Original Article Obesity

CLINICAL TRIALS AND INVESTIGATIONS

The Additional Effects of a Probiotic Mix on Abdominal

Adiposity and Antioxidant Status: A Double-Blind,
Randomized Trial
Aline Corado Gomes1, R avila Graziany Machado de Sousa1, Patrıcia Borges Botelho1, Tatyanne Letıcia Nogueira Gomes1,
2
Patrıcia Oliveira Prada , and Jo~ao Felipe Mota1

Objective: To investigate whether a probiotic mix has additional effects when compared with an isolated
dietary intervention on the body composition, lipid profile, endotoxemia, inflammation, and antioxidant
profile.
Methods: Women who had excess weight or obesity were recruited to a randomized, double-blind trial
and received a probiotic mix (Lactobacillus acidophilus and casei; Lactococcus lactis; Bifidobacterium
bifidum and lactis; 2 3 1010 colony-forming units/day) (n 5 21) or placebo (n 5 22) for 8 weeks. Both
groups received a dietary prescription. Body composition was assessed by anthropometry and dual-
energy X-ray absorptiometry. The lipid profile, lipid accumulation product, plasma fatty acids, lipopoly-
saccharide, interleukin-6, interleukin-10, tumor necrosis factor-a, adiponectin, and the antioxidant
enzymes activities were analyzed.
Results: In comparison with the dietary intervention group, the dietary intervention 1 probiotic mix group
showed a greater reduction in the waist circumference (23.40% vs. 25.48%, P 5 0.03), waist-height ratio
(23.27% vs. 25.00%, P 5 0.02), conicity index (22.43% vs. 24.09% P 5 0.03), and plasma polyunsatu-
rated fatty acids (5.65% vs. 218.63%, P 5 0.04) and an increase in the activity of glutathione peroxidase
(216.67% vs. 15.62%, P < 0.01).
Conclusions: Supplementation of a probiotic mix reduced abdominal adiposity and increased antioxidant
enzyme activity in a more effective way than an isolated dietary intervention.
Obesity (2017) 25, 30-38. doi:10.1002/oby.21671

Introduction
Obesity is considered a low-grade chronic and systemic inflamma-
tory disease (1) and results from complex interactions between genes
and environmental factors, such as diet, food components, and life-
style. It is characterized by increased visceral white adipose tissue
mass and associated with a greater propensity to develop type 2
diabetes, hypertension, dyslipidemia, cardiovascular disease, and
cancer (2).
Due to the comorbidities associated with obesity, great efforts have
been made to develop treatment strategies for weight reduction. Die-
tary manipulation remains the first-line treatment for people with
obesity (3). However, evidence suggests that the gut microbiota are
involved in the development of obesity and that their modulation
may aid in the treatment of this disease.
Manipulating the gut microbiota with probiotics affects the host
metabolism (4). Probiotics are defined as “live microorganisms
which when administered in adequate amounts confer a health bene-
fit to the host” (5). Certain strains of Lactobacillus and Bifidobacte-
rium seem to have beneficial effects on metabolism, improving glu-
cose homeostasis and inflammation, reducing body weight and fat
mass (FM) (4), and protecting against oxidative stress (6). These
effects may be related to a reduction in lipopolysaccharide (LPS)
translocation accompanied by a decrease in inflammatory cytokines,

1
Clinical and Sports Nutrition Research Laboratory (Labince), Faculty of Nutrition, Federal University of Goias, Goi^ania, Goias, Brazil. Correspondence:
Jo~ao Felipe Mota (jfemota@gmail.com) 2 School of Applied Sciences, State University of Campinas, Campinas, S~ao Paulo, Brazil.

Funding agencies: The study was supported by the Fundaça ~o de Amparo a  Pesquisa do Estado de Goia s (FAPEG), Brazil (grant no. 201200544230609).
Disclosure: The authors declared no conflict of interest.
Author contributions: ACG: conception and design of the study, acquisition of data, analysis and interpretation of data, drafting the manuscript; RGMS: conception and
design of the study, acquisition of data; PBB and TLNG: conception and design of the study, acquisition of data, revising critically for important intellectual content; POP:
analysis and interpretation of data; and JFM: revising critically for important intellectual content, final approval of the version to be submitted. All authors read and
approved the final manuscript.
Clinical trial registration: www.ensaiosclinicos.gov.br, U1111-1137-4566.
Received: 23 June 2016; Accepted: 25 August 2016; Published online 23 December 2016. doi:10.1002/oby.21671

