An Infant Milk Formula Supplemented with Heat-Treated Probiotic Bifidobacterium animalis subsp. lactis CECT 8145, Reduces Fat Deposition in C. elegans and Augments Acetate and Lactate in a Fermented Infant Slurry

Abstract

Pediatric obesity has a growing health and socio-economical impact due to cardiovascular and metabolic complications in adult life. Some recent studies suggest that live or heat-treated probiotics have beneficial effects in preventing fat deposition and obesity in preclinical and clinical sets. Here, we have explored the effects of heat-treated probiotic Bifidobacterium animalis subsp. lactis CECT 8145 (HT-BPL1), added as a supplement on an infant milk formula (HT-BPL1-IN), on Caenorhabditis elegans fat deposition and short-chain fatty acids (SCFAs) and lactate, using fermented baby fecal slurries. We have found that HT-BPL1-IN significantly reduced fat deposition in C. elegans, at the time it drastically augmented the generation of some SCFAs, particulary acetate and organic acid lactate. Data suggest that heat-treated BPL1 maintains its functional activities when added to an infant powder milk formula.

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Article
An Infant Milk Formula Supplemented with
Heat-Treated Probiotic Bifidobacterium animalis
subsp. lactis CECT 8145, Reduces Fat Deposition in
C. elegans and Augments Acetate and Lactate in a
Fermented Infant Slurry
Ángela Silva 1, †, Nuria Gonzalez 1, †, Ana Terrén2, Antonio García2,3,4,
Juan Francisco Martinez-Blanch 5, Vanessa Illescas 5, Javier Morales 6, Marcos Maroto 2,
Salvador Genovés1, Daniel Ramón1,5, Patricia Martorell 1and Empar Chenoll 1, 5, *
1Health & Wellness-ADM Nutrition-ADM Biopolis, 46980 Paterna, Spain; angela.silva@adm.com (Á.S.);
nuria.gonzalez@adm.com (N.G.); Salvador.genoves@adm.com (S.G.); daniel.ramonvidal@adm.com (D.R.);
patricia.martorell@adm.com (P.M.)
2Instituto Fundación Teófilo Hernando, 28029 Madrid, Spain; ana.terren@ifth.es (A.T.); agg@uam.es (A.G.);
marcos.maroto@ifth.es (M.M.)
3Departamento de Farmacología y Terapéutica, Universidad Autónoma de Madrid, 28029 Madrid, Spain
4Instituto de Investigación Sanitaria, Hospital Universitario de La Princesa, Facultad de Medicina,
28006 Madrid, Spain
5Health & Wellness-ADM Nutrition-ADM LIfesequencing, 46980 Paterna, Spain;
juan.martinezblanch@adm.com (J.F.M.-B.); Vanessa.illescas@adm.com (V.I.)
6Product Development Department, Alter Farmacia SA, 28880 Madrid, Spain; jmoraleso@grupo-alter.com
*Correspondence: maria.chenoll@adm.com
†These two authors have contributed equally to the present work.
Received: 20 April 2020; Accepted: 13 May 2020; Published: 19 May 2020


Abstract:
Pediatric obesity has a growing health and socio-economical impact due to cardiovascular
and metabolic complications in adult life. Some recent studies suggest that live or heat-treated
probiotics have beneficial effects in preventing fat deposition and obesity in preclinical and clinical
sets. Here, we have explored the effects of heat-treated probiotic Bifidobacterium animalis subsp.
lactis CECT 8145 (HT-BPL1), added as a supplement on an infant milk formula (HT-BPL1-IN),
on Caenorhabditis elegans fat deposition and short-chain fatty acids (SCFAs) and lactate, using
fermented baby fecal slurries. We have found that HT-BPL1-IN significantly reduced fat deposition
in C. elegans, at the time it drastically augmented the generation of some SCFAs, particulary acetate
and organic acid lactate. Data suggest that heat-treated BPL1 maintains its functional activities when
added to an infant powder milk formula.
Keywords:
heat-treated probiotic; Bifidobacterium animalis subsp. lactis; fat deposition; acetate; lactate;
C. elegans
1. Introduction
A pool analysis of 128.9 million children, adolescents, and adults reveals the huge health impact
of obesity [
1
]. This is illustrated by several studies. For instance, adults with a history of chilhood
obesity have higher prevalence of metabolic syndrome and cardiovascular risk [
2
,
3
]. Additional
complications of pediatric obesity are poor skeletal maturation [
4
], psychosocial inadaptation [
5
],
or early inflammatory status [6].
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Foods 2020,9, 652 2 of 13
During the last years, new strategies derived from the gut microbiome analysis are emerging
to understand and combat childhood obesity. Thus, an association of early life gut microbiota
with body mass index has been demonstrated in an observational study in 0–3 years age infants [
7
].
Such association seems to be linked to short-chain fatty acids (SCFA) binding G protein coupled receptor
(GPR41), which modulates host adiposity [
8
]. These findings point to a potential use of prebiotic,
probiotic or symbiotic products to modify in some way the gut microbiota in individuals prone to
obesity and to reduce the impact of this dysbiosis in childhood and later adulthood. Specific probiotic
supplements can impinge on lower incidence of obesity and hypertension [
9
–
12
]. Other studies have
also shown that specific probiotics impact on weight management in adults [10–12]
Recent studies have shown that heat-treated bacteria release metabolites or cell components
that can display functional actions, such as immunomodulation or antagonism of pathogens [
13
–
15
].
This has led to an increasing interest in using heat-treated probiotics [
13
–
16
]. In fact, a few clinical
trials have shown heat-treated bacteria functional activities in for instance infantile colic [
17
]) or in
functional bloating [18].
