Metformin-Induced Invertase Unfolding: Enzyme Kinetics and Activity Regulation.

The effects of metformin on invertase activity and its inhibition on sucrose digestion were studied. The rapid unfolding kinetics of invertases, followed a two-state model with an inactive intermediate formation. The dynamic interaction between metformin and invertase caused the secondary structure of the enzyme to become less ß-sheet, more α-helix, and random coiling oriented, which weakened the binding force between enzyme and its substrate. Metformin acted as a chaotrope and disrupted the hydrogen bonds of water, which facilitated the unfolding of invertase. However, some sugar alcohols, which promoted the H-bond formation of water, could repair the secondary structure of metformin-denatured invertase and therefore regulate the enzyme activity. This research enriches our understanding of the mechanism of enzyme unfolding induced by guanidine compounds. Moreover, because metformin and sugar substitutes are of concern to diabetes, this research also provides useful information for understanding the activity of the digestive enzyme that coexists with metformin and sugar alcohols.

Rapid Kinetic Interactions of Sugar and Sugar Alcohol with Sweet Taste Receptors on Live Cells Using Stopped-Flow Spectroscopy.

The subjective measurement of the dynamic perception of sweetness is a problem in food science. Herein, the rapid interactions of sugars and sugar alcohols with sweet taste receptors on living cells on a millisecond timescale were studied via stopped-flow fluorescence spectroscopy. According to the rapid-kinetic parameters, sweeteners were divided into two groups. Sweeteners in group I disrupted the hydrogen bond network structure of water, and the apparent rate constant (kobs) was in the range of 0.45-0.6 s-1. Sweeteners in group II promoted the hydrogen bond formation of water, and the kobs was mostly in the range of 0.6-0.75 s-1. For most sweeteners, the kobs of cell responses was negatively correlated with the apparent specific volume of sweeteners. The differences in the cellular responses may be attributed to the disturbance in the water structure. Experimental results showed that the kinetic parameters of sweet cell responses reflected the dynamic perception of sweetness. Rapid kinetics, solution thermodynamic analysis, and water structure analysis enriched the physicochemical study of the sweetness mechanism and can be used to objectively evaluate the dynamic perception of sweetness.

The effect of macromolecular crowding degree on the self-assembly of fatty acid and lipid hydrolysis.

Investigation on the physiochemical nature involved in the production of fatty acid catalyzed by the vesicles is of importance to understand the digestion of lipid. In this paper, the effects of crowding degree, which was constructed by polyethylene glycol (PEG), on the autocatalytic production of fatty acid with different chain lengths was studied. The results showed that the higher crowding degree led to the slower production rate of decanoic acid but the faster rate of oleic acid. The reason lies in that the presence of macromolecules resulted in the increased sizes of decanoic acid vesicles, but decreased sizes of oleic acid vesicles. Meanwhile, decanoic acid vesicles in more crowded medium exhibited viscous behavior, whereas oleic acid displayed elastic behavior. This research provides useful information for understanding the unusual autocatalyzed production of fatty acid in macromolecular crowding and may also draw an attention to the physiologically relevant lipid digestion.

Glutathione inhibited starch digestion: Structural and kinetic analysis of substrate and α-amylase.

Glutathione (GSH) is a natural antioxidant that helps fight free radicals. Whether it will affect the activity of amylase and starch digestion remains unknown. This research disclosed that GSH could interact with starch through hydrogen bonds, which accelerated the swelling of starch granules and promoted the formation of ordered double-helix crystalline, and therefore inhibited starch digestion. Moreover, pig pancreas α-amylase (PPA) which was incubated with GSH displayed a less stable conformation and decreased activity. However, in a crowded media constructed by sodium caseinate (NaCas), an antagonistic effect existed between GSH and NaCas. As the rate and extent of starch digestion have been linked with health aspects, this study suggests that GSH can be used in the formulation of diet foods. It also reminds us to consider the synergistic or antagonistic effects caused by the coexisted components in the complexed food matrix.

