C-Phycocyanin (C-PC) represents an alternative to artificial blue/green dyes in food products. This study characterized and gained insights into C-PC thermal stability mechanisms and provided a model to estimate its thermal degradation. Aqueous solutions of C-PC (0.3 μM, pH:6.1) were isothermally heated at 45-80 °C. C-PC degradation was monitored based on the photophysical properties of its lumiphores (phycocyanobilins and aromatic aminoacids-AAs). While C-PC was stable at 45 °C, less than 10 min at 80 °C sufficed to degrade most of it. The thermal degradation curves were characterized using the Weibull model, which was validated with data obtained under non-isothermal conditions. Deviations between estimated and experimental values were lower than 8%. Hypsochromic shifts of the AAs' spectra (from 340 to 315 nm) and increase (>30%) in anisotropy at λexc = 280 and 520 nm suggest that colour losses are not solely associated with alterations of the chromophore but also with conformational changes and possible aggregation of the protein subunits.

The molecular mobility of amorphous excipients is important for the stability of biomaterials during preservation, facilitating matrix formulation and product design. Phosphorescence spectroscopy is a sensitive optical method to study molecular mobility. However, there is a need to expand the pool of probes available for analysis since molecules differ in sensitivity. This research explored the feasibility and limitations of using riboflavin as a phosphorescent probe for monitoring matrix molecular mobility. Phosphorescence decays of riboflavin in four amorphous cryosolvents (aqueous solutions of glycerol, ethanol, sucrose, and dextran) were collected at 77 K to capture its natural phosphorescence lifetime (estimated at 170 ms). Decays were also collected during ballistic heating to assess the sensitivity of riboflavin towards changes in matrix molecular mobility. Riboflavin exhibited good sensitivity towards matrix secondary relaxations in the glass, indicating that riboflavin has excellent potential as an edible phosphorescent probe for molecular mobility in food and pharmaceutical products.

Luminescence spectroscopy coupled with molecular rotors was used in the TNO Intestinal Model-1 (TIM-1) to monitor in situ changes to luminal viscosity of three maize starch samples varying in the amylose-to-amylopectin ratio (AM: AP): normal, high amylose (AM) and high amylopectin (AP). The fluorescence intensity (FI) of Fast Green (FG), a proven micro (and bulk) viscosity probe, was monitored throughout digestion to track changes in the gastric viscosity. The FI of FG and the viscosity imparted by the starch followed a power-law relationship. The emission of the MR was unaffected by the composition of TIM-1 secretion fluids nor pH. Hence, direct measurements of digesta FI are sensitive to changing viscosity during the simulated digestion. The viscosity was highest for AP, followed by normal starch, and high AM had the lowest viscosity. In the TIM-1 gastric compartment, from highest to lowest FI, and thus viscosity was high AM > high AP > normal maize starches. We conclude the validity of the proposed method to facilitate the measurement of luminal viscosity, in vitro, when the microviscosity represents bulk viscosity (i.e., when the increase in bulk viscosity is a result of molecular crowding and the surrounding environment around the rotor is homogeneous). Careful consideration is required when foods are heterogeneous as molecular rotors report only on their local non-uniform environment.

Nobiletin has received much attention for its promising biological activities. Owing to its limited solubility, various encapsulation strategies have been developed to enhance nobiletin bioavailability. However, the understanding of the bioavailability and biotransformation of nobiletin in vivo and the correlation between in vitro and in vivo data remains limited. This study developed a high-loading nobiletin (1%) emulsion. The in vitro models, which combined pH-stat lipolysis with a Franz cell, showed very good correlation with in vivo data for the relative bioavailability. Rat studies showed that nobiletin had a high absolute bioavailability (≈20% for oil suspension). Besides, the emulsification improved the amount of bioavailable nobiletin and its major metabolite in the blood by about two times, as compared to an oil suspension. This work provides scientific insights into a rapid screening method for delivery systems and a better understanding of the biological fate of nobiletin in vivo.

