Obesity is one of the most widespread chronic diseases globally, resulting from a complex
interaction between dietary habits and environmental and genetic factors. According to
the statistics from the World Health Organization, more than 1.9 billion adults are
overweight, and approximately 650 million people suffer from obesity. Moreover, these
individuals are at a higher risk of developing numerous metabolic disorders, such as type
2 diabetes, atherosclerosis, cardiovascular diseases, non-alcoholic fatty liver disease,
reproductive issues, and some forms of cancer. Lifestyle and pharmacological
interventions are two of the most important strategies for treating obesity. However,
strict lifestyle changes are often only accepted by a limited number of individuals, and
anti-obesity drugs can have some adverse effects, while their efficacy is often
diminished after prolonged use. Therefore, a significant unmet need is the lack of
convenient and effective adjunctive therapies for treating obesity.

There is a bidirectional association between obesity and mood disorders such as
depression and anxiety. Obesity also has a negative effect on health and quality of life,
as well as on self-esteem. Specifically, it is estimated that approximately 50% of
individuals with obesity develop depressive and anxious traits, which are more common in
females and more frequent as the degree of obesity increases. Furthermore, people with
obesity often suffer a considerable emotional burden, increasing stress, daily worries,
and even experiencing difficulties in performing daily life activities. Added to the
stigma of the disease, which has a negative impact on self-esteem and body image, this
worsens the quality of life and emotional state of these individuals. Therefore, mental
health is closely interconnected with obesity, and addressing both the physical and
emotional aspects is crucial for promoting overall well-being. It is important to adopt a
holistic approach that includes interventions in diet, physical exercise, and emotional
and psychological support for those struggling to maintain a healthy weight and adequate
mental health.

The gut-brain axis is a bidirectional communication network between the gut and the
central nervous system, functioning through neuroimmune and neuroendocrine processes. Its
involvement in the onset of depression has been postulated and is mediated by molecules
such as short-chain fatty acids, gamma-aminobutyric acid (GABA), and tryptophan
metabolites originating from the gut microbiota. In situations of dysbiosis or
alterations in microbiota homeostasis, the gut-brain pathways can be found deregulated
and are associated with neuroinflammation and altered blood-brain barrier permeability.
Moreover, alterations in the gut microbiota may contribute to a depressive state by
directly affecting the release of neurotransmitters such as serotonin and dopamine,
influencing the stress response and the hypothalamic-pituitary-adrenal (HPA) axis,
affecting brain-derived neurotrophic factor (BDNF) levels, and triggering the release of
inflammatory cytokines. For example, depression is associated with an increased release
of C-reactive protein (CRP) and cytokines such as IL-1, IL-2, IL-6, IFN-y, and IL-1ß.

Obesity also causes inflammation at the intestinal level, insulin resistance, and body
fat deposits. Research in this field suggests that there is a bidirectional relationship
between the composition of the gut microbiota, obesity, and insulin resistance. It has
been observed that people with obesity have a different microbial composition compared to
those with a healthy weight. It is believed that this alteration in the gut microbiota
could further contribute to the progression of obesity by increasing the ability to
extract energy from food, promoting low-grade inflammation, increasing lipogenesis,
decreasing fatty acid oxidation, and increasing triglyceride accumulation at the hepatic
level, among other mechanisms.

Considering the role of the gut microbiota in regulating the immune system and
inflammation, it can be observed that chronic inflammation is linked to both obesity and
psychological disorders, including depression and anxiety. These changes in the gut
microbiota may contribute to systemic inflammation and immune dysfunction, which in turn
could affect both obesity and psychological disorders. Some gut bacteria can produce
neurotransmitters that are absorbable at the intestinal level, such as serotonin or GABA,
which are important for regulating mood and anxiety. It is believed that changes in the
composition of the gut microbiota could affect the production of these neurotransmitters,
and therefore, influence mental health.

Although lifestyle modifications, such as caloric restriction and physical exercise, are
considered the best therapies for weight loss, there are other adjunctive options, such
as the modulation of the gut microbiota, that could be useful for people with obesity.
Studies in which prebiotics, probiotics, and synbiotics were administered to individuals
with obesity have shown their beneficial effects on weight reduction and other metabolic
parameters through the modulation of the gut microbiota. Due to the emerging evidence
implicating the gut-brain axis in obesity and its relation to psychological disorders,
there has been increased interest in the development of these treatments as therapies for
restoring the gut microbiota.