30 Obesity | VOLUME 25 | NUMBER 1 | JANUARY 2017 www.obesityjournal.org

Original Article
CLINICAL TRIALS AND INVESTIGATIONS
but these mechanisms have yet to be elucidated. Furthermore, it has
been hypothesized that different microbial species might modulate
the fatty acid composition in tissues crucial for host metabolism,
increasing the concentrations of eicosapentaenoic acid and docosa-
hexaenoic acid, two omega-3 (x-3) polyunsaturated fatty acids with
important anti-inflammatory and lipid-lowering properties (7). This
study assessed whether a probiotic mix has additional effects when
compared with an isolated dietary intervention (DI) on (1) body
composition and lipid profile, (2) metabolic endotoxemia and
chronic inflammation, and (3) the antioxidant profile.
Methods
Participants
The participants were identified through advertisement flyers and
enrolled at the Clinics Hospital and Nutrition College at the Federal
University of Goias in May 2012. Eligible participants were women
with excess weight or obesity and were 20 to 59 years old with a
BMI (in kg/m2) from 24.9 to 40. The exclusion criteria were BMI
(in kg/m2) values less than 24.9 and higher than 40, participation in
a food restriction program or in a diet, the presence of an acute dis-
ease requiring treatment, chronic immunologic diseases, thyroid dis-
eases, pregnancy or plans to become pregnant, gastrointestinal sur-
gery, hormone replacement therapy, antibiotic treatment or treatment
with any drug known to affect the immune response, and regular
consumption of probiotic products and fermented food. We also
excluded chronic alcoholics and subjects who used insulin or nutri-
tional supplements. Participants were instructed to refrain from eat-
ing fermented dairy products as well as products containing probiot-
ics from the screening until the conclusion of the study.
Ethical approval was obtained from the Ethics Committee of the
Federal University of Goias (reference number 044/2012). The study
was registered at http://www.ensaiosclinicos.gov.br/as U1111-1137-
4566. All participants were informed about the study orally and in
writing and gave their written informed consent to participate.
Study design
The study was a randomized, double-blind, placebo-controlled, two-
arm, parallel-group study in women with excess weight or obesity.
The intervention lasted 8 weeks, and the evaluations were performed
at two time points, at the baseline and at the conclusion of the study.
The intervention period was selected based on clinical trials that eval-
uated the effect of administration of probiotics on other comorbidities
associated with obesity. These studies showed that 4 weeks of probi-
otic intervention is sufficient to cause benefits, as evidenced by the
meta-analyzes of Shimizu et al. (8) and Ruan et al. (9).
Random assignment and blinding
Potential female subjects were screened for eligibility and randomly
allocated 1:1 into two groups: DI or DI plus probiotic mix (DI 1 P).
The study staff allocated randomization numbers consecutively to
the subjects in the order that they attended the randomization visit.
The randomization list was provided by an independent research
group not involved in the study using the Excel program 1997 to
2003 (Microsoft, Redmond, WA, EUA). The blinding code was pro-
vided to the investigators after the statistical analyses were
completed.
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Obesity
Viability of strains
The resistance to acidic pH and bile salts was evaluated to test the
viability of bacteria in the gastrointestinal tract. The acid tolerance
of probiotic strains was studied according to Mishra and Prasad (10)
with minor modifications. The acid solutions were prepared with
potassium chloride (2 g/L) and pepsin (3 g/L), adjusting the pH, if
necessary, using 1N solutions of HCl and NaOH. Active cultures of
bacteria were transferred to 9 mL of each pH solution and subse-
quently subjected to shaking (150 rpm at 378C) in a thermoshaker
(Tecnal TE-420) for 120 min.
The resistance of the strains to bile was studied according to Mishra
and Prasad (10), with slight modifications. Active cells were added
to 0.3% bile salt solutions (Oxoid) and shaken (150 rpm at 378C) in
a thermoshaker (Tecnal TE-420) for 6 h. After both 3 and 6 h, an
aliquot (1 mL) of the culture solution with bile salts was removed
and inoculated into MRS broth for a 24-h growth at 378C, under
anaerobic conditions, and then the colony-forming units (CFU) were
measured. Analyses were performed in duplicate.
The aforementioned methods were repeated twice at 12-month inter-
vals and applied to the same batch of product stored at room tem-
perature (25 6 28C). The mix exhibited satisfactory growth and sta-
bility during the 12 and 24 months of analysis, maintaining an
average population of all strains above 7 3 1010 CFU/g of product
and above 2.3 3 1010 CFU/g after exposure to acid and bile salts.
Intervention/procedures
The sachet with probiotic mix contained maltodextrin (48.3%),
modified starch (24.21%), xylitol (24.21%), silicium dioxide
(0.97%), and 1 3 109 CFU of each of the probiotic strains: Lactoba-
cillus acidophilus LA-14, Lactobacillus casei LC-11, Lactococcus
lactis LL-23, Bifidobacterium bifidum BB-06, and Bifidobacterium
lactis BL-4 (DaniscoV R ). The women consumed four sachets daily
before breakfast for 8 weeks, totaling 2 3 1010 CFU/day. The pla-
cebo product was similar to the active product in appearance, smell,
and taste. Participants were instructed to ingest four sachets dis-
solved in liquid at room temperature each day after waking up.
Adherence to treatment was assessed by weekly phone calls,
monthly during regularly scheduled appointments, and by counting
empty sachets.
In the beginning of the study, participants received the prescription
of a normocaloric diet (25 to 30 kcal/kg) calculated by AVANUTRI,
version 3.1.5, as well as guidelines for healthy eating. The prescrip-
tion consisted of six meals, containing ingredients amounts, way of
cooking, and a food substitutes list. The diet was isocaloric and con-
tained the same quantity of protein, lipids, and carbohydrates
between groups. Participants were also instructed to maintain their
usual exercise program for the entire study. Physical activity was
assessed at both the beginning and the end of the study using the
International Physical Activity Questionnaire (short form, last 7
days, self-administered format) (11). The metabolic equivalent tasks
(METs) were calculated by International Physical Activity Question-
naire evaluation as follows:
1. Walking MET-minutes per week 5 3.3 3 walking minutes 3
walking days
Obesity | VOLUME 25 | NUMBER 1 | JANUARY 2017 31
Obesity
2. Moderate MET-minutes per week 5 4.0 3 moderate-intensity
activity minutes 3 moderate-intensity days
3. Vigorous MET-minutes per week 5 8.0 3 vigorous-intensity
activity minutes 3 vigorous-intensity days
4. Total physical activity MET-minutes per week 5 sum of wal-
king 1 moderate 1 vigorous MET-minutes per week scores
To transform the MET values in kilocalories (kcal), the following
equation was used: (MET 3 weight)/60 min.
Follow-up
Each follow-up visit consisted of data collected through a standar-
dized medical history, 3-day food records, anthropometry, body
composition, and blood collection.
Assessment of food intake
At the beginning and at the end of the study, dietary intake was
assessed using the 24-h dietary recall method, and participants were
instructed to record their daily dietary intake for 3 days, including a