In a set of preclinical studies, strain Bifidobacterium animalis subsp. lactis strain BPL1 was isolated
from infant feces and deposited in Spanish Type Culture Collection (CECT) under accession number
CECT 8145 and, in a massive screening, demonstrated its capacity to reduce fat deposition [
19
].
Initially, the strain was tested for its ability to reduce fat content in Caenorhabditis elegans model [
19
],
and later in two murine models of obesity; one carried out in conventional wistar rats fed on a cafeteria
diet [
20
] and the second based on the use of genetically obese rats Zücker rats [
21
]. In all three
studies, the animals were fed BPL1 from infancy/larvae state to young adulthood, and in all three cases,
an effect of lifetime probiotic consumption on adult fat was observed. In the two murine model studies,
it was also observed that probiotic intake in lean individuals had no effect on their body fat levels
or other measured parameters. Moreover, both in the C. elegans model and in the study in rodents
on a cafeteria diet, promising results were obtained with a thermally treated version of the probiotic
strain, pointing to a non-life-dependent mode of action, as a part of BPL1 effectiveness. However,
while these results showed an effect of BPL1 cells on fat deposition in both animal models and clinical
human trial, we were unable to study the potential involvement of the gut microbiome in the observed
functionality. This potential involvement is of great importance in order to be able to analyze in depth
and systematically the potential mechanisms of action of the probiotic strain (by direct action and by
the modulating effect of the microbiome). Thus, further to these results, in this study we considered to
evaluate whether the effect observed in animals and humans was due to a direct action of the cells,
or if there was an impact on the bacterial microbiome and compounds derived from its metabolism
that could have an effect on its functionality in the preclinical model of C. elegans. Secondly, and in
conjunction with the previous point, we defined as an objective to study whether the addition of the
probiotic to a commercial product (in this case infant milk formula) could have a positive or negative
impact (reinforcing or diminishing functionality respectively) on its effect.
In the present study, a mixed fecal slurry model was used to analyze the impact of HT-BPL1 strain
and infant formula containing the heat-treated probiotic on microbiome, and its potential synergistic
effect with the infant milk formula. Fecal slurry approach enabled us to ex vivo analyze together with
microbiome changes by massive sequencing, SCFAs production and, furthermore, the impact of this
microbiome modulation on C. elegans fat deposition.
2. Materials and Methods
2.1. In Vitro Fecal Fermentations
Bifidobacterium lactis BPL1 cells were grown anaerobically in de Man, Rogosa, and Sharpe medium
(MRS; Oxoid, Basingstoke, UK), supplemented with cysteine (0.05% wt/vol; Sigma, St. Louis, MO,
USA; MRS-C) at 37
◦
C for 18 h. Cells were then harvested by centrifuging and viability evaluated
by plate counting in MRS-C agar (anaerobically, 37
◦
C for 48 h). After that, cells were heat treated

Foods 2020,9, 652 3 of 13
(autoclaved at 121
◦
C for 20 min) and the absence of viability checked by broth culturing in MRS
anaerobically at 37
◦
C for 18 h. Fecal fermentations were carried out by using 3 mL fermentations in
10 mL fermentor (24 multi-well plate;
µ
-24 Bioreactor, Applikon Biotechnology, Delft, The Netherlands).
Fresh fecal samples came from 3 months babies fed with a milk formula free of probiotics. Immediately
after being obtained, stools were vacuum stored in refrigerated conditions until the fermentation
experiments were performed. Fresh feces were weighted and dissolved in pre-reduced Mc Bain
and MacFarlane medium [
22
] in a ratio 1:5 (feces:medium) to hydrate them. Three milliliters of
the homogenized mixture (fecal slurry) were inoculated in each fermentor, and heat-treated BPL1
(HT-BPL1) was added alone (HT-BPL1), or included in the infant formula Innova, from Nutriben,
Alter, Madrid, Spain (HT-BPL1-IN). Medium was previously degassed and headspace was removed
before starting fermentation with nitrogen. All the protocol was performed in an anaerobic cabinet.
All fermentors were maintained under a head space of oxygen-free nitrogen and were continuously
stirred. Anaerobiosis, temperature (37
◦
C) and pH (pH 5.5) were maintained constant along the
fermentation period that was run for 24 h. Slurry samples were anaerobically taken and aliquoted for
further processing and analysis.
2.2. Body Fat Monitoring in C. elegans
Fresh samples were analysed in C. elegans model. The effects of fecal slurry samples subjected
to different treatments, on body fat content in C. elegans, were analysed as previously described [
19
].
Briefly, concentrated C. elegans cultures (50
µ
L, OD =30) were added on top of a NGM surface
previously seeded with Escherichia coli OP50, to ensure adequate nutrition conditions. Positive
controls were NGM plates with anti-obesity drug Orlistat (6 µg/mL).
Lipid content in worms was determined by adding 0.05
µ
g/mL of Nile red to NGM agar plates
containing the different fecal treatments. Worms were incubated for 3 days at 20
◦
C, until they reach
the adult stage. Then, nematode samples placed in M9 buffer were assayed for their fluorescence in
a FP-6200 system (JASCO Analytical Instruments, Easton, MD, USA). Excitation wavelength was at
480 nm and emission was at 571 nm. A total of 120 worms per condition were analyzed in each of
2 different experiments.
2.3. Organic Acids Analysis
To analyze short chain fatty acids (SFCAs) acetate, propionate, butyrate and succinate, and organic
acid lactate at the end of the experiment, a volume of 500
µ
L of fermentation was centrifuged at
14,000 rpm for 60 min, filtered through a Millipore 0.45
µ
m pore-size filter (Billerica, MA, USA),
and diluted
1
/
2
in MilliQ quality water. The quantification was conducted on a HPLC Acquity equipped
with an Aminex HPX-87H 300
×
7.8 mm column (BioRad) under conditions defined by manufacturer.
