## 2 Materials and Methods
### 2.1 Oat Sample Preparation and Pulsed Electric fields (PEF) Treatment
Lab Technology Company, New Territories, Hong Kong). D-glucose (due to starch hydrolysis during digestion) content in the oat digest was determined using glucose oxidase/peroxidase (GOPOD) enzyme (K-GLUC, Megazyme, Bray, Ireland). Firstly, the collected supernatant from the digest was added with 0.05 mL amyloglucidase (3300 U/mL, Megazyme, Wicklow, Ireland), vortexed for 3 sec, and maintained at 50 \({}^{\circ}\)C (Grant Instruments water bath, Cambridgeshire, United Kingdom) for 60 min. Then, samples were diluted with distilled water to appropriate volume and centrifuged (1090\(\times\)_g_, 10 min, 4 \({}^{\circ}\)C). Supernatant (0.05 mL) was then reacted with 1.5 mL GOPOD, and kept at 50 \({}^{\circ}\)C for 20 min. Absorbance of the coloured mixture was determined using Ultrospec 3300 Pro UV/visible spectrophotometer (Amersham Biosciences, Uppsala, Sweden) at 510 nm. D-glucose (1 mg/mL) provided by Megazyme K-GLUC kit was used as standard. The measured glucose in the digest is expressed as mg glucose released per mL of digest. Three different starch fractions during _in vitro_ digestibility were estimated [19]: (1) readily digestible starch (RDS), defined as the amount of D-glucose (mg/mL digest) released after 20 min of _in vitro_ intestinal digestion (Glucose20 min), (2) slowly digestible starch (SDS), defined as amount of glucose (mg/mL digest) released between 20 and 120 min of _in vitro_ intestinal digestion (\(\Delta\)Glucose = Glucose120 min - Glucose20 min), and resistant starch (RS) were calculated using Equations (1)-(3), respectively. \[\text{RDS}\left(\%\right)=\frac{\text{Glucose20 min }\times 0.9}{\text{Total starch}}\times 100\% \tag{1}\]  
\[\text{SDS}\left(\%\right)=\frac{\Delta\text{Glucose }\times 0.9}{\text{Total starch}}\times 100\% \tag{2}\]\[\text{RS (\%)}=\text{Total starch}-\left(\text{RDS}+\text{SDS}\right) \tag{3}\]  
To convert the measured D-glucose in the digest based on the molecular mass ratio of starch to glucose (162/180), 0.9 factor value was used. Total starch content of the undigested samples was determined using the Megazyme kit (K-TSTA, Bray, Ireland) based on GOPOD format with modifications [20]. To estimate the extent (\(G_{f}\), mg/mL digest) and the rate of starch hydrolysis (\(k\), min\({}^{-1}\)) of oat flour during the 2 h _in vitro_ small intestinal digestion, the experimental data was fitted into a first-order fractional conversion kinetic model (Equation (4)). \[G\left(t\right)=G_{f}+\left(G_{0}-G_{f}\right)\ \times\ \exp^{\left(-k\ t \right)} \tag{4}\]  
where \(G(t)\) is the glucose released (mg/mL digest) at digestion time \(t\) and \(G_{0}\) is the amount of glucose (mg/mL digest) at the start of small intestinal phase (0 min). Three relevant starch digestion parameters \(k\), \(G_{0}\) and \(G_{f}\) were estimated using R function \"nls: Nonlinear Least Squares\" (version 4.0.4 2021). Approaches to evaluate the goodness of fit of the fractional conversion model (Equation (4)) to the experimental data obtained in this study include calculation of adjusted R\({}^{2}\), and visual assessment of residual and parity plots. R Studio (version 1.4.1103 2021) was used to execute the R programming language. Systematic Investigation to Understand the Mechanism How PEF Affects the Physicochemical Properties of Oat Flour  
The overview of the experimental design to understand the impact of PEF treatment application on oat flour properties is presented in Figure 1. Due to the heterogenous nature of oat flour, different oat flour segments were collected after PEF treatment and further analysed. Figure 1: (**a**) Experimental design used in understanding the impact of PEF treatment application on oat flour properties through a systematic evaluation of its flour segments. (**b**) Different flour segments observed after PEF treatment of oat flour. “SS” refers to Solid Segment.