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Silkworm (Bombyx mori) pupae is a by-product from the silk industry which is rich in protein. Hydrolysates from silkworm pupae generated using Alcalase®, Prolyve®, Flavourzyme® and Brewers Clarex® proteolytic prepa- rations were characterised. The antioxidant activity of the hydrolysates was investigated using in vitro antioxi- dant assays and an in situ assay for reactive oxygen species (ROS) reduction using hepatic HepG2. Overall, Alcalase and Prolyve hydrolysates had highest scavenging activities, however, Flavourzyme and Brewers Clarex hydrolysates had enhanced ferric reducing antioxidant power (FRAP) activity compared to the other samples. Furthermore, the Flavourzyme hydrolysate significantly reduced ROS by 40% compared to untreated control HepG2 cells. Peptides identified by LC-MS/MS were synthetised and then tested for their in vitro and in situ antioxidant activity. Peptides SWFVTPF and NDVLFF showed highest antioxidant activity (ROS reduction, su- peroxide dismutase (SOD) expression and glutathione (GSH) production activity) in HepG2 cells, and therefore may have potential as natural antioxidants.

peer-reviewed, The full text of this article will not be available in ULIR until the embargo expires on the 21/01/2021, Twenty-two novel dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides (with IC50 values <200 µM) and fifteen novel insulinotropic peptides were identified in a boarfish protein hydrolysate generated at semi-pilot scale using Alcalase 2.4L and Flavourzyme 500L. This was achieved by bioassay-driven semi preparative reverse phase-high performance liquid chromatography fractionation, liquid chromatography-mass spectrometry and confirmatory studies with synthetic peptides. The most potent DPP-IV inhibitory peptide (IPVDM) had a DPP-IV half maximal inhibitory concentration (IC50) values of 21.72 ± 1.08 µM in a conventional in vitro and 44.26 ± 0.65 µM in an in situ cell-based (Caco 2) DPP-IV inhibition assay. Furthermore, this peptide stimulated potent insulin secretory activity (1.6 fold increase compared to control) from pancreatic BRIN-BD11 cells grown in culture. The tripeptide IPV exhibited potent DPP-IV inhibitory activity (IC50: 5.61 ± 0.20 µM) comparable to that reported for the known DPP-IV inhibitor IPI (IC50: 5.61 µM). Boarfish proteins contain peptide sequences with potential to play a role in glycaemic management in vivo., peer-reviewed

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.

The current consumer demand for healthier diets, the growing interest in the search for new sources of protein, and the desire to reduce the negative effects on the environment have increased interest in the study of insect proteins. The present study focused on the technofunctional characteristics (interfacial and foaming properties) and the in-vitro antioxidant activity of a protein concentrate obtained from silkworm (Bombyx mori) pupae (SPC). The isoelectric point of the SPC was close to pH 4.0–5.0 as determined by protein solubility and z potential analysis. Given that the SPC had solubilities of ~50% and z potentials of ~20 mV at pH 2.0 and 8.0, it was decided to further study SPC properties at these pH values. The supernatant obtained after adjustment of SPC to pH 8.0 showed higher (p < 0.05) antioxidant activity than that at pH 2.0 when analysed by the ferric reducing antioxidant power (FRAP) assay (168.0 ± 3.0 V. 43.5 ± 8.1 μmol Trolox Eq. ·g−1 protein). However, no significant differences in antioxidant activity were found between pH 2.0 and 8.0 when using the oxygen radical absorbance capacity (ORAC) assay (1826.0 ± 131.9 vs. 1659.2 ± 46.8 μmol Trolox Eq. g−1 protein). The interfacial properties of SPC were determined at pH 2.0 and 8.0 during protein adsorption and after reaching the pseudo equilibrium state by means of dilatational and interfacial shear rheology following by foaming capacity and stability analyses. Faster adsorption kinetic values were obtained at pH 8 ( Vs. at pH 2.0). However, lower kinetic values at pH 2.0 increased the elastic behaviour of the viscoelastic interfacial film formed (E's ⁓ 30 mN/m at pH 2.0 V. E's ⁓ 20 mN/m at pH 8.0), which can be related with the higher protein sizes found at pH 2.0. These rearrangements of the SPC components appeared to increase its foaming capacity, whereas the foaming capacity of SPC adjusted to pH 8.0 was minimal.

peer-reviewed, Much interest is currently focused on plants such as legumes as an alternative source of protein for human nutrition. In this work a Vicia faba (fava bean, FB) protein concentrate was characterised for its ability to stabilise sunflower oil, oil-in-water (o/w) emulsions at three different pHs (3.0, 5.0 and 8.0). Emulsion microstructure was characterised by droplet size distribution (DSD) and optical microscopy, and resulting rheological properties were assessed. Smaller droplet sizes and better rheological properties (G′ and G″) were obtained at pH 3.0 and 8.0, i.e., at pH values away from the isoelectric point (pH 5.0). The in vitro bioactive properties of the emulsions at pH 3.0, 5.0 and 8.0 were tested using the ferric-reducing antioxidant power (FRAP) and the oxygen radical absorbance capacity (ORAC) antioxidant assays. The angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities were also determined. The highest antioxidant activity was observed when the emulsions were generated at pH 8.0 with FRAP and ORAC values of 935.7 ± 151.5 and 368.8 ± 2.3 μmol Trolox Eq. · g−1 of emulsion, respectively. Overall, the emulsions were poor inhibitors of ACE and DPP-IV. However, subjection of the emulsions to in vitro simulated gastro-intestinal digestion (SGID) released peptides which increased ACE and DPP-IV inhibitory activity (67.4 ± 2.7% and 11.2 ± 1.1%, respectively, for emulsions generated at pH 8). The emulsions generated at pH 3.0 displayed the lowest extent of lipid oxidation following 15 d storage. These results show the capability of FB protein concentrate to stabilise o/w emulsions while also demonstrating antioxidant along with ACE and DPP-IV inhibitory properties following gastrointestinal digestion.

