Hindawi

Mediators of Inflammation
Volume 2017, Article ID 5650627, 10 pages
https://doi.org/10.1155/2017/5650627

Clinical Study
Evidences of a New Psychobiotic Formulation on Body
Composition and Anxiety

Carmela Colica,1 Ennio Avolio,2 Patrizio Bollero,3 Renata Costa de Miranda,4,5

Simona Ferraro,6 Paola Sinibaldi Salimei,7 Antonino De Lorenzo,7 and Laura Di Renzo7
1
CNR, IBFM UOS of Germaneto, University “Magna Graecia” of Catanzaro, Campus “Salvatore Venuta”, Germaneto, 88100
Catanzaro, Italy
2
Comparative Neuroanatomy Laboratory of Ecology Department, University of Calabria, Ponte Pietro Bucci, Arcavacata di Rende,
87030 Cosenza, Italy
3
Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy
4
PhD School of Medical-Surgical Applied Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy
5
CAPES Scholarship (Proc No. BEX 13264/13-3), CAPES Foundation, Ministry of Education of Brazil, 70040-020 Brasília, DF, Brazil
6
Health Center Srl, Biomedical and Nutritional Center, 87100 Cosenza, Italy
7
Section of Clinical Nutrition and Nutrigenomic, Department of Biomedicine and Prevention, University of Rome Tor Vergata,
Via Montpellier 1, 00133 Rome, Italy

Correspondence should be addressed to Laura Di Renzo; laura.di.renzo@uniroma2.it

Received 1 June 2017; Accepted 3 August 2017; Published 24 September 2017

Academic Editor: Giovanni Gambassi

Copyright © 2017 Carmela Colica et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Gut microbiota is implied in obesity, because of its ability to harvest energy from diet, and in the regulation of
behavior. Given the link between gut microbiota, body composition, obesity, and anxiety, the aim of this study was to
evaluate the effects of a new psychobiotic formulation. Methods. Eligible patients were randomly divided into three groups:
psychobiotics oral suspension group (POSG); dietary treatment group (DTG); combined treatment group (CTG). All subjects
underwent body composition and psychological profile evaluation. Results. Significant changes in body composition
parameters in each group were relieved after all treatments. Hamilton anxiety rating scale (HAM-A) highlighted a significant
reduction of the total score for all study population after treatments in POSG (p = 0 01) and CTG (p = 0 04). A reduction of
HAM-A total score in anxious subjects in POSG or CTG and a significant reduction of positive subjects for HAM-A in
POSG (p = 0 03) and in CDG (p = 0 01) were shown. Discussion. Three-week intake of selected POS represents a good
approach to solve problems related to obesity and behavior disorders. However, new clinical trials need to be performed on
a larger population and for a longer period of treatment before definitive conclusions can be made. This trial is registered
with NCT01890070.

1. Introduction gut microbiota, both temporarily and definitely. An external

change of its composition could induce a dramatic variation
Gut microbiota is an ensemble of 100 trillion microorgan- on the hosts’ health [3, 4].
isms present in the gastrointestinal tract (GI), which A growing body of evidence demonstrated the possible
belong to more than 1000 species and 700 strains [1], involvement of gut microbiota in fat mass accumulation
and plays a crucial role in human’s physiology, due to and cardiometabolic disease onset. The World Health Orga-
its ability to maintain energy homeostasis and stimulate nization described obesity as a disorder with an excessive
immunity as an endocrine organ, in a symbiotic relationship body fat accumulation, abandoning the old definition, which
with the host [2]. Several factors like genetics, diet, infection, restricted this condition to the simple body mass index (BMI)
drug consumption, age, and sex could influence the nature of classification, to embrace a broader range of phenotypes,
2 Mediators of Inflammation

