International Journal of

Molecular Sciences

Article
In Vitro Evaluation of Different Prebiotics on the
Modulation of Gut Microbiota Composition and
Function in Morbid Obese and
Normal-Weight Subjects
Alicja M. Nogacka 1,2, * , Nuria Salazar 1,2 , Silvia Arboleya 1,2 , Patricia Ruas-Madiedo 1,3 ,
Leonardo Mancabelli 4 , Adolfo Suarez 2,5 , Ceferino Martinez-Faedo 6,7 , Marco Ventura 4 ,
Takumi Tochio 8 , Katsuaki Hirano 8 , Akihito Endo 9 , Clara G. de los Reyes-Gavilán 1,2 and
Miguel Gueimonde 1,2
1 Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de
Asturias (IPLA-CSIC), 33300 Villaviciosa, Asturias, Spain; [email protected] (N.S.);
[email protected] (S.A.); [email protected] (P.R.-M.);
[email protected] (C.G.d.l.R.-G.); [email protected] (M.G.)
2 Diet, Human Microbiota and Health Group, Institute of Health Research of the Principality of
Asturias (ISPA), 33011 Oviedo, Spain; [email protected]
3 Functionality and Ecology of Beneficial Microorganisms, Institute of Health Research of the Principality of
Asturias (ISPA), 33011 Oviedo, Spain
4 Laboratory of Probiogenomics, Department of Life Sciences, University of Parma, 43121 Parma, Italy;
[email protected] (L.M.); [email protected] (M.V.)
5 Digestive Service, Central University Hospital of Asturias (HUCA), 33011 Oviedo, Asturias, Spain
6 Endocrinology and Nutrition Service, Central University Hospital of Asturias (HUCA), 33011 Oviedo,
Asturias, Spain; [email protected]
7 Endocrinology, Nutrition, Diabetes and Obesity Group, Institute of Health Research of the Principality of
Asturias (ISPA), 33011 Oviedo, Spain
8 β-Food Sciences Co., Chita 478-0046, Japan; [email protected] (T.T.); [email protected] (K.H.)
9 Department of Food and Cosmetic Science, Tokyo University of Agriculture, Abashiri 099-2493, Japan;
[email protected]
* Correspondence: [email protected]




Received: 20 December 2019; Accepted: 28 January 2020; Published: 30 January 2020


Abstract: The gut microbiota remains relatively stable during adulthood; however, certain intrinsic
and environmental factors can lead to microbiota dysbiosis. Its restoration towards a healthy condition
using best-suited prebiotics requires previous development of in vitro models for evaluating their
functionality. Herein, we carried out fecal cultures with microbiota from healthy normal-weight and
morbid obese adults. Cultures were supplemented with different inulin-type fructans (1-kestose,
Actilight, P95, Synergy1 and Inulin) and a galactooligosaccharide. Their impact on the gut microbiota
was assessed by monitoring gas production and evaluating changes in the microbiota composition
(qPCR and 16S rRNA gene profiling) and metabolic activity (gas chromatography). Additionally, the
effect on the bifidobacterial species was assessed (ITS-sequencing). Moreover, the functionality of the
microbiota before and after prebiotic-modulation was determined in an in vitro model of interaction
with an intestinal cell line. In general, 1-kestose was the compound showing the largest effects. The
modulation with prebiotics led to significant increases in the Bacteroides group and Faecalibacterium
in obese subjects, whereas in normal-weight individuals, substantial rises in Bifidobacterium and
Faecalibacterium were appreciated. Notably, the results obtained showed differences in the responses
among the tested compounds but also among the studied human populations, indicating the need for
developing population-specific products.

Int. J. Mol. Sci. 2020, 21, 906; doi:10.3390/ijms21030906 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 906 2 of 17

Keywords: in vitro model; microbiota; prebiotics; gas production; obesity; functionality; HT29;
RTCA; SCFA; bifidobacterial-ITS