Prebiotics are functional foods, given their beneficial role in promoting health and
preventing disease. An example is tannins, considered bioactive compounds due to their
ability to modulate metabolic processes and promote health. Much of the tannins ingested
reach the large intestine, where the gut microbiota converts them into metabolites,
including short-chain fatty acids such as acetate, propionate, and butyrate. These
short-chain fatty acids are important metabolites that can have various beneficial
effects on the host's intestinal and overall health. They also exert a potential
antidiabetic effect through the following mechanisms: (i) Improvement of insulin and
proinsulin levels in the blood: the affinity of tannins for binding to polysaccharides
causes a delay and a decrease in the availability of glucose in the gastrointestinal
tract. Additionally, several studies have reported the potential for inhibiting the
activities of α-amylase and α-glucosidase through hydrolyzable tannins and condensed
tannins, respectively. (ii) Insulin-like effect on insulin-sensitive tissues:
procyanidins may act on specific components of the intracellular insulin signaling
pathway. (iii) Regulation of the antioxidant environment of pancreatic cells: oxidative
stress is believed to play a role in insulin resistance as it determines pancreatic cell
apoptosis. Additionally, the expression of genes related to antioxidant enzymes in the
pancreas is low. The high antioxidant capacity of tannins can counteract the pathogenesis
of insulin resistance, along with their anti-inflammatory properties (they decrease TNFα,
IL-1, IL-6 levels, etc.) and cardioprotective properties (they increase superoxide
dismutase, decrease ROS, etc.).

Specifically, high molecular weight tannins reach the gut microbiota in the colon showing
a prebiotic effect. In this case, the compounds are metabolized by microorganisms,
producing metabolites with different bioavailability, activity, or functional effects
compared to the original molecule. Finally, tannins can modulate the composition and
function of the gut microbiota, selectively inhibiting pathogens and promoting the growth
of beneficial bacteria.

Probiotics are preparations of microorganisms that, when administered in appropriate
conditions and amounts, improve the microbial balance of the gut. These microorganisms
have been shown to suppress inflammation and modulate the immune system by preventing the
induction of the IL-8 cytokine in the human colon epithelium, as well as reducing
intestinal permeability, inhibiting endotoxemia. Probiotic interventions can contribute
to the treatment of obesity and associated complications by improving the abundance and
function of the gut microbiota. Therefore, the combination of prebiotic and probiotic
interventions (synbiotics) can provide a synergistic and effective therapy for metabolic
disorders. Additionally, the positive role of synbiotic supplementation in mental
illnesses such as major depressive disorder has been documented. In this case, synbiotics
could improve depression symptoms by enhancing tryptophan metabolism and decreasing
dopamine metabolite concentrations in the amygdaloid cortex.

It has been suggested that synbiotics could help improve the type and functionality of
the microbiota, reduce intestinal inflammation, and promote satiety through increased
production of hormones that have this effect on the body. Therefore, this could be
beneficial in managing obesity. Synbiotics may play a crucial role in modulating
macronutrient metabolism by producing short-chain fatty acids, which bind to
G-protein-coupled receptors and increase the secretion of glucagon-like peptide 1 (GLP-1)
and peptide YY (PYY) from enteroendocrine L cells. These bindings can trigger insulin
production by pancreatic β-cells, inhibit glucagon secretion, decrease hepatic
gluconeogenesis, and increase insulin sensitivity. Synbiotics also improve intestinal
function, elevate mucin production, and reduce the number of pathogenic gram-negative
bacteria in the colon. These changes reduce the transmission of lipopolysaccharides (LPS)
across the mucosal wall and metabolic endotoxemia, which may ultimately lead to
improvements in insulin receptor function and lower insulin levels. Previous studies have
shown that their administration is beneficial for both obesity and typical psychological
symptoms of depression or anxiety, among others. A 6-week intervention with synbiotics
can significantly reduce depression symptoms compared to a placebo. In this field, a 2017
meta-analysis suggested that probiotics can reduce psychological symptoms, including
anxiety, depression, and perceived stress in healthy adult volunteers. The gut microbiota
can directly produce neurotransmitters like serotonin and influence its production. It
also has immunomodulatory functions and is capable of activating the
hypothalamic-pituitary axis through the production of proinflammatory cytokines IL-1 and
IL-6.

