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Modulation of Gut-Brain Axis Improves Microbiome, Metabolism, and Mood

Objective: There is a close bidirectional relationship between overweight/obesity and depression, which may be largely modified through the microbiome and the gut-brain axis. Previous research has shown targeted weight loss effects and anti-depressive benefits of diets high in fiber and phytonutrients and low in sugar and processed foods. Thus, our objective was to determine changes in parameters common to both obesity and depression (e.g., microbiome balance, metabolic biomarkers, and psychological mood state) following a coordinated supplementation regimen combining probiotics, prebiotics, and phytonutrients (“phytobiotics”). Methods: Thirty-three (33) healthy subjects participated in a 6-week supplementation trial (Amare “Project b3”) containing a targeted blend of probiotics, prebiotics, and phytobiotics. Microbiome balance was assessed in fecal samples using a novel PCR-based analysis (BiomeTracker) that has previously compared favorably to 16S sequencing. Biomarkers, including blood lipids, glucose, cortisol, and butyrate kinase, were assessed as indicators of effects on cardiovascular, inflammatory, and energy metabolism. Psychological mood state was assessed using the validated Profile of Mood States survey (POMS) to generate scores for Global Mood State and six sub-scales (Depression, Tension, Fatigue, Anger, Confusion, and Vigor). Results: Following supplementation, there was a significant increase in populations of “good” bacteria (+8% Bifidobacterium, +33% Lactobacillus, +62% S. Thermophilus, +90% Akkermansia) as well as bacterial ratios associated with a healthier “obesity-resistant” metabolism (+6% composite score, -11% Firmicutes, +6% Bacteroidetes, -14% F/B ratio). Metabolites associated with stress and glycemic control improved post-supplementation (-11% cortisol; +89% butyrate kinase, -6% glucose), as did body fat (-2%) and blood lipids (-8% total cholesterol, -5% LDL, +3% HDL, -23% triglycerides, -7% TC/HDL). Psychological indices were significantly improved postFunctional Foods in Health and Disease 2020; 10(1): 37-54 Page 38 of 54 supplementation for both positive (+17% Global Mood; +23% Vigor) and negative mood states (38% Depression; -41% Tension; -42% Fatigue; -31% Confusion; -39% Anger). Conclusions: These results demonstrate the close relationship between microbiome balance, systemic metabolism, and psychological parameters – and the utility of targeted supplementation to optimize gut-brain-axis balance for both improved metabolism and enhanced mental wellness.

Thirty-three (33) volunteers, free of prescription medications for depression, anxiety, diabetes, or obesity, signed informed consent documents after the study details were explained. Subjects selfadministered the supplements daily (Project b3; Amare Global, Irvine, CA) for 6-weeks and were contacted weekly to remind them to take their supplement daily ( Table 1). The 6-week duration was selected as more representative of persistent changes in mood state that may result from microbiome modulation and superior neurotransmitter balance, as opposed to short-term changes in emotions that may be more closely linked with stressors of daily living. Unfortunately, 6-weeks is often not a sufficient duration of time to observe significant changes in body weight, but since our main objective was not overt weight loss, but rather to assess obesity-related metabolic traits (e.g., microbiome, cholesterol, cortisol, etc.), the 6-week duration was judged to be sufficient to evaluate changes in these aspects of metabolism.

Microbiome Assessment
Microbiome analysis of fecal samples was carried out using the complete BiomeTracker system (Wasatch Scientific, Murray, UT). Briefly, fecal samples were obtained by nylon swab and placed into a preservative binding buffer to lock the composition of bacteria in place. DNA was then purified by following the recommended procedure and using the provided DNA columns. Reaction mixtures were set up as recommended with the components provided (WS#1-WS#8), and ~20ng of DNA from each sample was added to the reaction mixtures. Samples were processed using the recommended conditions on an ABI 7500 Fast (Applied Biosystems) instrument in duplicate.
Threshold values were input into the normalization/quantification template provided.