weekend day. Dietary caloric intakes and macronutrient values were
analyzed using AVANUTRI software.
Anthropometry and body composition
The height of the subjects was measured during the selection phase
to the nearest 0.5 cm with a stadiometer (Model Standard, Sanny).
Weight was measured, after voiding, with participants wearing light
clothing to the nearest 0.1 kg on a digital scale (Filizola, Brazil).
Waist circumference was measured on undressed subjects at the
midpoint between the lower margin of the last palpable rib and the
top of the iliac crest.
Dual-energy X-ray absorptiometry (DXA) assessments of FM, fat-free
mass, and body fat percentage as well as android and gynoid fat were
conducted using a GE Lunar densitometer (DPX NTV, GE) with
R
enCORE 2011 software (version 13.60, GE Healthcare). The coeffi-
cient of variation (CV) for the DXA tests of muscle and FM were
0.75% and 1.03%, respectively. Based on anthropometric data, the
body adiposity markers of interest were estimated using the formulas
described below:
1. BMI 5 weight (kg)/height2 (m)
2. Waist–height ratio 5 waist circumference (cm)/height (cm)
3. Conicity index 5 waist circumference (cm)/0.109 x 冑weight(kg)/
height(m)
Blood sampling and laboratory methods
Blood samples were drawn for each participant from the antecubital
vein in the arm after a 12-h fast. The serum samples were separated
from the whole blood by centrifugation at 3,500 rpm for 10 min at
48C (Combate, C.E.L.M) and frozen at 2808C until analysis. Uric
acid, c-glutamyl transferase, alanine aminotransferase, aspartate ami-
notransferase, urea, creatinine, serum total cholesterol (TC), high-
density lipoprotein (HDL) cholesterol, and triglycerides were deter-
R
mined by automated enzymatic methods on a VITROSV 950 Chem-
istry System (Johnson and Johnson). Glycated hemoglobin was
measured in the whole blood by turbidimetric methods with auto-
matic equipment (Analyzer A25, Biosystems), low-density lipopro-
tein (LDL) cholesterol concentrations were calculated by the
32 Obesity | VOLUME 25 | NUMBER 1 | JANUARY 2017
Probiotic Mix and Abdominal Adiposity Gomes et al.
Friedewald equation, and Castelli’s Risk Indexes were calculated as
described below (12):
1. Castelli’s Risk Index I (CRI I) 5 TC/HDL
2. Castelli’s Risk Index II (CRI II) 5 LDL/HDL
The lipid accumulation product (LAP) was obtained by the formula:
LAP 5 (waist circumference [cm] – 58) 3 triglyceride concentration
[mmol/L]) (13).
To determine the plasma fatty acid composition, the samples were
derivatized using direct esterification, as described by Shirai et al.
(14), and their composition was determined by gas chromatography
(Agilent 7890A gas chromatograph, Agilent Technologies Inc.,
Santa Clara). A fused silica capillary column (J&W DB-23 Agilent
122-236; 60 m 3 250 mm inner diameter) was used for injection.
High-purity helium was used as the carrier gas at a flow rate of
1 mL/min with a 50:1 split injection. The program for column tem-
perature ran as follows: started at 808C, heated at a rate of 58C/min
up to 1758C, followed by another gradient of 38C/min to 2308C, and
this temperature maintained for 5 min. The injector and detector
temperatures were 2508C and 2808C, respectively. The fatty acids
were identified by comparing the retention times with those of a
purified standard mixture of four fatty acid methyl esters (Sigma
Chemical Co.: 4-7801; 47085-U; 49453-U; and 47885-U). The
results were expressed as percentage of total fatty acids.
Inflammatory cytokines were determined in duplicate in 25 lL of plasma
R R
using immunoassays multiplex kit (MilliplexV MAP) and LuminexV
technology (Millipore), according to the manufacturer’s instructions. The
Human Cytokine/Chemokine Magnetic Bead Panel kit allowed the
simultaneous quantification of the following biomarkers: interleukin
(IL)26, IL-10, and tumor necrosis factor (TNF)-a, and the Human Adi-
pokine Magnetic Bead Panel 1 kit allowed quantification of adiponectin.
The CV% of the assay corresponded to the following: IL-6 (intra-assay
CV% 5 2.0; inter-assay CV%518.3); IL-10 (intra-assay CV% 5 1.6;
inter-assay CV% 5 16.8); TNF-a (intra-assay CV% 5 2.6; inter-assay
CV% 5 13.0); and adiponectin (intra-assay CV% 5 4.0; inter-assay
CV% 5 10.0).
The superoxide dismutase (SOD), catalase, and glutathione peroxi-
dase (GPx) activities of erythrocyte lysates was determined using a
microassay (15) by a multiwavelength detection microplate reader
(BioTek, Synergy HT, Winooski, VT). The serum malondialdehyde
concentration was determined using the thiobarbituric acid method
described by Bilici et al. (16) with modifications.
The LPS levels were determined in duplicate by the limulus amebo-
cyte lysateassay according to the manufacturer’s protocol (LAL
QCL-1000TM, Basal, Switzerland) in plasma samples diluted 1/5
with endotoxin-free water and heated to 708C for 10 min to inacti-
vate plasma proteins.
Statistical analysis
The power calculation was based on the results of a related study
(17) with the assistance of the G-Power software (version 3.0.10).
The difference in waist circumference between groups was the pri-
mary outcome. The power calculation indicated that 17 subjects per
were required (95% power; 5% type I error) to detect a difference of
www.obesityjournal.org
Original Article
CLINICAL TRIALS AND INVESTIGATIONS
Figure 1 Participant flow through the study. Values are expressed as the number of participants.
21.2 (21.5 to 20.9) cm in waist circumference. Assuming a 40%
dropout rate, target enrollment was set at 30 per group.
To ensure a normal distribution of variables, histogram and
Kolmogrov-Smirnov tests were applied. The results are expressed as
the mean 6 standard deviation or median. We used paired-samples t-
tests to identify within-group differences (before and after interven-
tion). Student’s t-test was used to detect differences between the two
groups (DI and DI 1 P). Wilcoxon’s rank test was used for skewed
variables. Potential confounders (covariates) that could affect bio-
chemical measures and body composition were examined in the
entire group. Significant covariates were identified using multiple
linear regression models with backward elimination of those that
were nonsignificant. Covariates remained in the model at P < 0.05.
Macronutrient intakes were adjusted for energy intake, and total
cholesterol and HDL were adjusted for age (P < 0.05; data not
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Obesity
shown). The effect of the dietary intervention plus probiotic mix on
the variables measured was examined using ANCOVA with adjust-
ment for the screening/baseline observation. The changes in the rela-
tionships between the different fatty acid intakes and their plasma con-
centrations were examined using Pearson’s or Spearman’s correlation
coefficient. P < 0.05 was considered statistically significant. All statis-
tical analyses were performed using STATA, version 12 (StataCorp,
College Station, TX). Outcome analyses were carried on intention-to-
treat (adjusted by last-observation-carried-forward analysis).
Results
Patient screening, enrollment, and retention by treatment group are
shown in Figure 1. The baseline body composition (Table 1) and
food intake (Table 2) of the two groups were similar. No adverse
Obesity | VOLUME 25 | NUMBER 1 | JANUARY 2017 33
34
TABLE 1 Body composition before and after treatment with dietary intervention or a dietary intervention plus probiotic mix in women with overweight and obesity