1. Introduction
The human intestinal microbiota represents a very complex and diverse microbial ecosystem that
remains relatively stable during adult life [1]. However, several intrinsic and environmental factors
can disrupt the microbiota composition, causing a microbiota “dysbiosis” [2]. Given the frequent
association of dysbiosis with different disease states, the restoration of the microbiota through dietary
modulation strategies could be a suitable approach. Among the different microbiota-modulation
strategies, the administration of prebiotic supplements has been associated with health benefits to
the gastrointestinal tract, cardiometabolism, mental health and mineral absorption, among others [3].
An international experts group has defined a prebiotic as a “substrate that is selectively utilized
by host microorganisms conferring a health benefit” [3]. Most often, these substrates are complex
carbohydrate moieties that, due to the presence of β-glycosidic bonds, are resistant to digestion during
their passage through the gastrointestinal tract, reaching almost intact the large intestine, where they
can be metabolized by the intestinal microbiota [4]. However, it is important to underline that resistance
to digestion is not enough, and, by definition, prebiotics substrate must be selectively utilized by the
microorganisms, with a subsequent promotion of health.
Traditionally, the selective nature of prebiotics have been specifically associated with the genera
Bifidobacterium and Lactobacillus [3]. Nevertheless, during the last two decades, the development of
culture-independent technologies has demonstrated that other intestinal microorganisms could be
affected as well. Among these, some butyrate producers, such as members of Clostridium cluster
XIVa and IV, have been found to be favored by prebiotic supplementation, and negative correlations
were also found with some pathogenic bacteria [3,5]. These results indicate more global changes
associated with prebiotics consumption than just the effects upon bifidobacteria and lactobacilli and
underline the importance of considering the total microbiota when screening compounds for their
prebiotic properties.
On the other hand, the metabolism of prebiotics leads to the generation, as main end-products, of
bacterial fermentation, of short-chain fatty acids (SCFA), among which are acetate, butyrate, propionate
and also branched SCFA (BSCFA: iso-valerate and iso-butyrate) and gases hydrogen, methane and
carbon dioxide [6]. These compounds are well-known mediators of the microbiota-host interaction,
playing an important role in host health [7,8].
To date, the most widely studied prebiotics include fructooligosaccharides (FOS) of variable
chain lengths, commercial preparations often containing a mixture of molecules [9], and
galactooligosaccharides (GOS), which are being often used in studies focusing on infants [10]. However,
the comparative studies on the impact of different prebiotics upon the microbiota in different population
groups are still scarce [11]. In this context, the assessment of the impact of prebiotics in microbiota
composition and metabolism using in vitro models as a tool for screening the most effective modulatory
strategies prior to accomplish expensive and complex human interventions, is valuable [12]. In vitro
models, such as fecal cultures, are broadly used. Moreover, complementing such models with gas
production assessment can be used for determining the fermentation profile of prebiotics by the
gut microbiota of different population groups [13–18]. Among these, obese subjects constitute an
interesting target, since some studies have shown that the use of prebiotics is an effective modulatory
strategy in obesity [19], and animal studies provide support for a potential beneficial effect on energy
homeostasis and weight loss [20]. In mice, an inverse relationship has been established between the
level of bifidobacteria and some features of the metabolic alterations linked to obesity (endotoxemia,
fat mass and glucose intolerance) [21]. Some of these were confirmed in human studies, such as the
increase of bifidobacterial levels after prebiotic treatment, with beneficial systemic consequences for
Int. J. Mol. Sci. 2020, 21, 906 3 of 17

obese individuals [21–24]. However, there is still limited evidence on the in vitro fermentation profiles
of different prebiotic compounds by the microbiota of obese humans [25–28] and its comparison with
that of normal-weight individuals. Moreover, the characteristics of the intestinal microbiota in the
extreme form of obesity (morbid obesity; MOB) (BMI ≥ 40 kg/m2 ) is still not completely known [29,30].
The variability in the response of the obese population to prebiotic and probiotic supplementation in
weight loss interventions [31] and the lower microbial richness generally characterizing the microbiota
of obese subjects [32] points to the gut microbiota as a target for investigation in this field.
Unfortunately, most often the in vitro screenings of prebiotic substrates have failed to consider
the microbiota complexity and the potential differences on the basal microbiota composition among
different human groups, with few studies selecting the best-suited compounds for defined population
groups [33]. The availability of fast, easy and cheap methods, considering the influence of the basal
microbiota, for assessing the fermentability and specificity of potential prebiotics would be of help in
the selection of prebiotics for specific applications to human groups. In this context, it is well-known
that the microbiota of obese subjects is different from that of normal-weight (NW) individuals [30,34,35],
suggesting that the impact of different prebiotic compounds in these human groups may also differ,
making advisable selecting the best-suited compounds for each of them. Therefore, in this study, we
aimed at the evaluation of fermentative dynamics of different prebiotic substrates and the assessment
of their impact on the composition and metabolic activity on the intestinal microbiota of lean and
extreme obese individuals.

2. Results

2.1. Gas Production and pH Variations during Fermentation

The check of gas production in real-time allowed us to assess the in vitro fermentative dynamics of
the different prebiotics. The decreases in pH and the gas formed by fecal microbiotas of NW and MOB
individuals after 24 h of incubation in the presence of different carbon sources are shown in Table 1.

Table 1. Cumulative gas produced (mL) and decreases of pH values (∆ pH) after 24 h of incubation in
fecal cultures with normal-weight (NW) and morbid obesity (MOB) microbiota. Kinetic parameters
were determined using the modified-Gompertz equation, in which “A” represents the upper asymptote
(mL) and “µ” is the rate of gas production (mL/h). The values not sharing the same superscript (a, b, c
or d) indicate significant differences (p-value < 0.05) among carbon sources for each population group
(NW or MOB).

Group Condition ∆ pH Cumulative Gas A µ R2

Control 0.10 a ± 0.06 5.10 a ± 0.65 5.111 0.39 0.979
Glucose b
−1.22 ± 0.43 b
18.44 ± 7.08 19.306 1.205 0.999
1-kestose −1.34 b ± 0.20 21.05 b ± 5.09 22.299 1.267 0.997
Actilight −1.37 b ± 0.18 19.80 b ± 5.40 20.486 1.163 0.998
MOB
GOS b
−1.37 ± 0.20 b
19.24 ± 4.89 20.394 1.293 0.998
Inulin −0.86 a ± 0.17 17.68 b ± 8.54 21.915 0.822 0.997
P95 −1.25 b ± 0.21 19.82 b ± 5.82 21.688 1.294 0.997
Synergy1 b
−1.18 ± 0.10 b
18.48 ± 8.32 18.68 1.189 0.997
Control 0.07 a ± 0.10 5.52 a ± 1.84 5.421 0.364 0.979
Glucose −1.16 c ± 0.31 25.62 c,d ± 6.38 27.399 1.589 0.999
1-kestose c
−1.28 ± 0.16 d 27.52 1.861 0.999
26.57 ± 5.87
Actilight −1.25 c ± 0.20 19.73 b,c ± 6.36 20.635 1.384 0.998
NW
GOS −1.28 c ± 0.25 22.20 b,c,d ± 5.63 22.641 1.761 0.998
Inulin −0.77 a,b ± 0.23 b
19.10 ± 6.51 21.662 0.962 0.997
P95 −1.24 c ± 0.16 23.67 b,c,d ± 5.76 23.952 1.721 0.998
Synergy1 −1.08 b,c ± 0.13 25.19 b,c,d ± 6.25 26.454 1.456 0.997
Int. J. Mol. Sci. 2020, 21, 906 4 of 17

