Phase-Separated Structures of Sake Flavors-Containing Cell Model Membranes.

Sake (a traditional Japanese alcoholic beverage) contains ethyl caproate (EC), which enhances its economic value. Isovaleraldehyde (IVA) is also a well-known flavoring agent in alcoholic beverages, which some people enjoy. Recently, studies revealed that EC decreased the size of homogenous 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes whereas IVA increased their size. Cholesterol (Chol) and ergosterol were previously referred to as animal and fungus sterols. For the first time, this study demonstrated the phase behavior of the membrane in cell-sized liposomes containing EC and IVA. After adding EC, the solid ordered/liquid disordered (Ld) and liquid ordered (Lo)/Ld phase separation in DOPC/dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol or ergosterol ternary membranes decreased, but the Lo/Ld phase separation decreased after adding IVA. Biophysics, physiological, and application aspects of EC and IVA evaluation were discussed. The findings of this study not only enhance our understanding of the function of flavors but also provide rapid and cost effective performance for the measurement.

Charged Lipids Influence Phase Separation in Cell-Sized Liposomes Containing Cholesterol or Ergosterol.

Positively charged ion species and charged lipids play specific roles in biochemical processes, especially those involving cell membranes. The cell membrane and phase separation domains are attractive research targets to study signal transduction. The phase separation structure and functions of cell-sized liposomes containing charged lipids and cholesterol have been investigated earlier, and the domain structure has also been studied in a membrane model, containing the yeast sterol ergosterol. The present study investigates phase-separated domain structure alterations in membranes containing charged lipids when cholesterol is substituted with ergosterol. This study finds that ergosterol increases the homogeneity of membranes containing charged lipids. Cholesterol-containing membranes are more sensitive to a charged state, and ergosterol-containing liposomes show lower responses to charged lipids. These findings may improve our understanding of the differences in both yeast and mammalian cells, as well as the interactions of proteins with lipids during signal transduction.

The Flavonoid Molecule Procyanidin Reduces Phase Separation in Model Membranes.

Procyanidin extracted from fruits, such as apples, has been shown to improve lipid metabolization. Recently, studies have revealed that procyanidin interacts with lipid molecules in membranes to enhance lipid metabolism; however, direct evidence of the interaction between procyanidin and lipid membranes has not been demonstrated. In this study, the phase behaviors and changes in the membrane fluidity of cell-sized liposomes containing apple procyanidin, procyanidin B2 (PB2), were demonstrated for the first time. Phase separation in 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol ternary membranes significantly decreased after the addition of PB2. The prospect of applying procyanidin content measurements, using the results of this study, to commercial apple juice was also assessed. Specifically, the PB2 concentrations were 50%, 33%, and 0% for pure apple juice, 2-fold diluted apple juice, and pure water, respectively. The results of the actual juice were correlated with PB2 concentrations and phase-separated liposomes ratios, as well as with the results of experiments involving pure chemicals. In conclusion, the mechanism through which procyanidin improves lipid metabolism through the regulation of membrane fluidity was established.

Phase Separation in Liposomes Determined by Ergosterol and Classified Using Machine Learning.

Recent studies indicated that ergosterol (Erg) helps form strongly ordered lipid domains in membranes that depend on their chemical characters. However, direct evidence of concentration-dependent interaction of Erg with lipid membranes has not been reported. We studied the Erg concentration-dependent changes in the phase behaviors of membranes using cell-sized liposomes containing 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). We observed the concentration range of phase separation in ternary membranes was significantly wider when Erg rather than cholesterol (Chol) was used as the sterol component. We used machine learning for the first time to analyze microscopic images of cell-sized liposomes and identify phase-separated structures. The automated method was successful in identifying homogeneous membranes but performance remained data-limited for the identification of phase separation domains characterized by more complex features.

Quality Evaluation of Drinks Based on Liposome Shape Changes Induced by Flavor Molecules.

