Modulation of Gut Microbiota through Low-Calorie and Two-Phase Diets in Obese Individuals

Different nutritional regimens have been reported to exert beneficial effects on obesity through the regulation of the composition and function of gut microbiota. In this context, we conducted in obese subjects two dietary interventions consisting of a low-calorie and two-phase (ketogenic plus low-calorie) diet for 8 weeks. Anthropometric and clinical parameters were evaluated at baseline and following the two diets, and gut microbiota composition was assessed by 16S rRNA gene sequencing. A significant reduction was observed for abdominal circumference and insulin levels in the subjects following the two-phase diet. Significant differences in gut microbial composition were observed after treatment compared to the baseline. Both diets induced taxonomic shifts including a decrease in Proteobacteria, which are recognized as dysbiosis markers and enrichment of Verrucomicrobiaceae, which has recently emerged as an effective probiotic. An increase in Bacteroidetes, constituting the so-called good bacteria, was observable only in the two-phase diet. These findings provide evidence that a targeted nutritional regimen and an appropriate use of probiotics can modulate gut microbiota to reach a favorable composition and achieve the balance often compromised by different pathologies and conditions, such as obesity.


Introduction
The gut microbiota (GM) is a complex microbial community including bacteria, fungi, viruses, and parasites that lives in symbiosis within the human gastrointestinal tract. It performs many important physiological functions that allow the host to achieve intestinal homeostasis [1,2]. In fact, GM is involved in several biological processes such as nutrient extraction, metabolism, immunity as well as biosynthesis of bioactive molecules such as vitamins, folate, riboflavin, biotin, amino acids, and lipids [3][4][5]. Additionally, GM exerts structural and protective functions to strengthen the intestinal epithelium of the host and protect him from pathogens [6]. Gut microbiota varies taxonomically and functionally along the gastrointestinal tract segments and undergoes significant intraindividual variations in composition over infant transition, weaning period, and age [7][8][9]. An increase in the gut community diversity and abundance has been observed throughout life. From three years to adulthood, the predominant phyla were Firmicutes, Bacteroidetes, and Actinobacteria; meanwhile, after the age of 70, a general decrease in Firmicutes and an increase in Bacteroidetes and Verrucomicrobia abundances have been observed [10,11].
Differences in the gut microbiota composition have also been observed among individuals. The origin of this inter-individual plasticity lies in the interplay among GM, dietary and cultural habits, host genetics, and pathological conditions as well as antibiotics use [12][13][14][15]. Many studies are proving that diet represents the main modulator of GM composition in the short and the long term by both directly introducing food-derived adopted, in which foods such as rice, whole grain bread, fruit, and legumes were gradually introduced to integrate the Carbolight products. In neither of the two diets did we vary the foods to the subjects, but in both, we adopted a balanced nutritional plan that reduced the caloric intake by preserving components that provide adequate amounts of carbohydrates, lipids, proteins, minerals, and vitamins. The daily meal plan of the two diets is schematized in Table S1.
The Ethical Committee of University of Calabria approved the study, and all subjects gave their written informed consent.

Anthropometric and Clinical Measurements
Anthropometric measurements including body weight (kg), height (cm), abdominal circumference (cm), fat mass, and muscle mass were collected at baseline and after diets. Body Mass Index (BMI) was calculated as the ratio between weight and squared height (kg/m 2 ). We adopted the classifications in use by the World Health Organization (WHO): underweight-BMI under 18.5 kg/m 2 , normal weight-BMI greater than or equal to 18.5 to 24.9 kg/m 2 , overweight-BMI greater than or equal to 25 to 29.9 kg/m 2 , obesity-BMI greater than or equal to 30 kg/m 2 .
Venous blood sample were drawn using a vacutainer, and clinical measurements such as glycemia, cholesterol, total triglycerides, insulin, cortisol, and glycated hemoglobin were analyzed.

