Measuring the impact of olive pomace enriched biscuits on the gut microbiota and its metabolic activity in mildly hypercholesterolaemic subjects
Measuring the impact of olive pomace enriched biscuits on the gut microbiota and its metabolic activity in mildly hypercholesterolaemic subjects
Lorenza Conterno 0 1 2
Francesca Martinelli 0 1 2
Matteo Tamburini 0 1 2
Francesca Fava 0 1 2
Andrea Mancini 0 1 2
Maddalena Sordo 0 1 2
Massimo Pindo 0 1 2
Stefan Martens 0 1 2
Domenico Masuero 0 1 2
Urska Vrhovsek 0 1 2
Claudia Dal Lago 0 1 2
Gabriele Ferrario 0 1 2
Mario Morandini 0 1 2
Kieran Tuohy 0 1 2
0 Genomics and Advanced Biology Unit, Research and Innovation Centre-Fondazione Edmund Mach , San Michele all'Adige , Italy
1 Department of Food Quality and Nutrition, Research and Innovation Centre-Fondazione Edmund Mach , San Michele all'Adige , Italy
2 Casa di Cura Eremo di Arco s.r.l. , Via XXI Aprile 1, 38062 Arco, TN , Italy
3 Kieran Tuohy
Purpose Olive pomace is a major waste product of olive oil production but remains rich in polyphenols and fibres. We measured the potential of an olive pomace-enriched biscuit formulation delivering 17.1 ± 4.01 mg/100 g of hydroxytyrosol and its derivatives, to modulate the composition and metabolic activity of the human gut microbiota. Methods In a double-blind, controlled parallel dietary intervention 62 otherwise healthy hypercholesterolemic (total plasma cholesterol 180-240 mg/dl) subjects were randomly assigned to eat 90 g of olive pomace-enriched biscuit (olive-enriched product, OEP) or an isoenergetic control (CTRL) for 8 weeks. Fasted blood samples, 24-h urine and faecal samples were collected before and after dietary intervention for measurement of microbiota, metabolites and clinical parameters. Results Consumption of OEP biscuits did not impact on the diversity of the faecal microbiota and there was no statistically significant effect on CVD markers. A trend towards reduced oxidized LDL cholesterol following OEP ingestion was observed. At the genus level lactobacilli and Ruminococcus were reduced in OEP compared to CTRL biscuits.
Olive product; Prebiotic; Polyphenols; Metabolomic; Tyrosol glucoside; Tyrosol group
-
OlioCRU s.r.l. Research and Development Group, Via Aldo
Moro 1, 38062 Arco, TN, Italy
Introduction
Olives and olive oil are important and characteristic
components of the Mediterranean diet, a dietary pattern shown
to improve on both physical and mental quality of life, and
reduce the risk of chronic diet-associated disease, especially
cardiovascular disease (CVD) [
1
]. Indeed, extra-virgin
olive oil, as part of a Mediterranean style diet significantly
reduced both the incidence of composite CVD end points
and total mortality in the PREDIMED study [
2
].
Polyphenols, complex aromatic plant secondary metabolites, are
independently linked to these health effects [
1, 3
]. Olives
and various olive oils and extracts have been shown to
mediate different health effects in humans, many associated
with CVD risk. Olive extracts have been reported to lower
systolic blood pressure (SBP) and diastolic blood pressure
(DBP) from baseline in both hypertensive and
pre-hypertensive individuals [
4–7
] and to improve plasma lipid profiles in
both normo-lipidaemic and hypercholesterolaemic subjects
[
4, 6, 8–10
]. Olive extracts have also been found to induce
acute reductions in arterial stiffness [
11
], which agrees with
data suggesting that olive oil and olive extract significantly
improve vascular function [
12, 13
] and blood pressure [14].
Currently, these improvements are thought to be associated
with polyphenol-rich olive oil fractions rather than other
bioactives which may be present [
15, 16
]. In contrast, other
studies have failed to demonstrate significant modulation of
CVD biomarkers upon olive extract ingestion. For example,
olive leaf extracts did not appear to improve plasma lipids
[
7, 17
], ambulatory blood pressure, cytokines and/or carotid
intima-media thickness [
17
] in different studies. However,
the European Food Safety Authority (EFSA) has recognized
a specific health claim for the polyphenol extract of olive
for protection of LDL cholesterol particles against oxidative
damage—although they also noted that there is a lack of
evidence for other health claims including maintenance of
normal blood pressure and HDL cholesterol levels, reduced
inflammation and improved gastrointestinal function [
18
].
EFSA considers that the claim that “consumption of olive
oil polyphenols contributes to the protection of blood lipids
from oxidative damage” reflects the scientific evidence, and
that a dose of 5 mg of hydroxytyrosol and its derivatives
(e.g. oleuropein complex and tyrosol) in olive oil should be
consumed daily for food products to bear the health claim.
In olive, the majority of polyphenols present belong to the
tyrosol group [hydroxytyrosol (HT), tyrosol (TYR) and
conjugated forms like oleuropein]. These conjugated forms
are extensively hydrolyzed in the stomach [
19
] to HT and
TRY, which are either absorbed in the small intestine and
undergo extensive phase I and II biotransformation or reach
the colon where they undergo biotransformation by the
resident microbiota [
19, 20
]. The most common derivatives are
small phenolic acids like homovanillic acid (HVA),
dihydroxyphenylacetic acids (DHPAA), hydroxyphenylacetic
acid (HPAA), protocatechuic acid and benzoic acids for
example. The impact of olives or their constituent parts on
the composition and metabolic activity of the human
intestinal microbiota is, however, poorly understood. One recent
study has shown that thyme phenolic compounds at
different doses in olive oil can induce a small increase in
bifidobacteria using the quantitative culture-independent method
fluorescent in situ hybridization (FISH), with the suggestion
that this change in microbiota could be related to improved
LDL cholesterol oxidative status [21]. Similarly, a sex effect
in terms of metabolism of HT and related compounds has
been observed in rats with the suggestion that differential
excretion of HT derivatives between male and female
animals might be due to sex-linked differences in enterohepatic
circulation [
22
]. However, no data are reported for
differences in metabolism of these compounds between men and
women or indeed, whether such differences if they do exist,
could be due to sex-specific differences in gut microbiota.
