*3.4. Bifidobacterium Pseudolongum Was the Potential Functional Intestinal Microorganism in JSRS Utilization*

Since there was a difference in the intestinal microbiota between NFD and HFD groups, the differential microorganisms at the genus levels in JSRS utilization were found on Mann–Whitney tests by combined NFD plus JSRS group and HFD plus JSRS group into the JSRS group. The remaining two groups were combined into the NFD and HFD group (N and H group), based on the high-throughput 16s rRNA sequencing abundance results at week 2 and week 4. Differential microorganisms at the species level in the eighth week were calculated by the same method, as the abundance results were derived from metagenomic sequencing data.

In the fourth week, the genus abundance of *Bifidobacterium*, *Parabacteroides*, *Akkermansia*, *Robinsoniella,* and *Unclassified Beijerinckiaceae* in the JSRS group was significantly higher than that in the N and H group, while the *Bifidobacterium* and *Parabacteroides* were also significantly different in the second week. The abundance of *B. pseudolongum*, *Akkermansia muciniphila*, and *Subdoligranulum unclassified* were significantly higher in the JSRS group than in the N and H group at week eight (Figure 3A). Overall, the average abundance of *Bifidobacterium* was significantly higher than that of the N and H group at weeks two and four, which highlighted the importance of the species belonging to the *Bifidobacterium* genus. Accordingly, for fecal samples collected at week eight, shotgun metagenomic sequencing was applied to identify the potential specific species of *Bifidobacterium*, which utilized JSRS in mice guts. Metagenomic species annotation results confirmed that *B. pseudolongum* was the dominant species capable of utilizing JSRS for their growth in mice gut (Figure 3A). Using the same grouping method, the top twenty metabolic pathways with significant baseline differences were screened (Figure 3B). Compared with the JSRS group, 6-hydroxymethyl-dihydropterin diphosphate biosynthesis I, anaerobic energy metabolism, anhydro-muropeptides recycling, chorismate biosynthesis from 3dehydroquinate, L-lysine biosynthesis, Purine nucleobases degradation, superpathway of N-acetylglucosamine, N-acetylmannosamine, N-acetylneuraminate degradation, and Superpathway of phospholipid biosynthesis were significantly more enriched in the N and H group. The remaining 12 pathways such as L-tryptophan biosynthesis and superpathway of pyrimidine ribonucleotides de novo biosynthesis were enriched in JSRS group. Collectively, the major differential metabolic pathways were more active in the JSRS group. JSRS significantly altered specific metabolic pathways in mice gut microbes.

**Figure 3.** Comparison of differential microbial and metabolic pathways. (**A**) Variations of differential bacteria in different groups at weeks 2, 4, and 8 in Stage I. The significant difference genera (week 2 and week 4) and species (week 8) were screened (Wilcoxon rank-sum test). The depth degree of color represents the relative abundance of the genus or species (blue indicates a small number, and red indicates a large number). (**B**) Metabolic pathway analysis was performed based on the shotgun metagenomic sequencing data in Stage I. Significantly different pathways with baseline ranking in the top 20 were screened out. Log2 foldchange value greater than 0 indicated that the enrichment abundance in the JSRS group (the combination of the HFD plus JSRS group and the NFD plus JSRS group) is greater than that in the N and H group (the combination of the NFD group and the HFD group). (JSRS, Jackfruit seed sourced resistant starch).

#### *3.5. The Ability of B. pseudolongum to Utilize the JSRS In Vitro*

Since we found *B. pseudolongum* in mice gut to be strongly correlated with JSRS intake, to test this hypothesis, we performed an in vitro validation experiment in Stage II. A strain of *B. pseudolongum* from the gut of cattle was used in an in vitro validation experiment. The glucose in the MRS agar medium was replaced entirely by JSRS (JSRS agar medium), and *B. pseudolongum* was inoculated. After 48 h, the average growth of *B. pseudolongum* in the starch agar medium reached 6Log CFU/mL, although the average growth of the control group was 7Log CFU/mL. The ratio of JSRS utilization circle reached 8.3 (Figure S3A). These results proved that *B. pseudolongum* could grow by using JSRS as the primary carbon source in vitro.

