## 2 Materials and Methods
### In Vitro Gastrointestinal Digestion (IVDG) Model with Dialysis Process  
In vitro gastrointestinal digestion (IVDG) was performed according to the harmonised protocol developed by the COST INFOGEST network, with slight modifications [31; 32]. The IVDG model consists of an oral phase, a gastric phase, and an intestinal phase. All solutions were preheated to 37 \({}^{\circ}\)C prior to use, and all the experimental conditions were performed in a pre-set 37 \({}^{\circ}\)C shaking water bath (Grant OLS-200, UK). Briefly, in the oral phase, weighed sample (1 g) in triplicate was mixed with 3 mL of water to create a 'food-like' paste. 50% (\(v/v\)) Simulated Salivary Fluid (SSF, pH 7) containing calcium chloride (CaCl\({}_{2}\)) (150 mM) and amylase (150 U/mL) was added to the paste and was allowed to mix for 2 min. Followed by mixing, the sample from the oral phase with the same volume of gastric phase, Simulated Gastric Fluid (SGF, pH 3) containing (16 mg/mL) and CaCl\({}_{2}\) (0.15 mM). After adjusting to pH 3.0 with 1 M HCl, the sample was incubated for 2 h stimulating gastric phase. Then the simulated food bolus was exposed to Simulated Intestinal Fluid (SIF, pH 7) at 50% (\(v/v\)) that contained pancreatin (without bile acids) and was incubated for further 3 h after the pH was brought to 7.0 with 1 M NaOH. The digesta from the IVDG were transferred to a 50 mL falcon tube and centrifuged (3200\(\times\)_g_, 5 min, 4 \({}^{\circ}\)C). The pellet was kept at \(-\)20 \({}^{\circ}\)C. The supernatant was transferred into a cellulose dialysis membrane (Cheshire biotech Cheshire, UK) with a molecular weight cut off of 12-14 kDa and dialysed against a 100-fold volume of denoised water for one hour at room temperature, repeated twice again. The dialysis fluid from outside of dialysis membrane was collected and combined, 30 mL aliquots were sampled from 4.5 L homogeneous dialysis fluid and analysed for metabolites by LC-MS/MS, which represent the small molecules likely absorbed in the small intestine. Aliquots of the dialysis fluid were freeze-dried and dissolved with 100 \(\mu\)L methanol for LC-MS analysis as detailed in 2.4 and 2.5. The digesta retained inside of dialysis membrane were combined with the saved pellets, stored at \(-\)70 \({}^{\circ}\)C and freeze-dried. This fraction represents the portion delivered to the colon and was used as the substrate for the faecal incubation. In Vitro Incubation of Hemp Samples Using Human Mixed Microbiota (Faecal Samples) 2.9.1. Human Faecal Incubation  
Freeze-dried digesta from IVDG were weighed (0.025 \(\pm\) 0.001 g) in six replicate pre-weighed Hungate tubes sealed with butyl rubber stoppers and screw caps (Bellco Glass, Shrewsbury, UK). A volume of approximately 7.5 mL M2 basal medium (containing no other carbohydrate energy source) was subsequently added and autoclaved. The faecal slurry was achieved by anaerobically mixing 2 g of fresh faeces (three healthy omnivore human donors) with 8 mL of anaerobic 50 mM phosphate buffer containing 0.05% cysteine under a CO\({}_{2}\) atmosphere and vortex mixing. After cooling, three tubes of six duplicates (three control tubes incubated with 100 \(\mu\)L buffer) were inoculated with the 100 \(\mu\)L faecal slurry under CO\({}_{2}\) and incubated at 37 \({}^{\circ}\)C. Samples for metabolite analysis by LC-MS/MS were recovered at 0 h and at 24 h, 48 h, and 72 h post-inoculation. Samples recovered at 0 h and 72 h were also used for the SCFA analysis. Written informed consent was obtained from each subject. The volunteers were healthy, consuming an omnivore diet, with no use of antibiotics for at least three months prior to the study. The study involved collection of faecal samples from human donors, followed all the principles of the Declaration of Helsinki and good clinical practice, and all procedures involving human subjects were reviewed and approved by the Human Studies Management Committee of the Rowett Institute, University of Aberdeen, UK, and the Rowett Institute Ethics Panel.