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Almonds are known to have a number of nutritional benefits, including cholesterol-lowering effects and protection against diabetes. They are also a good source of minerals and vitamin E, associated with promoting health and reducing the risk for chronic disease. For this study we investigated the potential prebiotic effect of almond seeds in vitro by using mixed fecal bacterial cultures. Two almond products, finely ground almonds (FG) and defatted finely ground almonds (DG), were subjected to a combined model of the gastrointestinal tract which included in vitro gastric and duodenal digestion, and the resulting fractions were subsequently used as substrates for the colonic model to assess their influence on the composition and metabolic activity of gut bacteria populations. FG significantly increased the populations of bifidobacteria and Eubacterium rectale, resulting in a higher prebiotic index (4.43) than was found for the commercial prebiotic fructooligosaccharides (4.08) at 24 h of incubation. No significant differences in the proportions of gut bacteria groups were detected in response to DG. The increase in the numbers of Eubacterium rectale during fermentation of FG correlated with increased butyrate production. In conclusion, we have shown that the addition of FG altered the composition of gut bacteria by stimulating the growth of bifidobacteria and Eubacterium rectale.
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2008, p. 4264–4270 Vol. 74, No. 14
0099-2240/08/$08.000 doi:10.1128/AEM.00739-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Potential Prebiotic Properties of Almond (Amygdalus communis L.) Seeds
G. Mandalari,
1,3
* C. Nueno-Palop,
2
G. Bisignano,
3
M. S. J. Wickham,
1
and A. Narbad
2
The Model Gut Platform, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom
1
;
Commensal and Microflora Programme, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA,
United Kingdom
2
; and Department of Pharmacobiology, University of Messina, Viale Annunziata, 98100 Messina, Italy
3
Received 28 March 2008/Accepted 15 May 2008
Almonds are known to have a number of nutritional benefits, including cholesterol-lowering effects and
protection against diabetes. They are also a good source of minerals and vitamin E, associated with
promoting health and reducing the risk for chronic disease. For this study we investigated the potential
prebiotic effect of almond seeds in vitro by using mixed fecal bacterial cultures. Two almond products,
finely ground almonds (FG) and defatted finely ground almonds (DG), were subjected to a combined
model of the gastrointestinal tract which included in vitro gastric and duodenal digestion, and the
resulting fractions were subsequently used as substrates for the colonic model to assess their influence on
the composition and metabolic activity of gut bacteria populations. FG significantly increased the popu-
lations of bifidobacteria and Eubacterium rectale, resulting in a higher prebiotic index (4.43) than was
found for the commercial prebiotic fructooligosaccharides (4.08) at 24 h of incubation. No significant
differences in the proportions of gut bacteria groups were detected in response to DG. The increase in the
numbers of Eubacterium rectale during fermentation of FG correlated with increased butyrate production.
In conclusion, we have shown that the addition of FG altered the composition of gut bacteria by
stimulating the growth of bifidobacteria and Eubacterium rectale.
Functional foods are known as dietary components that may
cause physiological effects on the consumer, leading to justifi-
able claims of health benefits (36). According to the European
consensus document “Scientific concepts of functional foods,”
a food ingredient may be regarded as functional if it benefi-
cially affects one or more target functions in the body, beyond
its nutritional effects, in order to improve the state of health
and/or reduce the risk of disease (9). A prebiotic is defined as
“a nondigestible food ingredient which beneficially affects the
host by selectively stimulating the growth and/or activity of one
or a limited number of bacteria in the colon and thus improv-
ing host health” (15). Prebiotics of proven efficacy are able to
modulate the gut microbiota by stimulating indigenous bene-
ficial flora while inhibiting the growth of pathogenic bacteria,
such as proteolytic bacteroides and clostridia (43). Bifidobac-
teria and lactobacilli are able to inhibit the growth of clostridia
and pathogenic Enterobacteriaceae by the production of short-
chain fatty acids and antimicrobial compounds, as well as by
competition for growth substrate and adhesion sites (16, 19,
23). As such, these beneficial gut bacteria, together with mu-
cins and antimicrobial peptides, represent part of the host’s
front line of defense against harmful microorganisms (24, 40).
In order to be effective as a prebiotic, an ingredient must
neither be hydrolyzed nor absorbed in the upper part of the
gastrointestinal tract (GIT). Although any dietary material that
enters the large intestine can be considered as potentially pre-
biotic, currently the best-known prebiotics are nondigestible
oligosaccharides (17). Different oligosaccharides with prebiotic
properties, such as inulin, fructooligosaccharides (FOS), galac-
tooligosaccharides, and lactulose, are commercially available,
but currently there is increasing interest in the identification
and development of new prebiotic compounds, perhaps with
added functionality (26, 29, 33, 42).
