Anaerobe 26 (2014) 1e6

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Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe

Clinical microbiology

Prebiotic effects of almonds and almond skins on intestinal microbiota

in healthy adult humans
Zhibin Liu a, Xiuchun Lin a, Guangwei Huang b, Wen Zhang a, Pingfan Rao a, Li Ni a, *
a
b

Institute of Food Science & Technology, Fuzhou University, No. 2 Xueyuan Road, Fuzhou 350108, PR China
Almond Board of California, 1150 9th Street, Suite 1500, Modesto, CA 95354, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 7 August 2013
Received in revised form
5 November 2013
Accepted 22 November 2013
Available online 3 December 2013

Almonds and almond skins are rich in ber and other components that have potential prebiotic properties. In this study we investigated the prebiotic effects of almond and almond skin intake in healthy
humans. A total of 48 healthy adult volunteers consumed a daily dose of roasted almonds (56 g), almond
skins (10 g), or commercial fructooligosaccharides (8 g) (as positive control) for 6 weeks. Fecal samples
were collected at dened time points and analyzed for microbiota composition and selected indicators of
microbial activity. Different strains of intestinal bacteria had varying degrees of growth sensitivity to
almonds or almond skins. Signicant increases in the populations of Bidobacterium spp. and Lactobacillus spp. were observed in fecal samples as a consequence of almond or almond skin supplementation.
However, the populations of Escherichia coli did not change signicantly, while the growth of the
pathogen Clostridum perfringens was signicantly repressed. Modication of the intestinal microbiota
composition induced changes in bacterial enzyme activities, specically a signicant increase in fecal bgalactosidase activity and decreases in fecal b-glucuronidase, nitroreductase and azoreductase activities.
Our observations suggest that almond and almond skin ingestion may lead to an improvement in the
intestinal microbiota prole and a modication of the intestinal bacterial activities, which would induce
the promotion of health benecial factors and the inhibition of harmful factors. Thus we believe that
almonds and almond skins possess potential prebiotic properties.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Almond
Almond skins
Prebiotics
Intestinal microbiota
Bacterial enzymes

1. Introduction
It is well established that the colonic microbiota have a profound inuence on health. The human gut contains a large variety
of bacterial genera, species, and strains, which are either benecial
(e.g., Bidobacterium spp. and Lactobacillus spp.) or harmful (e.g.,
Clostridium spp., Shigella spp., and Veillonella spp.) to host health.
The intestinal microbiota play an essential role in inuencing the
health of the host. They can greatly inuence the intestinal environment [1], contributing to the hosts health through a variety of
mechanisms such as activation of the immune response, production of bacteriocins, nutritional and physical competition with
pathogens, and maintenance of an acid environment.
Currently there is great interest in the use of prebiotics as
functional food ingredients to manipulate the composition of
colonic microbiota in order to improve health [2]. Many food oligosaccharides and polysaccharides (including dietary ber), such as
fructooligosaccharides, inulin, galactooligosaccharides, and other

* Corresponding author. Tel./fax: 86 591 22866378.

E-mail address: nili@fzu.edu.cn (L. Ni).
1075-9964/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.anaerobe.2013.11.007

related carbohydrates, are reported to have prebiotic properties. In

contrast to a probiotic that introduces exogenous bacteria into the
colonic microbiota, a prebiotic aims to stimulate the growth of one
or a limited number of the potentially health-promoting indigenous microorganisms, thus modulating the composition of the
natural ecosystem [3]. It is not the prebiotic by itself, but rather the
changes induced in the microbiota composition that are responsible for its effects. Besides, dietary prebiotics have the potential
advantage over some probiotics of not being susceptible to antibiotics [4].
Almonds (Prunus amygdalus L.) are a good source of nutrients
such as vitamin E, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), arginine, and magnesium [5]. Almonds also contain considerable amounts of potential prebiotic
indigestible carbohydrates. Almond skins, which are removed from
the almond kernel by hot water blanching, constitute 4% of the total
almond weight and are generally treated as a waste product [6].
However, an array of avonoids, including catechins, avonols, and
avanones in their aglycone and glycoside forms, have been identied in almond skin. These compounds may contribute to the
health benets associated with almond consumption. Many researchers have studied the functions of almonds in the diet,