The flavors of ethyl caproate and isoamyl acetate and their precursors are crucial in sake brewing for fermentation and evaluation of the corresponding quality of drinks. However, the quality evaluation of drinks containing these flavors is challenging. Therefore, sake quality was evaluated via dynamic membrane transformation on cell-sized liposomes while adding flavor-containing solutions. Flavor varieties have been reported to influence dynamic shape change patterns. This study reports the observed difference in the dynamic shape change of each flavor. Based on these results, proper quality evaluation of drinks is expected.

Effects of isovaleraldehyde on cell-sized lipid bilayer vesicles.

Membrane composition and components are intrinsic properties of a cell membrane. Any changes in lipid vesicle composition or any stimuli, such as heat, that affect molecular packing induce dynamic shape change. Dynamic shape changes allow the determination of structural organization changes upon a change in the membrane internal or external environment. In this study, we report how thermal stress can affect isovaleraldehyde (IVA) flavor compound-containing membranes. We revealed that (1) IVA-containing lipid vesicles are large and their increasing size results in increasing IVA/vesicle concentration; (2) IVA-containing lipid vesicles are less thermo-responsive and are affected by increasing IVA concentration; finally, we discussed (3) the molecular mechanisms behind membrane packing. We proposed that the characteristic of IVA-containing membranes could be used in evaluating drink quality. Our results would potentially contribute to the development of membrane technology and the progress in further understanding physiological processes, such as flavor sensation.

Freeze concentrated apple juice maintains its flavor.

Concentrated juices are sources of alcoholic drinks. Juice concentration may be achieved using different methods, such as freezing or heating. High temperatures in the process of juice concentration damage heat-sensitive components, such as aromatic compounds. Although the freezing process of juice concentration has been studied, analyses have been inadequate, particularly in addressing flavors. Therefore, we investigated the characteristics of freezing and heating during apple juice concentration in the context of flavor. We found that a total of 97 compounds were found in fresh juice, and freeze-concentrated juice retained 57 of these compounds. Interestingly, freezing led to the generation of 37 flavor compounds. Furthermore, people had difficultly differentiating between intact and frozen concentrated juice. The ratios were almost same between those who correctly identified (28%) and those who incorrectly identified fresh and reconstituted freeze-concentrated juice (25%). We discuss the mechanisms of flavor generation on freezing concentration with regard to the increases in enzymatic activity or other causes. Our study showed that the methods of juice concentration that utilize freezing retain flavor better. These data will benefit juice concentration processes of apples and other fruits in the future.

Effect of container shape on freeze concentration of apple juice.

Concentrating fruit juices by freezing supports the maintenance of both nutrients and flavors. Development of the freezing concentration process has introduced equipment such as centrifuge or block freezing systems, which are suitable for large-scale commercial processing. However, the necessary characteristics of freeze concentration methods for juices include simplicity and low cost. This study examined the effects of different container shapes on the processes of freezing and melting. The shape of the container was found to be more important than the melting temperature, across a relatively large scale. Furthermore, the nutrient procyanidin B2 and saccharides were concentrated. The methods concentrated juice components under low cost conditions without complex equipment. This research thus not only offers benefits for commercial juice preparation but also provides new insight into effects of shape differences in concentration technologies.

Size of Cells and Physicochemical Properties of Membranes are Related to Flavor Production during Sake Brewing in the Yeast Saccharomyces cerevisiae.

Ethyl caproate (EC) and isoamyl acetate (IA) are key flavor components of sake. Recently, attempts have been made to increase the content of good flavor components, such as EC and IA, in sake brewing. However, the functions of EC and IA in yeast cells remain poorly understood. Therefore, we investigated the effects of EC and IA using cell-sized lipid vesicles. We also investigated lipid vesicles containing EC and/or caproic acid (CA) as well as IA and/or isoamyl alcohol (IAA). CA and IAA are precursors of EC and IA, respectively, and are important flavors in sake brewing. The size of a vesicle is influenced by flavor compounds and their precursors in a concentration-dependent manner. We aimed to establish the conditions in which the vesicles contained more flavors simultaneously and with different ratios. Interestingly, vesicles were largest in a mixture of 50% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with 25% EC and 25% CA or a mixture of 50% DOPC with 25% IA and 25% IAA. The impact of flavor additives on membrane fluidity was also studied using Laurdan generalized polarization. During the production process, flavors may regulate the fluidity of lipid membranes.

