In order to investigate the morphological nature of the SDMCs, we used DIPA to characterize the four SDMCs species (Fig. 1). The SDMCs diameters and aspect ratios are summarized in Table S1. SDMCs particles that showed signs of cracks, noninherent large holes, or partial wall sectors were deemed broken. In comparison to the untreated group, the SDMCs incubated in SGF and SIF exhibited only slight differences in diameter, aspect and circularity. However, broken SDMCs can still be counted by visual inspection of the optical micrographs of each individual particle. As demonstrated in Fig. 1B, representative examples of intact and broken particles were assigned to each group. Cracks, large holes or partial wall sectors were all termed ‘broken’, and the ‘broken’ ratio was calculated for each group. Most of the broken camellia and cattail SDMCs remained as single particles, whereas the dandelion and lycopodium SDMCs were observed to break into several pieces (Fig. 1B).

Figure 1 DIPA analysis of the four SDMCs after incubation in simulated gastrointestinal fluids. (A) Boxplot representations of the broken particle population for the four SDMCs. Dots indicate the median and the whiskers indicate the highest and lowest points within 1.5 standard deviations. The broken particle population of untreated samples served as a control for each SDMCs species. (B) Representative DIPA images of intact and broken SDMCs. Full size image

The impressive morphological variation of pollen grains between various plant species was immediately evident upon the analysis of the physical characteristics of the four SDMCs. Of particular interest were the unique geometries of the apertures, which are the weakest site of the pollen outer wall40. Camellia SDMCs are triaperturate, with three cleavage planes (Fig. 2B). Cattail SDMCs are monopartite (Fig. 3B), dandelion SDMCs possess three endoapertures that are surrounded by ridges (Fig. 4B), and lycopodium SDMCs are trilete (Fig. 5B). Most breakages on the camellia SDMCs were observed along the aperture stripe, which suggests that the walls of the camellia SDMCs might be weakened and torn away during the incubation and washing processes, whereas only small holes were formed on the surface of the cattail SDMCs without bulk erosion. Given that the number, arrangement and shape of the apertures greatly influence the mechanical properties of the pollen grains40,41,42, it seems highly likely that the relative fragility of the camellia SDMCs can be attributed to its distinct aperture geometry.

Figure 2 SEM images of camellia SDMCs before and after degradation treatment with simulated gastrointestinal fluids (SGF and SIF). Surface morphology of (A) untreated camellia SDMCs and those treated for 24 h with (B) SGF or (C) SIF, at different magnifications. Full size image

Figure 3 SEM images of cattail SDMCs before and after degradation treatment with simulated gastrointestinal fluids (SGF and SIF). Surface morphology of (A) untreated cattail SDMCs and those treated for 24 h with (B) SGF or (C) SIF, at different magnifications. Full size image

Figure 4 SEM images of dandelion SDMCs before and after degradation treatment with simulated gastrointestinal fluids (SGF and SIF). Surface morphology of (A) untreated dandelion SDMCs and those treated for 24 h with (B) SGF or (C) SIF, at different magnifications. Full size image

Figure 5 SEM images of lycopodium SDMCs before and after degradation treatment with simulated gastrointestinal fluids (SGF and SIF). Surface morphology of (A) untreated lycopodium SDMCs and those treated for 24 h with (B) SGF or (C) SIF, at different magnifications. Full size image

The surface morphologies of the four SDMCs were examined for signs of surface degradation after SGF or SIF treatment. Camellia SDMCs exhibited a nanoparticle-assembled porous surface structure (Fig. 2C), cattail SDMCs exhibited a honeycombed surface with large irregular passages (Fig. 3C), dandelion SDMCs exhibited porous echinate ridges (Fig. 4C) and lycopodium SDMCs exhibited web-like microridges and a tripartite structure (Fig. 5C). We observed that the surface morphologies of the SDMCs remain largely unchanged after incubation for 24 h in SGF or SIF (Figs 1–4). This suggests that surface erosion does not play a major role in SDMCs degradation, concurring with previously reported findings on lycopodium SDMCs degradation in human blood plasma8,24.