Effect of Ionic and Non-Ionic Surfactants on the Pasting Characteristics and Digestive Properties of Regular and Frozen Starch for Oral Delivery.

Starch is an ideal wall material for controlled release in oral delivery systems due to its non-allergic properties, availability, and cheap price. However, because of its poor mechanical behavior and high water permeability, it is necessary to modify the amphiphilic nature of starch. Surfactants are essential components to emulsify the lyophobic food ingredients. However, the interaction of starch with emulsifiers and how they affect the pasting behavior and digestion of starch are not well understood. In this paper, surfactants, such as non-ionic Tween (TW) and ionic sodium fatty acid (NaFA), with varying hydrophobic carbon chain lengths, were selected as model amphiphiles to investigate the structural, pasting, rheological properties and in vitro digestibility of regular and frozen starch samples. The results showed that, in most cases, the addition of TW reduced the viscosity of starch. However, saturated medium-chain NaFA increased the starch viscosity and rheological modulus greatly. Both surfactants inhibited starch digestion. This paper presents a comparative investigation on the effect of ionic and non-ionic surfactant on the structure and properties of corn starch, and therefore the information is useful for structural-based formulation with starch for developing colloidal delivery systems. It is also helpful for developing functional food with controllable digestion properties.

l-Arginine inhibits the activity of α-amylase: Rapid kinetics, interaction and functional implications.

In this work, the rapid unfolding kinetics of pancreas α-amylase (PPA) induced by l-arginine and the interaction mechanism were investigated. The unfolding followed a first-level reaction kinetics equation, without intermediates. l-arginine interacted with PPA though diffusion-controlled process rather than complexion. The interaction between l-arginine and PPA resulted in a pronounced decrease in ß-sheet and a significant increase in random coil, and thereby the enzyme activity decreased. However, the unfolding of PPA could be compensated and the second structure change could be recovered to some extent by the macromolecular crowded medium of Pluronics. Further insight into the mechanism disclosed that the broken H-bond network of water may contribute to PPA unfolding. This work provides a new perspective on the interaction of l-arginine with digestive enzyme. The unfolding mechanism of enzymes by may help to understand the effects of other structurally similar drugs, which is of concern in food-drug interactions.

Rapid unfolding of pig pancreas α-amylase: Kinetics, activity and structure evolution.

α-Amylase plays an important role in food processing and in-vivo digestion. Many biological functions of α-amylase are affected by unfolding. The pre-steady-state rapid unfolding kinetics of α-amylase remains unknown. In this study, the rapid unfolding kinetics of porcine pancreatic α-amylase (PPA) with guanidine hydrochloride (GdmHCl) were investigated by stopped-flow spectroscopy. Structural characterization of PPA by fluorescence spectroscopy, and molecular dynamics simulation showed that the unfolding process of PPA might start from the internal active center, where the ß-sheet structure was destroyed, followed by the exposure of hydrophobic amino acid residues. Further research revealed that GdmHCl denaturized PPA not by complexing with PPA. The surrounding H-bond network of water was changed by GdmHCl. This research improves our understanding of the unfolding kinetics of the PPA on the microsecond scale. It also provides the evidence experimentally of the surrounding water contribution to protein denaturization.

Inhibition of starch digestion: The role of hydrophobic domain of both α-amylase and substrates.

The physicochemical mechanism of starch digestion is very complicated since it may be affected by the non-valence interactions of the amylase inhibitor with the substrate or the enzyme. The role of hydrophobic interaction in the process of starch digestion is not clear. In this study, pluronics (PLs) with different hydrophobicity were used as model amphiphilic compounds to study their inhibition on starch digestion using multi-spectroscopic methods. The results showed that the hydrophobic nature of PLs changed starch structure, but it had a greater effect on the structure of α-amylase by exposing more tryptophan residues and increasing α-helix and ß-sheet contents. Further investigation by using different chain-length fatty acids confirmed the results. The finding in this study is informative to design and fabricate α-amylase inhibitors for controlling starch digestion at the molecular level.