The almond nut (Amygdalus communis L.) is a species of
Prunus belonging to the family Rosaceae. The global pro-
duction of almonds is around 1.7 million metric tons, with
California producing 80% of the world’s almonds. Lipid, the
main storage component in almond seeds, constituting over
50% of the total weight of the seeds, is located as intracel-
lular oil bodies (35). Proteins comprise about 22 to 25% of
the seeds, while 11 to 12% is represented by dietary fiber.
Recent results have shown that the encapsulation of intra-
cellular lipids by the cell walls restricts their digestion in the
stomach and small intestine (13, 27). Furthermore, if undi-
gested lipid from almond tissue reaches the large intestine,
it could be used by resident microbiota, and evidence of
bacterial fermentation was previously shown (13). Almond
cell wall material contains pectic substances that are rich in
arabinose, and the observation of their partial degradation
by the gut microbiota in the fecal samples can be explained
by the erosion of the middle lamella (10, 13). The results of
our recent study have demonstrated that the bioaccessibility
of nutrients and phytochemicals from almond seeds is im-
proved by increased residence time in the gut and is regu-
lated by almond cell walls. The results of these in vitro and
ileostomy digestibility studies have shown high amounts of
lipid and protein remaining in the almond tissue after duo-
denal digestion and therefore available for fermentation in
the colon by the gut microbiota (27).
Here we describe the investigation of the potential prebiotic
effect of almond seeds by using a full model of GIT digestion,
including gastric and small intestinal environments and a co-
* Corresponding author. Mailing address: Institute of Food Re-
search, Norwich Research Park, Colney Lane, Norwich NR4 7UA,
United Kingdom. Phone: 44 1603 251405. Fax: 44 1603 507723. E-mail:
giusy.mandalari@bbsrc.ac.uk.
Published ahead of print on 23 May 2008.
4264
lonic model consisting of in vitro fermentation systems with
representative human gut bacteria.
MATERIALS AND METHODS
Almond products. Blanched finely diced and powdered almonds (Amygdalus
communis L; variety Nonpareil) were kindly provided by the Almond Board of
California. Commercial FG did not contain skin coat and had a mean particle
size of 200 m. DG was prepared by extracting 25 g of FG three times with 400
ml of n-hexane for2honeach occasion, as previously described (27).
Chemicals and enzymes. Egg
L--phosphatidylcholine (PC; lecithin grade 1,
99% purity) was purchased from Lipid Products (South Nutfield, Surrey, United
Kingdom). Porcine gastric mucosa pepsin (activity of 3,300 U/mg of protein
calculated by using hemoglobin as substrate), bovine -chymotrypsin (activity of
40 U/mg of protein using benzoyl-L-tyrosine ethyl ester as substrate), porcine
trypsin (activity of 13,800 U/mg of protein using benzoyl-
L-arginine ethyl ester as
substrate), porcine pancreatic lipase (activity of 25,600 U/mg protein), porcine
colipase, sodium taurocholate, and sodium glycodeoxycholate were obtained
from Sigma (Poole, Dorset, United Kingdom). The lipase for the simulated
gastric phase of digestion was a gastric lipase analogue from Rhizopus oryzae
(F-AP15; activity of 150 U/mg) obtained from Amano Enzyme (Nagoya,
Japan). All other chemicals were of Analar quality.
In vitro digestion studies. The protocol previously developed to study almond
digestion under gastric and duodenal conditions (27) was used to simulate gas-
trointestinal processing for both FG and DG. Each simulated digestion was
performed at least four times, and the solid material recovered for analysis.
Control digestions of the two almond products (FG and DG) were performed in
saline solution (150 mM NaCl, pH 2.5 or 6.5 for gastric and duodenal digestion,
respectively) without enzyme additions.
In vitro gastric digestion. Phospholipid vesicles were prepared as previously
described (27). Briefly, solvent was removed from 0.94 ml of PC stock solution
(63.5 mM), and the thin film of phospholipids was then suspended in 12.2 ml of
warmed saline (150 mM NaCl, pH 2.5, at 37°C). The suspension was then
sonicated at 5°C in a coolant-jacketed vessel, using a sonication probe (Status US
200; Avestin) with a pulsed cycle of 30% full power on for 0.9 s and off for 0.1 s.
The single-shelled liposome suspension was filtered through a 0.2-m nylon
syringe filter (Nalgene, United Kingdom) and equilibrated in an orbital shaking
incubator (170 rpm) at 37°C. Each almond product (1.5 g) was suspended in 12.4
ml acidic saline (150 mM NaCl, pH 2.5) in the presence of the PC vesicle
suspension, pepsin, and the gastric lipase analogue at concentrations of 2.4 mM,
146 U/ml, and 0.56 mg/ml, respectively. In vitro gastric digestion was performed
for2hatpH2.5.
