3.2.3. Fourier Transform Infrared Spectroscopy (FT-IR)

The FR-IR spectrum of HPIDF/F-HPIDF is shown in Figure 4. At 3353 cm<sup>−</sup>1, 2932 cm−1, 1737 cm<sup>−</sup>1, 1626 cm−1, 1418 cm−1, 1374 cm−1,1248 cm−1, 1057 cm−1, 706 cm−1, 623 cm−1, and 532 cm−1, both HPIDF/F-HPIDF showed significant absorption peak, which represented the composition of their polysaccharide functional groups. Actually, although HPIDF and F-HPIDF showed some differences in peak height, no differences in peak location were observed. In contrast with other studies [26–28], these absorption peaks represented O-H group in cellulose or hemicellulose, C-H stretching of -CH3 or =CH2 on carboxymethyl and methylene, oxygen (CO-OR) stretching vibration in hemicelluloses, C-O stretching vibration in the guaiacyl unit of lignin, glycuronic acid, etc. The other small peaks were not different from the previous study [16].

**Figure 4.** Fourier transform infrared spectrum (FTIR) of HPIDF/F-HPIDF. (Black: HPIDF; Red: F-HPIDF).

Although no differences were found in peak location, F-HPIDF had a markable characteristic in the FT-IR spectrum analysis. All peaks in F-HPIDF are higher than that in HPIDF, which means the fermentation increased the exposure of all functional groups. This result was consistent with the SEM results. The more complex the spatial structure, the greater the exposure of functional groups. This inference needs to be supported by the results of particle size and the specific surface area below.

To sum up, the fermentation might change the structure of HPIDF but did not change the composition, which would make the adsorption of F-HPIDF better than that of HPIDF, so that it may have better potential bioactivity in the colon.

#### 3.2.4. Particle Size and Specific Surface Area

Particle size and specific surface area of HPIDF/F-HPIDF are shown in Table 1,which both showed significant differences (*p* < 0.05). The smaller average particle size and the increase of specific surface area might lead to a higher adsorption capacity of harmful substances and metal ions. This result was in high agreement with the results of SEM and FTIR. These showed that the fermentation had made a great change in the structure of HPIDF. As for the composition, the analysis of monosaccharide composition of HPIDF, F-HPIDF, and hydrolysate was necessary.


**Table 1.** Particle size and specific surface area of HPIDF/F-HPIDF.

Different lowercase letters indicate a significant difference (*p* < 0.05).

#### *3.3. The Monosaccharide Composition of HPIDF, F-HPIDF, and Hydrolysate*

The monosaccharide composition of HPIDF, F-HPIDF, and hydrolysate is shown in Figure 5. The main constituents of HPIDF are shown in Figure 5a, which are same as the previous result [16]. With the progress of colonic fermentation, the monosaccharide composition of HPIDF changed significantly. F-HPIDF (Figure 5b) consisted of a less content of galactose (34.40%) and galacturonic acid (11.47%), meanwhile a higher content of arabinose (27.49%), rhamnose (5.32%), glucose (6.86%), and fucose (3.91%) than HPIDF. This showed that galactose and galacturonic acid were used as the carbon source of bacteria in the colon during fermentation.

**Figure 5.** The monosaccharide composition of HPIDF, F-HPIDF, and hydrolysate. (**a**: HPIDF; **b**: F-HPIDF; **c**: Hydrolysate).

As for the hydrolysate (Figure 5c), galactose (66.49%) and arabinose (15.00%) were the main components. The largest component in the hydrolysate should be considered as the monosaccharide that constituted the main component of HPIDF. As know, monosaccharides that make up hemicelluloses are mainly glucose, xylose, mannose, arabinose, and galactose [29]. This result proved that HPIDF is a dietary fiber with hemicellulose as the core. Then, during colonic fermentation, a large amount of hemicellulose was decomposed to free monosaccharides, which is a typical fermentation mode of hemicellulose [30].

The colonic fermentation model of HPIDF with hemicellulose as the main part should be regarded as one of the important findings of this study, which could help us to carry out more accurate research on the metabolic model of colonic microorganisms using HPIDF. On this basis, the metabolic pathway of free monosaccharides in the colon can be studied to make sure if they had any potential functional value. Even the modification of HPIDF

could be carried out based on the result, at least we can add modifiers to the functional groups of hemicellulose so that to make these components easier to release in the colon.

In this experiment, the microorganism used to ferment HPIDF was mainly *Lactobacillus* (genus level), and the main components of HPIDF were hemicellulose and lignin. The fermentation mode of hemicellulose by *Lactobacillus* is relatively clear. In short, it is a process of using hemicellulose as the carbon source to produce lactic acid in the colon [31,32]. An increase in lactic acid, as a precursor of propionic acid [33], results in an increase of shortchain fatty acid (SCFA). This phenomenon is beneficial to colon health and may even prevent colon cancer [34,35]. In contrast, the high level of lactic acid in the colon may also have adverse effects on intestinal health [36]. However, we believed that the lactic acid produced by dietary fiber fermentation should not pose this risk because they are ingested non-exogenously.

As the above process occurred, many new monosaccharides would appear in the colon, in which the composition was mainly galactose and arabinose. Galactose is a widely recognized functional monosaccharide with biological activities, such as being conducive to the appreciation of *Bifidobacterium* [37]. Galactose binding to lectin [38], a process that occurs in the colon before cancerization, proved that galactose plays an important role in intestinal health. Arabinose is also an intestinal prebiotic, which can prevent or even treat colitis by affecting the composition of intestinal flora directly [39]. In short, the monosaccharides produced by the fermentation of colonic flora would have a positive effect on intestinal health.