Detection was achieved by a refractive index detector. The eluent was H
2
SO
4
5 mM with an isocratic
flow rate of 0.6 mL/min. No internal standard was included. Quantification was performed by the use
of standard curves with correlation coefficient R2>0.999.
2.4. Microbiome Analysis by Deep Sequencing
Slurry samples were immediately frozen and stored at
−
20
◦
C until their processing. DNA from
samples was isolated following Yuang and co-workers [
23
] with minor modifications to avoid bias
in DNA purification toward misrepresentation of gram positive bacteria, with the aid of MagnaPure
Compact System (Roche Life Science). For massive sequencing, the hypervariable region V3-V4 of
the bacterial 16s rRNA gene was amplified using key-tagged eubacterial primers [24] and sequenced
with a MiSeq Illumina Platform, following the Illumina recommendations for Library preparation
and sequencing.
The resulting sequences were split, taking into account the barcode introduced during the PCR
reaction. Quality control of the sequences was performed in different steps. Firstly, quality filtering
(with a minimum threshold of Q20) was performed using fastx tool kit version 0.013, then primer

Foods 2020,9, 652 4 of 13
(16s rRNA primers) trimming and length selection (reads over 300 nts) was done with cutadapt version
1.2. (https://cutadapt.readthedocs.io/en/stable) [
25
]. FASTQ files were converted to FASTA files and
UCHIME program version 7.0.1001 [
26
] was used in order to remove chimeras that may arise during
the amplification and sequencing step. Then clean FASTA files were BLAST against NCBI 16s rRNA
database using blastn version 2.2.29+. The resulting XML files were processed using a python script
developed by Lifesequencing S.L. (Paterna, Valencia, Spain) in order to annotate each sequence at
different phylogenetic levels (phylum, family, genus and species). Alpha diversity was conducted
using specaccum program in the vegan package, as implemented for R version 3.2.3. Presence results
are expressed as the percentage of each group sequences.
2.5. Statistical Analysis
Results are given as the mean
±
standard deviation. Differences between the groups were
analyzed using Tukey’s multiple comparison tests. Data on fat deposition in C. elegans were analyzed
by Student’s ttest. Statistical analyses were performed with GraphPad Prism 8.4.1 software (San Diego,
CA, USA), setting the level of statistical significance at 5%.
3. Results
3.1. Effects of a Milk Powder Supplemented with Heat-Treated BPL1, on C. elegans Fat Deposition
First we explored whether an infant milk powder added alone (MP) or MP supplemented with
HT-BPL1 (HT-BPL1-IN, INNOVA), exerted a functional effect on C. elegans fat content. Worms were
incubated by 3 days with milk powder at increasing concentrations. Figure 1shows that fat body content
(monitored as relative fluorescence of Nile Red, ordinate) was unchanged at the four concentrations of
control MP. However, in the case of worms exposed to HT-BPL1-IN, there was a significant 12.5
±
1.7%
reduction (p<0.001) at the concentration of 1.80 mg/mL and 9.0
±
1.4% reduction at the concentration
of 3.60 mg/mL (p<0.001). This preliminary result prompted another set of experiments through the
incubation of fermented baby slurries in the presence of HT-BPL1-IN.
Foods 2020, 9, x FOR PEER REVIEW 10 of 13
version 1.2. (https://cutadapt.readthedocs.io/en/stable) [25]. FASTQ files were converted to FASTA
files and UCHIME program version 7.0.1001 [26] was used in order to remove chimeras that may
arise during the amplification and sequencing step. Then clean FASTA files were BLAST against
NCBI 16s rRNA database using blastn version 2.2.29+. The resulting XML files were processed using
a python script developed by Lifesequencing S.L. (Paterna, Valencia, Spain) in order to annotate each
sequence at different phylogenetic levels (phylum, family, genus and species). Alpha diversity was
conducted using specaccum program in the vegan package, as implemented for R version 3.2.3.
Presence results are expressed as the percentage of each group sequences.
2.5. Statistical Analysis
Results are given as the mean ± standard deviation. Differences between the groups were
analyzed using Tukey’s multiple comparison tests. Data on fat deposition in C. elegans were analyzed
by Student’s t test. Statistical analyses were performed with GraphPad Prism 8.4.1 software (San
Diego, CA, USA), setting the level of statistical significance at 5%.
3. Results
3.1. Effects of a Milk Powder Supplemented with Heat-Treated BPL1, on C. elegans Fat Deposition
First we explored whether an infant milk powder added alone (MP) or MP supplemented with
HT-BPL1 (HT-BPL1-IN, INNOVA), exerted a functional effect on C. elegans fat content. Worms were
incubated by 3 days with milk powder at increasing concentrations. Figure 1 shows that fat body
content (monitored as relative fluorescence of Nile Red, ordinate) was unchanged at the four
concentrations of control MP. However, in the case of worms exposed to HT-BPL1-IN, there was a
significant 12.5 ± 1.7% reduction (p < 0.001) at the concentration of 1.80 mg/mL and 9.0 ± 1.4%
reduction at the concentration of 3.60 mg/mL (p < 0.001). This preliminary result prompted another
set of experiments through the incubation of fermented baby slurries in the presence of HT-BPL1-IN.
Figure 1. Milk powder supplemented with heat-treated BPL1 (HT-BPL1-IN), reduced fat deposition
in C. elegans measured as fluorescence with red Nile, in a concentration-dependent manner. Worms
were incubated for 3 days with increasing concentrations (abscissa) of milk powder alone (MP) or
milk powder supplemented with HT-BPL1-IN. Data are means ± SD of 120 worms assayed in a
prototype experiment. * p < 0.001 with respect control.
