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

Angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory activities of transglutaminase treated sodium caseinate hydrolysates

Angiotensin converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities of crosslinked and non-cross-linked sodium caseinate (NaCN) hydrolysates were studied. Three different samples were generated: NaCN hydrolysed with Prolyve 1000 (TM) (Prolyve), NaCN cross-linked with transglutaminase (TGase) pre-Prolyve hydrolysis and NaCN cross-linked post-Prolyve hydrolysis. Gel filtration and reverse phase HPLC analysis of the resulting samples indicated that the hydrolysates had similar peptide profiles. Hydrolysates showed higher (p 0.05) differences in activity were found between cross-linked and non-cross-linked hydrolysate samples. Hydrolysate IC50 values for ACE and DPP-IV inhibition ranged from 0.10 to 0.17 mg mL(-1) and 0.85-1.18 mg mL(-1), respectively. Simulated gastrointestinal digestion had no significant (p > 0.05) effect on the bioactivities of the hydrolysates. The results demonstrated that incubation with TGase before or after NaCN hydrolysis with Prolyve had no effect on ACE or DPP-IV inhibitory activities. (c) 2017 Elsevier Ltd. All rights reserved

In vitro antioxidant and immunomodulatory activity of transglutaminase-treated sodium caseinate hydrolysates

Sodium caseinate (NaCN) was incubated prior to and after hydrolysis with a microbial transglutaminase (TGase) and hydrolysed with Prolyve 1000. The resultant hydrolysates were tested for their immunomodulatory and antioxidant activity. TGase-treated hydrolysates significantly reduced (p 0.05) differences were found between the TGase-treated and non-TGase-treated samples. In the presence of hydrogen peroxide, the non-TGase-treated sample exhibited the highest DNA protective effect in U937 cells. These findings suggest that NaCN derived hydrolysates with and without treatment with TGase may exert specific antioxidant, genoprotective and anti-inflammatory effects. (C) 2016 Elsevier Ltd. All rights reserved

Application of in silico approaches for the generation of milk protein-derived bioactive peptides

Milk protein derived peptides have numerous well-documented bioactive properties. The conventional approach for the generation, identification and validation of bioactive peptides (BAPs) has involved (i) protein hydrolysis, (ii) bioactivity screening and (iii) validation in vivo. The low potency (in comparison to conventional drugs), susceptibility to breakdown during gastrointestinal transit and low intestinal permeability are key challenges in the development of highly bioactive food protein hydrolysates/peptides. However, the generation of potent and effective health enhancing hydrolysates/peptides can benefit from a range of in silico techniques including the application of structure bioactivity relationship modelling (e.g., quantitative structure activity relationship (QSAR) modelling), molecular docking and design of experiments (DOE) approaches to optimise BAP production and identification. Some examples of how these approaches have been employed in BAP discovery and generation will be outlined

Role of carbohydrate conjugation on the emulsification and antioxidant properties of intact and hydrolysed whey protein concentrate

The conjugation of proteins with carbohydrates generates covalent bonds which may improve their techno- and biofunctional properties and therefore expand their applications in the food industry. In the present study, a design of experiments (DOE) approach was used to determine the effect of conjugation of whey protein concentrate (WPC) and WPC hydrolysates with carrageenan (CGN) on its emulsifying and antioxidant properties. The DOE was composed of 3 levels with 3 factors, i.e., WPC:CGN (1:1.0 1:3.5, 1:6.0), time of conjugation (6, 27 and 48 h) and degree of hydrolysis (DH; unhydrolysed, low DH and high DH). The conjugated samples were characterised for their oxygen radical absorbance capacity (ORAC) and emulsion activity (EA). Two samples, C3 (1:1.0, 6 h and unhydrolysed) and C4 (1:1.0, 6 h and low DH) with ORAC and EA values of 601.30 ± 14.71 and 709.32 ± 11.11 μmol Trolox equivalents g−1 freeze-dried powder and 0.51 ± 0.01 and 0.58 ± 0.01 absorbance units, respectively, were selected for further study. Emulsions were generated with WPC, non-conjugated (NC3 and NC4) and conjugated (C3 and C4) samples and their functional properties were compared. The NC3 and NC4 samples had higher viscoelastic moduli (G′ and G″) than C3, C4 and the WPC samples which indicated that more stable emulsions may be formed with non-conjugated samples. However, the NC3, NC4 and WPC samples showed low emulsion stability (ES) after 28 days storage with values ranging from 78.6 ± 3.5–85.6 ± 3.6% whereas the conjugated C3 and C4 samples had improved ES with significantly (p < 0.05) lower coalescence index values (64.2 ± 2.5 and 66.7 ± 3.7%, respectively). However, conjugation of the hydrolysate (C4) had a significant (p < 0.05) negative effect on lipid oxidation (0.62 ± 0.04 equivalents of 1,1,3,3-tetraethoxypropane (TEP)/kg emulsion) compared to the unhydrolysed conjugate C3 (0.42 ± 0.04 eq TEP/kg emulsion)

Effect of enzymatically hydrolysed brewers’ spent grain supplementation on the rheological, textural and sensory properties of muffins

Brewers’ spent grain (BSG) is the major co-product of the brewing industry having a high content of protein and fibre. Enzymatic modification improves the properties of different ingredients when they are incorporated into confectionary products. This study characterised the rheological, textural and sensory properties of unmodified BSG (BSGA) and enzymatically modified BSG (BSGB, i.e., proteinase and carbohydrase modified BSG) when substituted at different levels (5, 10 and 15%) in a muffin ingredient mix. Rheological assessment showed a lower viscosity for the BSGB compared to the BSGA batters. Baked BSGB containing muffins had lower hardness values compared to BSGA and control without added BSG. Sensory assessment showed no significant differences in liking of the different attributes tested (overall appearance, texture, smell, colour and taste) between the 5% BSGA and BSGB supplemented muffins. Incorporation of BSGB at 10% had no negative impact on the sensory attributes in comparison to BSGA 10%. Overall, low level substitution of enzymatic modified BSG beneficially reduced batter viscosity and muffin hardness and gave comparable sensory attributes to those of unmodified BSG supplemented muffins. Therefore, prior enzymatic modification can enhance the ingredient functionality of BSG in confectionary products

Enzymatic modification of porphyra dioica-derived proteins to Improve their antioxidant potential

Enzymatic hydrolysis has been employed to modify protein functional properties and discover new sources of antioxidants. In this study, the effect of different enzymatic treatments on antioxidant activity of Porphyra dioica (blades and protein isolate (PI)) was investigated. Protein nitrogen content of P. dioica blades and PI were 23 and 50% (dry weight), respectively. Blades and PI were hydrolyzed with Prolyve®and Prolyve®plus Flavourzyme®. Peptide profiles and molecular mass distribution of the hydrolysates were investigated. The hydrolysis promoted generation of peptides and low molecular mass components <1 kDa. Antioxidant activity was assessed using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH ) scavenging, 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS +) inhibition, and reactive oxygen species scavenging ability, i.e., oxygen radical absorbance capacity (ORAC) and hypochlorous acid (HOCl) scavenging assays. In general, enzymatic hydrolysis of P. dioica blades and PI enhanced the in vitro antioxidant activity. Direct hydrolysis of blades improved ORAC values up to 5-fold (from 610 to 3054 mol Trolox eq./g freeze dried sample (FDS). The simultaneous release of phenolic compounds suggested a potential synergistic activity (ORAC and ABTS + assays). Such hydrolysates may be of value as functional food ingredients