Development of an Antioxidative Pickering Emulsion Gel through Polyphenol-Inspired Free-Radical Grafting of Microcrystalline Cellulose for 3D Food Printing

The manufacture of next-generation 3D-printed foods with personalized requirements can be accelerated by in-depth knowledge of the development of a multifunctional biopolymeric-based ink. As a fat replacer in the food industry, microcrystalline cellulose (MCC) has the potential to address the growing need for sustainable healthy reduced-fat 3D printed foods. The modification of MCC structure by polyphenols gives the way to produce a multifunctional antioxidative Pickering emulsion with improved emulsifying properties. In this study, different types of polyphenols, including gallic acid (GA), tannic acid (TA), and cyanidin-3-O-glucoside (C3G), were individually used to synthesize the grafted MCC-g-polyphenol conjugates by the free-radical grafting method. Then, the antioxidative grafted microconjugates were added to a soy protein-based emulsion gel to partially substitute its oil, and each Pickering emulsion gel variant was printed through an extrusion-based 3D printing system. Emulsifying properties and antioxidant character of MCC were proven to be enhanced after the fabrication of grafted microconjugates. Compared to MCC-g-TA, MCC-g-GA and MCC-g-C3G could efficiently improve the stability of a reduced-fat soy-based emulsion gel upon storage. Moreover, the reduced-fat soy-based emulsion gel containing grafted microconjugates endowed a characteristic shear-thinning behavior with a gel-like structure and superlative thixotropic properties. Following the printing, the antioxidative Pickering emulsion gels containing grafted microconjugates produced well-defined 3D structures with superior lubrication properties. This study demonstrated that the grafting of polyphenols onto MCC could enhance bioactive properties and improve emulsifying performance of MCC, making it a useful component in the development of personalized functional foods.


S.1. Antioxidant activity and reducing power of "Pickering emulsion gels"
In addition to the grafted MCC compound, the antimicrobial activity and reducing power of all obtained Pickering emulsion gels (including control SPI, SP/MC, SP/MC/GA, SP/MC/TA, and SP/MC/C3G inks) were performed. Compared to the pristine MCC or grafted MCC-polyphenol conjugates, the antimicrobial activity and reducing power of the respected inks were slightly decreased ( Figure S.1). The data exposed that the control SPI-based ink and SP/MC had the lowest DPPH scavenging effect or reducing power, while SP/MC/C3G ink offered the highest antioxidant activities. These results were not surprising, as the incorporation of polyphenols onto MCC could rationally induce a versatile antioxidant property with a promising therapeutic application. These results are also in accordance with those obtained for the grafted MCC-polyphenol conjugates.

S.2. Effect of different levels of micro-conjugate variants on functionally of SPI-based emulsion
In the preliminary assays in order to produce 'reduced-fat' Pickering emulsion gels, five different levels from each MCC-polyphenol conjugate variant were incorporated into the control SPI-based emulsion (10 wt.% canola oil, 25.0 wt.% SPI, pH 5.6) to be replaced with the part of its oil, according to the Method stated in the main Manuscript (Section 2.4). In this regard, the levels of 1.05, 2.1, 3.15, 4.2, and 5.25 wt.% of each MCC-polyphenol conjugate (i.e., MCC-g-GA, MCC-g-TA, and MCC-g-C3G) were added to the SPI-based emulsion (10 wt.% canola oil, 25.0 wt.% SPI, pH 5.6) to be replaced with 15, 30, 45, 60, and 75% of the oil in the system, respectively.
Based on emulsion stability data and rheological results, the MCC-polyphenol conjugate variants in the ranges of 1.05-3.15 wt.% did not change the initial mean particle diameter (d 3,2 ), poly-dispersity index (PDI), TSI, delta-backscattering, and apparent viscosity parameters. In contrast, the concentration of 4.2 wt.% of MCC-polyphenol conjugates (i.e., 60% oil-reduced ink) significantly affected all these parameters (P < 0.05), showing a high stable emulsion. However, beyond this level (i.e., 5.25 wt.%), the droplet size and TSI value notably increased, promoting some levels of emulsion instability.
In this case, since the results of 60% reduced-fat SPI-based Pickering was affected by the presence of MCC-polyphenol conjugate variants (in the level of 4.2 wt.%), the instrumental results of this sample were discussed.
The detailed physical results for this point have been listed as follows:

S.2.1. Particle diameter and polydispersity index
The initial mean particle diameter ((d 3,2 ) = 61 μm) and polydispersity index (PDI = 0.39) of the dropletscoated SPI (control ink) showed the presence of some flocculated oil droplets having a non-uniform particle size distribution.  In the cases of (d 3,2 ) and PDI, the means inside each column with various letters (a-e) are significantly different (P < 0.05) according to Duncan's test.

S.2.2 TSI measurement
The TSI values of various emulsions were calculated and plotted as a function of time ( Figure S.2). Again, the MCC-polyphenol conjugates in the ranges of 1.05-3.15 wt.% did not change the TSI parameter of SPI-based emulsion. The MCC-polyphenol conjugate variants in a concentration of 4.2 wt.%, by contrast, developed a stable SPI-based emulsion with a high level of stability. The better emulsion stability provided by grafted micro-conjugates (i.e., MCC-g-GA, MCC-g-TA, and MCC-g-C3G) may be attributed to an increase in the viscosity of the aqueous phase and also increasing the internal friction of the fluid (Shahbazi et al., 2021). Again, the grafted MCC-polyphenol conjugates in the level of 5.25 wt.% produced an important increase in the TSI value. This might be attributed to oil droplets' flocculation as a result of introducing a high level of MCC-polyphenol conjugates, where they could not be well-dispersed in the system. Alternatively, the interaction of SPI with MCC-polyphenol conjugates might decrease the thickness and charge of the lipid droplets. This weakens the flocculation stability of droplets by decreasing the steric and electrostatic repulsion between them.

S.2.3 Emulsion stability by vertical laser profiling
The stability analysis of soy-based emulsion after the addition of different levels (1.05-5.25 wt.%) of each MCC-polyphenol conjugate after 4 h storage at 25 °C was also performed using vertical laser profiling based on the transmission (ΔT) and delta-backscattering (ΔBS) profiles ( Figure S.3). The ΔBS of control emulsion decreased overall with time, signifying an incremental increase in the particle size caused by flocculation or coalescence, which is also according to TSI results.

S.3. Side view of 3D printed objects
After 4 h of the printing process, the 3D printed structures were moved to a specific chamber (20 × 20 × 20) cm 3 to take the photos from their side view by a digital camera (Alpha 7M3 E-Mount, Full-Frame Mirrorless, 24.2 MP, Sony, Tokyo, Japan) ( Figure S.4).  The spindle speed of a jump between the end of one extrusion and the next Perimeter P 10 -Number of outline layers Infill density ρ infill 90 % Quantity of material filling the object Table S2. The obtained viscosity, flow behavior index, and yield stress of SPI-based ink variants.