In vitro duodenal digestion. Following gastric digestion, the pH was immedi-
ately raised to 6.5 in order to simulate the duodenal conditions. Bile salt solution
(4 mM sodium taurocholate and 4 mM sodium glycodeoxycholate), CaCl
2
(11.7
mM), and bis-Tris buffer, pH 6.5 (0.73 mM) were also added. Duodenal diges-
tions were initiated by the addition of -chymotrypsin (5.9 U/ml), trypsin (104
U/ml), colipase (3.2 g/ml), and pancreatic lipase (54 U/ml) and performed in a
shaking incubator (170 rpm) at 37°C for 1 h.
Lipid content determination. Total lipid and vitamin E extraction of FG and
of FG after in vitro gastric and gastric plus duodenal digestion was performed as
previously described (27).
Total protein assays. Original almond materials (FG and DG) and solid
residues recovered after in vitro gastric and duodenal digestion were analyzed for
total nitrogen by using the micro-Kjeldahl method, as previously reported (27).
Cell wall analysis. Cell wall material was prepared from FG and FG after
gastric plus duodenal digestion by using the method previously described (27).
The alditol acetates were quantified by gas-liquid chromatography, and total
uronic acids determined colorimetrically at 580 nm (2, 3).
Fecal batch culture fermentations. Water-jacketed fermenter vessels (300
ml) were filled with 135 ml of presterilized basal growth medium (2 g/liter
peptone water, 2 g/liter yeast extract, 0.1 g/liter NaCl, 0.04 g/liter K
2
HPO
4
,
0.04 g/liter KH
2
PO
4
, 0.01 g/liter MgSO
4
7H
2
O, 0.01 g/liter CaCl
2
6H
2
O, 2
g/liter NaHCO
3
, 2 ml Tween 80, 0.02 g/liter hemin, 10 l vitamin K
1
, 0.5
g/liter cysteine HCl, 0.5 g/liter bile salts, pH 7.0) and inoculated with 15 ml of
fecal slurry. Before the addition of the fecal slurry, prepared by homogenizing
10% (wt/vol) freshly voided fecal material from one healthy donor in 0.1 M
phosphate-buffered saline (PBS), pH 7.0, the almond extract (FG or DG after
gastric and duodenal digestion) or FOS was added to give a final concentra-
tion of 1% (wt/vol). Each vessel was magnetically stirred, the pH automati-
cally controlled and maintained at pH 6.8, and the temperature set at 37°C.
Anaerobic conditions were maintained by sparging the vessels with oxygen-
free nitrogen gas at 15 ml/min. Samples (5 ml) were removed over 24 h for the
enumeration of bacteria and short-chain fatty acid analysis. Fermentations
were run on three separate occasions.
Enumeration of bacteria. Bacteria were counted by using fluorescent in situ
hybridization (FISH) (37). Duplicate fermentation samples were diluted four
times in 4% (wt/vol) filtered paraformaldehyde and fixed overnight at 4°C.
Samples were then washed twice with filtered PBS (0.1 M, pH 7.0) and stored at
20°C in PBS-ethanol (1:1, vol/vol) until further analysis. Hybridization was
performed at an appropriate temperature by using genus-specific 16S rRNA-
targeted oligonucleotide probes labeled with the fluorescent dye Cy3 for the
different bacterial groups or with 4,6-diamidino-2-phenylindole (DAPI) for total
cell counts. The probes used were Bif164, specific for Bifidobacterium (22);
Bac303, specific for bacteroides (28); Lab158, specific for Lactobacillus/Entero-
coccus spp. (18); His150, specific for most species of the Clostridium histolyticum
group (Clostridium clusters I and II) (14); and EREC482, specific for most of the
Clostridium coccoides-Eubacterium rectale group (Clostridium clusters XIVa and
XIVb) (5). The hybridized mixture was then vacuum filtered using a 0.2-m
membrane filter (Millipore, Watford, United Kingdom), and the filter was
mounted on a microscope slide. At least 15 random fields were counted on each
slide by using a Nikon Microphot fluorescent microscope.
Short-chain fatty acid analysis. One-milliliter samples removed from the
batch culture fermenter were centrifuged at 15,000 g for 5 min, and 20 lof
the supernatant was injected into a high-pressure liquid chromatography system
equipped with a refractive index detector. We used an ion exclusion Aminex
HPX-87H column (7.8 by 300 mm; Bio-Rad, Watford, United Kingdom), main-
tained at 50
°
C,and5mMH
2
SO
4
as eluent at a flow rate of 0.6 ml/min. Quan
-
tification of the organic acids was carried out by using calibration curves of acetic,
propionic, butyric, and lactic acids in concentrations between 0.5 and 100 mM,
and the results expressed in mmol/liter (37).
Statistical analysis. Differences between bacterial numbers at 0, 8, and 24 h of
fermentation for each batch culture were checked for significance by paired t test,
assuming normal distribution and equal variances and considering both sides of
the distribution. The differences were considered significant when the P value
was 0.05.
RESULTS
Almond product characterization after in vitro digestion.
