3.1. SEM analysis
The SEM images of the SPs at 500 ×, 1000 ×, and 2000 × magnification are shown in Fig. 1. The surface morphology of the SPs was mostly aggregated and stacked together (Fig. 1A). At a higher magnification, the SPs surface morphology was irregular, dendritic, and fibrous (Fig. 1B and C), which could be attributed to the aggregation of branches or molecular clusters. The SPs surface was uneven, with noticeable folds and large inter-fiber spaces, which could be due to the complex structure and connection modes of the polysaccharides. Similar results were obtained by Su and Li [18] for the morphology of Auricularia polysaccharides. However, Gao et al. [19] found that polysaccharides extracted from Ulva pertusa exhibited flakes with smooth surfaces. The different polysaccharide surface morphologies may be attributed to the variations in the physicochemical properties and the specific types of extraction methods [20].
3.2. The Mw and monosaccharide composition of the SPs
No absorption peaks were evident at 260 nm or 280 nm in the ultraviolet spectra of the SPs (Fig. S2), indicating the absence of protein and nucleic acids [19]. The chromatogram showed a single symmetrical peak (Fig. 2), indicating that the polysaccharides were homogeneous. The standard curve was used to calculate the Mw of the SPs as 6.26 × 103 kDa with a retention time of 7.37 min (Fig. 2). Furthermore, high-Mw plant polysaccharides were also found in Ixeris polycephala (1.95×103 kDa) [21] and Dendrobium huoshanense (8.09×103 kDa) [22]. However, the Mw of sea buckthorn polysaccharides isolated by Shen et al. [16] was 26.4 kDa, which was distinctly lower than in this study. Wei, et al. [23] extracted polydisperse polysaccharides from sea buckthorn with Mw values of 9.68×102 kDa, 1.77×102 kDa, and 33.6 kDa. These variations might be due to the different sea buckthorn origins and extraction methods.
Further monosaccharide composition analysis showed that the SPs comprised Rha, Ara, Xyl, Man, Glu, Gal, and GalA at a molar ratio of 13.37 : 1.40 : 2.55 : 8.08 : 19.47 : 55.13 (Fig. 3, Table 1), indicating that the SPs was acidic heteropolysaccharides. GalA, belonging to a derivative of galactose and a common class of plant-derived polysaccharides, represented the main monosaccharide in the SPs [24]. Wei, Yang, Zhao, Wang, Zhao, Zhai, Zhang and Zhou [23] found that the polysaccharides from sea buckthorn berries were composed of Rha, Man, Glu, and Gal at a molar ratio of 1.00 : 6.89 : 1.62 : 13.52. Shen et al. [16] reported that sea buckthorn berry polysaccharides contained Man, Glc, Gal, and Ara, denoting neutral polysaccharides with no anionic groups. The differences between the polysaccharide Mw and monosaccharide compositions indicated that the SPs obtained in this study were new and not yet reported in previous research. These differences could be ascribed to varied sea buckthorn cultivation regions and polysaccharide extraction methods [25].
Table 1
The monosaccharide composition of the SPs (A) and standard mixture (B)
| Monosaccharide | Standard mixture Figure 3B | SPs sample Figure 3A |
| Peak | Retention time (min) | Peak | Retention time (min) | Peak area | Molar ratio |
| Rhamnose (Rha) | 1 | 22.716 | 1 | 23.453 | 4.609\(\times\)109 | 13.37 |
| Arabinose (Ara) | 2 | 23.499 | - | - | - | - |
| Xylose (Xyl) | 3 | 24.321 | 3 | 24.253 | 4.843 \(\times\)108 | 1.40 |
| Mannose (Man) | 4 | 27.904 | 4 | 27.786 | 8.805\(\times\)108 | 2.55 |
| Glucose (Glu) | 5 | 28.236 | 5 | 28.176 | 2.788\(\times\)109 | 8.08 |
| Galactose (Gal) | 6 | 28.471 | 6 | 28.372 | 6.713\(\times\)109 | 19.47 |
| Glucuronic acid (GluA) | 7 | 34.130 | - | - | - | |
| Galacturonic acid (GalA) | 8 | 34.522 | 8 | 34.519 | 1.901\(\times\)1010 | 55.13 |
3.3. FT-IR analysis of the SPs
The FT-IR spectra of the SPs is shown in Fig. 4. Peaks at 3279.68 cm− 1, 2920 cm− 1, 1700.56 cm− 1, 1615.20 cm− 1, 1413.64 cm− 1, 1095.1 cm− 1, 1016.55 cm− 1, and 629.62 cm− 1 were identified via FT-IR spectroscopy. The broad peak at 3279.68 cm− 1 represented the O-H stretching vibration absorption peak, demonstrating the intense inter- and intramolecular interactions between the polysaccharide chains [26]. The weak absorption at 2920 cm− 1 was ascribed to the antisymmetric stretching vibration of C-H [27]. The absorption peak at 1700.56 cm− 1 denoted the stretching vibration peak of C = O, while the strong absorption at the peak of 1615.20 cm− 1 signified the stretching vibration of a free carboxylic carbonyl group [28], and the absorption peak at 1413.64 cm− 1 represented C-O stretching vibration [29]. Furthermore, the significant absorption peaks at 1095.1 cm− 1 and 1016.55 cm− 1 suggested the possible existence of pyranose rings [30], while the weaker absorption peaks between 800 cm− 1 and 900 cm− 1 indicated the presence of α- and β-configurations [31]. These results revealed that the SPs displayed typical polysaccharide absorption peaks, denoting pyranose sugars.