Z. Liu et al. / Anaerobe 26 (2014) 1e6

including lipid regulation [7,8], antioxidant activity [9e11], weight

control [8,12], and Yin-Yang balance [13]. It is also reported that,
almond or almond skin can serve as a candidate food for potential
prebiotic effects [14e16]. Mandalari et al. [15,16] investigated the
prebiotic effects of almond seeds and almond skins in vitro by using
mixed fecal bacterial cultures, and found that after digested by
simulative in vitro gastric and duodenal digestion, almond seeds
and almond skins signicantly increased the populations of bidobacteria and Eubacterium rectale. The results were consistent
with our previous ndings. Male SPF Wistar rats were subjected to
daily oral treatment with raw almond or roasted almond for 4
weeks to assess the inuence of almond intake on the composition
of gut bacteria populations. After 4 weeks of treatment, the populations of Bidobacterium spp. and Lactobacillus spp. in feces and
ceca of the rats increased signicantly, while the populations of
Escherichia coli and Enterococus spp. in feces and ceca of the rats
decreased signicantly (Zhibin Liu, unpublished results).
Unfortunately, to the extension of our knowledge, there are no
available data from clinical studies concerning the prebiotic properties of almonds or almond skins consumption in healthy subjects.
The aim of present study was to investigate the effects of daily
consumption of either almonds or almond skins on the composition of the fecal microbiota and on selected indicators of microbial
activity (fecal pH and water content, activities of b-galactosidase, bglucuronidase, nitroreductase and azoreductase) in healthy adult
volunteers.
2. Materials and methods
2.1. Subjects
A total of 48 subjects (24 males and 24 females), 18e22 years of
age, were recruited from Fuzhou University. All subjects lived in
school dormitories with a similar environment throughout the
study period. The eligibility criteria for candidates included being of
good health; a nonsmoker; stable weight (deviation of <2.5 kg over
the previous 3 months); daily defecation; using no antibiotics,
laxatives or other gastrointestinal medications in the 3 months
prior to the beginning of the study; and no consumption of yogurt
or products containing bidobacteria, lactobacilli, or prebiotics
(oligosaccharide-containing products or supplements) in the 3
weeks before the beginning of the study. Written consent was
obtained from each person, and the study was approved by the
Human Industrial Examination Committee of the Institute of Food
Science and Technology of Fuzhou University.
2.2. Study design
The study included a 2-week run-in period, a 6-week treatment
period, and a 2-week wash-out period. During the run-in period the
48 volunteer subjects were instructed to start dietary restrictions
for 2 weeks. The diet was a basic diet provided by the school
canteen, which excluded peanuts or other nuts. The average energy
content of the basic diet was controlled as 10 MJ/d for male and
8 MJ/d for female subjects. This 2-week run-in period helped the
volunteers adapt to the dietary restrictions. Subsequently, the 48
subjects were divided into three groups of 16 participants each (8
males and 8 females): the control group, the almond skin group,
and the almond group. In addition to the routine basic diet
mentioned above, the subjects of the three groups were supplied
daily with (i) commercial fructooligosaccharides (FOS), 8 g/d (4 g/
serving for lunch and supper, which was dissolved in 100 mL
drinking water); (ii) almond skin powder, 10 g/d (5 g/serving for
lunch and supper, which was consumed after mix into the basic
diet); or (iii) roasted, unsalted whole almonds, 56 g/d (28 g/serving