To date, no studies have reported whether olives or olive
pomace can impact on the relative abundances of the human
gut microbiota, on the diversity of the gut microbiota and
only a few studies have specifically addressed the metabolic
end products produced by combined host–microbiota
cometabolism of olive polyphenols [
20, 21
].
Olives contain many potential biologically active
compounds such as polyphenols, dietary fibre (including pectin),
oleic acid, linoleic acid and other beneficial fats,
tocopherols, phytosterols and squalene. Olive oil is extracted from
the fruit of Olea europaea, leaving waste in the form of
olive water and solid olive pomace. The olive pomace and
wastewater produced from oil extraction processes contain
macromolecules such as polysaccharides, lipids, proteins
and polyphenolic compounds (mainly of the tyrosol group)
which can range from 1 to 8 g/l [
23
] in wastewater and 2.9
to 3.7 mg/l in olive pomace [
24
]. The annual worldwide
production of olive oil is about 2 million metric tons reported
for 2015/16 (average: COI, 2017). For each ton of olive oil
the waste produced is dependent on the fruit quality, ripeness
and extraction technology and typically ranges from 2.75 to
4 tons of olive pomace and 1–8 m3 of wastewater [
25–27
]. In
addition these olive mill wastes are produced in significantly
large quantities during the short olive production season and
because the waste cannot be disposed of through ordinary
waste treatment systems, disposal of wastewater and olive
pomace is a major environmental problem and cost to the
industry. Given the push towards a green economy, there is
a stimulus for the oil industry to move towards a circular
economic model, reducing waste production or adding value
to waste streams, developing added value secondary
product lines. To this end, we have developed an olive pomace
extract as a functional food ingredient. This olive pomace,
which delivers 17.1 ± 4.01 mg/100 g HT and its derivatives,
is also rich in fibres and our preliminary in vitro data showed
it was fermentable and leads to increased numbers of
bifidobacteria in pH-controlled faecal batch cultures (data not
shown). For this current study, we have incorporated this
olive pomace into a biscuit formulation. For flavour, texture
and constitutional reasons, other ingredients were also
necessary including extra virgin olive oil and flours from
chestnut, pea and buckwheat, but the olive pomace remained the
dominant ingredient and the dominant source of polyphenols
(> 90% of polyphenols present in the final biscuit). Here we
report the effect of this olive pomace-enriched biscuit on
the human gut microbiota, their metabolic output and on
various biomarkers of CVD and inflammation. The study
was conducted jointly by researchers at Fondazione Edmund
Mach (San Michele all’Adige, Italy), OlioCru s.r.l (Arco,
Italy) and the study centre was the Casa di Cura Eremo di
Arco s.r.l. (Italy), a private specialist clinic specialized in
treating CVD.
Materials and methods
Study design
The study was a double-blind, randomized, controlled,
parallel trial (Italy protocol Prebioil2 number: 6/2015,
Clinicaltrials.gov ID: NCT02664428, see Fig. 1 for the study design).
This study was conducted according to the guidelines laid
down in the Declaration of Helsinki, and all procedures
involving human subjects were approved by the Ethic
Committee for Clinical Trials of the Trento Azienda Provinciale
Servizi Sanitari (APSS). The clinical trial was carried out at
the Casa di Cura Eremo di Arco (Arco, TN, Italy) between
November 2015 and June 2016. The primary outcome
measure was the faecal microbiota analysis by Illumina
sequencing and fluorescence in situ hybridization (FISH) carried
out with probe for Bifidobacterium spp., Lactobacillus spp.
and Ruminococcus spp. The secondary outcome measures
were related to cholesterol analysis in plasma [total, LDL,
HDL cholesterol, oxidized LDL, triglycerides,
apolipoproteins A–I and B (APO A and APO B)] together with the
analysis of the variation of polyphenols and their metabolites
in plasma and urine. Additional measures were the analyses
of the anthropometric indices, the fasting plasma insulin,
glucose and C-reactive protein (CRP) and the analysis of
isoprostane F2 in urine. This study was powered for changes in
blood LDL cholesterol and changes in faecal bifidobacteria.
Since previous studies have shown that fewer individuals are
required for measuring changes in faecal bifidobacteria and
because of its clinical significance, the sample size
calculation was performed only for changes in LDL cholesterol
levels. Based on measures from a previous parallel trial design
using similar products [
28
], we assessed as clinical
significant end point the average LDL decrease of 15.44 mg/dl
with a standard deviation of 14.28 mg/dl. According to
Snedecor and Cochran equation with significance value α equal
to 0.05 and a 90% power (1 − β) [
29
], the minimum number
of subjects to enrol was 28. Taking into account possible
dropouts, a total of 73 subjects were enrolled and randomly
assigned to the dietary intervention with the product under
investigation (OEP) or to the control dietary intervention.
Intervention foods
The study product “PreBiÒ®”, herein called olive
pomaceenriched product (OEP), was a bakery product comprising
dehydrated food-grade olive powder, together with
chestnut, peas, buckweat flour, extra virgin olive oil (EVOO),
salt and sugar according to the OlioCRU proprietary recipe,
and the OlioCRU pending Patent Process. The product was
prepared at a local bakery. The product contained about
411 ± 25 mg/100 g of total biophenols measured according
to the COI method of which 17.1 ± 4.01 mg/100 g belong to
the tyrosol group polyphenols and were measured according
to Gasperotti et al. [
30
].
