Effects of Gallotannins on the Gut Microbiome and How it Affects Inflammatory Markers Among Obese Individuals.

Introduction Background Obesity is a serious, common, and expensive chronic disease, and according to a report by the Center for Disease Control and Prevention, more than 2 in 5 adults in the United States (US) live with obesity. The issue of obesity in the US is an increasing trend, as reports have shown that the prevalence of obesity among US adults aged 20 and above from 2017 to 2020 was 41.9%, rising from 30.5% in 2000. Concurrently, the prevalence of severe obesity also increased from 4.7% to 9.2%. Meaning over 100 million adults in the US are living with obesity, while more than 22 million people are living with severe obesity.

The main cause of obesity is abnormal and excessive lipid buildup in many organs and tissues, especially in the visceral or abdominal area, including white adipose tissue (WAT), which is referred to as abdominal obesity. Numerous important non-communicable diseases, such as diabetes mellitus, metabolic dysfunction-associated fatty liver disease (MAFLD), cardiovascular diseases, hypertension, and renal disorders, are significantly influenced by this type of obesity. One of the main pathogenic mechanisms in obesity has been proposed to be impaired adipogenesis, or the process of adipocyte differentiation.

Increased oxidative stress and inflammation are further characteristics of unhealthy WAT remodeling. Histological crown-like structures (CLSs) are formed when enlarged adipocytes release chemokines that attract immune cells, mainly macrophages, to encircle the adipocytes. By triggering pro-inflammatory regulators like nuclear factor-kappa B (NF-κB), which in turn boosts the production of inflammatory genes and overpowers anti-inflammatory regulators like interleukin 10 (IL10), this mechanism intensifies the hyperinflammatory state. Furthermore, reactive oxygen species (ROS)-induced oxidative stress can override antioxidant regulation, especially the nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2)-mediated expression of antioxidant genes.

Metabolic problems, such as insulin resistance, dyslipidemia, and disturbed adipogenesis, are caused by both oxidative stress and inflammation. Therefore, methods to avoid adipocyte hypertrophy, improve adipogenesis to create normal adipocytes, reverse harmful WAT remodeling, and limit oxidative stress and inflammation may be effective ways to fight obesity and metabolic syndrome.

The use of dietary intervention for obesity prevention and management has been used for many years and has been described by researchers as foundational. Consuming meals or natural products, including fruits and vegetables, that are low in energy density and high in bioactive chemicals has been suggested as a way to manage obesity and its associated issues. Edible wild fruits and vegetables from different areas are known to contain a variety of bioactive substances, including phenolics, which may have anti-inflammatory, antioxidant, and metabolic-regulating effects. Fruit extracts that contain flavonoids and polyphenols, such as quercetin, catechin, and rutin, have demonstrated positive benefits on markers of metabolic dysfunction. In animal models of obesity, they include decreases in adipocytes and macrophages, lipid buildup, visceral adipose tissue, and markers of inflammation and oxidative stress.

The high concentration of phenolic substances (such as gallic acid, gallotannin, galloyl glycosides, and flavonoids) in mangoes (Mangifera indica L.) suggests that they have strong antioxidant and anti-inflammatory potential, indicating that polyphenols produced from mangoes can aid in the prevention of chronic diseases linked to obesity. Mango consumption decreased obesity and enhanced lipid profiles, glucose tolerance, and inflammatory cytokine expressions in rats given a high-fat diet in preclinical research. The reduction of mitotic clonal growth, a crucial stage in the commencement of adipocyte differentiation, is most likely linked to the inhibition of adipogenesis by mango extracts, according to an in vitro model employing 3T3-L1 preadipocyte cells.

Human clinical investigations have also shown that mango supplementation lowers blood glucose in obese people, individuals with type 2 diabetes, and healthy adults' lipid profiles and antioxidant capacity. In overweight people with hyperglycemia, natural phytochemicals from mango fruits and their metabolites, such as mangiferin, have been demonstrated to raise HDL levels and lower triglyceride and free fatty acid levels. According to these results, polyphenols extracted from mangos have strong anti-inflammatory and anti-lipogenic properties that may help prevent chronic disorders linked to obesity.