#### *3.6. The Synergistic Effect of JSRS and B. pseudolongum Suppressed Hyperlipidemia in Mice*

Based on the results above, another hypothesis in the present study is that the synergistic effect of JSRS and *B. pseudolongum* could suppress hyperlipidemia in mice. Therefore, we performed an in vivo validation experiment to test this hypothesis (Figure 1B). At week three, there was no significant difference in body weight between the TR and HFD groups, both of which were significantly higher than the NFD group. The body weight of the TR group was significantly higher than the NFD group (Figure 4A), although there was no significant difference in body weight between the two groups. After another three weeks, the body weight of the TR group not only gradually decreased but also was lower than the NFD group (Figure 4B). Moreover, the body weight of the PR group was consistently lower than the HFD group, even lower than the NFD group. The synergistic effect of JSRS and *B. pseudolongum* prevented high fat diet-induced obesity in mice.

Data on serum lipid levels and abdominal fat of mice further verified that the synergistic effect could reduce abdominal fat and blood lipid in mice. At week six, with the synergistic effect of both, the abdominal white fat weight was effectively reduced and controlled in PR and TR groups, which was significantly lower than that in the HFD group. TR group was slightly higher than the NFD group (Figure 4C), but no significant differences existed. The same trend was found in the anatomy of mice (Figure S3B). Similarly, the level of blood lipids of mice reinforced the conclusion that JSRS and *B. pseudolongum* worked together to reduce body weight and serum lipid levels in mice. The levels of TC, TG, and LDL showed the highest trend in the HFD group (Figure 4D). The levels of these three blood lipid parameters were significantly reduced by the synergistic effect, although there was no significant difference in HDL-C level between the four groups. TC and TG levels were lower in the TR group than in the NFD group, while they were not statistically significantly different in LDL-C levels between the two groups.

The histopathology results showed that the liver structure of the NFD group was normal, and the liver lobules were clearly visible. The liver cells were intact, without rupture or autolysis, and closely arranged. No fatty vacuoles were observed. In contrast, the liver of the HFD group mice had many fatty vacuoles and disturbed hepatic cord distribution. Hepatocytes were loosely arranged and had fuzzy margins. Evidently, the HFD caused severe pathological changes in the liver of mice. The fatty infiltration of hepatocytes was significantly reduced in the TR group, although the arrangement was slightly disturbed. Compared with the HFD group, the number of fatty vacuoles in the liver of the PR group was significantly reduced, and the hepatocytes had a clearer contour and tighter structure (Figure 4E).

**Figure 4.** Effect of Jackfruit seed sourced resistant starch (JSRS)plus *Bifidobacterium pseudolongum* on hyperlipidemia in mice. (**A**) Body weight of four groups at each time point in Stage II. (Wilcoxon rank-sum test, \* *p* < 0.05, \*\* *p* < 0.01 compared with HFD; # *<sup>p</sup>* < 0.05, ## *<sup>p</sup>* < 0.01 compared with NFD, error bar: mean <sup>±</sup> SE) (**B**) Weight gain was compared between the four groups during the first three weeks of the study at each time point in Stage II (Wilcoxon rank-sum test, \* *p* < 0.05, \*\* *p* < 0.01, NS means no significant difference, error bar: mean ± SE) (**C**) Comparison of the weight of white fat in the abdomen of four groups of mice. (Wilcoxon rank-sum test, \* *p* < 0.05, \*\* *p* < 0.01, error bar: mean ± SE) (**D**) Comparison of TC, TG, LDL-C, and HDL-C levels among the four groups in Stage II. (Wilcoxon rank-sum test, \* *p* < 0.05, \*\* *p* < 0.01, error bar: mean ± SE) (**E**) Histopathological analysis of the liver sections of mice in four groups at 200× and 400× magnification in Stage II. ("-a" and "-b" were liver section images at the same position, but with different magnification. "-a" means 200×; "-b" means 400×).