peer-reviewed, The influence of dairy cow feeding regime was investigated using H-1 nuclear magnetic resonance (NMR). Two different NMR analytical systems were deployed: high field H-1 NMR to investigate the influence on milk metabolomics and low field NMR to characterise proton relaxation linked to changes in the state of mozzarella cheese moisture during ripening. The metabolomics results showed that grass-based feeding increased the concentration of a biological marker that signifies near-organic milk production conditions. On the other hand, the investigation of cheese moisture distribution showed that grass-based diets reached final moisture partitioning in a shorter time, which implied the formation of a more compact protein structure in the cheese matrix. These results indicate that pasture-based dairying may be differentiated in terms of the provenance of milk produced along with the accrual of additional benefits during ripening of the resulting mozzarella cheeses. (C) 2018 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, 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., PUBLISHED, peer-reviewed

peer-reviewed, A blue whiting (Micromesistius poutassou) protein hydrolysate generated using Alcalase 2.4L and Flavourzyme 500L and its simulated gastrointestinal digestion (SGID) sample was assessed for antidiabetic potential in vitro and in vivo. In addition to inhibiting dipeptidyl peptidase-IV (DPP–IV), the hydrolysates mediated insulin and glucagon-like peptide-1 (GLP-1) release from BRIN-BD11 and GLUTag cells, respectively. No significant difference was observed in insulinotropic and DPP-IV inhibitory activity following SGID, while GLP-1 secretion increased significantly (p < 0.01). SGID resulted in a significant increase in membrane potential, intracellular calcium and cyclic AMP concentration (p < 0.001) versus a glucose control, indicating that insulin secretion may be mediated by the KATP channel-dependent and the protein kinase A pathways. Additionally, acute (90–120 min) and persistent (4 h) glucose-lowering effects of the blue whiting hydrolysate were observed in normal healthy mice. These results demonstrate that the blue whiting protein hydrolysate had significant metabolic effects relevant to glucose control in vivo., ACCEPTED, peer-reviewed

peer-reviewed, Proteins derived from the macroalgal species Palmaria palmata have emerged as potential substrates for the generation of bioactive peptides. The aim of this study was to fractionate, identify and characterize antioxidant peptides from a P. palmata protein hydrolysate. The P. palmata protein hydrolysate generated with the food-grade proteolytic enzyme Corolase PP was sequentially fractionated using solid phase extraction and semi-preparative (SP) RP-HPLC. The most active SP-RP-HPLC peptide fraction (SP-RP-HPLC-30-F26) was analysed by ESI-MS/MS. Seventeen novel peptide sequences were identified in this fraction. Of the peptides selected for synthesis, Ser-Asp-Ile-Thr-Arg-Pro-Gly-Gly-Asn-Met, showed the highest oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) activity with values of 152.43 ± 2.73 and 21.23 ± 0.90 nmol TE/μmol peptide, respectively. The results presented herein indicate that P. palmata derived peptides may have potential applications as health enhancing ingredients and as food preservatives due to their antioxidant activity., ACCEPTED, peer-reviewed

peer-reviewed, β-Lactoglobulin hydrolysates (βlgHs) were generated using elastase at enzyme-to-substrate ratios (E:S) of 0.5, 1.0 and 1.5% in order to reach target degree of hydrolysis (DH) values of 9 and 13%. The impact of different E:S during manufacture on hydrolysates having similar DHs was assessed. Samples with similar DHs generated with different E:S showed comparable molecular mass distribution profiles and in vitro dipeptidyl peptidase IV (DPP-IV) inhibitory activities (p > 0.05). Liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis showed that 62 and 84% of the peptides identified were common within hydrolysates having a similar DH of 9 or 13%, respectively. Differences in the peptides identified within hydrolysates having similar DHs may be due to E:S dependent modifications in specificity and enzyme kinetics. Overall, this study showed that reduction in E:S while targeting the development of a similar DH for βlgHs may be employed to reduce the cost of hydrolysate production without having an adverse impact on the bioactivity and physicochemical properties studied herein, ACCEPTED, peer-reviewed

peer-reviewed, Whey protein isolate (WPI) solutions (100 g L−1 protein) were subjected to a heat-treatment of 80 °C for 10 min. Unheated and heat-treated WPI solutions were hydrolysed with Corolase® PP at pilot-scale to either 5 or 10% degree of hydrolysis (DH). Hydrolysates were subsequently processed via cascade membrane fractionation using 0.14 μm, and 30, 10, 5 and 1 kDa cut-off membranes. The compositional and molecular mass distribution profiles of the substrate hydrolysates and membrane processed fractions were determined. Whole and fractionated hydrolysates were assayed for both angiotensin-I-converting enzyme (ACE) inhibitory activity and ferrous chelating capabilities. A strong positive correlation (P < 0.01) was established between the average molecular mass of the test samples and the concentration needed to chelate 50% of the iron (CC50) in solution. The lowest ACE inhibition concentration (IC50 = 0.23 g L−1 protein) was determined for the 1 kDa permeate of the heat-treated 10% DH hydrolysate.