for lunch and supper, consumed directly), respectively. The subjects

were instructed to refrain from consuming any additional foods or
snacks during the treatment period, which lasted for 6 weeks. Then
all subjects underwent a subsequent 2-week wash-out period,
during which they were asked to return to their usual lifestyle and
diet.
2.3. Collection of fecal samples
Fecal samples were collected at the end of the run-in period, and
were labeled as Baseline (week 0). During the treatment period,
fecal samples were collected at the end of week 1, week 3, and week
6 and were labeled as Ingestion (week 1, 3, or 6). At the end of the
wash-out period, fecal samples were collected and labeled as
Evacuation (week 8). Subjects were instructed to pick a small part
of their fresh stool (approximately 10 g) with a small stick at
dened time points, and place it in a sterilized sealed specimen cup.
And then the specimen cups were collected. For one individual
specimen cup, we weighted three samples, 1 g each, for measurement of fecal pH and water content, enumeration of fecal bacteria
and bacterial enzyme assays, respectively.
2.4. Measurement of fecal pH and water content
To prepare fecal samples for pH measurement, 1 g of feces was
immediately diluted with 10 mL of deionized water and dispersed
by vortexing. A laboratory pH meter with a protein-resistant electrode at room temperature was used to measure pH. Fecal water
content was determined for 1-g samples, which were weighed
before and after drying in a vacuum oven at105  C by an infrared
moisture gauge.
2.5. Enumeration of fecal bacteria
From each fecal sample, 1 g of feces was dissolved in 99 mL of
sterile water and mixed thoroughly, and a series of 10-fold dilutions
(102 to 107) were prepared. A 0.1-mL aliquot of each dilution was
used to inoculate plates of four selective media, including
Rafnose-Bidobacterium (RB) agar, lactobacilli select (LBS) agar,
alizarin-b-galactosidase (ALIZ-GAL) agar, and sulteepolymyxine
sulfadiazine (SPS) agar used for enumeration of Bidobacterium
spp., Lactobacillus spp., E. coli, and Clostridum perfringens, respectively. The inoculated plates were incubated anaerobically at 37  C
for 48 h for Bidobacterium spp. and Lactobacillus spp. and for 24 h
for E. coli and C. perfringens. All plating was done in triplicate. After
incubation, plates were examined for bacterial colonies and the
results of the microbial counts were expressed as colony-forming
units (CFU) per gram of wet feces.
2.6. Enzyme assays
The fecal samples collected at the end of the run-in period
(Baseline, week 0) and treatment period (Ingestion, week 6) were
used for quantitative determinations of b-galactosidase, b-glucuronidase, nitroreductase and azoreductase enzyme activities. To
prepare samples for enzyme assay, 1 g of feces was diluted 10-fold
in 0.1 M potassium phosphate buffer. The specimens were homogenized in a blender, then sonicated for 15 min, and centrifuged
at 12,000 rpm for 10 min at 4  C. The supernatant fractions were
used for measuring the bacterial enzyme activities, following the
instructions of the respective commercial assay kits (human ELISA
kits for b-galactosidase, b-glucuronidase, nitroreductase and azoreductase; R&D Systems, USA). One unit of b-galactosidase, bglucuronidase, and nitroreductase was dened as the amount that
released 1 mmol of o-nitrophenyl, p-nitrophenyl, and p-