Problem statement Globally, obesity has become an epidemic of complicated, multivariate metabolic problems that are causing an increase in the prevalence of chronic conditions like type 2 diabetes, heart disease, and several types of cancer. An altered gut microbial ecology, or dysbiosis, and a persistent low-grade inflammatory state are key components of the pathophysiology of obesity. Reduced populations of beneficial bacteria that produce short-chain fatty acids (SCFAs), a rise in pro-inflammatory taxa, and a decrease in microbial diversity are common characteristics of this dysbiotic condition, which can all contribute to the maintenance of metabolic dysfunction and systemic inflammation. To reduce inflammation and restore microbial balance in obese people, there is increasing interest in creating dietary interventions.

Polyphenols are well known for their anti-inflammatory, antioxidant, and prebiotic-like properties, especially gallotannins (GTs), which are present in mangoes. After being broken down by gut microorganisms, GTs may promote the growth of good bacteria and inhibit the growth of bad ones, however the exact effects may depend on the makeup of each individual's microbiota. Similarly, Lactiplantibacillus pentosus enhances gut mucosal integrity and modulates immune function, though its effects in isolation are often inconsistent.

Lactiplantibacillus pentosus LPG1 (hence referred to as LPG1) is a ferment derived from table olive biofilms that has demonstrated probiotic properties in vitro and in vivo (murine model) investigations. This strain possesses anti-inflammatory and phytase properties, can lower cholesterol levels, inhibits food-borne infections, attaches to Caco-2 cells, and has no hemolytic activity, among other characteristics. Furthermore, clinical investigations have demonstrated its safety in people. In this regard, a recent study discovered that the L. pentosus LPG1 genome lacks antibiotic resistance and virulence genes while containing numerous possible beneficial bacteria, bacteriocin, exopolysaccharides, folate production cluster genes, and digestive enzymes that are capable of breaking down complex carbohydrates such as galactose, glycogen, starch, cellulose, or xylan.

Lactiplantibacillus strains' probiotic capacity has already been studied in human clinical trials. Kotani investigated the ability of L. pentosus b240, originally isolated from fermented tea leaves, could stimulate salivary immunoglobulin A secretion in the elderly. Wang investigated the impact of oral Lactiplantibacillus pentosus Lp-8 ingestion on the makeup of the fecal microbiota. de Vos studied the effects of oral ingestion of several L. pentosus strains on immunological response, whereas Rudzki investigated the effect of oral L. pentosus 299's effect on cognitive skills in depressed patients. Recently, Ahn investigated the effects of an L. pentosus strain isolated from kimchi, on children with allergen-sensitive atopic dermatitis and showed improved symptoms in the participants.

Although it has not been explored empirically in obese people, the possibility of synergistic regulation of metabolites obtained from microorganisms or attenuation of inflammatory indicators is particularly intriguing. By assessing the effects of GT extract, with or without L. pentosus supplementation, on the gut microbiota and inflammation in obese adults in vitro, this pilot study aims to close this gap. The results will guide future approaches in personalized nutrition and obesity management by shedding light on whether polyphenol-probiotic combos can provide more health advantages than single-agent therapy.

Significance of the study Chronic low-grade inflammation and disturbed gut microbial ecology are now understood to be key factors in the development and progression of obesity and related metabolic diseases. The advancement of obesity prevention and treatment efforts depends on the effective management of these interconnected dysfunctions. A popular tropical fruit, mangoes (Mangifera indica L.) are particularly high in gallotannins (GTs), a subclass of hydrolyzable polyphenols that have been shown to have anti-inflammatory, antioxidant, metabolic-regulating, and prebiotic-like properties. However, the biotransformation of GTs into absorbable, health-promoting metabolites by gut microbes is a major factor in their bioactivity. The potential advantages of polyphenols generated from mangoes are not consistently achieved across populations because microbial composition differs among individuals.