Fecal Sample Collection and Coproculture Analysis
Fecal samples were collected in sterile plastic cups at the beginning of the study (baseline) and after the 8 weeks of the diet plan. They were inoculated onto selective and differential growth media: Salmonella Shigella (SS) agar for the isolation of Salmonella spp and some strains of Shigella spp, McConkey agar for the detection of Enterobacteriaceae, and Man Rogosa Sharpe (MRS) agar for the detection of Lactobacillus. To generate an anaerobic environment, a BD GasPak EZ system was used.

Microbial DNA Extraction
Microbial DNA was extracted from feces by a PureLink Microbiome DNA Purification Kit (ThermoFisher Scientifics) according to the manufacturer's recommendations. Briefly, 0.2 g of feces was resuspended by vortexing in 600 µL of S1-Lysis Buffer and, subsequently, in 100 mL of S2-Lysis Enhancer. Samples were incubated at 65 • C for 10 min, homogenized by bead beating on vortex for 10 min, and centrifuged at 14,000× g for 5 min. Then, 400 µL of the supernatants was transferred to a new microcentrifuge tube in the presence of 250 µL of S3-Cleanup Buffer and centrifuged at 14,000× g for 2 min, and 500 µL of the isolated supernatants was vortexed in 900 µL of S4-Binding Buffer. Afterwards, 700 µL of samples was loaded onto a spin column-tube and centrifuged at 14,000× g for 1 min. Then, 500 µL of S5-Wash Buffer was added to each sample, and columns were centrifuged at 14,000× g for 1 min. Microbial DNA samples were eluted by a centrifugation at 14,000× g for 1 min in 100 µL of S6-Elution Buffer. The purity and concentration of the DNA obtained were determined through 260/280 nm absorbance measures using the NanoDrop spectrophotometer.

Microbiome Analysis by Next-Generation Sequencing
The variable V3-V4 region of the bacterial 16S rRNA gene (16S ribosomal ribonucleic acid) was sequenced by the company BMR Genomics of Padua through the MiSeq platform (Illumina).

Data Processing and Analysis
The raw data set reads of the full processing of amplicons (fastaq files) were imported using QIIME 2.0 tools version 2021.4.0. Raw reads were pre-processed using Cutadapt. Paired-end reads were demultiplexed and featured tables were constructed by using the Divisive Amplicon Denoising Algorithm (DADA2). Taxonomic assignment was obtained using trained sequences (Operational Taxonomic Units, OTUs at 99%) from the GreenGenes database version 13-8 by the q2-feature-classifier QIIME 2 plugin. To visualize microbiota composition, stacked bar plots were constructed with ggplot2 R-package.
Alpha diversity was assessed using the alpha_rarefaction.py script in QIIME to determine the Shannon index. Alpha diversities were compared using the Wilcoxon paired test. Beta-diversity was calculated in R-vegan package (2.6.0) using the Bray-Curtis index. Statistical significance of beta diversity was determined through the permutational multivariate analysis of variance (PERMANOVA).
To analyze the different OTUs, we used the "edgeR" package in R for the empirical analysis of differential gene expression (DGE). This package uses the relative log expression (RLE) as the default normalization method and assumes a negative binomial distribution model for the counts. The zeros present in count data are modeled using point mass at zero, while remaining log-transformed counts follow a normal distribution.
Statistical analyses were performed using SPSS 20.0 statistical software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) and Student's t-test were adopted. A p value ≤ 0.05 has been considered statistically significant.

Characteristics of the Study Participants
The anthropometric and clinical characteristics of the study participants at baseline and after the two diets are presented in Tables 1 and S2. No significant variations in the anthropometric values were observed after 8 weeks of the low-calorie diet, although there is a tendency for all the parameters to decrease after the diet. A statistically significant decrease in the abdominal circumference was found following the two-phase diet (p-value = 0.041). Additionally, a trend toward a reduction was observed for all other parameters. As for the clinical parameters, in individuals following the low-calorie diet, no changes were observed in the values of glycemia, cholesterol, total triglycerides, insulin, cortisol, and glycated hemoglobin. We found similar results in individuals consuming the twophase diet with the only exception being that insulin levels decreased significantly after the diet (p-value = 0.040; Tables 2 and S3).