In this context, an intervention with a synbiotic composed of tannins and beneficial
intestinal strains in individuals with obesity could have beneficial effects on health by
increasing antioxidant capacity and short-chain fatty acid production, modulating the
production of neurotransmitters involved in satiety and mood, and promoting gut
microbiota balance, mucosal protection, and improved intestinal permeability.

Therefore, combining synbiotic interventions could provide a synergistic and effective
therapy for both metabolic and psychological disorders. Addressing obesity
comprehensively, including medical treatment, psychological support for mental health,
and synbiotic treatment, could improve emotional state, obesity, and psychological
disorders. Ultimately, it could enhance the quality of life for affected individuals.
While research in this area is still ongoing and more evidence is needed to understand
these interactions underfully, there is growing evidence suggesting that gut microbiota
health can influence both physical and mental health in complex and significant ways.
Promoting a healthy gut microbiota through a balanced diet, intake of fiber-rich foods
and probiotics, as well as stress reduction, may be beneficial for addressing both
obesity and psychological disorders, even synergistically with other treatments.

Therefore, our goal is to determine whether the intake of a synbiotic composed of lactic
acid bacteria, bifidobacteria, and tannin-based phytocomplexes, is capable of improving
anxious and depressive symptoms, glycemic profile, and insulin resistance in patients
with obesity, depending on the presence or absence of psychological disorders, through
the improvement of the inflammatory profile, appetite control, neurotransmitter
production, and the differential alteration of the gut microbiota.

A clinical-basic, prospective, randomized, double-blind, placebo-controlled intervention
study is proposed for patients with obesity who present depressive and/or anxiety
disorders.

To achieve the proposed objectives, 120 patients with obesity who present depressive
and/or anxiety disorders (n=60), and those without disorders (n=60) will be included. The
diagnostic criteria for depression and anxiety will be those established in the DSM-5.
Furthermore, each group will be randomly divided into 2 subgroups, one of which will
receive the synbiotic and the other will receive the corresponding placebo (n=30 per
group) for 12 weeks. The randomization process will be carried out using a software
program for random assignment. Patients will be enrolled sequentially in the study as
they attend the Obesity Unit in the Endocrinology and Nutrition consultations at Dr.
Peset Hospital and meet the following criteria:

- Inclusion criteria: patients with a BMI of 30-40 kg/m², aged between 18 and 65
years, with a known disease duration of more than five years. Additionally, all
patients will maintain a stable weight (<5% fluctuation) during the 3 months before
the study.

- Exclusion criteria: patients who have taken antibiotics 3 months before the start of
the study, as well as prior use of supplements containing probiotics and/or
prebiotics. Also, patients diagnosed with intestinal diseases, and those who have
undergone previous gastrointestinal surgery. Secondary causes of obesity, type 1
diabetes, pregnancy or lactation, active neoplastic disease, and those with
established liver or renal failure will be excluded. Finally, patients with other
diagnosed psychiatric disorders different from anxiety and/or depression, and those
who are undergoing treatment with antidepressants before the start of the study will
also be excluded.

Before starting the study, the patients, or their legal representative, will sign an
informed consent form after being explained the study's objective and resolving any
possible doubts. This study adheres to the ethical guidelines established for human
experimentation in Helsinki and its subsequent updates; it will also have approval from
the Ethics Committee of the Hospital. The medical information and all data collected
during the study will be kept confidential following the Organic Law 15/1999 on Personal
Data Protection and the corresponding Royal Decree 1720/07.

