Cell-sized liposome, method for estimating ethyl caproate concentration using cell-sized liposome, method for estimating ethyl caproate concentration using observation results and calibration curve of cell-sized liposome, and method for measuring ethyl caproate concentration

Abstract

To provide a simple ethyl caproate analyzing method capable of being performed at a low cost and having a form like preliminary analysis or screening for selecting a sample to be subjected to precise analysis by GC (gas chromatography).SOLUTION: The cell-sized liposome 110 is a cell-sized liposome composed of a lipid component system in which one or two or more kinds of saturated phospholipids 13 are used in addition to one or two or more kinds of unsaturated phospholipids 12, and a sample whose ethyl caproate concentration is to be estimated or ethyl caproate 18, and generates a different phase-separated domain generation pattern depending on the concentration of the ethyl caproate 18, and thus can be used for estimating the ethyl caproate concentration from the phase-separated domain generation pattern. The lipid component system of the cell-sized liposome 110 may include a sterol-based lipid such as cholesterol. Further, a calibration curve can be used for concentration estimation.SELECTED DRAWING: Figure 1-3

Description

[Technical Field]

[0001] The present invention relates to cell-sized liposomes, a method for estimating the concentration of ethyl caproate using cell-sized liposomes, a method for estimating the concentration of ethyl caproate using observation results of cell-sized liposomes and a calibration curve, and a method for measuring the concentration of ethyl caproate, and in particular to a technology that can estimate the concentration of ethyl caproate contained in sake and the like at low cost and in a relatively short time. [Background technology]

[0002] Yeast requires long-chain fatty acids to form biomembranes, but it is believed that by-products include caproic acid, which has a shorter lipid chain. Ethyl caproate, produced from caproic acid, is a major component of ginjo aroma in sake, along with isoamyl acetate. Because the ginjo aroma components of sake increase its commercial value, there has long been demand for yeast that produces a high amount of ethyl caproate. Furthermore, even when the same yeast is used, the concentration of ethyl caproate in the sake produced will vary depending on factors such as temperature during fermentation.

[0003] In light of the above, with the aim of increasing the commercial value of sake, tests and research are being conducted on themes such as how differences in yeast and manufacturing test conditions affect the concentration of ethyl caproate.

[0004] Gas chromatography (GC) is used to measure the concentration of ethyl caproate, but this equipment is not only very expensive to purchase, but also has the drawback of high running costs because high-purity analytical gas is used for each measurement.In addition, it takes several hours to set up the equipment, and multiple standard samples must be run to create a calibration curve before the concentration can be measured, which takes a considerable amount of time.

[0005] Patent applications have been filed for techniques for measuring ethyl caproate. For example, Patent Document 1, listed below, discloses a quantitative method for accurately measuring ethyl caproate in sake, in which a free fatty acid coloring agent produced by an acyl-activating enzyme is used, and the color intensity corresponding to the concentration is quantified by absorptiometry. Furthermore, Non-Patent Document 1, written by the present inventor, discloses a technique for estimating the concentration of ethyl caproate by adding sake during the lipid film preparation stage and observing the state of the phase-separated domains when preparing cell-sized liposomes. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2012-157349 A: "Simple method for measuring the concentration of ethyl caproate contained in sake" [Non-patent literature]

[0007] [Non-Patent Document 1] Chemistry & Biodiversity (2023)Vol.22,p. e202200750 Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, ethyl caproate contained in sake increases its commercial value. Therefore, if a measurement method were simple, it would be possible to rapidly and inexpensively investigate production conditions. However, GC, which is used to measure ethyl caproate, has problems such as high initial and running costs, as well as considerable labor and time. Furthermore, although the technology disclosed in Patent Document 1 uses a relatively inexpensive absorptiometer, it has the disadvantage of requiring the preparation of multiple reagents, such as enzymes and colorants, during measurement. Therefore, it would be convenient to have a simple analytical method for preliminary analysis, or what is called screening, to select samples for precision analysis by GC, which can be performed at the lowest possible cost.