including subjects within the normal BMI range but with a Given the link among gut microbiota, body composition,
critical percentage of body fat mass [5–10]. Obesity condition obesity, and the risk of developing anxiety, the aim of this
seems to be mostly caused by the obesogenic environment study was to evaluate the differences deriving from the
[11], which consists of a series of bad lifestyle habits from combination of 3-week administration of a new psychobiotic
the disproportionate intake of calories, especially from sim- formulation, (psychobiotics oral suspension, POS) with or
ple carbohydrates, and the reduction of physical activity. without dietary treatment (DT), consisting of a hypocaloric
Moreover, obese subjects could present some behavioral dis- diet, on general population.
orders [12], and some genetic profiles seem to be associated The evaluation was performed based on anthropo-
with the body weight regulation [13–16]. metric, bioimpedance analysis (BIA), dual X-ray absorpti-
Gut microbiota seems to be implicated in obesity onset ometry (DXA) measurements, and anxiety assessment with
given its ability to harvest energy from the diet, through its Hamilton anxiety rating scale (HAM-A).
influence on gut epithelium and motility [17], and increase
triglyceride storage in the host adipocytes, inhibiting the 2. Methods
fasting-induced adipose factors [18]. Furthermore, gut
microbiota influences several metabolic processes such as 2.1. Study Design and Subjects. This research was con-
lipogenesis, fatty acid oxidation, triglycerides, and cholesterol ducted using a prospective intervention study design,
production [19]. Numerous in vivo studies observed that between January 2017 and April 2017. Forty-five subjects
microbiota plays a strong role in adipose tissue accumula- were recruited sequentially within a routine medical check-
tion. In fact, germ-free mice, although they ingest more calo- up program at the Section of Clinical Nutrition and Nutrige-
ries than their littermates, result leaner than conventional nomics, Department of Biomedicine and Prevention of the
mice. At the same time, microbiota transplantation from University of Rome “Tor Vergata.” POS was administrated
conventional to germ-free mice highlights a dramatic 1 time/day, 2 h before lunch in order to ensure gastrointesti-
increase in body fat, triglyceride production, and insulin nal transit and absorption.
resistance, without changing their food habits [17, 20, 21]. Eligible patients were randomly divided into three groups
In recent years, researchers focused their attention on the (1 : 1 : 1 ratio): (1) psychobiotics oral suspension group
relationship between gut microbiota and brain development (POSG), subjects took daily note 1 bag of 3 g of POS, and they
and function, discovering a bidirectional communication did not change their ordinary diet; (2) dietary treatment
pathway between them, defined as microbiota-gut-brain axis. group (DTG), subjects followed a hypocaloric diet; (3)
Several studies highlighted the role of microbiota in the combined treatment group (CTG), subjects followed the
regulation of mood and behavior, like stress, anxiety, depres- hypocaloric diet and took daily note 1 bag of 3 g of POS.
sion, and autism [22–24], as well as the potential therapeutic Each group followed the assigned treatment consecutively
effects deriving from its modulation. for 3 weeks. At the beginning and at the end of each treat-
Due to the psychotropic effects in animal models and ment, body composition evaluation and psychodiagnostic
human clinical trials, the term “psychobiotics” was intro- tests were performed.
duced [25]. In fact, it has been demonstrated that treatment Subjects were asked to maintain their usual lifestyle
with probiotics formulation, containing Lactobacillus aci- habits and to report any illness or abnormality arisen during
dophilus, Lactobacillus casei, and Bifidobacterium bifidum, the study.
showed positive effects on psychological distress [26, 27]. The primary outcome of this study was the evaluation of
In vivo studies observed a relation between altered gut nutritional status according to body composition changes
microbiota composition and anxiety related behavior, with measured by anthropometry, BIA, and DXA, due to the
increased exploration of aversive zones and improved different treatments. The secondary outcome was the evalua-
serotoninergic function in germ-free mice when compared tion of anxiety disorder through the HAM-A test, pre- and
to specific pathogen-free counterparts [24, 28, 29]. posttreatment each.
Moreover, the anxiety-like behavior increases during All participants recruited in the study authorized their
pathogen infection and GI inflammation in animal participation by reading and signing the informed con-
models [30]. sent, drafted in accordance with the provisions of the
At the same time, anxiety and other psychological Ethics Committee of Medicine, University of Rome Tor
disorders seem to be related to body composition and obesity Vergata and with the Helsinki Declaration of 1975 as
[31]. Several studies observed learning, memory, and func- revised in 1983. This trial is registered with NCT01890070,
tion deficits in obese subjects, linking obesity to the exacerba- http://www.ClinicalTrials.gov.
tion of depression and anxiety disorders [32–35]. Vice versa,
depression disorders have a strong positive association with 2.2. Exclusion Criteria. Exclusion criteria included age < 20 y
eating behaviors and fat mass, especially in subjects who do or >75 y, pregnancy, breastfeeding, type 1 diabetes, presence
not follow a Mediterranean-like eating pattern [36]. of intestinal bacterial overgrowth, characterized by high
Up today, very few studies demonstrated the beneficial levels of hydrogen and methane production in the small
effects of psychobiotics on the health status of obese subjects. bowel, acute diseases, endocrine disorders, liver, heart or kid-
An improvement of psychosocial behavior was seen in ney dysfunctions, history of chronic medication, antibiotic
subjects with a fat mass surplus that underwent weight therapy up to ten days before enrollment, smoke, drug or
loss dietary treatments [37]. alcohol abuse, and participation in another diet trial. No
Mediators of Inflammation 3

