Measuring the Impact of Dietary Supplementation With New Baking Product PreBIOil on Gut Microbiota and Cholesterol Metabolism

Study hypothesis: Diet integrated with food prepared with olive, buckwheat, peas and chestnut flour as in PreBIOil product combination can modify the gut microbiota and the cholesterol metabolism.

Primary objectives of the study are to assess whether the test product to be able to change:

1. fecal microbiota profile;
2. Plasma cholesterol LDL, total, total LDL and HDL ratio;
3. plasma triglycerides;
4. Apolipoprotein ApoA-I, ApoB, and Lp.

Secondary objectives of the study are:

1. anthropometric indices;
2. secondary metabolites of polyphenols in human biofluids;
3. mass spectrometry plasma and urine metabolite profile;
4. blood glucose and fasting insulin levels; 5,6) C-reactive protein (PRC or hsPRC);

7. urinary isoprostane F2; 8) oxidized LDL in plasma.

Study Design: placebo-controlled, randomized, double-blind parallel trial.

Inclusion criteria: Aged 30-65 years; BMI 20-29,9 kg/m^2. Total Cholesterol 180-240 mg/dl

Exclusion criteria: Fasting blood glucose >150 mg/dl; triglycerides >500 mg/dl; uncontrolled hypertension (blood pressure [BP] >160/100 mm Hg under antihypertensive therapy); any long term medical therapy; food intolerances; alcohol intake >5 drinks per day or use of narcotic substances; use of dietary supplements, pro or pre- biotics; special diet; pregnancy, tobacco smoking.

Methodology: Determination of eligibility: for each volunteer aged between 30 and 65 years, will undergo to clinical and biochemistry evaluation. Clinical visit, clinical tests, and blood drawing will be performed after an overnight fasting at visit T-1 at the Casa di Cura Eremo di Arco (TN). In this T-1 visit the eligibility will be established and the participants will be randomized to receive supplementation with either PreBIOil biscuit (90g/day) or Control for 8 weeks in a double-blind manner. Clinical tests, blood drawing, and stool and urine collection will be performed during visits at the beginning and end of each treatment period (T0 and T1). A 4 day-food diary record will be collected before visits T0, at the beginning of T0 and before T1.

Efficacy Assessments Arterial BP; BMI; ratio of waist to hip circumference; food questionnaires; blood sample analysis (total cholesterol, triglycerides, HDL and LDL cholesterol, oxidized LDL, serum glucose and insulin, C-RP, Apolipoproteins, Isoprostane; urinary and plasma metabolite profiling; fecal microbiota analysis.

Safety assessments. Adverse events registration.

Statistical analyses. The differences between the two group will be evaluated with univariate and multivariate statistical methods. The medium before and after the intervention will be compared with the General Linear Model (ANOVA) for repeated measures or for paired data. Simple and multiple linear regressions will be performed to determine the relationships between independent variables; also we will run the t-test to evaluate differences in compliance detected in the types of supplementation. The results are expressed as mean +/- SEM and the differences will be considered significant when P <0.05.

Duration: Subjects will make three visits (visit T-1, beginning visit T0, and end of treatment period visit T1, week 8 from T0). The treatment duration is 8 weeks; a daily portion of about 90 g of biscuit is introduced.