Z. Liu et al. / Anaerobe 26 (2014) 1e6

aminobenzoic acid per minute, respectively. One unit of azoreductase activity was dened as1 mmol of amaranth metabolized
per minute. Results were expressed as units per gram (U/g) of wet
feces.
2.7. Statistical analysis
Results were expressed as mean  SD (standard deviation) or
mean  SEM (standard error of the mean). Paired-sample t-tests
were applied to compare the data among study groups. The statistical analysis of the results was performed by the SPSS 13 software and the signicance threshold was set at 5% (P < 0.05).
3. Results
3.1. Characteristics of the study subjects
Throughout the whole experimental period, there were no
serious adverse reactions to the diets reported by the subjects,
except for 4 subjects in the FOS control group reported mild diarrhea on the rst week of FOS ingestion, however they recovered
naturally a few days later. All of the 16 subjects in each group were
in good health, except for two subjects, one in control group and
the other in the almond group who failed to nish the study for
personal reasons. The weight, BMI, body fat percentage, body water
content and muscle mass were measured at the beginning (Baseline, week 0) and after 6 weeks of treatment (Ingestion, week 6).
Daily water intake and defecation frequency per week were
recorded by the subjects themselves. Details of the study group
members physical characteristics, water intake and defecation are
provided in Table 1. There were no statistically signicant differences in these parameters within and among the groups.
3.2. Water content and pH of feces
Changes in the fecal moisture and fecal pH of the subject groups
are shown in Table 2. Overall, no signicant differences were
observed in fecal moisture and fecal pH levels throughout the
experiment, except for the control group, which showed a signicant decrease in fecal pH (P < 0.05) at week 3. Also, in the rst week
of daily consumption of FOS by the control group, the fecal samples
seemed to be moister and 4 subjects reported mild diarrhea. Fecal
moisture in the almond group tended to (P < 0.1) decrease at week
3 and week 6.
3.3. Bacterial populations
The effects of almonds and almond skins intake on the
composition of fecal microbiota in the healthy adult volunteers
are shown in Table 3. Bidobacteria levels increased signicantly
after 6 weeks of ingesting FOS (P < 0.01), almond skins (P < 0.01)
or almonds (P < 0.05). The control and almond skin groups

maintained a high level of viable counts of bidobacteria during

the whole ingestion period (P < 0.01 at weeks 1, 3 and 6). Even in
the evacuation period, the bidobacteria populations were still
higher than the baseline level (P < 0.01 at week 8). Almond
intake did not bring about a signicant change in bidobacteria
populations for the rst 3 weeks; however, at the end of 6
weeks, a signicant increase was observed (P < 0.05), and the
trend was maintained for the next 2 weeks of evacuation
(P < 0.05 at week 8).
Lactobacilli levels also increased signicantly after 6 weeks of
ingesting FOS (P < 0.05), almond skin (P < 0.01) or almond
(P < 0.01). For groups ingesting FOS or almonds, no signicant
changes were observed for the rst 3 weeks, but at the end of 6
weeks the viable counts of lactobacilli had increased signicantly
(P < 0.05 and P < 0.01, respectively). The almond skin group
maintained signicantly higher levels of lactobacilli during the
entire ingestion period (P < 0.05 at week 1, P < 0.01 at week 3 and
6). As with the bidobacteria levels, the lactobacilli levels remained
high during the evacuation period for the control group (P < 0.05 at
week 8) and the almond skin group (P < 0.01 at week 8), but for the
almond group the lactobacilli levels decreased down to the initial
level.
No signicant differences were observed in fecal enterobacter
levels, specically E. coli, during intake of FOS, almond skins or
almonds. Clostridia levels, specically C. perfringens, decreased
signicantly at week 6 in the control group (P < 0.05) and the
almond skin group (P < 0.05), and tended to decrease (P < 0.1) at
week 6 in almond group. In the evacuation period, the clostridia
populations raised to the baseline level.
3.4. Fecal bacterial enzymes
Six weeks of intake of FOS, almond skins or almonds altered the
activities of b-galactosidase, b-glucuronidase, nitroreductase and
azoreductase in the feces of all healthy adult volunteers, as shown
in Fig. 1. FOS consumption resulted in a signicant increase
(P < 0.01) in fecal b-galactosidase activity. Signicant increases in
fecal b-galactosidase activity were also found over 6 weeks of
almond skin consumption (P < 0.01), and an increased trend
(P 0.07) was observed with almond consumption. A decreased
trend in fecal b-glucuronidase activity was observed after 6 weeks
of FOS or almond intake (P 0.08 and 0.1, respectively), and for
almond skin intake, fecal b-glucuronidase activity decreased
signicantly (P < 0.01). A signicant decrease of nitroreductase
activity was observed in the FOS control group (P < 0.01), almond
skin group (P < 0.01) and almond group (P < 0.05) over the 6
weeks. After six weeks of FOS intake, the nitroreductase activity
was lower than after almond skin intake (4.20%) or almond intake
(10.16%). And after six weeks of FOS, almond skin or almond
intake, the fecal azoreductase activity tended to decrease (P 0.07,
0.22 and 0.12, respectively), although no statistically signicant
differences were observed.