The compositions of the two almond extracts (FG and DG)
obtained after in vitro gastric plus duodenal digestion are
shown in Table 1. These fractions were subsequently used as
TABLE 1. Chemical composition of FG and DG before and after in vitro gastric plus duodenal digestion
a
Time of analysis
Almond
product
used
Amt of nutrient per 100 gm
Lipid (g) Vitamin E (mg) Protein (g) Dietary fiber (g)
Before digestion FG 54.9 2.5 28.2 1.5 25.9 2.6 9.8 1.3
DG 0 0 57.2 2.4 32.5 1.1
After digestion FG 57.4 3.8 34.0 1.8 24.7 1.9 13.3 0.9
DG 0 0 58.0 2.8 31.2 0.8
a
Values are the means standard deviations of the results. Other components include carbohydrates, minerals, and vitamins.
VOL. 74, 2008 PREBIOTIC EFFECT OF ALMOND SEEDS 4265
substrates for the colonic model. As previously reported (13,
27), almond cell walls are not degraded in the upper GIT, and
therefore, the mass loss during digestion is mostly related to
loss of intracellular components, such as lipid and protein. The
simulated gastric digestion step was responsible for the major-
ity of the gravimetric losses and the highest extent of lipolysis
and proteolysis (27). FG after duodenal digestion still con-
tained 57% of the start lipid and 34 mg of vitamin E per 100 g
total almond mass, of which 96% was -tocopherol, 1.3% -to-
copherol, and the remainder -tocopherol. The sugar compo-
sition indicated that almond cell walls are mainly composed of
arabinose-rich polysaccharides, including the pectic sub-
stances, encasing cellulose microfibrils. The monomeric sugar
concentrations did not change significantly after digestion: the
sugar contents (as percentage of the total) were 39.9 and 39.5
arabinose, 12.0 and 12.6 xylose, 4.7 and 4.7 galactose, 16.7 and
16.8 glucose, and 21.1 and 20.7 galacturonic acid for FG and
for FG after gastric plus duodenal digestion, respectively. This
suggests that almond cell walls were not degraded during di-
gestion.
Batch culture fermentations. Batch fermentations were used
to monitor the effects of predigested FG, DG, and FOS on the
growth of a mixed bacterial population of the human colon.
Samples were removed at intervals, and FISH was used to
quantify the levels of different bacterial groups. The results
shown in Table 2 indicate that a significant increase in the level
of total bacteria was seen with both FG and FOS after 8 and
24 h of incubation, whereas the total bacterial number was
largely unaffected by the addition of DG. Generally, an in-
crease in the numbers of bifidobacteria, lactobacilli, and Eu-
bacterium rectale was observed in response to the addition of
FG and FOS at both the 8- and 24-h incubation time points.
Compared to the control vessel, the bacteroides population
with FG decreased significantly after 24 h, whereas their num-
bers were similar to those of the control at 8 and 24 h of
incubation in the presence of DG. An increase in the number
of Eubacterium rectale was observed in the presence of both
FOS and FG, the latter showing a greater increase after 24 h of
incubation. Both fractions also stimulated the growth of bi-
fidobacteria, with a 0.61 and 0.68 log increase in their numbers
at 24 h with FG and FOS, respectively. The effect of FG on
bifidobacteria, lactobacilli, and Eubacterium rectale numbers
was optimal after8hofincubation and did not evolve toward
the end of incubation, whereas a smaller prebiotic effect was
observed with FOS after 24 h than after 8 h. This suggests a
slower fermentation with FG, which will further the prebiotic
effect in the colon. In comparison to the results with the con-
trol, the addition of DG did not alter the bacterial numbers of
any of the groups examined. The relative changes in the num-
bers of different groups of bacteria after8hand24hof
incubation as a result of FG and FOS addition are shown in
Fig. 1.