CONSORT 2010 Flow Diagram

Enrollment
Assessed for eligibility (n = 45)

Excluded (n = 12)
(i) Not meeting inclusion criteria (n = 5)
(ii) Declined to participate (n = 7)
(iii) Other reasons (n = 0)

Randomized (n = 33)

Allocation

Allocated to intervention POS (n = 11) Allocated to intervention DT (n = 11) Allocated to intervention CT (n = 11)
(i) Received allocated intervention (n = 11) (i) Received allocated intervention (n = 11) (i) Received allocated intervention (n = 11)
(ii) Did not received allocated (ii) Did not received allocated (ii) Did not received allocated
intervention (n = 0) intervention (n = 0) intervention (n = 0)

Follow-up

Lost to follow-up (n = 0) Lost to follow-up (n = 1) Lost to follow-up (n = 2)

Subject voluntarily drop out the study Subject voluntarily drop out the study Subject voluntarily drop out the study
discontinued intervention (n = 0) discontinued intervention (n = 0) discontinued intervention (n = 0)

Analysis

Analysed (n = 11) Analysed (n = 10) Analysed (n = 9)

(i) Excluded from analysis (n = 0) (i) Excluded from analysis (n = 0) (i) Excluded from analysis (n = 0)

Figure 1: Study flow diagram according to Consort, 2010.

subjects with known alterations of intestinal transit following and National Institute of Health Guidelines, waist circumfer-
organic pathologies (abdominal surgery, diabetes mellitus, ence (WC) and hip circumference (HC) were taken using a
scleroderma, hypothyroidism, etc.) were included in the flexible steel metric tape to the nearest 0.5 cm. Body weight
study. Subjects were advised not to consume any antibiotics (Kg) was measured to the nearest 0.1 Kg, using a technical
or probiotics for the month prior to study initiation and to balance (Invernizzi, Rome, Italy). Waist/hip ratio (WHR)
avoid using it for all the duration of the study. was also evaluated in relation to clinical risk thresholds, that
is, WHR > 1 for men and WHR > 0.9 for women. Height (m)
2.3. Psychobiotics Oral Suspension Composition. 1 bag of POS was measured to the nearest 0.1 cm using a stadiometer
of 3 g contained: 1.5 × 1010 colony-forming unit CFU of (Invernizzi, Rome, Italy). BMI was calculated using the
Streptococcus thermophilus (CNCM strain number I-1630), formula: BMI = body weight/height2 (Kg/m2).
1.5 × 1010 colony-forming unit CFU of Lactobacillus bulgari-
cus (CNCM strain numbers I-1632 and I-1519); 1.5 × 1010 2.5. Bioelectrical Impedance Analysis (BIA). Resistance, reac-
colony-forming unit CFU of Lactococcus lactis subsp. lactis tance, impedance, phase angle, total body water (TBW),
(CNCM strain number I-1631); 1.5 × 1010 colony-forming intracellular water (ICW), and extracellular water (ECW)
unit CFU of Lactobacillus acidophilus; 1.5 × 1010 colony- were assessed by BIA phase sensitive system (BIA 101S,
forming unit CFU of Streptococcus thermophiles; 1.5 × 1010 Akern/RJL Systems, Florence, Italy) [38, 39]. Impedance
colony-forming unit CFU of Lactobacillus plantarum; index (II) was evaluated with the following formula [40]:
1.5 × 1010 colony-forming unit CFU of Bifidobacterium lactis
(CNCM I-2494); 1.5 × 1 1010 colony-forming unit CFU of height2 cm2
II = 1
Lactobacillus reuteri (DSM 17938), maltodextrin from corn, resistance Ω
anticaking agent (silica), casein, lactose, and gluten < 3 ppm
LLOQ (lower limit of quantitation), (Biocult strong, HOME- Measurements were taken according to Di Renzo
OSYN, Rome, Italy). et al. [39].
2.4. Anthropometric Analysis. According to the International 2.6. Dual X-Ray Absorptiometry (DXA). Body composition
Society for the Advancement of Kinanthropometry protocol analysis was assessed by DXA (i-DXA, GE Medical Systems,
4 Mediators of Inflammation

