*3.6. Enzyme-Treated Bee Pollen Regulates the Composition of Gut Microbial Structures*

Furthermore, food allergy can induce an imbalance of gut microbiota associated with the immune system. As shown in Figure 5A,C, the Ace, Chao and Shannon indices were higher in the BP, 2E-BP and 3E-BP groups than that in the OVA groups, indicating that bee pollen can increase α-diversity of gut microbiota in mice. As shown in Figure 5D,E, 2E-BP and 3E-BP groups were well-separated with OVA and BP groups, suggesting the significant changes in β-diversity of gut microbiota. Additionally, clustering analysis showed that the OVA and BP groups had similar microbial structures, while the CK, 2E-BP and 3E-BP groups exhibited analogous microbial structures (Figure 6A). In consideration of the above findings, a severe allergy was induced by OVA and BP but was alleviated by 2E-BP and 3E-BP, thereby leading to a similar microbial structure among CK, 2E-BP and 3E-BP groups. Kruskal–Wallis H test and LEfSe analysis showed that *Lachnospiraceae*, *Marinifilaceae* and *Helicobacteraceae* were significantly more abundant in the CK, 2E-BP and 3E-BP groups than in the OVA and BP groups; while, the abundance of *Bacillaceae* and *Akkermansiaceae* was significantly lower in the CK, 2E-BP and 3E-BP groups than in the OVA and BP groups (Figure 6B,C).

**Figure 5.** The alpha-diversity and beta-diversity analysis of different samples at ASV level, including (**A**) Ace index analysis; (**B**) Chao index analysis; (**C**) Shannon index analysis; (**D**) PCoA analysis; (**E**) Typing analysis. Asterisk \* represents *p* < 0.05, \*\* represents *p* < 0.01, and \*\*\* depicts *p* < 0.001.

**Figure 6.** Heatmap analysis of gut microbiota community at ASV level (**A**). Kruskal–Wallis H test analysis of gut microbiota at family level (**B**). Cladogram based on LEfSe analysis of gut microbiota from phylum to class level (**C**). Asterisk \* represents *p* < 0.05, \*\* represents *p* < 0.01, and \*\*\* depicts *p* < 0.001.

As reported, *Lachnospiracea* could be involved in food allergies [52]. Its abundance was significantly increased after the allergic mice received an allergen-specific Treg cell therapy compared with the no-treatment group [53]. *Lachnospiraceae* level was also significantly reduced in the gut microbiota of allergy sufferers compared with healthy population [54]. *Marinifilaceae* can be affected by intenstinal inflammation. Its abundance was reduced in the gut microbiota of colitis mice but recovered following anti-inflammatory therapy [55]. *Helicobacteraceae* shows beneficial effects against food allergy, for instance, the neutrophilactivating protein of *Helicobacter pylori* can inhibit peanut allergy by up-regulating the production of Tregs [56]. The level of *Bacillaceae* is related to gut inflammatory diseases [57]. It presented a higher level in the gut microbiota of Crohn's disease patients compared with healthy people [58]. Additionally, the level of *Akkermansiaceae* increased due to inflammatory gut injury and other gastorintestinal diseases [59]. Therefore, our findings correspond with those of previous reports. Enzyme-treated bee pollen can alleviate allergic reactions and regulate the composition of microbial gut structures.