In order to obtain a general quantitative measure of the
prebiotic effect, a prebiotic index (PI) was calculated for the
oligosaccharide fractions (31). The PI equation is described
as follows: PI (Bif/total) (Lac/total) Erec/total)
(Bac/total) (Clos/total), where Bif is bifidobacterial num-
bers at sample time divided by numbers at inoculation, Lac
is lactobacilli numbers at sample time divided by numbers at
inoculation, Erec is Eubacterium rectale numbers at sample
time divided by numbers at inoculation, Bac is bacteroides
numbers at sample time divided by numbers at inoculation,
Clos is clostridia numbers at sample time divided by num-
bers at inoculation, and total is total bacteria number at
TABLE 2. Changes in bacterial populations in batch cultures after 8 and 24 h of incubation
a
Organism(s)
Incubation
time (h)
No. (log 10 cells/ml) with:
Control FOS FG DG
Total bacteria 0 9.20 0.03
8 9.17 0.04 9.36 0.11 9.39 0.05
b
9.23 0.02
24 9.19 0.02 9.47 0.01
c
9.40 0.01
c
9.21 0.01
Bifidobacteria 0 8.03 0.03
8 8.14 0.06 8.81 0.05
b
8.54 0.02
b
8.22 0.05
24 7.99 0.04 8.67 0.01
c
8.60 0.11
c
8.01 0.11
Bacteroides 0 8.23 0.01
8 8.30 0.04 8.39 0.01 8.31 0.02 8.31 0.06
24 8.34 0.12 8.42 0.04
c
8.24 0.01
c
8.38 0.06
Clostridia 0 7.47 0.09
8 7.54 0.07 7.18 0.04
b
7.29 0.07
b
7.51 0.03
24 7.54 0.02 7.37 0.04 7.52 0.02 7.57 0.07
Eubacterium rectale 0 8.28 0.01
8 8.33 0.01 8.64 0.13 8.97 0.08
b
8.45 0.04
b
24 8.34 0.08 8.64 0.08
c
8.91 0.02
c
8.46 0.08
c
Lactobacilli 0 7.58 0.02
8 7.72 0.09 8.02 0.01
b
7.73 0.04 7.66 0.05
24 7.68 0.07 7.90 0.04
c
7.63 0.01 7.66 0.08
a
Bacterial counts were obtained by using FISH. Values are the means standard deviations of the results.
b
Significantly different from control at8h(P 0.05).
c
Significantly different from control at 24 h (P 0.05).
4266 MANDALARI ET AL. APPL.ENVIRON.MICROBIOL.
sample time divided by numbers at inoculation. The PI
represents a comparative relationship between the growth
of “beneficial” bacteria, such as bifidobacteria, lactobacilli,
and Eubacterium rectale, and that of the “less desirable”
ones, such as clostridia and bacteroides, in relation to the
change in the total number of bacteria (Fig. 2). For both
substrates, the PI values obtained at8hofincubation were
higher than those at 24 h. The FOS fraction produced the
highest PI value after8hofincubation, 6.36, with that of FG
being 4.98, whereas FG produced the highest PI value at the
24-h time point, 4.43, with that of FOS being 3.49. Low PI
values were obtained with DG and the control at both the 8-
and 24-h incubation time points.
Short-chain fatty acid production during fermentation. The
concentrations of lactic, acetic, propionic, and butyric acids
produced during fermentation are shown in Table 3. FOS gave
the highest total short-chain fatty acid production at all time
points tested. However, butyrate production significantly in-
creased after8hofincubation with FG and peaked at 24 h,
coinciding with the highest number of Eubacterium rectale bac-
teria. Fermentation with FOS resulted in the highest produc-
tion of lactic and acetic acids: their concentrations increased at
4 h and remained elevated up to 24 h. These increases corre-
lated with changes in the numbers of bifidobacteria and lacto-
bacilli. The concentrations of propionic and butyric acids were
higher after 8 and 24 h of fermentation with FG and DG, again
correlating with Eubacterium rectale population changes. In the
absence of the added carbon source, an increase in acetic acid
was observed after 24 h, although the amounts of the other
organic acids did not change significantly.
FIG. 1. Differences in the bacterial population sizes (black bars, FOS; white bars, predigested FG) compared to the total numbers of bacteria
counted at 8 h [(selected bacterial numbers at 8 h/total bacteria counted at 8 h) (selected bacterial numbers at 0 h/total bacteria counted at 0 h)]
(A) and 24 h [(selected bacterial numbers at 24 h/total bacteria counted at 24 h) (selected bacterial numbers at 0 h/total bacteria counted at
0 h)] (B). Error bars show standard deviations.
FIG. 2. Prebiotic index (PI) scores from batch cultures at 8 h (black
bars) and 24 h (white bars) using FOS, predigested FG, DG, and
untreated (control) cultures. Error bars show standard deviations.
VOL. 74, 2008 PREBIOTIC EFFECT OF ALMOND SEEDS 4267
DISCUSSION
In the present study, we have demonstrated the prebiotic
potential of almond seeds. As far as we are aware, this is the
first study that has used combined models of human digestion
which include gastric and duodenal digestion followed by co-
lonic fermentation to study the effects of almond extracts on
the modulation of gut microbiota. Commercially available pre-
biotics (such as FOS and galactooligosaccharides) are not sen-
sitive to gastric acid and do not serve as substrate for hydrolytic
enzymes in the upper digestive tract. The evaluation of novel
prebiotic compounds should take into account the available
ingredients which can be digested by human enzymes and
adsorbed, thus entering into intermediary metabolism. On the
contrary, food components able to reach the colon can poten-
tially provide the body with additional energy via microbial
fermentation, and the production of short-chain fatty acids
may have potential prebiotic functionality.