Synergistic interaction between exogenous and endogenous emulsifiers and its impact on in vitro digestion of lipid in crowded medium.

Control of lipid digestibility by various food components has received great attention in recent decades. However, there is limited literature on investigating the synergistic effect of exogenous emulsifiers and endogenous sodium cholate (SC) on lipid digestion in a simulated physiological crowded medium. In this work, the synergistic interaction of Tween80 and SC according to the regular solution theory, and the hydrolysis of lipid emulsions containing tricaprylin, glyceryltrioleate or soybean oil in crowding medium was studied. The results show that emulsions stabilized by a combination of Tween80 and SC showed higher digestion rate and transformation than those with Tween80 or SC. The digestion rate could be increased by polyethylene glycols (PEGn) with varying crowding degree. The denaturation temperature of the lipase was increased in macromolecular crowded medium. This work allows for better understanding of the interaction between the amphiphiles and the macromolecular crowding effect on lipase digestion in the physiological environment.

The relationship between alkylamide compound content and pungency intensity of Zanthoxylum bungeanum based on sensory evaluation and ultra-performance liquid chromatography-mass spectrometry/ mass spectrometry (UPLC-MS/MS) analysis.

BACKGROUND: Zanthoxylum bungeanum originating in different places varies in alkylamide content and pungency intensity. RESULTS: The pungency intensity of 19 Zanthoxylum bungeanum samples was first determined with Scoville pungency units (SPUs). The SPUs were found to range from 3.80E + 04 to 5.40E + 05. The chemical compositions and contents were measured next, using the ultra-performance liquid chromatography-mass spectrometry/ mass spectrometry (UPLC-MS/MS) method. The total alkylamide content ranged from 9.83 ± 0.15 to 89.98 ± 1.35 g kg-1 . Hydroxy-ϵ-sanshool, hydroxy-α-sanshool, hydroxy-ß-sanshool, hydroxy-γ-sanshool, bungeanool, and isobungeanool were found to be the key pungent compounds, ranging in proportion from 92.65% to 97.69%. The relationship between alkymide compound content and pungency intensity was also analyzed by ridge regression, and it was found that the ß values of independent variables were stable when k was more than 0.6. The regression coefficients of hydroxy-ϵ-sanshool, hydroxy-α-sanshool, hydroxy-ß-sanshool, hydroxy-γ-sanshool, bungeanool, isobungeanool, and other alkylamides were 0.105, 0.177, 0.386, -0.166, -0.006, 0.005, and -0.018, respectively. CONCLUSION: Hydroxy- sanshool compounds were important in determinant the pungency intensity of Z. bungeanum. Knowledge of the relationship between alkymide compound content and pungency intensity will assist in the creation of new methods to determine pungency intensity and provide a scientific basis for flavor design, development of pungent food products, and consumer choice evaluations. © 2018 Society of Chemical Industry.

Difference in Binding of Long- and Medium-Chain Fatty Acids with Serum Albumin: The Role of Macromolecular Crowding Effect.

Fatty acids (FAs) are transported by serum albumin in plasma. Studies have been undertaken to address the binding of MCFAs or LCFAs to human plasma albumin (HPA) and bovine serum albumin (BSA) by characterizing the binding affinities. Previous research on FA binding to serum albumin was usually performed in dilute solutions that are not sufficiently concentrated for the interpretation of the significance of the results under normal physiological conditions. How macromolecular crowded media affect fatty acids and bovine serum albumin (BSA) binding remains unknown. In this article, we investigated the mechanism of FA-BSA binding in a polyethylene glycol crowding environment by using thermodynamic and spectroscopic methods. Molecular crowding increased the binding constant for saturated medium-chain fatty acids (MCFAs) but significantly decreased the binding constant for unsaturated long-chain FAs. The binding sites tended to increase in all the cases. Further investigation revealed that crowding media might loosen the structure of BSA, facilitating MCFA-BSA binding. This research is useful for understanding the transportation of FAs by BSA under physiological conditions and may also help to control digestion by the eventual incorporation of macromolecular crowding agents into food formulations.