The limited stability of Spirulina protein extract towards environmental factors limits its application in food formulations. This study evaluates the characteristics and efficacy of different delivery systems composed of pure trehalose and trehalose-maltodextrin mixtures at different ratios (50:50; 20:80) to encapsulate Spirulina extract. The delivery systems were obtained through conventional amorphization techniques as freeze- and spray-drying and novel ones such as co-milling. Among the studied techniques, freeze-dried samples, regardless of the matrix composition, exhibited the highest carrying capacity with a residual amount of phycocyanin >89% after encapsulation. The use of ball co-milling for encapsulation caused a complete degradation of the core compound when applied using processing times applied of 6 h and 12 h. The glass transition temperature of the different samples, determined by differential scanning calorimetry, was affected by the carrier composition, increasing with increasing amounts of maltodextrin present in the matrix. When samples were exposed to high temperature during storage the delivery systems containing maltodextrin were more effective in preventing thermal degradation of the Spirulina extract and preserving its colouring ability.

Digestion is the process of breaking down food into smaller nutrient components which can be easily absorbed in the intestinal tract. The aim of this study was to experimentally investigate the influence of bolus (gastric content) viscosity on digestion and nutrient absorption processes, using an in vitro gastrointestinal model, the TIM-1 system. Two types of simple carbohydrates, namely, glucose and maltodextrin, were used as model foods. The initial bolus viscosity was varied (∼1 mPa·s, ∼15 mPa·s, and ∼100 mPa·s) using different glycerol-water proportions. A fluorescent molecular rotor compound (Fast Green For Coloring Food) was used to monitor viscosity changing patterns of the gastrointestinal content during digestion in the in vitro stomach and small intestinal sections. The digested-nutrient absorption data indicated that the initial bolus viscosity did not significantly affect the glucose absorption process in the small intestine. However, an increase in the initial bolus viscosity from ∼1 mPa·s to ∼15 mPa·s reduced the maltodextrin to glucose conversion by 35%. A further increase in the initial bolus viscosity from ∼15 mPa·s to ∼100 mPa·s did not significantly reduce the maltodextrin to glucose conversion.

Milk protein concentrate (MPC; 0 g, 5 g, and 10 g) was added to two commercially available oat products (instant oats and steel cut oats) to examine how MPC addition, and consequent changes in meal formulation, manipulates both gastric lumen viscosity and intestinal carbohydrate-digestion kinetics, in vitro. We used the TNO Intestinal Model-1 (TIM-1) to simulate gastrointestinal digestion of the oats-based meals. Meals containing 5 g or 10 g MPC yielded significantly less total bioaccessible sugar compared with those containing 0 g MPC, while the rate of starch digestion was significantly higher in meals containing 5 g or 10 g MPC. The TIM-1 was coupled with fluorescence spectroscopy and a luminescent molecular rotor to report changes in gastric viscosity in situ, showing that the gastric viscosity was higher in the meals containing MPC. Those findings suggest that MPC in oats-based meals significantly modifies the kinetics of carbohydrate digestion and increases gastric viscosity. (c) 2018 Elsevier Ltd. All rights reserved.

Allura Red AC (AR) is a synthetic color approved for use in foods and pharmaceuticals in the US. The sensitivity of this dye as a fluorescence probe of microviscosity has recently been reported. The dye fluorescence is weak, often undetectable, in low viscosity fluids; however, its quantum yield, emission intensity and lifetime increase significantly with an increase in medium viscosity. It thus exhibits presumptive molecular rotor behavior. To facilitate the operationalization of AR as a probe of food quality, its photophysical properties and performance in model food systems were studied. The absorption, emission and excitation spectra of AR was measured in water, glycerol and their mixtures. Additionally, the fluorescence quantum yields and lifetimes were determined as a function of increasing viscosity (0.6 to 15 mPa s) and temperature (−4 to 40°C) in water, ethanol and glycerol. AR showed a large Stokes shift (∼2500 cm−1) and a moderate sensitivity to polarity. Its sensitivity to molecular crowding was characterized using the Förster-Hoffmann equation (x ∼ 0.40). The fluorescence lifetime of AR was extremely short in fluid environments (12 ps in water at room temperature) and increased over 10-fold when dissolved in glycerol (231 ps). The temperature dependence of AR's quantum yield in ethanol and glycerol followed Arrhenius behavior with a lower activation energy in ethanol (3.2 kcal/mol) than in glycerol (7.6 kcal/mol), providing additional support for molecular rotor behavior. AR also showed high sensitivity to the bulk rheological properties of gelatin desserts and to changes in consistency of custards during cooking. This study makes a significant contribution to the currently incomplete photophysical characterization of this safe-to-ingest fluorophore and its application as a sensor in edible products.