Milwaukee, WI, USA) according to the previously described Table 1: Overall description of anthropometric, body composition,
procedures [12]. and anxiety data.
Total body fat (TBFat), total body lean (TBLean),
android body fat (ABF), and gynoid body fat (GBF) were Overall
n = 30
expressed as a percentage (%) of the total body mass.
Median (min–max)
TBFat percentage was estimated by the ratio between the
TBFat (Kg) and the sum of TBFat (Kg), TBLean (Kg) and Age (years) 45.00 (21.00–72.00)
bone mineral content (BMC) (Kg) multiplied by 100. The Weight (Kg) 77.75 (50.30–121.00)
intermuscular adipose tissue (IMAT) was evaluated accord- Height (cm) 165.00 (150.00–186.00)
ing to Bauer et al. [41]. BMI (Kg/m2) 26.87 (20.12–39.93)
WC (cm) 89.25 (69.50–122.50)
2.7. Dietary Intervention. The energy intake for DT was
calculated based on basal metabolic rate (BMR) of each sub- HC (cm) 108.50 (85.50–132.00)
ject, with Weir’s formula: BMR = [(3.94 × VO2) + (1.106 × WHR 0.82 (0.69–1.09)
VCO2)] × 1.44 VO2, where VO2 is the volume of oxygen Phase angle (°) 7.05 (5.10–930)
uptake (mL/min), estimated by the following formulas: II (cm2/Ω) 53.97 (39.35–94.28)
TBFat (Kg) 28.45 (14.03–44.64)
VO2 woman = TBLean DXA × 4 5,
2 ABFat (Kg) 2.50 (0.70–4.38)
VO2 man = TBLean DXA × 5 3, GBFat (Kg) 5.20 (2.72–7.70)
IMAT 1.21 (0.46–2.04)
and VCO2 is the volume of carbon dioxide output (mL/min),
evaluated with the following formula: TBLean (Kg) 43.23 (35.63–58.13)
HAM-A (points) 11.00 (0.00–30.00)
VCO2 = VO2 × 0 85 3 All results were expressed as median (minimum–maximum). BMI: body
mass index; WC: waist circumference; HC: hip circumference; WHR:
Protein intake was determined considering 2 g of protein/ waist-to-hip ratio; II: impedance index; TBFat: total body fat; ABFat:
Kg of TBLean, representing 21–26% of daily caloric intake. android body fat; GBFat: gynoid body fat; IMAT: intermuscular adipose
tissue; TBLean: total body lean; HAM-A: Hamilton anxiety rating scale
Carbohydrate intake was between 44% and 51% of total
(negative test (nonanxious subjects) if total score < 18 and positive test
energy intake, and fat intake was between 27% and 31% of (anxious subjects) if total score ≥ 18).
daily caloric intake (<10% of saturated fatty acids, <300 mg/
day of cholesterol). The fiber intake was 30 g/day [42]. 3. Results
2.8. Psychodiagnostic Instruments. Hamilton anxiety rating
scale (HAM-A) was administered by instructed physicians Out of forty-five subjects recruited, twelve were excluded
and was used to measure the severity of anxiety symptoms. from the trial: five did not meet inclusion criteria, seven
The 14 items on the scale define several symptoms and mea- declined to participate, and other three subjects voluntarily
sure psychic and somatic anxiety. Each item has a score from stopped the treatment (Figure 1).
0 to 4, respectively, from the absence to the severe presence of During the trial, three subjects dropped out of the study
the related symptom. The total score, which has a range from and, finally, thirty patients between 21 and 72 years old with
0 to 56, describes three different scenarios: <17 indicates mild a BMI between 18.5 and 39.9 Kg/m2 and without metabolic
anxiety severity, 18–24 from mild to moderate anxiety sever- complications met the inclusion criteria and completed the
ity, and 25–30 from moderate to high anxiety severity. In this trial. No changes to trial outcomes occurred after it com-
work, we considered anxious subjects those who had a score menced. The median age of subjects was 45 years, 83.3%
higher than or equal to 18 [43, 44]. female and 16.7% male (Table 1). At baseline, no statistical
significance was observed between groups (Table 2).
2.9. Statistical Analysis. Nonparametric tests for asymmetri- Significant changes in body composition parameters in
cally distributed data were conducted in all analyses and pre- each group were relieved after all treatments. In fact, POSG
sented as median (minimum and maximum). Kruskal Wallis and CTG showed a significant reduction in II (p = 0 03 and
test was carried out to compare the three groups at baseline. p = 0 01, resp.), whereas both DTG and CTG highlighted a
To evaluate differences before and after 21 days of treatment, significant reduction in weight (p = 0 01), BMI (p = 0 01),
Wilcoxon test was performed in each group. To describe, waist circumference (p = 0 01 and p = 0 04, resp.), TBFat
quantitatively, variable change after treatments, we used a (Kg) (p = 0 03 and p = 0 04, resp.), and IMAT (p = 0 03;
ratio of the absolute variation to the baseline value (percent p = 0 04, resp.). Significant reduction in hip circumference
variation = Δ%). Categorical variables were compared among (p = 0 02) and TBLean (Kg) (p = 0 02) were observed only
groups by Chi-square (χ2) or Fisher’s test. McNemar test was in DTG, whereas waist/hip ratio (p = 0 04), PA (p = 0 02),
used for the comparison between groups at baseline (T0) and ABFat (Kg) (p = 0 04), and GBFat (Kg) (p = 0 04) parameters
after treatment (T1). Statistical analyses were carried out were significantly reduced in CTG (Table 3).
using IBM SPSS 21.0 for Windows (Armonk, NY: IBM Corp. The HAM-A test performed on POSG and CTG
USA). In all statistical tests performed, the null hypothesis highlighted a significant reduction in the total score for
was rejected at the 0.05 level of probability. all study population after both treatments (p = 0 01 and
Mediators of Inflammation 5

