Phil M. Kelly

Molecular characterization of whey protein hydrolysate fractions with ferrous chelating and enhanced iron solubility capabilities.

The ferrous (Fe2+) chelating capabilities of WPI hydrolysate fractions produced via cascade membrane filtration were investigated, specifically 1 kDa permeate (P) and 30 kDa retentate (R) fractions. The 1 kDa-P possessed a Fe2+ chelating capability at 1 g L(-1) equivalent to 84.4 μM EDTA (for 30 kDa-R the value was 8.7 μM EDTA). Fourier transformed infrared (FTIR) spectroscopy was utilized to investigate the structural characteristics of hydrolysates and molecular interactions with Fe2+. Solid-phase extraction was employed to enrich for chelating activity; the most potent chelating fraction was enriched in histidine and lysine. The solubility of ferrous sulfate solutions (10 mM) over a range of pH values was significantly (P<0.05) improved in dispersions of hydrolysate fraction solutions (10 g protein L(-1)). Total iron solubility was improved by 72% in the presence of the 1 kDa-P fraction following simulated gastrointestinal digestion (SGID) compared to control FeSO4·7H2O solutions.

Anti‐inflammatory effects of a casein hydrolysate and its peptide‐enriched fractions on TNFα‐challenged Caco‐2 cells and LPS‐challenged porcine colonic explants

Bioactive milk peptides are reported to illicit a range of physiological benefits and have been proposed as potential functional food ingredients. The objective of this study was to characterize the anti-inflammatory properties of sodium caseinate (NaCAS), its enzyme hydrolysate (EH) and peptide-enriched fractions (5 kDa retentate [R], 1 kDaR and 1 kDa permeate [P]), both in vitro using a Caco-2 cell line, and also ex vivo using a porcine colonic tissue explant system. Caco-2 cells were stimulated with tumour necrosis factor alpha (TNFα) and co-treated with casein hydrolysates for 24 h. Following this, interleukin (IL)-8 concentrations in the supernatant were measured using enzyme-linked immunosorbent assay. Porcine colonic tissue was stimulated with lipopolysaccharide and co-treated with casein hydrolysates for 3 h. The expression of a panel of inflammatory cytokines was measured using qPCR. While dexamethasone reduced the IL-8 concentration by 41.6%, the 1 kDaR and 1 kDaP fractions reduced IL-8 by 68.7% and 66.1%, respectively, relative to TNFα-stimulated Caco-2 cells (P < 0.05). In the ex vivo system, only the 1 kDaR fraction elicited a decrease inIL1-α,IL1-β,IL-8,TGF-β andIL-10 expression (P < 0.05). This study provides evidence that the bioactive peptides present in the 1 kDaR fraction of the NaCAS hydrolysate possess anti-inflammatory properties in vitro and ex vivo. Further in vivo analysis of the anti-inflammatory properties of the 1 kDaR is proposed.

Recent advances in spray drying relevant to the dairy industry: A comprehensive critical review

ABSTRACT Milk is extremely perishable, and yet it has to be preserved for later consumption. In this view, membrane filtration, vacuum concentration lactose crystallization, homogenization, and spray-drying dehydration are valuable techniques to stabilize most dairy ingredients. Considering the increasing development of dairy trade, there is a need for the dairy industry to improve its understanding of how these concentration and spray-drying processes affect the quality of the resulting dairy powders, so to control it. However, the residence time of the droplet and the powder in the spray dryer is so short that it is very difficult to implement studies on the mechanisms of the structural changes in the protein without fundamental research into the process/product interactions. Moreover, several authors have reported the crucial and specific role of dairy components in the mechanisms of water transfer during drying and rehydration. The aim of this paper is to review the present and recent advances in knowledge and innovations, on the properties of spray-dried dairy products, on the modeling and simulation of water transfer processes (drying and rehydration), and on spray-drying equipment and energy consumption.

Anticancer Activity of Buttermilk Against SW480 Colon Cancer Cells is Associated with Caspase-Independent Cell Death and Attenuation of Wnt, Akt, and ERK Signaling

