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

but these mechanisms have yet to be elucidated. Furthermore, it has Viability of strains
been hypothesized that different microbial species might modulate The resistance to acidic pH and bile salts was evaluated to test the
the fatty acid composition in tissues crucial for host metabolism, viability of bacteria in the gastrointestinal tract. The acid tolerance
increasing the concentrations of eicosapentaenoic acid and docosa- of probiotic strains was studied according to Mishra and Prasad (10)
hexaenoic acid, two omega-3 (x-3) polyunsaturated fatty acids with with minor modifications. The acid solutions were prepared with
important anti-inflammatory and lipid-lowering properties (7). This potassium chloride (2 g/L) and pepsin (3 g/L), adjusting the pH, if
study assessed whether a probiotic mix has additional effects when necessary, using 1N solutions of HCl and NaOH. Active cultures of
compared with an isolated dietary intervention (DI) on (1) body
bacteria were transferred to 9 mL of each pH solution and subse-
composition and lipid profile, (2) metabolic endotoxemia and
quently subjected to shaking (150 rpm at 378C) in a thermoshaker
chronic inflammation, and (3) the antioxidant profile.
(Tecnal TE-420) for 120 min.

The resistance of the strains to bile was studied according to Mishra


Methods and Prasad (10), with slight modifications. Active cells were added
to 0.3% bile salt solutions (Oxoid) and shaken (150 rpm at 378C) in
Participants a thermoshaker (Tecnal TE-420) for 6 h. After both 3 and 6 h, an
The participants were identified through advertisement flyers and aliquot (1 mL) of the culture solution with bile salts was removed
enrolled at the Clinics Hospital and Nutrition College at the Federal and inoculated into MRS broth for a 24-h growth at 378C, under
University of Goias in May 2012. Eligible participants were women
anaerobic conditions, and then the colony-forming units (CFU) were
with excess weight or obesity and were 20 to 59 years old with a
measured. Analyses were performed in duplicate.
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 The aforementioned methods were repeated twice at 12-month inter-
a food restriction program or in a diet, the presence of an acute dis- vals and applied to the same batch of product stored at room tem-
ease requiring treatment, chronic immunologic diseases, thyroid dis-
perature (25 6 28C). The mix exhibited satisfactory growth and sta-
eases, pregnancy or plans to become pregnant, gastrointestinal sur-
bility during the 12 and 24 months of analysis, maintaining an
gery, hormone replacement therapy, antibiotic treatment or treatment
average population of all strains above 7 3 1010 CFU/g of product
with any drug known to affect the immune response, and regular
and above 2.3 3 1010 CFU/g after exposure to acid and bile salts.
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-
Intervention/procedures
ing fermented dairy products as well as products containing probiot-
The sachet with probiotic mix contained maltodextrin (48.3%),
ics from the screening until the conclusion of the study.
modified starch (24.21%), xylitol (24.21%), silicium dioxide
(0.97%), and 1 3 109 CFU of each of the probiotic strains: Lactoba-
Ethical approval was obtained from the Ethics Committee of the
cillus acidophilus LA-14, Lactobacillus casei LC-11, Lactococcus
Federal University of Goias (reference number 044/2012). The study
lactis LL-23, Bifidobacterium bifidum BB-06, and Bifidobacterium
was registered at http://www.ensaiosclinicos.gov.br/as U1111-1137-
lactis BL-4 (DaniscoV R ). The women consumed four sachets daily
4566. All participants were informed about the study orally and in
before breakfast for 8 weeks, totaling 2 3 1010 CFU/day. The pla-
writing and gave their written informed consent to participate.
cebo product was similar to the active product in appearance, smell,
and taste. Participants were instructed to ingest four sachets dis-
Study design solved in liquid at room temperature each day after waking up.
The study was a randomized, double-blind, placebo-controlled, two- Adherence to treatment was assessed by weekly phone calls,
arm, parallel-group study in women with excess weight or obesity. monthly during regularly scheduled appointments, and by counting
The intervention lasted 8 weeks, and the evaluations were performed empty sachets.
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- In the beginning of the study, participants received the prescription
uated the effect of administration of probiotics on other comorbidities of a normocaloric diet (25 to 30 kcal/kg) calculated by AVANUTRI,
associated with obesity. These studies showed that 4 weeks of probi- version 3.1.5, as well as guidelines for healthy eating. The prescrip-
otic intervention is sufficient to cause benefits, as evidenced by the tion consisted of six meals, containing ingredients amounts, way of
meta-analyzes of Shimizu et al. (8) and Ruan et al. (9). 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
Random assignment and blinding usual exercise program for the entire study. Physical activity was
Potential female subjects were screened for eligibility and randomly
assessed at both the beginning and the end of the study using the
allocated 1:1 into two groups: DI or DI plus probiotic mix (DI 1 P).
International Physical Activity Questionnaire (short form, last 7
The study staff allocated randomization numbers consecutively to
days, self-administered format) (11). The metabolic equivalent tasks
the subjects in the order that they attended the randomization visit.
(METs) were calculated by International Physical Activity Question-
The randomization list was provided by an independent research
naire evaluation as follows:
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 1. Walking MET-minutes per week 5 3.3 3 walking minutes 3
completed. walking days

