vessels (300 mL working
volume) were prepared and aseptically filled with 135 mL of sterile
basal nutrient medium, prepared as reported by Guergoletto et al.
(2016). Once in the fermentation vessels, sterile medium was main-
tained under anaerobic conditions by sparging the vessels with O
2
-free
N
2
overnight (15 mL/min). The temperature was held at 37 °C using a
circulating water bath and pH values controlled between 6.7 and 6.9
using an automated pH controller (Fermac 260; Electrolab, Tewkes-
bury, UK) which added acid or alkali as required (0.5 M HCl and 0.5 M
NaOH).
Seven vessels (a–g below) containing faecal slurry were prepared for
treatments with: (a) maltodextrin (1% w/v), (b) FOS (1% w/v), (c) B.
lactis Bb12 (10
6
CFU/mL), (d) B. lactis HN019 (10
6
CFU/mL), (e) FOS
combined with B. lactis Bb12 (Syn 1), (f) FOS combined with B. lactis
HN019 (Syn 2) at the same concentrations as when used alone, and (g)
a negative control (without any added substrate or microorganism).
Maltodextrin was tested alone since this carbohydrate is commonly
used as placebo/control for studies testing the effects of prebiotics in
humans. Seven batch culture fermenters were thus run in parallel and
the experiment was performed in triplicate, using one faecal sample
from a different donor each time. The probiotics and carbohydrates
were added to each vessel just before the addition of 15 mL (1:10, w/v)
of fresh faecal slurry. Batch cultures experiments were conducted for
48 h, and 4 mL samples obtained from each vessel at 0, 8, 24 and 48 h
for analyses.
2.4. Enumeration of bacterial populations by FISH analysis
Fluorescence in situ hybridisation (FISH) analysis by flow cytometry
was performed as described by Daims, Stoecker, and Wagner (2005).
Briefly, aliquots (750 μl) of batch culture samples were centrifuged at
1136gfor 5 min. Pellets were suspended in 375 μlfiltered 0.1 M Phos-
phate-buffered saline (PBS) solution. Filtered 4% paraformaldehyde
(PFA) (1125 µl) was added and samples were stored at 4 °C for 4 h.
Samples were then washed twice with PBS to remove PFA and re-sus-
pended in a mixture containing 300 µl PBS and 300 µl 99% ethanol.
Samples were then stored at −20 °C, until used for hybridization as
described by Wang et al. (2019). The probes used (Sigma Aldrich Ltd,
Poole, UK) are listed in Table 1.
2.5. Short-chain Fatty Acids (SCFAs) analysis
SCFAs were measured by gas chromatography as previously re-
ported by Richardson, Calder, Stewart, and Smith (1989). Aliquots of
1 mL of sample supernatant were transferred into glass tubes with 50 μl
of 2-ethylbutyric acid (0.1 M, internal standard) (Sigma, Poole, UK).
500 μl of concentrated hydrochloric acid (HCl) and 2 mL of diethyl
ether (Sigma Aldrich Ltd., Poole, UK) were added to each glass tube and
F.C. Henrique-Bana, et al. Food Research International 128 (2020) 108776
2

samples vortexed for 1 min. Samples were centrifuged at 2000gfor
10 min. The diethyl ether (upper) layer of each sample was transferred
to a labelled clean glass tube. A second extraction was conducted by
adding another 0.5 mL diethyl ether, followed by vortexing and re-
centrifugation. The diethyl ether layers were pooled. 400 μl of pooled
ether extract and 50 μl N-(tert-butyldimethylsilyl)-N-methyltri-
fluoroacetamide (MTBSTFA) (Sigma-Aldrich, Poole, UK) were added
into a GC screw-cap vial. Samples were left at room temperature for
72 h to allow SCFAs in the samples to completely derivatise.
An Agilent/HP 6890 Gas Chromatograph (Hewlett Packard, UK)
fitted with a HP-5MS 30 m × 0.25mm column with a 0.25 μm coating
(Crosslinked (5%-Phenyl)-methylpolysiloxane, Hewlett Packard, UK)
was used for analysis of derivatised SCFAs and BCFAs. Temperatures of
injector and detector were 275 °C. The column was programmed to
increase in temperature from 63 °C to 190 °C at a rate of 15 °C min
−1
and was then held at 190 °C for 3 min. Helium was the carrier gas (flow
rate 1.7 mL min
−1
; head pressure 133 KPa). A split ratio of 100:1 was
used. Quantification of the samples was obtained through calibration
curves of lactic, acetic, propionic, butyric, valeric, iso-butyric and iso-
valeric acids at concentrations between 12.5 and 100 mM.