Table 2: Anthropometric, body composition, and anxiety analysis of the 3 groups.

POSG DTG CTG

n = 11 n = 10 n=9 p
Median (min–max) Median (min–max) Median (min–max)
Age (years) 42.00 (26.00–65.00) 36.50 (21.00–72.00) 51.00 (44.00–72.00) 0.12
Weight (Kg) 67.80 (50.30–111.80) 77.00 (62.20–99.00) 79.50 (57.20–121.00) 0.45
Height (cm) 166.00 (155.00–186.00) 166.50 (150.00–173.00) 165.00 (161.00–185.00) 0.81
BMI (Kg/m2) 25.11 (20.12–39.45) 28.60 (26.10–38.60) 29.20 (22.07–39.93) 0.10
WC (cm) 77.00 (69.50–121.00) 94.25 (85.00–106.50) 90.00 (74.00–122.50) 0.27
HC (cm) 104.50 (85.50–132.00) 108.00 (102.00–120.00) 112.25 (89.00–132.00) 0.27
WHR 0.77 (0.69–1.05) 0.86 (0.80–0.94) 0.82 (0.71–1.09) 0.25
Phase Angle (°) 6.50 (4.60–10.20) 7.05 (5.10–9.30) 6.90Z (4.90–9.00) 0.71
II (cm2/Ω) 61.54 (39.35–69.05) 57.12 (42.79–82.53) 52.76 (45.40–94.28) 0.91
TBFat (Kg) 23.29 (14.03–24.57) 29.52 (23.00–44.64) 34.39 (15.05–44.19) 0.13
ABFat (Kg) 1.36 (0.70–2.40) 2.60 (1.29–4.23) 2.53 (0.82–4.38) 0.17
GBFat (Kg) 3.90 (2.97–4.95) 5.70 (4.35–7.70) 5.86 (2.72–7.05) 0.25
IMAT 0.89 (0.46–2.00) 1.22 (0.87–1.79) 1.47 (0.50–2.04) 0.25
TBLean (Kg) 35.88 (34.43–51.36) 43.23 (35.63–58.13) 41.58 (39.59–54.96) 0.35
HAM-A (points) 11.00 (3.00–28.00) 13.50 (7.00–30.00) 6.00 (00.00–23.00) 0.06
All parameters were evaluated at baseline among the 3 groups, by Kruskal Wallis test. All results were expressed as median (minimum–maximum). Statistical
significance attributed to results with ∗ p < 0 05. POSG: psychobiotics oral suspension group; DTG: dietary treatment group; CTG: combined treatment group;
BMI: body mass index; WC: waist circumference; HC: hip circumference; WHR: waist-to-hip ratio; II: impedance index; TBFat: total body fat; ABFat: android
body fat; GBFat: gynoid body fat; IMAT: intermuscular adipose tissue; TBLean: total body lean; HAM-A: Hamilton anxiety rating scale (negative test
(nonanxious subjects) if total score < 18 and positive test (anxious subjects) if total score ≥ 18).

