In Vitro Bioaccessibility of 3D-Printed Snacks Containing Spirulina

Growing interest in sustainable food systems has increased the demand for alternative protein sources. Spirulina (Limnospira spp; SP) is a protein-rich cyanobacterium that can also be used as a source of high-value compounds, such as C-phycocyanin. Following their extraction, the residual biomass (Spirupower^®; SPW) retains nutritional components to innovative foods. In this context, 3D food printing provides an opportunity to develop foods incorporating alternative protein sources. Therefore, this study aimed to evaluate in vitro protein bioaccessibility of 3D-printed snacks containing 5% (w/w) SP or SPW. Printing was performed at 40 mm.min⁻¹ using a 0.8 mm nozzle, 0.35 mm layer height, and 50% infill, with 100% infill every three layers (Genesis, 3D Biotechnology Solutions, Brazil). Printed snacks were baked at 175 °C for 20 min and dried at 50 °C for 20 min. Proximate composition was determined, and simulated gastrointestinal digestion was performed according to the INFOGEST protocol. Protein bioaccessibility was calculated from the difference between the initial protein content and the protein remaining after digestion, as determined by the Dumas method. Incorporation of SP and SPW significantly increased (p < 0.05) protein content from 4.76 ± 0.09 g.100 g^-1 in the control to 9.81 ± 0.02 and 11.53 ± 2.22 g.100 g^-1, respectively, corresponding to 2.06- and 2.42-fold increases. Protein bioaccessibility was 46.68 ± 5.34% and 48.21 ± 6.24% for SP- and SPW-enriched snacks, respectively, compared with 95.37 ± 1.12% for the control. These values were also lower than the approximately 56% previously reported for the respective raw biomasses using INFOGEST. Considering the snack matrix containing oat flour and cornstarch, the bioaccessibility reduction following SP and SPW incorporation may be associated with processing-induced interactions and/or physical entrapment of proteins within the matrix, limiting their release during gastrointestinal digestion. These matrix effects may occur in addition to cell walls that can restrict protein accessibility to digestive enzymes. The substantially higher bioaccessibility of the control, however, indicates that the carbohydrate matrix alone does not explain this effect, suggesting a matrix-dependent behavior of SP and SPW proteins. Nevertheless, SPW showed protein enrichment and bioaccessibility comparable to SP, supporting its valorization as an alternative protein ingredient within a circular food production approach.

Acknowledgments: This study was financed, in part, by the São Paulo Research Foundation (FAPESP), Brazil, process Number 2024/19218-0; 2023/00857-0. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant number 405238/2025-8, and A.R.C.B (PQ 305518/2024-0) was a recipient of a research productivity fellowship from CNPq. VIIAFOOD PRR - Plataforma de Valorização, Industrialização e Inovação comercial para o AgroAlimentar (C644929456-00000040) – project AltProtein (SONAE); FCT (strategic project UID/AGR/04129/2020 (LEAF)).