Nutrition 32 (2016) 716719

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Nutrition
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Brief report

Benecial effects of Bidobacterium lactis on lipid prole and

cytokines in patients with metabolic syndrome: A randomized
trial. Effects of probiotics on metabolic syndrome
Luciana Jesus Bernini M.Sc. a, Andrea Name Colado Sima ~o Ph.D. b,
Daniela Frizon Aleri M.Sc. , Marcell Alysson Batisti Lozovoy Ph.D. b,
b

Naiara Lourenco Mari M.Sc. b, Cnthia Hoch Batista de Souza Ph.D. a,

Isaas Dichi M.D., Ph.D. c, Giselle Nobre Costa Ph.D. a, *
a
Program of Master in Dairy Science and Technology, University of North ParanadUnopar, Londrina, Parana, Brazil
b
Department of Pathology, Clinical Analysis and Toxicology, University of Londrina, Londrina, Parana, Brazil
c
Department of Internal Medicine, University of Londrina, Londrina, Parana, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Objective: Human studies have shown the benecial effects of probiotic microorganisms on the
Received 25 July 2015 parameters of metabolic syndrome (MetS) and other cardiovascular risks, but to our knowledge the
Accepted 2 November 2015 effect of Bidobacterium lactis has not yet been reported. The aim of this study was to evaluate the
effect of consumption of milk containing the probiotic B. lactis HN019 on the classical parameters of
Keywords: MetS and other related cardiovascular risk factors.
Metabolic syndrome
Methods: Fifty-one patients with MetS were selected and divided into a control group (n 25) and
Probiotic
a probiotic group (n 26). The probiotic group consumed fermented milk with probiotics over the
Bidobacterium lactis
Cytokines course of 45 d. The effects of B. lactis on lipid prole, glucose metabolism, and proinammatory
Cardiovascular risk factors cytokines (tumor necrosis factor-a and interleukin-6) were assessed in blood samples of the in-
dividuals at the baseline and after 45 d.
Results: Daily ingestion of 80 mL fermented milk with 2.72  1010 colony-forming units of B. lactis
HN019 showed signicant reduction in body mass index (P 0.017), total cholesterol (P 0.009),
and low-density lipoprotein (P 0.008) compared with baseline and control group values.
Furthermore, a signicant decrease in tumor necrosis factor-a (P 0.033) and interleukin-6
(P 0.044) proinammatory cytokines was observed.
Conclusion: These data showed potential effects of B. lactis HN019 in reducing obesity, blood lipids,
and some inammatory markers, which may reduce cardiovascular risk in patients with MetS.
2016 Elsevier Inc. All rights reserved.

Introduction

Metabolic syndrome (MetS) is a set of disorders, whose risk

LJB was responsible for recruiting the patients, interpretation of the results,
and writing the manuscript. CHBS was responsible for writing of the manuscript.
DFA and MABL were responsible for recruiting the patients and the laboratory
analysis. ANCS and ID were responsible for interpretation of the results and
writing the manuscript. GNC was responsible for the original concept of the
study, the study design, interpretation of the results, and writing of the
manuscript. All authors read and approved the nal manuscript. The authors
acknowledge the nancial support of the Development National Foundation of
Private Universities of Brazil.
* Corresponding author. Tel.: 55 433 371 7723; fax: 55 433 371 7834.
E-mail address: gcnobre@gmail.com (G. N. Costa).
factors are atherogenic dyslipidemia (hypertriacylglycerols, high
levels of apolipoprotein B, small, dense particles of low-density
lipoprotein cholesterol, and low high-density lipoprotein
cholesterol levels), hypertension, hyperglycemia, and a proin-
ammatory state [1].
Recent studies have associated the development of obesity in
humans to the relative proportions of some major phyla of bac-
teria of the intestinal microbiota, such as Bacteroidetes, Firmi-
cutes, and Actinobacteria, suggesting that the metabolic activity
of these intestinal microbiota facilitates the extraction and/or

http://dx.doi.org/10.1016/j.nut.2015.11.001
0899-9007/ 2016 Elsevier Inc. All rights reserved.
 L. J. Bernini et al. / Nutrition 32 (2016) 716719 717

