**4. Discussion**

The grea<sup>t</sup> majority of obese subjects are in the class I obese category, which considerably increases morbidity and public health expenses. In the US Centers for Disease Control and Prevention [30], epidemiological data indicate that approximately 2/3 of obese men and 50% of obese women are in this group. Although the mortality rate within class I obesity is similar to normal weight, the risk of developing T2DM, hypertension, dyslipidemia, metabolic syndrome, obstructive sleep apnea, cancer, and non-alcoholic fatty liver disease is notoriously elevated [31]. Therefore, the evidence calls attention to finding more effective and safe therapies for these patients. In this regard, PENS of dermatome T6 has been proposed as an alternative to pharmacological products and surgical procedures to decrease appetite and weight loss, allowing a better compliance of hypocaloric diets. PENS was initially applied to morbidly obese patients awaiting bariatric surgery, in order to reduce the pre-surgery body weight [10]. Later, we and others extended this technique to patients with overweight and mild-to-moderate obesity. PENS or the hypocaloric diet induced by themselves a significant but slight reduction in body weight (3.6 and 5.6 kg, respectively). However, the combination of both PENS and a diet revealed a mean of weight loss over 10–14 kg, with maintained effects for at least one year after therapy [8,9,32]. Now, in class I obese individuals, we show that ten weeks of PENS-Diet displayed a similar reduction in body weight (11.1 kg) and an improvement of blood pressure and the glycemic and lipid profiles. These effects could be justified by the caloric restriction and by the neurostimulation of the gastric wall and promotion of early satiety [9]. However, an alteration in gu<sup>t</sup> microbiota could have also played a key role. In this regard, the etiopathogenetic of obesity is multifactorial and data from literature sugges<sup>t</sup> a contribution of intestinal dysbiosis in obesity development [33]. Our obese subjects unveiled a microbiota alteration, with a reduction of muconutritive and immunomodulatory bacteria such as *Akkermansia muciniphila, Faecalibacterium sp,* and *Bifidobacterium spp*. In consonance, the Firmicutes/Bacteroidetes ratio was considerably elevated. Importantly, these variations in microbiota may have influenced on obesity development [34,35]. However, PENS-Diet tended to enrich *Prevotella spp* and consequently, the balance of Firmicutes/Bacteroidetes was slightly lessened. The amelioration of this ratio has been frequently linked with an improvement of weight loss and intestinal inflammation and permeabilization [36], and although a precise taxonomic characterization of the bacteria would have been discerned between species, *Prevotella spp* can lead to beneficial effects on mucin regulation, glucose metabolism, and hepatic glycogen storage [37]. Undoubtedly, several lifestyle factors (e.g., smoking, sedentarism, stress, circadian rhythms, personal hygiene, ovarian cycle) may have also altered intestinal microbiota. In this

sense, a 20% rate of menopause (without hormonal supplementation) was described in both groups of patients.

