Pathogens 2021 , 10 , 235
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4.3. In vivo Study This was a randomised, single-blind, placebo-controlled, and cross-over study to assess the effect of hot cooked OFO on a range of variables including faecal bacterial composition and plasma SCFAs, in a sample of healthy adults with elevated cholesterol levels within the same range as the in vitro study. There were six visits in total with two phases to data collection (Figure S1). Each phase consisted of a 10-week period. The trial phase comprised of a six-week intervention period (Week 0–Week 6) after which subjects completed a four-week washout (Week 10). In total, 34 randomised subjects (aged 18–65 years old, BMI of 18.5–30 kg/m 2 , fasting blood glucose (FBG) of 3.0–6.0 mmol/L) were included in the analysis. To enrol in the trial, subjects provided written informed consent, and then completed the required screening procedures to evaluate their eligibility for the trial. At screening (Visit 1), fasting blood samples were collected to examine potential subject’s safety and blood lipid profiles, the subjects were also administered a food frequency questionnaire (FFQ); this information determined eligibility. The study participants were required to have a low to moderate consumption of dietary fibre based on data from the 2008–2010 Irish National Adult Nutri- tion Survey [53] (Low- fibre diet: ≥ 9.9g/dand ≤ 17 g/d for males; 8.2 g/d and ≤ 14.3g/d for females; moderate extends to 25.1 g/d for males and to 22.3 g/d for females), and elevated total and LDL cholesterol levels (total cholesterol ≥ 5.5 and <7 mmol/L and LDL ≥ 3.4 mmol/L and ≤ 4.9 mmol/L). The trial included a two-week run-in period to washout possible pre-trial prebiotics prior to baseline assessment. Eligible subjects were then randomised in a 1:1 ratio at Visit 2 to receive either treatment or control product in Phase 1 (Baseline: Visit 2; Week 0—End of Phase 1: Visit 3 at Week 6; Washout period: Visit 4 at Week 10), and they would then cross-over to consume the alternative product for Phase 2 (Baseline: Visit 4 at Week 10; Week 10—End of Phase 2: Visit 5 at Week 16; Washout period: Visit 6 at Week 20). Group 1 received OFO during the first intervention period and Cream of Rice during the second intervention period, while Group 2 received Cream of Rice during the first intervention period and OFO during the second intervention period. Subjects were provided with a stool collection kit at visits 1, 2, 3, 4, and 5 and instructed to collect a sample at home (24 hours prior to their visit) and bring it to the clinic at their next visit. Samples were kept chilled preceding analysis. The subjects consumed the study product once daily, at breakfast, for six weeks starting on Day 1, the day after Visit 2. The subjects were asked not to consume any other prebiotic/probiotics and fibre supplements or whole-grain oat products throughout the duration of the trial (22 weeks). They were also asked to maintain their habitual lifestyle in relation to physical activity level and diet. Ethical approval was granted by the Clinical Research Ethics Committee, Cork Ireland, prior to the study starting with code ECM 5 (6) 17/01/18. The study was conducted according to the principles of ICH-GCP and the Declaration of Helsinki. 4.4. Microbial Metabolic Activity During the in vitro SHIME ® experiment, samples for analysis of microbial metabolic activity were collected three times per week during the control and treatment period from each colonic reactor. Short-chain fatty acid (SCFA) measurements were performed as described by De Weirdt et al. [54] and included acetate, propionate, butyrate, and branched- chain fatty acids (isobutyrate, isovalerate, and isocaproate; bCFA). Lactate levels were determined using a commercially available enzymatic assay kit (R-Biopharm, Darmstadt, Germany) according to the manufacturer’s instructions. Ammonium determination was conducted as previously reported by Duysburgh et al. [55]. During the in vivo experiment, samples for analysis of plasma SCFA were collected at each scheduled visit, starting from Visit 2. Four plasma samples were collected in total per participant, i.e., one sample per baseline and endpoint of Phase 1 and Phase 2 of the in vivo experiment. Samples were prepared, extracted, and subjected to Ultra Performance
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