2.6. Statistical analysis
Differences within each treatment were evaluated after 8, 24 and
48 h of fermentation compared with baseline (0 h of fermentation)
using paired Student's t-tests and were considered significantly different
from baseline when P ≤0.05. The data were then analysed to compare
different treatments at the same time point using the post hoc analysis
(Tukey test) and P ≤0.05 to indicate significance. Analyses were per-
formed using Statistica software version 10.0 (Statsoft South America).
3. Results
3.1. Bacterial enumeration by FISH
Bacterial counts in control and treatment groups are shown in
Figs. 1 and 2. Increases in total bacterial levels (Fig. 1A) were observed
after 8 h for maltodextrin, FOS, Syn 1 and Syn 2 compared to probiotics
alone (P = 0.014, P = 0.034, P = 0.013 and P = 0.032, respectively).
The highest number of bacteria was detected in the fermentation with
Bb12 + FOS (Syn 1) at 8 h, Log
10
8.34 ± 0.18 CFU/mL, while at
baseline counts were Log
10
7.56 ± 0.38 CFU/mL.
A significant increase in Bifidobacterium spp. numbers (Fig. 1B) was
also observed for maltodextrin, FOS, Syn 1 and Syn 2 (P = 0.022,
P = 0.038, P = 0.034 and P = 0.023, respectively) compared to pro-
biotics and the negative control. Bifidobacteria counts ranged from
Log
10
5.64 ± 0.32 at baseline to 7.77 ± 0.52 CFU/mL after 8 h of
fermentation with Syn 1. Regarding Lactobacillus-Enterococcus group
(Fig. 1C), a rise was observed, but the increase was not significant at all
time points (P > 0.10), the highest number was detected in the FOS
culture at 24 h.
Levels of Clostridium coccoides–Eubacterium rectale group (Fig. 1E)
significantly increased at 8 and 24 h of fermentation with maltodextrin
(P = 0.028 and P = 0.038, respectively) (Log
10
6.61 ± 0.23 to
7.63 ± 0.53 CFU/mL). An increase in Atopobium cluster (Fig. 2A) was
observed with FOS fermentation at 8 h (Log
10
5.12 ± 0.59 to
7.02 ± 0.54 CFU/mL) (P = 0.034). Because of high variations among
the volunteers at 8 and 24 h, the increase in Atopobium cluster was not
significant for maltodextrin and synbiotic mixtures compared to base-
line.
No significant differences were found for the other bacterial groups
analysed, including Bacteroides spp.-Prevotella group, Roseburia genus,
Clostridial cluster IX populations, F. prausnitzii group, Desulfovibrio
genus, Clostridium histolyticum group and Cytophaga-Flexibacter-
Bacteroides.
3.2. SCFAs concentrations
Following administration of all treatments, acetate was the main
end product of microbial fermentation (Table 2). Acetate concentra-
tions were highest with maltodextrin, FOS and synbiotic mixtures
(P < 0.0002) at all time points (8, 24 and 48 h) compared to the ne-
gative control and probiotics; however, there were no significant dif-
ferences among these four treatments (maltodextrin, FOS, Syn 1 and
Syn 2). Acetate levels ranged from 4.12 ± 1.57 mM at baseline to
77.34 ± 9.14 mM after 48 h of fermentation with FOS.
Supplementation with maltodextrin had the largest effect in in-
creasing the concentration of butyrate at 8 h (1.58 ± 0.60 mM), which
was significantly higher (P < 0.011) than the negative control
(0.36 ± 0.07 mM) and other treatments. After 24 h of fermentation,
the concentration of butyrate was also higher in vessels with FOS
(1.93 ± 0.13 mM, P < 0.004), Syn 1 (2.49 ± 0.55 mM, P < 0.002)
and Syn 2 (2.56 ± 1.03 mM, P < 0.001) compared to the negative
control (0.60 ± 0.09 mM) and probiotics (0.59 ± 0.12 mM with Bb12
Table 1
Name, sequence, and target group of oligonucleotide probes used for Fluorescence in situ hybridization of bacterial enumeration. +: These probes are used together
in equimolar concentration of 50 ng/μL.