peer-reviewed, 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., peer-reviewed

peer-reviewed, This review, focusing on studies published between 2005 and 2017, analysed the literature on the generation of bioactive peptides (BAPs) from edible insect proteins following enzymatic hydrolysis. The protein extraction and quantification methodologies used for edible insects varied considerably. While several edible insects have been evaluated for their ability to release BAPs, silkworm (Bombyx mori) is currently the most studied. Specifically, the angiotensin converting enzyme (ACE) inhibitory, antioxidant and antidiabetic properties of edible insect protein enzymatic hydrolysates have been studied. Potent in vitro ACE inhibitory and antioxidant hydrolysates/peptides have been reported. In certain instances, these properties were validated in small animal studies (i.e. hypotensive effects). Enzymatic hydrolysis of edible insect proteins may also enhance technofunctional properties (i.e. solubility). The wider application of enzymatic hydrolysis protocols to edible insect proteins may ultimately allow for the increased discovery and utilisation of novel BAPs as sustainable protein/peptide sources for human nutrition., ACCEPTED, peer-reviewed

peer-reviewed, Nine novel dipeptidyl peptidase IV (DPP-IV) inhibitory peptides (FLQY, FQLGASPY, ILDKEGIDY, ILELA, LLQLEAIR, LPVP, LQALHQGQIV, MPVQA and SPVVPF) were identified in camel milk proteins hydrolysed with trypsin. This was achieved using a sequential approach combining liquid chromatography tandem mass spectrometry (LC-MS/MS), qualitative/quantitative structure activity relationship (QSAR) and confirmatory studies with synthetic peptides. The most potent camel milk protein-derived DPP-IV inhibitory peptides, LPVP and MPVQA, had DPP-IV half maximal inhibitory concentrations (IC50) of 87.0 ± 3.2 and 93.3 ± 8.0 µM, respectively. DPP-IV inhibitory peptide sequences identified within camel and bovine milk protein hydrolysates generated under the same hydrolysis conditions differ. This was linked to differences in enzyme selectivity for peptide bond cleavage of camel and bovine milk proteins as well as dissimilarities in their amino acid sequences. Camel milk proteins contain novel DPP-IV inhibitory peptides which may play a role in the regulation of glycaemia in humans., ACCEPTED, peer-reviewed

peer-reviewed, A multifactorial [temperature (40, 50 and 60 °C), hydrolysis time (60, 150 and 240 min) and enzyme to substrate ratio (E:S; 1.0, 1.5 and 2.0%)] design of experiments (DOE) was used to optimise the release of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides during hydrolysis of bovine milk protein isolate (MPI) with Neutrase 0.8L™, yielding 15 hydrolysates (H1-H15). Variation in temperature and time had a significant effect on DPP-IV inhibitory properties (p < 0.05) in contrast with E:S (p > 0.05). The DPP-IV half maximal inhibitory concentration (IC50) of H4, a potent sample, was maintained following simulated gastrointestinal digestion (SGID, DPP-IV IC50 = 0.60 ± 0.06 vs. 0.58 ± 0.09 mg ml−1, p > 0.05). Several peptides with DPP-IV inhibitory features or known activity were identified by liquid chromatography-tandem mass spectrometry (LC–MS/MS) within the hydrolysates. MPI hydrolysates may have potential for use as dietary ingredients with serum glucose lowering activity in humans., ACCEPTED, peer-reviewed

peer-reviewed, The full text of this article will not be available on ULIR until the embargo expires on the 9/2/2018, The release of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from bovine milk protein isolate (MPI) during trypsin hydrolysis was studied using a design of experiments (DOE) approach. A 3 factor×3 level DOE including temperature (40, 50 and 60°C), enzyme to substrate ratio (E:S; 0.50, 1.25 and 2.00% (w/w)) and hydrolysis time (60, 150 and 240min) was used during the generation of 15 hydrolysates (H1-H15). The degree of hydrolysis (DH) varied between 6.98±0.31 (H8) to 12.75±0.62% (H10). The DPP-IV half maximal inhibitory concentration (IC50) ranged from 0.68±0.06 (H11)/0.68±0.10 (H4) to 1.59±0.11mgmL-1 (H8). Temperature had no effect (p>0.05) on the DPP-IV IC50 value, while an increase in E:S or time significantly decreased DPP-IV IC50 value (p<0.05). The DPP-IV IC50 value of 0.69mgmL-1, predicted by response surface methodology (RSM), to be obtained with an hydrolysate generated at 50.5°C, 2% ES and 231min (H16) was similar to the experimentally obtained value (DPP-IV IC50=0.66±0.10mgmL-1, p>0.05, n=3). Following simulated gastrointestinal digestion (SGID) of H16 (H16_CorPP), the DPP-IV IC50 value increased (p<0.05) to 0.90±0.07mgmL-1. There was no significant difference between the DPP-IV IC50 value of the SGID of MPI (MPI_CorPP, 0.89±0.11mgmL-1) and that of H16_CorPP. Potent known DPP-IV inhibitory peptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) within H16, some of which were also present within H16_CorPP. MPI hydrolysates may be of interest for serum glucose regulation in humans., ACCEPTED, peer-reviewed

peer-reviewed, Background: The widespread application of protein-derived bioactive peptides (BAPs) with health promoting properties in human nutrition is currently limited. This may be due to the fact that several challenges exist in the discovery and identification of BAPs both in vitro and in vivo. Scope and approach: To date, most BAP studies have been conducted following a so-called conventional approach. This is based on the non-targeted release of BAPs in vitro, followed, in certain instances, with their subsequent evaluation in vivo. However, more targeted approaches have recently been described for the release of specific BAPs in a more predictable and efficient manner. These targeted approaches are mostly based on in silico protocols (e.g., peptide cutters, molecular docking, quantitative structure activity relationship (QSAR) models) aimed at predicting the release and/or the bioactivity of specific peptides. Key findings: Targeted approaches have, in certain instances, resulted in the development of particularly potent BAPs/hydrolysates and the discovery of novel BAP sequences. In addition, significant progress has been made in the identification of short peptides, involving the utilisation of multi-stage processes combining various physicochemical, analytical and in silico tools. This has allowed identification of novel sequences which are more relevant to human health from a bioavailability and stability perspective. BAPs have successfully been detected and quantified in human samples (e.g., serum, intestinal contents and urine) using different liquid chromatography-mass spectrometric (LC-MS) methodologies. In addition, human dose-response studies have allowed determination of their in vivo potency and efficacy, which in turn contributes to the development of scientific dossiers for regulatory approval., ACCEPTED, peer-reviewed