[Assessment of Left Atrial Appendage Size and Morphology by Enhanced Cardiac Computed Tomography in Patients with Non-valvular Atrial Fibrillation].

OBJECTIVE: This study was designed to investigate left atrial appendage (LAA) size and morphology characteristics in patients with non-valvular atrial fibrillation (NVAF) by enhanced cardiac computed tomography (CT) scanning,and to evaluate the predictive value of these parameters for thromboembolic risk. METHODS: A total of 189 NVAF patients were prospectively enrolled during December 2012 to January 2014 in West China Hospital of Sichuan University. Fifty-two atrial tachycardia patients without organ dysfunction were also recruited as controls. All subjects were scheduled for radiofrequency ablation and underwent enhanced cardiac CT scan. The clinical characteristics,comorbid diseases,anticoagulant therapy,and AF chronicity were collected and CHA2DS2-VASc score was calculated for the assessment of thromboembolic risk. The NVAF cohort was also divided further into LAA thrombus present (n=13) and absent (n=176) subgroup according to LAA with thrombus or not. Differencesin size and morphology characteristics of the LAA were compared among controls and different CHA2DS2-VASc thromboembolic risk subgroups,and between LAA thrombus and non-thrombus group. In addition,logistic regression analysis was used to identify whether these parameters predict thrombus formation independent of the CHA2DS2-VASc score in NVAF patients. RESULTS: Compared with controls,NVAF patients had significantly bigger orifice size. Windsock and cactus-type LAA were main morphology in controls,while the distribution of the four kinds of LAA types were similar in NVAF patients. The orifice and volume of the LAA were increased with increasing CHA2DS2-VASc riskscore,and chicken wing-type LAA was the predominant prevalence in low-intermediate thromboembolic risk subgroup while cauliflower in high thromboembolic risk subgroup. Compared with non-thrombus patients,LAA-thrombussubgroup exhibited a significantly increased orifice and volume,and higher prevalenceof the cauliflower shaped LAA. Logistic regression showed that the LAA orifice and cauliflower shape were independent predictors for thrombus formation in patients with NVAF following adjustment for the CHA2DS2-VASc score. CONCLUSION: Cardiac images obtained with CTis feasible for the analysis of LAA size and morphology,and these parameters provide incremental value in assessing the LAA thrombus risk in patients with NVAF.

Thermodynamics and Structural Evolution during a Reversible Vesicle-Micelle Transition of a Vitamin-Derived Bolaamphiphile Induced by Sodium Cholate.

Interaction of endogenous sodium cholate (SC) with dietary amphiphiles would induce structural evolution of the self-assembled aggregates, which inevitably affects the hydrolysis of fat in the gut. Current work mainly focused on the interaction of bile salts with classical double-layered phospholipid vesicles. In this paper, the thermodynamics and structural evolution during the interaction of SC with novel unilamellar vesicles formed from vitamin-derived zwitterionic bolaamphiphile (DDO) were characterized. It was revealed that an increased temperature and the presence of NaCl resulted in narrowed micelle-vesicle coexistence and enlarged the vesicle region. The coexistence of micelles and vesicles mainly came from the interaction of monomeric SC with DDO vesicles, whereas micellar SC contributed to the total solubilization of DDO vesicles. This research may enrich the thermodynamic mechanism behind the structure transition of the microaggregates formed by amphiphiles in the gut. It will also contribute to the design of food formulation and drug delivery system.

Influence of carboxymethyl cellulose and sodium alginate on sweetness intensity of Aspartame.

Sensory evaluation of Aspartame in the presence of sodium carboxymethyl cellulose (CMC-L) and sodium alginate (SA) revealed that only CMC-L showed a suppression effect, while SA did not. By using an artificial taste receptor model, we found that the presence of SA or CMC-L resulted in a decrease in association constants. Further investigation of CMC-L solution revealed that the decrease in water mobility and diffusion also contribute to the suppression effect. In the case of SA, the decreased viscosity and comparatively higher amount of free water facilitated the diffusion of sweetener, which might compensate for the decreased binding constant between Aspartame and receptor. This may suppress the impact of SA on sweetness intensity. The results suggest that exploring the binding affinity of taste molecules with the receptor, along with water mobility and diffusion in hydrocolloidal structures, provide sufficient information for understanding the mechanism behind the effect of macromolecular hydrocolloids on taste.