ABSTRACT Buttermilk is a rich source of milk fat globule membrane (MFGM) fragments assembled from bioactive polar lipids and proteins that originate from bovine mammary epithelial cells. The objective of this study was to examine growth-modulatory effects of experimental buttermilks varying in sphingolipid and phospholipid composition on a colon cancer cell line of human origin. Buttermilks were prepared from washed and unwashed cream using gravity or centrifugation. Compositional analysis showed that sphingomyelin (SM) (10.4–29.5%) and lactosylceramide (LacCer) (1.2–44.3%) were the predominant sphingolipids detected. Experimental samples inhibited in vitro growth of SW480 colon cancer cells in a dose-dependent manner. Antiproliferative activity was selective toward cancer cells. A fraction enriched in LacCer (44.3%), obtained by microfiltration induced caspase-independent cell death as evident by phosphatidylserine externalization, increased percentage of degraded DNA, and loss of mitochondrial membrane potential in SW480 cells. This fraction downregulated growth-signaling pathways mediated by β-catenin, phosphorylated Akt (serine/threonine-specific protein kinase), ERK1/2 (extracellular signal–regulated kinase), and c-myc. This study is to our knowledge the first to screen buttermilk samples that vary in polar lipid composition for antiproliferative activity in vitro.

Effects of depleting ionic strength on 31P nuclear magnetic resonance spectra of micellar casein during membrane separation and diafiltration of skim milk

Membrane separation processes used in the concentration and isolation of micellar casein-based milk proteins from skim milk rely on extensive permeation of its soluble serum constituents, especially lactose and minerals. Whereas extensive literature exists on how these processes influence the gross composition of milk proteins, we have little understanding of the effects of such ionic depletion on the core structural unit of micellar casein [i.e., the casein phosphate nanocluster (CPN)]. The 31P nuclear magnetic resonance (NMR) is an analytical technique that is capable of identifying soluble and organic forms of phosphate in milk. Thus, our objective was to investigate changes to the 31P NMR spectra of skim milk during microfiltration (MF) and diafiltration (DF) by tracking movements in different species of phosphate. In particular, we examined the peak at 1.11 ppm corresponding to inorganic phosphate in the serum, as well as the low-intensity broad signal between 1.5 and 3.0 ppm attributed to casein-associated phosphate in the retentate. The MF concentration and DF using water caused a shift in the relevant 31P NMR peak that could be minimized if orthophosphate was added to the DF water. However, this did not resolve the simultaneous change in retentate pH and increased solubilization of micellar casein protein. The addition of calcium in combination with orthophosphate prevented micellar casein solubilization and simultaneously contributed to preservation of the CPN structure, except for overcorrection of retentate pH in the acidic direction. A more complex DF solution, involving a combination of phosphate, calcium, and citrate, succeeded in both CPN and micellar casein structure preservation while maintaining retentate pH in the region of the original milk pH. The combination of 31P NMR as an analytical technique and experimental probe during MF/DF processes provided useful insights into changes occurring to CPN while retaining the micellar state of casein.

pH-stat vs. free-fall pH techniques in the enzymatic hydrolysis of whey proteins.

Enzymatic hydrolysis of a commercial whey protein isolate (WPI) using either trypsin or Protamex® was compared using controlled (pH-stat) and uncontrolled (free-fall) pH conditions. pH-stat control at the enzymes optimum value led to a more rapid rate of WPI hydrolysis by trypsin, while the opposite was the case when Protamex® was used. Furthermore, the choice of alkaline solution used to maintain constant pH during pH-stat experiments appeared to affect the reaction rate, being higher when KOH is added to the reaction mixture instead of NaOH. It would appear that potassium may play a role as co-factor or activator for the activity of this particular protease preparation. Although pH-stat techniques are usually considered to yield better hydrolysis kinetics, these findings suggest that the response of proteolytic enzyme preparations to static or free-fall pH control should be checked in advance, particularly when undertaking large scale production of WPI hydrolysates.

Invited review: Whey proteins as antioxidants and promoters of cellular antioxidant pathways

Oxidative stress contributes to cell injury and aggravates several chronic diseases. Dietary antioxidants help the body to fight against free radicals and, therefore, avoid or reduce oxidative stress. Recently, proteins from milk whey liquid have been described as antioxidants. This review summarizes the evidence that whey products exhibit radical scavenging activity and reducing power. It examines the processing and treatment attempts to increase the antioxidant bioactivity and identifies 1 enzyme, subtilisin, which consistently produces the most potent whey fractions. The review compares whey from different milk sources and puts whey proteins in the context of other known food antioxidants. However, for efficacy, the antioxidant activity of whey proteins must not only survive processing, but also upper gut transit and arrival in the bloodstream, if whey products are to promote antioxidant levels in target organs. Studies reveal that direct cell exposure to whey samples increases intracellular antioxidants such as glutathione. However, the physiological relevance of these in vitro assays is questionable, and evidence is conflicting from dietary intervention trials, with both rats and humans, that whey products can boost cellular antioxidant biomarkers.