peer-reviewed, Dipeptidyl peptidase IV (DPP-IV) inhibitory peptides were identified in silico within camel milk proteins. Camel milk was hydrolysed with trypsin using a design of experiments (DOE, temperature (40–60 C), enzyme to substrate (E:S) ratio (0.50–2.00% (w/w)) and time (60–240 min)). Fifteen hydrolysates (H1–H15) having DPP-IV half maximal inhibitory concentration (IC50) values between 0.52 ± 0.06 (H9) and 1.26 ± 0.13 (H1) mg mL 1 were produced. Camel and bovine milk proteins hydrolysed at 40 C, 1.8% E:S and 218 min had DPP-IV IC50 values of 0.68 ± 0.08 and 0.85 ± 0.10 mg mL 1 (p < 0.05), respectively. Potent and unique DPP-IV inhibitory peptides (Leu-Pro-Val-Pro-Gln and Trp-Lys) were identified in camel milk protein hydrolysates, which were not present in bovine milk protein hydrolysates. The DPP-IV inhibitory properties of camel milk peptides were reported for the first time in this study. Camel milk is an interesting substrate to further investigate for its antidiabetic potential., ACCEPTED, peer-reviewed

peer-reviewed, Short peptides in food protein hydrolysates are of significant interest as they may be highly bioactive whilst also being bioavailable. A dipeptidyl peptidase IV (DPP-IV) inhibitory whey protein hydrolysate (WPH) was fractionated using nanofiltration (NF) with a 200 Da MWCO membrane. The DPP-IV half maximal inhibitory concentration of the NF permeate (IC50 = 0.66 ± 0.08 mg protein equivalent mL− 1) was significantly more potent (P > 0.05) than that of the starting WPH (IC50 = 0.94 ± 0.24 mg protein equivalent mL− 1) and associated retentate (IC50 = 0.82 ± 0.13 mg protein equivalent mL− 1). This confirmed the contribution of short peptides within the NF permeate to the overall DPP-IV inhibitory activity. An hydrophilic interaction liquid chromatography (HILIC-) and reverse-phase (RP-) liquid chromatography tandem mass spectrometry (LC–MS/MS) strategy, based on two retention time models, allowed detection of eight free amino acids and eight di- to tetrapeptides in the NF permeate. The potential sequences of the peptides within the NF permeate were then ranked on the basis of their highest probability of occurrence. A confirmatory study with synthetic peptides showed that valine–alanine (VA), valine–leucine (VL), tryptophan–leucine (WL) and tryptophan–isoleucine (WI) displayed DPP-IV IC50 values < 170 μM. The NF and LC–MS strategies employed herein represent a new approach for the targeted identification of short peptides within bioactive food protein hydrolysates., ACCEPTED

peer-reviewed, The potential of quinoa to act as a source of dipeptidyl peptidase IV (DPP-IV) inhibitory and antioxidant peptides was studied. A quinoa protein isolate (QPI) with a purity of 40.73 ± 0.90% was prepared. The QPI was hydrolysed at 50 °C for 3 h with two enzyme preparations: papain (P) and a microbial papain-like enzyme (PL) to yield quinoa protein hydrolysates (QPHs). The hydrolysates were evaluated for their DPP-IV inhibitory and oxygen radical absorbance capacity (ORAC) activities. Protein hydrolysis was observed in the QPI control, possibly due to the activity of quinoa endogenous proteinases. The QPI control had significantly higher DPP-IV half maximal inhibitory concentrations (IC50) and lower ORAC values than QPH-P and QPH-PL (P < 0.05). Both QPH-P and QPH-PL had similar DPP-IV IC50 and ORAC values. QPH-P had a DPP-IV IC50 value of 0.88 ± 0.05 mg mL−1 and an ORAC activity of 501.60 ± 77.34 μmol Trolox equivalent (T.E.) g−1. To our understanding, this is the first study demonstrating the in vitro DPP-IV inhibitory properties of quinoa protein hydrolysates. QPHs may have potential as functional ingredients with serum glucose lowering properties., ACCEPTED

peer-reviewed, An in silico approach was developed to predict the potential of 72 dietary proteins to act as a source of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. The model takes 68 DPP- IV inhibitory peptides (having an IC50 value < 2000 μM) and the specific contribution of their amino acids into account. Bovine α-lactalbumin (α-La) and κ-casein (CN) displayed the highest protein coverage (PC, 43.9 %) and potency index (PI, 17.9 10-6 μM-1 g-1), respectively for DPP-IV inhibitory peptides. Sequence alignment of 39 DPP-IV inhibitory peptides having IC50’s < 200 μM revealed the frequent occurrence of Trp at the N-terminus and Pro at position 2. Canola, chicken egg, oat and wheat were identified as potential sources of DPP-IV inhibitory peptides. In silico approaches may assist in the selection of food proteins for the enzymatic release of DPP-IV inhibitory peptides. The results are relevant to the generation of biofunctional ingredients for glycaemic management., ACCEPTED