Metabolic Assessments
Lipid Profile (including total cholesterol/TC, low-density lipoproteins/LDL, high-density lipoproteins/HDL, TC/HDL ratio) and blood glucose were assessed by fingerstick with all subjects in an overnight fasted condition (Cholestech LDX Analyzer, Abbott/Alere, Charlottesville, VA). Cortisol was measured as free cortisol in first-morning saliva samples (Salimetrics, Carlsbad, CA).
Body fat percentage was assessed by bioelectrical impedance analysis (BIA) in a fasted condition before and after 6-weeks of supplementation (InBody, Seoul, South Korea).

Psychological Mood Assessment
We employed the Profile of Mood States (POMS) questionnaire to measure six primary psychological factors (tension, depression, anger, fatigue, vigor, or confusion) plus the combined global mood state as an indication of subjective well-being. The POMS methodology has been used in ~3,000 studies, and its validity is well established [75]. The POMS profile uses 65 adjective-based intensity scales scored on a 0-4 hedonic scale (e.g., "not at all" to "extremely"). The 65 adjective responses are categorized into the six mood factors (tension, depression, anger, fatigue, vigor, or confusion), tabulated, scored, and analyzed. The output of the POMS questionnaire is an assessment of the positive and negative moods of each subject at baseline and post-supplementation (6 weeks).

Data Management and Analysis
All questionnaires were collected and stored in a central location and transcribed to a central database. Data were identified by subject number and examined for accuracy and completeness. Tabulated data were analyzed with JMP 14.0 (SAS Institute, Cary, NC) using standard parametric paired t-tests, and significance was assessed with a 2-tailed alpha level set at 0.05.

Body Composition
As expected, following the short 6-weeks of dietary supplementation, with no major alterations in dietary intake or physical activity patterns, there was no significant change in average body weight (~176 lbs) or muscle mass (~71 lbs), but there was a surprising and meaningful drop of 2% in total body fat levels from 27% to 25% (p<0.05; Figure 1).