Dietary intervention (n 5 22) Dietary intervention 1 probiotic mix (n 5 21) Intervention effect
Obesity

Mean Mean
Baseline Week 8 change Pa Baseline Week 8 change Pa Pb Effect (95% CI) Pc
BMI (kg/m2) 33.34 6 4.69 32.61 6 4.53 20.72 0.00 31.70 6 3.90 31.24 6 3.96 20.45 0.01 0.41 20.49 (23.24 to 2.25) 0.71
Weight (kg) 83.55 6 13.44 82.59 6 13.00 20.95 0.02 76.58 6 9.94 75.60 6 10.24 20.98 0.02 0.18 23.64 (211.24 to 3.95) 0.33
WC (cm) 97.50 6 10.40 94.18 6 10.24 23.32 0.00 93.95 6 8.37 88.80 6 7.26 25.14 0.00 0.33 1.81 (0.13 to 3.50) 0.03
Body fat (%) 48.82 6 4.39 48.03 6 4.18 20.79 0.02 48.56 6 4.28 47.37 6 5.00 21.19 0.00 0.91 21.09 (23.91 to 1.72) 0.43
FM (kg) 39.73 6 8.23 39.05 6 7.35 20.68 0.29 36.30 6 7.32 34.96 6 7.52 21.33 0.00 0.60 21.93 (27.02 to 3.15) 0.44
FFM (%) 49.68 6 4.33 50.36 6 4.09 0.68 0.04 49.79 6 4.09 50.92 6 4.76 1.13 0.00 0.79 20.94 (21.79 to 3.69) 0.48
FFM (kg) 41.37 6 6.74 41.46 6 6.53 0.09 0.77 37.86 6 3.65 38.20 6 4.20 0.33 0.26 0.00 21.13 (24.68 to 2.41) 0.52
Android (%) 54.36 6 3.35 53.05 6 3.23 21.31 0.00 54.80 6 3.36 53.50 6 4.14 21.30 0.02 0.98 0.03 (22.32 to 2.39) 0.97
Gynoid (%) 53.98 6 5.31 52.75 6 5.42 21.22 0.00 53.95 6 4.66 53.13 6 4.70 20.81 0.00 0.56 21.92 (5.12 to 1.26) 0.23
WHR 0.61 6 0.06 0.59 6 0.06 20.02 0.00 0.60 6 0.06 0.57 6 0.05 20.03 0.00 0.87 0.01 (0.00 to 0.02) 0.02
Conicity index 1.23 6 0.07 1.19 6 0.07 20.03 0.00 1.22 6 0.07 1.16 6 0.06 20.05 0.92 0.91 0.02 (0.00,0.04) 0.03