peer-reviewed, Enzymatic hydrolysis of whey protein (WP) was carried out under pH-controlled and non pH-controlled conditions using papain and a microbial-derived alternative (papain-like activity). The impact of such conditions on physicochemical and bioactive properties was assessed. WP hydrolysates (WPH) generated with the same enzyme displayed similar degree of hydrolysis. However, their reverse-phase liquid chromatograph mass spectrometry peptide profiles differed. A significantly higher oxygen radical absorbance capacity (ORAC) value was obtained for WP hydrolysed with papain at constant pH of 7.0 compared to the associated WPH generated without pH regulation. In contrast, there was no significant effect of pH regulation on dipeptidyl peptidase IV (DPP-IV) properties. WP hydrolysed with papain-like activity under pH regulation at 7.0 displayed higher ORAC activity and DPP-IV inhibitory properties compared to the associated WPH generated without pH regulation. This study has demonstrated that pH conditions during WPH generation may impact on peptide release and therefore on WPH bioactive properties. (c) 2015 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, Xanthine oxidase (XO) and dipeptidyl peptidase IV (DPP-IV) inhibition by amino acids and dipeptides was studied. Trp and Trp-containing dipeptides (Arg-Trp, Trp-Val, Val-Trp, Lys-Trp and Ile-Trp) inhibited XO. Three amino acids (Met, Leu and Trp) and eight dipeptides (Phe-Leu, Trp-Val, His-Leu, Glu-Lys, Ala-Leu, Val-Ala, Ser-Leu and Gly-Leu) inhibited DPP-IV. Trp and Trp-Val were multifunctional inhibitors of XO and DPP-IV. Lineweaver and Burk analysis showed that Trp was a non-competitive inhibitor of XO and a competitive inhibitor of DPP-IV. Molecular docking with Autodock Vina was used to better understand the interaction of the peptides with the active site of the enzyme. Because of the non-competitive inhibition observed, docking of Trp-Val to the secondary binding sites of XO and DPP-IV is required. Trp-Val was predicted to be intestinally neutral (between 25% and 75% peptide remaining after 60 min simulated intestinal digestion). These results are of significance for the reduction of reactive oxygen species (ROS) and the increase of the half-life of incretins by food-derived peptides. (C) 2013 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, Background: The workflow used for the discovery of novel milk protein-derived bioactive peptides (BAPs) has significantly evolved over the past number of years. Many of the approaches currently described in the literature for the study of milk protein-derived BAPs have taken similar routes as those conventionally used in drug discovery. However, because milk protein-derived BAPs are prepared from dietary sources and in a food-grade manner, they differ from drugs in that they are generally present in a complex mixture as opposed to drug active compounds which are generally synthetically produced, having a relatively high level of purity.Scope and approach: This review assesses the utilisation of drug discovery methods for the identification of novel milk protein-derived BAPs. Different methods relevant to drug discovery have already proven effective for the discovery of new BAPs from milk proteins.Key findings and conclusions: However, reversing the workflow, starting from peptide identification in vivo to in vitro screening followed by conventional drug discovery approaches may have several merits. These include (a) the design of more targeted studies, (b) the development of novel approaches for the generation of milk, protein hydrolysates and (c) the discovery of milk BAPs which may be bioavailable in humans. These BAPs may find applications in the commercial generation of dietary ingredients with health promoting effects in humans. (C) 2016 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, Quantitative structure activity type models were developed in an attempt to predict the key featuresof peptide sequences having dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The models were thenemployed to help predict the potential of peptides, which are currently reported in the literature to bepresent in the intestinal tract of humans following milk/dairy product ingestion, to act as inhibitors ofDPP-IV. Two models (z- and v-scale) for short (2–5 amino acid residues) bovine milk peptides, behavingas competitive inhibitors of DPP-IV, were developed. The z- and the v-scale models (p < 0.05, R2of 0.829and 0.815, respectively) were then applied to 56 milk protein-derived peptides previously reported inthe literature to be found in the intestinal tract of humans which possessed a structural feature of DPP-IV inhibitory peptides (P at the N2position). Ten of these peptides were synthetized and tested fortheir in vitro DPP-IV inhibitory properties. There was no agreement between the predicted and experi-mentally determined DPP-IV half maximal inhibitory concentrations (IC50) for the competitive peptideinhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitorswas conserved. Furthermore, potent in vitro DPP-IV inhibitory activity was observed with two peptides,LPVPQ (IC50= 43.8 ± 8.8 M) and IPM (IC50= 69.5 ± 8.7 M). Peptides present within the gastrointestinaltract of human may have promise for the development of natural DPP-IV inhibitors for the managementof serum glucose., ACCEPTED, peer-reviewed

peer-reviewed, The influence of different substrates and the effect of pH regulation during enzymatic hydrolysis of whey protein on glucose-stimulated insulin secretion by BRIN-BD11 pancreatic beta cells were studied. No significant differences (P >= 0.05) were detected in terms of glucose-stimulated insulin secretion by BRIN-BD11 pancreatic beta cells exposed to whey protein hydrolysates (WPH1 and WPH2) obtained with two different whey protein substrates. The whey protein hydrolysate (WPH3) obtained without pH regulation during hydrolysis, had a significantly lower insulinotropic potential in BRIN-BD11 cells than the WPH1 hydrolysate that was manufactured with pH regulation. Fractionation of WPH1 was carried out to enrich bioactive peptides. Comparing the different fractionation techniques studied (solid-phase extraction and semi-preparative reverse phase-high performance liquid chromatography), the most potent insulinotropic fraction was enriched in free amino acids and contained relatively hydrophilic peptides. This indicates that amino acids and hydrophilic peptides may be involved in the insulinotropic effect of WPH1. (C) 2013 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, The in vitro dipeptidyl peptidase IV (DPP-IV) inhibitory activity of a whey protein hydrolysate (WPH) generated with a food-grade pancreatic enzyme preparation was studied. The 50 % inhibitory concentration (IC50) value in the presence of WPH was 1.34 ± 0.11 mg.mL-1. Ultrafiltration (UF) fractionation of WPH allowed enrichment in DPP-IV inhibitory peptides. The permeates generated by UF through 5 and 2 kDa membranes along with the hydrophilic fraction isolated by solid-phase extraction were significantly more potent DPP-IV inhibitors than WPH. These samples respectively had IC50 values of 0.95 ± 0.16, 0.48 ± 0.01 and 1.11 ± 0.09 mg.mL-1. Simulated gastrointestinal digestion (SGID) of WPH resulted in an increased DPP- IV inhibitory potency (IC50 value of 1.02 ± 0.05 mg.mL-1). Competitive inhibition of DPP-IV was observed with WPH and all its fractions, indicating a direct interaction of the bioactive peptides therein with the active site of DPP-IV. Combinations of sitagliptin, a conventional drug-inhibitor of DPP-IV, and whey-derived peptides resulted in an additive effect on DPP-IV inhibition., ACCEPTED, peer-reviewed