storage of calories ingested [2,3]. Moreover, imbalance in this
ecosystem can lead not only to obesity, as well as the develop-
ment of insulin resistance [46].
In this context, probiotic microorganisms have demonstrated
health-improving effects on the hosts. The Firmicutes and Acti-
nobacteria include the Lactobacilli and Bidobacteria, respec-
tively, whose genera are the most used and often are related to
these benecial probiotic effects [7,8].
Although some studies have reported the benecial effects of
species of Lactobacilli in patients with MetS, we are not aware of
any report on the effects of Bidobacterium lactis in this condition
and it is important to emphasize that the effects of probiotics are
highly strain-dependent [9].
Therefore, the aim of this study was to evaluate the inuence
of fermented milk with B. lactis HN019 on lipid prole, glucose
metabolism, and cytokines in patients with MetS.
Participants and methods
The study was conducted according to the guidelines in the Declaration of
Helsinki and approved by the Ethical Committee involving humans of the Uni-
versity of Londrina, Parana, Brazil, protocol number 185/2013. Patients were
recruited between March and May 2013 in the city of Jataizinho, Parana State,
Brazil. Inclusion criteria were being between ages 18 and 60 y, and meeting MetS
diagnosis criteria, according to the National Cholesterol Education Program,
Adult Treatment Panel III (NCEP/ATP III) [1]. Fifty-one patients with MetS were
randomized by drawing and began to participate in the study Exclusion criteria
involved patients with chronic diseases or individuals with use of drugs that
interfere in the lipids and/or glycemic prole, antibiotics, and anti-inammatory
agents, vitamins and foods with probiotic microorganisms, besides lactose-
intolerant individuals. The patients were instructed to maintain their usual di-
ets, level of physical activity or other lifestyle factors throughout the intervention
period.
Fifty-one patients with MetS agreed to participate in the study by written
consent. The participants were divided into a control group or untreated (n 25)
and a probiotic group (n 26). The diet of the probiotic groups included the daily
ingestion of 80 mL of the probiotic milk containing on average 3.4  108 colony-
forming units (CFU)/mL of B. animalis ssp. lactis ssp. nov. HN019.
The probiotic milk was produced and packaged aseptically at the Laboratory
of Microbiology of the University of North Parana, according the current Brazilian
legislation. The probiotic milk ready for consumption showed high probiotics
counts with 3.4  108 CFU/mL, 0.38% acidity, pH 5.15, 2.80% protein, 7.54% car-
bohydrates, 0.0% fat, 12.08% total solids, and 0.48% ash that results in 33.09 cal-
ories per bottles. Additionally, the nal product contained 0.01% (v/v) orange,
vanilla, or both avors. Pathogens were not detected, as required by legislation
[10], thus the product was characterized as suitable for consumption and seven
bottles of 80 mL were distributed weekly to each volunteer until they reached
45 d of intake. Patients were asked to drink one bottle every morning.
Anthropometric measurements and biochemical parameters were assessed
at the beginning of the study (T0) and after 45 d (T45). Waist circumference (WC),
body weight, and height were measured, and body mass index (BMI) was
calculated (kg/m2). Three blood pressure measurements taken at 1-min intervals
after the participant had been seated were recorded. The mean of these mea-
surements was used.
After fasting for 12 h, serum or plasma samples were obtained and patients
underwent the following laboratory blood analysis: glucose, total cholesterol
(TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein
cholesterol (LDL-C), and triacylglycerols (TGs). Levels were evaluated by a
biochemical auto-analyzer (Dimension Dade AR Dade Behring, Deereld, IL,
USA), using Dade Behring kits; plasma insulin level was determined by chem-
iluminescence microparticle immunoassay (Architect, Abbott Laboratory, Abbott
Park, IL, USA). Homeostasis model assessment insulin resistance (HOMA-IR) was
used as a surrogate measure of insulin sensitivity [11]. Tumor necrosis factor
(TNF)-a levels were measured by a sandwich enzyme-linked immunosorbent
assay (ELISA) using a commercial immunoassay ELISA (Ready-Set Go Set, e-
Bioscience, San Diego, CA, USA).
The sample size was calculated to obtain a statistically signicant result when
changes of the parameters evaluated were at 10% level. This study was per-
formed to obtain 80% statistic power. Mann-Whitney test was performed to
compare differences between parameters of groups at baseline and differences
across treatment groups (intergroup changes). Wilcoxon matched pairs test was
performed to verify changes from baseline (intragroup changes). Data are pre-
sented as median (25%75%). A statistical analysis program (Graph Pad Prism
version 4.0) was used for evaluations. Signicance was set at P < 0.05.
Results
During the proposed study period, only one of the partici-
pants failed to join the group, claiming typical effects of lactose
intolerance, although the recruitment survey excluded in-
dividuals with this characteristic.
There were no differences between control and probiotic
groups with respect to age, sex, medication, and antihyperten-
sive parameters at baseline.
No signicant differences were observed intra- or intergroup
for WC, systolic and diastolic blood pressure levels, TGs, HDL-C,
glucose, insulin, and the HOMA (Table 1). The probiotic group
showed signicant decreases in BMI (P 0.017), TC (P 0.009),
and LDL-C (P 0.008) compared with baseline values. Regarding
intergroup changes, there was a signicant decrease (P < 0.05) in
BMI, TC, and LDL-C veried after 45 d in the probiotic group
compared with the control group. The control group had a sig-
nicant increase in BMI and HDL-C, and a signicant decrease in
TC and LDL-C compared with the baseline values of the probiotic
group (Table 1; P < 0.05).