Coproculture Analysis
Coproculture analysis showed the prevalence in the growth media of Lactobacilli and Bifidobacteria, although the presence of Bacteroides and Clostridia has also been detected. It is interesting to observe that in some subjects, Lactobacilli appear long and filamentous, while in others, it was more compact. We also noted the detection of Akkermansia municiphila in some fecal samples.

OTU Analysis and Microbiota Species Diversity
The total number of Operational Taxonomic Units (OTUs) was equal to 1645 with an average of 104 ± 40 for each sample (ranging from 50 to 188) in the baseline group and of 97 ± 36 (ranging from 48 to 166) in the low-calorie diet. Regarding the two-phase diet, the baseline group was characterized by 134 ± 37 OTUs for each sample (ranging from 80 to 225); meanwhile, the diet group was by 129 ± 47 OTUs (ranging from 54 to 220). As shown in Figure 1, the rarefaction curves of each sample tend to plateau as the sequencing depth increases, demonstrating that the sample sequencing in both low-calorie ( Figure 1A) and two-phase ( Figure 1B) diets is adequate to capture the entire microbial community, thus guaranteeing the reliability of our research.
The analysis of the relative abundance of OTUs for the two dietary regimens revealed 120 and 122 significantly different OTUs after the low-calorie diet and the twophase diet (p < 0.05), respectively, and bacteria were distributed among five phyla, namely Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Particularly, Proteobacteria and Verrucomicrobia phyla showed a marked decrease and an increase, respectively, after the low-calorie diet, meanwhile the other phyla have shown greater variability after the two-phase diet. The list and the relative abundance of the significant OTUs after the low-calorie diet and the two-phase diet are reported in Tables 3 and 4, respectively.  The analysis of the relative abundance of OTUs for the two dietary regimens revealed 120 and 122 significantly different OTUs after the low-calorie diet and the two-phase die (p < 0.05), respectively, and bacteria were distributed among five phyla, namely Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Particularly Proteobacteria and Verrucomicrobia phyla showed a marked decrease and an increase respectively, after the low-calorie diet, meanwhile the other phyla have shown greater variability after the two-phase diet. The list and the relative abundance of the significan OTUs after the low-calorie diet and the two-phase diet are reported in Table 3 and Table  4, respectively.           Alpha diversity, which reflects the species diversity of the community, was assessed using the Shannon index. Although the index of the low-calorie diet group was higher and that of the two-phase diet group was lower than that of the baseline groups (Figure 2), the differences were not statistically significant (p = 0.83 and p = 0.55, respectively). Alpha diversity, which reflects the species diversity of the community, was assessed using the Shannon index. Although the index of the low-calorie diet group was higher and that of the two-phase diet group was lower than that of the baseline groups ( Figure  2), the differences were not statistically significant (p = 0.83 and p = 0.55, respectively). No significant results in low-calorie ( Figure 3A) and two-phase ( Figure 3B) diets were also observed for beta diversity assessed using the Bray-Curtis index to evaluate differences in species diversity among samples (p-value = 1).

Figure 2.
Alpha diversity evaluated by Shannon index at baseline and after the low-calorie and two-phase diets.
No significant results in low-calorie ( Figure 3A) and two-phase ( Figure 3B) diets were also observed for beta diversity assessed using the Bray-Curtis index to evaluate differences in species diversity among samples (p-value = 1). Alpha diversity, which reflects the species diversity of the community, was assessed using the Shannon index. Although the index of the low-calorie diet group was higher and that of the two-phase diet group was lower than that of the baseline groups ( Figure  2), the differences were not statistically significant (p = 0.83 and p = 0.55, respectively). No significant results in low-calorie ( Figure 3A) and two-phase ( Figure 3B) diets were also observed for beta diversity assessed using the Bray-Curtis index to evaluate differences in species diversity among samples (p-value = 1). Figure 3. Principal Coordinate Analysis of Bray-Curtis distance for beta-diversity evaluation between baseline (blue samples) and low-calorie (on the left) and two-phase (on the right) diets (violet sample). PC1 and PC2 represent the top two principal coordinates that captured most of the diversity.