peer-reviewed, Dipeptides with a C terminal Pro inhibit dipeptidyl peptidase IV (DPP-IV), a key enzyme in incretin hormone processing. It was hypothesised that tri- and tetrapeptides with a proline at the C-terminus may also be DPP-IV inhibitors. Therefore, an in silico hydrolysis approach was used to release short(4 ≤ amino acids) C terminal Pro peptides from the individual caseins which constitute Pro rich substrates. This was achieved using theoretical digestion of caseins with a prolyl oligopeptidase activity. Fifteen peptides were subsequently selected for in vitro DPP-IV inhibitory analysis. Stability of these peptides to gastrointestinal enzymes was also evaluated in silico and the predicted breakdown peptides were assessed for their DPP-IV inhibitory and antioxidant potential. New DPP-IV inhibitors were identified, the most potent being Phe-Leu-Gln-Pro (IC50 65.3 +/- 3.5 mu M). A low in vitro antioxidant (2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging) activity was also associated with the peptides studied. The strategy presented highlights the utility of employing an in silico approach for the prediction of food-derived peptides with a potential role in glycaemic management for subsequent development of functional foods. (C) 2013 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, Of twelve dipeptides tested, only the Trp containing peptides Val-Trp and its reverse peptide Trp-Val showed a xanthine oxidase (XO) inhibitory activity. Studies with Val and Trp revealed that XO inhibition was mainly attributed to the Trp residue. No significant difference (P >= 0.05) was found for the XO inhibitory potency (IC50) values for Trp, Val-Trp and Trp-Val, which were about 200 times higher than that for Allopurinol. Lineweaver and Burke analysis demonstrated that Trp, Val-Trp and Trp-Val were non-competitive inhibitors while Allopurinol was a competitive inhibitor. Of the different milk-protein substrates hydrolyzed with gastro-intestinal enzyme activities, only lactoferrin (LF) hydrolyzates displayed XO inhibition. Peptides present in a LF hydrolyzate (GLF-240 min) were adsorbed onto activated carbon followed by subsequent desorption with stepwise elution using acetonitrile (ACN). Separation and detection of Trp containing peptides within the different fractions were achieved using RP-HPLC coupled with fluorescence detection. The desorbed fractions displayed different XO inhibitory properties, with no inhibition in the unbound fraction and highest inhibition in fractions eluted with 30, 40 and 70% ACN. The fraction eluting at 40% ACN was significantly more potent (19.1 +/- 2.3% inhibition at 1.25 mg mL(-1)) than the GLF-240 min hydrolyzate (13.4 +/- 0.4% inhibition at 1.25 mg mL(-1)), showing the potential for enrichment of the bioactive peptides on fractionation with activated carbon. (C) 2012 Elsevier Inc. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, This study investigates the ability of milk protein-derived peptides to specifically activate the serotonin 2C (5-HT2C) receptor, a key receptor in central regulation of food intake. A dose dependent 5-HT2C receptor activation by the 1 kDa ultrafiltration permeates of a sodium caseinate (NaCNH-1 kDa permeate) and a whey protein hydrolysate (WPH-1 kDa permeate) was demonstrated using an intracellular calcium mobilization assay in human embryonic kidney (Hek) cells expressing the 5-HT2C receptor. Both samples activated the 5-HT2C but not the 5-HT2A and 5-HT2B receptors. NaCNH-1 kDa permeate significantly (p < 0.01) reduced cumulative food intake when administered to male mice (C57Bl/6) by intraperitoneal injection at 500 mg kg-1 body weight. In contrast, no effect of WPH-1 kDa permeate could be seen on food intake in vivo. These results demonstrate the promising appetite-suppressing potential of NaCN-derived peptides, targeting the 5-HT2C receptor., ACCEPTED, peer-reviewed

peer-reviewed, Selected synthetic dipeptides and milk protein hydrolysates were evaluated for their dipeptidyl peptidase IV (DPP-IV) inhibitory properties, and their superoxide (SO) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities. DPP-IV inhibition was seen with eight out of the twelve dipeptides and 5 of the twelve hydrolysates studied. Trp-Val inhibited DPP-IV, however, inhibition was not observed with the reverse peptide Val-Trp. The most potent hydrolysate inhibitors were generated from casein (CasH2) and lactoferrin (LFH1). Two Trp containing dipeptides, Trp-Val and Val-Trp, and three lactoferrin hydrolysates scavenged DPPH. The dipeptides had higher SO EC50 values compared to the milk protein hydrolysates (arising from three lactoferrin and one whey protein hydrolysates). Higher molecular mass fractions of the milk protein hydrolysates were associated with the SO scavenging activity. Trp-Val and one lactoferrin hydrolysate (LFH1) were multifunctional displaying both DPP-IV inhibitory and antioxidant (SO and DPPH scavenging) activities. These compounds may have potential as dietary ingredients in the management of type 2 diabetes by virtue of their ability to scavenge reactive oxygen species and to extend the half-life of incretin molecules. (C) 2012 Elsevier Inc. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, Natural bioactives are an excellent source of carotenoids for the production of nutraceuticals, functional foods and food additives. The extraction efficiencies of solid-liquid extraction (SLE), supercritical CO2, supercritical CO2 with ethanol as co-solvent for the recovery of the carotenoids, xanthophyll and fucoxanthin, from two brown macroalgae Fucus serratus and Laminaria digitata were explored. The extraction efficiency was measured using both purity and yield of target compounds. Solid liquid extraction. (hexane/acetone (70:30) at 50 degrees C for 24 h produced the greatest yield of carotenoid rich extracts from F. serratus. Optimal conditions in terms of carotenoid yield using supercritical CO2 were 50 degrees C, 300 Atm with an extraction time of 60 min. SCO2 yielded a higher purity of fucoxanthin while SLE resulted in a higher purity of xanthophyll. Seasonal/spatial variation based on the purity and yield was also investigated to provide valuable information on optimal harvest time for these compounds. (C) 2016 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, 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., ACCEPTED, peer-reviewed

