*3.7. The Potential Mechanism of the Synergistic Effect of JSRS and B. pseudolongum for Improving the Symptom of Hyperlipidemia in Mice*

The previous analysis revealed the differential bacteria and metabolic pathways. To better reveal the correlation of JSRS, metagenomic species, microbial metabolic pathways, and serum lipid indicators, we calculated Spearman's rank correlation coefficients based on the data from Stage I of the experiment and constructed correlation networks for them (Figure 5). *B. pseudolongum*, *Subdoligranulum unclassified,* and *Akkermansia muciniphila* were positively correlated with JSRS, and *B. pseudolongum* had the highest correlation with JSRS

compared to the other two bacteria. The adenosylcobalamin salvage from cobinamide I (M) as well as TG was positively correlated with *B. pseudolongum;* conversely, the thiamin salvage (E), putrescine biosynthesis (P), and superpathway of polyamine biosynthesis (S) were negatively correlated. The network diagram showed the interconnections and interactions among JSRS, differential strains, differential metabolic pathways, and TG.

**Figure 5.** The correlation network among Jackfruit seed sourced resistant starch (JSRS), differential species, metabolic pathways, and TG. The R-value less than −0.4 or greater than 0.4 was selected. The width and color (red indicates a positive correlation, while blue indicates a negative correlation) of the edge are proportional to the correlation intensity. The green node represents JSRS, purple nodes represent species, light blue nodes represent metabolic pathways, and light red nodes represent TG. The node size is proportional to the mean abundance in the respective population.

### **4. Discussion**

The results of this study show that only 10% of JSRS had a limited preventive effect against hyperlipidemia in mice. On the other hand, the combination of JSRS and *B. pseudolongum* improved not only body weight, abdominal white fat, and serum lipid levels, but also had preventive and therapeutic effects on hepatic steatosis in liver cells. In previous studies, JSRS was able to regulate blood glucose levels and lipid metabolism [30], and prevent and treat obesity [31]. However, it has also been shown that the effects of JSRS on body weight and visceral fat were limited [12,32]. Similarly, this study shows that the addition of 10% JSRS to diet did not significantly reduce body weight and serum lipid levels in mice. The possible reason for this result may be that a high-fat diet could minimize the RS fermentation, resulting in lower levels of propionic acid and butyric acid [33]. SCFAs are closely related to the host's health, especially some metabolic diseases [34,35]. This condition led to an attenuated beneficial effect of RS in rats on a 20% high-fat diet, compared to a low-fat diet [33]. Another possible reason may be the structural differences of RS. It has been reported that the RS with lower crystallinity and double helix content had a poor effect on the body weight and serum TC content of mice induced by a high-fat diet. Resistant starch with high crystallinity and double helix content had a more stable structure, resulting in a more stable and slow fermentation in the intestine, allowing the substance to be evenly distributed in the intestine and meeting the needs of the distal colon [17]. Moreover, the variation of RS polymorphism would also lead to ecological changes in the microbial community structure of the colon. Different types of RS produce slightly different proportions of SCFAs and might undergo different fermentation patterns in the gut of obese mice, resulting in different effects on intestinal health [36]. However, our results indicated that JSRS ingestion could significantly elicit a response from *B. pseudolongum*. Furthermore, we observed that the major differential metabolic pathways such as L-tryptophan biosynthesis, gluconeogenesis III and thiamin salvage II were more active in the JSRS group. It has been shown that L-tryptophan can regulate immunity [37] and intestinal homeostasis [38]. Proper nutritional supplementation with tryptophan can prevent or reduce inflammation of the gut [39]. Bacterial fermentation of fiber can improve glycemic control by producing