Discussion
The diet greatly influences the composition, diversity, and functional activity of gut microbiota, significantly affecting human health [15]. Different factors determine perturbations that induce the onset of dysbiosis phenomena, which is characterized mainly by a lowering of microbial diversity and an alteration in the symbiotic relationship with the guest [36]. Dysbiosis in the microbiota appears strongly connected to numerous chronic pathologies from metabolic, inflammatory, neurological, cardiovascular, and respiratory disorders [37]. Since the well-being of the intestinal microbiota generally reflects that of its host, numerous therapeutic interventions are aimed at improving dysbiosis conditions and, therefore, pathological conditions, including the use of probiotics. Obesity is a complex, multifactorial disease due to various factors including the host genetic background, decreased physical activity, and excess food intake [38,39]. Changes in bacterial abundance of more than 1.5-fold ratio induced by the diet with respect to the baseline were also considered relevant. We found that the low-calorie diet induces, with respect to the baseline, an enrichment, at Phylum level, in Verrucomicrobia (3.9-fold) and a decrease in Proteobacteria (6.1-fold) ( Figure 4A). At the level of Family, we observed that the diet induces, with respect to the baseline, enrichment in Lactobacillaceae (1.5-fold), Turicibacteraceae (1.8-fold), and Verrucomicrobiaceae (3.9-fold), and a reduction in Enterobacteriaceae (6.4-fold), and Prevotellaceae (2.8-fold) ( Figure 4B).