The highest level of cumulative gas was reached with 1-kestose in both groups of individuals and
the lowest with inulin (Table 1). Notably, in fecal cultures of MOB subjects, all prebiotics led to similar
gas production (p > 0.05), whereas fecal cultures from NW adults showed higher heterogeneity, with
significant differences in production among several substrates. The determination of kinetic parameters
by the modified-Gompertz equation confirmed different dynamics of gas production between fecal
cultures of MOB and NW subjects. For all prebiotics, production rates were lower in fecal cultures of
MOB individuals (Table 1). In accordance with the results obtained from gas production, inulin was
the substrate inducing the lower decline in pH in both population groups. Interestingly, the drops in
pH did not totally mirror the increases in gas production, suggesting that differences among prebiotics
are not only due to differences in their utilization yields but may also involve different microorganisms
or catabolic pathways.

2.2. Impact of Prebiotics on Microbiota Composition

The microbiota composition was evaluated at the relative (16S rRNA gene profiling; Figure 1) and
absolute level (quantification of representative microbial groups by qPCR; Figure 2) before (time 0) and
after 24 h of incubation of fecal cultures with the carbohydrates.

Figure 1. Microbial composition (relative abundance %) evaluated by 16S rRNA gene profiling at
family levels in basal conditions (time 0: T0) and after 24 h of incubation in fecal cultures with several
carbon sources and without an external carbon source added (Control) in morbid obesity (MOB) and
normal-weight (NW) groups.

The assessment of the microbial composition of fecal preparations before incubation (time 0)
evidenced a high variability (Supplementary Table S1), which is an inherent feature derived from
the different microbiota composition of fecal donors [36]. In spite of this, the carbon sources tested
(prebiotics and glucose) displayed differential effects on the microbiota, as depending on the substrate
itself and on the groups of fecal donors, MOB or NW.
Regarding the comparison among prebiotics, in fecal cultures of MOB subjects, none of the
compounds affected the overall microbiota composition, without noticing any statistically significant
differences among them at phyla levels (Supplementary Table S1). At family levels, significant
differences with regard to the control culture were found for some minority microbial groups. These
included reduced levels of the family Eggerthellaceae, belonging to Actinobacteria phylum, and an
increase of the Tannerellaceae family, belonging to Bacteroidetes, in all carbon sources. Moreover, a
nonsignificant trend towards higher levels of Bacteroidetes phylum and the Bacteroidaceae family were
also found (Supplementary Table S1). As with regard to qPCR data, all substrates but inulin led to a
significant increase of the absolute levels of the Bacteroides group, as compared to the control. This effect
Int. J. Mol. Sci. 2020, 21, 906 5 of 17

was more pronounced with 1-kestose and GOS (8.13 ± 0.51 and 8.17 ± 0.55 Log CFU/mL, respectively)
(Figure 2). An increase in the absolute levels of Faecalibacterium were also obtained with all substrates,
whereas the genus Bifidobacterium was not significantly affected by any prebiotic or glucose. In spite of
this, when looking at specific bifidobacterial species, the absolute levels determined by qPCR of the
species Bifidobacterium longum were found to be increased after incubation with glucose, 1-kestose and
GOS (6.52 ± 0.82, 6.66 ± 1.27 and 7.13 ± 1.03 Log CFU/mL, respectively), as compared to the control
(5.47 ± 0.31 Log CFU/mL) (Figure 2). Moreover, ITS-sequencing allowed detecting a decrease of the
initial higher relative abundances of Bifidobacterium animalis subsp. lactis and Bifidobacterium crudilactis
after incubation with all prebiotics tested and with glucose (Supplementary Table S1). These results
indicate that in spite of no variations found at the genus Bifidobacterium levels, some changes occurred
in the species profiles after incubation with the prebiotic carbohydrates.

Figure 2. Absolute levels (Log CFU/mL) of fecal microbial groups determined by qPCR after fecal
cultures of (A) MOB and (B) NW subjects. For each microbial group, the box and whiskers plot
represent median, interquartile range and minimum and maximum values obtained in each human
group (NW or MOB). Different letters above the boxes indicate significant differences (p-value < 0.05)
among carbon sources for the microbial groups considered.
Int. J. Mol. Sci. 2020, 21, 906 6 of 17