Experiments were done at two concentrations of HT-BPL1, equivalent to 10
8
and 10
9
cells per
reactor (Figure 2). Worms were grown for 3 days in 24-h fermented fecal slurries from two different
infants. The antiobesity drug Orlistat was used as a positive control. In the presence of HT-BPL1, fat
reduction accounted for 17.8 ± 1.1% (p < 0.05) in baby 1 slurry and 20.2 ± 6.0% (p < 0.05) in baby 2. In
Figure 1.
Milk powder supplemented with heat-treated BPL1 (HT-BPL1-IN), reduced fat deposition in
C. elegans measured as fluorescence with red Nile, in a concentration-dependent manner. Worms were
incubated for 3 days with increasing concentrations (abscissa) of milk powder alone (MP) or milk
powder supplemented with HT-BPL1-IN. Data are means
±
SD of 120 worms assayed in a prototype
experiment. * p<0.001 with respect control.

Foods 2020,9, 652 5 of 13
Experiments were done at two concentrations of HT-BPL1, equivalent to 10
8
and 10
9
cells per
reactor (Figure 2). Worms were grown for 3 days in 24-h fermented fecal slurries from two different
infants. The antiobesity drug Orlistat was used as a positive control. In the presence of HT-BPL1,
fat reduction accounted for 17.8
±
1.1% (p<0.05) in baby 1 slurry and 20.2
±
6.0% (p<0.05) in baby 2.
In HT-BPL1-IN assays, reductions in fat deposition with respect to control were 21.3
±
1.6% (p<0.05)
in baby 1 and 22.8
±
7.9% in baby 2 (p<0.05). In spite of those pronounced reductions, they did not
reach the level of statistical significance compared to HT-BPL1.
Foods 2020, 9, x FOR PEER REVIEW 10 of 13
HT-BPL1-IN assays, reductions in fat deposition with respect to control were 21.3 ± 1.6% (p < 0.05) in
baby 1 and 22.8 ± 7.9% in baby 2 (p < 0.05). In spite of those pronounced reductions, they did not reach
the level of statistical significance compared to HT-BPL1.
Figure 2. Effects of heat treated BPL1 (HT-BPL1) alone, or added on the infant milk formula Innova
(HT-BPL1-IN), on fat deposition of C. elegans grown for 3 days on the fermented slurries (24 h) from
two different healthy babies. Fat deposition was monitored with relative fluorescence (ordinates) at
the end of the incubation periods, expressed as % reduction of fluorescence compare to control worms
(worms grown on DMSO). Antiobesity drug Orlistat was added to a well at 6 µg/mL as positive
control (DMSO, the solvent for Orlistat). (A, C) experiments done with 108 HT-BPL1 cells/reactor in
slurries from babies 1 (A) and 2 (C). (B, D), experiments done with 109 HT-BPL1/reactor with innova
milk formula (HT-BPL1-IN) or HT-BPL1 alone, in fermented slurries from baby 1 (B) or baby 2 (D).
Data are mean ± SD of 120 worms per well in each condition * p < 0.05, with respect to control. * p <
0.001, with respect to control.
The reduction in fat deposition observed in worms grown in the presence of 109 heat-treated
bacteria was more notable. In all cases, HT-BPL1 reduction was close to Orlistat results. HT-BPL-1
reduced fat deposition by 39.8 ± 5.1% (p < 0.001) and 36.2 ± 1.0% in the presence of HT-BPL1-IN (p <
0.001). In baby 2 slurry, decreased fat deposition was 39.8 ± 4.6% for HT-BPL1, and 37.8 ± 2.7% for
HT-BPL1-IN (p < 0.001 for the two treatments).
3.2. Effects of HT-BPL1, Alone or Included in an Infant Milk Formula, on the Production of Short-Chain
Organic Fatty Acids
This study was aimed at finding out how HT-BPL1 could affect the level of SCFAs and organic
acids generation in fecal slurries from healthy infants, before (0 h) and after fermentation for 24 h.
Table 1 summarizes the results obtained. Figure S1 shows representative chromatograms
obtained in assays 1 and 2. Note that neither propionic acid nor butyric acid underwent significant
changes with respect to control (0 h), both in assay 1 and assay 2. Neither, slurries incubated with
HT-BPL1 alone for 24 h, experimented any change with respect to control, both in the case of acetic
acid or lactic acid. However, when fermentation was done in the presence of HT-BPL1-IN, acetic acid
augmented over 3-fold compared to 0 h samples (9.89 ± 0.02; p < 0.001). The same occurred with lactic
acid that rose from near undetectable levels at 0 h (0.08 ± 0.11 g/L) to 8.34 ± 0.20 g/L (p < 0.001). In
assay 2 these differences were maintained even though in this assay acetate and lactate baseline levels
were higher in the control samples (Table 1). In Figure 3, the net increases of acetate in fermented
fecal slurries, are plotted.
Figure 2.
Effects of heat treated BPL1 (HT-BPL1) alone, or added on the infant milk formula Innova
(HT-BPL1-IN), on fat deposition of C. elegans grown for 3 days on the fermented slurries (24 h) from
two different healthy babies. Fat deposition was monitored with relative fluorescence (ordinates) at the
end of the incubation periods, expressed as % reduction of fluorescence compare to control worms
(worms grown on DMSO). Antiobesity drug Orlistat was added to a well at 6
µ
g/mL as positive control
(DMSO, the solvent for Orlistat). (
A
,
C
) experiments done with 10
8
HT-BPL1 cells/reactor in slurries
from babies 1 (
A
) and 2 (
C
). (
B
,
D
), experiments done with 10
9
HT-BPL1/reactor with innova milk
formula (HT-BPL1-IN) or HT-BPL1 alone, in fermented slurries from baby 1 (
B
) or baby 2 (
D
). Data are
mean
±
SD of 120 worms per well in each condition * p<0.05, with respect to control. ** p<0.001, with
respect to control.