[0009] Liposomes, the most simplified model of biological membranes used in many experiments and research, are formed by the self-assembly of phospholipids, which have both hydrophilic and hydrophobic fatty chains, with the hydrophilic portion facing outward to form a lipid bilayer membrane, which then closes into a sac-like shape. Liposomes with diameters ranging from a few micrometers to several tens of micrometers are roughly the same size as living cells and are called cell-sized liposomes (giant liposomes). Cell-sized liposomes are useful for research into the elucidation of biological membrane control mechanisms, due to their advantages, such as their ability to be directly observed with an optical microscope and their use in creating experimental models that mimic cells.

[0010] Furthermore, it has been revealed that the cell membrane contains phase-separated domains (raft domains) rich in saturated lipids and cholesterol, which are responsible for signal transduction and other functions. Raft domains are thought to move, adhering and separating in response to intercellular signaling. Similar domain structures have also been observed in artificially constructed liposomes from lipids, and research in this area is also in full swing. These studies using biological model membranes (artificial cell membranes) and human-derived cells have revealed that the three-dimensional dynamics and two-dimensional (domain structure) assembly dynamics of membranes are related to signal transduction using intracellular calcium ions as an indicator, and that altering the structure of lipids and sterols alters the temperature response of the domain structure.

[0011] The inventors have previously conducted research to clarify the function of cell membranes by observing the movements and phase-separated structures of biological model membranes and cell-sized liposomes under a microscope, and have published the results. For example, they discovered that the frequency of dynamics occurrence in biological model membranes in response to aroma components of alcohol differs depending on the type of aroma (T. Yoda et al., ACS. Omega 2022).

[0012] The inventors have also demonstrated that domain structures form in membranes containing antioxidants when subjected to oxidative stress. Specifically, mixing cell-sized liposomes with solutions of polyphenols known for their antioxidant properties, such as resveratrol and theaflavin, induces membrane dynamics, such as membrane contraction and fluctuation, resulting in the emergence of phase-separated domains during observation (HHT. Phan, T. Yoda et al., Biochim. Biophys. Acta, Biomembr. 2014). Furthermore, fluorescence microscopy has revealed that concanavalin A, a functional component found in sword beans that activates the immune system, induces the accumulation of domain structures on the cell membrane and activates intracellular signaling (S. Yabuuchi et al., 2017 J. Biosci. Bioeng.).

[0013] When cell-sized liposomes are prepared by mixing equal amounts of the unsaturated phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the saturated phospholipid dipalmitoylphosphatidylcholine (DPPC), they undergo phase separation into a DOPC-rich liquid disordered phase domain (Ld domain) and a DPPC-rich solid ordered phase domain (So domain). Furthermore, when cholesterol is added to this mixture, many cell-sized liposomes are observed that phase separate into a DOPC-rich liquid disordered phase domain (Ld domain) and a DPPC- and cholesterol-rich liquid ordered phase domain (Lo domain).

[0014] In Non-Patent Document 1, the inventors reported a technique for estimating the concentration of cell-sized liposomes by adding sake during the lipid film preparation stage and observing the state of the phase-separated domains. However, these methods involve a step of vacuum drying the organic solvent after film preparation, which poses a risk of volatilizing and vaporizing ethyl caproate.

[0015] The problem to be solved by the present invention is to provide a simple analytical method for ethyl caproate concentration, based on the progress and results of previous research using liposomes and the state of the art in measurement. This method can be performed as a preliminary analysis for selecting samples for precise analysis by GC, i.e., a so-called screening method, at low cost and in a short time, although the accuracy may not be as high as that of GC. In other words, the present invention provides a simple method for easily distinguishing between cell-sized liposomes and a simple measurement method that utilizes clear differences characterized by observation due to the interaction between ethyl caproate and membranes. Furthermore, the present invention also provides a method for suppressing volatilization and evaporation of ethyl caproate by adding a substance to be measured for ethyl caproate concentration, such as sake, at a stage other than film production. [Means for solving the problem]

[0016] The inventors have carried out research with the above problem in mind. To prepare cell-sized liposomes, we attempted to add sake during the hydration process. First, we attempted to prepare cell-sized liposomes using DOPC alone. We found that adding sake during hydration resulted in micrometer-scale liposomes. We also found that adding sake during hydration to prepare cell-sized liposomes using DOPC alone resulted in significantly smaller liposomes than those obtained by hydration with pure water. Furthermore, adding a solution of sake with ethyl caproate added beforehand during hydration further reduced the size. These findings are consistent with a report that showed that adding ethyl caproate to films beforehand reduced the size depending on the concentration of the added ethyl caproate (T. Yoda et al., Biomimetics 2020).