In our previous study, we showed that relatively small
amounts of almond lipids and proteins are bioavailable during
gastric and small intestinal digestion and that nutrient encap-
sulation by cell walls is likely to prevent their digestion in the
upper GIT (27). However, bioaccessibility is improved when
the number of fractured cells is increased by processing or by
increased residence time in the gut. Evidence of bacterial fer-
mentation of the almond tissue was provided by micrographs
of fecal samples collected from healthy subjects consuming an
almond-rich diet (13). By using transmission electron micros-
copy, it was possible to document the presence of bacteria both
on the cell wall surface and within the cells. Therefore, almond
intracellular lipids, together with nonstarch polysaccharides
from cell walls, which are known to be metabolized to a vari-
able degree in the large intestine, could represent a suitable
carbon source for bacterial fermentation (12, 13). The erosion
of the middle lamella observed in fecal samples was considered
to be further evidence of pectic degradation by gut microbiota.
Our data presented in this study show that when almond lipids
are available for fermentation in the large bowel, modulation
of the composition of the bacterial population is observed, with
a significant increase in the numbers of bifidobacteria and
Eubacterium rectale (Fig. 1). However, when the almond lipid
source was removed (defatted almond product) no significant
changes in the bacterial population were detected (Table 1).
These results suggest that the lipid component of almond seeds
is relevant in the alteration of bacterial growth and metabo-
lism.
The unique role of dietary fiber, essentially the plant cell
wall, has been evaluated in many physiological processes and in
disease prevention (7). Pectins with different degrees of ester-
ification have previously been shown to increase Eubacterium
rectale numbers, and this group of gut bacteria is known to
produce relatively large amounts of butyrate (1, 11). The anti-
inflammatory (4) and antineoplastic (25, 39) properties of bu-
tyrate have been demonstrated on cell tissue cultures in vitro,
increasing the interest in the potential effect of butyrate in
inflammatory bowel disease and colorectal cancer. However,
butyrate production from oligofructose fermentation was
shown to be mainly derived from interconversion of extracel-
lular lactate and acetate (30). In the present study, FG pro-
duced more butyrate than FOS and can therefore be consid-
ered a butyrogenic prebiotic.
The kinetics of almond lipid digestion and absorption is an
important factor for postprandial lipemia and has implications
for the regulation of body weight (41). The results of a dose-
response study have shown that almond consumption im-
TABLE 3. Concentrations at 0, 4, 8, and 24 h of short-chain fatty acids and lactate produced during fermentation
Fatty acid(s)
Incubation
time (h)
Concn (mM)
a
with:
Control FOS FG DG
Total fatty acids 0 4.19 5.58 4.10 4.22
4 7.44 40.35 14.44 11.29
8 14.80 74.02 39.13 33.21
24 24.20 91.23 62.36 61.36
Lactic acid 0 0.53 0.03 0.54 0.05 0.25 0.02 0.22 0.05
4 0.51 0.29 12.23 0.36 4.96 0.03 1.18 0.20
8 0.68 0.21 16.58 1.36 4.79 0.27 0.45 0.38
24 0.69 0.25 16.91 2.02 7.18 0.44 1.58 0.17
Acetic acid 0 1.42 0.22 2.15 0.28 1.24 0.32 1.16 0.28
4 3.38 1.03 21.97 0.36 5.25 1.06 7.00 0.88
8 8.81 1.89 46.15 1.21 14.09 0.16 20.77 1.72
24 15.05 2.43 50.77 3.91 26.88 0.01 34.79 2.90
Propionic acid 0 0.90 0.21 1.05 0.16 1.27 0.04 1.21 0.06
4 1.80 0.56 2.67 0.36 2.44 1.06 1.04 0.06
8 2.42 0.19 4.00 0.62 8.09 1.31 7.06 1.60
24 3.90 0.70 11.11 2.27 12.09 2.23 14.77 0.70
Butyric acid 0 1.34 0.04 1.85 0.21 1.35 0.04 1.63 0.08
4 1.75 0.66 3.47 0.36 1.79 0.54 2.07 0.09
8 2.89 0.25 7.29 0.71 12.16 0.33 4.93 0.24
24 4.56 0.17 12.44 4.73 16.21 0.87 10.22 1.07
a
Values for individual acids are means standard deviations of the results.
4268 MANDALARI ET AL. APPL.ENVIRON.MICROBIOL.
proved the serum profile of healthy and middle-hypercholes-
terolemic adults, thus reducing risk factors for coronary heart
disease (38). The predominant fatty acid of almond triacyl-
glycerols is oleic acid, comprising more than 65% of the total
oil fraction and contributing to the high monounsaturated fat
content of almonds. Prebiotics have also been reported to
indirectly lead to a reduction in serum triglyceride levels (44),
and short-chain fatty acid production can modulate the expres-
sion of multiple genes involved in the atherosclerosis process
(32). An in vivo murine study investigating the effects of pre-
biotics on atherosclerotic plaques showed that inulin and FOS
were able to reduce plasma and hepatic cholesterol (34). In the
present study, we have shown that almond extracts can act as
prebiotics, and this may add functionality to almonds in im-
proving serum cholesterol levels.
