Ionic calcium determination in skim milk with molecular probes and front-face fluorescence spectroscopy: simple linear regression

The purpose of this study was to determine if the ionic calcium content of skim milk could be determined using molecular probes and front-face fluorescence spectroscopy. Current methods for determining ionic calcium are not sensitive, overestimate ionic calcium, or require complex procedures. Molecular probes designed specifically for measuring ionic calcium could potentially be used to determine the ionic calcium content of skim milk. The goal of the current study was to develop foundation methods for future studies to determine ionic calcium directly in skim milk and other dairy products with molecular probes and fluorescence spectroscopy. In this study, the effect of pH on calcium-sensitive fluorescent probe (Rhod-5N and Fluo-5N) performance using various concentrations of skim milk was determined. The pH of diluted skim milk (1.9 to 8.9% skim milk), was adjusted to either 6.2 or 7.0, after which the samples were analyzed with fluorescent probes (1 μM) and front-face fluorescence spectroscopy. The ionic calcium content of each sample was also determined using a calcium ion-selective electrode. The results demonstrated that the ionic calcium content of each sample was highly correlated (R² > 0.989) with the fluorescence intensities of the probe-calcium adduct using simple linear regression. Higher than suggested ionic calcium contents of 1,207 and 1,973 μM were determined with the probes (Fluo-5N and Rhod-5N) in diluted skim milk with pH 7.0 and 6.2, respectively. The fluorescence intensity of the probe-calcium adduct decreased with a decrease in pH for the same ionic calcium concentration. This study demonstrates that Fluo-5N and Rhod-5N can be used to determine the ionic-calcium content of diluted milk with front-face fluorescence spectroscopy. Furthermore, these probes may also have the potential to determine the ionic calcium content of undiluted skim milk.     

Application of fluorescence spectroscopy for monitoring changes in nonfat dry milk during storage

The objective of this study was to determine if fluorescence spectroscopy could be used to characterize the biochemical characteristics of nonfat dry milk (NDM) caused by manufacturing and storage conditions. Nine low-heat NDM samples were collected from 3 manufacturers and stored at 4 temperatures (4, 22, 35, and 50°C) for 8 wk. The spectra of Maillard products, tryptophan, and riboflavin were recorded and analyzed with principal components analysis. Colorimetric indices L*, a*, and b* were also determined. The before-storage NDM samples collected from each manufacturer had different fluorescent characteristics. Inconsistency was observed for the NDM samples collected from 1 manufacturer, whereas the samples from the other 2 manufacturers displayed consistent fluorescence characteristics. Biochemical reactions, such as Maillard reaction, modification of the tryptophan environment, and degradation of riboflavin occurred during the manufacturing process. For each of the data collections, discrimination of the NDM samples stored at 50°C from the samples stored at 4, 22, and 35°C was observed in the similarity maps. The factor loadings of the first 2 principal components for the fluorescence spectra of the samples before storage were similar to the principal components analysis results of the samples during storage. It appears that similar factors are responsible for the variation in the samples before storage and their changes during storage. Additionally, storage of the samples at 50°C accelerated these reactions. The results demonstrate that front-face fluorescence spectroscopy, coupled with multivariate statistical methods, can be utilized as an analytical technique to monitor variation in NDM samples from different manufacturers and changes during storage.     

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: pH buffering properties of cheese

The pH buffering capacity of cheese is an important determinant of cheese pH. However, the effects of different constituents of cheese on its pH buffering capacity have not been fully clarified. The objective of this study was to characterize the chemical species and chemical equilibria that are responsible for the pH buffering properties of cheese. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), residual lactose (2.4 vs. 0.78%), and salt-to-moisture ratio (6.4 vs. 4.8%) were manufactured. The pH-titration curves for these cheeses were obtained by titrating cheese:water (1:39 wt/wt) dispersions with 1 N HCl, and backtitrating with 1 N NaOH. To understand the role of different chemical equilibria and the respective chemical species in controlling the pH of cheese, pH buffering was modeled mathematically. The 36 chemical species that were found to be relevant for modeling can be classified as cations (Na⁺, Ca²⁺, Mg²⁺), anions (phosphate, citrate, lactate), protein-bound amino acids with a side-chain pKa in the range of 3 to 9 (glutamate, histidine, serine phosphate, aspartate), metal ion complexes (phosphate, citrate, and lactate complexes of Na⁺, Ca²⁺, and Mg²⁺), and calcium phosphate precipitates. A set of 36 corresponding equations was solved to give the concentrations of all chemical species as a function of pH, allowing the prediction of buffering curves. Changes in the calculated species concentrations allowed the identification of the chemical species and chemical equilibria that dominate the pH buffering properties of cheese in different pH ranges. The model indicates that pH buffering in the pH range from 4.5 to 5.5 is predominantly due to a precipitate of Ca and phosphate, and the protonation equilibrium involving the side chains of protein-bound glutamate. In the literature, the precipitate is often referred to as amorphous colloidal calcium phosphate. A comparison of experimental data and model predictions shows that the buffering properties of the precipitate can be explained, assuming that it consists of hydroxyapatite [Ca₅(OH)(PO₄)₃] or Ca₃(PO₄)₂. The pH buffering in the region from pH 3.5 to 4.5 is due to protonation of side-chain carboxylates of protein-bound glutamate, aspartate, and lactate, in order of decreasing significance. In addition, pH buffering between pH 5 to 8 in the backtitration results from the reprecipitation of calcium and phosphate either as CaHPO₄ or Ca₄H(PO₄)₃.     