Influence of Ethyl Caproate on the Size of Lipid Vesicles and Yeast Cells.

Ethyl caproate (EC) is a key flavor component of sake. Recently, in sake brewing, an effort has been underway to increase the content of aromatic components such as EC. However, the function of EC in yeast cells remains poorly understood. Therefore, we investigated the effects of EC using cell-sized lipid vesicles. We found that vesicle size decreases in a concentration-dependent manner when EC is contained in lipid vesicles. Furthermore, yeast experiments showed that a strain producing high quantities of EC in its stationary phase decreased in size during EC production. Given caproic acid's (CA) status as the esterification precursor of EC in yeast, we also compared lipid vesicles containing CA with those containing EC. We found that CA vesicles were smaller than EC vesicles of the same concentration. These results suggest that EC production may function apparently to maintain cell size.

Effects of Capsaicin on Biomimetic Membranes.

Capsaicin is a natural compound that produces a warm sensation and is known for its remarkable medicinal properties. Understanding the interaction between capsaicin with lipid membranes is essential to clarify the molecular mechanisms behind its pharmacological and biological effects. In this study, we investigated the effect of capsaicin on thermoresponsiveness, fluidity, and phase separation of liposomal membranes. Liposomal membranes are a bioinspired technology that can be exploited to understand biological mechanisms. We have shown that by increasing thermo-induced membrane excess area, capsaicin promoted membrane fluctuation. The effect of capsaicin on membrane fluidity was dependent on lipid composition. Capsaicin increased fluidity of (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes, while it rigidified DOPC and cholesterol-based liposomes. In addition, capsaicin tended to decrease phase separation of heterogeneous liposomes, inducing homogeneity. We imagine this lipid re-organization to be associated with the physiological warming sensation upon consumption of capsaicin. Since capsaicin has been reported to have biological properties such as antimicrobial and as antiplatelet, the results will help unravel these biological properties.

Structure-dependent interactions of polyphenols with a biomimetic membrane system.

Polyphenols are naturally-occurring compounds, reported to be biologically active, and through their interactions with cell membranes. Although association of the polyphenols with the bilayer has been reported, the detailed mechanism of interaction is not yet well elucidated. We report on spatio-temporal real-time membrane dynamics observed in the presence of polyphenols. Two distinct membrane dynamics, corresponding to the two classes of polyphenols used, were observed. Flavonoids (epi-gallocatechin-3-gallate, gallocatechin, theaflavin and theaflavin-3-gallate) caused lipid membrane aggregation and rigidification. As simple structural modification through opening of the aromatic C-ring into an olefin bond, present in trans-stilbenes (resveratrol and picead), completely changed the membrane properties, increasing fluidity and inducing fluctuation. There were differences in the membrane transformations within the same class of polyphenols. Structure-dependent classification of membrane dynamics may contribute to a better understanding of the physicochemical mechanism involved in the bioactivity of polyphenols. In general, an increase in the number of hydrophilic side chains (galloyl, hydroxyl, glucoside, gallate) increased the reactivity of the polyphenols. Most notable was the difference observed through a simple addition of the gallate group. Unraveling the importance of these polyphenols, at a functional group level further opens the key to tailored design of bioactive compounds as potential drug candidates.

The effect of oxysterols on the interaction of Alzheimer's amyloid beta with model membranes.

The interaction of amyloid beta (Aß) peptide with cell membranes has been shown to be influenced by Aß conformation, membrane physicochemical properties and lipid composition. However, the effect of cholesterol and its oxidized derivatives, oxysterols, on Aß-induced neurotoxicity to membranes is not fully understood. We employed here model membranes to investigate the localization of Aß in membranes and the peptide-induced membrane dynamics in the presence of cholesterol and 7-ketocholesterol (7keto) or 25-hydroxycholesterol (25OH). Our results have indicated that oxysterols rendered membranes more sensitive to Aß, in contrast to role of cholesterol in inhibiting Aß/membrane interaction. We have demonstrated that two oxysterols had different impacts owing to distinct positions of the additional oxygen group in their structures. 7keto-containing cell-sized liposomes exhibited a high propensity toward association with Aß, while 25OH systems were more capable of morphological changes in response to the peptide. Furthermore, we have shown that 42-amino acid Aß (Aß-42) pre-fibril species had higher association with membranes, and caused membrane fluctuation faster than 40-residue isoform (Aß-40). These findings suggest the enhancing effect of oxysterols on interaction of Aß with membranes and contribute to clarify the harmful impact of cholesterol on Aß-induced neurotoxicity by means of its oxidation.