Discussion
The diet greatly influences the composition, diversity, and functional activity of gut microbiota, significantly affecting human health [15]. Different factors determine perturbations that induce the onset of dysbiosis phenomena, which is characterized mainly by a lowering of microbial diversity and an alteration in the symbiotic relationship with the guest [36]. Dysbiosis in the microbiota appears strongly connected to numerous chronic pathologies from metabolic, inflammatory, neurological, cardiovascular, and respiratory disorders [37]. Since the well-being of the intestinal microbiota generally reflects that of its host, numerous therapeutic interventions are aimed at improving dysbiosis conditions and, therefore, pathological conditions, including the use of probiotics. Obesity is a complex, multifactorial disease due to various factors including the host genetic background, decreased physical activity, and excess food intake [38,39]. A series of microbiota markers associated with this pathology have been identified. Recently, research efforts have focused on identifying bacterial taxa involved in the development of obesity.
In this study, we report changes in the composition of fecal bacteria of obese individuals fed with two different dietary regimens: an 8-week low-calorie diet and a two-phase diet in which the initial phase of 4 weeks consisted of a ketogenic diet and the second 4 weeks consisted of a low-calorie diet. Furthermore, in the two-phase dietary intervention, low in sugar, source of protein, and rich in fiber foods were given along with a multivitamin to make up for the lack of fruit consumption in the ketogenic diet. A probiotic containing Lactobacillus and Bifidobacteria was also administered during the two dietary regimens.
The low-calorie diet is commonly considered optimal for managing obesity for its versatility and flexibility and may be helpful in restoring the gut microbiome dysbiosis in obese patients.
The two-phase diet we adopted combines the well-known benefits of a ketogenic diet on weight loss with the previously described advantages of the low-calorie-diet to prevent the outbreak of some negative effects correlated to a long-time ketogenic diet, such as increased risk of kidney stones, hypoproteinemia, and osteoporosis, and increased blood levels of uric acid [40]. Two-phase dietary approaches have already been described in the literature, although differences in terms of duration of each phase, caloric intake and macroand micro-nutritional supplementation make their generalization difficult [41][42][43]. In this context, we also opted for the administration of Carbolight Products from the LightFlow Company, which are poor in carbohydrates and relatively rich in proteins of vegetable origin, to enrich the nutritional regimen with fibers [44]. It is interesting to note that the two-phase nutritional approach we adopted, compared with the low-calorie diet, was more effective in inducing a decrease in abdominal circumference and in insulin levels.
The gut microbiota composition of healthy non-obese individuals consists, in order of relative abundance, of Bacteroidetes (73%), Firmicutes (22%), Proteobacteria (2%) and Actinobacteria (1.8%) [45]. In our study, we found that the obese subject constituting the baseline group exhibited an increased abundance of Firmicutes (70%) at the expense of Bacteroidetes (4%), further reinforcing evidence already reported in the literature that consider the high ratio of Firmicutes: Bacteroidetes as a hallmark of obesity [39,46,47]. It has been proposed that Firmicutes take out energy from foods more effectively than Bacteroidetes, thus supporting the efficient absorption of calories with subsequent weight gain. In line with Turnbaugh et al., besides the above two phyla, we observed in the same persons high levels of Actinobacteria and Proteobacteria [48]. Furthermore, as stated by Clarke et al., obese participants in our study contained a lower proportion of Verrucomicrobia [49].
In addition, the abundance of gut microbiota in individuals subjected to the two nutritional regimens was different from that of the same individuals before starting the diet.
Interestingly, after the two nutritional regimens, the amount of Firmicutes remains globally unchanged, although we observed variations relating to specific families, according to the type of diet that are unrepresentative in terms of the percentage of the entire community. Still, an increase in Bacteroidetes occurred after the two-phase diet, which could be explained considering the high uptake of proteins in the first phase followed by an increase administration in soluble fiber intake in the second. Therefore, it is plausible to hypothesize that the two-phase diet is more efficient than the low-calorie diet in promoting the abundance of members of Bacteroidetes, so-called good bacteria because they produce favorable metabolites, such as SCFAs. The data obtained in our study demonstrated that both nutritional regimens decrease the abundance of Proteobacteria but do not affect that of Actinobacteria. This result seems very interesting, since an increased prevalence of Proteobacteria in the gut microbiome is a potential diagnostic signature of dysbiosis and risk of disease [50]. Proteobacteria is the phylum most conditioned by the Western diet rich in fats, sugars, and animal proteins, and, simultaneously, it is more linked to the metabolic and inflammatory states of the host [51]. Indeed, in obese subjects, the gut-derived endotoxin lipopolysaccharide (LPS), of which Proteobacteria is a major source, binds to the TLR-CD14-MD-2 complex and activates the Toll-like receptor 4 (TLR4) signaling, resulting in the activation of the expression of IFN inducible genes and pro-inflammatory mediators [52]. What is more, Alexander et al. demonstrated the beneficial effect of the natural fiber inulin in decreasing the abundance of Proteobacteria and in increasing the abundance of some Firmicutes [53]. The decrease in the relative abundance of Proteobacteria we observed following the two diets suggests that the reduced fat uptake associated with the consumption of fiber and probiotics could reduce and/or eliminate the chronic inflammatory state of the body. Furthermore, resistance to commonly used antibiotics, a problem that has been assuming enormous importance from some years, characterizes many members of this phylum. Therefore, the decrease in Proteobacteria, more specifically of the Enterobacteriaceae, which is highlighted as the adoption of a probiotic in association with nutritional regimens of only 8 weeks, can be considered a starting point for the eradication of many infections and their complications.