Obesity | VOLUME 25 | NUMBER 1 | JANUARY 2017

Values are presented as mean 6 standard deviation.
a
Difference between baseline and end point. P value obtained from paired t-test/Wilcoxon matched-pairs signed-rank test for the within-group comparisons.
b
Homoscedasticity test between groups on baseline.
c
Obtained from unpaired Student’s t-test or Mann–Whitney test, as statistical difference between changes (DI vs. DI 1 P). The effect of the probiotic mix associated with dietary intervention on the variables measured
was examined using ANCOVA with adjustment for the screening/baseline observation.
FFM, free-fat mass; FM, fat mass; WC, waist circumference; WHR, waist-to-height ratio.

TABLE 2 Dietary intake before and after treatment with dietary intervention or a dietary intervention plus probiotic mix in women with overweight and obesity

Dietary intervention (n 5 22) Dietary intervention 1 probiotic mix (n 5 21) Intervention effect
Mean Mean
Baseline Week 8 change Pa Baseline Week 8 change Pa Pb Effect (95% CI) Pc

Energy (kcal) 1,451.86 6 327.51 1,417.86 6 228.09 234.00 0.65 1,420.94 6 215.17 1,265.52 6 50.05 2155.42 0.01 0.07 121.42 (274.43 to 317.27) 0.21
Protein (%) 20.94 6 5.49 20.97 6 5.67 0.02 0.98 20.18 6 8.51 20.21 6 6.67 0.03 0.98 0.05 18.00 (13.05 to 22.94) 0.69
CHO (%) 57.25 6 13.83 55.18 6 12.61 22.07 0.61 57.11 6 9.83 52.21 6 17.24 24.90 0.28 0.16 2.83 (29.34 to 15.00) 0.63
Total fat (%) 26.38 6 8.26 25.22 6 6.26 21.16 0.53 31.79 6 8.41 28.44 6 8.92 23.34 0.15 0.93 2.18 (23.67 to 8.04) 0.45
SFAs (%) 34.68 6 7.12 33.00 6 11.18 21.67 0.53 31.85 6 7.14 27.66 6 6.68 24.19 0.03 0.91 2.52 (23.91 to 8.96) 0.43
MUFAs (%) 25.66 6 6.89 24.28 6 8.58 21.38 0.56 26.36 6 6.08 24.58 6 6.50 21.77 0.28 0.61 0.38 (5.43 to 6.20) 0.89
PUFAs (%) 12.60 6 5.22 18.39 6 9.00 5.79 0.00 18.78 6 9.58 25.65 6 12.48 6.87 0.01 0.00 21.07 (27.40 to 5.25) 0.73
Fiber (g) 16.77 6 5.18 16.04 6 5.92 20.73 0.53 14.70 6 5.05 12.43 6 4.57 22.26 0.18 0.91 1.53 (22.52 to 5.60) 0.44

Values are presented as mean 6 standard deviation. Macronutrients were adjusted for energy intake.
a
Difference between baseline and end point. P value obtained from paired t-test/Wilcoxon matched-pairs signed-rank test for the within-group comparisons.
b
Homoscedasticity test between groups on baseline.
c
Obtained from unpaired Student’s t-test or Mann–Whitney test, as statistical difference between changes (DI vs. DI 1 P).
CHO, carbohydrates; SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids.

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Probiotic Mix and Abdominal Adiposity Gomes et al.
Original Article Obesity
CLINICAL TRIALS AND INVESTIGATIONS

events were reported by groups. Blood test results for uric acid, c-

0.25
0.64
0.82
0.37
0.25
0.24
0.13
0.62
0.51

CRI, Castelli’s Risk Index I; CRII, Castelli’s Risk Index II; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very-low-density lipoprotein; LAP, lipid accumulation product;
Obtained from unpaired Student’s t-test or Mann–Whitney test, as statistical difference between changes (DI vs. DI 1 P). The effect of the probiotic mix associated with dietary intervention on the variables measured
Pc
glutamyl transferase, alanine aminotransferase, aspartate aminotrans-
Intervention effect ferase, urea, and creatinine did not show physiological differences
TABLE 3 Blood lipid parameters and glycated hemoglobin before and after treatment with dietary intervention or a dietary intervention plus probiotic mix in

throughout the study (data not shown). The participants showed no

5.55 (211.41 to 22.53)

20.07 (20.51 to 0.36)

20.10 (20.28 to 0.07)

20.22 (20.62 to 0.16)
Effect (95% CI)
0.26 (20.72 to 0.20)

0.02 (20.16 to 0.20)

0.15 (20.20 to 0.51)

0.67 (20.21 to 1.56)

0.18 (20.58 to 0.95)
change in the level of physical activity (DI: M0, 5,181.54 kcal/wk;
M2, 4,448.99 kcal/wk, and DI 1 P: M0, 5,386.51 kcal/wk; M2,
5,155.00 kcal/wk). Moreover, no differences between groups were
observed (P > 0.05).