Probe name Sequence (5′–3′) Target group References
Non Eub ACTCCTACGGGAGGCAGC Control probe complementary to EUB338 Wallner, Amann, and Beisker (1993)
Eub338 I + GCT GCC TCC CGT AGG AGT Most bacteria Daims, Brühl, Amann, Schleifer, and Wagner
(1999)
Eub338 II + GCA GCC ACC CGT AGG TGT Planctomycetales Daims et al. (1999)
Eub338 III + GCT GCC ACC CGT AGG TGT Verrucomicrobiales Daims et al. (1999)
Bif164 CAT CCG GCA TTA CCA CCC Bifidobacterium spp. Langendijk et al. (1995)
Lab158 GGTATTAGCAYCTGTTTCCA Lactobacillus and Enterococcus Harmsen, Elfferich, Schut, and Welling (1999)
Bac303 CCA ATG TGG GGG ACC TT Most Bacteroidaceae and Prevotellaceae, some
Porphyromonadaceae
Manz, Amann, Ludwig, Vancanneyt, and Schleifer
(1996)
Erec482 GCT TCT TAG TCA RGT ACCG Most of the Clostridium coccoides-Eubacterium rectale group (Clostridium
clusters XIVa and XIVb)
Franks et al. (1998)
Rrec584 TCA GAC TTG CCG YAC CGC Roseburia genus Walker, Duncan, Leitch, Child, and Flint (2005)
Ato291 GGT CGG TCT CTC AAC CC Atopobium cluster Harmsen, Wildeboer-Veloo, Grijpstra, Knol, and
Welling (2000)
Prop853 ATT GCG TTA ACT CCG GCAC Clostridial cluster IX Walker et al. (2005)
Fprau655 CGCCTACCTCTGCACTAC Faecalibacterium prausnitzii and related sequences Suau et al. (2001)
DSV687 TAC GGA TTT CAC TCC T Desulfovibrio genus Ramsing, Fossing, Ferdelman, Andersen, and
Thamdrup (1996)
Chis150 TTATGCGGTATTAATCTYCCTTT Most of the Clostridium histolyticum group (Clostridium clusters I and II) Franks et al. (1998)
CFB286 GTAGGGGTTCTGAGAGGA Cytophaga-Flexibacter-Bacteroides O’Sullivan, Weightman, and Fry (2002)
F.C. Henrique-Bana, et al. Food Research International 128 (2020) 108776
3

and 0.52 ± 0.14 mM with HN019 strain).
Propionate was increased in all vessels compared to baseline
(P < 0.046); for instance, values ranged from 0.17 ± 0.07 to
18.55 ± 9.54 mM after 48 h of fermentation with FOS. However, be-
cause of high variations among volunteers, increase of this SCFAs in all
treatments was not significant compared to the negative control
(P > 0.15).
On the other hand, isovalerate, a branched-chain fatty acid (BCFA)
product of amino acid metabolism was higher in the probiotic vessels
(0.12 ± 0.05 mM with Bb12 and 0.11 ± 0.05 mM with HN019,
P < 0.009) and the negative control (0.17 ± 0.03 mM, P < 0.016)
after 24 and 48 h of fermentation compared to the carbohydrate-con-
taining vessels. Finally, to a lesser extent, valerate and isobutyrate
concentrations were higher (P < 0.033 and P < 0.049, respectively)
after all treatments compared to baseline, but no statistical difference
was found among treatments (P > 0.38).
4. Discussion
In this study, the experimental design compared probiotics and
prebiotics alone and in combination in a controlled setting. Our novel
results show that there were marked changes in total bacteria and
Fig. 1. Total bacterial (A), Bif164 (B), Lab158 (C), Bac303 (D), Erec482 (E) and Rrec584 (F) changes over time as log
10
CFU/mL in anaerobic batch culture as
analysed by Fluorescence in situ hybridization (FISH). Values are mean ± standard deviation at four time points from batch cultures of faeces from three healthy
donors. *Mean values were significantly different from baseline (P < 0.05) within the same treatment. Different letters in the same time of different treatments are
shown when significantly different (P < 0.05) by Tukey's test.