The product corresponded to the energy intake of
434 Kcal/100 g (average of three replicates).
Subjects were provided with single 90 g daily doses
and instructed to consume one each day of the 8 weeks of
intervention. The control product comprised wheat flour,
sugar, salt and low-polyphenol EVOO, food colourings in
safety-approved quantities to match the test product OEP as
closely as possible in appearance, taste, texture. The product
contains less than 1 mg/100 g of total biophenols measured
according to the IOC official method (IOC 2009) of which
0.7 ± 0.5 mg/100 g belong to the tyrosol group. The product
corresponds to an energy intake of 419 Kcal/100 g (average
of three replicates). Table 1 shows the gross nutritional
composition of the OEP and control (CTRL) biscuits.
Healthy volunteer were recruited through advertisement
in the geographical area of Arco (TN) in the northeast of
Italy. Advertisement was carried out via flyer, posters and
e-mails. Individuals who answered the call were asked their
weight and height and those who corresponded to BMI in
the range established by the inclusion criteria were given an
appointment for the general health assessment to determine
conformity with other inclusion/exclusion criteria. At the
clinic a health and lifestyle questionnaire was completed,
eco-cardiogram and physical examinations were performed
together with collection of urine and fasted blood samples.
The inclusion criteria were non-smoking status, age between
30 and 65 years, BMI between 20 and 29.9 kg/m2, plasmatic
total cholesterol between 180 and 240 mg/dl, being free from
chronic disease, including cardiovascular disease, diabetes,
cancer, inflammatory or digestive disorders. Pregnancy or
breastfeeding, and individuals consuming more than 21 U/
week of alcohol were excluded. Subjects were excluded if
taking statins or other medication or dietary supplements
that may affect lipids. Uncontrolled hypertension was an
exclusion criteria, and to be included the subjects were either
not hypertensive or under hypertensive medication and
presenting with average SBP below 121 mmHg and average
DBP 90 mmHg. Subjects with food allergies or intolerances
were also excluded.
Randomisation and blinding
Treatment allocation was done using a random block design.
A six-digit code was assigned to each recruited individual.
The products was randomly assigned to the subject code by
an external individual by picking codes form a bag in a blind
manner but matching groups by age and sex. Each daily dose
was prepared in a food-grade sealed box enveloped in dark
green paper sachet. The treatment codes were kept offsite
and not released until statistical analysis was complete.
Therefore, allocation concealment was achieved and both
researchers and subjects were blinded to which product was
being consumed at which time.
Screening
Clinical visits took place at the beginning of week 1, and
at the end of week 8 at the Casa di Cura Eremo di Arco
s.r.l., Via XXI Aprile 1, 38062 Arco (TN) Italy. Subjects
arrived for screening fasted and measurements of height and
weight were taken in a Kern scale (Kern & Sohn, Balingen,
Germany) with stand and height rod (Mod MPB300K100P)
to calculate BMI. Blood pressure was measured after 5-min
rest, seated and with the subject’s dominant arm resting on
a table, using an Omron digital blood pressure equipment
(HEM-705 CP).
Three readings were taken 60 s apart and averaged.
Subjects were not permitted to talk during measurements. Blood
pressure was measured at the screening visit (T-1) and later
at the beginning (T0) and at the end of the treatment (T1).
Blood samples obtained via single venepuncture were
collected into heparin and EDTA vacutainers (BD) and used
for the analysis established for health. A total of 73 suitable
subjects were identified and accepted onto the trial.
Each subject was informed about the study aims and
procedure to allow them to sign and informed consent. Eligible
participants were asked to provide written informed consent
to take part in the study.
Compliance measures
Subjects were asked to return all remaining full or empty
daily packages of test product after 8 week intervention.
Remaining material were weighed and recorded. Subjects
were asked to complete weekly online questionnaires and
supplied with daily tick sheets.
Faecal sample collection
Volunteers were provided with a sealable pot, and sterile bag
to collect the stool sample, each of them were instructed to
collect the stool sample in the sterile bag, put it into the pot
and, prior to sealing the pot, to add an atmosphere
generation system AnaeroGen Compact (Thermo Scinetific). This
ensured an anaerobic environment during sample transport.
Samples were collected and treated for further analysis or
stored at − 80 °C within 24 h.
Urine sample collection
Volunteers were supplied 3-l sterile containers to collect the
24-h urine samples. Each container was added with 15 ml of
3M hydrochloridric acids as a preservative. After collection,
the total urine volume was measured and the samples for
further analysis were prepared and stored at − 80 °C.
Biochemical measures
Blood collected in EDTA and heparin vacutainers was
centrifuged at 1550×g for 15 min to separate plasma. Plasma
was immediately analysed or stored in low-binding
Eppendorf tubes (Axygen, Tewksbury MA, USA) at − 80 °C until
analysis. Total cholesterol (TC), HDL cholesterol (HDL),
LDL cholesterol (LDL), apolipoprotein A1 (Apo A1) and B
SD
0.01
0.72
Values below the limit of quantification are not shown
(Apo B) triglycerides (TG), CRP, glucose (GLU) and insulin
(Ins) were measured at Clinic Casa di Cura Eremo
Laboratory (Arco, TN, Italy), using ILab 650 chemistry analyser
(Instrumentation Laboratories UK Ltd, Warrington, United
Kingdom) for all measures except insulin which was
measured using a Roche COBAS 6000.
Oxidized LDL was measured in duplicate via an ELISA
kit (Mercodia, Sweden).
F2 isoprostane urinary total (conjugated and
non-conjugated) were measured using the enzyme immunoassay
based kit Urinary Isoprostane Elisa Kit (Oxford
Biomedical Research, USA).
Metagenomic analysis
A whole fresh stool sample was collected at T0 and T1.
The stool was stored at − 80 °C. DNA was extracted from
100 mg stool aliquots using Power faecal DNA extraction
kit (MOBio), following the manufacturer’s instructions.