The reduction in fat deposition observed in worms grown in the presence of 10
9
heat-treated
bacteria was more notable. In all cases, HT-BPL1 reduction was close to Orlistat results. HT-BPL-1
reduced fat deposition by 39.8
±
5.1% (p<0.001) and 36.2
±
1.0% in the presence of HT-BPL1-IN
(p<0.001). In baby 2 slurry, decreased fat deposition was 39.8
±
4.6% for HT-BPL1, and 37.8
±
2.7% for
HT-BPL1-IN (p<0.001 for the two treatments).
3.2. Effects of HT-BPL1, Alone or Included in an Infant Milk Formula, on the Production of Short-Chain
Organic Fatty Acids
This study was aimed at finding out how HT-BPL1 could affect the level of SCFAs and organic
acids generation in fecal slurries from healthy infants, before (0 h) and after fermentation for 24 h.
Table 1summarizes the results obtained. Figure S1 shows representative chromatograms obtained
in assays 1 and 2. Note that neither propionic acid nor butyric acid underwent significant changes
with respect to control (0 h), both in assay 1 and assay 2. Neither, slurries incubated with HT-BPL1
alone for 24 h, experimented any change with respect to control, both in the case of acetic acid or lactic
acid. However, when fermentation was done in the presence of HT-BPL1-IN, acetic acid augmented
over 3-fold compared to 0 h samples (9.89
±
0.02; p<0.001). The same occurred with lactic acid that

Foods 2020,9, 652 6 of 13
rose from near undetectable levels at 0 h (0.08
±
0.11 g/L) to 8.34
±
0.20 g/L (p<0.001). In assay 2 these
differences were maintained even though in this assay acetate and lactate baseline levels were higher
in the control samples (Table 1). In Figure 3, the net increases of acetate in fermented fecal slurries,
are plotted.
Table 1.
Levels of short-chain organic fatty acids in slurry fermentations in the absence (control 24 h) or
the presence of HT-BPL1 alone or with milk powder supplemented with HT-BPL1 (HT-BPL1-IN).
Organic Acids Concentration (g/L)
Acetic Acid Lactic Acid Propionic Acid Butyric Acid
Experiment 1
Control (0 h) 2.88 ±0.44 0.08 ±0.11 0.87 ±0.14 1.01 ±0.20
Control (24 h) 3.59 ±0.11 <LOQ 11.22 ±0.10 1.94 ±0.16 *
HT-BPL1 (1 ×108)3.49 ±0.29 <LOQ 11.19 ±0.16 1.90 ±0.24 *
HT-BPL1-IN (1 ×108)9.89 ±0.02 *** 8.34 ±0.20 *** 1.11 ±0.08 0.77 ±0.03
Experiment 2
Control (0 h) 7.98 ±0.27 3.37 ±0.21 0.78 ±0.07 1.00 ±0.07
Control (24 h) 8.18 ±0.13 0.29 ±0.03 * 1.35 ±0.06 3.92 ±0.03 **
HT-BPL1 (1 ×109)7.55 ±0.34 0.10 ±0.05 * 1.32 ±0.28 3.78 ±0.21 **
HT-BPL1-IN (1 ×109) 14.66 ±1.03 ** 10.62 ±0.97 *** 0.85 ±0.01 0.80 ±0.03
Data are means
±
SD of 2 experiments in triplicate; *** p<0.001; ** p<0.01; * p<0.05, with respect control.
1
LOQ
(Limit of Quantification) 0.05 g/L.
Foods 2020, 9, x FOR PEER REVIEW 10 of 13
Table 1. Levels of short-chain organic fatty acids in slurry fermentations in the absence (control 24 h)
or the presence of HT-BPL1 alone or with milk powder supplemented with HT-BPL1 (HT-BPL1-IN).
Organic Acids Concentration (g/L)
Acetic Acid Lactic Acid Propionic Acid Butyric Acid
Experiment 1
Control (0 h) 2.88 ± 0.44 0.08 ± 0.11 0.87 ± 0.14 1.01 ± 0.20
Control (24 h) 3.59 ± 0.11 <LOQ
1
1.22 ± 0.10 1.94 ± 0.16 *
HT-BPL1 (1·10
8
) 3.49 ± 0.29 <LOQ
1
1.19 ± 0.16 1.90 ± 0.24 *
HT-BPL1-IN (1·10
8
) 9.89 ± 0.02 *** 8.34±0.20 *** 1.11 ± 0.08 0.77 ± 0.03
Experiment 2
Control (0 h) 7.98 ± 0.27 3.37 ± 0.21 0.78 ± 0.07 1.00 ± 0.07
Control (24 h) 8.18 ± 0.13 0.29 ± 0.03 * 1.35 ± 0.06 3.92 ± 0.03 **
HT-BPL1 (1·10
9
) 7.55 ± 0.34 0.10 ± 0.05 * 1.32 ± 0.28 3.78 ± 0.21 **
HT-BPL1-IN (1·109) 14.66 ± 1.03 ** 10.62 ± 0.97 *** 0.85 ± 0.01 0.80 ± 0.03
Data are means ± SD of 2 experiments in triplicate; *** p < 0.001; ** p < 0.01; * p < 0.05, with respect
control.
1
LOQ (Limit of Quantification) 0.05 g/L.
