*2.1. Starch Oxidation*

Purified starch from pea pod powder was enzymatically oxidized by using fungal laccase and the mediator TEMPO in mild reaction conditions and at a variable molar ratio (see Table 1), as described in Section 4.2.


1 Laccase was dissolved in 1 mL of water prior use. 2 Sample D was not enzymatically oxidized.

*Trametes versicolor* was chosen as a source of laccase because its carboxyl content was the largest compared to that of other enzymes [27] and because it is able to promote the oxidation of the mediator TEMPO in an unbu ffered water medium. During oxidation, the mediator TEMPO is converted into an oxonium ion, able to selectively act on the primary hydroxyl groups present on pea pod starch (PS) chains, thus generating aldehyde groups. The chemical nature of PS derivatives was deeply investigated by mono- and two-dimensional NMR spectroscopy. Results were perfectly in line with the literature data, supporting experimentally the occurrence of the oxidation in low yield (10%) as observed for other polysaccharides, such as polygalactomannans [25] arabinoxylan and konjac glucomannans [10], for which a maximum of 12% of oxidation degree was reached. As a consequence, the resonances of the partially oxidized PS, in the proton spectrum of Figure 1, have small intensities overlapping with those of the native PS. The obtainment of aldehyde derivatives was confirmed by the appearance in the proton spectrum of characteristic resonances at 9.23 and 9.28 ppm in the 1H spectrum of Figure 1. Moreover, 1H chemical shifts of partially oxidized PS were assigned and are listed in Table S1 according to available literature data and two-dimensional experiments. Some peculiar signals in the proton spectrum were very close to those reported for acetylated starches [28,29] and oxidized polygalactomannans [24,25].

**Figure 1.** 1H NMR spectrum of partially oxidized pea starch (sample C), recorded at 298 K, in D2O (for simplicity, only the numbering scheme of the amylopectin derivative is illustrated; the assignment refers to partially oxidized starch).

In the 13C spectrum of partially oxidized PS in Figure S3, the signals of produced acid residues are scarcely detectable because of the low yield of the oxidization reaction and especially the absence of NOE signal enhancement together with a long relaxation delay. In addition, the carbonyl signal expected in the 190–200 ppm region in the 13C spectrum cannot be easily detected, thus indicating that the aldehyde groups are hydrated or forming hemiacetals with the hydroxyl groups [30]. Nevertheless, the formation of a carboxyl carbon derivative for the modified PS was confirmed by the HMBC spectrum in Figure 2; this is because of the major sensitivity of the experiment along the proton dimension. Analyzing in detail the carboxyl region in Figure 2 (at 170–190 ppm), it is possible to observe a cross peak at 177.9 ppm referring to a *3JCH* correlation of a proton at 4.1 ppm with the carboxyl atom. This cross peak was assigned to the newly formed carboxyl group at the C6 position of the modified PS correlating through three bonds with the H4 proton. The enhanced technique also allowed easier detection of minor peaks attributable to the partially oxidized PS. Indeed, signals related to hemiacetalic derivatives were revealed from the HMBC analysis (at ca. 80–90 ppm), in agreemen<sup>t</sup> with literature data [25,30,31] and confirming the 13C NMR data. These derivatives can be justified from the chemo-enzymatic oxidation mechanism that supports the creation of a crosslinked network between the newly formed carbonyl and free hydroxyl groups. These groups are able to form intraand inter-chain hemiacetalic bonds that are finally responsible for the modified material behavior.

**Figure 2.** 1H–13C HMBC spectrum of partially oxidized pea pod starch (PS) from sample C in D2O at 298 K. Some characteristic resonances are highlighted and refer to minor components from the oxidation process assigned with this technique.

Finally, the entire spin system was verified by 2D 1H–1H double quantum filtered-total correlation spectroscopy (TOCSY) experiment reported in Figure S4.