peer-reviewed, Many studies have demonstrated that milk protein consumption has benefits in terms of promoting human health. This review assesses the intervention studies which have evaluated potential health enhancing effects in humans following the ingestion of milk proteins. The impact of milk protein ingestion has been studied to asses their satiating, hypotensive, antimicrobial, anti-inflammatory, anticancer, antioxidant and insulinotropic properties as well as their impact on morphological modifications (e.g., muscle and fat mass) in humans. Consistent health promoting effects appear to have been observed in certain instances (i.e., muscle protein synthesis, insulinotropic and hypotensive activity). However, controversial outcomes have also been reported (i.e., antimicrobial, anti-inflammatory, anticancer and antioxidant properties). Several factors including interindividual differences, the timing of protein ingestion as well as the potency of the active components may explain these differences. In addition, processing conditions have been reported, in certain instances, to affect milk protein structure and therefore modify their bioactive potential. It is thought that the health promoting properties of milk proteins are linked to the release of bioactive peptides (BAPs) during gastrointestinal digestion. There is a need for further research to develop a more in-depth understanding on the possible mechanisms involved in the observed physiological effects. In addition, more carefully controlled and appropriately powered human intervention studies are required to demonstrate the health enhancing properties of milk proteins in humans. (C) 2015 Elsevier Inc. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, Since drug based inhibition of dipeptidyl peptidase IV (DPP-IV) is employed in type 2 (T2D) diabetes therapy, food protein hydrolysates which inhibit DPP- IV may also have potential in the management of T2D. Specific peptide motifs, consisting of an N-terminal Trp and/or a Pro at position 2, have been associated with relatively potent inhibition of DPP-IV. Different modes of inhibition which may, or may not, involve the active site of DPP-IV have been identified. Animal studies have shown that food protein hydrolysates having in vitro DPP-IV inhibitory activity generally yield antidiabetic effects in vivo. However, clear evidence of such effects in humans is still required in order to establish the potential role of food protein hydrolysates in the management of T2D., ACCEPTED, peer-reviewed

peer-reviewed, Milk proteins are a good source of bioactive peptides (BAPs). BAPs can positively affect various health biomarkers in vitro. The role of milk protein-derived BAPs in humans was reviewed herein. To date, a limited number of BAPs have been identified in the gastrointestinal tract of humans and in the circulation at concentrations which may be sufficient to mediate in vivo bioactivity. While the outcomes of several human intervention studies have shown promising results, some studies have failed to demonstrate additional health benefits. This may be related to a low potency and bioavailability of the BAPs evaluated as well as confounding factors (i.e. genotype, health status and other sources of inter-individual variability). Universal guidelines for the evaluation of BAPs in humans such as adequately powered double-blind randomised clinical trials are needed. (C) 2015 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

peer-reviewed, In silico digestion of milk protein-derived peptides with gastrointestinal enzyme activities was used to predict the release of peptides with a Pro residue at position 2 from the N terminus. These peptides are known to act as preferred dipeptidyl peptidase IV (DPP-IV) substrates. Five casein-derived synthetic peptides (Ile-Pro-Ile-Gin-Tyr, Leu-Pro-Leu-Pro-Leu, Tyr-Pro-Tyr-Tyr, Leu-Pro-Tyr-Pro-Tyr and Ile-Pro-Ile) and a casein (CasH), whey (WPH) and lactoferrin hydrolysate (LFH) generated with gastrointestinal enzymes were incubated with DPP-IV at 37 degrees C for 18 or 24 h. Peptide breakdown was evident following incubation with DPP-IV. Different modes of DPP-IV inhibition were observed depending on the test compound. Ile-Pro-Ile-Gln-Tyr, Tyr-Pro-Tyr-Tyr and Leu-Pro-Tyr-Pro-Tyr were substrate-, Leu-Pro-Leu-Pro-Leu and CasH were prodrug- while WPH and LFH were true DPP-IV inhibitors. These results are relevant for the bioactivity and bioavailability of functional foods targeting DPP-IV inhibition with potential blood glucose regulatory properties in humans. (C) 2013 Elsevier Ltd. All rights reserved., ACCEPTED, peer-reviewed

The influence of different substrates and the effect of pH regulation during enzymatic hydrolysis of whey protein on glucose-stimulated insulin secretion by BRIN-BD11 pancreatic beta cells were studied. No significant differences (P ≥ 0.05) were detected in terms of glucose-stimulated insulin secretion by BRIN-BD11 pancreatic beta cells exposed to whey protein hydrolysates (WPH1 and WPH2) obtained with two different whey protein substrates. The whey protein hydrolysate (WPH3) obtained without pH regulation during hydrolysis, had a significantly lower insulinotropic potential in BRIN-BD11 cells than the WPH1 hydrolysate that was manufactured with pH regulation. Fractionation of WPH1 was carried out to enrich bioactive peptides. Comparing the different fractionation techniques studied (solid-phase extraction and semi-preparative reverse phase-high performance liquid chromatography), the most potent insulinotropic fraction was enriched in free amino acids and contained relatively hydrophilic peptides. This indicates that amino acids and hydrophilic peptides may be involved in the insulinotropic effect of WPH1.