F.C. Henrique-Bana, et al. Food Research International 128 (2020) 108776
4

bifidobacterial numbers in human faecal slurry in response to incuba-
tion with either FOS, maltodextrin or either synbiotic mixture.
Bifidobacteria are recognised as one of the most important bacterial
groups associated with human health, providing beneficial effects in the
large intestine (Russel, Ross, Fitzgerald, & Stanton, 2011) and it is well
established that an increase in bifidobacterial numbers is favoured by
the presence of (mainly) fermentable carbohydrates. In fact, a simple
bifidobacterial community may co-operate between themselves as well
as with other members of the gut microbiota in the utilization of
specific glycans, by means of cross-feeding activities, so as to provide
growth benefits to one or both members of such a community as well as
with the other members of the gut microbiota (Turroni et al., 2018).
Surprisingly our data also show that there was increased growth of
bifidobacteria with maltodextrin supplementation. Whereas FOS (along
with GOS) is the dietary prebiotic most extensively documented to
confer health benefits in humans, maltodextrin is a mal-
tooligosaccharide not usually considered to be a prebiotic. FOS is pre-
ferentially metabolized by bifidobacteria, since its β(1 →2)-glycosidic
Fig. 2. Ato291 (A), Prop853 (B), Fprau655 (C), DSV687 (D), Chis150 (E) and CFB286 (F) changes over time as log10 CFU/mL in anaerobic batch culture as analysed
by Fluorescence in situ hybridization (FISH). Values are mean values ± standard deviation at four time points from batch cultures of faeces from three healthy
donors. *Mean values were significantly different from baseline (P < 0.05) within the same treatment. Different letters in the same time of different treatments are
shown when significantly different (P < 0.05) by Tukey's test.
F.C. Henrique-Bana, et al. Food Research International 128 (2020) 108776
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bonds can be readily degraded by β-fructanosidase enzyme (prevalent
in bifidobacteria) (Gibson et al., 2017). It was postulated that enhanced
growth of bifidobacteria in the presence of maltodextrin was probably
due to the ability of some bifidobacterial species to produce the enzyme
that hydrolyses maltodextrin to glucose for growth (Yeo & Liong, 2010),
confirming the bifidogenic activity of maltodextrin in vitro. However, it
is important to emphasize that maltodextrin is a common placebo for
studies testing the effects of prebiotics in humans (Costabile et al.,
2010; Kolida, Meyer, & Gibson, 2007; Pedersen et al., 2016; Ramnani
et al., 2015). Since maltodextrins are partially depolymerized starch
granules, digestion of maltodextrin occurs through the same starch
digesting enzymes in humans (α-amylase and maltase). The glucose and
maltose obtained from maltodextrin digestion is readily absorbed in the
small intestine and subsequently used in metabolism (Hofman, Van
Buul, & Brouns, 2016), and therefore, no intact substrate reaches the
colon to be fermented by the intestinal bacteria such as bifidobacteria.
The impact of bifidobacteria in the breakdown of dietary carbohy-
drates is crucial for the establishment and reinforcement of trophic
relationships among members of the gut microbiota (Turroni et al.,
2018). In particular, bifidobacteria possess a range of cell-associated
and extracellular glycosidases and specific transport systems enabling
them to rapidly assimilate low-molecular weight sugars (Rivière, Selak,
Geirnaert, Van den Abbeele, & De Vuyst, 2018). On the other hand,
other microorganisms are adept at breaking down high molecular
weight polysaccharides and this pathway from a polysaccharide to a
SCFA is a complex and indirect network of metabolism (Sanders et al.,
2019). Since lactate and acetate are utilized by other microorganisms to
produce propionate and butyrate, probable ecological networks in-
volved in the metabolism of carbohydrates have been elucidated
(Rivière et al., 2018; Scott, Martin, Duncan, & Flint, 2014), although
the extent to which they operate in the gut is not clear at the present
time. In this respect, lactate and acetate produced by bifidobacteria
may be substrates for butyrate-producing colon bacteria such as Eu-
bacterium rectale (Moens, Verce, & De Vuyst, 2017). Numbers of Clos-
tridium coccoides-Eubacterium rectale group were increased in the mal-
todextrin vessel, possibly due to a cross-feeding between species. E.
rectale is recognised as one of the most prolific butyrate producers in the
human colon. Coinciding with the increase in E. rectale numbers, the
highest concentration of butyrate after 8 h was found in the vessel with
maltodextrin. FOS and synbiotic mixture fermentations also increased
butyrate concentrations after 24 and 48 h. Butyrate also has an im-
portant role in the colon because of its beneficial effects in the colonic
epithelium (Canani et al., 2011).