We performed a comprehensive analysis of the FTIR spectra collected for the untreated and incubated SDMCs in order to identify any chemical changes and possible pathways of degradation (Fig. 6). To remove the effects of the sample thickness on the peak height, all the FTIR spectra were standardized to zero mean and unit variance (z-scores)35. The resultant functional group peaks for the SDMCs were assigned according to the literature35,37,43,44,45,46,47,48,49 and are summarized in Table 1. Briefly, a common peak at 1,516 cm−1 is observed for all four species of SDMCs, which is an attribution of UV sensitive aromatic rings in spropollenin35. Several peaks attributed to functionalized carbohydrates (e.g., C–O–C, C–OH) were observed between 1,200 and 900 cm−1, with the peak shapes varying between the different species. A strong peak at 1,745 cm−1, representing lipids, is observed only for the lycopodium SDMCs. Overall, as expected, the spectral differences between the four species of SDMCs, especially between 800 and 1,800 cm−1, were attributed to their different compositions.

Figure 6 FTIR spectra of SDMCs before and after incubation in SGF/SIF. The spectra presented are the means of six replicates. (A) Camellia, (B) cattail, (C) dandelion, and (D) lycopodium. Full size image

After incubation treatment, no significant changes in peak shape or number were observed, except for the 1,516 cm−1 peak in camellia SDMCs, which was absent after 1 h of incubation in SGF. The absorbance difference spectra, as presented in Fig. 7, further highlight the more noticeable change in the camellia SDMCs relative to the other SDMCs. Here, the FTIR absorbance difference spectrum of the camellia SDMCs was the most changed upon degradation treatment, with its hydroxyl, aliphatic and aromatic group peaks exhibiting significant decreases in peak intensity, whereas the carboxyl group peaks and the peaks in the 1,400–1,000 cm−1 range were seen to increase, likely due to the formation of new bonds, such as C–O bond. Similar results regarding the formation of new C–O bonds during the acetolysis of sporopollenin have been reported37. Cattail SDMCs appear to be the most stable and show the least fluctuation in their FTIR absorbance difference spectra. The hydroxyl groups in the lycopodium SDMCs were relatively stable in both SGF and SIF, whereas the other peaks presented a decreasing trend. These results demonstrate that the chemical signatures of SDMCs are altered to various degrees during incubation in gastrointestinal fluids, most likely owing to their species-dependent chemical composition.

Figure 7 FTIR absorbance difference spectra of the four species of SDMCs after degradation treatment. Each plot was produced with the treated sample mean spectrum minus the mean untreated spectrum. The gray dashed lines indicate the0 value. (A) Camellia, (B) cattail, (C) dandelion, and (D) lycopodium. Full size image

Semiquantitative analysis of peak heights was further performed by calculating the peak height ratios for different SDMCs functional groups (Fig. 8). Different internal standard peaks were used for each species, and the peak that showed the least variation in peak height and wavenumber was chosen. Typically, the 1,421 cm−1 peak was set as the internal standard for camellia, 989 cm−1 for cattail, 1,378 cm−1 for dandelion and 994 cm−1 for lycopodium. The peak heights for C = O, C = O, C–H, C–O–C and O–H groups were then standardized relative to the corresponding internal standard. The peak ratios were normalized relative to the untreated SDMCs, and a heat map was generated to better compare the chemical stability of all four SDMCs (Fig. 9).

Figure 8 Boxplot representations of peak height ratios for the different functional groups in the four species of SDMCs before and after incubation. Internal standard peaks were set as 1,421 cm−1 for camellia, 989 cm−1 for cattail, 1,378 cm−1 for dandelion, and 994 cm−1 for lycopodium. The dots indicate the median and whiskers indicate the highest and lowest points within 1.5 standard deviations. Full size image

Figure 9 Comparison of the normalized peak height ratios for different functional groups in the four species of SDMCs after different treatments. The color of the bar changes from red to blue, indicating that the peak ratio values increase from the minimum (0.314) to the maximum (1.153). For each SDMCs species, the peak ratio of each corresponding functional group was set as 1 (not shown). The peak ratio was the mean value of six replicates. Full size image