peer-reviewed, The full text of this article will not be available in ULIR until the embargo expires on the 29/09/2019, 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).

peer-reviewed, Oil-in-water (O/W) emulsions were prepared using bovine milk protein isolate (MPI) or MPI hydrolysates containing 1.60% (w/v) protein, 3.60% (w/v) lipid blend and 7.00% (w/v) lactose. The role of enzymatic treatment of MPI with Flavourzyme™, Neutrase™ and Protamex™ at two hydrolysis time points per enzyme [ranging in degree of hydrolysis (DH) from 15.6 to 37.1%] on the emulsifying properties was determined. All emulsion samples had mean fat globule size (d43) values < 1 μm after homogenisation. The hydrolysate containing emulsions displayed reduced emulsion stability under accelerated storage conditions, i.e., following an applied centrifugal force, and reduced heat stability at pH 7.0 compared with emulsions formed using intact MPI. The results demonstrated that hydrolysis reduced emulsion stability in the model system studied. Emulsion instability was hydrolytic enzyme dependent, increased with increasing DH and increasing content of peptides <1 kDa.

peer-reviewed, This study investigated the antihyperglycaemic effectiveness of an oral Palmaria palmata protein hydrolysate (PPPH), versus metformin, upon metabolic control in streptozotocin (STZ)-induced diabetic mice. Mice were administered PPPH (50 mg/kg bodyweight) or metformin (200 mg/kg bodyweight) by oral gavage twice-daily for 18 days. Blood glucose and plasma insulin were measured every third day. PPPH caused a significant reduction in blood glucose (p < 0.001) and a significant increase in plasma insulin (p < 0.001) versus STZtreated saline controls. PPPH treatment reduced energy intake (p < 0.05), bodyweight (p < 0.01) and total plasma glucagon-like peptide-1 (p < 0.01) after 18 days. Terminal oral glucose tolerance (Day 18, p < 0.05), fasting blood glucose (p < 0.001), HbA1C (p < 0.01), plasma cholesterol (p < 0.01) and plasma triglycerides (p < 0.05) were significantly improved versus STZ-treated saline controls. All groups showed significant increases in pancreatic islet area, β-cell area, and β:α cell ratio. PPPH demonstrated potent antidiabetic potential in vivo through reduced food intake and improved beta-cell function.

peer-reviewed, Milk protein concentrate-85 (MPC85) is a dairy ingredient which has a diverse range of applications in food products. The technofunctional properties of two MPC85 samples having similar gross composition but different levels of native whey protein (WP), i.e., MPC85S1 and MPC85S2 with 16.6 and 6.0 g native WP/100 g protein, respectively, were compared. Rheometeric analysis showed that under an applied normal stress of 1.0–15.0 kPa, the compressibility, the air permeability and the cohesiveness of MPC85S2 was higher compared to MPC85S1. Differential scanning calorimetry showed that protein denaturation in MPC85S1 began at 63 ◦C while for MPC85S2 it began at 70 ◦C. The heat coagulation time (HCT at 140 ◦C) for 4.2% (w/v, on a protein basis) reconstituted MPC85S1 and MPC85S2 was 2.2 and 2.7 min, respectively. While a higher lightness for MPC85S1 was evidenced using colourimeter analysis, the colour stability on oven drying at 95 ◦C for MPC85S2 was higher than MPC85S1. The emulsion produced with MPC85S1 flocculated after 1 d and phase separation occurred after 14 d. In the case of MPC85S2, flocculation began after 4 d while phase separation was observed at 33 d. The viscosity of MPC85S2 (4.2% (w/v) protein) was higher than MPC85S1. This study showed differences between the flowability, viscosity, colour properties, thermal stability (in powder and in reconstituted format), emulsification and buffering capacity for MPC samples having two different levels of WP denaturation. The results demonstrated that the MPCs studied having two different levels of WP denaturation could be targeted for different functional applications. The minimal/maximum level of denaturation required to induce technofunctional property differences requires further study.

peer-reviewed, Two bovine milk protein concentrate (MPC85) samples containing 85% protein were produced using different heat treatments. The physicochemical and water absorption properties (i.e., the solubility, wettability, water holding capacity (WHC) and particle size on reconstitution) of these two MPC85 samples with two different levels of undenatured whey protein (WP, i.e., MPC85S1 and MPC85S2) were investigated. The undenatured WP level in MPC85S1 and MPC85S2 was 16.6 and 6.0 g/100 g total protein, respectively. The calcium ion activity of MPC85S1 and MPC85S2 was 2.93 and 2.64 mmol/L, respectively (p < 0.05). The surface elemental profile, as determined using X-ray photoelectron spectroscopy, showed that protein (84%) and lipid (16%) were the dominant macromolecules on the surface of both MPC85 powder particles. Scanning electron microscopy analysis showed that MPC85S1 powder particles were relatively spherical while MPC85S2 particles appeared agglomerated. Particle size analysis of 5% aqueous suspensions showed a broader size distribution for MPC85S1 compared to MPC85S2. The cold solubility of the samples rehydrated at 4 ◦C after 192 h storage at 4 ◦C was higher for MPC85S1 than MPC85S2, while on increasing the temperature of rehydration from 4◦ to 75 ◦C the solubility increased for both MPC85 samples. A lower contact angle, and thus higher wettability was observed for MPC85S2 in comparison to MPC85S1. While the MPC85 samples studied herein had similar gross composition, major differences in the level of heat induced WP denaturation (~2.5 times) and a 10% difference in ionic calcium content existed. Consequently differences in the ionic calcium and more importantly the differences in the interactions between casein and native/denatured WP are considered to have resulted in altered physico-chemical properties and thereby different aqueous phase interactions.

peer-reviewed, 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.