Regarding the comparison among prebiotics in fecal cultures of NW people, the 16S rRNA gene
profiling evidenced very low abundances of Fusobacteria and Fusobacteriaceae in the negative control,
which were practically undetectable after incubation with prebiotics (Supplementary Table S1). qPCR
analyses showed that all carbon sources (prebiotics and glucose) caused a significant increase of the
absolute levels of Faecalibacterium and Bifidobacterium at 24 h of incubation but did not significantly
affect the population of Bacteroides (Figure 2). In addition, the absolute levels of the species B. longum
increased significantly in fecal cultures of NW people with all substrates tested, and the same was
true for Bifidobacterium adolescentis, with the exception of the prebiotic inulin (Figure 2B). Moreover,
ITS-sequencing evidenced a reduction of Bifidobacterium breve relative abundances after incubation of
NW fecal cultures with all substrates assayed (Supplementary Table S1).
Focusing on the comparison among fecal cultures of NW and MOB subjects, the absolute
quantification (qPCR) of the main bacterial groups evidenced significant differences in the basal
microbiota composition (time 0), for most microorganisms analyzed, between MOB and NW individuals
(Supplementary Table S2). Moreover, the alpha-diversity (Chao-1 index) determined with the 16S
rRNA gene profiling data demonstrated a reduced diversity (p < 0.05) in MOB subjects, as compared
to NW (132.85 ± 43.39 vs. 169.19 ± 21.29, respectively). In the cultures with glucose, the differences in
favor of the NW cultures for the microbial groups Akkermansia, Faecalibacterium, B. adolescentis and
Clostridium cluster XIVa were maintained along incubation, giving rise to significantly higher counts of
these microorganisms in NW cultures, as compared to MOB. Among prebiotics, inulin contributed to
maintain differences already existing in the basal population for Bifidobacterium catenulatum in favor
of the fecal cultures of NW individuals. The genus Akkermansia was significantly higher at 24 h of
incubation in the cultures of NW individuals with respect to MOB in all conditions, these differences
being not evident in cultures of the basal microbiota with no carbohydrates added (negative control).
However, the most noticeable effect among prebiotics was that promoted by GOS on the Bacteroides
group in MOB. Interestingly, ITS analysis showed a clear differential pattern of abundances of several
bifidobacterial species between fecal cultures of NW and MOB subjects after incubation with different
carbon sources (Supplementary Figure S1). The comparison between fecal samples of both human
population groups in basal conditions (time 0, before incubation) reflected higher abundances of
Bifidobacterium mongoliense and B.crudilactis in NW individuals (2.78 ± 4.13 % and 14.98 ± 17.04 %,
respectively) than in MOB subjects (0.16 ± 0.16 % and 1.96 ± 1.46 %, respectively) and a lower species
richness (number of species of bifidobacteria detected by ITS-sequencing) in the NW fecal cultures at
time 0 and after 24 h incubation in all conditions assayed (Supplementary Table S3). Moreover, the
qPCR quantification of bifidobacterial species confirmed higher (p < 0.05) levels of B. longum (NW:
5.85 ± 0.48; MOB: 5.60 ± 0.32) and B. catenulatum (NW: 6.80 ± 0.70; MOB: 5.34 ± 1.10) in NW subjects.
All these results point to substantial differences at the species level in the microbiota of NW and MOB
subjects that are conditioning differences among fecal cultures from groups NW and MOB subjects
after the incubation with prebiotics.

2.3. Production of Short-chain Fatty Acids

In a similar way as for microbiota composition, differences on the levels of SCFA were determined
in fecal cultures depending on the prebiotic tested and the population group considered (Figure 3).
Focusing on the comparison among prebiotics, GOS and 1-kestose were the substrates promoting
the highest increase of total SCFA at 24 h of incubation in NW and MOB fecal cultures, respectively.
1-kestose gave rise to the highest increase of acetate and propionate among prebiotics tested in fecal
cultures of MOB subjects, whereas GOS was the main promoter of acetate production in cultures from
NW individuals. Inulin and Synergy1 were the prebiotics with a lower impact on the production
of acetate and propionate in fecal cultures of both MOB and NW people. All prebiotics enhanced
butyrate production in fecal cultures of NW individuals, with no clear differences among the different
compounds (Figure 3B). In contrast, not statistically significant increases of butyrate were evidenced in
fecal cultures of MOB subjects (Figure 3A), which could be due to the high variability in the production
Int. J. Mol. Sci. 2020, 21, 906 7 of 17

of this compound by the fecal cultures analyzed. In spite of that indicated above, no significant
differences were obtained in the increments of acetic, propionic, butyric, BSCFA and total SCFA between
fecal cultures of MOB and NW individuals at 24 h of incubation (Mann Whitney U test, p-value > 0.05).

Figure 3. Increments in ascending order, with respect to time 0, in the concentration (mM) of the major
short-chain fatty acids (acetic, propionic and butyric) after 24 h of incubation with different carbon
sources in fecal cultures from MOB (A) and NW (B) groups. Differences are shown for each short-chain
fatty acid (SCFA); columns that do not share the same letter are significantly different (p < 0.05).

2.4. Interaction of the Isolated Microbiotas and Supernatants from the Fecal Cultures with HT29 Cells
Functional differences of fecal supernatants (FS) and isolated microbiotas (IM) collected before and
after incubation with representative prebiotics (1-kestose, Actilight, inulin and GOS) were evaluated
through an in vitro model using the HT29 intestinal cell line (Figure 4). Regarding FS, a significant
decrease of the Area Under the Curve (AUC) values was evidenced after incubation of the HT29 cell
line with samples from fecal MOB cultures added with prebiotics (AUC values ranging between −0.07
and 0.04), with respect to the value before the addition of substrates (0.63 ± 0.42). The only exception
to this was inulin, for which no significant variations were obtained (Figure 4A). It is interesting to
note that AUC determined with FS from cultures of MOB subjects added with prebiotics resemble
those obtained with FS of NW subjects. Notably, in these last samples, the AUC before incubation with
HT29 were lower than in MOB cultures. These data suggest that the functionality of the FS from MOB
subjects could be restored (becoming similar to that of NW subjects) after culturing with some of the
prebiotics studied (1-kestose, Actilight and GOS). In co-cultures of HT29 with IM from fecal cultures of
MOB and NW groups, all prebiotics promoted significant increases of AUC values, with the exception
of inulin in both population cohorts.
Int. J. Mol. Sci. 2020, 21, 906 8 of 17

Figure 4. Real-time monitoring the interaction between (A) fecal supernatants and (B) isolated
microbiota obtained before and after incubation with prebiotics and HT29 intestinal epithelial cells.
Values (media ± SD) correspond to the Area Under the Curve (AUC) resulting from monitoring the
cell index (CI) during 10 h. Significant differences (p-value < 0.05) represent the comparison of results
before and after prebiotics addition in each condition.

