Contribution of transglutaminase cross-linking to the physicochemical, bioactive properties and bioavailability of sodium caseinate hydrolysates

Various enzymatic modifications are applied to proteins and peptides during food processing. Treatment with microbial transglutaminase (TGase) can lead to improvements in the physicochemical properties of food proteins by introducing inter- and intra-molecular cross-links between amino acids. However, the effect of the polymerization of proteins/peptides with TGase on their biofunctional properties is still relatively unexplored. The aim of this research was to determine the effect of TGase treatment in conjunction with enzymatic hydrolysis on the physicochemical and bioactive properties of sodium caseinate (NaCN). NaCN was treated with TGase prior to and after hydrolysis with a Bacillus proteinase, Prolyve, leading to hydrolysate samples TGase/Prolyve and Prolyve/TGase, respectively. The non-TGase-treated sample was submitted to hydrolysis with Prolyve (Prolyve) and intact NaCN was used as a control. The degree of hydrolysis (DH) of the samples was ~ 17%. The physicochemical properties were characterized chromatographically, using gel permeation (GPC) and reverse phase (RP) approaches, showing similar profiles between the three hydrolysates generated. The hydrolysates were submitted to simulated gastrointestinal digestion and reached DH% values of ~ 33% showing no significant differences between samples. The in vitro dipeptidyl peptidase IV (DPP-IV) and angiotensin converting enzyme (ACE) inhibitory activity of the hydrolysates was significantly higher in comparison with intact NaCN. However, no significant differences were found between the TGase treated and non-TGase treated samples. Similar results were found with the in vitro antioxidant activities. At a cellular level some specific differences were found in cell proliferation depending on the cell line studied. The three hydrolysates had the ability to inhibit the production of the interleukin-6 (IL-6) cytokine, which suggested an anti-inflammatory activity. The three hydrolysates demonstrated cytotoxic properties in the carcinogenic Caco-2, HepG2 and MCF-7 cell lines and induced apoptosis in the U937 cell line. The hydrolysates were tested for their transport/bioavailability using the Caco-2 cell monolayer model. The percentage peptide permeation observed was between 2-5%, which is similar to the results reported in literature. The contents in the basolateral chambers of the cells incubated for 2 h with the hydrolysates were collected and freeze-dried. RP-HPLC analysis showed hydrolysate-dependent differences in the profiles. The highest oxygen radical antioxidant capacity (ORAC) activity was observed in the basolateral fluid obtained from the Prolyve/TGase treated cells. Ultra-performance liquid chromatography (UPLC) - mass-spectrometry analysis (MS/MS) of the basolateral contents of the Prolyve/TGase treated cells revealed that a high number of peptides were translocated across the Caco-2 cell membrane. Among these, a total of 124 peptides were previously reported as bioactive mainly displaying antioxidant, ACE and DPP-IV inhibitory activity. The results presented above indicate that, depending on the order of cross-linking, prior or following to hydrolysis, TGase-treated NaCN hydrolysates may have potential as multifunctional food ingredients.