Legumes are recognized for their distinctive nutritional profile, rich in plant-based proteins, low-glycemic-index carbohydrates, fiber, B vitamins, minerals, and polyphenols. Due to their protein content and amino acid composition, legumes in combination with grains can effectively replace meat and its derivatives. Despite worldwide nutritional guidelines recommending legumes as the predominant source of dietary protein, the consumption of red meat and meat products remains high and may have negative consequences for public health. Indeed, epidemiological evidence indicates that long-term consumption of increasing amounts of red and processed meats is associated with a higher risk of mortality, cardiovascular diseases, colon cancer, and type 2 diabetes. Furthermore, a recent study has shown that higher intake of red meat and choline is associated with higher concentrations of trimethylamine-N-oxide (TMAO), a gut microbiota byproduct that has been associated with a higher incidence of adverse cardiovascular events. Numerous research studies indicate that the consumption of plant-based foods brings health benefits for humans and supports the recommendation of international guidelines to modify dietary habits towards a diet richer in plant-based products. In addition, epidemiological studies show a possible association between high legume consumption and a decrease in coronary heart disease and colorectal adenoma, while the evidence for a protective role of legumes against cardiovascular diseases is less strong due to heterogeneity in results and/or potential confounding factors. The ability of legumes to reduce cardiometabolic risk factors is also supported by various scientific evidence from clinical trials. These studies demonstrate that legume consumption has a positive effect on lipid profile, glucose metabolism, blood pressure, body weight, oxidative stress, and inflammatory status.

Despite the recognized health benefits of consuming legumes regularly, there is still a limited understanding of the underlying physiological mechanisms that drive these positive effects. An observational study conducted in Italy shed some light on this issue by demonstrating that individuals who closely adhere to the Mediterranean diet, which emphasizes reducing red meat consumption and increasing the intake of fruits, vegetables, and legumes, have gut microbiota characterized by a higher abundance of fiber-degrading bacteria. These individuals also exhibit higher levels of short-chain fatty acids in their feces and lower concentrations of TMAO in their urine. Furthermore, a randomized controlled trial conducted on individuals at risk of cardiovascular diseases due to an unhealthy lifestyle revealed that shifting from a typical Western diet to a more Mediterranean-like pattern led to an increase in the presence of fiber-degrading bacterial species in the gut microbiota. This dietary change also resulted in elevated circulating microbial metabolites associated with improved inflammatory status. While there is still a lack of comprehensive in vivo studies assessing the bioavailability of nutrients from legumes, a few clinical trials have investigated the influence of legumes on the intestinal microbiome. Nevertheless, the available literature indicates that legumes have the ability to influence the human microbiota. However, it is important to note that the specific effects of legumes on the microbiota can vary significantly across different studies, making it difficult to generalize these findings to all types of legumes.

In this framework, the present project will focus on the evaluation of the effect of replacing red meat with pulses (PulD) or a combination of pulses and plant-based meat substitutes (PPD) on the cardiometabolic health of individuals with unhealthy habits and sedentary lifestyles via the modification of intestinal microbial communities. Additionally, it seeks to investigate the effects on health outcomes, with a primary focus on evaluating changes in inflammatory, oxidative, immune, and hormonal status. The study will include the establishment of a 2-month dietary intervention with an isocaloric and isoprotein pulses diet (PulD) and a plant proteins diet (PPD). Coupled with detailed host phenotyping and gut microbiota profiling during and after the intervention, this will allow assessment of the causal effects of a diet rich in plant-based proteins (mainly from pulses) and the gut microbiome in populations at high risk for cardiovascular disease (CVD).

The potential eligibility of subjects to participate in this study will be assessed through pre-recruitment questionnaires. These questionnaires will collect personal and socio-demographic data of volunteers, general health information (including anthropometry, health status, medical history, smoking and alcohol consumption habits), details about individual dietary habits using the Food Frequency Questionnaire (FFQ), information about eating behavior through the Three Factor Eating Questionnaire (TFEQ), and levels of physical activity using the International Physical Activity Questionnaire (IPAQ). Subjects in the PulD group and PPD group will be assigned a personalized diet prepared on the basis of own eating habits as established by 7-day food diary recalls. Energy values and whole macronutrient composition of habitual diets will be kept unchanged during PulD and PPD intervention. However, changes in carbohydrate (dietary fibre vs. starch), dietary fat (saturated vs. mono/polyunsaturated fatty acids), and protein (vegetable vs. animal) composition will be applied as a consequence of replacing meat with pulses (PulD group) or with a mix of pulses and plant-based meat substitutes (PPD group). Control subjects will not change their habitual diet (HabD) during intervention. All subjects will be requested not to change physical activity levels during the 8 weeks intervention period. Compliance will be assessed every 2 weeks with a phone interview in order to evaluate the dietary intake and physical activity during the previous week. At each intervention time-point (baseline, 4 weeks, 8 weeks), for the nutritional check, subjects will complete 7-day food diaries and associated questionnaires on appetite (Visual Analog Scale, VAS) related to the previous week before the nutritional analysis. Additionally, measurements of blood pressure, weight, circumferences (waist and hips), and body composition through bioimpedance testing will be conducted. During the intervention period, subjects will be asked to fill out the International Physical Activity Questionnaire (IPAQ), questionnaires on quality of life (QoL), on depression, anxiety and stress (DASS), the King's Stool Chart (KSC) to evaluate frequency, weight, and consistency of feces, along with the Pittsburgh Sleep Quality Index (PSQI) to evaluate the quality of sleep.

Further analysis of compliance will be conducted based on metabolomics, allowing discrimination of animal/vegetable protein intake. Metabolomes (well known to reflect both diet and microbial metabolism) will also be compared between categories in order to identify protective or risk profiles using both bioinformatics and chemometrics approaches. Metagenomes will be analyzed following Standard Operating Procedures (SOPs) utilized in landmark studies already published. Comparison of predefined groups of individuals will allow identification of microbial genes that have different abundance in the groups. Furthermore, genes will be associated with continuous variables of clinical and nutritional interest (e.g., intake of specific dietary components, insulin sensitivity) by covariance analysis. Concatenated datasets of physiological output data, metagenomic and metabolome profiles from the intervention studies will be used to predict subsets of features by multivariate analysis (PLS-DA) that can classify subjects according to their relative adherence to a PulD or PPD. The profile will be used to probe the microbiome for specific alterations as a function of the interventions.

The sample size needed to detect an effect of PulD and/or PPD on individual TMAO levels is defined based on previous study from Crimarco and colleagues. It was calculated that a sample of 28 participants per group would allow detecting a minimum difference of approximately -1.3 μM (-38%) in TMAO and approximately -0.9 mM (-18%) in cholesterol between each of the 2 test treatments vs. control and between the two test treatments, with a power of 80% and an α-error 0.017 to account for multiple comparisons (T-test).