Pathogens 2021 , 10 , 235
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OFOs were produced by steaming the whole groats to make them soft and pliable and then pressed to flatten them obtaining a particle size of 0.51 − 0.76 mm. POF was produced using PepsiCo’s proprietary process providing a ready-to-use format with improved dispersibility. The granulation range for POF ranged from 50–250 µ m with the targeting of 178–250 µ m. The daily dose of both oat ingredients during the in vitro experiment was standardised to provide 1.4g β -glucan once per day. For POF, two additional test doses were investigated— standardised to provide 0.3 g β -glucan twice per day (to total 0.6 g β -glucan per day) and 1.0g β -glucan once per day. In order to mimic the in vivo conditions as close as possible, the test ingredient OFO was cooked according to the manufacturer’s instructions prior to use in the in vitro experiment. During the in vivo study, dry OFO was supplied to the subjects and cooked according to the manufacturer’s instructions prior to consumption. The consumption of cooked OFO was investigated during an intervention period of six weeks, providing a daily one serving dose of 40 g oats containing 1.4 g β -glucan. The control test product during the in vivo trial was Cream of Rice (Nabisco, East Hanover, NJ, USA), a cooked cereal containing no fibre or β -glucan and was supplemented at a dose of 40 g per day. 4.2. Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) The reactor setup simulating the human gastrointestinal tract was derived from the SHIME ® (ProDigest and Ghent University, Ghent, Belgium) as described by Molly et al. [23]. To optimally address the research questions, the SHIME ® setup was adapted from a single SHIME configuration (including one SHIME ® arm) to a TripleSHIME ® configuration (in- cluding three SHIME ® arms). Each arm of the TripleSHIME ® consisted of a succession of three reactors simulating the different regions of the gastrointestinal tract. The first reactor mimicked the upper gastrointestinal tract with the subsequent simulation of a gastric and small intestinal phase. The two subsequent colonic reactors simulated the proximal colon (PC), operated at pH 5.6–5.9 with a retention time of 20 h, and the distal colon (DC), operated at pH 6.6–6.9 with a retention time of 32 h. In order to simulate both the luminal and mucus-associated microbial community in the colonic reactors, mucin-covered beads were included as described by Van den Abbeele et al. [25]. Inoculum preparation, temperature settings, feeding regime, and reactor feed composition were adopted from Possemiers et al. [52]. To evaluate the properties of four different test ingredients and/or concentrations, four parallel TripleSHIME ® experiments were executed using the micro- biota of three healthy adult human donors with elevated total and low-density lipoproteins (LDL) cholesterol levels (total ≥ 5.5 mmol/L and <7 mmol/L and LDL ≥ 3.4mmol/L and ≤ 4.9 mmol/L) and a BMI between 20 kg/m 2 and25kg/m 2 (donor A: F, 25 y; donor B: F, 30 y; Donor C: M, 35 y), meaning that each test ingredient/concentration was tested in a separate TripleSHIME ® experiment including the microbial community of three dif- ferent donors. Upon inoculation with the faecal inoculum from the different donors, a two-week stabilisation period was initiated allowing the faecal microbiota to differentiate in the colonic reactors depending on the local environmental conditions. Subsequently, the baseline microbial community composition and activity were determined in the PC and DC during a two-week control period, followed by an evaluation of the effects of repeated daily administration of the test products during a three-week treatment period. The test products were pre-digested prior to administration to the colonic reactors in order to produce relevant product fractions that would reach the colonic environment. Pre- digestion was performed as previously described by Van den Abbeele et al. [7], with some minor modifications. During the oral phase, the test ingredients were diluted to obtain a concentration mimicking the daily test doses of interest, corrected for moisture content. Furthermore, the intestinal dialysis approach included a 4.5-hour incubation, during which the dialysis fluid was replaced once every 45 minutes with fresh fluid.
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