Another group of bacteria stimulated by FOS was Atopobium, com-
monly isolated from healthy human faeces, which belongs to the
Collinsella genus (Thorasin, Hoyles, & McCartney, 2015). From the
present study, it is not possible to distinguish whether the Atopobium
group can ferment FOS, although it is able to metabolize fructose
(Moore, Cato, & Holdeman, 1971) or if the increase in numbers is due to
cross-feeding between different bacterial groups. An increase in this
group has already been reported in vitro with FOS (Saulnier, Gibson, &
Kolida, 2008); but the role of Atopobium in the human colon is still
unclear.
Although B. lactis fermentation in probiotic vessels had no sig-
nificant effect upon relevant microbial populations at the level of genus,
we can speculate that an interplay between B. lactis and other strains
may have occurred. This outcome is plausible due to the highly com-
petitive environment that favours autochthonous strains (Walter et al.,
2018) and/or the numbers of probiotic bacteria that were added was
possibly too low to have a discernible effect. Since most commercial
probiotic strains belong to species that are allochthonous to the human
gastrointestinal tract (such as B. lactis), and lack the required traits to
Table 2
Short-chain fatty acids (SCFAs) concentrations (mM) in pH-controlled batch cultures at 0, 8, 24 and 48 h (n = 3).
Time point Mean SCFA concentration (mM) in treatment ( ± SD)
Maltodextrin FOS Bb12 HN019 Bb12 + FOS HN109 + FOS Negative control
Acetate 0h 4.22 ± 1.50
a
4.12 ± 1.57
a
4.30 ± 1.63
a
4.37 ± 1.62
a
4.21 ± 1.41
a
4.24 ± 1.36
a
4.22 ± 1.68
a
8 h 61.15 ± 10.34*
a
61.03 ± 9.55*
a
10.94 ± 0.76*
b
8.94 ± 4.28*
b
58.59 ± 14.48*
a
65.84 ± 9.47*
a
12.57 ± 0.74*
b
24 h 68.38 ± 7.11*
a
66.75 ± 14.33*
a
15.30 ± 0.07*
b
14.90 ± 2.89*
b
70.75 ± 4.42*
a
71.39 ± 2.58*
a
16.30 ± 0.84*
b
48 h 69.60 ± 10.01*
a
77.34 ± 9.14*
a
15.64 ± 0.75*
b
15.38 ± 0.76*
b
72.16 ± 3.52*
a
70.38 ± 2.05*
a
16.81 ± 1.54*
b
Propionate 0 h 0.23 ± 0.12
a
0.17 ± 0.07
a
0.23 ± 0.08
a
0.20 ± 0.06
a
0.24 ± 0.12
a
0.23 ± 0.07
a
0.22 ± 0.05
a
8 h 8.29 ± 3.88
a
6.52 ± 4.85
a
2.21 ± 0.26*
a
1.61 ± 1.06
a
5.95 ± 1.84*
a
5.52 ± 2.08*
a
2.45 ± 0.07*
a
24 h 17.40 ± 8.29*
a
15.07 ± 8.81
a
3.44 ± 0.63*
a
3.15 ± 0.17*
a
17.96 ± 7.31*
a
15.07 ± 7.55
a
4.03 ± 0.28*
a
48 h 17.58 ± 8.35*
a
18.55 ± 9.54*
a
3.54 ± 0.41*
a
3.27 ± 0.37*
a