Food intake and body composition

No difference was noted in FM (DI 1 P: 23.66%; DI: 21.71%,
95% CI: 27.02 to 3.15, P 5 0.44) between groups, but in intragroup
0.00
0.09
0.35
0.10
0.05
0.05
0.46
0.41
0.88
Pb

analysis, only DI 1 P reduced FM (P 5 0.00). Participants taking the

probiotic mix had a greater decrease in waist circumference
Dietary intervention 1 probiotic mix (n 5 21)

(23.40% vs. 25.47%, P 5 0.03), waist-height ratio (23.27% vs.

0.00
0.32
0.34
0.00
0.31
0.31
0.04
0.00
0.04
Pa

25.00%, P 5 0.02), and conicity index (22.43% vs. 24.09%

Difference between baseline and end point. P value obtained from paired t-test/Wilcoxon matched-pairs signed-rank test for the within-group comparisons. P 5 0.03) than the DI group (Table 1).
change
21.07
20.13
20.05
20.28
0.05
0.11
20.31
20.37
211.95

There was no difference between groups for energy or the percen-

Mean

tages of fat, protein, carbohydrate, and fiber intake (Table 2).

Lipid profile, fatty acid composition, and

48.72 6 18.12
5.00 6 0.36
5.16 6 0.82
2.03 6 2.15
3.24 6 0.68
0.73 6 0.30
1.62 6 0.66
4.60 6 1.03
2.88 6 0.86

glycated hemoglobin
Week 8

No difference was noted in LDL (DI 1 P: 27.95%; DI: 23.76%, 95%

CI: 20.20 to 0.51, P 5 0.37), LAP (DI 1 P: 219.69%; DI: 210.61%,
95% CI: 211.41 to 22.53, P 5 0.51), and Castelli’s Risk Index I
(DI 1 P: 26.30%; DI: 1.34%, 95% CI: 20.21 to 1.56, P 5 0.13) and
Index II (DI 1 P: 211.38%; DI: 3.97%, 95% CI: 20.58 to 0.9,
60.67 6 35.79

P 5 0.62) between groups (Table 3). Regarding fatty acid profile, no

6.07 6 0.42
5.30 6 0.90
2.08 6 2.13
3.52 6 0.70
0.68 6 0.26
1.50 6 0.58
4.92 6 1.10
3.25 6 0.86
Baseline

difference was noted in monounsaturated fatty acids (MUFAs)

(DI 1 P: 31.11%; DI: 4.11, 95% CI: 29.72 to 3.55, P 5 0.35), x-3
Values are presented as mean 6 standard deviation. Total cholesterol and HDL were adjusted for age.

fatty acids (DI 1 P: 95.86%; DI: 22.91%, 95% CI: 232.58 to 2.87,
P 5 0.09), and x-6 fatty acids (DI 1 P: 213.53%; DI: 3.42%, 95%
CI: 22.75 to 39.93, P 5 0.08) between groups. Compared with the DI
0.00
0.23
0.68
0.44
0.47
0.48
0.82
0.69
0.24

group, the DI 1 P group had decreased polyunsaturated fatty acids

Pa

was examined using ANCOVA with adjustment for the screening/baseline observation.

(PUFAs) (5.65% vs. 218.63%, P 5 0.04) (Table 4). The saturated fat
(r 5 20.24, P 5 0.12), monounsaturated fat (r 5 20.08, P 5 0.61), and
change

polyunsaturated fat intake (r 5 20.00, P 5 0.99) after 8 weeks did not

Mean

0.06
0.10
Dietary intervention (n 5 22)

21.06
20.20
20.03
20.12
20.05
20.11

26.39

correlate with their respective plasma concentrations. There was no

difference in glycated hemoglobin between groups.
53.87 6 30.40

Antioxidant enzymes and inflammatory markers

5.40 6 0.66
4.88 6 1.17
3.03 6 2.90
3.07 6 0.91
0.67 6 0.35
1.48 6 0.77
4.51 6 1.37
2.88 6 1.28
Week 8

At the conclusion of treatment, no difference was noted in the activity

of antioxidant enzyme SOD (DI 1 P: 36.44%; DI: 6.80%, 95% CI:
Homoscedasticity test between groups on baseline.

22.79 to 1.54, P 5 0.56) between groups. Compared with the DI

women with overweight and obesity

group, the DI 1 P group had increased GPx activity (216.67% vs.

15.62%, P < 0.01) (Table 5). Compared with the DI group, the DI 1 P
group had increased TNF-a (9.30% vs. 216.86%, P 5 0.01) (Table
60.27 6 37.23
6.46 6 0.85
5.09 6 1.25
3.06 6 2.85
3.19 6 1.02
0.72 6 0.40
1.60 6 0.90
4.45 6 0.93
2.77 6 0.72

TC, total cholesterol; TG, triglyceride.

Baseline

4). No changes were observed in cytokines and LPS concentrations

(Table 5).