Supplementing with the well-characterized probiotic Lactiplantibacillus pentosus presents a viable way to address the heterogeneity in polyphenol metabolism. This probiotic may help break down complex phenolic chemicals like GTs, improve gut barrier integrity, and regulate immunological responses. L. pentosus may work in tandem with GTs to increase the bioavailability of advantageous metabolites and have a good impact on the gut microbial ecology. This could then intensify thermogenic and anti-inflammatory effects, which could show up as better gut health, lower levels of inflammatory biomarkers, and improved metabolic profiles, particularly in obese people.

This discovery has important ramifications for a number of fields. By assessing a combined polyphenol-probiotic approach designed to alter host metabolism through the gut microbiota, nutrition science advances the growing field of functional foods and nutraceuticals. It provides information to support individualized dietary therapies for obesity based on microbial composition and individual metabolic response, which is consistent with the tenets of precision nutrition. From the perspective of public health, the results can help develop food-based, scalable methods to lower the risk of chronic diseases in susceptible groups. Obesity remains a significant risk factor for cardiovascular disease, type 2 diabetes, and some types of cancer. Therefore, finding naturally derived, culturally accepted, and easily accessible dietary interventions that enhance metabolic and inflammatory outcomes can be an economical way to prevent and control disease, particularly in underprivileged settings.

For chronic illness research, the work provides mechanistic insights into the role of dietary polyphenols and probiotics in influencing systemic inflammation and metabolic dysfunction. It could help guide future clinical trials aiming at incorporating microbiome-targeted treatments into standard care models for obesity and other noncommunicable illnesses. The study also has implications for the fruit and agriculture industries, particularly in promoting the functional properties of mangoes and other polyphenol-rich fruits. If successful, this study will increase mangoes' marketability as a health-promoting food, drive demand, and encourage the development of polyphenol-enhanced food products. Furthermore, the findings can inform agricultural practices aimed at increasing bioactive compound content in fruit crops.

Beyond mangoes, this study lays the groundwork for investigating comparable interactions in other polyphenol-rich fruits, such as sumac, widening the study's relevance to diverse cultural and geographical dietary situations. In the field of microbiome research, this study advances our understanding of how dietary bioactive compounds interact with specific microbial taxa to produce functional metabolites that affect host health. It furthers our understanding of microbial diversity, SCFA-producing bacteria, and inflammation-related microbial pathways in obese people. Finally, the study contributes to educational and learning objectives by providing a practical example of translational nutrition research. It combines molecular nutrition, microbiology, and behavioral science, making it an excellent case study for training students and practitioners in interdisciplinary public health and biomedical sciences. It promotes the systems-thinking approach required to solve complex health concerns using integrative, food-based solutions.

Research Questions

To guide this investigation, the study focuses on understanding how GT extract from mango and Lactiplantibacillus pentosus supplementation affect gut microbiota and inflammation in obese individuals through the following questions:

• How does treatment with GTs, with or without L. pentosus, influence the gut microbial composition in adults with obesity?
• What is the impact of GT and L. pentosus supplementation on inflammatory biomarkers in obese individuals?
• Does genetic variation among individuals with obesity account for metabolizing gallotannins.
• Does the combination of GTs and L. pentosus exhibit synergistic effects in modulating specific microbial taxa associated with obesity and inflammation compared to either intervention alone?

Hypothesis

1. It is expected that there is an inverse correlation between body mass index (BMI) and plasma concentration of GTs and metabolites, as well as thermogenic and anti-inflammatory activities.
2. L. pentosus is expected to increase the beneficial effects of gallotannins

Aim and Objectives Main Aim The primary objective of this pilot study is to investigate the effects of a gallotannins (GTs) from mango, with or without supplementation of the probiotic L. pentosus, on gut microbiota composition and inflammatory markers in obese individuals in vitro.