3. Discussion
The definitive way to prove the impact of prebiotics on gut microbiota and health is through
human intervention studies. However, these studies are expensive and time-consuming, and it is
advisable to perform an initial screening of the different candidate substrates by using affordable
in vitro models that could help to predict in vivo functionality [12]. In a previous work, we described an
in vitro model that allowed us to predict the functionality of IM and FS of different human populations
groups [37]. Its application in the present work for assessing the impact of several prebiotics in NW
and MOB microbiota highlighted potential modulatory benefits on the gut microbiota of some FOS
and GOS. Particularly, real-time monitoring the interaction with the HT29 intestinal cell line of FS from
fecal cultures of MOB subjects added with different prebiotics evidenced a response that approaches
values obtained of FS from NW cultures in the same conditions. It suggests a possible restoration of
the unbalanced functionality of the microbiota by some of the prebiotic substrates tested. Nevertheless,
inulin preserved a behavior more similar to the initial situation in NW and MOB groups, probably
due to the fact that inulin was the prebiotic with less marked effects on microbiota composition and
activity, which is consistent with previous reports by other authors [14,16].
Another functional approach tested in this study was the application of a gas monitoring profiling
system during fecal cultures of NW and MOB microbiotas with different prebiotics. Although this
system has been recently applied to human fecal cultures [18,38], this is the first report on their
use to prebiotics evaluation. Differences in gas production ability among prebiotic substrates can
be partly explained by possible differences in the fermentability among compounds but also by
differential effects of these substrates on the intestinal microbiota. In fact, prebiotics may differ in
their ability to modulate the growth and activity of those microorganisms with limited gas production
and/or to up-regulating microbial gas-consuming reactions (methanogenesis, sulfate reduction and
acetogenesis) [39]. In addition to the potential direct impact on gas-producing bacteria, prebiotics are
also known to affect other microorganisms of the intestinal microbiota, such as bifidobacteria, not
releasing gas but producing acetate and lactate; these compounds could be involved in cross-feeding
mechanisms with gas-producing microorganisms such as Clostridium spp. or sulphate-reducing
bacteria [40]. Therefore, the final gas formed will depend, not only on the chemical and physical
structure of the prebiotic, but on several other factors related with the composition and metabolic
activity of the intestinal microbiota. In this context, different fermentation dynamics of fecal cultures
from MOB and NW people were demonstrated in this study. A lower rate of gas production was
appreciated with the MOB microbiota in all tested carbon sources. This feature suggests a metabolically
less active microbiota.
Int. J. Mol. Sci. 2020, 21, 906 9 of 17

In the context of obesity, an inverse association between body mass index and H2 and CH4
gas detection in breath tests has been reported [41,42], which is in good agreement with the lower
cumulative gas in MOB cultures obtained herein. Although emphasis has been given to the potential
inflammatory or carcinogenic properties of colonic gases, emerging evidence suggests that these gases
might have a beneficial effect in colonic health [43]. One of the main gases produced by anaerobic
fermentation is H2 [44], and an imbalance in its metabolism (H2 -producing and H2 -consuming bacteria)
might facilitate inflammation [43]. It is intriguing to consider whether the promotion of gas production
in MOB subjects could improve antioxidant and antiapoptotic status than contributing to decrease
inflammation [45].
The prebiotics used in the present work have previously proven efficacy for modulating the
microbiota of the general or specific population groups, both in in vivo and in vitro models [21]. In our
case, the most pronounced effects in NW and MOB fecal cultures were obtained with 1-kestose and GOS.
In this way, 1-kestose has been found to be metabolized by different intestinal microorganisms [46,47].
Moreover, a recent study reported that the administration to healthy volunteers of 1-kestose (5 g/day)
during eight weeks promoted an increase of the intestinal populations of Faecalibacterium prausnitzii
and Bifidobacterium spp. [48]. The potential beneficial changes promoted by GOS and FOS on the
intestinal microbiota found in the present work are in good agreement with the widely reported effects
of these substrates on the microbiota composition of the general population, assessed in clinical trials
and used in vitro models [21,49].
It is important to emphasize that in the present work we have focused on morbid obese subjects,
which could make the comparison with other studies difficult since, in the literature available, obese
individuals are not often subcategorized. Moreover, the obesity-associated microbiota shifts are still not
completely known, and several confounding factors often make this task difficult [32,50]. In this regard,
the modulation of the microbiota of obese subjects by prebiotics has produced contradictory results on
the genus Bacteroides, with increases reported after the administration of α-glucooligosaccharide and
arabinogalactan [25,26] and decreases with FOS [23]. Discrepancies on experimental results are likely
due to a differential effect depending on the type of prebiotics, the experimental design and the analytical
techniques used to determine the composition of the intestinal microbiota by different authors. It is also
necessary to point out that in the present work all prebiotics tested were able to up-modulate the levels
of the genus Faecalibacterium, a microorganism with well-known anti-inflammatory properties [51]
and, therefore, interesting in the context of obesity, which is generally accompanied by a low-grade
inflammation [52,53]. A bifidogenic effect was observed only for the species B. longum after the addition
of the prebiotics 1-kestose and GOS to fecal cultures. These observations and the inverse association
of this species with serum lipopolysaccharides and endotoxemia [22,54] suggests that some of these
prebiotics, specially 1-kestose and GOS, could be good candidates to modulate the microbiota in the
context of obesity.
A broad prebiotic enhancement of absolute levels of the genus Bifidobacterium was seen in fecal
cultures of the NW group, in contrast to the absence of effect in cultures from MOB subjects. In order
to expand the study of the prebiotic impact on bifidobacteria, we performed an ITS-region profiling.
To our knowledge, this is the first study going in detail on bifidobacteria species variation in NW and
MOB fecal cultures. Even though, regarding the influence of prebiotics, the ITS-profiling only stood
out as a decrease after the supplementation with carbon sources of B. animalis subsp. lactis and B.
crudilactis in NW cultures and of B. breve in cultures of MOB subjects, a clear distinction between MOB
and NW microbiotas was still evident. Firstly, we noticed a greater richness of bifidobacteria species in
MOB microbiota. Additionally, differentially higher levels of B. monogoliense and B. crudilactis were
present in the microbiota of the NW group, which may be explained by a higher consumption of dairy
fermented foods [55,56].
The production of SCFA in fecal cultures is in good agreement with variations in pH, gas
production and impact caused on the microbiota composition by the different substrates. Thus,
differences in the production of propionate between fecal cultures of MOB and NW promoted by
Int. J. Mol. Sci. 2020, 21, 906 10 of 17