succinate and thus activating intestinal gluconeogenesis [40]. The thiamin, as vitamin B1, is a cofactor for many enzymes indispensable for glucose and energy metabolism [41]. Apparently, JSRS can significantly improve the metabolism of intestinal microorganisms in mice and may be involved in related pathways such as host immunity and energy metabolism. Based on these findings, we hypothesized that the synergistic effect of JSRS and *B. pseudolongum* could prevent hyperlipidemia in mice. The hypothesis was then validated in the in vitro and in vivo experiments in Stage II. JSRS is not only a prebiotic but also a carbon source that promotes the growth of certain beneficial microorganisms in the gut. The pairing of prebiotics and probiotics is also gradually emerging in many studies. Ma et al. [25] found that constant supplementation with low galactose improves the stability of the intestinal microbes. Thus, probiotics may be able to work more reliably or better in the gut if they are supported by their substrates. Numerous studies have shown that the combined effects of probiotics and prebiotics can positively impact a wide range of human diseases, such as enhancing Disease Activity Score (DAS 28) and visual analog scores in rheumatoid patients, modulating plasma nitric oxide, rising glutathione [42], improving some symptoms of type 2 diabetes and quality of life [43], prolonging life, and reducing liver cancer cell proliferation in mice with malignant leukemia [44]. Research of synbiotics is expected to be promoted more with the updated definition of synbiotics [45]. *Bifidobacteria* are major degraders of RS, producing butyrate during the breakdown of RS to improve host health [46,47], and RS is the prebiotic capable of regulating changes in gut microbes [48]. There is no doubt that the combination of JSRS and *B. pseudolongum* as potential "synbiotics" can have a far more positive impact on the gut and health of mice than only JSRS supplementation.

In this research, differential microbes capable of utilizing JSRS in the intestine of mice on HFD and HFD were found. Interestingly, although 10% JSRS did not significantly improve body weight and serum TG level in mice, it was still able to regulate certain critical intestinal microbes, such as *Akkermansia*, *Ruminococcus,* and *Bifidobacterium*, which could promote the production of SCFAs that affect metabolic diseases positively to the host [49,50].

#### **5. Conclusions**

JSRS maintained the homeostasis of the intestinal microbes by correcting the damaging effects of a high-fat diet on gut microbes. As expected, *B. pseudolongum*, which are differential microorganisms at weeks two, four, and eight, could use JSRS to grow both in vitro and in vivo, and exerted excellent synergistic effects to treat and prevent hyperlipidemia and had a positive effect on hepatic steatosis in mice. Collectively, this study provided evidence on the suppression of hyperlipidemia in mice by the synergistic effects of JSRS and *B. pseudolongum*, which can support the development of "synbiotics" for the treatment of hyperlipidemia in the future. Collectively, this study provided evidence on the suppression of hyperlipidemia in mice by the synergistic effects of JSRS and *B. pseudolongum*, which can support the development of "synbiotics" for the treatment of hyperlipidemia in the future. By understanding the effects of JSRS on the gut microbiome and hyperlipidemia, it will help to obtain better applications in different fields, and provide a reference for the functional development of new varieties in the starch industry. We suggest the application of JSRS in combination with *B. pseudolongum* (synbiotics) to target the intestinal microbiota to alleviate hyperlipidemia in order to achieve the high value utilization of JSRS as a functional health food.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/foods10061431/s1, Figure S1. Effects of Jackfruit seed sourced resistant starch (JSRS) on body weight and serum lipid levels of mice in Stage I. Figure S2. Alpha diversity analysis at different time points among the four groups in Stage I. Figure S3. Verification experiment of *Bifidobacterium pseudolongum* in vitro and the anatomy of mice in Stage II.

**Author Contributions:** Conceptualization, J.Z. and R.J.; Methodology, J.Z.; Formal Analysis, Z.Z., Y.W., C.M. and S.J.; Investigation, H.C., Resources, Y.Z., J.Z. and W.L.; Data Curation, Z.Z. and Y.W.; Writing—Original Draft Preparation, Z.Z. and Y.W.; Writing—Review and Editing, Z.Z., K.C., J.Z. and R.J.; Visualization, Z.Z., D.H., and Y.W.; Supervision, J.Z.; Project Administration, J.Z., Funding Acquisition, J.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Natural Science Foundation of Hainan Province (No. 320RC513) and Key R & D programs in Hainan (No. ZDYF2019150 and ZDYF2018111).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Hainan University (AWER Issue No. HNU-EC-FS39, 09 May 2019).

**Data Availability Statement:** The sequence data reported in this paper have been deposited in the NCBI database (metagenomic sequencing data: PRJNA669624, PRJNA669572).

**Acknowledgments:** We sincerely appreciate Yanjun Zhang from Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, who supplied the jackfruit seed sourced resistant starch for the present study.

**Conflicts of Interest:** The authors declare no conflict of interest.
