Thao T. Le

Chemical and Physical Changes in Milk Protein Concentrate (MPC80) Powder during Storage

The solubility and chemical changes due to the Maillard reaction were investigated in milk protein concentrate powder containing 80% protein (MPC80) during storage at temperatures and relative humidities in the ranges of 25-40 °C and 44-84%, respectively. The Maillard reaction was studied by measuring furosine (a product of lactosylated protein after digestion with acid) and free hydroxymethylfurfural (HMF) contents by HPLC and L*, a*, b* values with a color-meter. Furosine, free HMF, and browning in MPC80 increased during storage, whereas the solubility decreased. The correlation between the Maillard reaction and solubility loss was explored in modified MPC80 to which glucose was added to enhance the rate of the Maillard reaction. More furosine and brown pigments were observed in the glucose-containing MPC80 than in MPC80 with added lactose. The opposite trend occurred for solubility, suggesting that the Maillard reaction may be a cause of solubility loss in MPC powder.

Maillard Reaction and Protein Cross-Linking in Relation to the Solubility of Milk Powders

Protein changes in relation to solubility, Maillard reaction (MR), and protein cross-linking in whole milk powder (WMP), skim milk powder (SMP), and whey protein concentrate (WPC) stored at different relative humidities (RHs) were investigated by chemical and electrophoretic methods. WMP and SMP reached minimum solubility rapidly, while WPC showed no change in solubility. The loss of solubility corresponded with development of high-molecular-weight protein complexes observed by two-dimensional electrophoresis. The maximal MR rate occurred at 66% RH for WMP and SMP (high lactose/protein ratios) and 84% RH for WPC (low lactose/protein ratios) based on the furosine and hydroxymethylfurfural contents. However, browning was greatest at 84% RH in all powders. The minimum solubility corresponded with the casein and fat contents. The retention of solubility and minimal protein cross-linking of WPC compared to casein-containing powders suggest that the casein content and cross-linking strongly influence the decrease in the solubility of milk powder.

A proteomic approach to detect lactosylation and other chemical changes in stored milk protein concentrate.

Milk proteins undergo chemical changes such as lactosylation, deamidation and protein cross-linking during processing and storage of milk products. A proteomic technique combining two-dimensional gel electrophoresis and mass spectrometry was used to investigate chemical modifications to proteins, in milk protein concentrate (MPC80), during storage. Lactosylation, deamidation and protein cross-linking were observed on 2-DE gels. They were storage temperature-, humidity- and time-dependent. Lactosylated whey proteins were well separated on 2-DE in vertical stacks of spots. The masses of the spots varied by multiples of 324, indicating the attachment of lactose to lysine residues in the proteins. The trypsin-digested spots of α-lactalbumin were analysed by MALDI-TOF mass spectrometry, which indicated multiple lactosylation sites. The lactose adducts on gels were quantified by image analysis, allowing development of adducts over time to be monitored. The results show that proteomics can be used for the detection and quantification of chemical modifications to proteins in stored MPC80.

Determination of minimal sequence for binding of an aptamer. A comparison of truncation and hybridization inhibition methods

Nucleic acid aptamers are attracting increasing interest as sensing and therapeutic molecules as a consequence of their high affinity and specificity for their target molecules. Aptamers are selected from large random libraries and where structural data are available it appears that only a small fraction of the sequence is actually involved in direct contact with the target. As there are many advantages to minimizing the size of the aptamer a rapid method that can determine those parts of the sequence critical for the target binding would be very useful. In this paper we describe mapping the effective binding region of an aptamer selected against electric eel acetylcholinesterase. As originally selected this aptamer is 77 nucleotides in length and was shown to bind the target with a high affinity (Kd = 174 ± 27 pM). Truncation to 39 nucleotides enhanced affinity for its target by an order of magnitude (Kd = 14 ± 1 pM). To further probe the relationship between sequence and affinity, we used two approaches: truncation and hybridization inhibition. Binding assays were performed with a number of truncated variants to determine a minimal binding sequence. A similar set of measurements for hybridization inhibition was also performed to allow a comparison of the two approaches. In general hybridization inhibition resulted in comparable conclusions to those found by truncation. The exception to this was where the former resulted in steric clashes between double stranded DNA regions and the target. In this case the effect on affinity was less pronounced than with truncation.

Quantification of lactosylation of whey proteins in stored milk powder using multiple reaction monitoring

Lactosylation in stored milk powder was quantified by multiple reaction monitoring (MRM), a mass spectrometry-based quantification method. The MRM method was developed from a knowledge of peptide fragmentation. The neutral losses of 162Da (cleavage of galactose) and 216Da (the formation of furylium ion) which were representative of lactosylated peptides were specifically selected as MRM transitions. Quantification of lactosylated protein was based on the peak areas of these fragmentation ions. The MRM results showed an increase in peak areas of the two transition fragments from tryptic digests of whey proteins in stored milk protein concentrate powder. A good correlation between the MRM and furosine results indicated that MRM based on tryptic digests of whole products was a feasible method for quantification of modified milk proteins.

High efficiency acetylcholinesterase immobilization on DNA aptamer modified surfaces.