Discussion
VLDL (mmol/L)
HDL (mmol/L)
LDL (mmol/L)

TG (mmol/L)
TC (mmol/L)

To our knowledge, this was the first randomized, double-blind clini-

HbA1c (%)

cal trial to evaluate the effects of a probiotic mix containing Lacto-

bacillus acidophilus LA-14, Lactobacillus casei LC-11, Lactococcus
CRII
LAP
CRI

lactis LL-23, Bifidobacterium bifidum BB-06, and Bifidobacterium

b
a

www.obesityjournal.org Obesity | VOLUME 25 | NUMBER 1 | JANUARY 2017 35

36
TABLE 4 Distribution of plasma fatty acids (%) before and after treatment with dietary intervention or a dietary intervention plus probiotic mix in women with
Obesity

overweight and obesity

Dietary intervention (n 5 22) Dietary intervention 1 probiotic mix (n 5 21) Intervention effect

Mean Mean
Baseline Week 8 change Pa Baseline Week 8 change Pa Pb Effect (95% CI) Pc

SFAs 43.20 6 10.05 37.97 6 12.09 25.23 0.18 35.41 6 13.73 36.90 614.97 1.49 0.65 0.05 23.93 (213.80 to 5.92) 0.42
MUFAs 22.86 6 7.45 23.80 6 8.72 0.94 0.71 16.10 6 32.97 21.12 6 9.28 5.01 0.04 0.96 23.08 (29.72 to 3.55) 0.35
PUFAs 32.51 6 11.47 34.35 6 16.35 1.84 0.64 46.69 6 18.47 37.98 619.38 28.70 0.01 0.97 13.85 (22.53 to 30.24) 0.04
x-3 30.19 6 22.99 29.31 6 22.25 0.88 0.64 11.84 6 8.24 23.20 6 23.75 11.35 0.04 0.00 214.85 (232.58 to 2.87) 0.09
x-6 71.98 6 25.12 74.44 6 24.45 2.45 0.55 90.29 6 8.99 78.07 6 28.33 212.22 0.03 0.03 18.58 (22.75 to 39.93) 0.08

Values are presented as mean 6 standard deviation.

Obesity | VOLUME 25 | NUMBER 1 | JANUARY 2017

a
Difference between baseline and end point. P value obtained from paired t-test/Wilcoxon matched-pairs signed-rank test for the within-group comparisons.
b
Homoscedasticity test between groups on baseline.
c
Obtained from unpaired Student’s t-test or Mann–Whitney test, as statistical difference between changes (DI vs. DI 1 P). The effect of the probiotic mix associated with dietary intervention on the variables measured
was examined using ANCOVA with adjustment for the screening/baseline observation.
SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; x-3, omega-3; x-6, omega-6.

TABLE 5 Oxidative and inflammatory parameters before and after treatment with dietary intervention or a dietary intervention plus probiotic mix in women with
overweight and obesity

Dietary intervention (n 5 22) Dietary intervention 1 probiotic mix (n 5 21) Intervention effect
Mean Mean
Baseline Week 8 change Pa Baseline Week 8 change Pa Pb Effect (95% CI) Pc
CAT (U/mg) 8.10 6 2.01 7.17 6 2.05 20.92 0.15 8.20 6 1.26 7.71 6 1.15 20.49 0.65 0.06 0.30 (20.50 to 0.11) 0.44
SOD (U/mg) 5.88 6 1.94 6.28 6 1.90 0.40 0.55 5.35 6 2.50 7.31 6 6.29 1.95 0.00 0.10 20.62 (22.79 to 1.54) 0.56
GPx (U/mg) 0.42 6 0.12 0.34 6 0.10 20.07 0.00 0.32 6 0.13 0.37 6 0.11 0.05 0.04 0.31 20.12 (20.22 to 0.02) 0.01
MDA (nmol/mL) 0.16 6 0.04 0.16 6 0.04 0.00 0.83 0.18 6 0.25 0.21 6 0.28 0.02 0.39 0.00 20.01 (20.13 to 0.10) 0.81
LPS (EU/mL) 0.85 6 0.44 0.95 6 0.60 0.09 0.49 0.67 6 0.11 0.65 6 0.11 0.02 0.34 0.00 0.08 (20.26 to 0.44) 0.62
IL-10 (pg/mL) 4.65 6 4.34 4.53 6 3.14 20.12 0.88 3.57 6 1.66 3.96 6 1.84 0.39 0.15 0.00 21.29 (23.34 to 0.74) 0.20
IL-6 (pg/mL) 1.35 6 1.81 0.80 6 1.81 20.54 0.07 0.58 6 0.27 0.58 6 0.31 0.00 0.94 0.00 21.98 (24.15 to 0.18) 0.07
TNF-a (pg/mL) 2.49 6 0.89 2.07 6 0.78 20.42 0.07 1.72 6 0.44 1.88 6 0.37 0.16 0.24 0.00 20.84 (21.47 to 20.20) 0.01
ADIPO (ug/mL) 35.59 6 20.76 38.44 6 29.93 2.84 0.65 33.72 6 29.78 36.33 6 27.46 2.61 0.63 0.11 3.92 (215.27 to 23.11) 0.68

Values are presented as mean 6 standard deviation.

a
Difference between baseline and end point. P value obtained from paired t-test/Wilcoxon matched-pairs signed-rank test for the within-group comparisons.
b
Homoscedasticity test between groups on baseline.
c
Obtained from unpaired Student’s t-test or Mann–Whitney test, as statistical difference between changes (DI vs. DI 1 P). The effect of the probiotic mix associated with dietary intervention on the variables measured
was examined using ANCOVA with adjustment for the screening/baseline observation.
ADIPO, adiponectin; CAT, catalase; SOD, superoxide dismutase; GPx, glutathione peroxidase; IL, interleukin; LPS, lipopolysaccharide; MDA, malondialdehyde, TNF-a, tumor necrosis factor-a.