[0017] From the above, we speculated that ethyl caproate in sake could be incorporated into cell-sized liposomes during the formation stage by adding sake during hydration, similar to the method disclosed in Non-Patent Document 1, i.e., adding sake during film preparation. We then observed the phase-separated domain structure of the cell-sized liposomes formed when sake was added during hydration and when a solution in which part of the sake had been previously replaced with ethyl caproate was added, and found that the ratios were different. Based on this, we found that the ethyl caproate content in a sample can be determined by the ratio of solid-ordered to liquid-disordered phases, i.e., the phase separation ratio.

[0018] In addition to the above findings obtained through experiments using sake, we also confirmed the cost-reduction effect through trial calculations, demonstrating the usefulness of cell-sized liposomes as a tool for analyzing ethyl caproate concentrations. Specifically, to analyze ethyl caproate in sake and other beverages, we added sake during hydration to prepare cell-sized liposomes containing unsaturated lipids, more preferably cell-sized liposomes containing unsaturated and saturated lipids, and then observed the ratio of domain structures to quickly estimate the relative concentration of ethyl caproate. (Note: The use of "unsaturated lipids" and "saturated lipids" here, rather than "unsaturated lipids such as DOPC" and "saturated lipids such as DPPC," indicates that the cell-sized liposomes of the present invention can also be prepared using lipids other than the phospholipids described herein, such as glycolipids. These lipids are also within the scope of the present invention.) The present invention, thus completed, i.e., the invention claimed in this application as a means for solving the above-mentioned problems, or at least the invention disclosed therein, is as follows:

[0019] [1] A cell-sized liposome comprising a lipid component system using one or more types of unsaturated phospholipids and a sample for estimating the concentration of ethyl caproate or ethyl caproate, wherein the cell-sized liposome generates a phase-separated domain generation pattern that varies depending on the concentration of ethyl caproate, and is therefore used to estimate the concentration of ethyl caproate from the phase-separated domain generation pattern. [2] The cell-sized liposome according to [1], characterized in that the lipid component system contains one or more saturated phospholipids. [3] The cell-sized liposome according to [2], characterized in that the lipid component system contains one or more sterol-based lipids. [4] The cell-sized liposome according to [1], characterized in that the lipid component system is composed of an unsaturated phospholipid, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), and a saturated phospholipid, Dipalmitoylphosphatidylcholine (DPPC). [5] The cell-sized liposome according to [1], characterized in that the lipid component system is composed of an unsaturated phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a saturated phospholipid dipalmitoylphosphatidylcholine (DPPC), and a sterol lipid cholesterol.

[0020] [6] The cell-sized liposome according to any one of [2], [3], [4], and [5], characterized in that, when used at room temperature, the molar composition of unsaturated phospholipids and saturated phospholipids in the lipid component system is unsaturated phospholipids:saturated phospholipids=75 to 10:25 to 90. [7] The cell-sized liposome according to any one of [2], [3], [4], and [5], characterized in that, when used at room temperature, the molar concentration composition of unsaturated phospholipids and saturated phospholipids in the lipid component system is equal. [8] The cell-sized liposome according to any one of [1], [2], [3], [4], and [5], characterized in that a fluorescent reagent for detecting phase-separated domains is added. [9] The cell-sized liposome according to [8], characterized in that the fluorescent reagent used is one or more of a reagent for staining a solid-ordered phase (So phase), a reagent for staining a liquid-disordered phase (Ld phase), or a reagent for staining a sterol-based lipid.

[10] The cell-sized liposome according to any one of [1], [2], [3], [4], and [5], characterized in that it is produced by a static hydration method.

[11] The cell-sized liposome according to

[10] , wherein after the lipid film is prepared, a sample whose concentration is to be estimated is added during hydration of the cell-sized liposome.