In order to further normalize and visualize the changes in peak height ratios, a heat map was constructed to represent the data (see Fig. 9). For the treated samples, the peak height ratios of the abovementioned functional groups were normalized to that of the untreated samples. C = C groups were seen to be unstable in all four SDMCs when incubated in SGF for 1 h. The greatest change was observed for camellia, which presented a decrease of ≈ 0.3 in the peak height ratio. A similar phenomenon has been reported for lycopodium sporopollenin, in which UV-B-absorbing compounds that contain double bonds were removed by oxidation49. This suggests that enzymatic lysis in gastric fluids could play a similar role for the plant SDMCs. However, upon treatment in SIF, the changes in the peak height ratios are more complex. While cattail and dandelion presented an increase in the C = C peak height ratio, a decrease was observed for camellia SDMCs, and no change was observed for lycopodium SDMCs. These results demonstrate that even though the main sporopollenin component of the SDMCs is common to a range of plant species, the diversity in the quantity and chemistry of its functional groups significantly affect its biodegradability.

Finally, PCA of the FTIR spectra was performed to observe data clustering for the untreated, SGF-treated and SIF-treated samples (Fig. 10). PCA is an ordinary multivariate analysis technique for data dimensionality reduction, thereby identifying the differences and similarities among the samples and the variables that constitute the modeled data50,51. The aim of PCA is to obtain a small set of principal components (PCs) that explain the most variable parameters of the data sets. This method has proved useful in the interpretation of FTIR spectra, which show band diversity and complication depending on the source of the sample52,53. In this work, standardized FTIR data sets, which consist of the wavenumber and the corresponding absorbance, were used to perform PCA. The first PC describes as much of the variability (variation of absorbances) in the data as possible, the second PC, orthogonal to the first, accounts for as much of the remaining variability as possible, and so forth49,52,53. We observed that the first two components account for more than 60% of the total variance for each species.

Figure 10 PCA of SDMCs before and after incubation in gastrointestinal fluids. (A) Camellia, (B) cattail, (C) dandelion, and (D) lycopodium. The cyan dots denote untreated samples, the blue dots denote samples treated with SIF for 1 h, the green dots denote samples treated with SIF for 24 h, the red dots denote samples treated with SGF for 1 h, and the black dots denote samples treated with SGF for 24 h. The ellipses represent 95% confidence intervals for each group. Full size image

The results of the PCA are presented in Fig. 10. Compounding our earlier results, only camellia shows a clear difference in IR spectra between the untreated control and the SGF- or SIF-treated SDMCs. Here, the first component (PC1) accounts for 85% of the variance in the FTIR database, thereby separating the untreated control from the SGF and SIF groups, mostly owing to a decrease in the 3,370 cm−1 and 1,516 cm−1 peaks (Fig. 7). The second component (PC2) accounted for only 4.3% of the variance, relating to fluctuations in the 3,370 cm−1 band and some weak peaks between 600 and 800 cm−1.

For cattails, although the SGF/SIF groups cannot be separated from the untreated control, SGF and SIF were separated at the 5% level of significance. The sums of variance, as presented by PC1 and PC2, are 54% and 16.7% (Fig. 10). Here, a clear separation is seen between SGF and SIF degradation (Fig. 10). For dandelion and lycopodium, all three groups overlap, indicating that the chemical changes here were not significant enough to be separated out by PCA. Thus, it may be inferred that dandelion and lycopodium SDMCs were stable during incubation with simulated gastrointestinal fluids. Finally, it was observed that prolonged incubation times in both SGF and SIF have no further significant effects on the SDMCs chemical signatures, indicating that most of the chemical changes/degradation occured within the first hour.

Thus, combining the results of PCA and peak height ratio calculations, we conclude that the stability and quantity of functional groups are dependent on differences in the composition and properties of the SDMCs obtained from different plant species. Intriguingly, although the FTIR and PCA analysis showed that chemical changes occur within the first hour of incubation (e.g., camellia SDMCs), the morphology of these SDMCs remained consistent. As surface erosion leads to the loss of surface material, while bulk erosion results in rapid internal degradation54, we propose that bulk erosion is the main mechanism for the degradation of SDMCs in SGF and SIF54,55.