1-kestose could be directly related with its higher capacity (together with GOS) to differentially promote
the increase of Bacteroides (the main propionate producer in the human colon and a producer of acetate)
in fecal cultures of MOB individuals. Interestingly the intestinal microbiota, mainly that from MOB
subjects, showed a better ability to produce SCFA with some of the prebiotics tested than from glucose.
This could be related with the enrichment of the microbiome in some metabolic pathways involved
in the initial steps of breaking down indigestible dietary polysaccharides, including pathways for
starch/sucrose metabolism, galactose metabolism and butanoate metabolism, previously reported in
the obese population [57].
It is also worth mentioning that the basal differences in the metabolic activity and microbiota
composition, added to the specific effects of prebiotics found in the present work depending on the
donor population, highlights the importance of selecting the best-suited compounds for the desired
target population and the potential limitations of extrapolating conclusions from one population group
to another.
To summarize, the present study provides evidence about the in vitro fermentation profiles of
different prebiotics by microbiotas from NW and MOB individuals. In our study, we did not perform
total metagenome analyses, but instead, we performed a microbiota characterization by 16S rRNA
gene profiling and complemented it with two functional tests, gas production and interaction with an
intestinal line, allowing the assessment of both microbiota compositions and some functional properties
of these microbiotas. By monitoring gas production along fermentation, we found a higher capacity of
gas production by fecal cultures of NW subjects than from MOB individuals. 1-kestose was the fructan
showing the highest gas accumulation and largest microbiota modulation activity in MOB subjects,
together with GOS, underlining the utilization of this compound by the intestinal microbiota of these
individuals. The fecal cultures incubated with some of the prebiotics tested also showed differences
at the functional level when assessed upon epithelial cell lines. Even though the in vitro models
present inherent limitations and a difficult interpretation with respect to physiological conditions,
the application of in vitro models to the analysis of microbiota composition and functionality could
allow the selection of the most suitable prebiotics for different populations prior to their assessment
in human intervention studies. Moreover, our results underline the interest of further exploring the
prebiotic role of 1-kestose due to their modulatory capacity of the microbiota composition and activity
in MOB subjects.

4. Materials and Methods

4.1. Prebiotics and Carbon Sources

Two types of prebiotics, based on their monosaccharide’s composition (fructose or galactose),
were evaluated. Among FOS, the trisaccharide 1-kestose (>99%; β Food Science Co. Ltd., Chita, Japan);
Actilight® (DP = 3–5, enzymatically produced, 95% purity; Beghin Meiji, Lila, France); P95 (DP =
2–8, obtained by hydrolysis, 95% purity; Beneo-Orafti, Oreye, Belgium); Synergy1 (FOS plus inulin in
proportion 1:1, 92% purity; Beneo-Orafti, Oreye, Belgium) and the long-chain fructan–inulin (DP>36;
Sigma-Aldrich, Madrid, Spain) extracted from dahlia tubers were included in the study. A GOS from
the brand Bimuno Daily (Clasado, Shinfield, England) with 79.70% (w/w) of purity was also evaluated.
Glucose (Fluka Analytical, Madrid, Spain) was also used as a nonprebiotic universal carbon source.
Sterilization of all substrates was carried out by filtration through a pore size of 0.45 µm, except for the
inulin, which was autoclaved.

4.2. Volunteers and Fecal Sample Collection

Fecal samples were obtained from nine healthy NW adults (77.78% women; BMI <25 kg/m2 ) and
nine MOB volunteers (75% women; BMI ≥40 kg/m2 ) recruited at the Digestive and Endocrinology
Services, respectively, of Asturias Central University Hospital (HUCA, Asturias, Spain). The mean age
of the volunteers was 38 ± 9 and 45 ± 10 for NW and MOB subjects, respectively. All participants have
Int. J. Mol. Sci. 2020, 21, 906 11 of 17

followed an unrestricted diet and have not taken antibiotics during the previous six months. The study
was approved by the Regional Ethical Committee of Asturias Public Health Service (Ref. Nº 120/13, 20
November 2013), and an informed written consent was obtained from each volunteer. Samples were
collected and immediately introduced into anaerobic jars (Anaerocult A System; Merck, Darmstadt,
Germany) for transportation to the laboratory within 1 h after collection. A 1/10 (w/v) dilution was
made in prereduced PBS solution and homogenized in a Lab-Blender 400 stomacher (Seward Medical,
London, UK) for 5 min.