Comparison of antioxidant activities of bovine whey proteins before and after simulated gastrointestinal digestion

Oxidative stress caused by free radicals has been implicated in several human disorders. Dietary antioxidants can help the body to counteract those reactive species and reduce oxidative stress. Antioxidant activity is one of the multiple health-promoting attributes assigned to bovine whey products. The present study investigated whether this activity was retained during upper gut transit using a static simulated in vitro gastrointestinal digestion (SGID) model. The capacity to scavenge free radicals and reduce ferric ion of whey protein isolate (WPI), individual whey proteins, and hydrolysates pre- and post-SGID were measured and compared using various antioxidant assays. In addition, the free AA released from individual protein fractions in physiological gut conditions were characterized. Our results indicated that the antioxidant activity of WPI after exposure to the harsh conditions of the upper gut significantly increased compared with intact WPI. From an antioxidant bioactivity viewpoint, this exposure negates the need for prior hydrolysis of WPI. The whey protein α-lactalbumin showed the highest antioxidant properties post-SGID (oxygen radical absorbance capacity = 1,825.94 ± 50.21 μmol of Trolox equivalents/g of powder) of the 4 major whey proteins tested with the release of the highest amount of the antioxidant AA tryptophan, 6.955 μmol of tryptophan/g of protein. Therefore, α-lactalbumin should be the preferred whey protein in food formulations to boost antioxidant defenses.

Technical note: Fourier transform infrared spectral analysis in tandem with 31P nuclear magnetic resonance spectroscopy elaborates detailed insights into phosphate partitioning during skimmed milk microfiltration and diafiltration

Our previous study identified peaks in the 31P nuclear magnetic resonance (31P NMR) spectra of skim milk, denoting the interaction of different phosphate species such as inorganic and casein-associated phosphate during the separation of colloidal and serum phases of skim milk by microfiltration (MF) and diafiltration (DF). In the current study, we investigated the same samples generated by the aforementioned separation using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy analysis. The results confirmed that the technique was not only capable of differentiating between the mineral equilibrium of the casein phosphate nanocluster (CPN) and milk serum, but also complemented the application of 31P NMR. An ATR-FTIR broad band in the region of 1,055 to 1,036 cm-1 and a specific band at 1,076 cm-1 were identified as sensitive to the repartitioning of different phosphate species in milk in accordance with the 31P NMR signals representing casein-associated phosphate and inorganic phosphate in the serum. A third ATR-FTIR signal at 1,034 cm-1 in milk, representing precipitated inorganic calcium phosphate, had not previously been detected by 31P NMR. Thus, the results indicate that a combination of ATR-FTIR and 31P NMR spectroscopies may be optimally used to follow mineral and protein phase changes in milk during membrane processing.

A Dairy-Derived Ghrelinergic Hydrolysate Modulates Food Intake In Vivo

Recent times have seen an increasing move towards harnessing the health-promoting benefits of food and dietary constituents while providing scientific evidence to substantiate their claims. In particular, the potential for bioactive protein hydrolysates and peptides to enhance health in conjunction with conventional pharmaceutical therapy is being investigated. Dairy-derived proteins have been shown to contain bioactive peptide sequences with various purported health benefits, with effects ranging from the digestive system to cardiovascular circulation, the immune system and the central nervous system. Interestingly, the ability of dairy proteins to modulate metabolism and appetite has recently been reported. The ghrelin receptor (GHSR-1a) is a G-protein coupled receptor which plays a key role in the regulation of food intake. Pharmacological manipulation of the growth hormone secretagogue receptor-type 1a (GHSR-1a) receptor has therefore received a lot of attention as a strategy to combat disorders of appetite and body weight, including age-related malnutrition and the progressive muscle wasting syndrome known as cachexia. In this study, a milk protein-derivative is shown to increase GHSR-1a-mediated intracellular calcium signalling in a concentration-dependent manner in vitro. Significant increases in calcium mobilisation were also observed in a cultured neuronal cell line heterologously expressing the GHS-R1a. In addition, both additive and synergistic effects were observed following co-exposure of GHSR-1a to both the hydrolysate and ghrelin. Subsequent in vivo studies monitored standard chow intake in healthy male and female Sprague-Dawley rats after dosing with the casein hydrolysate (CasHyd). Furthermore, the provision of gastro-protected oral delivery to the bioactive in vivo may aid in the progression of in vitro efficacy to in vivo functionality. In summary, this study reports a ghrelin-stimulating bioactive peptide mixture (CasHyd) with potent effects in vitro. It also provides novel and valuable translational data supporting the potential role of CasHyd as an appetite-enhancing bioactive. Further mechanistic studies are required in order to confirm efficacy as a ghrelinergic bioactive in susceptible population groups.