Metagenomic sequencing was performed to evaluate
microbiota diversity and genus-level abundances. Using the
specific bacterial primer set 341F (5′
CCTACGGGNGGCWGCAG 3′) and 806R (5′GACTACNVGGGTWTCTAATCC
3′) with overhang Illumina adapters, total genomic DNA was
subjected to PCR amplification by targeting a~ 460-bp
fragment of the 16S rRNA variable region V3–V4. PCR
amplification of each sample was carried out using 25-μl reactions
with 1 μM of each primer. Specifically 12.5 μl of 2× KAPA
HiFi HotStart ReadyMix, 5 μl forward primer, 5 μl reverse
primer were used in combination with 2.5 μl of template
DNA (5 ng/μl). All PCR amplifications were carried out
using a GeneAmp PCR System 9700 (Thermo Fisher
Scientific) and the following steps—melting step; 94 °C for
5 min (one cycle), annealing step; 95 °C for 30 s, 55 °C for
30 s, 72 °C for 30 s (30 cycles), extension step; 72 °C for
5 min (1 cycle). The PCR products were checked on 1.5%
agarose gel and cleaned from free primers and primer dimer
using the Agencourt AMPure XP system (Beckman Coulter,
Brea, CA, USA) following the manufacturer’s instructions.
Subsequently dual indices and Illumina sequencing adapters
Nextera XT Index Primer (Illumina) were attached by
sevencycle PCR (16S Metagenomic Sequencing Library
Preparation, Illumina). The final libraries, after purification by the
Agencourt AMPure XP system (Beckman), were analysed
on a Typestation 2200 platform (Agilent Technologies,
Santa Clara, CA, USA) and quantified using the Quant-IT
PicoGreen dsDNA assay kit (Thermo Fisher Scientific) by
the Synergy 2 microplate reader (Biotek). Finally all the
libraries were pooled in an equimolar way in a final
amplicon library and analysed on a Typestation 2200 platform
(Agilent Technologies, Santa Clara, CA, USA). Bar-coded
library were sequenced on an Illumina® MiSeq (PE300)
platform (MiSeq Control Software 2.0.5 and Real-Time
Analysis software 1.16.18). Differences in relative
abundance after intervention (V2–V1) with treatment W or P
were analysed using non-parametric t test (Mann–Whitney
U test).
Quantitative microbial molecular techniques
Flow cytometry (FCM) fluorescent in situ hybridization
(FISH)
1:10 dilution (wt:vol) of the faecal sample was prepared by
weighting out 2–3 g of faecal sample and diluting it with
1M PBS on the scale (e.g. 3 g of sample + 27 g of PBS,
considering that 1 ml of 1M PBS weights 1 g). The diluted
sample was homogenized using a Stomacher 400 (Seward)
at the speed of 230 rpm for 2 min or until it appeared
homogeneous. Ten millilitre of the suspension was transferred
into a 15-ml falcon tube containing glass beads; the tube
was mixed by vortexing for about 30 s, then centrifuged at
1100 rpm for 2 min to pellet fibrous particles. Then 375 μl
of the suspension were transferred into a 1.5-ml Eppendorf
tube containing 1125 μl of 4% paraformaldehyde (PFA).
The suspension was fixed in 4% PFA for 4–24 h at 4 °C.
After fixation the tubes were centrifuged at 13,000 rpm
for 5 min and the pellet were resuspended in 1 ml of
filtersterilized 1M PBS. This washing procedure was repeated
twice, then the tubes were centrifuged again at 13,000 rpm
for 5 min, supernatant was carefully removed and the
pellet was finally resuspended in 150 μl of filter-sterilized 1M
PBS. One hundred and fifty (150) microlitre of absolute
ethanol was added and the samples were mixed by
inversion and immediately stored at − 20 °C. For FCM-FISH
analysis, 10 μl of the fixed faecal sample was resuspended
in 190 μl of PBS 1X sterile. Every step was done in 96-well
plates. After resuspending, the sample was centrifuged at
4000 rpm for 15 min. The sample was resuspended in 200 μl
of Tris–EDTA buffer and then centrifuged another time at
4000 rpm for 15 min. For hybridisation with the probes
specific for Lactobacillus/Enterococcus spp. [
31
], Bifidobac‑
terium spp. [
32
] and Ruminococcus spp. [
33
] that needed
lysozyme treatment to render the cell wall more
permeable to the probes, we resuspended the sample in 200 μl of
Tris–EDTA containing 1 mg/ml of lysozyme and incubated
for 10 min at room temperature. After that period, sample
was washed with centrifugation and resuspended in 50 μl
of hybridization buffer [0.9 M NaCl, 20 mM Tris–HCl (pH
7.5), 0.1% [wt/vol] sodium dodecyl sulphate (SDS)] with
5 μl of 50 ng/μl fluorescently Cy5-labelled oligonucleotide
probe and incubated at the appropriate hybridization
temperature (Table 2). Washing was repeated and the sample was
resuspended in 200 μl of hybridization buffer without SDS
and incubated at the appropriate wash temperature (Table 2).
After washing, sample was resuspended in SYBR Green,
used to enumerate the total cells. SYBR Green binds to DNA
and the resulting complex absorbs blue light (λmax = 497 nm)
and emits green light (λmax = 520 nm). A blank sample
(without the fluorescently Cy5-labelled oligonucleotide probe and
without the SYBR Green) was prepared and for every
sample, following the same steps as per the hybridized sample,
to set the threshold of the gates of the flow cytometer that
permits the revelation of the microbial species and exclude
the false positives due to the potential autofluorescence of
the sample. FCM was performed using Guava easyCyte™
Single Sample Flow Cytometer (Millipore) with a single
blue (488 nm), dual blue and red (642 nm), or triple blue,
red, and violet (405 nm) excitation lasers that provided 12
simultaneous detection parameters, including 10 fluorescent
colours plus forward and side scatter for size and
granularity determination. The FCM parameters were adjusted
to give particle counts of 1000 events in total. Data were
analysed by the InCyte software, version 4.1.1. To avoid
loss of the signal intensity of hybridized cells, the samples
were kept in the dark until the FCM analysis. Results were
expressed as the percentage of cells hybridized with the
group-specific-Cy5 probe calculated on the total bacteria,
counted after SYBR Green staining. Also absolute numbers
were obtained.