Despite extensive experimental and computational efforts to understand the nature of the hierarchy of protein fluctuations and the modulating role of the protein hydration shell, a detailed microscopic description of the dynamics of the protein-solvent system has yet to be achieved. By using single tryptophan protein phosphorescence, we follow site-specific internal protein dynamics over a broad temperature range and demonstrate three independent dynamic processes. Process I is seen at temperatures below the bulk solvent Tg, has low activation energy, and is likely due to fast vibrations that may be enabled by water mobility on the protein surface. Process II is observed above 170 K, with activation energy typical of β relaxations in a glass; it has the same temperature dependence as fluctuations of hydration shell waters. Process III is observed at T > 200 K; it has super-Arrhenius temperature dependence and closely follows the primary relaxation of the bulk. The fluorescence of pyranine bound to the protein reports on the mobility of water in the hydration shell; it reveals a shift in emission spectra with increasing temperature, indicative of a changing H-bond network at the surface of the protein. These results support a model of solvent-slaved protein dynamics.

Three commercially available oat products instant oats, steel cut oats, and oat bran-were studied using the TNO Intestinal Model-1 (TIM-1) coupled with fluorescence spectroscopy and molecular rotors to evaluate carbohydrate digestion and in vitro gastric viscosity as a function of time. A proportional relationship between total bioaccessible sugars and the concentration of available carbohydrates was observed for the different oat-based foods. The rate of starch digestion was greatest for instant oats and lowest for steel cut oats. beta-glucan, starch, and total carbohydrate concentrations were proportional to the initial gastric viscosity. Overall, gastric viscosity differed considerably between samples. Instant oat and oat bran viscosities were highest at the onset of digestion and decreased with time, whereas the viscoity of steel cut oats at the onset of digestion was the lowest viscosity observed, increasing with time. These findings suggest that modification of food form and formulation during processing alters sugar bio-accessibility and luminal viscosity. (C) 2017 Elsevier Ltd. All rights reserved.

Protein-based particles are very promising colloidal systems for protection and controlled release applications in the food, cosmetics and pharmaceutical sector. One technique to produce these protein colloidal particles is liquid antisolvent precipitation (LAS). Despite the simplicity and versatility of LAS, not much is known about the protein conformational changes and interactions that are at the basis of the particle formation process. In this study, steady state fluorescence experiments using intrinsic fluorophores were evaluated as a tool to unravel the dynamics of the protein nanoparticle formation. Colloidal whey protein isolate and gliadin particles were produced by LAS. Changes in particle diameter (distribution), polydispersity index and photophysical properties of intrinsic fluorophores were monitored as a function of antisolvent concentration. By combining dynamic light scattering with photophysical data, a model of the changes occurring during particle formation and disintegration could be proposed. The results suggest that particle formation and disintegration are fully reversible processes during which the main changes in protein conformation (around the fluorescent probes) occur at the same antisolvent concentrations. In principle, steady state fluorescence measurements using intrinsic probes can indeed be used to effectively report on (part of the) conformational changes for both protein systems under study.

Fluorescent molecular rotors (MRs) are compounds whose emission is modulated by segmental mobility; photoexcitation generates a locally excited (LE), planar state that can relax either by radiative decay (emission of a photon) or by formation of a twisted intramolecular charge transfer (TICT) state that can relax nonradiatively due to internal rotation. If the local environment around the probe allows for rapid internal rotation in the excited state, fast non-radiative decay can either effectively quench the fluorescence or generate a second, red-shifted emission band. Conversely, any environmental restriction to twisting in the excited state due to free volume, crowding or viscosity, slows rotational relaxation and promotes fluorescence emission from the LE state. The environmental sensitivity of MRs has been exploited extensively in biological applications to sense microviscosity in biofluids, the stability and physical state of biomembranes, and conformational changes in macromolecules. The application of MRs in food research, however, has been only marginally explored. In this review, we summarize the main characteristics of fluorescent MRs, their current applications in biological research and their current and potential applications as sensors of physical properties in food science and engineering.