[0021]

[12] A method for estimating the relative concentration of ethyl caproate in a target substance for concentration estimation, comprising using a cell-sized liposome according to any one of [1], [2], [3], [4], and [5], which is configured using an ethyl caproate standard having a known concentration, as a liposome for obtaining phase-separation domain information, and using the liposome for obtaining phase-separation domain information, comprising: Observing the cell-sized liposomes for which ethyl caproate concentration is to be estimated using a sample, the cell-sized liposomes being the cell-sized liposomes according to any one of [1], [2], [3], [4], and [5], under a fluorescent microscope; The phase separation domain generation pattern, i.e., whether or not a phase separation domain is generated, or the pattern when a phase separation domain is generated, is read; Referring to the ethyl caproate concentration dependency information of the phase-separation domain generation pattern in the liposome for obtaining phase-separation domain information, Estimate the relative concentration of ethyl caproate contained in the target substance. A method for estimating the concentration of ethyl caproate, comprising:

[13] The method for estimating the concentration of ethyl caproate according to

[12] , wherein a calibration curve based on the observation results of the liposome for obtaining phase-separation domain information is used in estimating the concentration of ethyl caproate.

[14] The method for estimating the concentration of ethyl caproate according to either

[12] or

[13] , wherein the subject of concentration estimation is sake or a processed sake product.

[15] A method for measuring the concentration of ethyl caproate, comprising: first estimating the relative concentrations of ethyl caproate for a plurality of samples by the method for estimating the concentration of ethyl caproate according to either

[12] or

[13] ; and then precisely measuring the concentrations of ethyl caproate by gas chromatography for some of the samples that have been subjected to the ethyl caproate concentration estimation process.

[16] The method for measuring the concentration of ethyl caproate according to

[15] , wherein the object of concentration measurement is sake or a processed sake product. [Effects of the Invention]

[0022] The cell-sized liposomes, the method for estimating ethyl caproate concentration using cell-sized liposomes, the method for estimating ethyl caproate concentration using observation results of cell-sized liposomes and a calibration curve, and the method for measuring ethyl caproate concentration of the present invention are configured as described above, and therefore provide a simple measurement method that utilizes clear differences characterized by observation due to the interaction between ethyl caproate and a membrane, and a simple discrimination method using cell-sized liposomes. Specifically, ethyl caproate concentration can be analyzed at significantly lower cost and in a shorter time than conventional methods. In particular, the method for estimating ethyl caproate concentration using a calibration curve can further improve the accuracy and precision of concentration estimation and measurement.

[0023] Conventional measurement methods, which require expensive GC, have the problem of requiring a long time for equipment startup, etc. However, the concentration estimation method of the present invention makes it possible to use a relatively inexpensive fluorescence observation microscope as the measurement device, reducing not only the initial cost but also the running cost, making it easy to analyze the ethyl caproate concentration in sake and the like, and also enabling measurements to be performed quickly.

[0024] In particular, the present invention involves a method in which sake containing the ethyl caproate to be measured is added during hydration to incorporate the ethyl caproate into cell-sized liposomes, which are then observed under a fluorescence microscope.With this method, once the optimal conditions for the combination of lipids that make up the cell-sized liposomes have been determined, the concentration of ethyl caproate contained can be estimated simply by short-term observation.