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Obesity Probiotic Mix and Abdominal Adiposity Gomes et al.

2. Moderate MET-minutes per week 5 4.0 3 moderate-intensity Friedewald equation, and Castelli’s Risk Indexes were calculated as
activity minutes 3 moderate-intensity days described below (12):
3. Vigorous MET-minutes per week 5 8.0 3 vigorous-intensity
activity minutes 3 vigorous-intensity days
1. Castelli’s Risk Index I (CRI I) 5 TC/HDL
4. Total physical activity MET-minutes per week 5 sum of wal-
2. Castelli’s Risk Index II (CRI II) 5 LDL/HDL
king 1 moderate 1 vigorous MET-minutes per week scores
The lipid accumulation product (LAP) was obtained by the formula:
To transform the MET values in kilocalories (kcal), the following
LAP 5 (waist circumference [cm] – 58) 3 triglyceride concentration
equation was used: (MET 3 weight)/60 min.
[mmol/L]) (13).

Follow-up To determine the plasma fatty acid composition, the samples were
Each follow-up visit consisted of data collected through a standar- derivatized using direct esterification, as described by Shirai et al.
dized medical history, 3-day food records, anthropometry, body (14), and their composition was determined by gas chromatography
composition, and blood collection. (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.
Assessment of food intake High-purity helium was used as the carrier gas at a flow rate of
At the beginning and at the end of the study, dietary intake was 1 mL/min with a 50:1 split injection. The program for column tem-
assessed using the 24-h dietary recall method, and participants were perature ran as follows: started at 808C, heated at a rate of 58C/min
instructed to record their daily dietary intake for 3 days, including a up to 1758C, followed by another gradient of 38C/min to 2308C, and
weekend day. Dietary caloric intakes and macronutrient values were this temperature maintained for 5 min. The injector and detector
analyzed using AVANUTRI software. temperatures were 2508C and 2808C, respectively. The fatty acids
were identified by comparing the retention times with those of a
Anthropometry and body composition purified standard mixture of four fatty acid methyl esters (Sigma
The height of the subjects was measured during the selection phase Chemical Co.: 4-7801; 47085-U; 49453-U; and 47885-U). The
to the nearest 0.5 cm with a stadiometer (Model Standard, Sanny). results were expressed as percentage of total fatty acids.
Weight was measured, after voiding, with participants wearing light
clothing to the nearest 0.1 kg on a digital scale (Filizola, Brazil). Inflammatory cytokines were determined in duplicate in 25 lL of plasma
R R