3.4. The effect of the SPs on mice displaying gut imbalance
3.4.1. The effect of the SPs on the body weights and colon lengths of the mice
The colon lengths and body weights of the mice are shown in Table 2. No significant differences (p ＞ 0.05) were evident between the colon lengths of the CON, NR, and SPs groups, indicating the SPs did not affect the colon lengths of the mice. After cefixime gavage, the body weights of the mice decreased significantly (p ＞ 0.05), while no significant differences were apparent between the body weights of the SPs and CON groups. This showed that the SPs prevented the decline in weight caused by cefixime, consequently maintaining a normal body weight, which could be due to the protective effect of the SPs on the gut of the mice. Similarly, coix seed polysaccharides mitigated the significant loss in body weight caused by streptozotocin [32], while walnut green husk polysaccharides prevented abnormal weight gain in rats during a 50-d treatment period [33].
Table 2
The lengths of the colons and body weights of the three groups
| Treatment | The length of the colon (cm) | Body weight (g) |
| CON | 8.53 ± 0.11 | 27.05 ± 1.23 |
| NR | 8.23 ± 0.09 | 26.38 ± 1.84* |
| SPs | 8.91 ± 0.09 | 27.28 ± 2.00 |
| CON: Control group; NR: Natural recovery group; SPs: SPs group. Data are presented as the mean ± SD. * p < 0.05, compared to CON |
3.4.2. The effect of the SPs on the SCFAs in the mouse feces
SCFAs represent the primary metabolic products of polysaccharides fermented by the microbiota in the gut and have been proven to play multiple roles during energy metabolism [34, 35]. As shown in Fig. 5, seven different SCFAs were identified in the feces of the mice, with acetic acid, propionic acid, and butyric acid the most abundant. The acetic acid, propionic acid, isobutyric acid, isovaleric acid, and valeric acid levels in the SPs group were significantly higher than in the NR group (p ＜ 0.05), while no significant differences were evident between the SPs and CON groups (p ＞ 0.05). However, the butyric acid and caproic acid concentrations did not exhibit substantial changes after treatment with SPs compared with the NR group (p ＞ 0.05).
The results demonstrated that acetic acid repaired the intestinal barrier and reduced diabetic blood sugar levels while providing energy to intestinal epithelial cells and regulating immune cell activity [32]. Propionic acid, considered an important energy substrate for colon cells, participated in the gluconeogenesis pathway and inhibited fat and cholesterol production [36]. Butyric acid is also a significant energy source for intestinal epithelial cells and has a beneficial immunoregulatory effect on these and other mucosal cell populations [37]. This study indicated that the SCFAs content in the SPs group was generally higher than in the NR group, which could be attributed to the fermentation of the SPs into SCFAs by intestinal bacteria [33], benefitting gut recovery in the mice.
3.4.3. The effect of the SPs on microbial diversity
To determine the microbial community changes at the OTU level, a Venn diagram was created to show overlapping OTU data in the CON, NR, and SPs groups (Fig. 6A). A total of 733 OTUs were identified in the feces of the experimental mice, of which 563, 382, and 358 were represented in the CON, SPs, and NR groups, respectively. Here, 204 OTUs were shared by the three groups, while the 134 OTUs identified in the SPs group were not found in the NR group. The results showed that the number of unique OTUs in the SPs group (69) exceeded those in the NR group (57), indicating that the SPs improved and maintained the stability of the microbial environment [38].
To further analyze the effect of the SPs on the richness and diversity of the gut microflora in the mice, alpha diversity analysis was performed to obtain the main Chao1, Shannon, Simpson, and PD indexes. As shown in Fig. 6B, no significant differences were apparent between the four indexes of the CON, NR, and SPs groups (p ＞ 0.05), demonstrating that the SPs did not alter the diversity of the gut microbiota. Similarly, Sun, Duan, Liu, Luo, Ma, Song and Ai [8] found that the Chao1, Shannon, and Simpson indexes were not altered by Gracilaria lemaneiformis polysaccharides. No significant changes were observed in the Chao1 index of the gut microbiota in mice treated with pumpkin polysaccharides compared with the control group [9]. The differences between the gut microbiota of the three groups were also analyzed via beta diversity metrics. However, principal component analysis (PCA) showed that the microbial composition of the SPs group was similar to the CON group (Fig. 6C), suggesting that the SPs promoted gut microbiota recovery by modifying the gut microbial structure without affecting the diversity [39]. Previous studies showed that coix seed and walnut green husk polysaccharides could change the gut microbial structure [32, 33].