www.obesityjournal.org
Probiotic Mix and Abdominal Adiposity Gomes et al.
Original Article
CLINICAL TRIALS AND INVESTIGATIONS
lactis BL-4 on the body composition, lipid and fatty acid profiles,
metabolic endotoxemia, inflammatory cytokines, antioxidant
enzymes, and malondialdehyde. Our results show that this treatment
improves body composition and antioxidant enzyme activity more
effectively than dietary intervention alone, but these results were not
accompanied by a decrease in inflammatory markers.
The gut microbiota produce active signaling molecules that interact
with the metabolism of the host (18). The short-chain fatty acids
(SCFAs) are produced by fermentation of dietary fibers by gut bac-
teria. SCFAs interact with G protein-coupled receptors and affect
insulin sensitivity in adipocytes and peripheral organs, thus regulat-
ing energy metabolism (19). In this study, we observed that the
DI 1 P group, compared with the DI group, had decreased waist cir-
cumference, waist-height ratio, and conicity index, which are all var-
iables related to fat storage. The improvement in body composition
due to the administration of probiotics may be a consequence of
fasting-induced adipose factor suppression in the gut, which would
modulate the production of SCFAs (19). Kadooka et al. (17) exam-
ined the antiobesity effects of the probiotic Lactobacillus gasseri
LG2055 in healthy adults and also observed a reduction in waist cir-
cumference and body FM, but the participants were instructed to
maintain their diet and no dietary intervention was made.
In this study, inflammatory and anti-inflammatory cytokine concen-
trations did not change, nor did LPS concentrations. However, our
study did not induce the inflammatory processes that accompany the
consumption of a high-fat diet as in the experimental studies that
showed the reduction of inflammatory cytokines after supplementa-
tion with probiotics (20). Thus, we believe that the improvement in
inflammatory profile achieved by probiotics occurs when there are
more inflammatory processes, such as high-fat diet consumption,
and there is no additional effect when compared with dietary
intervention.
Bifidobacteria species have been linked to alterations in the fatty acid
profile of tissues. At the conclusion of this study, the DI 1 P group
showed lower PUFA concentrations compared with the DI group due
to a reduction in the proportion of x-6. However, no studies evaluat-
ing the effect of probiotic supplementation on the proportion of fatty
acids in human plasma were performed. A previous study using feces,
colon, and cecal contents from germ-free, gnotobiotic, and conven-
tional rodents indicated that gut microbes were capable of transform-
ing linoleic acid (LA) into conjugated linoleic acids (CLAs) during in
vitro incubations (21), suggesting the influence of gut microbiota on
the profile of fatty acids in various tissues. Bioactive isomers of CLA
can exert antidiabetogenic, antiobesogenic, antiatherogenic, hypocho-
lesterolemic, hypotriglyceridemic, and immunomodulatory actions
(22). The mechanisms by which certain bacteria induce changes in x-
3 fatty acid composition has yet to be elucidated, but it may be asso-
ciated with the use or assimilation of certain polyunsaturated fatty
acids, such as a-linolenic acid, or the modulation of dietary fatty acid
uptake in the intestine (23). The modulation of gut microbiota can
regulate desaturase activity involved in the metabolism of fatty acids
to their longer chain derivatives. Indeed, it has been shown that a
variety of commensals increased the activity of rat liver d-6-
desaturase, which resulted in increased amounts of arachidonic acid
derived from linoleic acid (24).
Special strains of lactic acid bacteria also have antioxidant proper-
ties. The antioxidative mechanisms of probiotics could be ascribed
www.obesityjournal.org
Obesity
to reactive oxygen species scavenging, metal ion chelation, enzyme
inhibition, and activity reduction and inhibition of ascorbate autoxi-
dation (25). In our study, the antioxidant enzyme activities of SOD
and GPx were increased in the DI 1 P group. This effect was
observed by Naruszewicz et al. (26) and Songisepp et al. (27) in
humans supplemented with Lactobacillus plantarum 299v and L. fer-
mentum ME-3, respectively. The increased antioxidant enzyme
activity from probiotic supplementation can be derived from the res-
toration of gut microbiota (28) and the induction of genes involved
in the biosynthesis of glutathione in the intestinal mucosa (29) and
in pancreatic cells (30). Furthermore, probiotics may also enhance
the absorption of micro- and macronutrients, including antioxidants,
or finally reduce postprandial lipids, which are connected with oxi-
dative damage and are often responsible for some food-related path-
ologies (31).
Conclusion
This study was sufficiently powered and adequately blinded. The
intervention was noninvasive and short in duration, yielded no
adverse effects on the participants, and had good compliance and
acceptability in this population. The intervention protocol also
enabled an assessment of the importance of probiotics in enhancing
the benefits resulting from dietary intervention. Furthermore, the
wide scope of data collection allowed numerous outcomes to be
examined. One limitation of this study was that we did not evaluate
the gut microbiome, which may have indicated the effects of probi-
otic mix consumption on the gut microflora and confirmed our sug-
gested mechanism of action.
In conclusion, in the present study, supplementation with a probiotic
mix reduced abdominal fat and increased antioxidant enzyme activ-
ity in a more effective way than an isolated dietary intervention.O
C 2016 The Obesity Society
V
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