p = 0 04, resp.). No significant difference was seen in the [11]. It is well known that the increase of body fat mass
HAM-A of DTG (Table 3). However, according to the total represents a strong risk factor for the development of
score, the sample was divided into anxious (total score ≥ 18) metabolic and cardiovascular diseases [46–48].
and nonanxious subjects (total score< 18) within each group. Gut microbiota could play an essential role in fat mass
At baseline, no statistical significance was observed between increase and obesity development by invading the intesti-
POSG, DTG, and CTG for anxious and nonanxious subjects nal mucosa and causing systemic inflammation. On the
(p = 0 06). A notable reduction in the HAM-A total score in other hand, the integrity of the intestinal barrier and a
anxious subjects that had undergone the POS or combined healthy intestinal microflora induce an anti-inflammatory
treatment (Δ = −5 points and Δ = −9.5 points, resp.) was effect that causes a consequent reduction in fat mass body
highlighted, while anxious DTG patients had HAM-A total composition [49].
score increased (Figure 2). Gut microbiota has been studied for decades in order to
Table 4 shows absolute numbers of anxious and nonan- evaluate its impact on different aspects of human health
xious subjects in the 3 groups, before and after treatment. and body composition [18–20], and recently, the role of pro-
Furthermore, a significant reduction in the number of biotics with or without diet has been evaluated in terms of
anxious subjects was observed in POSG (p = 0 03; Δ% = changing the overall health status [50, 51], body weight, body
−39.3%), as well as in CTG, where all anxious subjects composition, and obesity [52, 53].
became nonanxious (p = 0 01; Δ% = −100%) (Figure 3). In our study, we enrolled normal weight, preobese, and
obese up to the second-degree patients based on BMI, and,
4. Discussion at the same time, we performed DXA to evaluate body com-
position. At baseline, the population resulted homogeneous
Neurotransmitters and neuromodulators, secreted by in the three groups for the studied variables as reported in
bacteria, are able to modulate the state of the hosts’ Table 2. On average, the population chosen had a TBFat
mood: gamma-aminobutyric acid is produced by certain percentage over 30%, who are considered obese according
Lactobacillus and Bifidobacterium species; norepinephrine to De Lorenzo et al. [54].
is released by Escherichia, Bacillus, and Saccharomyces spp.; The statistical comparison among the three groups
5-hydroxytryptamine is released by Candida, Streptococcus, exhibited that the subjects who belong to POSG did not
Escherichia, and Enterococcus spp.; and dopamine is pro- report significant differences between time T0 and T1, in
duced by Bacillus and acetylcholine by Lactobacillus [45]. weight, waist and hip circumference, and body composi-
Fat mass increase in humans is related to several environ- tion, except for the II (p = 0 03, Δ% = −1.92%). In accor-
mental factors, especially bad lifestyle habits, like sedentary dance with the literature [55], the DTG demonstrated
living and excess of daily caloric, carbohydrate, and fat intake significant reductions in weight (p = 0 01; Δ% = −2.27%),
 6

Table 3: Comparison between before and after treatment in each group.

POSG (n = 11) DTG (n = 10) CTG (n = 9)

T0 T1 p T0 T1 p T0 T1 p
Median (min–max) Median (min–max) Median (min–max) Median (min–max) Median (min–max) Median (min–max)
Weight (Kg) 67.80 (50.30–111.80) 66.30 (49.70–107.90) 0.39 77.00 (62.20–99.00) 75.25 (60.50–93.30) 0.01∗ 79.50 (57.20–121.00) 76.50 (56.10–119.50) 0.01∗
BMI (Kg/m2) 25.11 (20.12–39.45) 24.75 (20.12–38.52) 0.39 28.60 (26.10–38.60) 27.57 (23.90–35.80) 0.01∗ 29.20 (22.07–39.93) 28.10 (21.64–38.58) 0.01∗
∗
WC (cm) 77.00 (69.50–121.00) 78.00 (68.00–121.00) 0.07 94.25 (85.00–106.50) 88.50 (80.00–100.00) 0.01 90.00 (74.00–122.50) 86.50 (71.00–115.00) 0.04∗
HC (cm) 104.50 (85.50–132.00) 105.00 (87.00–121.50) 0.40 108.00 (102.00–120.00) 104.50 (96.00–116.00) 0.02∗ 112.25 (89.00–132.00) 109.50 (88.00–119.00) 0.17
WHR 0.77 (0.69–1.05) 0.76 (0.66–1.05) 0.28 0.86 (0.80–0.94) 0.84 (0.76–0.93) 0.09 0.82 (0.71–1.09) 0.81 (0.74–1.00) 0.04∗
°
Phase angle ( ) 6.50 (4.60–10.20) 6.30 (4.90–8.60) 0.15 7.05 (5.10–9.30) 6.50 (4.70–8.60) 0.06 6.90 (4.90–9.00) 5.70 (5.10–6.60) 0.02∗
II (cm2/Ω) 51.52 (39.35–69.05) 50.53 (39.35–66.92) 0.03∗ 57.12 (42.79–82.53) 53.43 (40.99–76.08) 0.06 55.11 (45.40–94.28) 51.93 (43.42–91.02) 0.01∗
TBFat (Kg) 23.29 (14.03–24.57) 22.70 (14.43–25.50) 0.59 29.52 (23.00–44.64) 29.13 (22.76–40.33) 0.03∗ 34.39 (15.05–44.19) 28.18 (13.60–43.05) 0.04∗
ABFat (Kg) 1.36 (0.70–2.40) 1.24 (0.67–2.33) 0.11 2.60 (1.29–4.23) 2.65Z (1.23–3.60) 0.07 2.53 (0.82–4.38) 1.81 (0.61–4.23) 0.04∗
GBFat (Kg) 3.90 (2.97–4.95) 3.91 (2.90–4.87) 0.28 5.70 (4.35–7.70) 4.90 (4.10–7.77) 0.07 5.86 (2.72–7.05) 5.20 (2.43–6.76) 0.04∗
IMAT 0.89 (0.46–2.00) 0.85 (0.47–1.18) 0.59 1.22 (0.87–1.79) 1.20 (0.86–1.69) 0.03∗ 1.47 (0.50–2.04) 1.15 (0.44–1.72) 0.04∗
TBLean (Kg) 35.88 (34.43–51.36) 35.87 (35.39–50.63) 1.00 43.23 (35.63–58.13) 42.61 (35.62–57.21) 0.02∗ 41.58 (39.59–54.96) 40.42 (39.51–54.11) 0.22
∗
HAM-A 11.00 (3.00–28.00) 7.00 (1.00–28.00) 0.01 13.50 (7.00–30.00) 10.00 (6.00–31.00) 0.72 6.00 (00.00–23.00) 5.00 (0.00–19.00) 0.04∗
All parameters were evaluated before and after treatments by Wilcoxon test. All results were expressed as median (minimum–maximum). Statistical significance attributed to results with ∗ p < 0 05 between T0 and
T1. POSG: psychobiotics oral suspension group; DTG: dietary treatment group; CTG: combined treatment group; BMI: body mass index; WC: waist circumference; HC: hip circumference; WHR: waist-to-hip ratio;
II: impedance index; TBFat: total body fat; ABFat: android body fat; GBFat: gynoid body fat; IMAT: intermuscular adipose tissue; TBLean: total body lean; Ham-A: Hamilton anxiety rating scale (negative test
(nonanxious subjects) if total score < 18 and positive test (anxious subjects) if total score ≥ 18).
Mediators of Inflammation
Mediators of Inflammation 7