Oxygen radical absorption capacity (ORAC) and cellular antioxidant activity (CAA) of cocoa pulp, as affected by single and double pasteurization treatments (both at 85C for 60 s), were studied to understand its potential health benefits. Single and double pasteurization decreased total phenolic content by 25 and 38%, respectively. ORAC values were not significantly different between unpasteurized, single and double pasteurized pulp samples. However, significant increase in CAA values of double pasteurized pulp was observed in comparison to unpasteurized pulp. Stability of total phenolics and ORAC were monitored for 8 weeks at 4, 25 and 37C. Overall, single and double pasteurized pulps showed minimal changes in total phenolic content and ORAC values during storage at 4 and 25C. However, at 37C, single pasteurization resulted in 50% relative loss in total phenolics and 40% reduction in ORAC values, whereas double pasteurization resulted in less total phenolic loss and ORAC value reduction.

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme's absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.

Foods contain a plethora of aromatic molecules-natural colors, synthetic dyes, flavors, vitamins, antioxidants, etc.-that are luminescent, exhibiting prompt fluorescence or delayed phosphorescence. Although food autofluorescence has been used to detect specific contaminants (e.g., aflatoxins) or to authenticate specific foods (olive oil), much of the potential of using the optical luminescence of intrinsic molecules for sensing properties of foods is unrealized. We summarize here work characterizing the photophysical properties of some edible, and potentially GRAS (generally-recognized-as-safe), chromophores and especially their sensitivity to, and thus potential for sensing, various physical-viscosity, mobility/rigidity-or chemical-polarity, pH-properties of food known to reflect or be indicative of food quality, stability, and safety. A thoroughgoing characterization of and robust protocols for interpretation of the luminescent signals from edible chromophores can expand the repertoire of analytical techniques available to monitor quality, and even safety, of the food supply at various stages of production, distribution and storage or even at point of sale.

Riboflavin fluorescence has been characterized profusely, however little attention has been devoted to riboflavin phosphorescence. We have characterized steady state and time resolved phosphorescence of riboflavin from 77K to 333K in solvent mixtures (water:alcohol, water:glycerol) and amorphous solids (glucose, glucose oligomers, sucrose, dextran) to expand our knowledge on riboflavin photophysical properties and assess its potential use in quality monitoring. When excited at 440nm, the emission spectra of riboflavin exhibited maxima at 515nm (delayed fluorescence) and 620nm (phosphorescence). Deconvolution of the spectra to two individual bands characterized by asymmetric lognormal functions (I(ν)=I0∗exp{-ln(2)(ln[1+2b(ν-νp)/Δ2]/b)} ) facilitated the extraction of parameters and the analysis of their temperature dependence. Time resolved intensity decays were fitted using stretch, multi-exponential and distribution (by MEM) models. Riboflavin exhibited a lifetime of 184ms at 77K in glycerol-water. The lifetime decreased slowly below the solvent mixture's Tg (170K) and abruptly above Tg; e.g., an increase of 20K above Tg reduced the lifetime from 100ms to 10ms. This reduction was associated with molecular motions in the matrix. Molecular mobility was also modulated by changing the composition of the matrix, by using components of different molecular sizes or by adding plasticizers. Riboflavin phosphorescence also exhibited good sensitivity towards molecular mobility changes driven by composition. This suggests its use as a GRAS optical probe for molecular mobility and its potential application to optimize matrix composition in food and pharmaceutical products to enhance stability of micro and bioactive components. Continuous monitoring of riboflavin phosphorescence during heating and cooling cycles revealed differences in the delayed luminescence emission spectra, likely due to a higher rate of irreversible photodegradation of riboflavin at high temperatures. If properly characterized, the thermal dependence of riboflavin photodegradation can potentially be operationalized in sensors for temperature abuse.