In this study, we have not investigated whether oleic acid,
which is present in the FG fraction, was utilized by the gut
bacteria. However, there is evidence to indicate that oleic acid
may be metabolized by the ruminal bacterium Selenomonas
ruminantium and strains of Streptococcus, Enterococcus, and
Lactobacillus (20, 21). Linoleic acid, another unsaturated fatty
acid, was also metabolized in the human colon by a number of
Roseburia species (8). In addition, by being incorporated into
the bacterial membrane of specific gut bacteria, oleic acid may
contribute to their increased survival rate in gastric juice (6).
In conclusion, we have shown that almond seeds exhibited
the potential to be used as a novel source of prebiotics, in-
creasing the populations of bifidobacteria and Eubacterium
rectale with the subsequent increase in butyrate concentrations.
More-detailed studies on the digestibility of almonds and the
role played by lipids in the potential prebiotic effect need to be
performed using human volunteers.
ACKNOWLEDGMENTS
We gratefully acknowledge the help provided by Yvan Lemarc
(IFR) with statistic analyses. We thank Karen Lapsley (ABC) for
providing the almond products and for useful discussions.
This research was funded by the Almond Board of California
(ABC).
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The prebiotic effects of lactulose were monitored in a human feeding study. Prebiotics are dietary carbohydrates that have a selective microbial metabolism in the gut, directed towards bacteria seen as bene cial, examples being bi dobacteria and:or lactobacilli. The study was conducted in a double blind, placebo controlled manner. A dose of 10 g per day, half the pharmacological dose, was fed to 10 healthy adult volunteers. In parallel, 10 persons were fed a placebo (glucose:lactose). Both culture based methodologies and genetic probing, based around uorescent in situ hybridization were used to determine bacterial populations. Faecal water genotoxicity was assessed using the Comet assay to investigate the ability of lactulose to protect against DNA damage. Bi dobacteria showed a statistically signi cant increase during lactulose intake, whilst genetic probing showed a concomitant decrease in clostridia. Viable plate counts of lactobacilli increased when lactulose was fed, but this was not replicated by the genetic probing. During the trial, none of the recruits experienced any signi cant adverse gastrointestinal symptoms. Single-cell gel electrophoresis, used to assess faecal water genotoxicity, did not reveal that lactulose intervention reduced genotoxicity. The prebiotic nature of 10 g:day lactulose towards the human gut microbiota has been clearly demonstrated in this study. Lactulose is shown to be an effective food-grade prebiotic for healthy adults particularly in sections of the community with low bi dobacterial populations. The value of the more direct, culture independent FISH technique in the microbial ecology of the gut has been demonstrated in this study.
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Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 x 10(10) cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 x 10(10) cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 x 10(7) to 7 x 10(8) per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.
Article
1. The Functional Food Science in Europe (FUFOSE) project was introduced, evaluated and accepted by the EU DG XII FAIR Programme as a Concerted Action. Its aim was to develop and establish a science-based approach for the emerging concepts in functional food development. Over the last three years of this EU Concerted Action co-ordinated by ILSI Europe, scientific data have been evaluated and new concepts have been elaborated. This Consensus Document is the culmination of the EU Concerted Action and its key points and recommendations are summarized here. It is by no means the end of the process, but, rather, an important starting point and the stimulus for functional food development. 2. Considerable progress has been made in scientific knowledge leading to the identification of functional food components which might eventually lead to an improved state of health and well-being and/or reduction of risk of disease. Consumers are becoming more aware of this development as they seek a better-quality, as well as a longer, life. The food industry has an opportunity to provide products that are not only safe and tasty, but also functional. The originality of the approach in this EU Concerted Action is that it is function-based, rather than product-based. The latter approach would have to be influenced by local considerations of different cultural as well as dietary traditions, whereas the function-based approach starts from the biologically based science that is universal. Furthermore, and most importantly, the function-based approach in this EU Concerted Action has allowed the development of ideas that suggest a unique way in which to link this scientific basis of functional foods with the communication about their possible benefits to consumers. 3. This EU Concerted Action has adopted the following working definition, rather than a firm definition, for functional foods: A food can be regarded as 'functional' if it is satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate nutritional effects in a way that is relevant to either an improved state of health and well-being and/or reduction of risk of disease. 4. Functional foods must remain foods and they must demonstrate their effects in amounts that can normally be expected to be consumed in the diet. They are not pills or capsules, but part of a normal food pattern. A functional food can be a natural food, a food to which a component has been added, or a food from which a component has been removed by technological or biotechnological means. It can also be a food where the nature of one or more components has been modified, or a food in which the bioavailability of one or more components has been modified; or any combination of these possibilities. A functional food might be functional for all members of a population or for particular groups of the population, which might be defined, for example, by age or by genetic constitution. 