Specific Objectives

1. To evaluate the changes in gut microbiota composition in obese participants following treatment with GT extract, with or without L. pentosus supplementation.
2. To assess the anti-inflammatory potential of GTs and L. pentosus individually and in combination.
3. To identify potential synergistic effects between GTs and L. pentosus by comparing microbial diversity, abundance of SCFA-producing bacteria, and inflammatory marker changes across intervention groups.

Justification Disrupted gut microbial ecology and chronic low-grade inflammation are closely associated with obesity, and both have a role in the development and progression of metabolic disorders. To effectively manage obesity, it is imperative to address these interconnected metabolic abnormalities. Mangoes are a rich source of gallotannins (GTs), which have been demonstrated to have anti-inflammatory, metabolic-regulating, and prebiotic-like qualities. However, the host's capacity to convert them into bioactive derivatives-a capability that varies according to the makeup of the gut microbiota-is crucial to their health advantages.

Lactiplantibacillus pentosus probiotic supplementation presents a strong approach to overcoming this metabolic constraint. By enhancing mucosal barrier function, regulating immunological responses, and maybe aiding in polyphenol metabolism, L. pentosus has shown promise in promoting gut health. Therefore, combining this probiotic with mango that is high in GT may increase systemic exposure to advantageous GT metabolites. As seen in plasma and adipose tissue samples, the combination action of microbial regulation and enhanced metabolite bioavailability may produce more noticeable thermogenic and anti-inflammatory effects, particularly in obese people.

Despite the expected advantages, it is important to recognize that any changes in inflammation or metabolism that are seen after taking L. pentosus supplements might not be solely due to elevated GT metabolite levels. The probiotic itself may have biological effects on its own, such as modifying the host's immune system or directly competing with bacteria that cause inflammation. Therefore, the purpose of this study is to investigate the functional interactions between probiotic supplementation and GT-rich meals in a practical, integrative setting rather than to identify GT-specific pathways.

Considering the growing interest in gut microbiome-targeted therapies and personalized nutrition, this study is both necessary and timely. It will offer fundamental information about the ways in which interactions between polyphenols and probiotics affect important metabolic and inflammatory markers in the groups most at risk for chronic illness. In the end, the results might influence dietary recommendations and supplement plans designed to enhance health outcomes in obese people using microbiome-aware techniques.

Methodology Study Design This study is a human clinical trial. The study design is a randomized, double-blinded, placebo-controlled. Participants will be asked to provide human samples such as saliva, blood, and stool. The study has been approved by the Institutional Review Boards (IRB) of Prairie View A\&M University (PVAMU) and Texas A\&M University and will be registered at www.Clinicaltrials.gov.

Study population A total of 20 volunteers will be recruited for this study, with 15 people living with obesity as part of the intervention group, while 5 lean and healthy individuals will form part of the control group. For the intervention group, the 15 volunteers will be randomized into 3 groups, with n=5 participants per group. Recruitment will be done by sharing recruitment flyers at key areas such as grocery shops, supermarkets, churches, community centers, and other event centers in Waller County and other surrounding counties. Participants will be asked screening questions before recruiting them into the study.

Instruments A range of instruments will be used to ensure accurate data collection and analysis. Buccal cells will be collected with Isohelix SK-1 DNA buccal swabs, and biological samples (plasma, urine, and stool) will be stored at -80˚C under appropriate conditions. Inflammatory and metabolic biomarkers will be measured using a multiplex bead-based assay with analysis on the Luminex L200 system and xPONENT software. Genotyping will be conducted with TaqMan assays to detect selected SNP variants. Microbiome profiling will be performed by DNA extraction with the Qiagen QIAamp DNA Stool Mini Kit, sequencing on Illumina platforms (MiSeq or NovaSeq), and analysis with QIIME2, SILVA/Greengenes databases, LEfSe, and PICRUSt2. Chemical analysis of gallotannin metabolites will be carried out using HPLC-ESI-MSn on a Thermo Finnigan Altis triple quadrupole and HPLC-MS high-resolution ion trap. Finally, ex vivo immune assays will involve stimulating peripheral blood mononuclear cells with LPS, and cytokine levels will be quantified with the xMAP Multiplex Assay.