Table 1
Body composition, water intake and defecation of study volunteers at start and end of 6-week study.a
Parameter

FOS group (control) (n 15)

Weight (kg)
BMI (kg/m2)
Body fat percentage (%)
Body water content (%)
Muscle mass (%)
Daily water intake (ml)
Defecation (no.) per week

58.56
21.22
20.61
54.26
43.98
884
6

Baseline, week 0

Values are expressed as mean  SD.









9.39
2.13
8.07
4.52
8.52
478
2

Almond skin group (n 16)

Ingestion, week 6
57.09
20.70
19.19
55.20
43.63
955
7









8.62
1.99
8.06
4.39
7.87
473
2

Baseline, week 0
57.41
20.83
19.90
54.51
43.66
914
7









7.99
1.74
7.01
4.40
8.26
300
1

Almond group (n 15)

Ingestion, week 6
55.98
20.33
18.31
55.62
43.43
1184
7









7.60
1.63
6.71
4.12
8.08
651
2

Baseline, week 0
57.59
20.53
21.06
53.98
43.62
956
6









8.10
2.06
5.66
3.32
8.46
558
2

Ingestion, week 6
56.77
20.25
18.96
55.06
43.69
1491
7









8.14
2.01
5.64
2.84
8.26
1060
3

Z. Liu et al. / Anaerobe 26 (2014) 1e6

Table 2
Changes in fecal moisture and pH during the treatment period for all subjects.a
Feces characteristic

Water content (%)

FOS control (n 15)
Almond skin (n 16)
Almond (n 15)
pH
FOS control (n 15)
Almond skin (n 16)
Almond (n 15)
a
b

Baseline

Ingestion

Week 0

Week 1

Week 3

Week 6

Week 8

Evacuation

80.53  5.60
80.64  6.60
79.53  8.89

83.10  4.47
80.95  5.43
78.67  6.40

82.81  3.86
80.36  6.15
76.60  6.75

80.36  5.87
79.53  7.28
76.73  5.67

80.71  6.77
77.76  8.42
76.69  7.67

6.36  0.58
6.31  0.69
6.16  0.67

6.11  0.61
6.16  0.81
6.24  0.74

5.57  0.55*b
6.06  0.80
6.26  0.52

6.04  0.83
6.25  0.63
6.30  0.54

5.75  0.69
6.12  0.65
6.32  0.40

Values are expressed as mean  SD.

Symbol (*) indicates a signicant difference from the value (within row) on week 0 (baseline) at P < 0.05.

4. Discussion
Healthy adult humans were used as the subjects in this study to
evaluate the impact of the consumption of almonds and almond
skins on human intestinal microbiota. A total of 48 healthy college
students were involved in an intervention trial including a 2-week
run-in period, a 6-week treatment period, and a 2-week wash-out
period. The 48 subjects were not selected randomly. A screening
procedure was carried out rst. The criteria were the stability of
intestinal bacteria of subjects. In the screening procedure, 80
healthy volunteers recruited from our campus. They all lived in
school dormitories with a similar environment, and were instructed to start dietary restrictions for 2 weeks. During this time, they
were offered the basic diet we mentioned above and not allowed to
have any other food. Following the methods mentioned above, we
collected the fecal samples of all the 80 volunteers on 0th, 7th and
14th day of the 2-week screening procedure period, and then we
enumerated the populations of Bidobacterium spp., Lactobacillus
spp., E. coli and C. perfringens in the stools (data were not shown).
Based on the stability of intestinal bacteria, 48 subjects were
selected from the 80 volunteers. We assumed that the selected 48
subjects would remain stable in the short term (for example, not
more than ten weeks) if they remained in the same environment
and kept the dietary restriction. Based on this assumption, we
dened the populations of the four intestinal bacteria as the
baseline, and considered this 2-week screening procedure as the

