Resistant Starch

Towards the end of the baseline assessment period (April 2009-September 2010), the UK Biobank started collecting detailed dietary intake measures using a web-based self-administered 24 h dietary assessment, the Oxford WebQ. The Oxford WebQ was completed by 70,724 participants attending their baseline assessment. Additionally, between 2011 and 2012 all participants with valid email addresses (n = 331,013) were invited to complete the Oxford WebQ on four separate occasions. This was done every 3–4 months on variable days to maximise the coverage by season and day of the week (cycle 1: February 2011 to April 2011; cycle 2: June 2011 to September 2011; cycle 3: October 2011 to December 2011; cycle 4: April 2012 to June 2012). Approximately 53% of the participants (n = 176,012) who were contacted by email completed at least one assessment, with a total of 211,050 participants completing at least one dietary assessment either online or at the baseline assessment [6 (link)].
The Oxford WebQ collects information on foods and beverages consumed over the previous day. Participants were presented with a list of up to 206 foods and 32 beverages commonly consumed in the UK and selected the number of portions consumed from each food. This food list was constructed using information from the UK National Diet and Nutrition Survey (NDNS) as well as a pilot study [5 (link)]. At the start of the questionnaire, participants indicated whether their diet over the previous day was typical and if they were following a special diet. Descriptions and help sections were used to help estimating portion sizes of foods (e.g. slices, cups, servings) and participants were asked to report ingredients of composite dishes separately. Total energy and nutrient intakes were generated by multiplying the number of portions consumed by the set quantity of each food portion size and its nutrient composition obtained from the UK Nutrient Databank Food Composition Tables (FCT) from survey year 6, (2012–2013 and 2013–2014) [9 –11 ]. Dietary fibre was calculated using the Englyst method [12 ], which includes non-starch polysaccharides but not lignin and resistant starches.

The defined medium (DM) was purchased from ProDigest (Ghent, Belgium). It contained (in gL^-1) arabinogalactan (1.2), pectin (2.0), xylan (0.5), resistant starch (4.0), glucose (0.4), yeast extract (3.0), peptone (1.0), mucin (3.0), and cysteine (0.5). The defined medium was adjusted to pH 2 prior to autoclaving. Pancreatic juice contained (in gL^-1) NaHCO₃ (12.5) (Sigma-Aldrich, Saint Louis, MO), bile salts (6.0) (BD, Franklin Lakes, NJ), and pancreatin (0.9) (Sigma-Aldrich, Saint Louis, MO). Mucin-Agar containing carriers were prepared by dipping plastic, hollowed carriers (DI, 5 mm; ProDigest) into a mucin-agar solution. The Mucin-Agar solution was prepared by boiling 1% bacterial agar in autoclaved MilliQ water three times, to dissolve 5% type II porcine mucin (Sigma-Aldrich). The filled carriers were allowed to solidify under laminar flow in a biosafety cabinet at room temperature and stored at -4 °C until use.
Fecal samples (Microbiome Health Research Institute; Boston, MA) were harvested from an individual American, randomly selected from a pool of typical, Western diet consumers, between the ages of 21 and 45 years old, with an average Body Mass Index (BMI), who had been antibiotics-free for at least 1 year. According to the provider, the donor of the fecal sample was required to fill out a medical history questionnaire and interviewed in a process similar to that used for blood donors. A series of tests were performed on both fecal and blood samples to screen for any potentially infectious pathogens before deposition of fecal material. Sixty days post donation, a second round of tests were performed on both the fecal and blood samples. Upon both sets of tests proving negative for the presence of pathogens, the fecal sample thus collected was homogenized to make a 10% feces in glycerol buffer solution. The homogenate was then aliquoted into 250 mL containers, frozen, and stored at -80°C (http://www.Openbiome.org, cited December 01, 2016). The same homogenate was used to inoculate both systems, simultaneously, for each experiment.

Whole grain samples were digested following established procedures.^{64 (link)} Briefly, 5 g of whole grain were ground into fine powder using Geno/Grinder 2025 (SPEX SamplePrep) at 1600 rpm for 10 min. Then, 2.5 g of sample was mixed with 30 mL of water in a 50 mL Falcon tube for 20 min until fully dispersed. Tubes containing the slurries were immersed in boiling water for 20 min with constant shaking. The slurries were then placed on an orbital shaker (200 rpm) and incubated at 37°C for 40 min. The pH was adjusted to 2.5 with 1 M HCl followed by the addition of 1 mL of 10% (wt/vol) pepsin (P7000; Sigma, St. Louis, MO) in 50 mM HCl. The slurry was then incubated on an orbital shaker (200 rpm) at 37°C for 60 min. The pH was adjusted to 6.9 with 0.5 M NaHCO₃ and 5 mL of 12.5% (wt/vol) pancreatin (P7545; Sigma, St. Louis, MO) in 0.1 M sodium maleate buffer, and 0.2 mL of amyloglucosidase (E-AMGDF, 3,260 U/mL, Megazyme) was added. The slurry was incubated for 6 h at 37°C with orbital shaking at 200 rpm. Following digestion, the material was transferred to dialysis tubing (molecular weight cutoff of 1000 Dalton) and dialyzed against distilled water for 3 days at 4°C with a water change every 12 h. The retentate from the dialysis was collected, freeze-dried and resuspended in 30 mL of sterile distilled water. The resistant starch concentration was determined with a commercial kit using the protocol variation entitled ‘Determination of total starch content of samples containing resistant starch’ (K-TSTA, Total Starch Assay Kit (AA/AMG), Megazyme).