• Assessment of RNAlater® as a Potential Method to Preserve Bovine Muscle Proteins Compared with Dry Ice in a Proteomic Study

      Zhu, Yao; Mullen, Anne Maria; Rai, Dilip K.; Kelly, Alan L.; Sheehan, David; Cafferky, Jamie; Hamill, Ruth; Teagasc Walsh Fellowship Programme; NFFQ0017 (MDPI, 2019-02-05)
      RNAlater® is regarded as a potential preservation method for proteins, while its effect on bovine muscle proteins has rarely been evaluated. Bovine muscle protein samples (n = 12) collected from three tender (Warner–Bratzler shear force: 30.02–31.74 N) and three tough (Warner–Bratzler shear force: 54.12–66.25 N) Longissimus thoracis et lumborum (LTL) samples, preserved using two different sampling preservation methods (RNAlater® and dry ice), at two post mortem time points (day 0 and day 14), were characterized using one-dimensional electrophoresis. Fourteen bands with molecular weights ranging from 15 to 250 kDa were verified, both in the dry ice and RNAlater® storage groups, at each time point, using image analysis. A shift from high to low molecular weight fragments, between day 0 and day 14, indicated proteolysis of the muscle proteins during post mortem storage. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and database searching resulted in the identification of 10 proteins in four bands. Protein profiles of muscle preserved in RNAlater® were similar to those of muscle frozen on dry ice storage, both at day 0 and day 14. The results demonstrate that RNAlater® could be a simple and efficient way to preserve bovine muscle proteins for bovine muscle proteomic studies
    • The effect of direct and indirect heat treatment on the attributes of whey protein beverages

      Kelleher, Clodagh M.; O'Mahony, James A.; Kelly, Alan L.; O'Callaghan, Donal; Kilcawley, Kieran; McCarthy, Noel; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship programme; 10 RD TMFRC 703 (Elsevier, 2018-06-11)
      Thermal processing of ready-to-drink high protein beverages can have a substantial impact on the physical and sensory properties of the final product for long-life milks such as extended shelf life and ultra high temperature processed products. Direct and indirect heat treatment technologies were applied to whey protein isolate (WPI) -based beverages containing 4, 6 or 8% (w/w) protein. Lower levels of protein denaturation (66–94%) were observed using direct heating compared with indirect heating (95–99%) across protein levels and heating temperatures (121 and 135 °C final heat). Direct heat treatment resulted in significantly lower viscosity and less extensive changes to the volatile profile, compared with indirect heat treatment. Overall, the application of direct and indirect heat treatment to WPI solutions resulted in significantly different final products in terms of appearance, physical characteristics and volatile profile, with direct heating resulting in many enhanced properties compared with conventional indirect heat treatment.
    • Effect of milk centrifugation and incorporation of high heat-treated centrifugate on the microbial composition and levels of volatile organic compounds of Maasdam cheese

      Lamichhane, Prabin; Pietrzyk, Anna; Feehily, Conor; Cotter, Paul D.; Mannion, David T.; Kilcawley, Kieran; Kelly, Alan L.; Sheehan, Diarmuid (JJ); Dairy Levy Trust; Teagasc Walsh Fellowship Programme; et al. (Elsevier, 2018-03-15)
      Centrifugation is a common milk pretreatment method for removal of Clostridium spores which, on germination, can produce high levels of butyric acid and gas, resulting in rancid, gassy cheese. The aim of this study was to determine the effect of centrifugation of milk, as well as incorporation of high heat-treated centrifugate into cheese milk, on the microbial and volatile profile of Maasdam cheese. To facilitate this, 16S rRNA amplicon sequencing in combination with a selective media-based approach were used to study the microbial composition of cheese during maturation, and volatile organic compounds within the cheese matrix were analyzed by HPLC and solid-phase microextraction coupled with gas chromatography–mass spectrometry. Both culture-based and molecular approaches revealed major differences in microbial populations within the cheese matrix before and after warm room ripening. During warm room ripening, an increase in counts of propionic acid bacteria (by ∼101.5 cfu) and nonstarter lactic acid bacteria (by ∼108 cfu) and a decrease in the counts of Lactobacillus helveticus (by ∼102.5 cfu) were observed. Lactococcus species dominated the curd population throughout ripening, followed by Lactobacillus, Propionibacterium, and Leuconostoc, and the relative abundance of these accounted for more than 99% of the total genera, as revealed by high-throughput sequencing. Among subdominant microflora, the overall relative abundance of Clostridium sensu stricto was lower in cheeses made from centrifuged milk than control cheeses, which coincided with lower levels of butyric acid. Centrifugation as well as incorporation of high heat-treated centrifugate into cheese milk seemed to have little effect on the volatile profile of Maasdam cheese, except for butyric acid levels. Overall, this study suggests that centrifugation of milk before cheesemaking is a suitable method for controlling undesirable butyric acid fermentation without significantly altering the levels of other volatile organic compounds of Maasdam cheese.
    • Influence of chaperone-like activity of caseinomacropeptide on the gelation behaviour of whey proteins at pH 6.4 and 7.2.