DNA extraction was performed using the FastDNA™ SPIN
Kit for Feces (MP Biomedicals). Amplifications were
performed with sets of primers specific for Bifidobacterium
Dietary supplement
Dietary supplement
OEP
T0
p value is relative to one-way ANOVA on the difference between T0 and T1
SYS systolic pressure, DIA diastolic pressure, WM waist measure, HM hips measure, BMI body mass index,
TC plasma total cholesterol, HDL plasma high-density lipoproteins, LDL plasma low-density lipoproteins,
TG plasma triglycerides, Apo Apolipoproteins in plasma, Glu plasma glucose, Ins plasma insulin, CRP
plasma reactive C protein, oxLDL plasma oxidized LDL, F2 Isp total 24-h urine Isoprostane F2
ANOVA
p value
spp. [Bif F: TCG CGT C(C/T)G GTG TGA AAG; Bif R:
CCA CAT CCA GC(A/G) TCC AC] and for total bacteria
(Bact 1369: CGG TGA ATA CGT TCC CGG; Prok 1492
TAC GGC TAC CTT GTT ACG ACTT). Reactions were
performed at the specified conditions (see reference) using
SsoFAST Evagreen SupemixKit (BIO RAD) and a
Lightcycler 480 PCR machine (Roche). Quantifications were
done using standard curves obtained by amplifying pure
cultures of Bb12 which had been previously quantified by
plate counting. For total bacteria a mixture of bacterial DNA
was obtained by pooling the total faecal genomic DNA from
four faecal samples, which had been previously enumerated
using FCM-FISH.
Metabolite analysis
Targeted metabolomics analysis by UHPLC–ESI-MS/MS
was carried out as previously described [
30, 34
] on 24-h
urine and fasted blood samples. After methanol extraction
of polyphenols, analysis was performed by an
ultra-performance liquid chromatographic system (UHPLC) coupled
with a tandem mass spectrometer. The system used was an
ACQUITY UPLC system coupled to a Xevo TQ triple
quadrupole via an electrospray (ESI) interface (Waters, Milford,
MA, USA). The separation was performed with a Waters
ACQUITY UPLC column HSS T3 (100 mm × 2.1 mm,
1.8 μm) equipped with a guard column. The injection
volume was 5 μl. Mobile phases of 0.1% formic acid in
MilliQ water (A) and 0.1% formic acid in acetonitrile (B) were
used. Chromatographic separation was performer using
a gradient as follows: 0 min, 5% B; 0–3 min, 5–20% B;
3–4.30 min, 20% B; 4.30–9 min, 20–45% B; 9–11 min,
45–100% B; 11–14 min, 100% B; and 14.01–17 min, 5%
B as equilibration time. For calibration, a standard mixture
of polyphenol metabolites was serially diluted in aqueous
methanol (50:50) at a concentration range of 0.01–20 mg/l.
Quantitative data were processed withTargetlynx software
(Masslynx, Waters).
Statistical analysis
Statistical analysis was performed using STATISTICA 13.1
statistics software for data analysis. Data were checked
for normality using the Kolmogorov–Smirnov and
Shapiro–Wilk tests. Treatment effects were assessed using
oneway analysis of variance, or non-parametric Mann–Whitney
test. Treatments were compared to each other using a paired
Student’s t test. p values < 0.05 were deemed statistically
significant. qPCR data analysis was performed using
factorial ANOVA (factors: treatment, time) with FDR correction.
Urine and plasma metabolite data analysis was performed
using factorial ANOVA (factors: treatment, time) with FDR
correction.
Results
Study report
A total of 73 suitable subjects were identified and were
accepted onto the trial to begin the dietary intervention.
Eight did not finish the study. Two subjects dropped out
because of illness not related to the intervention (flue and
surgery), one for family-related issues, five did not like the
taste of the product and dropped out. Exclusion occurred
because of deviation from the protocol: one volunteer
declared after finishing the study to have taken
antibiotics and two subjects were excluded because they did not
consume the product as directed (more than 25% returned
unconsumed). In total 62 people completed the study
successfully and were included in the statistical analysis. In
detail, 32 females and 30 males, between 30 and 65 years
old, with BMI from 20 to 29.9 (average 24 ± 3.4) and total
cholesterol ranging between 180 and 240 mg/dl completed
the study. Female group had an average age of 48 years
(± 8.5), while the male group average age was 49 (± 9.6).
Between the treatment groups at baseline, blood pressure
(average 120/74 and 122/74), BMI (average 24 and 24) and
total cholesterol (average 204 and 217), no significant
differences were measured (Table 2).
Faecal microbiota analysis
16S rRNA gene community analysis
A total of 8,924,305 reads was generated, with an average
of 68,648.5 ± 36,038.2, mean ± SD, high-quality 16S rRNA
gene sequences per stool sample. Microbiota diversity was
evaluated for alpha diversity (diversity within a sample,
Chao index) and beta diversity (diversity between samples,
Bray–Curtis dissimilarity) using QIIME (https://www.qiime.
org, [
35
]). Sequences with expected error rate > 1.5% and
length < 400 bp were removed from analysis. After filtering
and chimera removal, 1418.2 ± 623.9, mean ± SD,
Operational Taxonomic Units (OTU) were obtained on average per
sample analysed. OTUs that were present in less than 25%
of the samples were removed.
The two intervention treatments did not show statistically
significant differences in alpha diversity (2634.54± 943.69
vs 2682.012 ± 1177.8 and 2419.24 ± 1170.06 vs
2753.09 ± 875.9 Chao index, V1 vs V2, OEP and CTRL,
respectively; p = 0.29) and beta diversity (0.433 ± 0.087 vs
0.43 ± 0.064 Bray–Curtis dissimilarity index, mean ± SD,
OEP vs CTRL, respectively; p = 0.66) (Fig. 2a, b).
At the phylum level, although there was a trend towards
increased Actinobacteria with both treatments, there was no
statistically significant change within or between treatments
over the course of the trial (Fig. 3).