Figure 3. Net increase of acetate in fecal baby slurries fermented for 24 h, in the absence (control) and
the presence of HT-BPL1 alone or HT-BPL1 included in an infant milk formula (Innova)
supplemented with HT-BPL1 cells (HT-BPL1-IN). (A) experiment 1 done with a baby slurry in the
presence of 10
8
HT-BPL1 cells; (B), experiment done with a baby slurry in the presence of 10
9
HT-
BPL1 cells. Data calculated from Table 1, by subtracting the background level of acetate in a non-
fermented slurry (0 h in the table).
3.3. Effects of a Milk Powder Supplemented with Heat-Treated BPL1, on Microbiome
Microorganisms present in fecal slurries were identified and quantified in terms of relative
abundance. Diversity by Shannon Index at genus level was evaluated (Supplementary Figure S2),
identifying some differences between babies after 24 h of fermentation in some of the trials, and
showing a later homogeneity once BPL1-HT or BPL1-HT-IN is added, with slight differences between
them.
When we analyzed the potential effect on the regulation of microbiome at genus level in
assays·with 10
8
cells HT-BPL1 incorporated (Figure 4A), we observed that fermentation decreased
the presence of genera Bacteroides and Prevotella, both in majority presence, and increased the
presence of the genus Fecalibacterium and a non-assigned OTU (OTU 94). The comparison of the
results obtained in baby faeces fermented with HT-BPL1 (both HT-BPL1 and HT-BPL1-IN) showed
the highest differences. When comparing HT-BPL1-IN with the rest of the 24 h trials, a trend towards
a higher percentage of the genera Fecalibacterium, Bifidobacterium, Lactobacillus, Flintibacter and OTU94,
whereas lower percentages of Prevotella, Coprococcus and Eubacterium were observed. In the case of
assays with 1·10
9
cells HT-BPL1 per fermentation added (Figure 4B) OTU94, and genera
Fecalibacterium and Flintibacter increased in fecal slurries fermented with HT-BPL1-IN while genera
Eubacterium and Dialister decreased. In the case of Coprococcus, slurry fermentations caused an
elevation of this genus, except in the case of HT-BPL1-IN, with very similar levels to those of fecal
material before being fermented. On the contrary, Akkermansia levels dropped in all fermentations,
except in the case of trials with HT-BPL1-IN assayed.
Figure 3.
Net increase of acetate in fecal baby slurries fermented for 24 h, in the absence (control) and the
presence of HT-BPL1 alone or HT-BPL1 included in an infant milk formula (Innova) supplemented with
HT-BPL1 cells (HT-BPL1-IN). (
A
) experiment 1 done with a baby slurry in the presence of 10
8
HT-BPL1
cells; (
B
), experiment done with a baby slurry in the presence of 10
9
HT-BPL1 cells. Data calculated
from Table 1, by subtracting the background level of acetate in a non-fermented slurry (0 h in the table).
3.3. Effects of a Milk Powder Supplemented with Heat-Treated BPL1, on Microbiome
Microorganisms present in fecal slurries were identified and quantified in terms of relative
abundance. Diversity by Shannon Index at genus level was evaluated (Supplementary Figure S2),
identifying some differences between babies after 24 h of fermentation in some of the trials, and showing
a later homogeneity once BPL1-HT or BPL1-HT-IN is added, with slight differences between them.
When we analyzed the potential effect on the regulation of microbiome at genus level in assays
with 10
8
cells HT-BPL1 incorporated (Figure 4A), we observed that fermentation decreased the presence
of genera Bacteroides and Prevotella, both in majority presence, and increased the presence of the genus
Fecalibacterium and a non-assigned OTU (OTU 94). The comparison of the results obtained in baby
faeces fermented with HT-BPL1 (both HT-BPL1 and HT-BPL1-IN) showed the highest differences.
When comparing HT-BPL1-IN with the rest of the 24 h trials, a trend towards a higher percentage
of the genera Fecalibacterium, Bifidobacterium,Lactobacillus,Flintibacter and OTU94, whereas lower
percentages of Prevotella,Coprococcus and Eubacterium were observed. In the case of assays with
1×109cells
HT-BPL1 per fermentation added (Figure 4B) OTU94, and genera Fecalibacterium and
Flintibacter increased in fecal slurries fermented with HT-BPL1-IN while genera Eubacterium and

Foods 2020,9, 652 7 of 13
Dialister decreased. In the case of Coprococcus, slurry fermentations caused an elevation of this genus,
except in the case of HT-BPL1-IN, with very similar levels to those of fecal material before being
fermented. On the contrary, Akkermansia levels dropped in all fermentations, except in the case of trials
with HT-BPL1-IN assayed.
Foods 2020, 9, x FOR PEER REVIEW 10 of 13
Figure 4. Percentage of identified microorganisms by 16s rRNA by Illumina V3-V4 sequencing. (A)
experiments run with two different baby slurries in the presence of 108 HT-BPL1 cells; (B) experiments
run with two different baby slurries in the presence of 109 HT-BPL1 cells.