Lipids of Thermococcus hydrothermalis, an archaea isolated from a deep-sea hydrothermal vent

The membrane lipids of a deep-sea hydrothermal vent archaea, Thermococcus hydrothermalis, were isolated, purified, and structurally characterized. On the basis of acid methanolysis and spectroscopic studies, the polar lipids, amounting to 4.5% (w/w) of the dry cells, comprised diphytanyl glycerol diethers and dibiphytanyldiglycerol tetraethers, in a 45∶55 ratio. No cyclopentane ring was present in the tetraethers. From the neutral lipids, accounting for 0.4% (w/w) of the dry cells, besides low amounts of di- and tetraethers occurring in a free form, four acyclic tetraterpenoid hydrocarbons, di- and triunsaturated were identified. All were structurally related to lycopane. The presence of these hydrocarbons provides some evidence that lycopane, widely distributed in oceans, could be derived, at least partially, from the hydrocarbons synthesized by some thermophilic Archaea. Finally, analysis of the uninoculated culture medium indicates that fatty acid derivatives and some steroid and triterpenoid compounds identified in the lipidic extract of the archaea originated from the culture medium.     

Lewis acid induced additions to unsaturated fatty compounds IV: Synthesis of cyclopentenones from Friedel- Crafts acylation products of unsaturated fatty compounds with α,β-unsaturated acyl chlorides

The ethylaluminium dichloride induced Friedel- Crafts acylation of unsaturated fatty compounds such as oleic acid ( 1a ), methyl oleate ( 1b ) and 10-undecenoic acid ( 9b ) and furthermore of 1-octene ( 9a ) with α,β-unsaturated acyl chlorides e.g. crotonic acid chloride ( 2a ) and acrylic acid chloride ( 2b ) gave the corresponding allyl vinyl ketones. Nazarov cyclizations of the acylation products 3a/4a, 3b/4b, 10a and 10b afforded the alkyl substituted 2-cyclopentenones 5a/6a, 5b/6b, 11a/12a and 11b/12b . Catalytic hydrogenation of 5b/6b and 11b/12b gave the respective saturated cyclic products 7b/8b and 13b/14b as diastereomeric mixtures.     

Development and Characterization of a 96-Well Exposure System for Safety Assessment of Nanomaterials

In this study, a 96-well exposure system for safety assessment of nanomaterials is developed and characterized using an air–liquid interface lung epithelial model. This system is designed for sequential nebulization. Distribution studies verify the reproducible distribution over all 96 wells, with lower insert-to-insert variability compared to non-sequential application. With a first set of chemicals (TritonX), drugs (Bortezomib), and nanomaterials (silver nanoparticles and (non-)fluorescent crystalline nanocellulose), sequential exposure studies are performed with human lung epithelial cells followed by quantification of the deposited mass and of cell viability. The developed exposure system offers for the first time the possibility of exposing an air–liquid interface model in a 96-well format, resulting in high-throughput rates, combined with the feature for sequential dosing. This exposure system allows the possibility of creating dose-response curves resulting in the generation of more reliable cell-based assay data for many types of applications, such as safety analysis. In addition to chemicals and drugs, nanomaterials with spherical shapes, but also morphologically more complex nanostructures can be exposed sequentially with high efficiency. This allows new perspectives on in vivo-like and animal-free approaches for chemical and pharmaceutical safety assessment, in line with the 3R principle of replacing and reducing animal experiments.