4.3. Fecal Batch Culture Fermentation

Independent batch fermentations were performed at pH-uncontrolled in a carbohydrate-free basal
medium (CFBM) [58], with feces from different human donors and different carbohydrates added.
Briefly, CFBM was prepared and reduced overnight in an anaerobic chamber MG500 (Don Whitley
Scientific, West Yorkshire, UK) one day before the sample processing. On the day of the assay, fresh
fecal samples, collected and homogenized as stated above, were added (10% v/v) to the reduced CFBM
and then were distributed into 100 mL bottles of the ANKOMRF system (ANKOM Technology, USA).
An overnight incubation in anaerobic conditions was performed at 37 ◦ C prior to the addition of carbon
sources in order to allow microbiota to stabilize in the culture medium.
A set of independent fermentations were performed with feces from each donor, using as carbon
sources either inulin-type fructans (1-kestose, Actilight, P95, Synergy1 and inulin), GOS or glucose
(nonprebiotic positive control) at a final concentration of 0.3% (v/v). A bottle with no carbon source
added was used as a control. Fermentations were carried out under anaerobic conditions at 37 ◦ C
during 24 h. The pH of cultures was determined with a pHmeter (SensION + PH3, Hach; Barcelona,
Spain) and was considered as an indicator of the progression of fermentation. Samples (1 mL) were
taken in duplicate before incubation (time 0) and considered as basal conditions and at 24 h of
incubation. Samples were centrifuged at full speed for 10 min, and supernatants and pellets were
stored separately at −20 ◦ C until their use for microbiota and metabolite analyses.

4.4. Gas Monitorization

The cumulative gas produced along the different fermentations was monitored in real-time by
using the ANKOM RF system. The system provides increases in pressure (psi), which can be converted
to mL of gas produced using the ideal gas equation.

V = Vj · Ppsi · 0.068004084 (1)

where V = gas volume at 39 ◦ C in mL, Vj = headspace of digestion jar (glass bottle) in mL and Ppsi =
cumulative pressure recorded by Gas Monitor System software.
The data of gas production were fitted to a modified-Gompertz equation, a model frequently used
to fit data of bacterial, plant growth, tumor proliferation and gas production [59], by using the formula:

µ×e
  
y = A × exp − exp (λ − t) + 1 (2)
A

in which variable “A” represents the upper asymptote (mL), “µ” is the rate of gas production (mL/h)
and “λ” is the time lag before the exponential phase (h).

4.5. Microbiota Composition and SCFA Quantification

DNA was extracted from the pellets harvested using the QIAamp DNA Stool Mini kit (Qiagen
GmbH; Hilden, Germany), as previously described [60], and the isolated DNA was stored at −20 ◦ C
until use for qPCR analyses and 16S ribosomal and intergenic ribosomal transcriber spaces (ITS).
Int. J. Mol. Sci. 2020, 21, 906 12 of 17

4.5.1. qPCR Analyses

Absolute levels of some relevant intestinal microbial groups (Bacteroides–Prevotella–Porphyromonas
group, Lactobacillus group, Akkermansia, Clostridium cluster XIVa, Bifidobacterium and Faecalibacterium
genus), as well as total bacteria, were determined at 0 and 24 h of fermentation by qPCR using
previously described primers and conditions [31,61]. Variations in the levels of the species B. longum,
B. catenulatum and B. adolescentis were assessed as described elsewhere [22].

4.5.2. 16S rRNA Gene Based Microbiota Profiling

Purified DNA was used as a template for amplification of partial 16S rRNA gene sequences
by PCR using the primers and conditions described by Milani and coworkers [62]. The obtained
amplicons were then sequenced by using the MiSeq (Illumina) platform at GenProbio srl (Italy). The
individual reads obtained were filtered, trimmed and processed [63]. 16S rRNA operational taxonomic
units were defined at ≥ 97% sequence homology using the UCLUST tool developed by Edgar [64]. All
reads were classified to the lowest possible taxonomic rank using QIIME and a reference dataset from
the SILVA database [65].

4.5.3. ITS Region-Based Profiling of Bifidobacterial Microbiota

To gain further insight into the fecal bifidobacterial populations present in the samples and how
the different prebiotics affected them, the 16S–23S internal transcriber spaces of the ribosomal DNA
(ITS region) was amplified by PCR using the primer pair Probio_bif_uni/Probio_bif_rev. and further
sequenced as indicated in the previous section. An improved bifidobacterial ITS database, containing
all publicly available bifidobacterial genomes and a custom bioinformatics script [66], were used.
Relative abundance of bifidobacterial composition filtered by a minimum presence (≥1%) of each
species in all databases were represented by a heatmap following instructions described elsewhere [67],
centered and scaled by the “scale” function in RStudio version 1.2.5001.