Waist circumference was measured on undressed subjects at the using immunoassays multiplex kit (MilliplexV MAP) and LuminexV
midpoint between the lower margin of the last palpable rib and the technology (Millipore), according to the manufacturer’s instructions. The
top of the iliac crest. Human Cytokine/Chemokine Magnetic Bead Panel kit allowed the
simultaneous quantification of the following biomarkers: interleukin
Dual-energy X-ray absorptiometry (DXA) assessments of FM, fat-free (IL)26, IL-10, and tumor necrosis factor (TNF)-a, and the Human Adi-
mass, and body fat percentage as well as android and gynoid fat were pokine Magnetic Bead Panel 1 kit allowed quantification of adiponectin.
conducted using a GE Lunar densitometer (DPX NTV, GE) with
R
The CV% of the assay corresponded to the following: IL-6 (intra-assay
enCORE 2011 software (version 13.60, GE Healthcare). The coeffi- CV% 5 2.0; inter-assay CV%518.3); IL-10 (intra-assay CV% 5 1.6;
cient of variation (CV) for the DXA tests of muscle and FM were inter-assay CV% 5 16.8); TNF-a (intra-assay CV% 5 2.6; inter-assay
0.75% and 1.03%, respectively. Based on anthropometric data, the CV% 5 13.0); and adiponectin (intra-assay CV% 5 4.0; inter-assay
body adiposity markers of interest were estimated using the formulas CV% 5 10.0).
described below:
The superoxide dismutase (SOD), catalase, and glutathione peroxi-
dase (GPx) activities of erythrocyte lysates was determined using a
1. BMI 5 weight (kg)/height2 (m) microassay (15) by a multiwavelength detection microplate reader
2. Waist–height ratio 5 waist circumference (cm)/height (cm)
(BioTek, Synergy HT, Winooski, VT). The serum malondialdehyde
3. Conicity index 5 waist circumference (cm)/0.109 x 冑weight(kg)/
concentration was determined using the thiobarbituric acid method
height(m)
described by Bilici et al. (16) with modifications.

Blood sampling and laboratory methods The LPS levels were determined in duplicate by the limulus amebo-
Blood samples were drawn for each participant from the antecubital cyte lysateassay according to the manufacturer’s protocol (LAL
vein in the arm after a 12-h fast. The serum samples were separated QCL-1000TM, Basal, Switzerland) in plasma samples diluted 1/5
from the whole blood by centrifugation at 3,500 rpm for 10 min at with endotoxin-free water and heated to 708C for 10 min to inacti-
48C (Combate, C.E.L.M) and frozen at 2808C until analysis. Uric vate plasma proteins.
acid, c-glutamyl transferase, alanine aminotransferase, aspartate ami-
notransferase, urea, creatinine, serum total cholesterol (TC), high-
density lipoprotein (HDL) cholesterol, and triglycerides were deter- Statistical analysis
R
mined by automated enzymatic methods on a VITROSV 950 Chem- The power calculation was based on the results of a related study
istry System (Johnson and Johnson). Glycated hemoglobin was (17) with the assistance of the G-Power software (version 3.0.10).
measured in the whole blood by turbidimetric methods with auto- The difference in waist circumference between groups was the pri-
matic equipment (Analyzer A25, Biosystems), low-density lipopro- mary outcome. The power calculation indicated that 17 subjects per
tein (LDL) cholesterol concentrations were calculated by the were required (95% power; 5% type I error) to detect a difference of

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


Original Article Obesity
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% shown). The effect of the dietary intervention plus probiotic mix on
dropout rate, target enrollment was set at 30 per group. the variables measured was examined using ANCOVA with adjust-
ment for the screening/baseline observation. The changes in the rela-
To ensure a normal distribution of variables, histogram and tionships between the different fatty acid intakes and their plasma con-
Kolmogrov-Smirnov tests were applied. The results are expressed as centrations were examined using Pearson’s or Spearman’s correlation
the mean 6 standard deviation or median. We used paired-samples t- coefficient. P < 0.05 was considered statistically significant. All statis-
tests to identify within-group differences (before and after interven- tical analyses were performed using STATA, version 12 (StataCorp,
tion). Student’s t-test was used to detect differences between the two College Station, TX). Outcome analyses were carried on intention-to-
groups (DI and DI 1 P). Wilcoxon’s rank test was used for skewed treat (adjusted by last-observation-carried-forward analysis).
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
Results
were nonsignificant. Covariates remained in the model at P < 0.05. Patient screening, enrollment, and retention by treatment group are
Macronutrient intakes were adjusted for energy intake, and total shown in Figure 1. The baseline body composition (Table 1) and
cholesterol and HDL were adjusted for age (P < 0.05; data not food intake (Table 2) of the two groups were similar. No adverse

www.obesityjournal.org 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.