run-in adaption process for the following almond or almond skin

intervention trial for the 48 subjects. Since a relatively stable
baseline was assumed, and also for the considerations of workload,
blank control group wasnt include in this study. The treatments of
almond and almond skin were visually quite different, so it was
difcult to prepare the corresponding placebos. And for nutrition
studies of conventional food which were participated by healthy
population, placebo effect should be very week. So placebo group
was not included in this study too. However, a positive control
group was included. A soluble commercial fructooligosaccharides
(FOS) product, which was considered to be prebiotic, was employed
as positive control of the treatment. So, after the 2-week run-in
period (screening procedure), the 48 subjects were divided into
three groups, FOS control group, almond skin group and almond
group. Then, a short term (6 weeks) intervention effect of almond
and almond skin on intestinal microbiota was investigated. In order
to check the persistence of prebiotic effect of almond and almond
skin, the subjects were observed for an extra 2 weeks.
During the entire trial, all the subjects were in good health. No
subject indicated a signicant change in body weight, BMI, body fat
percentage, body water content or muscle mass after almond skin,
almond or FOS intervention. Thus, in the short term, almond and
almond skin would not bring signicant changes in the conventional characteristics of the subjects. However, 4 subjects in the FOS
control group reported mild diarrhea on the rst week of FOS
ingestion, but they recovered naturally a few days later. Also, the

Table 3
Effect of almond skin and almond intake on fecal microbiota populations in healthy human subjects.a
Fecal organism

Bidobacterium spp.
FOS control (n 15)
Almond skin (n 16)
Almond (n 15)
Lactobacillus spp.
FOS control (n 15)
Almond skin (n 16)
Almond (n 15)
E. coli
FOS control (n 15)
Almond skin (n 16)
Almond (n 15)
C. perfringens
FOS control (n 15)
Almond skin (n 16)
Almond (n 15)

Microbial populations in wet feces (log CFU/g)

Baseline

Ingestion

Week 0

Week 1

Week 3

Week 6

Week 8

Evacuation

9.84  0.10
9.64  0.10
9.75  0.16

11.01  0.11**b
10.16  0.10**
9.75  0.12

11.04  0.20**
10.40  0.11**
9.96  0.06

10.90  0.15**
10.15  0.12**
10.29  0.14*

10.38  0.12**^
^
10.22  0.15**
10.47  0.17**^
^

8.99  0.19
8.77  0.14
8.92  0.12

9.18  0.15
9.19  0.16*
9.05  0.11

9.35  0.13
9.38  0.12**
8.91  0.17

9.54  0.19*
9.57  0.12**
9.39  0.12**

9.53  0.14*
9.34  0.12**
9.13  0.23

9.28  0.18
9.23  0.16
9.38  0.13

9.19  0.15
9.34  0.11
9.28  0.09

9.38  0.12
9.24  0.11
9.35  0.16

9.30  0.13
9.30  0.12
9.41  0.09

9.39  0.12
9.31  0.13
9.56  0.12

6.24  0.21
6.27  0.25
6.64  0.19

5.77  1.13
6.03  0.42
6.24  0.35

5.89  0.33
6.14  0.31
6.36  0.37

5.44  0.37*
5.66  0.28*
6.19  0.24

6.33  0.28^
6.13  0.23^
6.27  0.22

Within rows, symbol (*) indicates signicant difference from the value on week 0 (baseline) at P < 0.05, (**) indicates signicant difference from the value on week 0 (baseline)
at P < 0.01, and (^) indicates signicant difference from the value on week 6 (Ingestion) at P < 0.01.
a
Values are expressed as the mean  SEM.