In this line, the addition of a multistrain probiotic to the PENS-Diet could have further enhanced these favorable effects. Adomelle® is formulated by *Lactobacillus plantarum LP115, Bifidobacterium brevis B3,* and *Lactobacillus acidophilus LA14*. Supplementation of *L. plantarum* in humans and mice induced a body fat decrease and muscle mass increase, enhancing energy harvest and anti-fatigue effects [38,39]. In obese mice, *L. plantarum* also reduced insulin resistance, plasma triglycerides and proinflammatory factors [40]. *L. acidophilus*, when combined with phenolic compounds or other probiotics, induced weight loss in overweight adults [41]. Moreover, it promoted a significant improvement of glucose homeostasis and cholesterol metabolism in obese mice, by gene downregulation of glucose transporters, cholesterol precursors, and immune factors [42]. Finally, administration of *B. breve-B3* ameliorated the body fat in obese individuals and rats due to its ability to conjugate linoleic acid from diets [43,44]. In our study, the addition of these probiotics to the PENS-Diet further improved the body weight, plasma A1C, triglycerides, and HDL-cholesterol. Moreover, a clear tendency was found for fasting glucose, TC and LDL-cholesterol, which could reach statistical significance in a larger group of patients. Previous randomized controlled trials showed that administration of probiotics alone slightly reduced the body weight and BMI (−0.55 kg and −0.3 kg/m2, respectively) in parallel with fasting glucose (−0.35 mg/dL) and lipids (total cholesterol, −0.43 mg/dL and LDL-cholesterol, −0.41 mg/dL) [19]. Particularly, single probiotics such as *Lactobacillus gasseri, Bifidobacterium animalis*, and *Pediococcus pentosaceus* achieved higher benefits than multiple probiotics (i.e., combinations of *Bifidobacterium spp., Lactobacillus spp.*, and/or *Lactococcus spp.*) Thus, a combination of diverse anti-obesity strategies could lead to better outcomes against obesity. In particular, probiotics may induce summative effects when administered together with PENS and a hypocaloric diet. In fact, this triple intervention exhibited a synergic action on reduction of body weight and cardiovascular risk factors. Likely, the promotion of early satiety induced by PENS could be helped by the ingestion of a salutary diet of low caloric intake, and by the balance of healthy microbiota. In this sense, the addition of probiotics further enhanced *Prevotella spp*, *Bifidobacterium spp*, and *Akkermansia muciniphila*, promoting a more muconutritive and immunomodulatory microbiota. *Bifidobacterium spp* have been demonstrated to be positive for the gastrointestinal barrier function and for immunoregulation [45]. By increasing the abundance of *Bifidobacterium spp* (i.e., with prebiotic oligofructose), gu<sup>t</sup> permeability was reduced in obese mice, in correlation with a decrease in LPS and inflammatory markers [46]. In these animals, when combined with *L. acidophilus*, *Bifidobacterium spp* also enriched microbiota composition [47]. Moreover, *Bifidobacterium spp* produced lactate, which is transformed into butyrate by butyrate-producing bacteria in the intestine (i.e., *Prevotella ruminicola*) [48]. These SCFAs play a crucial role in cardiovascular homeostasis and lipid and glucose metabolism by supplying energy and producing glucagon-like peptide-1, peptide YY, and leptin [49]. Furthermore, butyrate induces mucin synthesis and protects intestine integrity by increasing tight junction assembly. In addition, *A. muciniphila* can also regulate gu<sup>t</sup> permeability [50]. Its abundance was inversely correlated with adipose tissue inflammation and insulin resistance in mice and humans [51,52]. In obese hyperlipidemic mice, *A. muciniphila* also improved metabolic endotoxemia, vascular inflammation, and atherosclerotic lesions [53]. Altogether, the enrichment of the muconutritive and immunomodulatory bacteria observed in our patients could also participate in the improvement of their plasma metabolic and cardiovascular factors, and in the attenuation of their body weight.

#### *Limitations of the Study*

In this pilot study, we evaluated the addition of probiotics to an anti-obesity strategy with the PENS-Diet. Probiotics administration further reduced body weight, but its effect on the waist circumference was not evaluated. The abdominal obesity might be affected by microbiota changes, and its quantification would also add key information on the risk of cardiovascular disease. In addition, since multiple factors could influence the effects of probiotics, our data should be taken with care. Unknown comorbidities or habits may alter bacterial distribution and probiotics action. Furthermore, di fferent physical levels and skills could have a ffected the practice of daily exercise and subsequent weight loss in patients. A personalized control of these practices by an exercise specialist might also improve the adherence and outcomes of this work. Finally, for a group of subjects who follow only a diet regime, PENS intervention or probiotics intake could o ffer interesting and comparative data about potential changes in microbiota distribution. Therefore, all these variables will be considered in a future study. In this line, the estimation of the sample size per group of obese patients will be at least thirty (two-side significance level; α = 0.05 and power 1 – β = 0.8), following the published formula [54]. According to previous works, the di fference of BMI among treatments would be at least 3.0 ± 4.87 kg/m<sup>2</sup> [55,56].