DISCUSSION
The novelty of the current study is its combination of a number of previously-researched natural ingredients to positively and holistically influence the entire gut-brain axis (gut/microbiome; brain/mood; axis/metabolites) in a population of healthy-stressed subjects. Our subjects represent perhaps the largest subset of the American population who routinely complain of symptoms related to high stress, day-time fatigue, night-time restlessness, low mood, suppressed libido, brain fog, lack of enjoyment (anhedonia/burnout), and general malaise. Such individuals are poor candidates for prescription medications intended for severe disease states such as major depressive disorder and generalized anxiety disorders (and their wide range of attendant side effects). As such, there is a pressing need for natural functional food approaches to improve psychological mood stateand the emerging understanding of the gut-brain axis in modulating mental wellness represents a promising non-drug approach to help people feel better. Obesity increases the risk of multiple co-morbidities, including cardiovascular disease, diabetes, hypertension, depression/anxiety, and several cancers, and carries an associated reduction in predicted lifespan. While full obesity reversal is often impractical, even modest, sustained weight loss of ~5% reduces morbidity and mortality risk.
Potential benefits of gut-brain axis modulation for enhancing weight loss and/or improving mood are often confounded by the multi-factorial nature of each condition, as well as by the disparate nature of different probiotic strains (e.g., single or multi-strain formulation); structure of prebiotic fibers (e.g., preferential usage by certain bacterial species); supplementation dose; and duration of intervention, among others. Nevertheless, a number of recent meta-analyses have reported on dozens of clinical trials across thousands of individuals showing an overall positive benefit of microbiome modulation for weight loss [76][77][78][79] and mood improvement [80,81].
The anti-obesity and anti-depressive activity of gut-brain-axis interventions may be associated with their ability to alter both the structure and function of the microbiome (and associated metabolites including neurotransmitters, hormones, cytokines, short-chain fatty acids, etc.); remodeling of energy metabolism; modulation of gene expression related to appetite and thermogenesis; glucose homeostasis; lipid metabolism; and attenuation of HPA axis activity, among others [76]. The brain sends a variety of signals to the gut via efferent vagal and neuroendocrine pathways, including serotonin, dopamine, and GABA that can modify the intestinal environment, including mucus secretion, immune responses, gut motility, and intestinal barrier functioneach of which can influence microbial composition and function [79]. Similarly, the gut sends a variety of signals to the brain via afferent vagal pathways and a wide variety of blood-borne substances (e.g., ~70-95% of the body's serotonin and dopamine are gut-derived) including numerous bioactive molecules involved in appetite and energy balance (e.g., peptide YY, glucagon-like peptide-1, cholecystokinin, and ghrelin) [79].
The two largest phyla making up the gut microbiome in humans are Firmicutes and Bacteroidetes, representing ~90% of all gut microbiota. The relationship between these two large groups, expressed as the Firmicutes/Bacteroidetes (F/B) ratio, has been associated with a number of pathological conditions, including obesity, type 2 diabetes, systemic inflammation, and depression (specifically a greater abundance of Firmicutes and/or a drop in Bacteroidetes, thus an increase in the F/B ratio). A number of studies have shown that obese populations tend to have a significantly higher level of Firmicutes and a significantly lower level of Bacteroidetes compared to normal-weight and lean adults [2, 3, 82-84]. Recent interventional trials have shown that the administration of Lactobacillus and Bifidobacterium species can reestablish the F/B ratio and inflammatory tone, thus potentially affecting both mood disorders and obesity through the common gut-brain axis pathways [85,86].
Highly processed Western-style diets, typically high in fat and sugar and low in fiber and micronutrients, have long been associated with inflammatory diseases, including obesity, diabetes, and depression. Public health recommendations to increase fiber intake, reduce processed food consumption, and increase whole food intake have a positive impact on gut health by stimulating the growth and activity of beneficial gut bacteria [87]. In traditional hunter-gatherer societies, high fiber intakes of 50-150 g/day yield a much more diverse gut microbiota with higher colonization by beneficial bacterial species (e.g., Lactobacillus, Bifidobacterium, and Prevotella) and corresponding metabolite richness with higher SCFA production that are associated with lower levels of systemic inflammation [87].
A recent 6-month trial has shown that treatment with a probiotic supplement (10B CFU B. lactis B420), alone or in combination with a prebiotic fiber (12g Litesse Ultra polydextrose), controlled body fat mass, trunk fat mass, and waist circumference, and reduced energy intake in overweight adults [88,89]. The B420 strain increases the relative abundance of Akkermansia muciniphila in the gut microbiota, a bacterium linked with improved metabolic health and anti-inflammatory properties. The relative change in body fat mass over six months was -3.0% with the probiotic alone, and -4.5% with the synbiotic combination of probiotic + prebiotic, which compares favorably with pharmaceutical interventions, in which 3-5% body weight changes are typically observed for 6-month treatment (e.g., orlistat/Alli/Xenical, liraglutide/Victoza, lorcaserin/Belviq). Reduced energy intake has been observed with supplementation of B420 alone (-300kcal/day) or in combination with polydextrose (-210 kcal/day), possibly related to the observed increase in microbiome-derived short-chain fatty acids [90]. Unfortunately, the 6-month intervention timeframe, while delivering promising benefits for subjects who successfully complete the fullduration of intervention, is often plagued by poor subject compliance (e.g., ~40% drop out rate), possibly due to the close association between obesity and depression.
One shorter study showed that 4-months of supplementation with Lactobacillus gasseri BNR17 significantly reduced visceral adipose tissue and waist circumference in obese adults [90], while another 4-month supplementation with Lactobacillus rhamnosus CGMCC1.3724, combined with probiotics (inulin and oligofructose), reduced both body weight and hunger scores in obese women [12].
The present study, investigating a very short 1.5-months (6-weeks) of supplementation with targeted probiotic strains, matched prebiotic fibers, and a blend of research-validated phytonutrients targeting gut-brain-axis modulation, showed meaningful improvements in both microbiome structure (bacterial abundance) and function (metabolic activity), as well as positive changes in parameters associated with both physical health (body fat percentage, blood lipids/glucose, and cortisol exposure) and mental health (reduced negative mood states and enhanced positive mood states).

CONCLUSIONS
The World Health Organization has identified both metabolic issues (e.g., diabetes/obesity) and psychological issues (e.g., depression/anxiety/stress) as urgent public health epidemics threatening global well-being and increasing global health burden. These results demonstrate the close relationship between microbiome balance and both physical and mental health. Importantly, these results also describe the effectiveness of multi-factorial targeted supplementation to positively influence the gut-brain axis for simultaneously improving mental wellness and physical healthowing in large part to the close bi-directional connections across the gut-brain axis and the long-observed associations between obesity and depression.