Fig. 2 Alpha- (a) and
betadiversity (b) indexes after 16S
rRNA metagenomic analysis of
faecal samples collected before
(T0) and after (T1) dietary
intervention. Center lines show
the medians; box limits indicate
the 25th and 75th percentiles
as determined by R software;
whiskers extend 15 times the
interquartile range from the
25th and 75th percentiles;
outliers are represented by dots
At a genus taxonomic level, a significant although very
small increase of Lactobacillus and Ruminococcus was
observed after intervention with CTRL compared to OEP
(0.22 ± 1.21 and 0.27 ± 1.45 vs 0.07 ± 0.33 and 0.05 ± 0.31,
p = 0.042 for Lactobacillus; 0.54 ± 0.60 and 0.70 ± 0.76 vs
0.77 ± 0.93 and 0.43 ± 0.38 for Ruminococcus, p = 0.0191,
percentage relative abundance, V1 and V2, CTRL vs OEP,
respectively). Also very small changes in relative
abundance of the less dominant bacterial genera were observed
(0.0007 ± 0.002 and 0.0009 ± 0.001 vs 0.0011 ± 0.002 and
0.0003 ± 0.0007, for an unknown genus Gemellaceae
family, p = 0.017; and 0.0011 ± 0.0026 and 0.0047 ± 0.0102 vs
0.0015 ± 0.0031 and 0.0011 ± 0.0023, p = 0.032 for Anaero‑
fustis, percentage relative abundance, V1 and V2, CTRL vs
OEP, respectively). Figure 3a shows the change in relative
abundance at genus level for Bifidobacterium, Ruminococcus
and Lactobacillus for OEP and CTRL treatments between
V1 and V2.
Culture‑independent targeted quantitative enumeration
of faecal bacteria
No significant differences were observed between
treatment OEP and CTRL compared to baseline
values (1.85 ± 2.89 and 2.17 ± 3.33 vs 1.39 ± 1.70 and
2.20 ± 3.88) for Bifidobacterium spp. (1.07 ± 1.57
and 1.06 ± 1.31 vs 0.53 ± 0.78 and 0.61 ± 1.11) for
Lactobacillus/Enterococcus spp. (0.58 ± 0.97 and
0.54 ± 0.54 vs 0.50 ± 0.79 and 0.59 ± 0.87, for Rumino‑
coccus obeum group V1 and V2, OEP vs CTRL,
respectively, p > 0.05 factorial ANOVA). Figure 4 shows the % of
bacteria enumerated with oligonucleotidic probes specific
for Bifidobacterium spp., Lactobacillus/Enterococcus spp.
and Ruminococcus spp.
No significant changes in faecal bifidobacteria (6.34± 0.95
and 6.62 ± 0.92 vs 6.67 ± 0.74 and 6.72 ± 0.74, V1 and
V2, P vs W, respectively, p > 0.05, factorial ANOVA) or
in total faecal bacteria (10.08 ± 0.04 and 10.08 ± 0.05 vs
10.07 ± 0.05 and 10.07 ± 0.04, V1 and V2, OEP vs CTRL,
respectively, p > 0.05, factorial ANOVA) were observed
after qPCR analysis (data not shown).
Urinary metabolites quantified by LC–MS
The results of targeted urinary polyphenols are shown in
Table 3. Metabolites which were below the detection limit
in the majority of samples were excluded from further
analysis. Statistical analysis (factorial ANOVA) showed
that a number of polyphenol metabolites were
significantly higher after OEP treatment compared to CTRL. In
particular, 3-3-hydroxyphenyl propanoic acid (p = 0.009),
3,4-dihydroxyphenyl acetic acid (p < 0.001), hippuric
acid (p = 0.014), caffeic acid (p = 0.003), homovanillic
acid (p < 0.001), 3-hydroxyphenyl acetic acid (p = 0.001),
sinapic acid (p = 0.002), scopoletin (p = 0.001).
2,4-Dihydroxybenzoic acid (p < 0.001), 2,5-dihydroxybenzoic acid
(p = 0.022), 3-(3-hydroxyphenyl) propionic acid (p = 0.009),
were increased after OEP feeding.
Plasma metabolites quantified by LC–MS
The results of targeted quantification of plasma
metabolites by LC–MS are shown in Table 4. Metabolites which
were below the detection limit in the majority of samples
Fig. 4 Difference in percentage
relative abundance of relevant
bacterial genera before (T0) and
after (T1) dietary intervention
with olive-enriched product
(OEP) or control product
(Ctrl). p = 0.73, p = 0.034
and p = 0.02, respectively, for
Bifidobacterium, Lactobacillus
and Ruminococcus genera, after
comparison of the difference
T1–T0 between olive-enriched
product (OEP) and control
product (Ctrl), according to
Mann–Whitney U test. Center
lines show the medians; box
limits indicate the 25th and 75th
percentiles as determined by R
software; whiskers extend 1.5
times the interquartile range
from the 25th and 75th
percentiles; outliers are represented
by dots
were excluded from further analysis. Statistical analysis
(factorial ANOVA) showed that 3,4-dihydroxyphenyl
acetic acid (p = 0.002) and homovanillic acid (p = 0.003)
were significantly higher after OEP treatment compared
to CTRL. Most of the polyphenol metabolites were
present at very low concentrations in plasma compared
to urine since the plasma samples were taken in a fasted
state.