      Gaspard, Sophie J.; Sharma, Prateek; Fitzgerald, Ciarán; Tobin, John T.; O’Mahony, James A.; Kelly, Alan L.; Brodkorb, Andre; Dairy Research Ireland; Teagasc Walsh Fellowship Programme; European Union; et al. (Elsevier, 2020-08-15)
      The effect of caseinomacropeptide (CMP) on the heat-induced denaturation and gelation of whey proteins (2.5–10%, w/v) at pH 6.4 and 7.2, at a whey protein:CMP ratio of 1:0.9 (w/w), was investigated using differential scanning calorimetry (DSC), oscillatory rheology (90 °C for 20 min) and confocal microscopy. Greater frequency-dependence in the presence of CMP suggested that the repulsive interactions between CMP and the whey proteins affected the network generated by the non-heated whey protein samples. At pH 6.4 or 7.2, CMP increased the temperature of denaturation of β-lactoglobulin by up to 3 °C and increased the gelation temperature by up to 7 °C. The inclusion of CMP strongly affected the structure of the heat-induced whey protein gels, resulting in a finer stranded structure at pH 6.4 and 7.2. The presence of CMP combined with a lower heating rate (2 °C/min) prevented the formation of a solid gel of whey proteins after heating for 20 min at 90 °C and at pH 7.2. These results show the potential of CMP for control of whey protein denaturation and gelation.
    • Influence of herd diet on the metabolome of Maasdam cheeses

      Panthi, Ram R.; Sundekilde, Ulrik; Kelly, Alan L.; Hennessy, Deirdre; Kilcawley, Kieran; Mannion, David T.; Fenelon, Mark; Sheehan, Diarmuid (JJ); Dairy Research Ireland.; Teagasc Walsh Fellowship Programme; et al. (Elsevier, 2019-05-20)
      The untargeted metabolic profiles of ripened Maasdam cheese samples prepared from milk derived from three herd groups, fed: (1) indoors on total mixed ration (TMR), or outdoors on (2) grass only pasture (GRA) or (3) grass and white clover pasture (CLO) were studied using high resolution nuclear magnetic resonance (1H NMR), high resolution magic angle spinning nuclear magnetic resonance (1H HRMAS NMR) and headspace (HS) gas chromatography mass spectrometry (GC-MS). A total of 31 compounds were identified using 1H NMR and 32 volatile compounds including 7 acids, 5 esters, 4 alcohols, 4 ketones, 4 sulfur compounds, 2 aldehydes, 3 hydrocarbons, 2 terpenes and a lactone were identified using GC–MS in Maasdam cheeses ripened for 97-d. On comparing the 1H NMR metabolic profiles, TMR-derived cheese had higher levels of citrate compared to GRA-derived cheese. The toluene content of cheese was significantly higher in GRA or CLO compared to TMR cheeses and dimethyl sulfide was identified only in CLO-derived cheese samples as detected using HS GC–MS. These compounds are proposed as indicator compounds for Maasdam cheese derived from pasture-fed milk. Clear differences between outdoor or indoor feeding systems in terms of cheese metabolites were detected in the lipid phase, as indicated by principal component analysis (PCA) from 1H HRMAS NMR spectra, although differences based on PCA of all 1H NMR spectra and HS-GC–MS were less clear. Overall, this study presented the metabolite profile and identified specific compounds which may be useful for discriminating between ripened Maasdam cheese and related cheese varieties manufactured from indoor or outdoor herd-feeding systems.
    • Self-association of bovine β-casein as influenced by calcium chloride, buffer type and temperature

      Li, Meng; Auty, Mark; Crowley, Shane V.; Kelly, Alan L.; O'Mahoney, James A.; Brodkorb, Andre; Irish Dairy Levy Research Trust; Teagasc Walsh Fellowship Programme; MDDT 6261 (Elsevier, 2018-09-25)
      The aim of this study was to investigate the aggregation behaviour of a pure β-casein (β-CNpure) and a β-casein concentrate (β-CNconc) as a function of temperature, buffer type (pH 6.8) and the presence of CaCl2. The particle size distribution and turbidity of β-casein (β-CN) dispersions were measured by dynamic light-scattering (DLS) and UV/vis spectroscopy between 4 and 55 °C. Upon heating (4–55 °C), the particle size of both β-CN samples increased, indicating self-association via hydrophobic interactions. It was shown that the self-association of β-CN increased with increasing β-CN concentration and that β-CNpure self-associated at significantly lower concentration than β-CNconc. Both turbidity and particle size measurements showed that the β-CN samples had similar aggregation behaviour in water and imidazole buffer (pH 6.8) but differed in sodium phosphate buffer (pH 6.8), especially at higher ionic calcium concentrations. Fourier Transform Infrared (FTIR) spectroscopy revealed very little change in the secondary structure of β-CN during heating (4–55 °C). The microstructure of β-CN aggregates was monitored during heating from 10 to 55 °C, followed by cooling to 10 °C, using polarised light microscopy. Spherical and heterogeneous aggregates were observed when heated at temperatures above 37 °C, which were reversible upon cooling. This study confirmed that β-CN undergoes self-association on heating that reverses upon cooling, with the aggregation process being highly dependent on the purity of β-CN, the solvent type and the presence of ionic calcium.