Since the health benefits exerted by the administration of probiotics in human health have been extensively described, both nutritional treatments were supplemented by Lactobacillus acidophilus and Bifidobacterium lactis Bi-07, representing the most studied bacterial species recommended for dietary use [54]. In vivo studies carried out in different mice models revealed that the administration of these probiotics induces an improvement in insulin sensitivity and lipid profile with the decreased level of total cholesterol, LDL cholesterol, and plasma TG, the reduction of pro-inflammatory genes including IL-6, tumor necrosis factor-a, IL-1b, and IL-17, and the increase in IL-10 [55]. The supplementation of overweight and obese adults with Lactobacilli and Bifidobacteria significantly reduced body weight, BMI, abdominal circumference, and waist-to-height ratio in a free-living overweight/obese population and improves well-being [56]. It has been reported that some Bifidobacterium spp. and Lactobacillus spp. promote the synthesis of conjugated linoleic acid (CLA), which has been shown to modulate body weight by reducing energy intake and improving metabolic rate and lipolysis [57]. Additionally, the administration of the prebiotic 2 -fucosyllactose, the most prevalent human milk oligosaccharide (HMO) present in human breast milk, has been demonstrated to counteract gut permeability and insulin resistance, improve lipid utilization, and decrease de novo lipogenesis, thus reducing the obesityassociated steatosis [58]. It follows that the combined use of pro-and prebiotics, which are directly involved in the reduction of the state of chronic systemic inflammation and in the promotion of lipolysis as well as associated with the two nutritional approaches used in this study for the treatment of obesity, appears particularly effective not just for weight loss but for a global restoration of systemic well-being, acting through the improvement of gut microbiota.
Despite the oral consumption of probiotics, we did not find significant abundance changes for Bifidobacteriaceae, thus confirming similar evidence reported in the literature [59]. An increase in Lactobacillales was observed after the sole low-calorie diet. However, the presence of these bacteria is evident in the coprocultures we carried out.
Furthermore, the significant increase in Verrucomicrobiaceae, more specifically in Akkermansia muciniphila, a mucin-degrading bacterium, in subjects administered with both nutritional regimens is of particular interest. It seems to play a key role in metabolic and gastrointestinal pathologies by mainly improving the functionality of the intestinal barrier [60]. Note that due to its highly promising activities against obesity and diabetes, Akkermansia has drawn intensive interest for research so much that it was recently marketed as a probiotic. Similarly, after the administration of the two-phase diet, the significant increase we observed in the genus Roseburia, a butyrate-producing bacteria, appears of relevance. Some evidence reported that butyrate, by the activation of AMPK, the increasing ATP consumption, and the induction of PGC-1α activity, promotes mitochondrial function and the expression of genes involved in lipolysis and fatty acid β-oxidation [61]. Therefore, the rise in the Roseburia genus seems directly involved in the increase in fat mobilization and the promotion of energy expenditure, suggesting that the assumption of this dietary regimen represents an effective strategy for the control and treatment of obesity. Additionally, the rise of Roseburia abundance has been found to exert protective effects on the development of type 2 diabetes by increasing insulin sensitivity, as well as against all inflammatory pathologies, by inhibiting the synthesis of proinflammatory cytokines and the balance of the immune system [62].

Conclusions
The results we obtained demonstrate that the adoption of specific nutritional interventions associated with the administration of effective probiotics, in just 8 weeks, may modify the structure of the gut microbiota, affecting bacteria whose functions have been demonstrated to be correlated with the health status in humans. Particularly, the increase in the phylum Bacteroidetes, with a shift of the ratio Firmicutes:Bacteroidetes toward values closer to that found in non-obesity conditions, associated with the increase in genera Akkermansia and Roseburia, let us consider the two-phase nutritional approach as the most effective in restoring the balance at the level of the gut microbial community in obesity. It follows that an adequate combination of nutritional intake and probiotics may modify the intestinal microbiota by enhancing those species, genera, and families, which is useful to contrast the dysbiosis and weaken the state of chronic systemic inflammation that characterize different systemic pathologies.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/nu15081841/s1, Table S1: Daily meal plain in the low-calorie and two-phase diets; Table S2: Anthropometric characteristics of the participants before (baseline) and after low-calorie and two-phase diets; Table S3: Clinical characteristics of the participants before (baseline) and after low-calorie and two-phase diets.