⁎ ⁎
8
100
4
HAM-A score (points)

0 80

Frequency of subjects (%)

−4
60
−8

−12 40

−16
POSG DTG CTG 20
−20
Nonanxious variation score 0
Anxious variation score Negative Positive Negative Positive Negative Positive
test test test test test test
Figure 2: Hamilton anxiety rating scale (HAM-A) score variation POSG DTG CTG
before and after treatments in anxious and nonanxious subjects.
Nonanxious subjects (negative test) if total score < 18 and anxious T0
subjects (positive test) if total score ≥ 18. Variation score is shown T1
as median, minimum, and maximum. 609 statistical significance
attributed to results with p < 0 05 by Kruskal Wallis test. Anxious Figure 3: Frequency change of anxious subjects in POSG, DTG, and
variation score among groups: p = 0 10 and nonanxious variation CTG after treatment. Frequency of anxiety was evaluated before and
score among groups: p = 0 67. POSG: psychobiotics oral after treatment in POSG, DTG, and CTG. Negative test (nonanxious
suspension group; DTG: dietary treatment group; CTG: combined 619 subjects) if total score < 18 and positive test (anxious subjects)
treatment group. if total score ≥ 18. Statistical significance attributed to results with
∗
p < 0 05 between T0 and T1 by McNemar test. POSG p = 0 03∗ ;
DTG: p = 0 10; CTG: p = 0 01∗ . POSG: psychobiotics oral
Table 4: Absolute numbers of anxious and nonanxious subjects in suspension group; DTG: dietary treatment group; CTG: combined
all groups, before and after treatment. treatment group.
POSG DTG CTG
HAM-A score n = 11 n = 10 n=9 (p = 0 04, Δ% = −21.77%). This result is the first evidence
T0 T1 T0 T1 T0 T1 reported in the literature, and we speculate that it could
HAM-A < 18 7 9 8 7 8 9 be attributed to the capacity of probiotics to decrease the
HAM-A ≥ 18 4 2 2 3 1 0
intestinal permeability with a consequent reduction of lipo-
polysaccharide and inflammatory cytokine levels [58].
Frequency of anxiety subjects was evaluated before and after treatment in
The improvement of inflammatory state and oxidative
POSG, DGT, and CTG. Negative test (nonanxious subjects) if total
score < 18 and positive test (anxious subjects) if total score ≥ 18. HAM-A: status induced by probiotics administration [59, 60] could
Hamilton anxiety rating scale; POSG: psychobiotics oral suspension group; be able to contribute to the proper fatty acids and glucose
DTG: dietary treatment group; CTG: combined treatment group. metabolism, with the improvement of insulin resistance
related to a better IMAT [61]. In CTG, the significant reduc-
tion of ABFat and waist/hip ratio, both related to insulin
BMI (p = 0 01; Δ% = −3.60%), waist circumference (p = 0 01; resistance and cardiovascular risk [62], could be due to an
Δ% = −6.10%), hip circumference (p = 0 02, Δ% = −3.24%), improvement of insulin profile.
TBFat (Kg) (p = 0 03, Δ% = −.32%), IMAT (p = 0 03; Δ% = BIA results show that POS intervention led to a signifi-
−1.64%), and TBLean (Kg) (p = 0 02, Δ% = −1.64). cant reduction in II and, consequently, a resistance increase.
In the CTG, a higher variation of TBFat loss (p = 0 04, The same trend was marked by Valentini Neto et al. [63],
Δ% = −18.06%) and a statistically significant difference in even if not significant.
the reduction of android (Δ% = −28.46%) and gynoid fat However, a significant decrease of phase angle was
mass (Δ% = −11.46%) (p = 0 04) were highlighted. This pre- observed in CTG, probably due to body water loss which
liminary data underlines the role of probiotics as a supple- translates into a resistance increase, despite of TBFat loss.
ment for diet, as described by Kim et al. [56]. The 21-day Since in POSG we observed a phase angle reduction and a
period treatment is probably not enough to point out the significant reduction of II, we can hypothesize a synergic
positive effect of probiotic treatment alone on the improve- action between diet and POS.
ment of weight and body composition, taking into account Multiple studies have demonstrated the existence of a
that most pharmacological treatments for obesity have a clear link between gut microbiota and brain function. Given
duration of at least three months, and in all cases, it is advised this, the gut microbiota appears to be a key regulator of mood
that they are coupled with lifestyle changes [57]. and behavior [22]. Moreover, probiotics due to their effects
IMAT was significantly reduced in subjects treated only on gut microbiota seem to have a positive impact on the
with diet (p = 0 03, Δ% = −1.64%), whereas the group under management of psychological disorders, such as anxiety. In
diet with probiotic intake showed a greater IMAT reduction our study, POS supplementation led to a significant decrease
8 Mediators of Inflammation