www.obesityjournal.org
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 Obesity
CLINICAL TRIALS AND INVESTIGATIONS

lactis BL-4 on the body composition, lipid and fatty acid profiles, to reactive oxygen species scavenging, metal ion chelation, enzyme
metabolic endotoxemia, inflammatory cytokines, antioxidant inhibition, and activity reduction and inhibition of ascorbate autoxi-
enzymes, and malondialdehyde. Our results show that this treatment dation (25). In our study, the antioxidant enzyme activities of SOD
improves body composition and antioxidant enzyme activity more and GPx were increased in the DI 1 P group. This effect was
effectively than dietary intervention alone, but these results were not observed by Naruszewicz et al. (26) and Songisepp et al. (27) in
accompanied by a decrease in inflammatory markers. humans supplemented with Lactobacillus plantarum 299v and L. fer-
mentum ME-3, respectively. The increased antioxidant enzyme
The gut microbiota produce active signaling molecules that interact activity from probiotic supplementation can be derived from the res-
with the metabolism of the host (18). The short-chain fatty acids toration of gut microbiota (28) and the induction of genes involved
(SCFAs) are produced by fermentation of dietary fibers by gut bac- in the biosynthesis of glutathione in the intestinal mucosa (29) and
teria. SCFAs interact with G protein-coupled receptors and affect in pancreatic cells (30). Furthermore, probiotics may also enhance
insulin sensitivity in adipocytes and peripheral organs, thus regulat- the absorption of micro- and macronutrients, including antioxidants,
ing energy metabolism (19). In this study, we observed that the or finally reduce postprandial lipids, which are connected with oxi-
DI 1 P group, compared with the DI group, had decreased waist cir- dative damage and are often responsible for some food-related path-
cumference, waist-height ratio, and conicity index, which are all var- ologies (31).
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- Conclusion
ined the antiobesity effects of the probiotic Lactobacillus gasseri
LG2055 in healthy adults and also observed a reduction in waist cir- This study was sufficiently powered and adequately blinded. The
cumference and body FM, but the participants were instructed to intervention was noninvasive and short in duration, yielded no
maintain their diet and no dietary intervention was made. adverse effects on the participants, and had good compliance and
acceptability in this population. The intervention protocol also
In this study, inflammatory and anti-inflammatory cytokine concen- enabled an assessment of the importance of probiotics in enhancing
trations did not change, nor did LPS concentrations. However, our the benefits resulting from dietary intervention. Furthermore, the
study did not induce the inflammatory processes that accompany the wide scope of data collection allowed numerous outcomes to be
consumption of a high-fat diet as in the experimental studies that examined. One limitation of this study was that we did not evaluate
showed the reduction of inflammatory cytokines after supplementa- the gut microbiome, which may have indicated the effects of probi-
tion with probiotics (20). Thus, we believe that the improvement in otic mix consumption on the gut microflora and confirmed our sug-
inflammatory profile achieved by probiotics occurs when there are gested mechanism of action.
more inflammatory processes, such as high-fat diet consumption,
and there is no additional effect when compared with dietary In conclusion, in the present study, supplementation with a probiotic
intervention. mix reduced abdominal fat and increased antioxidant enzyme activ-
ity in a more effective way than an isolated dietary intervention.O
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 C 2016 The Obesity Society
V
showed lower PUFA concentrations compared with the DI group due
to a reduction in the proportion of x-6. However, no studies evaluat- References
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