At the beginning of the study, the dietitian-nutritionist will carry out an
individualized nutritional assessment to calculate the resting energy expenditure and
personalized hypocaloric diets will be created, reducing 500 kcal from each individual's
total daily energy expenditure, while maintaining the recommended intake of each
macronutrient (50-55% carbohydrates, 30-35% fats, and 15% proteins). Dietary monitoring
will help avoid bias and reinforce adherence to the diet, along with monitoring side
effects. The treatment group will receive a capsule with a synbiotic formulation, and the
control group will receive a placebo capsule (maltodextrin), and both groups will be
instructed to take it daily for 12 weeks. The synbiotic will consist of a probiotic part
composed of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium lactis
(2×10⁹ CFU/g each), and a prebiotic part composed of phytocomplexes based on tannins (350
mg) extracted from various sources. The placebo will look identical to the synbiotic
capsule. To maintain blinding for the staff participating in the trial, the preparation
of the study products and the filling of the containers will be done by the companies
SILVATEAM and SACCO, which will operate under unblinded conditions.

At the beginning of the intervention, a structured psychological interview will be
conducted to gather information about the patient's current condition; history and
evolution of the disease (obesity and psychological disorder), eating patterns, sleep
quality, associated psychological comorbidities, family and social areas, motivation for
change, and expectations. The impact of obesity on the patient's quality of life
(emotional, social, and personal) will be evaluated. Psychological follow-up will take
place at 4, 6, and 12 weeks of the study. During the interventions, motivation status,
eating habits, lifestyle, self-control, cognitive-behavioral change, and relapse
prevention will be assessed.

At the beginning of the study and after the dietary intervention, fasting blood samples
(12 hours), saliva, urine (24 hours), and stool samples will be collected. The following
parameters will be determined:

1. Anthropometric Parameters and Body Composition Weight and height will be measured,
and BMI will be calculated (weight / (height)²). Waist and hip circumference, as
well as blood pressure, will also be determined. Additionally, a vector analysis
using bioelectrical impedance (seca mBCA 550®) will be performed both at the
beginning and the end of the intervention.

Patients will complete a 3-day dietary log (including 1 weekend day) at the
beginning, 6 weeks, and 12 weeks, along with the IPAQ questionnaire to monitor
physical activity. They will also complete a questionnaire on product adherence and
compliance, and if they have experienced any adverse effects after taking the
product.

2. Psychological Evaluation First, the investigators will apply the Depression Anxiety
and Stress Scale 21 (DASS-21) questionnaire. Based on the initial results, stress,
anxiety, and depression will be evaluated using three validated scales: the Beck
Depression Inventory (BDI-2), the Perceived Stress Scale (PSS), the State-Trait
Anxiety Inventory (STAI), and self-esteem using the Rosenberg questionnaire. This
evaluation will allow the subject's classification into both groups (obesity with
and without anxiety and/or depression), and check if there is a significant
improvement after the synbiotic intake group.

3. Biochemical Parameters A complete lipid profile will be assessed, determining total
cholesterol (TC) and triglycerides (TG) using enzymatic methods, HDL-C (direct
precipitation method), LDL-C (calculated using Friedewald's formula), and VLDL-C
(TG/5). Apolipoproteins B and A-I will be determined by nephelometry. Non-HDL
cholesterol (TC-HDL) and the atherogenic plasma index (API = log (TG/HDL)) will be
calculated. Fasting blood glucose and insulin will be measured using the hexokinase
enzymatic method and chemiluminescence, respectively, and the HOMA index will be
calculated to assess insulin resistance. Liver and kidney function, complete blood
count, coagulation, and hormonal profile will be evaluated using the following
markers: AST, ALT, GGT, LDH, alkaline phosphatase, creatinine, glomerular filtration
rate, A1c, and thyroid function (TSH, free T4). All of these parameters will be
determined in the Clinical Analysis Service of the Dr. Peset University Hospital.

4. Biomarker Determination d.1) Inflammatory Parameters and Antioxidant Capacity The
inflammatory state will be assessed by determining the concentrations of
ultrasensitive C-reactive protein, IL6, TNF, IL1b, adiponectin, PAI-1, and IL10,
using Luminex xMAP-Multiplex technology. Sample processing and data analysis will be
performed following the manufacturer's instructions. Serum levels of LPS will be
determined by ELISA. Total antioxidant capacity in serum will be determined using
the E-BQC system (Bioquochem).

d.2) Determination of Peptides and Intestinal Function Serum levels of intestinal
hormones (GIP, GLP1, PYY, ghrelin, leptin, CCK) will be determined using Luminex X-MAP
technology. To evaluate intestinal permeability and microbial translocation, plasma
levels of zonulin, occludin, LBP, FABP2/I-FABP, and Reg3A will be measured using ELISA
Kits. Detection of β-glucan in plasma will be performed using the Limulus Amebocyte
Lysate (LAL) assay.