16.15 ± 6.44*
a
17.41 ± 9.11
a
4.20 ± 0.64*
a
Butyrate 0 h 0.02 ± 0
a
0.02 ± 0
a
0.02 ± 0
a
0.02 ± 0
a
0.02 ± 0
a
0.02 ± 0
a
0.02 ± 0
a
8 h 1.58 ± 0.60*
a
0.68 ± 0.61
b
0.27 ± 0.10*
b
0.24 ± 0.21
b
0.77 ± 0.19*
b
0.66 ± 0.21*
b
0.36 ± 0.07*
b
24 h 2.56 ± 0.16*
a
1.93 ± 0.13*
a
0.54 ± 0.07*
b
0.46 ± 0.16*
b
2.49 ± 0.55*
a
2.56 ± 1.03
a
0.56 ± 0.09*
b
48 h 2.68 ± 0.05*
a
2.47 ± 0.28*
a
0.59 ± 0.12*
b
0.52 ± 0.14*
b
2.55 ± 0.50*
a
2.79 ± 0.73*
a
0.60 ± 0.09*
b
Lactate 0 h 0.01 ± 0
a
0.01 ± 0
a
0.01 ± 0
a
0.01 ± 0
a
0.01 ± 0a 0.01 ± 0
a
0±0
a
8 h 0.45 ± 0.40
a
1.05 ± 0.57
a
0±0
a
0 ± 0.01
a
0.72 ± 0.46
a
1.02 ± 0.41
a
0±0
a
24 h 0 ± 0
a
0.15 ± 0.24
a
0±0
a
0±0
a
0±0
a
0±0
a
0±0
a
48 h 0 ± 0
a
0±0
a
0.01 ± 0.01
a
0±0
a
0±0
a
0±0
a
0±0
a
Isobutyrate 0h 0 ± 0
a
0.01 ± 0.02
a
0±0
a
0±0
a
0±0
a
0±0
a
0.02 ± 0.04
a
8 h 0.12 ± 0.06
a
0.07 ± 0.08
a
0.07 ± 0.02*
a
0.08 ± 0.01*
a
0.07 ± 0.03*
a
0.07 ± 0.03
a
0.10 ± 0.01
a
24 h 0.55 ± 0.13*
a
0.39 ± 0.30
a
0.45 ± 0.34
a
0.59 ± 0.36
a
0.59 ± 0.21*
a
0.55 ± 0.24
a
0.74 ± 0.27*
a
48 h 0.68 ± 0.08*
a
0.65 ± 0.25*
a
0.51 ± 0.31
a
0.79 ± 0.25*
a
0.71 ± 0.20*
a
0.67 ± 0.25*
a
0.82 ± 0.25*
a
Isovalerate 0 h 0 ± 0
a
0±0
a
0±0
a
0±0
a
0±0
a
0±0
a
0±0
a
8 h 0.01 ± 0*
a
0±0
a
0.01 ± 0*
a
0.01 ± 0*
a
0.01 ± 0*
a
0.01 ± 0*
a
0.02 ± 0*
a
24 h 0.04 ± 0.01*
b
0.02 ± 0.01
b
0.10 ± 0.05
a
0.08 ± 0.03*
a
0.04 ± 0.01*
b
0.04 ± 0*
b
0.16 ± 0.04*
a
48 h 0.07 ± 0.01*
b
0.05 ± 0.02*
b
0.12 ± 0.05*
a
0.11 ± 0.05*
a
0.06 ± 0.01*
b
0.05 ± 0.01*
b
0.17 ± 0.03*
a
Valerate 0 h 0 ± 0
a
0±0
a
0±0
a
0±0
a
0±0
a
0±0
a
0±0
a
8 h 0.09 ± 0.09*
a
0.07 ± 0.11*
a
0.09 ± 0.12*
a
0.02 ± 0.03*
a
0.08 ± 0.10*
a
0.07 ± 0.08*
a
0.13 ± 0.11*
a
24 h 0.26 ± 0.11
a
0.18 ± 0.14
a
0.23 ± 0.18
a
0.20 ± 0.18
a
0.29 ± 0.11*
a
0.27 ± 0.12
a
0.34 ± 0.04*
a
48 h 0.34 ± 0.04*
a
0.29 ± 0.15
a
0.27 ± 0.19
a
0.23 ± 0.19
a
0.35 ± 0.07*
a
0.33 ± 0.09*
a
0.36 ± 0*
a
* Mean values were significantly different from baseline (p < 0.05) within the same treatment. Different letters in the same line (same time) are significantly
different (P < 0.05) by Tukey's test.
F.C. Henrique-Bana, et al. Food Research International 128 (2020) 108776
6

successfully colonize gut ecosystems, the potential of using auto-
chthonous members of the human microbiome to develop next-gen-
eration probiotics and bio-therapeutics is increasingly recognized
(O'Toole, Marchesi, and Hill (2017)).
