Effects of protein and peptides obtained from Okara, a by-product of soy-milk production, on human colon cancer Caco-2 cells

Beyond the nutritive role, recent research pointed out the potential beneficial effects on human health of foods proteins and peptides. These, may exert bioactivities, such as antioxidant and immunomodulating, relevant for maintaining well-being and to prevent diseases. Soybean seed proteins have been extensively reported in this respect. Okara is a prime agri-waste generated by soybean milk processing industry that still contains high amounts of nutrients, including proteins (25.4-28.4%) and polyphenols (up to 40 %) [1], [2] . The aim of this study was to investigate the possible effects on inflammation and anti-proliferative mechanisms of okara proteins (OP) and derived peptides, by using intestinal tumoral Caco-2 cells. OP were obtained by selective solubilization, separated from the insoluble fiber fraction by centrifugation, and concentrated by isoelectric precipitation at pH 4.5. Either the resulting supernatant (SN) and precipitate (PP) were used for subsequent analyses. Okara peptides were generated by proteolysis with pancreatin (OPEP1), and with pepsin followed by pancreatin (OPEP2), to simulate the gastro-intestinal digestion. Protein and peptide fractions were characterized by using electrophoretic and chromatographic methodologies. The anti-proliferative potential was investigated by MTT assay, after incubation of Caco-2 cells with okara samples. IL-8 gene expression was also assessed to evaluate the anti-inflammatory capacity of okara products. Its expression, downstream the activation of NF-kB, the major regulator of cellular inflammatory status was considered as a marker of inflammation [3] . The results indicated that PP, OPEP1 and OPEP2 can cause cell suffering and death in a dose dependent relation, whereas SN seems to exert no effects. No IL8 expression was observed in all cases. Interestingly, treatment with okara samples strongly enhanced the expression of IL8 on cells elicited by IL1, suggesting a synergic effect. By and large, the observed cytotoxic effects may be due to the presence of peptides already present in okara that may originate during the production of soybean drink. [1] S. P. Stanojevic, M. B. Barac, M. B. Pesic, V. S. Jankovic, e B. V. Vucelic-Radovic, «Bioactive proteins and energy value of okara as a byproduct in hydrothermal processing of soy milk», J. Agric. Food Chem., vol. 61, n. 38, pagg. 9210–9219, set. 2013, doi: 10.1021/jf4012196. [2] A. Orts et al., «Protease technology for obtaining a soy pulp extract enriched in bioactive compounds: isoflavones and peptides», Heliyon, vol. 5, n. 6, pag. e01958, giu. 2019, doi: 10.1016/j.heliyon.2019.e01958. [3] J. Capraro et al., «Characterization of Chenopodin Isoforms from Quinoa Seeds and Assessment of Their Potential Anti-Inflammatory Activity in Caco-2 Cells», Biomolecules, vol. 10, n. 5, Art. n. 5, mag. 2020, doi: 10.3390/biom10050795.