Chain-length-dependent autocatalytic hydrolysis of fatty acid anhydrides in polyethylene glycol.

Autocatalytic hydrolysis of fatty acid anhydrides induced by the spontaneously formed vesicles has been studied for years. However, whether the reaction autocatalyzed by vesicles formed in diluted solutions applies also to macromolecular crowded conditions remains unknown. The aim of this study is to characterize hydrolysis behavior of fatty acid anhydrides and formation of vesicles in crowded media. Inert macromolecular crowding agents such as polyethylene glycol (PEG) and Dextran were used to probe the impact of external crowding on the autocatalytic hydrolysis of fatty acid anhydrides with varied hydrophobic chain length. Under stringent conditions of crowding, hydrolysis rates of octanoic anhydride, nonanoic anhydride, and decanoic anhydride were found to decrease, but the rates of lauric anhydride and oleic anhydride increased. These results suggest that the effect of the crowding agent on the hydrolysis of fatty acid anhydrides was chain-length-dependent. Characterization of the size and polydispersity of vesicles formed from hydrolyzed fatty acid anhydrides in crowding revealed that long-chain fatty acids formed monodisperse vesicles easier at lower concentrations of PEG. Measurement of the critical aggregation concentration of ionized fatty acid in the presence of PEG showed that crowding media promoted vesicle formation from long-chain fatty acids but inhibited those from fatty acids with fewer carbon atoms. Further investigation of the diffusion property of ionized fatty acids in crowding agents suggested that PEG might create more hydrophobic areas for long-chain fatty acids anhydrides, which subsequently promoted the unreacted anhydride in the aqueous phase to be solubilized in the formed vesicles. This research provides information for understanding the autocatalytic reaction accompanied by self-producing aggregates and the behavior of fatty acids in crowding media.

Controllable self-assembly of sodium caseinate with a zwitterionic vitamin-derived bolaamphiphile.

The control of self-assembly of sodium caseinate (SC) including the formation of mixed layers, microspheres, or nanoparticles is highly relevant to the microstructure of food and the design of promising drug delivery systems. In this paper, we designed a structure-switchable zwitterionic bolaamphiphile, 1,12-diaminododecanediorotate (DDO), from orotic acid, which has special binding sites and can guide the self-assembly of SC. Complexation between SC and DDO was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, and fluorescence spectra measurements. Monomeric DDO was bound to the negatively charged sites on the SC micelle and made the structure of SC more compact with decreased electrostatic repulsion between the head groups. Vesicular DDO led to reassociation of vesicles with enlarged size via preferable hydrophobic interactions. Moreover, the aggregation between SC and DDO was found to be temperature-dependent and reversible. This research provides an effective way to control the reversible self-assembly of SC by the zwitterionic vitamin-derived bolaamphiphile.

Thermodynamics of the enantiomers of amino acid and monosaccharide binding to fullerenol used as an artificial sweet taste receptor model.

Fullerenol was used as sweet taste receptor model to investigate the binding affinities of structurally related pairs of enantiomers by isothermal titration calorimetry (ITC). It reveals that amino acid binding with fullerenol are enthalpy-cost and entropically-driven processes, whereas enthalpy contributes to monosaccharide binding to fullerenol. Spontaneous binding of amino acids was found through two sequential steps in which the sweeter enantiomer displays larger binding constants. Association of the d-form of fructose and l-form galactose with fullerenol suggested that, the higher the perceived sweetness intensity of the enantiomer, the larger was the binding constant with respect to their antipodes. Further investigation by molecular dynamic simulation showed that the binding energy and the perceived sweetness intensity were well correlated. The preliminary results of this biomimetic research cover the lack of information about the thermodynamic basis of sweet sensation and the underlying principles of sweetness differences between the enantiomers of amino acids and monosaccharides.

