## 4 Discussion  
Our results show that each donor manifested different fecal microbial community types according to the Dirichlet multinomial mixtures model, which implies individual discrepancies in their ability to metabolize specific foods. We also observed different levels of SCFA production in each fecal fermentation, suggesting that individual differences in the microbiota affect fecal microbial metabolic activities. In this study, a significant amount of SCFAs were produced following 90 min of fecal fermentation in the presence of digested beef powder (\(p<0.05\)), with specific variations in each SCFA (Figure S2). In addition, the microbiome was shown to be relatively stable during these 90 min fermentations (AMOVA, \(p>0.05\)), although 5 to 12 genera showed significant differential abundance in each subject (Figure S1). These results suggest that 90 min of fecal fermentation could be used to investigate how individual microbiomes metabolize digested beef while minimizing the environmental effects of the in vitro incubation. However, the incubation time may vary depending on the food materials. Given the fact that the gut microbiota plays an important role in digesting food and providing nutrients to hosts, differences in these microbiomes often result in varying effects of diets. Unlike animal models, this study used human microbiomes to investigate the effects of dietary intervention. We observed that fecal fermentation using beef as the carbon  
Figure 5: Comparison of PICRUS-predicted metabolic activities before and after fecal fermentation with digested beef. Principal component analysis (PCA) with red and blue letters indicating subjects before and after fecal fermentation (**A**). The number of differentially abundant metabolic activities after the addition of the digested beef powder (**B**). source produced SCFAs in most of the subjects and increased the abundance of butyrate-producing bacteria such as _Bifidobacterium_, _Ruminococcus_, and _Blautia_. Digested beef has been reported to act as an important protein and nutrient source for _Bifidobacterium_[2; 21; 22] and _Ruminococcus_[23]. [PERSON] et al. [24] reported that meat proteins increase the abundance of _Lactobacillus_ and the amount of SCFAs produced in the gut of rats, highlighting the types of differences associated with evaluations in human or rat models. On the other hand, we did not observe a significant increase in butyric acid in half of the subjects, which could be due to a significant decrease in the abundance of _Faecalibacterium_ (\(p<0.05\)), a major butyrate producer in the gut, which requires complex carbohydrates for butyrate generation [25]. In addition, the abundance of _Prevotella_ decreased in all subjects except for subject 6, who did not have any _Prevotella_ even at initiation. _Faecalibacterium_ and _Prevotella_ have been reported to be positively associated with complex carbohydrate degradation [25; 26]. We observed that nearly all the subjects showed significantly reduced numbers of both _Prevotella_ and _Bacteroides_ after 90 min of fecal fermentation using the beef powder as the substrate, which could be due to the lack of fermentable dietary fiber (carbohydrate) necessary to support their growth [27]. Metabolic activity prediction analysis showed that fecal fermentation of beef powders shifted metabolic activity profiles in the same direction (Figure 5A), while differential abundance analysis indicated varying numbers of changes in the metabolic activity of each subject. While there were not many commonly enriched metabolic activities, several metabolic activities were depleted in more than three subjects (Table 1). These depleted metabolic activities were largely linked to long-chain fatty acid biosynthesis, such as oleate, palmitoteate, and stearate, which are known to play protective roles against heart disease. For example, palmitoteate and oleate are monounsaturated fatty acids and are known to reduce the risk of coronary artery disease [28; 29].