Z. Liu et al. / Anaerobe 26 (2014) 1e6

15.00

fecal -galactosidase activities

fecal -glucuronidase activities

**

Units /g wet feces

Units /g wet feces

**

15.00

14.50

14.00

13.50

14.50

14.00

13.50

13.00

13.00
FOS Control

Almond Skin

FOS Control

Almond

A
**

Almond

15.00

fecal azoreductase activities

*
14.00

52.00

Units /g wet feces

Units /g wet feces

**

Almond Skin

fecal nitroreductase activities

54.00

50.00
48.00
46.00
44.00
42.00

13.00
12.00
11.00
10.00
9.00

40.00
FOS Control Almond Skin

Almond

8.00
FOS Control

Almond Skin

Almond

Fig. 1. Effects of almond skin and almond consumption on fecal bacterial enzyme activities in healthy human subjects. Fecal samples were examined at the beginning (week 0) and
after 6 weeks (week 6) of FOS (control), almond skin or almond ingestion. Values are means  SD; symbol (*) indicates signicantly different means at P < 0.05; symbol (*) indicates
signicantly different means at P < 0.01.

fecal pH and water content did not change signicantly for any
subject. Fecal pH may not accurately reect the pH in the colon and
depends on absorption of short chain fatty acids (SCFAs) and bicarbonate secretion. Bouhnik et al. [17], who studied the prolonged
administration of low-dose inulin that stimulates the growth of
bidobacteria in humans, found no change in fecal pH and SCFA
levels.
After six weeks of almond skin or almond ingestion, the populations of Bidobacterium spp. and Lactobacillus spp. increased
signicantly. Bidobacteria and lactobacilliare are predominant
members of the intestinal microbiota of humans. Bidobacteria are
thought to stimulate the immune system, produce B vitamins,
inhibit pathogen growth, reduce blood ammonia and blood
cholesterol levels, and help to restore the normal ora after antibiotic therapy [18]. Lactobacilli may aid digestion of lactose in
lactose-intolerant individuals, reduce constipation and infantile
diarrhea, help resist infections such as salmonellae and help to
relieve irritable bowel syndrome [19]. The results of the present
study indicated that almond skins and almonds possess bidobacteria and lactobacili stimulation effects. Also, we found that the
stimulation effects of almond skin and almond intake on bidobacteria and lactobacilli were different. Almond skin intake induced
a prompt increase of bidobacteria and lactobacilli, similar to FOS
intake, although the bidogenic effect was not as great as FOS
intake (P < 0.05), and a high level of viable bidobacteria and
lactobacilli remained for 2 weeks after the ingestion of the almond
skins. By contrast, the stimulation effect of almond intake was not
obvious until the end of 6 weeks. However, eventually (week 6) the
populations of bidobacteria or lactobacilli in almond skin group
and almond group reached a similar level (no signicant difference). Almond skin or almond ingestion for 6 weeks also induced a

decreasing trend of the viable counts of C. perfringens, an organism

known for causing histotoxin and gastrointestinal diseases in
humans. However, during the evacuation period of the study, the
fecal C. perfringens populations raised to the initial level. No signicant changes were observed in the populations of fecal E. coli
throughout all the trials.
These results indicated the stimulation effects of almond skin
and almond intake were typical prebiotic effects. Any food or ingredients that reach the colon without being digested are prebiotic
candidates; nondigestible carbohydrates, in particular FOS, are
authentic prebiotics. Almond skins contain approximately 50% dietary ber and almonds contain about 12% dietary ber [10]. Dietary ber is resistant to digestive enzymes and passes undigested
to the large intestine where it interacts with the intestinal mucosa
and microbiota to enhance gut health. Non-starch polysaccharides
are the major carbohydrate components of dietary ber. Reilly et al.
[20] found that oat-based diets possessed a high ability to enhance
benecial microbial populations, thus indicating the prebiotic potential of oats.
Almonds and almond skins also contain signicant quantities of
polyphenols. Dietary polyphenols are often absorbed slowly and
largely incompletely, apparently remain unabsorbed in the gut
lumen, and they may become concentrated in the ileal and colorectal lumen. Parkar et al. [21] investigated the effect of common
dietary polyphenols on the growth of human gut bacteria and their
adhesion to enterocytes and noted that polyphenols appeared to
have the potential to alter the gut microecology and, by affecting
the total number of benecial microbiota in the gut, may confer
positive gut health benets. So the abundance of dietary ber and
polyphenols may correlate to the prebiotic effects of almond skins
and almonds.