[0025] As an effective example of its use, the present invention can be used for preliminary analysis to select samples for precision analysis by GC, or for simple screening-type determination of ethyl caproate concentration, which is highly useful. Therefore, by combining the method of the present invention with GC, a rapid, low-cost, and precise analytical system can be realized. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 is a conceptual diagram showing the basic structure of the cell-sized liposome of the present invention. [Figure 1-2] FIG. 1 is a photograph showing an example of a pattern of phase-separated domains formed in the cell-sized liposomes of the present invention. [Figure 1-3] FIG. 1 is a conceptual diagram showing another basic structure of the cell-sized liposome of the present invention, which also contains saturated phospholipids. [Figure 2] FIG. 1 is a conceptual diagram showing another configuration of the cell-sized liposome of the present invention, which also contains a sterol lipid. [Figure 3] 1 shows the chemical structural formulas of lipids and ethyl caproate, which are examples of constituent elements of the cell-sized liposome of the present invention. [Figure 4] FIG. 1 is an explanatory diagram showing an example of a method for producing the cell-sized liposome of the present invention. [Figure 5] FIG. 1 is an explanatory diagram conceptually illustrating the configuration of a method for estimating the relative concentration of ethyl caproate according to the present invention. [Figure 6] FIG. 1 is a flow chart showing the configuration of a method for measuring the concentration of ethyl caproate according to the present invention. [Figure 6-2] 1 is a flow chart showing the configuration of the method for estimating the concentration of ethyl caproate according to the present invention (the following figures relate to examples). [Figure 7] 1 is a graph showing the structural composition of phase-separated domains in each cell-sized liposome according to Examples (Cases 1, 2, and 3). [Figure 8] 1 is a graph showing a calibration curve created based on the phase-separated domain structure composition when the lipid composition is 1 in cell-sized liposomes for estimating the concentration of ethyl caproate in Examples (Cases 1, 2, and 3). [Figure 9] 1 is a graph showing a calibration curve created based on the phase-separated domain structure composition when the lipid composition is 2 in cell-sized liposomes for estimating the concentration of ethyl caproate in Examples (Cases 1, 2, and 3). [Figure 10] 1 is a graph showing a calibration curve prepared based on the phase-separated domain structure composition when the lipid composition is 3 in cell-sized liposomes for estimating the concentration of ethyl caproate in Examples (Cases 1, 2, and 3). [Figure 11] 1 is a graph showing the results of measuring the phase transition temperature, i.e., the domain disappearance temperature, of each cell-sized liposome according to Examples (Cases 1, 2, and 3). DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will be described in detail below with reference to the drawings. FIG. 1 is a conceptual diagram showing the basic structure of the cell-sized liposome of the present invention. As shown in (a) of the figure, the cell-sized liposome 10 is a cell-sized liposome composed of a lipid component system using one or more types of unsaturated phospholipids 2 (the figure shows an example in which only one type of unsaturated phospholipid 2 is used; the desired effect of the present invention can be sufficiently achieved with just one type) and a sample for which ethyl caproate concentration is to be estimated or ethyl caproate 8. The cell-sized liposome generates different phase-separated domain formation patterns depending on the concentration of ethyl caproate 8, and can be used to estimate the concentration of ethyl caproate from the phase-separated domain formation patterns. (b) of the figure shows a cell-sized liposome 10' of the present invention containing a fluorescent reagent 9 as a component, which will be described later.

[0028] With this configuration, the cell-sized liposomes 10 exhibit different phase-separation domain generation patterns depending on the concentration of ethyl caproate 8 contained therein, and the ethyl caproate concentration can be estimated using these phase-separation domain generation patterns. At the very least, it is possible to determine whether the ethyl caproate content of a sample is relatively high or low by observing the phase-separation domain generation pattern.

[0029] The cell-sized liposome of the present invention having the structure shown in FIG. 1 will now be described in more detail. The ability to observe phase-separated domains under fluorescence observation with a single unsaturated lipid, such as DOPC (described below), is believed to be due to reactive oxygen species generated during fluorescence observation. Specifically, during fluorescence observation, excitation light is applied to observe the fluorescence emitted from the fluorescent dye, which generates reactive oxygen species that oxidize and cleave the unsaturated bond, i.e., part of the double bond, of the unsaturated lipid. As a result, two types (or several types) of lipids exist in the system: unoxidized unsaturated lipids and oxidized saturated lipids. This is observed as phase separation in the lipid membrane.

[0030] In fact, unsaturated lipids such as DOPC have a low phase transition temperature, allowing liposomes to be prepared without heating, and the formation of phase-separated domains can be easily confirmed. However, saturated lipids such as DPPC do not have unsaturated bonds and are therefore not susceptible to oxidation by reactive oxygen species during fluorescence observation. Therefore, liposomes composed solely of saturated phospholipids are unlikely to produce the desired phase-separated domains.

[0031] Figures 1-2 are photographs showing examples of phase-separated domain formation patterns in cell-sized liposomes of the present invention. These are micrographs showing typical patterns, of which A is an So (solid-ordered phase) / Ld (liquid-disordered phase) domain structure. B is a uniform membrane vesicle. Observations using a fluorescence microscope have shown that in the present invention, the So / Ld domain structure pattern shown in A can be particularly suitably used for concentration estimation. Observations using a fluorescence microscope will be discussed further below.