Sample collection Plasma samples will be collected at baseline, week 1 after an overnight fast. Aliquots of plasma and will be prepared immediately after they are obtained and stored at -80˚C until analysis. Stool will be collected as available and immediately anaerobically treated. Participants will collect buccal cell samples at baseline using Isohelix SK-1 DNA buccal swabs and Isohelix dried-capsules.

Sample analysis Inflammatory and metabolic markers Blood plasma samples will be analyzed to assess levels of various inflammatory biomarkers. These will include inflammatory cytokines such as interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1); and the cardiovascular marker C-reactive protein (CRP). All measurements will be performed using a multiplex bead-based assay (Millipore, Billerica, MA).

Genotyping DNA and genotyped specific loci will be extracted using TaqMan genotyping assays to provide bi-allelic scoring of single nucleotide polymorphisms for the following genes: fat mass and obesity-associated [FTO (rs9939609)] and fatty acid desaturase 1 [FADS1 (rs174546)].

Microbiome assay Microbial DNA will be extracted from stool samples using the Qiagen QIAamp DNA Stool Mini Kit. 16S rRNA gene sequencing (V3-V4 region) will be conducted using the Illumina MiSeq platform. For deeper analysis, shotgun metagenomic sequencing will be considered using the Illumina NovaSeq platform. Sequence data will be processed using Quantitative Insights Into Microbial Ecology (QIIME)2. Taxonomic classification will utilize the SILVA or Greengenes databases.

Diversity indices:

• Alpha diversity will be calculated using Shannon and Chao1 indices.
• Beta diversity will be assessed using Bray-Curtis and UniFrac distances.

Differential abundance analysis will be performed using LEfSe, and predicted microbial functions will be inferred via Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) using R studio.

Chemical analysis GT metabolites will be isolated by first centrifuging to remove biomass and then extracted with ethyl acetate using ethyl gallate as an internal standard. MS parameters will be optimized using standards of the predicted metabolites when available for the identification of parent/daughter fragments with the highest intensity. Target metabolites will be quantified using full scan, SRM (single ion), and MRM (multiple reaction monitoring) on an HPLC-ESI-MSn using a Thermo Finnigan Thermo Finnigan Altis triple quadrupole using internal standards. Untargeted metabolites or low concentration metabolites will be identified/quantified in the Texas A\&M metabolomics core laboratory using HPLC-MS high-resolution ion trap

Statistical Analysis All statistical testing will be performed at the 0.05 level and will be two-sided. Data will be examined for normality, and all nonnormal variables will be log10 transformed. Analysis will be done on the basis of intention to treat (ITT). ANOVA technique for crossover designs and, as necessary, supplemented by equivalent Wilcoxon rank test. Absolute change from baseline will be tested, and secondary analyses will be performed based on the percent change from baseline. The primary outcome is the change in inflammation biomarkers in plasma, since this outcome applies to lean and obese individuals. All other outcomes investigated will be treated as secondary outcomes. Further secondary analysis of treatment effects will use the Analysis of Covariance based on patient baseline characteristics. We will assign one-half of the patients at random to each of the two treatment sequences to maintain a balanced experimental design. Block-randomization will be as mixed block numbers to protect randomization and balance in treatment assignment, eliminate possible biases, and preserve blinding. Each block will consist of an individual.

Ethical Considerations Ethical approval has been sought and approved from the institutional review board (IRB) of Texas A\&M University and Prairie A\&M University before proceeding with recruitment and data collection. This is important since this study involves human subject and it is important to protect their rights and also maintain total confidentiality. To maintain confidentiality, a consent form will be signed by participants who qualify to participate in the study. The consent form will contain the overview of the study, participant information that will be collected, and biological samples to be collected.