Table 1
Anthropometric, blood pressure, and laboratory parameters in control and probiotic groups of patients with metabolic syndrome

Parameters Control (n 25) Probiotic (n 26)

T0 T45 P value T0 T45 P value

BMI (kg/m2)*,y 35.8 (33.444.5) 35.5 (32.240.7) 0.966 30.8 (27.233.7) 29.5 (25.933.3) 0.017
WC (cm) 107 (101128) 109 (99.5124.5) 0.084 105 (98.5112.5) 107 (98.5115) 0.208
SBP (mm Hg) 125 (112.8139) 121 (110131) 0.135 130 (120150) 140 (127.5150) 0.974
DBP (mm Hg) 77.5 (7090) 72.5 (63.582.3) 0.191 80 (8097.5) 90 (80100) 0.842
TG (mg/dL) 199 (103.3251.3) 168.5 (113.5221.3) 0.597 163 (128314.5) 174 (124296) 0.808
TC (mg/dL)*,y 199 (166.5208.5) 205 (173220.5) 0.506 209 (183.8249.8) 194 (168251) 0.009
HDL (mg/dL)* 42 (3547.5) 39 (35.547) 0.617 40.5 (3049.5) 38.5 (31.346) 0.820
LDL (mg/dL)*,y 117 (83142) 115 (95146) 0.820 128.5 (103152.8) 111 (93.6134.6) 0.008
Glucose (mg/dL) 98 (94115.5) 99 (91113.0) 1.000 94 (82.5115.5) 97 (88.3124.3) 0.424
Insulin (uU/mL) 13.90 (9.9317.78) 13.65 (9.4621.78) 0.107 13.4 (8.1817.3) 15.3 (10.217.4) 0.171
HOMA 3.63 (2.094.44) 3.23 (2.335.55) 0.119 3.43 (2.294.77) 4.09 (3.375.87) 0.196