4. Discussions
We focused this investigation to find out whether a heat-treated choose Bifidobacterium animalis
subsp. lactis CECT 8145 (BPL1) probiotic preserved some of its functional properties exhibited when
alive, in a fecal model that mimics microbial fermentation in the gut. Initially, this strain showed
activity lowering fat deposition both alive and heat treated in the pre-clinical C. elegans model [19]
0
10
20
30
40
50
60
70
80
Control oh Control 24h HT-BPL1 HT-BPL1-IN
% sequences
OTU_133
OTU_122
OTU_129
Flintibacter
Desul fovib rio
Anaerostipes
Blautia
Gemmiger
Dial ister
Collinsella
Methanobrevib acter
Ruminococcus
Oscillibacter
Faecal ibac terium
Akkermansia
Parabacteroides
Eubacteri um
OTU_137
Coprococcus
Alistipes
No Hit
Prevotella
Bifidobacteriu m
OTU_94
Bacteroid es
0
10
20
30
40
50
60
70
80
90
Control oh Control 24h HT-BPL1 HT-BPL1-IN
% sequences
Flintib acter
Desulfovibrio
Anaerostip es
Blautia
Gemmiger
Dialister
Colli nsella
Methano brevibacte r
Ruminococcus
Osci llibac ter
Faecal ibacterium
Akkermansia
Parabacteroi des
Eubact erium
OTU_137
Copro coccus
Alistipes
No Hi t
Prevotella
Bifidobacterium
OTU _94
Bacteroides
A
B
Figure 4.
Percentage of identified microorganisms by 16s rRNA by Illumina V3-V4 sequencing.
(
A
) experiments run with two different baby slurries in the presence of 10
8
HT-BPL1 cells; (
B
)
experiments run with two different baby slurries in the presence of 109HT-BPL1 cells.

Foods 2020,9, 652 8 of 13
4. Discussion
We focused this investigation to find out whether a heat-treated choose Bifidobacterium animalis
subsp. lactis CECT 8145 (BPL1) probiotic preserved some of its functional properties exhibited when
alive, in a fecal model that mimics microbial fermentation in the gut. Initially, this strain showed
activity lowering fat deposition both alive and heat treated in the pre-clinical C. elegans model [
19
]
demonstrating the usefulness of the model for probiotic evaluation in fat deposition. After that,
the activity in both formats was validated in two different pre-clinical murine models [
20
,
21
] and finally
in a randomized controlled clinical trial done in adult abdominally obese subjects in which the probiotic
validated previous results and showed to improve the anthropometric obesity biomarkers [
27
]. In the
light of these positive outcomes, in this work we attempted to study the effect of strain HT-BPL1 in
infant milk product.
Since the probiotic was to be incorporated into an infant milk, it was initially studied whether this
strain retained its functionality in this matrix. These trials were conducted in the nematode C. elegans.
The small size, short lifespan, complete genetic information and easy maintenance in the laboratory
make C. elegans a valuable animal model to research. It is noteworthy that many of the genes involved
in the synthesis, degradation, and transport of fats are conserved in mammals and have been identified
by RNAi [
28
–
33
], being its capacity of fat accumulation dependent on the homeostasis of lipogenesis
and lipolysis [
34
]. This nematode conserves 65% of the genes associated with human disease, and in
the case of lipid and energy metabolism, many of the proteins involved in synthesis, degradation
and transporting fats are highly conserved between C. elegans and mammals. Genetic screenings
have identified many gene inactivations that cause fat reduction or fat accumulation [
31
]. This has
led to the use of C. elegans used as a model to evaluate potential obesity therapeutics, and obtain
mechanistic information behind single-gene mutations related to obesity, and define the mechanistic
details of fat metabolism [
28
–
33
,
35
]. In this sense, compounds targeting conserved pathways regulating
lipids in nematodes are potential successfully candidates for testing in human trials. Trials conducted
in C. elegans model have both confirmed a dose-dependent effect in fat deposition with HT-BPL1
and that HT-BPL1 kept this property when incorporated into the infant milk product (HT-BPL1-IN).
This initial result led us to further develop the model, applying added fecal slurry from the different
products to analyze the evolution of the microbiome, the production of organic acids and its effect
on the fat deposition model of C. elegans both with BPL1 alone (HT-BPL1), and with infant milk
(HT-BPL1-IN), in two different cell concentrations (10
8
cells/reactor and 10
9
cells/reactor). In an attempt
to “humanize” the intestine of C. elegans, nematodes have been treated with baby fermented fecal
samples and results confirmed its fat-reducing effect in treated nematodes, particularly at 10
9
cells
concentration. These results suggest that microbiome impacts the physiology of the worm, influencing
in health parameters. This is consistent with other C. elegans studies showing host-microbiome
interactions, that are well stablished, like those related with oxidative stress or life-span [
36
]. From the
metabolomic analysis, the results obtained using the fecal slurry model showed that SCFA acetate and
the organic acid lactate were considerably augmented by HT-BPL1-IN, during a 24-h fermentation
period. This augmentation was not observed when HT-BPL1 was added alone, suggesting that either
there may be some kind of synergy between the milk powder and HT-BPL1 or most probably that
the milk powder contains compounds that act on the growth and/or metabolism of different acetate
and lactate producing microorganisms in the fermented infant slurries. Infant milk powder contains
lactose which is can be homo-fermented or hetero-fermented by taxons such as Bifidobacterium animalis
subsp. lactis, producing thus lactic and acetic acid respectively [37].
Whatever the mechanism involved, it seems that the increase in acetate has relevant impact in the
context of infant obesity. SCFAs are metabolic products of microbial activity that contribute to intestinal
homeostasis [
38
,
39
]. The most abundant are acetate, propionate and butyrate, that constitute more than
95% of all SCFAs [
38
,
40
]. While acetate and propionate are found in both small and large intestines,
butyrate is mainly present in colon and cecum [
40
]. Acetate also exerts anti-inflammatory activity in
C. elegans via NF-
κ
B [
41
], also presenting functionality as immunomodulator [
42
]. NF-
κ
B signaling is

Foods 2020,9, 652 9 of 13
directly involved in the relationship of inflammation and obesity [
43
]. In a previous work with BPL1
in C. elegans model [
19
], it was observed that this one presented, besides a marked functionality as for
reduction of the corporal fat, antioxidant and antiinflammatory activity. In the context of infant obesity,
some functional observations with acetate deserve a comment. For example, the oral administration of
acetate to mice fed a high-fat diet, reduced body weight, improved insulin sensitivity, decreased total
body fat content and hepatic fat accumulation without changing food intake or physical activity [
44
].
