*3.5. Effect of GCP on the Composition and Diversity of Gut Microbiota* 3.5.1. Diversity Analysis of the Structure of Gut Microbiota

Through α-diversity analysis, a series of statistical analysis indexes was used to estimate and reflect the abundance and diversity of microbial communities. Sobs, ACE and Chao index reflected the species richness of operational taxonomic units (OTUs) in the gut microbiota, while the Simpson and Shannon indexes reflected the differences in species diversity in the gut microbiota [22]. As shown in Table 3, the Sobs, ACE, Chao and Shannon index of the gut microbiota in the experimental groups showed an increasing trend (*p* < 0.05) in a concentration-dependent manner compared to the control group, and the Simpson index showed a downward trend (*p* < 0.05). This indicates that GCP increased the species richness and diversity of the gut microbiota in the cecum contents of mice.


**Table 3.** Effect of GCP on the α-diversity of gut microbiota (*n* = 10).

Different letters (a–d) represent significant differences, *p* < 0.05.

The different numbers of OTUs are illustrated among the four groups by the Venn diagram in (Figure 3). Among all the OTUs in this work, 371 were shared by all groups. Meanwhile, the numbers of OTUs shared by experimental groups and control group were 414 (low-dose group), 441 (middle-dose group) and 425 (high-dose group). Furthermore, 88 OTUs were detected in the low-dose group but not in the control group, and 77 and 88 OTUs were detected separately in the middle-dose group and high-dose group. Furthermore, a different group had its own separate set of OTUs: 2 in the control group, 8 in the low-dose group, 7 in the middle-dose group and 12 in the high-dose group. However, the total numbers of OTUs in the control group, low-dose group, middle-dose group and high-dose group were 472, 502, 518 and 513, respectively. This suggested that GCP increased the species richness of the gut microbiota in the cecum of mice.

**Figure 3.** Venn diagram of colon gut microbiota (*n* = 10). CON: control group; LOW: 100 μg/mL GCP; MID: 200 μg/mL GCP; HIG: 400 μg/mL GCP.

3.5.2. Composition Analysis of the Gut Microbiota

The relative abundance of cecum gut microbiota composition of each group is shown in Figure 4. At the phylum level, the gut microbiota in the four groups mainly consisted of *Bacteroidetes*, *Firmicutes*, *Proteobacteria*, *Spirochaetae* and *Verrucomicrobia*, and these five phyla in all groups represented approximately ≥95% of the sequences. The relative abundances of *Bacteroidetes* in the four groups were 62.41%, 55.38%, 54.05% and 58.97%, and those of *Firmicutes* were 32.28%, 33.81%, 39.12% and 38.36%. Notably, th *Firmicutes*/*Bacteroidetes* (F/B) ratio increased from 0.5172 in the control group to 0.6105 (low-dose group), 0.7238 (middledose group) and 0.6505 (high-dose group), showing an increasing trend. The increased dose of GCP might have an effect on the change in ratio of F/B in mice gut microbiota, but as the two main communities that affect energy metabolism homeostasis [36], researchers have different opinions on the functions of *Firmicutes* and *Bacteroidetes*. Wu et al. [22] found that *Cyclocarya paliurus* polysaccharides alleviated the liver inflammation of mice by increasing the F/B ratio in their gut microbiota. However, Yang et al. [29] demonstrated that *Linum usitatissimum* L. polysaccharides mitigated the high-fat diet-induced metabolic syndrome in mice, which did not affect the relative abundance of *Bacteroidetes*, but reduced the relative abundance of *Firmicutes*. At the same time, a few researchers have suggested that obesity is not associated with the ratio of F/B [37,38]. The utilization and digestion of polysaccharides by gut microbiota are affected by many factors, including monosaccharide composition, linkage mode, chain type, molecular weight, sulfate content, etc. [4,29]. In future work, we will investigate the effect of high-dose GCP with a relatively low F/B ratio (0.6505), compared with the 0.7238 of the middle-dose group.

### **4. Conclusions**

The toxicity of antibacterial polysaccharides to the body and gut microbiota is poorly understood. The results in the present work show that the antibacterial polysaccharide of GCP was safe for Caco-2 cells and mice. Meanwhile, GCP reduced the body weight gain of mice and increased the SCFAs concentration in the colon. Furthermore, GCP increased the diversity of gut microbiota and the *Firmicutes*/*Bacteroidetes* ratio. In future work, the specific bacteria in the microbiota of a gut affected by GCP, especially *Firmicutes* and *Bacteroidetes*, will be investigated.

**Author Contributions:** Conceptualization and methodology, X.S.; software and validation, X.H.; investigation, resources and data curation, C.Z.; writing—original draft preparation, X.Z.; writing review and editing, Z.W.; visualization and supervision, H.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is supported by Natural Science Foundation of Henan Province (212300410131), Henan major public welfare project (201300110100), Henan Province Human Resources and Social Security department Science and Sechnology Activities Programs for overseas students, Henan Key Laboratory of Cold Chain Food Quality and Safety Control (CCFQ2020-YB-18), Henan major public welfare project (201300110100), and The major breeding project of Zhengzhou university of light industry (2020ZDPY0102).

**Institutional Review Board Statement:** The experiments were performed strictly according to the guidelines for the care and use of laboratory animals of Henan University of Technology.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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