Z. Liu et al. / Anaerobe 26 (2014) 1e6

The studies on the specic components in diet which could

modulate the intestinal microbiota were inconclusive [22]. For
example, Jaquet et al. [23] evaluated the impact of moderate consumption of an instant coffee product on both the metabolic activity and bacterial composition of the intestinal microbiota which
might be related to other constituents, such as alkaloids, chlorogenic acids and other phenolic compounds, ber, and minerals.
Besides the original components in diet, the conversion of dietary
components by intestinal bacteria leads to the formation of a large
variety of compounds, which may further change the structure of
intestinal bacteria.
As a result of changes in the intestinal microbiota induced by
almond or almond skin consumption, the activities of fecal bacterial
enzymes changed. In particular, the activity of b-galactosidase
increased and the activities of b-glucuronidase, nitroreductase and
azoreductase decreased. These four bacterial enzymes are synthesized by colonic bacteria. b-galactosidaseis mainly synthesized by
bidobacteria and lactobacilli. It plays a positive role in the human
gut: the impairment of glycosidase activity may induce a failure in
the metabolism of unabsorbed carbohydrates, such as that reported
in ulcerative colitis and Crohns disease [24]. By contrast, b-glucuronidase, nitroreductase and azoreductaseare synthesized by
harmful bacteria (e.g., Clostridium, Shigella, and Veillonella), and
they are known to produce mutagens, carcinogens, and various
tumor promoters [25]. b-glucuronidase activity antagonizes hepatic metabolism by hydrolyzing the biliary conjugates, and delays
their excretion. Moreover, the increased incidence of colon-rectal
tumors in experimental studies was associated with high levels of
b-glucuronidase activity [26]. Nitro- and azoreductases are key
enzymes responsible for the metabolic activation of many procarcinogens [25]. Bacterial ora can reductively hydrolyze many
aromatic compounds to genotoxic metabolites by azoreductase and
nitroreductase activities. Azoreductase can also reduce food dyes to
substituted phenyl- and naphthyl-amines [27]. Nitroreductase
causes the formation of reactive N-nitroso and N-hydroxy intermediates, thereby converting aromatic nitro compounds into
potentially harmful amines [28].
In this study the increase in b-galactosidase activity was presumably a consequence of elevated populations of bidobacteria
and lactobacilli, which have high levels of this enzyme. And the
decreases in b-glucuronidase, nitroreductase and azoreductase
activities may be associated with the reduction of viable counts of
clostridia or other harmful bacteria.
In conclusion, in the present study we showed that almond skin
and almond ingestion may lead to an improvement of the intestinal
microbiota prole and modify the intestinal bacterial activities,
which induce the promotion of health benecial factors and inhibition of harmful factors. Thus, we believe that almond skins and
almonds possess potential prebiotic properties. The abundance of
dietary ber and polyphenols may be associated with the prebiotic
effects observed upon ingestion of almond skins and almonds.
However, further study is necessary to explore the specic prebiotic
components in almonds and almond skins.
Acknowledgments
We are grateful to the Almond Board of California for the
nancial support of this research. We value the leadership of Dr.
Karen Lapsley, Chief Scientic Ofcer of the Almond Board of California, and Sam Cunningham, chair of the Nutrition committee at

the Almond Board of California. The authors thank all volunteers

from Fuzhou University for assisting in this project.

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