Table 3 Urinary polyphenol concentration (µM) quantified by mass
spectrometry and normalized according to 24-h urine volume
T1
Data represent the mean and standard deviation (SD) at the beginning
(T0) and at the end (T1) of dietary intervention with olive-enriched
product (OEP) or control product (Ctrl), and the relative p value after
factorial ANOVA with Bonferroni’s correction. Cinnamic acid,
caftaric acid, cis-piceide, luteolin, hesperidin, catechin, epicatechin,
procyanidin B1, procyanidin B2 + B4, procyanidin B3, quercetin-3-Rha,
kaempferol-3-Glc, kaempferol-3-rutinoside, dihydrokaempferol,
quercetin-3-glucuronide, kaempferol-3-glucuronide, arbutin,
p-coumaric acid, o-coumaric acid, gallic acid, ellagic acid, pyrocatechol,
urolithin B, epigallocatechin gallate, epicatechin gallate,
quercetin3-Glc + quercetin-3-gal, isorhamnetin-3-Glc, rutin, salidroside are not
shown, since the levels fell below limit of quantification
Clinical measures of CVD risk and inflammation
Subjects in either group, OEP or CTRL, were matched
for age and sex. Little difference was observed in baseline
clinical parameters between the groups before dietary
intervention. After 8 weeks of treatment with either biscuit, no
significant change in CVD or inflammatory makers was
observed (Table 2). There was a trend towards reduced
oxidized LDL cholesterol in the OEP-treated group, but this
was not significant either with respect to time or compared
to the control treatment.
Sex impacted on urinary metabolite profiles
Although sex did not appear to influence to response to OEP
ingestion in urine or in plasma, differences were observed
in the concentrations of small phenolic acids excreted by
women compared to men after the OEP treatment. Higher
amounts of 3,5-diOH-benzoic acid, t-coutaric acid,
naringenin, 4-hydroxybenzoic acid, 4-hydroxyphenyl acetic acid
were excreted by male subjects compared to female subjects
after the OEP treatment (Table 5). Since the gut microbiota
is intricately involved in the metabolism of complex
polyphenolic compounds and especially in the production of
small phenolic acids, we measured whether the gut
microbiota of male and female subjects differed after OEP ingestion.
Statistically significant differences in the relative abundance
of Akkermansia, Bifidobacterium, Bacteroides, Prevotella,
Table 4 Plasma polyphenol concentration (µM) quantified by mass
spectrometry
Data represent the mean and standard deviation (SD) at the beginning
(T0) and at the end (T1) of dietary intervention with olive-enriched
product (OEP) or control product (Ctrl), and the relative p value after
factorial ANOVA with Bonferroni’s correction
Chlorogenic acid, cis-piceide, quercetin-3-Rha,
quercetin-3-glucuronide, kaempferol-3-glucuronide, t-ferulic acid, ellagic acid,
protocateTable 4 (continued)
chuic acid, cryptochlorogenic acid, quercetin-3-Glc + quercetin-3-gal,
hydroxytyrosol and tyrosol are not shown, since the levels fell below
limit of quantification
Table shows the p values after factorial ANOVA with Bonferroni’s
correction. Data represent mean and standard deviation (SD) of
urinary concentrations (µM), after normalization according to 24-h urine
volume
Rikenellaceae, Barnesiellaceae, and Enterobacteriaceae
were observed between the faecal microbiota of men and
women (Fig. 5). These differences were statistically
significant after correction for repeated measures (Mann–Whitney
U test, 2*1 exact p value) (Fig. 6).
Discussion
The primary objective of this study was to measure the
impact of an olive pomace-enriched product (OEP) on the
composition and metabolic output of the human gut
microbiota. Considering the accepted physiological relevance
of olive polyphenols, their apparent ability to protect LDL
cholesterol particles from oxidative damage, and the fact
that the gut microbiota appears to be intimately related
to their metabolism in vivo, we measured changes in key
olive-derived polyphenols, including tyrosol and HT, and
their derived catabolites using a quantitative LC–MS-based
strategy. The OEP biscuits did not have a major impact
on the composition of the gut microbiota, but did induce
subtle changes in relative abundances of certain bacteria.
Significant differences in relative abundance of Lactoba‑
cillus, Ruminococcus, Gemellaceae and Anaerofustis were
observed between treatments using community level 16S
rRNA profiling. More quantitative analysis using flow
cytometry-coupled fluorescent in situ hybridization did not
confirm statistically significance for bifidobacteria,
lactobacilli or the Ruminococcus obeum-like bacteria. However,
a trend was apparent, consistent between 16S rRNA gene
sequencing, the probe-based FISH and qPCR, showing a
small increase in bifidobacteria.
In terms of metabolic output, LC–MS-based targeted
metabolomics confirmed that ingestion of the OEP biscuits
resulted in a significant increase in urinary excretion of small
phenolic acids derived from the metabolism of olive
polyphenols. These small phenolic acids derive from the
combined activities of human phase I and II biotransformation
and the action of the gut microbiota. OEP ingestion resulted
in a significant increase in excretion of homovanillic acid,
3,4-dihydroxyphenyl acetic acid, scopoletin, protocatechuic
acid, sinapic acid, 3-hydroxyphenyl acetic acid, isoferulic
acid, caffeic acid, hippuric acid, 3,3-hydroxyphenyl acetic
acid, 2,5-dihydroxybenzoic acid and 2,4-dihydroxybenzoic
acid. Many of these compounds derive from the breakdown
pathways of the tyrosol group enriched in olives and/or the
hippuric acid pathway, a pathway common to many classes
of polyphenols. Both involve steps mediated by the gut
microbiota and these catabolites and similar small phenolic
acids have been reported to be excreted following ingestion
of olive or olive fractions in previous studies [
20–22, 36,
37
]. Few studies have reported the profile of metabolites
present in fasted blood samples after chronic ingestion of
olive pomace. Here we found that ingestion of the OEP
biscuits for 8 weeks resulted in a significantly higher level of
tyrosol metabolites, homovanillic acid and
3,4-dihydroxyphenyl acetic acid (DOPAC). Homovanillic acid increased
more then twofold in the OEP-treated group and was also
about double the concentration after the CTRL treatment.
