Partitioning of starter bacteria and added exogenous enzyme activities between curd and whey during Cheddar cheese manufacture

peer-reviewedPartitioning of starter bacteria and enzyme activities was investigated at different stages of Cheddar cheese manufacture using three exogenous commercial enzyme preparations added to milk or at salting. The enzyme preparations used were: Accelase AM317, Accelase AHC50, Accelerzyme CPG. Flow cytometric analysis indicated that AHC50 or AM317 consisted of permeabilised or dead cells and contained a range of enzyme activities. The CPG preparation contained only carboxypeptidase activity. Approximately 90% of starter bacteria cells partitioned with the curd at whey drainage. However, key enzyme activities partitioned with the bulk whey in the range of 22%–90%. An increased level of enzyme partitioning with the curd was observed for AHC50 which was added at salting, indicating that the mode of addition influenced partitioning. These findings suggest that further scope exists to optimise both bacterial and exogenous enzyme incorporation into cheese curd to accelerate ripening.Department of Agriculture, Food and the Marin

In silico approaches applied to the study of peptide analogs of Ile-Pro-Ile in relation to their dipeptidyl peptidase IV inhibitory properties

Inhibition of dipeptidyl peptidase IV (DPP-IV) may be exploited to maintain the incretin effect during the postprandial phase. As a result, glycemic regulation and energy homeostasis may be improved. Food protein-derived peptides have been identified as natural agents capable of inhibiting DPP-IV. Ile-Pro-Ile is the most potent DPP-IV inhibitory peptide identified to date. A minimum analog peptide set approach was used to study peptide analogs of Ile-Pro-Ile. The DPP-IV half maximal inhibitory concentration (IC50) values of the 25 peptides evaluated ranged from 3.9 ± 1.0 μM (Ile-Pro-Ile) to 247.0 ± 32.7 μM (Phe-Pro-Phe). The presence of Pro at position 2 of tripeptides was required to achieve high DPP-IV inhibition. Most peptides behaved as competitive inhibitors of DPP-IV with the exception of peptides with a N-terminal Trp, which were mixed-type inhibitors. While possessing the structure of preferred DPP-IV substrates, most peptides studied were particularly stable during 30 min incubation with DPP-IV. Molecular docking revealed that Ile-Pro-Ile and its peptide analogs interacted in a very similar manner with the active site of DPP-IV. In addition, no correlation was found between the Hydropathic INTeraction score and the DPP-IV IC50 values of the peptides studied. This outcome suggests that free energy may not be directly responsible for enzyme inhibition by the peptides. Finally, novel DPP-IV inhibitory peptides were identified using the strategy employed herein. These results may be relevant for the development of food protein-derived peptides with serum glucose lowering and food intake regulatory properties in humans

Identification of short peptide sequences in the nanofiltration permeate of a bioactive whey protein hydrolysate

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

A Whey Protein Hydrolysate Promotes Insulinotropic Activity in a Clonal Pancreatic β-Cell Line and Enhances Glycemic Function in ob/ob Mice

Whey protein hydrolysates (WPHs) represent novel antidiabetic agents that affect glycemia in animals and humans, but little is known about their insulinotropic effects. The effects of a WPH were analyzed in vitro on acute glucose-induced insulin secretion in pancreatic BRIN-BD11 β cells. WPH permeability across Caco-2 cell monolayers was determined in a 2-tiered intestinal model. WPH effects on insulin resistance were studied in vivo following an 8-wk oral ingestion (100mg/kg body weight) by ob/ob (OB-WPH) and wild-type mice (WT-WPH) compared with vehicle control (OB and WT groups) usinga 2 3 2 factorial design, genotype 3 treatment. BRIN-BD11 cells showed a robust and reproducible dose-dependent insulinotropic effect of WPH (from 0.01 to 5.00 g/L). WPH bioactive constituents were permeable across Caco-2 cell monolayers. In the OB-WPH and WT-WPH groups, WPH administration improved glucose clearance after a glucose challenge (2 g/kg body weight), as indicated by differences in the area under curves (AUCs) (P ≤ 0.05). The basal plasma glucose concentration was not affected by WPH treatment in either genotype. The plasma insulin concentration was lower in the OB-WPH than in the OB group (P ≤ 0.005) but was similar between the WT and WT-WPH groups; the interaction genotype 3 treatment was significant (P ≤ 0.005). Insulin release from pancreatic islets isolated from the OB-WPH group was greater (P ≤ 0.005) than that from the OB group but did not differ between the WT-WPH and WT groups; the interaction genotype 3 treatment was not significant. In conclusion, an 8-wk oral administration of WPH improved blood glucose clearance, reduced hyperinsulinemia, and restored the pancreatic islet capacity to secrete insulin in response to glucosein ob/ob mice. Hence, it may be useful in diabetes management

Insulinotropic properties of whey protein hydrolysates and impact of peptide fractionation on insulinotropic response

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