Realization of the reversible vesicle-micelle transition of vitamin-derived bolaamphiphiles by heat change monitoring.

The real-time energetics involved in the structural change of a zwitterionic vitamin-derived bolaamphiphiles (DDO) vesicles, which were induced by conventional surfactants, such as hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and Triton X-100 (TX100), was characterized by isothermal titration calorimetry (ITC). Interactions of both CTAB and SDS with DDO were accompanied with considerable heat release whereas the interaction energetics between TX-100 and the vesicles were small. However, the transition of DDO vesicles to micelles did occur upon the addition of all of the three surfactants. Fine inflection points were observed in heat flow enthalpograms, which indicated systematically the change of vesicle structure. By monitoring the interaction of CTAB with DDO, we found that heat release kept constant over a certain concentration range at higher temperatures. The repairing effect of heating was revealed and a reversible transition from micelles to vesicles of DDO was thus realized. Further encapsulation of fluorescein in DDO vesicles proved that the reversible vesicle-micelle transition was controllable. This research demonstrates that ITC combined with complementary analytical methods such as dynamic light scattering (DLS) and transmission electron microscopy (TEM) helps to get the real-time information of the structural changes of vesicles. It also shows that these synthetic novel bolaamphiphiles offer great promise for designing controllable release system.

Thermodynamics of the interaction of sweeteners and lactisole with fullerenols as an artificial sweet taste receptor model.

The thermodynamics of the mimetic interaction of lactisole and sweeteners with fullerenols as a synthetic sweet receptor model was elucidated by Isothermal Titration Calorimetry (ITC) technique. The presence of lactisole resulted in great differences in thermodynamics of the sweeteners binding with fullerenols in which lactisole led to much more entropy contribution to the free energy compared with the interaction of sweeteners with fullerenols. Two interaction equilibrium states were found in ITC titration profiles and competitive binding of lactisole and sweeteners with fullerenols was disclosed. Our results indicated that the larger value of the ratio of two equilibrium constant K1/K2, the more effectively lactisole inhibited the sweetness of the sweetener. The combined results of sensory evaluation and ITC thermodynamics revealed that introducing a synthetic receptor model to interact with the sweeteners and inhibitors helps to understand the inhibition mechanism and the thermodynamic basis for the initiation of sweetness inhibition.

Molecular recognition of melamine by vesicles spontaneously formed from orotic acid derived bolaamphiphiles.

Molecular recognition by means of multiple hydrogen bonds is of great importance in biological functions. In this paper, an orotic acid derived bolaamphiphile 1,12-diaminododecane diorotate (DDO) with molecular recognition function moieties was designed. Both self-aggregation behavior and molecular recognition with melamine were extensively examined. This bolaamphiphile itself can form vesicles easily in aqueous solutions at 25 °C. Steady-state fluorescence was used to characterize the detailed molecular recognition process. The fluorescence of melamine was quenched more effectively by the spontaneously formed vesicles than by the monomers of the surfactant. Two mechanisms were involved in the fluorescence quench process. At lower concentration, the fluorescence of melamine was found to be quenched by static complex formation. While at higher concentration, both static and dynamic quenching mechanisms coexisted in interaction process. Thermodynamic parameters measured by isothermal titration calorimetry showed that the free energy (ΔG) is negative, indicating that binding of DDO molecules with melamine is favorable energetically. Hydrogen-bonded interactions contribute comparatively a lot for the DDO monomer binding with melamine; at the higher concentration above its critical aggregation concentration, the dissociation of the aggregates take place and lead to an entropically driven molecular recognition process. As complicated binding sites can be constructed through self-assembly at the vesicle interface rather than simple molecular modules, this bolaamphiphile with the molecular recognition functional group may make it possible to generate well-defined recognition sites to mimic biomolecular receptors. Moreover, the present research will give a guide to design chemosensors for melamine detection based on molecular recognition.