POSG: Psychobiotics oral suspension group

DTG: Dietary treatment group
CTG: Combined treatment group
WC: Waist circumference
HC: Hip circumference
WHR: Waist-to-hip ratio
II: Impedance index
TBFat: Total body fat
TBLean: Total body lean
ABF: Android body fat
GBF: Gynoid body fat
BMC: Bone mineral content
IMAT: Intermuscular adipose tissue
BMR: Basal metabolic rate.

Conflicts of Interest
The authors declare that there is no conflict of interest
regarding the publication of this article.

Authors’ Contributions
Figure 4: Gut-microbiota axis. Carmela Colica contributed to the interpretation of the
data and drafted the manuscript. Patrizio Bollero, Ennio
Avolio, Simona Ferarro, and Paola Sinibaldi Salimei col-
of HAM-A score both in POSG than in CTG. This could be lected the data and performed the experiments. Renata
attributed to the effects of POS on molecular pathways in Costa de Miranda analyzed the data. Antonino De Lorenzo
the central nervous system, which could also act on gut had primary responsibility for the final content. Laura Di
microbiota [23, 24]. We did not observe any significant dif- Renzo conceived, designed the experiments, and drafted
ference in DTG on HAM-A score. This confirms that POS the manuscript. All the authors read and approved the
might have played a role in anxiety rather than only diet. final manuscript.
Based on the positive anxious score (total score ≥ 18), we
observed that in both groups with POS, there was a signifi-
cant decrease in the number of subjects that had a positive Acknowledgments
HAM-A test, with a score improvement of 5 points in POSG The authors are indebted to all the subjects who volunteered
and 9.5 points in CTG. These results confirm that POS in the study. The authors thank the entire medical team
supplementation is associated with a reduction of anxiety, from the Section of Clinical Nutrition and Nutrigenomic,
as shown by Wang et al. [64]. However, a balanced diet, asso- University of Rome Tor Vergata, Rome: Gemma Lou De San-
ciated with POS, seems to have a greater effect on the tis, Lorenzo Romano, Paola Gualtieri, Giorgia Cioccoloni,
improvement of anxiety symptoms (Figure 4). Magda Kavvadia, and Benedetta Primiceri. The authors also
thank Mario Bigioni for painting Figure 4. This study was
5. Conclusions supported by grants from the Ministry of Agriculture, Food
and Forestry (D.M.: 2017188 03/24/2011).
Despite the limitations of our study, related to the study
design and the low sample size, our results highlighted that
a three-week intake of selected psychobiotics represents a References
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