Moreover, oral acetate gavage in an obese and diabetic strain of rats reduced weight gain and improved
glucose tolerance [
44
,
45
]. Furthermore, in mice C-acetate can cross the blood-brain barrier to be taken
up into the hypothalamus that resulted in decreased food intake through appetite suppression [
44
,
45
],
accompanied by increased lactate production [
44
]. There are also some studies in humans, suggesting
that acetate may affect the central regulation of appetite with concomitant reduction of body weight [
44
].
Concomitantly, acetate stimulates leptin secretion from adipocytes to regulate energy balance and
appetite [
46
]. Furthermore, acetate inhibits intracellular lipolysis with reduced lipid overflow and
decreased ectopic accumulation of fat [
44
]. This effect may reduce free fatty acid flux to the liver;
in doing so, acetate decreases fatty liver risk, thus mitigating deterioration of glucose homeostasis [
46
].
Since SCFAs are produced as a result of the bacteria’s own metabolism of the digestive system,
it is to be expected that the greatest changes at the level of bacterial populations would be observed
in the fermented with HT-BPL1-IN assays. So that, microbiome study was done in order to evaluate
how both HT-BPL1 and HT-BPL1-IN impact on bacterial composition. The analysis of the changes
reflects a modulating effect of the microbial populations of INNOVA milk product. Although diversity
varied between different samples in the control after 24 h of fermentation probably due to different
initial microbial populations that evolve differentially in the fermentation process, the addition of
HT-BPL1 or HT-BPL1-IN resulted in greater uniformity in diversity within each trial. Among increased
groups, elevated levels of bifidobacteria and lactobacilli can enable the production of lactate and
acetate. In this context, the huge increase in HT-BPL1-IN assays may be produced by the use of milk
carbohydrates such us lactose in organic acids production. Some Bifidobacterium species preferentially
use lactose over glucose as a carbon source when grown in the presence of both sugars [
37
,
47
].
Genus Lactobacillus was in HT-BPL1-IN fermentations although at low percentage levels, being L. gasseri
its representative (data not shown). In the case of this species, there are studies that relate strains of
L. gasseri with regulation of gut environment and function in a postbiotic form [
48
]. Finally, in both
HT-BPL1 concentrations assayed, an increase in Faecalibacterium has been found. This genus is related
to protection against inflammation and related with a healthy human gut microbiome [
49
], so an
increase may have a protective effect against inflammatory dysbiosis and related to this, on metabolic
syndrome, and thus it may be an indicator in this model of incidence in the bacterial microbiome and
modulation of functionality.
The results obtained in this study show a significant effect of the product HT-BPL1-IN on fat
deposition C. elegans model. In previous studies, preclinical obese rodents both in wistar rats fed on a
cafeteria diet [
20
] and with genetically obese rats Zücker rats [
21
] confirmed the results obtained in
C. elegans hat were later observed in humans in a clinical study, randomized double-blind study in
obese patients. All in all, the current study has shown us the effect not only of the probiotic but also of
the product included in its matrix, both in the microbiota and in the fat deposition of C. elegans, which
predict, based on the previous “from worm to human” assays done, a potential effect in humans.
An ongoing clinical trial in infants fed with a milk formula supplemented with heat treated BPL1,
will probably define to what extent this formula is helping to prevent the future development of obesity
in those infants. An increasing number of studies have analyzed the effects of different bacterial strains
on SCFA and organic acids production and infant obesity [
50
,
51
]. Our preclinical present study add
evidence in favor of the potential beneficial effects of probiotics and postbiotics on the prevention of
obesity, when added as supplements to milk infant formulas.

Foods 2020,9, 652 10 of 13
5. Conclusions
In conclusion, here we show that heat-treated probiotic strain BPL1, reduces fat deposition in
C. elegans when added to a milk infant formula as a supplement. Although there is an increase in
acetate production, results here reported evidence that other mechanism underlying such effect is
present. The study confirm postbiotic HT-BPL1 effect in milk formula.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2304-8158/9/5/652/s1,
Figure S1: SCFAs and lactate determination by HPLC; Alpha-diversity reflected as Shannon Index; Figure S2:
Alpha-diversity reflected as Shannon Index.
Author Contributions:
Conceptualization, J.M., S.G., D.R., P.M. and E.C.; Methodology, J.F.M.-B. and P.M.;
Validation, A.T., A.G., M.M. and E.C.; Formal analysis, J.F.M.-B., M.M., P.M. and E.C.; Investigation,
Á
.S., N.G.
and V.I.; Data curation, J.F.M.-B. and P.M.; Writing—original draft preparation,
Á
.S., N.G., M.M. and E.C.;
Writing—review and editing, A.G., J.M., S.G., D.R., P.M. and E.C.; Supervision, A.T., A.G., D.R. and P.M.; Project
administration, A.T., S.G., D.R. and P.M.; Funding acquisition, J.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was supported by Cátedra UAM-Alter Nutrinfant, Madrid, Spain.
Conflicts of Interest:
A.T., M.M., A.G., declare no conflicts of interest;
Á
.S., N.G., S.G., D.R., P.M. and E.C. are
employees of ADM Biopolis; J.F.M.-B. and V.I. are employees of ADM Lifesequencing; J.M. is employee of Alter
Farmacia SA; The funders had no role in the design of the study; in the collection, analyses, or interpretation of
data; in the writing of the manuscript, or in the decision to publish the results.
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