For DOPAC, OEP induced more than tenfold increase in
fasted blood concentrations, and a similar difference in
magnitude was observed compared to the control group. As
well as being associated with the protection of LDL
cholesterol particles from oxidative damage, both homovanillic
acid and DOPAC have been reported to transiently associate
with LDL cholesterol particles in blood thereby mediating
their antioxidative effect. While most studies report these
molecules to be relatively rapidly cleared from the blood
[
38
], our study is one of the very few to record high levels of
these antioxidants in fasted blood samples, many hours after
ingestion of the olive-enriched food. This is an important
observation for the possible functional activities of the OEP
biscuit since persistence of these tyrosol-derived metabolites
Fig. 5 Bacterial populations
enumerated by FCM-FISH (%
of total bacteria enumerated by
SYBR green staining) in faecal
samples collected before (T0)
and after (T1) dietary
intervention with olive-enriched product
(OEP) or control product (Ctrl).
Center lines show the medians;
box limits indicate the 25th and
75th percentiles as determined
by R software; whiskers extend
1.5 times the interquartile range
from the 25th and 75th
percentiles; outliers are represented
by dots
in blood, or indeed within the intestine, could prime
antioxidant defences and/or absorption of oxidized LDL cholesterol
upon fat meal challenge. However, our current study was
not designed to measure the effect of olive polyphenols on
oxidized LDL cholesterol levels and therefore, any potential
beneficial effect of the OEP biscuit awaits confirmation in
an acute, postprandial study. Similarly, homovanillic acid
and DOPAC are both involved in the dopamine pathway
and have been shown to mediate other physiological effects
including ameliorating age-related decline in muscle
Fig. 6 Significant differences between male (M) and female (F) in
percentage relative abundance of bacterial genera after dietary
intervention with olive-enriched product (OEP), according to Mann–
Whitney U test. Center lines show the medians; box limits indicate
function [
39
] and brain function [
40
]. In addition, DOPAC
has been shown to impact on the inflammatory response of
immune cells to lipopolysaccharide or endotoxin, an
inflammatory microbially derived signal associated with increased
risk of metabolic disease [
41
]. However, any ability of the
OEP biscuits to mediate such health effects awaits
specifically and appropriately designed human studies.
In this current study, we also measured the ability of the
OEP biscuit to modulate blood lipid profiles. Previous
studies with whole plant foods or oat-derived beta-glucan in
particular, have shown significant and clinically meaningful
reductions in cholesterol upon ingestion [
42
]. However, the
mechanisms by which these foods mediate their cholesterol
lowering effects are still very much unclear, with different
mechanisms suggesting involving phytosterols, gel-forming
and cholesterol binding activities, modified bile acid profiles
and/or prebiotic type modulation of the gut microbiota [
43,
44
]. Our OEP, not containing the pit pulp of the olive, did
not contain large amounts of plant phytosterols, and only
had minor impact on microbiota composition. Although
in this case, the olive pomace did not change blood lipid
the 25th and 75th percentiles as determined by R software; whiskers
extend 15 times the interquartile range from the 25th and 75th
percentiles; outliers are represented by dots
profiles, further studies, possibly with larger sample size
be warranted, especially since the ability of olive and olive
extracts in general to modulate blood lipid profiles remains
to be convincingly established as per the EFSA statement on
olive polyphenol extract health claims [18].
The quantities of small phenolic acids in urine differed
between men and women upon OEP ingestion. Men excreted
significantly more 3,5-diOH-benzoic acid, t-coutaric acid,
naringenin, 4-hydroxybenzoic acid, 4-hydroxyphenyl acetic
acid than women. A sex bias in polyphenol metabolism has
been reported previously. Zamora-Ros et al. [
45
]
analysing the EPIC cohort study reported a significant sex bias
in excretion of dietary polyphenols. Moreover, the sex
differences in dietary polyphenol metabolism also appears to
be reflected in the concentration and profiles of
polyphenols or their derivatives in different tissues and organs, as
shown for grape seed flavanols in rats [
46
]. Such sex effects
could have important implications for the biological activity
of these compounds, especially since a sex effect has also
been reported in response to dietary interventions measuring
physiological change and/or reduced risk of chronic disease
upon intervention with polyphenol-rich foods [
47
]. The
contribution of the intestinal microbiota to any sex-specific
“metabotype” in terms of polyphenol metabolism remains
very much unexplored. In our study, faecal samples collected
from men and women post-OEP ingestion differed
significantly in the relative abundance of Akkermansia, Bifidobac‑
terium, Bacteroides, Prevotella, Rikenellaceae,
Barnesiellaceae, and Enterobacteriaceae. Some of these bacteria are
linked to host physiology and protection from metabolic and
cardiovascular disease (Akkermansia and Bifidobacterium
in particular), but also Bacteroides and Prevotella in
relation to obesity and traditional dietary paradigms [
48–50
].
Similarly, the enterobacteria, which includes many intestinal
pathogens, appear particularly susceptible to the
antibacterial activities of polyphenols [
51
]. The role of the human gut
microbiota in determining the profile and quantities of
different polyphenol breakdown products, their bioavailability,
bioactivity and nutri-kinetics may constitute and important
new compounding factor to be taken into consideration when
designing human dietary interventions where polyphenols
are considered mediators of physiological effect. Further
data from similar human studies are required to confirm the
involvement of the gut microbiota in gender-specific
polyphenol metabolism and possible implications sex-specific
response to diet.
In conclusion, ingestion olive pomace extract-enriched
biscuits mediated small changes within the composition of
the gut microbiota. Delivering 17.1 ± 4.01 mg/100 g HT
and its derivatives, the OEP biscuits induced a significant
increase in excretion of small phenolic acids in urine,
indicative of up-regulation of microbial polyphenol
biotransformation in the intestine. Quantities of some small phenolic
acids differed in urine of men and women, as did relative
abundances of important members of the gut microbiota.
OEP also led to a significant increase in homovanillic acid
and DOPAC in fasted plasma samples, indicating related
clearance of these compounds from the blood or extended
release and uptake from the intestine. In either case, higher
levels of these biologically active compounds mediated by
OEP ingestion warrant further investigation in acute or
postprandial studies specifically targeting LDL cholesterol
oxidation and cognitive function.
Open Access This article is distributed under the terms of the
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(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
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the Creative Commons license, and indicate if changes were made.
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