• Encapsulation of a Lactic Acid Bacteria Cell-Free Extract in Liposomes and Use in Cheddar Cheese Ripening

      Nongonierma, Alice B.; Abrlova, Magdalena; Kilcawley, Kieran; Department of Agriculture, Food and the Marine, Ireland (MDPI AG., Basel, Switzerland, 13/03/2013)
      A concentrated form of cell free extract (CFE) derived from attenuated Lactococcus lactis supsb. lactis 303 CFE was encapsulated in liposomes prepared from two different proliposome preparations (Prolipo Duo and Prolipo S) using microfluidization. Entrapment efficiencies of 19.7 % (Prolipo S) and 14.0 % (Prolipo Duo) were achieved and the preparations mixed in the ratio 4 (Prolipo Duo):1 (Prolipo S). Cheddar cheese trials were undertaken evaluating the performance of CFE entrapped in liposomes, empty liposomes and free CFE in comparison to a control cheese without any CFE or liposomes. Identical volumes of liposome and amounts of CFE were used in triplicate trials. The inclusion of liposomes did not adversely impact on cheese composition water activity, or microbiology. Entrapment of CFE in liposomes reduced loss of CFE to the whey. No significant differences were evident in proteolysis or expressed PepX activity during ripening in comparison to the cheeses containing free CFE, empty liposomes or the control, as the liposomes did not degrade during ripening. This result highlights the potential of liposomes to minimize losses of encapsulated enzymes into the whey during cheese production but also highlights the need to optimize the hydrophobicity, zeta potential, size and composition of the liposomes to maximize their use as vectors for enzyme addition in cheese to augment ripening.
    • Identification of short peptide sequences in the nanofiltration permeate of a bioactive whey protein hydrolysate

      Le Maux, Solene; Nongonierma, Alice B.; Murray, Brian A.; Kelly, Philip; Fitzgerald, Richard J.; Enterprise Ireland; TC2013-0001 (Elsevier, 16/09/2015)
      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.
    • Partitioning of starter bacteria and added exogenous enzyme activities between curd and whey during Cheddar cheese manufacture

      Doolan, I. A.; Nongonierma, Alice B.; Kilcawley, Kieran; Wilkinson, M.G.; Department of Agriculture, Food and the Marine, Ireland; 04/R&D/C/238 (Elsevier, 26/07/2013)
      Partitioning 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.