d.3) Neurotransmitter Peptide Analysis Cortisol, dopamine, serotonin, and oxytocin levels
in plasma, 24-hour urine, or saliva will be determined using ELISA kits or Luminex X-MAP
technology.

d.4) Metabolomic Analysis NMR (Nuclear Magnetic Resonance) will be applied to obtain
extensive metabolic profiles from large, well-phenotyped patient cohorts. Initially, all
samples will be analyzed by NMR, obtaining a global metabolic profile of up to 40
metabolites including amino acids, sugars, phospholipids, inflammation markers,
glycoproteins, lipoparticles, and some microbial co-metabolites.

The statistical significance of the different metabolites will be analyzed using
covariance tests corrected for potential false discovery rate (FDR) using SPSS and Matlab
software. Chemometric analysis to construct diagnostic, classification and predictive
models will be performed using custom Matlab scripts and PLS Toolbox for PCA, projection
to latent structure-discriminant analysis (PLS-DA), hierarchical cluster analysis (HCA),
and similar approaches on spectral data to classify groups based on clinical and
intervention parameters. Metabolic networks will be built using CytoScape and the Kyoto
Encyclopedia of Genes and Genomes (KEGG) for genomics, integrating metabolomic results
and other molecular outcomes of the project. Finally, all results will be evaluated
through cross-validation, and the discriminatory/predictive power of the different
metabolites and scores will be analyzed using receiver operating characteristic (ROC)
curves.

d.5) Study of the Microbiota Nucleic acid purification, 16S rRNA gene amplification, and
sequencing First, the stool samples will be triturated, homogenized, and lysed using a
Fastprep system (MP Biomedicals). Total DNA will be extracted following the automated
Magna Pure LC protocol (Roche, Manheim, Germany) according to the manufacturer's
instructions. Metagenomic libraries will be obtained using the NEXTERA XT kit following
Illumina's protocol. The V3-V4 regions of the 16S rRNA will be amplified, and the
libraries will be purified following Illumina's protocol.

MiSeq sequencing will be performed using the V3 kit (2x300 cycles), generating 300-bp
reads every 65 hours, starting from each end of the amplicon. Ultra-deep sequencing will
be performed using an Illumina sequencer at the FISABIO-Salud Pública Center.

Phylogenetic analysis of 16S rRNA will be carried out using QIIME software. Functional
assignment of metagenomic reads will be performed using dedicated bioinformatics
pipelines developed at FISABIO (integrating cutting-edge methodologies in the field with
customizable scripts).

Microbial community analyses (biodiversity, clustering, heat maps) and statistical
analysis will be performed using R software. For biomarker discovery analysis, the LEfSe
platform will be used.

d.6) Gene Expression Analysis Changes in gene expression levels will be evaluated using
Nanostring® technology for nCounter®, which will allow us to identify messenger molecules
that are altered between obese patients with/without psychological disorders. This system
enables the hybridization of each target molecule with a fluorescent probe for individual
identification without retrotranscription or amplification, using minimal sample amounts.
Total RNA will be extracted from PBMCs using the GeneAll® RibospinTM total kit, and from
just 100-200 ng of total RNA, the multiplex metabolism panel will assess 768 genes,
focusing on inflammatory and metabolic pathways. The results will be analyzed using the
nSolver tool. These experiments will be subcontracted to Diagnostica Longwood, S.L.

d.7) In Vitro Study of the Mechanism of Action of Colonic Digestate on Complex Cellular
Models Simulating the Gut-Brain Axis The use and design of an organ-on-chip (OoC) model
is proposed, with intestinal cells in contact with the fermentation digestate (from
patient feces) and neuroblastoma cells in contact with the absorbed material,
specifically designed to study the mechanism of action of the synbiotic. For biomarker
analysis, changes at the intestinal level in inflammatory cytokines and neuronal cell
receptor changes will be observed, which can later be related to the in vivo results. The
design will include obtaining sufficient control sample replicates, with colonic
digestate after stabilization, and with colonic digestate after treatment with placebo or
synbiotic.