4.5.4. SCFA Analyses

The analysis of SCFA was performed by gas chromatography (GC) in the fecal culture supernatants
in order to determine the molar concentrations of three main compounds: acetate, propionate and
butyrate. The remaining BSFA, namely isobutyrate and isovalerate, were also quantified and summed
up for further analysis. Briefly, culture supernatants collected during the fecal fermentation (0.250
mL) were mixed with 0.3 mL methanol, 0.05 mL internal standard solution (2-ethylbutyric 1.05 mg/
mL) and 0.05 mL of 20% formic acid. This mixture was centrifuged, and the supernatant was used
for quantification of SCFA by GC, as described previously [37]. Samples were analyzed in triplicate.
Increments in molar concentrations of the main SCFA and BSCFA with respect to the time 0 were
calculated for each fermentation batch with the different carbon sources.

4.6. Monitoring the Interaction of Isolated Microbiotas (IM) and Fecal Supernatants (FS) with HT29 Cells
Briefly, the behavior of HT29 cells monolayers in a confluent state was assessed upon exposure to
IM and FS collected after incubation of fecal samples with different carbon sources by using a real-time
cell analyzer (RTCA-DP) xCelligence apparatus (ACEA Bioscience Inc., San Diego, CA, USA). The
culture conditions and the maintenance of the intestinal epithelial cell line HT29 (ECACC 91072201)
are detailed in a previous work where the functional model was developed [37]. IM were purified
from 10-fold concentrated fecal cultures by using a density gradient method previously described [68].
Purified microbiotas were inactivated by UV light exposure (15 W; Selecta, Barcelona, Spain) and
adjusted to 1 × 108 bacteria/mL using a Neubauer-improved camera (Blau Brand, Germany).
For the functional assessment of IM and FS, HT29 monolayers in a confluent state were coincubated
with 6.5 × 107 bacteria/mL of UV-inactivated purified microbiotas in McCoy´s medium (MM) (bacteria
to cell ratio 10:1). In the case of fecal supernatants, the behavior of HT29 cells monolayers was assessed
Int. J. Mol. Sci. 2020, 21, 906 13 of 17

with filtered fecal supernatants (pH adjusted to 7.55 ± 0.05) and diluted 40% with MM. Additionally,
a negative control consisting of MM without bacteria or fecal supernatants was included in each
experiment. Each sample was tested in duplicate using two independent E-plates. The monitoring was
followed for every 10 min under standard incubation conditions. CI values recorded were normalized
by the time of the sample addition and by the control sample, as previously described [69]. For
statistical comparison purposes, the “Area Under the Curve” (AUC), representing the CI values along
10 h of incubation for each sample, was calculated as explained in [37].

4.7. Statistics Analyses

Unless otherwise specified, all experimental data are reported as mean ± standard deviation.
Statistical analysis of results was performed using the software SPSS v.25 (SPSS Inc., Chicago, USA).
Data were compared for the effect caused on the parameters analyzed by the addition of different
carbon sources in fecal cultures from each population cohort (NW and MOB) at the end of fermentation
(24 h). For variables with a normal distribution (Shapiro-Wilk test) and homoscedasticity (Levene test),
one-way ANOVA followed by post hoc LSD comparison were conducted (Supplementary Table S4). In
the remaining cases (variables showing non-normal distribution), a Kruskal-Wallis test followed by a
post hoc Dunn’s test of pairwise comparisons were applied when necessary (Supplementary Table S4).
A significant p-value of 0.05 was used for the interpretation of results. For two-group comparisons
between MOB and NW (at time 0 and after incubation with all conditions), a two-tailed Student’s t-test
or Mann-Whitney U test was conducted for the evaluation of data by parametric or nonparametric
contrast, respectively.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/3/906/s1.

Author Contributions: Conceptualization, A.E., T.T., K.H., C.G.R.-G. and M.G.; volunteers recruitment, A.S. and
C.M.-F.; methodology, A.N., N.S., S.A., P.R-M., L.M., A.S. and C.M.-F.; formal analysis, A.N., N.S., S.A., L.M., M.V.,
C.G.R.-G. and M.G.; writing—original draft preparation, A.N., C.G.R.-G. and M.G.; writing—review and editing,
A.N., N.S., S.A., P.R.-M., L.M., A.S., C.M.-F., M.V., T.T., K.H., A.E., C.G.R.-G. and M.G. All authors have read and
agreed to the published version of the manuscript.
Funding: The present work was financed by the project AGL2013-43770-R (receiving funds from the Ministry of
Economy and Competitiveness—MINECO and the European Union FEDER funds) and by a contract with the
company β- Food Sciences (Chita, Japan). Alicja M Nogacka was the recipient of a predoctoral contract granted by
MINECO (reference BES-2014-068796), and S.A. received a postdoctoral Juan de la Cierva contract (Ministry of
Science, Innovation and Universities, Spain, Ref. IJCI-2017-32156). Nuria Salazar is the recipient of a postdoctoral
contract awarded by the Biosanitary Research Foundation in Asturias (FINBA, Spain).
Acknowledgments: We wish to thank the volunteers participating in the study and the Scientific-Technical Service
of IPLA-CSIC for carrying out the SCFA determination.
Conflicts of Interest: Takumi Tochio and Katsuaki Hirano are employees at the company β- Food Sciences.

Abbreviations
AUC Area Under the Curve
CI Cell Index
FS Fecal Supernatant
GC Gas Chromatography
IM Isolated Microbiota
ITS 16S-23S internal transcriber spaces
LSD Least Significant Difference post-hoc
MM McCoy´s medium
MOB Morbid Obesity
NW Normal-Weight Adults
qPCR Real-time polymerase chain reaction
rRNA Ribosomal ribonucleic acid
RTCA Real-time Cell Analyzer
Int. J. Mol. Sci. 2020, 21, 906 14 of 17

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