Foods 2020 , 9 , 1147
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task a score of 2 was given, if the subject held the stance for 3–9.99 s, a score of 1 was given, and for < 3 s, a score of 0 was given. The sum of these scores was calculated for an overall score for each subject. The gait speed test was used to assess how a subject normally walked over a four meter distance. A cane or walking aid was allowed if normally required. Subjects were instructed to walk at their normal pace and the test was demonstrated by the assessor. The assessor always walked beside the subject, but at the pace of the subject. The time taken to cover four-meters was recorded, from the time the subject entered the course until one foot completely exited. Scoring was assessed as follows; > 8.70 s, 1 point; 6.21 to 8.70 s, 2 points; 4.82 to 6.20 s, 3 points; < 4.82 s, 4 points. In the SPPB, two di ff erent chair stand tests were used, the single and repeated chair tests. For the single chair test subjects were asked to stand up from a chair without using their arms. Again, the test was demonstrated by the assessor and the subject was asked if they felt safe to perform the test. Subjects were instructed to fold their arms across their chest and sit so that their feet were on the floor; then to stand up while keeping their arms folded across your chest. If the subject could not rise without using arms, it was assessed if they could rise from the chair using their arms. The result was recorded. In the repeated chair stand test, subjects were asked if they thought it would be safe for them to try to stand up from a chair five times without using their arms. The correct procedure was demonstrated. Subjects were instructed to stand up as quickly as they could five times, without stopping in between. After standing up each time, they must completely sit down and then stand up again while keeping their arms folded across their chest. The time to complete this was recorded. The test was immediately stopped and recorded if the subject became tired or out of breath. The test was also stopped if the subject used their arms to rise from the chair, of if the test was not completed within 1 min. Scoring in the SPPB was assessed as follows; if the subject used their arms to stand in the single test 0; if the subject stood without using their arms, they progressed to the repeated chair stand test, which was scored as follows; subject unable to complete 5 chair stands or completes stands in > 60 s, 0 points; 16.70 s or more, 1 point; 13.70 to 16.69 s, 2 points; 11.20 to 13.69 s, 3 points; > 11.19 s, 4 points. An overall SPPB score was obtained for all activities and recorded. 2.5.2. Glucose Tolerance Test Subjects fasted for at least ten hours prior to the visit, with only water allowed to be consumed within that period. A cannula was inserted into the subject’s non-dominant arm and an initial baseline 2 mL blood sample was collected. The subject drank 410 mL of Lucozade (70 kcal / 100 mL, equivalent to 75 g of anhydrous glucose). The load was consumed within 5 min. The time to finish the load was recorded and additional blood samples were taken at 30, 60, 90, and 120-min post completion of the drink. This test was performed at baseline and week 12. 2.5.3. Serum Cytokine Concentrations Serum concentrations of TNF- α , IL-1 β , IL-6 and C reactive protein (CRP) were measured using the Milliplex ™ MAP Human Cytokine / Chemokine Magnetic Bead Panel (EMD Millipore Corporation, Billerica, MA, USA) according to the manufacturer’s instructions. Plates were read using the Luminex MAGPIX ® system and concentrations were calculated using xPONENT software (Luminex, Austin, TX, USA). HDL and LDL concentrations were also measured in the serum samples. 2.6. Statistics For in vitro experiments, statistical analyses were performed using the statistical computing software R [42], significant di ff erences from untreated controls were determined by one-way ANOVA followed by a Dunnett’s test. Data are presented as a percentage of untreated controls (mean ± SEMof at least 3 independent experiments). For physicochemical analysis, following LC-MS / MS of rice NPN, all data were collected, and analyses carried out using in-house Python scripts. Graphs illustrating peptide physiochemical properties (amino acid length, overall charge, and percentage of hydrophobic residues) were created using the “ggplot2” R package.
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