3.4.4. The effect of SPs on microbial species
The gut microbial composition in the three groups at the phylum level is shown in Fig. 7A. Bacteroidetes and Firmicutes represented the main gut microbiota in the CON group. Compared with the NR group, the relative abundance of Verrucomicrobia and Actinobacteriota increased in the SPs group, while the abundance of Proteobacteria was similar to the CON group. However, the Epsilonbacteraeota abundance was reduced in the SPs group. Furthermore, Verrucomicrobia played a crucial role in the intestinal microbial ecosystem, with some of its members displaying polysaccharide hydrolase activity [40], while Proteobacteria was negatively correlated with diabetic phenotypes [41]. Previous studies confirmed that the Firmicutes/Bacteroidetes (F/B) ratio was a key indicator of significant gut microbial changes, which was closely linked to obesity and other metabolic diseases [42, 43]. In this study, the NR group exhibited a lower F/B ratio than the CON group. The SPs regulated the cefixime-induced decrease in the F/B ratio, rendering it similar to the CON group, indicating that the SPs could maintain the stability of the gut microbiota. Higher F/B ratios were also evident in Lentinula edodes [44] and Flammulina velutipes polysaccharides [45].
Moreover, the microbial gut compositions on the genus level varied between the different groups (Fig. 7B). SPs treatment reversed the abundance of Ralstonia to a level close to the CON group, increased the abundance of Akkermansia, Faecalibaculum, and Allobaculum, and decreased that of Helicobacter. Additionally, aloe [46], Gracilaria lemaneiformis [8], and walnut green husk polysaccharides also display the ability to increase these bacteria [33]. Akkermansia represents a key gut bacterium that improves the metabolic status, consequently mitigating T2DM [47–49]. Faecalibacterium is a butyrate-producing bacterium that plays a vital role in diabetes [50], while Faecalibacterium prausnitzii transplantation is a practical therapeutic approach for treating diabetes and the related complications [51]. Allobaculum is considered a producer of SCFAs [48, 52]. In conclusion, after SP treatment, the abundance of the microorganisms related to the metabolism of polysaccharides and SCFAs increased, while the number of harmful microorganisms was inhibited, suggesting that SPs displayed the potential to maintain gut health.
3.4.5. The relationship between SCFAs and microbiota
The relationship between metabolic SCFAs and gut microbiota (top 50) was investigated, as shown in the heatmap in Fig. 8. Except for Ruminococcus torques (p < 0.05), no significant correlation was evident between isovaleric acid and gut microbiota. Nine genera were positively correlated with SCFAs, while Rikenellaceae RC9 was significantly correlated to propionic and butyric acid (p < 0.001). Briefly, the Spearman correlation coefficient indicated that key communities were strongly correlated with the SCFAs following SP supplementation [9, 46].
This study showed that SPs treatment increased the SCFAs content, and the relative abundance of the SCFAs-producing bacteria (Allobaculum and Akkermansia) in the gut was also increased, indicating that SPs may upregulate the SCFAs content by increasing the number of bacteria. Spearman correlation analysis showed that key communities were correlated with SCFAs following SPs treatment, further proving the hypothesis mentioned above. Moreover, similar functionality was observed in aloe polysaccharides [46]. Recent research revealed a correlation between gut microbiota, SCFAs levels, and the intestinal barrier [32]. SCFAs are essential in maintaining intestinal barrier function, while the balance of the colonic mucosal environment largely depends on the SCFAs concentrations [53]. Furthermore, the gut microbiota is closely related to host cell immunity and various physiological responses by producing metabolites, such as SCFAs and bile acids [54]. Moreover, SCFAs are not only affected by gut microbiota but can also be produced via the fermentation of polysaccharides themselves by gut bacteria [33]. In this study, cefixime decreased the SCFAs content and changed the gut microbial structure in the mice, disrupting the intestinal microbiota. Gut microbial dysregulation activated the associated inflammatory pathways and exacerbated gut damage. The subsequent release of several intestinal toxins increased the permeability of the intestinal mucosa and destroyed the intestinal barrier, leading to gut microbial disorder, further damaging the intestinal barrier and forming a continuous cycle [32]. According to the results, SPs may regulate the SCFAs and gut microbial content to break this cycle, consequently restoring intestinal microbial order in the mice exposed to cefixime.