FOS, maltodextrin and synbiotic mixtures induced modulation of
faecal bacteria to increase acetate and butyrate production. Acetate is
produced mainly through the fructose-6-phosphate phosphoketolase
pathway by bifidobacteria (Miller & Wolin, 1996) and along with bu-
tyrate is considered beneficial to the human gut (Gibson et al., 2017). In
this manner, the greater concentration of acetate is most likely related
to the increase of bifidobacteria in the FOS, maltodextrin and synbiotic
vessels and their production of acetate from carbohydrates (Rivière
et al., 2018).
In contrast to these findings, the presence of maltodextrin and FOS
inhibited production of the BCFA isovalerate perhaps due to the in-
crease of carbohydrate availability which reduces the relative protein
availability for bacterial fermentation. Since the end products of pro-
teolytic fermentation include toxic metabolites (such as certain phe-
nolic compounds, amines, and ammonia) (Gibson, 2004), this beneficial
effect may be mediated by decreased microbial proteolysis. Moreover, it
is important to emphasize that it is also possible for prebiotics, such as
FOS, to exert microbiota-independent effects (Bindels et al., 2017; Wu
et al., 2017). The hypothesis underlying much research on prebiotics
(and barrier function and inflammation) is that fermentation products
such as SCFA probably mediate the beneficial effects through several
mechanisms (Sanders et al., 2019).
Finally, the synbiotics tested in this study did not have superior
efficacy in vitro, compared to the prebiotic component alone, in mod-
ulating either the faecal microbiota towards a purportedly healthy
composition or the production of SCFAs. Therefore, our findings are of
value to science and society since it was possible to establish that the
improvement of outcomes in the synbiotics tested was induced by the
prebiotic component. However, that said, it is plausible that there could
be strain-specific changes and other health benefits beyond SCFA pro-
duction that we have not measured. Given that no synergism was ob-
served between FOS and the probiotic strains, future trials may explore
higher doses of probiotics in synbiotic combinations. Additionally, in
vivo effects of the synbiotic supplement (B. lactis Bb12 and FOS) on
changes in gut microbiota through 16S rRNA gene sequencing have
been looked at (Scorletti et al., 2018). Hopefully together with the re-
sults showed here, this will help to better understand effects of this
specific synbiotic on dysbiosis. Synbiotic treatment is safe and well
tolerated; therefore if it can be shown that these agents have efficacy to
ameliorate liver fat issues, they could be used in primary care settings to
treat patients with NAFLD who are in the early stages of liver disease.
Our data highlights the need for a careful assessment of specific
combinations, dosages and outcome measures used in synbiotic in-
vestigations of health and disease-related outcomes, since overall in-
terventions of synbiotics showed mixed findings (Freitas et al., 2018;
Kim et al., 2018; Krumbeck et al., 2018). Further well-designed ran-
domised controlled trials are needed, testing individual components
and probiotic/prebiotic combinations.
5. Conclusion
In the current study, maltodextrin, FOS and synbiotics led to
changes in relevant microbial populations and SCFAs concentrations,
compared with the B. lactis strains alone. Beneficial modulations were
observed in terms of higher levels of bifidobacteria and Clostridium
coccoides-Eubacterium rectale group combined with significant increases
also in acetate and butyrate concentrations. Neither bacterial changes,
at the level of bacterial genus, nor SCFAs production were different
with the synbiotics compared with the prebiotic alone. These data
suggest that metabolic effects of the synbiotics tested (FOS combined
with either B. lactis Bb12 or B. lactis HN019) are largely induced by the
prebiotic component.
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgements
We would like to thank Chr. Hansen Holding A/S, Boege Alle 10-12,
2970 Hoersholm, Denmark, who provided materials at no cost.
Funding
This work was supported by the National Institute of Health
Research through the NIHR Southampton Biomedical Research Centre
and by the Parnell Diabetes Trust. Also, this work was supported by a
scholarship to F.H.B from Coordination for the Improvement of Higher
Education Personnel (CAPES –Brazil) (process number 88881.131985/
2016-01).
Author contributions
FCHB was responsible for laboratory analyses, recruitment of do-
nors and writing the manuscript. XW was responsible for laboratory
analyses and interpretation of the results. GNC, WAS and LHSM were
responsible for interpretation of the results and writing the manuscript.
GRG was responsible for the original concept of the study, the study
design and for supervising the work. ES, PCC, CDB and GRG made
extensive restructuring and revisions of the manuscript.
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