## 2 Materials and Methods  
### In Vitro Gastrointestinal Digestion and Fecal Fermentation  
The frozen beef used in these experiments was obtained from the food market and stored at \(-80\)\({}^{\circ}\)C. All chemicals and enzymes used in these experiments were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA). All of the procedures in this study which involved human participants were conducted in accordance with the institutional ethical standards of Jeju National University, Korea (IRB approval number JJNU-IRB-2018-040-001) and the Helsinki declaration of 1964 and its later amendments or comparable ethical standards. Fecal samples were collected from six healthy adult donors (S1-6) and used to perform fecal fermentation. Donors were not on any medication for at least four weeks prior to taking part in the study. The frozen meat was thawed and then minced to 2-3 mm [10,11] before heating to 60 \({}^{\circ}\)C for 2 min to complete the cooking process. The cooked meat was then used at 0.1% _w/o_ to prepare 100 mL of fermentation broth, before gastrointestinal digestion was performed according to the established INFOGEST method [9]. The cooked meat was incubated in 5 mL phosphate-buffered saline (PBS) at 37 \({}^{\circ}\)C for 2 min to normalize the temperature and then mixed with 3.5 mL of simulated salivary fluid (1500 U\(\cdot\)mL\({}^{-1}\)\(\alpha\)- amylase (0.5 mL), 25 uL of 0.3 mol\(\cdot\)L\({}^{-1}\) CaCl\({}_{2}\))) and the volume was made up to 10 mL using distilled water. The enzymatic contact time was 2 min at 37 \({}^{\circ}\)C with constant shaking at 100 rpm. For simulated gastric digestion, the prepared bolus (10 mL) was then mixed with 7.5 mL of simulated gastric fluid (1.6 mL of 25,000 U\(\cdot\)mL\({}^{-1}\)epsin, 5 uL of 0.3 mol\(\cdot\)L\({}^{-1}\) CaCl\({}_{2}\)) and 200 uL of 4 mol\(\cdot\)L\({}^{-1}\) HCl to adjust the pH to between 2.5 and 3. Distilled water was added to a final volume of 20 mL. Gastric digestion was then simulated for 2 h, with constant shaking at 100 rpm. Then, to simulate intestinal digestion, the gastric chrome (20 mL) was mixed with 11 mL of simulated intestinal fluid (5 mL of 800 U\(\cdot\)mL\({}^{-1}\) pancreatic, 2.5 mL of 0.160 mol\(\cdot\)L\({}^{-1}\) bile salt, 40 uL of 0.3 mol\(\cdot\)L\({}^{-1}\) CaCl\({}_{2}\)) and 150 uL of 1 mol\(\cdot\)L\({}^{-1}\) NaOH to normalize the pH to 7. This mixture was then made up to a final volume of 40 mL using distilled water. Intestinal digestion was performed for 2 h at 100 rpm to thoroughly mix the components. The digested fluid was rapidly frozen in liquid nitrogen to stop the enzymatic reaction and then stored at \(-20\)\({}^{\circ}\)C. Further, digested fluid was freeze-dried to obtain a dried beef powder to be used in fecal fermentation. Basal fecal fermentation medium was prepared in triplicate as previously described [12], with one minor modification: both the peptones and yeast extracts were excluded from this broth as they may serve as alternate carbon sources. The basal fermentation media and the PBS buffers were placed in an anaerobic chamber for one day to remove any oxygen from their composition. Fresh feces were collected from six adult healthy male donors of age 27-45 years, mixed with PBS (10% \(w/v\)), and then sieved using 250 \(\upmu\)m, 150 \(\upmu\)m, and 25 \(\upmu\)m pore size filters. The extracted fecal slurries were then immediately transferred into an anaerobic chamber (Bactron II, SHEL LAB, Cornelius, North Carolina, USA) and inoculated into the prepared medium (10% \(v/v\)). Digested beef powder was then added to the beef treatment group, and inoculated mixtures were incubated at 37 \({}^{\circ}\)C and 100 rpm for 90 min, during which samples were collected at four time points (0, 30, 60 and 90 min) along with their respective control (B0, B30, B60 and B90), where the content of controls was the same as beef-treated samples, except they were did not receive beef.