We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE). One selected aptamer sequence (R15/19) has a high affinity towards the enzyme (Kd = 157 ± 42 pM). Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM), however significant reduction in affinity occurred at high ionic strength (~1.2 M). In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.

Dual Recognition Element Lateral Flow Assay Toward Multiplex Strain Specific Influenza Virus Detection

Different influenza virus strains have caused a number of recent outbreaks killing scores of people and causing significant losses in animal farming. Simple, rapid, sensitive, and specific detection of particular strains, such as a pandemic strain versus a previous seasonal influenza, plays a crucial role in the monitoring, controlling, and management of outbreaks. In this paper we describe a dual recognition element lateral flow assay (DRELFA) which pairs a nucleic acid aptamer with an antibody for use as a point-of-care platform which can detect particular strains of interest. The combination is used to overcome the individual limitations of antibodies’ cross-reactivity and aptamers’ slow binding kinetics. In the detection of influenza viruses, we show that DRELFA can discriminate a particular virus strain against others of the same subtype or common respiratory diseases while still exhibiting fast binding kinetic of the antibody-based lateral flow assay (LFA). The improvement in specificity that DRELFA exhibits is an advantage over the currently available antibody-based LFA systems for influenza viruses, which offer discrimination between influenza virus types and subtypes. Using quantitative real-time PCR (qRT-PCR), it showed that the DRELFA is very effective in localizing the analyte to the test line (consistently over 90%) and this is crucial for the sensitivity of the device. In addition, color intensities of the test lines showed a good correlation between the DRELFA and the qRT-PCR over a 50-fold concentration range. Finally, lateral flow strips with a streptavidin capture test line and an anti-antibody control line are universally applicable to specific detection of a wide range of different analytes.

Digestibility of Glyoxal-Glycated β-Casein and β-Lactoglobulin and Distribution of Peptide-Bound Advanced Glycation End Products in Gastrointestinal Digests

This work reports the influence of glyoxal (GO)-derived glycation on the gastrointestinal enzymatic hydrolysis of β-lactoglobulin and β-casein. Reduced digestibility of glycated proteins was found in both gastric and intestinal stage. Distribution of Maillard reaction products in digests with different molecular weight ranges was investigated subsequently. The colorless and brown MRPs largely presented in the digests smaller than 20 kDa. However, the resistance of fluorescent advanced glycation end products (AGEs) to enzymatic hydrolysis gradually increased during glycation, rendering fluorescent AGEs largely present in the digests larger than 20 kDa. No free N (ε)-carboxymethyllysine (CML) was detected in digests. The relative amount of CML in digests larger than 1 kDa was higher than that of Lys, demonstrating the hindrance of CML to enzymatic hydrolysis. This study highlights the resistance of GO-derived AGEs to digestive proteases via blockage of tryptic cleavage sites or steric hindrance, which is a barrier to the absorption of dietary AGEs.

Streptavidin binding bifunctional aptamers and their interaction with low molecular weight ligands

This paper describes the measurement of the binding affinities of two bifunctional RNA aptamers to their respective ligands. The aptamers comprise either a theophylline or malachite green binding sequence fused to a streptavidin binding sequence. These bifunctional aptamers are shown to bind simultaneously to both the small ligand and to streptavidin whether in free solution or on gold surfaces. Binding isotherms for both interactions were measured by different physiochemical techniques: surface plasmon resonance, fluorescence spectroscopy and dynamic light scattering. Both qualitatively and quantitatively there is little difference in binding affinities between the bifunctional aptamers and their monofunctional components. The respective K(d) values for streptavidin binding in the monofunctional aptamer and in the theophylline bifunctional aptamer were 12 nM and 65 nM, respectively whilst the K(d) values for theophylline binding in the monofunctional aptamer and the streptavidin bifunctional aptamer were 300 nM and 120 nM. These results are consistent with treating each aptamer sequence as a module that can be combined with others without significant loss of function. This allows for the use of streptavidin based immobilization strategies without either the cost of biotinylated dNTPs or the variable yields associated with the chemical biotinylation of RNA.

Effect of glycation derived from α-dicarbonyl compounds on the in vitro digestibility of β-casein and β-lactoglobulin: A model study with glyoxal, methylglyoxal and butanedione

α-Dicarbonyl compounds, which are widely found in common consumed food, are one of the precursors of advanced glycation end products (AGEs). In this study, the effect of glycation derived from glyoxal (GO), methylglyoxal (MGO) or butanedione (BU) on the in vitro digestibility of β-casein (β-CN) and β-lactoglobulin (β-Lg) was investigated. Glycation from α-dicarbonyl compounds reduced the in vitro digestibility of studied proteins in both gastric and intestinal stage. In addition, glycation substantially altered the peptides released through gastric and gastrointestinal digestion, as detected by liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS). Crosslinked glycation structures derived from BU considerably reduced the sensitivity of glycated β-Lg towards digestive proteases, albeit to a lesser degree in glycated β-CN due to its intrinsic unordered structure. By contrast, non-crosslinked AGEs that formed adjacent to enzymatic cleavage sites did not block the enzymatic reaction in several cases, as evidenced by the corresponding digested peptides modified with glycation structures. These findings expand our understanding of the nutritional influence of α-dicarbonyl compounds and health impact of relevant dietary AGEs.