BMI, body mass index; DBP, diastolic blood pressure; HDL, high-density lipoprotein; HOMA, homeostatic model assessment; LDL, low-density lipoprotein; SBP, systolic
blood pressure; TC, total cholesterol; TG, triacylglycerol; WC, waist circumference
MannWhitney test was performed to compare differences between the baseline values and across treatment groups (intergroup changes). Wilcoxon matched pairs test
was performed to verify changes from baseline (intragroup changes). Data are expressed as median (25%75%)
* Differences of baseline groups, P < 0.05.
y
Percentage of change from baseline (P < 0.05).
718 L. J. Bernini et al. / Nutrition 32 (2016) 716719
The cytokines TNF-a (P < 0.05) and interleukin (IL)-6
(P < 0.05) decreased after 45 d of probiotic intake compared with
baseline values. Differences between treatment groups veried a
statistically signicant decrease in TNF-a and IL-6 (P < 0.05)
when the probiotic group was compared with the control group
(Fig. 1A, B).
Discussion
The main ndings of the present study are related to the
benecial effects of B. lactis on BMI, lipid prole, and cytokine
levels in patients with MetS. A recent report demonstrated that
fermented milk with Lactobacillus plantarum decreased TC,
glucose, and IL-6 levels. Meanwhile, there was a trend for de-
creases in LDL-C, although there were no signicant differences
in BMI [12].
Several studies have associated phyla and species of intestinal
microbiota to obesity or weight loss in humans and high levels of
bidobacteria in the gastrointestinal tract are related to weight
loss [46]. Although the fecal microbiota was not characterized
in the present study, the high daily intake of B. lactis as
allochthonous microbiota can positively affect the microbial
balance [13] leading to an improvement of the lipid metabolism
and weight loss. The mechanisms involved in body weight
reduction are not clear, but studies have pointed to the reduction
of adipocyte size, inhibition of adipogenesis, and suppression of
energy intake [9,14].
The reduction of TC and LDL-C in the probiotic group cor-
roborates others reports [9,15]. A meta-analysis of randomized
controlled trials to evaluate the effects of probiotics observed a
decrease LDL and TC concentrations in individuals with normal,
borderline high, and high cholesterol levels [15]. The mecha-
nisms proposed for these effects are associated with the assim-
ilation of cholesterol during bacterial multiplication or
cholesterol binding to the surface of the cell wall of these bac-
teria, preventing the absorption by the intestinal tract into the
bloodstream [16,17]. There are mechanisms related to the ability
of some probiotic microorganisms to produce hydrolase that
deconjugate bile acids. These compounds, when deconjugated,
are absorbed less efciently, resulting in increased excretion in
the feces. Another hypothesis is the reduction of serum choles-
terol as a function of lower lipid absorption, due to the
Fig. 1. Concentration of tumor necrosis factor (TNF)-a and interleukin (IL)-6 in the control (CONT) and probiotic (PROB) groups evaluated at the beginning of the study (T0)
and after 45 d (T45) of milk ingestion. MannWhitney test was performed to compare differences between the baseline values and across treatment groups (inter-group
changes). Wilcoxon matched pairs test was performed to verify changes from baseline (intragroup changes). Data are expressed as median (25%75%). *Differences of baseline
groups, P < 0.05. yPercentage of change from baseline (P < 0.05).
coprecipitation of cholesterol together with the deconjugated
bile acids, which are less effective in the solubilization and ab-
sorption of lipids from diet [18]. Additionally, the use of pro-
biotics has been associated with increased levels of short-chain
fatty acids (SCFA) such as propionate, which improve lipid
metabolism by acting directly on the liver by inhibiting
hydroxymethylglutaryl coenzyme A reductase, a rate-limiting
step of the cholesterol synthesis pathway [19].
Studies evaluating probiotic intake and markers of inam-
mation are controversial. Some studies with Lactobacilli showed
a decrease in inammatory markers [20], whereas others did not
report such ndings [21,22]. In a recent study, the effects of a
multispecies probiotic supplement containing L. acidophilus, L.
casei, L. rhamnosus, L. bulgaricus, B. breve, B. longum, and Strep-
tococcus thermophilus on high-sensitivity C-reactive protein
(hs-CRP) in patients with diabetes have been reported. The au-
thors found a signicant decrease in serum hs-CRP levels [20].
Results from another study demonstrated the effects of L. plan-
tarum on the classical parameters related to the MetS and other
parameters related to cardiovascular risk [12]. The authors found
a signicant decrease in IL-6 between the control (P 0.032) and
the intervention (P 0.001) groups. Therefore, it is difcult with
these sparse data to support a strain specicity related to in-
ammatory status. The metabolic activity of gut microbiota is
associated with the inammatory responses originated from
high-fat diets [14]. Bacterial lipopolysaccharides derived from
intestinal microorganisms can act in this process triggering in-
ammatory response and consequently may predispose to the
occurrence of MetS.
In this study, we observed the reduction on levels of TNF-a
and IL-6, cytokines that have been widely studied and related to
obesity. Fat accumulation caused by obesity provokes increased
production of these adipokines, which stimulate the generation
of hepatic acute-phase proteins, leading to a proinammatory
condition associated with the development of obesity comor-
bidities as insulin resistance [23].
In this study, the small number of participants (N 51) and
the differences at the baseline values in some parameters be-
tween the groups limited the interpretation of the results. For
example, decreases in TC, LDL-C, TNF-a, and IL-6 levels can be
related to reduction in BMI. Nevertheless, the percentage of
changes from baseline (intergroup differences) and intragroup
 L. J. Bernini et al. / Nutrition 32 (2016) 716719
differences attenuated this limitation. Additionally, the mecha-
nisms that underlie the current ndings are certainly needed,
such as characterization of fecal microbiota and modications of
the genome. There were some strengths, however. To our
knowledge, this is the rst study to evaluate the effects of a
species of Bidobacterium on patients with MetS. Few studies
have demonstrated the inuence of probiotics on cytokines. We
rigorously tried to ensure that the patients did not take any drug
or presented any disease that could interfere with the results.
Conclusion
Data from the present study showed that regular consump-
tion of B. lactis HN019 may contribute to the reduction of the
characteristic parameters of MetS and obesity. Although the re-
lationships between microbiota, obesity, diabetes, and MetS are
increasingly evident, there remain many mechanisms that need
to be explored. Currently, the main challenge is to identify bac-
teria that help to control obesity and related metabolic disorders.
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