Dynamic Morphological Changes Induced By GM1 and Protein Interactions on the Surface of Cell-Sized Liposomes.

It is important to understand the physicochemical mechanisms that are responsible for the morphological changes in the cell membrane in the presence of various stimuli such as osmotic pressure. Lipid rafts are believed to play a crucial role in various cellular processes. It is well established that Ctb (Cholera toxin B subunit) recognizes and binds to GM1 (monosialotetrahexosylganglioside) on the cell surface with high specificity and affinity. Taking advantage of Ctb-GM1 interaction, we examined how Ctb and GM1 molecules affect the dynamic movement of liposomes. GM1 a natural ligand for cholera toxin, was incorporated into liposome and the interaction between fluorescent Ctb and the liposome was analyzed. The interaction plays an important role in determining the various surface interaction phenomena. Incorporation of GM1 into membrane leads to an increase of the line tension leading to either rupture of liposome membrane or change in the morphology of the membrane. This change in morphology was found to be GM1 concentration specific. The interaction between Ctb-GM1 leads to fast and easy rupture or to morphological changes of the liposome. The interactions of Ctb and the glycosyl chain are believed to affect the surface and the curvature of the membrane. Thus, the results are highly beneficial in the study of signal transduction processes.

Thermo-induced vesicular dynamics of membranes containing cholesterol derivatives.

Membrane structural organization is an intrinsic property of a cell membrane. Any changes in lipid composition, and/or any stimuli that affect molecular packing induce structural re-organization. It membrane dynamics provide a means by which changes in structure organization can be determined, upon a change in the membrane internal or external environment. Here, we report on the effect of thermo-stress on membranes containing cholesterol liquid crystal (LC) compounds cholesterol benzoate (BENZO) and oxidized cholesterols. We have (1) revealed that lipid vesicles containing this artificial cholesterol derivative (BENZO) is thermo-responsive, and that this thermo-sensitivity is significantly similar to naturally oxy-cholesterols (2) elucidated the mechanism behind the membrane perturbation. Using Langmuir monolayer experiments, we have demonstrated that membrane perturbation was due to an increase in the molecular surface area, (3) discussed the similarities between cholesterol benzoate in the cholesterol LC state and in lipid bilayer membranes. Last, (4) drawing from previously reported findings, our new data on membrane dynamics, and the discussion above, we propose that artificial cholesterol derivatives such as BENZO, open new possibilities for controlled and tailored design using model membrane systems. Examples could include the development of membrane technology and provide a trigger for progress in thermo-tropical liquid crystal engineering.

The effect of oxycholesterols on thermo-induced membrane dynamics.

The effect of temperature change(s) on the dynamics of giant unilamellar vesicles containing oxidized and non-oxidized cholesterol was investigated and characterized. We have demonstrated that (i) major cholesterol auto-oxidation products, 7ß-hydroxycholesterol (7ß) and 7-ketocholesterol (7keto), rendered vesicles more responsive to temperature changes; (ii) 7keto imparted greater thermo-induced membrane dynamics than 7ß; (iii) 7ß and 7keto vesicles synergistically were more thermo-responsive than the individual oxysterols; (iv) the thermo-responsiveness of 7keto-containing vesicles was equivalent to that of 25 hydroxycholesterol (25OH)-containing vesicles; and (v) we have characterized the observed membrane dynamics. The results provide a new plausible mechanism: oxidative-stressed membranes in conjunction with temperature change induce membrane dynamics. These findings improve the mechanisms reported previously that attributed the induced dynamics solely to membrane oxidation.