5. The development of functional foods must rely on basic scientific knowledge of target functions in the body that are relevant to an improved state of health and well-being and/or the reduction of risk of diseases, the identification of validated markers for these target functions and the evaluation of sound scientific data from human studies for their possible modulation by foods and food components. This EU Concerted Action has proposed that markers can be classified according to whether they are markers of exposure to the functional food component whether they are markers that relate to target function or biological response or whether they are intermediate markers of the actual disease endpoint or health outcome. 6. Consumers must be made aware of the scientific benefits of functional foods and this requires clear and informative communication through messages (claims) on products and in accompanying materials. This EU Concerted Action has identified two types of claims that are vital to functional food development and has provided a scientific basis for them to help those who have to formulate and regulate the claims. Claims for 'Enhanced Function Claims' (Type A) should require that evidence for the effects of the functional food is based on establishment and acceptance of validated markers of Improved Target Function or Biological Response, while claims for the Reduced Risk Of A Disease (Type B) should require that evidence is based on the establishment and acceptance of Markers of Intermediate Endpoints of Disease. These markers must be shown to be significantly and consistently modulated by the functional food or the functional food component for either type of claim to be made. This EU Concerted Action has therefore proposed a scheme whereby the scientific basis of functional food development can be linked to the communication of their benefits to the public. If the principles of such a scheme can be universally adopted then this should ultimately improve communication to consumers and minimize their confusion. 7. Functional foods must be safe according to all standards of assessing food risk and new approaches to safety might need to be established. This EU Concerted Action proposes that the development of validated markers as described above should, if possible, be used and integrated in the safety assessment with particular attention being paid to long-term consequences and interactions between components. 8. The development of functional foods, with their accompanying claims, will proceed hand in hand with progress in food regulation, which is the means to guarantee the validity of the claims as well as the safety of the food. Science in itself cannot be regulated and functional food science provides only the scientific basis for these regulations. 9. The Individual Theme Group papers, which are the science base for this Concerted Action, represent the critical assessment of the literature by European experts.
Chapter
The colonic microflora is of crucial importance to any consideration of the role of dietary fibre in health and disease since many of the physiological effects of fibre on the gut are dependent on, or are influenced by, the activities of the colonic bacteria. This area of interaction between bacteriology and gut physiology is still poorly understood, mainly because of the inaccessibility of the proximal colon.
Article
Dietary fibre, principally the non-starch polysaccharides of the plant cell wall, is an important component of our diet. After more than 30 years of research into the many and varied claims for its benefits, it is now clear that fibre has uniquely significant physical effects in the gut and in addition through fermentation is a major determinant of large bowel function and bowel habit. Its physical properties in the small bowel effect lipid absorption and the glycaemic response. Fibre has some modest effects on appetite. These benefits feed through into a protective role in large bowel cancer, diabetes and coronary heart disease.
Article
Oligofructose and inulin, which are increasingly used in human food preparations, are now recognised as important prebiotic agents influencing the microbial composition of the gastrointestinal tract of the host. The specific objective of this study was to investigate the effect of ingesting a low dose of oligofructose (5 g/day) by healthy human subjects on the faecal microflora, especially bifidobacteria, and to compare it with the ingestion of a placebo (sucrose). In a placebo-controlled study design, faecal samples were collected in the morning from 8 healthy human subjects, who were not on any medication, and immediately enumerated for bifidobacteria, Bacteroides, coliforms, total anaerobes and total aerobes. Subjects first took sucrose (placebo) daily (5 g) for 3 weeks with their normal diet except for known sources of oligofructose and inulin and subsequently were administered oligofructose (5 g) daily for 3 weeks. Faecal samples were collected after 11 days and after 3 weeks. At 2 weeks post ingestion of oligofructose, another set of faecal samples was taken.All samples were subjected to immediate microbial enumeration. Ingestion of sucrose (5 g/day) was without effect on all faecal bacteria enumerated, whereas consumption of oligofructose (5 g/day) for 11 days resulted in close to one log cycle increase in bifidobacteria numbers. No further increase was observed after the next 10 days. At 2 weeks after termination of oligofructose ingestion, bifidobacteria numbers had decreased to almost that of the period before treatment. Increases in numbers of Bacteroides and total anaerobic bacteria but not in aerobic bacteria also occurred.
Article
A group-specific 16S rRNA-targeted oligonucleotide probe S-G-Lab-0158-a-A20 (Lab158) was designed and validated to quantify species of the phylogenetic group lactobacilli-enterococci. The Lab158 probe detects nearly all species of the genera Lactobacillus, Enterococcus, Pediococcus, Weissella, Vagococcus, Leuconostoc and Oenococcus. The specificity of the probe was tested on various species of the target group and on a range of common intestinal bacteria. For these experiments, procedures to permeabilize these groups of Gram-positive bacteria were optimized and fluorescent in situ hybridization (FISH) conditions for maximum specificity were determined. In addition, we showed that it is possible to distinguish the predominant gut-enterococci i.e. E. faecalis among the Lab158 probe-positive cells with the E. faecalis