[0032] 1-3 are conceptual diagrams showing another basic structure of the cell-sized liposome of the present invention. As shown in (a) of the figure, the cell-sized liposome 110 is composed of a lipid component system that uses one or more types of unsaturated phospholipids 12 (the figure shows an example in which only one type of unsaturated phospholipid 12 is used) and one or more types of saturated phospholipids 13 (the figure shows an example in which only one type of saturated phospholipid 13 is used; the desired effect of the present invention can be sufficiently achieved with just one type), and a sample whose ethyl caproate concentration is to be estimated or ethyl caproate 18. The cell-sized liposome generates different phase-separation domain formation patterns depending on the concentration of ethyl caproate 18, and is therefore primarily used to estimate the concentration of ethyl caproate from the phase-separation domain formation patterns.

[0033] The present cell-sized liposomes 110 having such a configuration also produce and exhibit different phase-separation domain generation patterns depending on the concentration of ethyl caproate 18 contained therein, and the concentration of ethyl caproate contained therein can be estimated using this phase-separation domain generation pattern. At the very least, it is possible to determine whether the ethyl caproate content of a sample is relatively high or low by observing the phase-separation domain generation pattern. Furthermore, in order to more fully achieve the intended object of the present invention, the present cell-sized liposomes 110, which are composed of unsaturated lipids and saturated lipids, are more desirable than the cell-sized liposomes 10 shown in FIG. 1.

[0034] 2 is a conceptual diagram showing another example of the cell-sized liposome of the present invention, which also contains a sterol-based lipid. As shown in (a) of the figure, the cell-sized liposome 210 comprises a lipid component system containing one or more unsaturated phospholipids 22 (the figure shows an example in which only one type of unsaturated phospholipid 22 is shown), one or more saturated phospholipids 23 (the figure shows an example in which only one type of saturated phospholipid 23 is shown), and one or more sterol-based lipids 25 (the figure shows an example in which only one type of sterol-based lipid 25 is shown; the desired effect of the present invention can be sufficiently achieved with just one type), and a sample for which ethyl caproate concentration is to be estimated or ethyl caproate 28. The cell-sized liposome generates different phase-separation domain formation patterns depending on the concentration of ethyl caproate 28, and is therefore primarily intended for use in estimating the concentration of ethyl caproate from the phase-separation domain formation patterns.

[0035] The cell-sized liposome 210 having such a configuration also produces and exhibits different phase-separation domain generation patterns depending on the concentration of ethyl caproate 18 contained therein. Therefore, the concentration of ethyl caproate contained therein can be estimated using this phase-separation domain generation pattern, and at least, it is possible to determine whether the ethyl caproate content of a sample is relatively high or low by observing the phase-separation domain generation pattern.

[0036] Sterol lipids are not essential elements for constructing the cell-sized liposomes of the present invention. However, there are advantages to using sterol lipids such as cholesterol. First, the phase transition temperature is lowered, allowing for a lower hydration temperature. In the present invention, heating is performed during hydration to prepare cell-sized liposomes, but by performing hydration at a lower temperature, the volatilization and evaporation of the fragrance component ethyl caproate can be suppressed.

[0037] Furthermore, sterol lipids such as cholesterol have the effect of increasing the fluidity of phospholipids, resulting in better mixing of lipids. This reduces the bias in the composition of each liposome produced, increasing the uniformity of the liposomes produced. As a result, the reproducibility of the method for estimating the relative concentration of ethyl caproate is improved. There are also other advantages when using specific fluorescent reagents (discussed below).

[0038] On the other hand, there are advantages to intentionally not including cholesterol in the lipid component system. This is the case when primarily observing changes in the So / Ld phase-separated structure, as in the method for estimating the relative concentration of ethyl caproate of the present invention, which will be explained later with reference to Figure 5 and other figures. By not including cholesterol in the lipid component system, almost all of the resulting cell-sized liposomes are So / Ld. Therefore, when primarily observing changes in the So / Ld phase-separated structure, the phase-separated domain formation pattern can be clearly distinguished. When the concentration of added cholesterol is low, the So / Ld structure is dominant, but some Lo / Ld phase separation is mixed in, making it more complicated to interpret the distinguishing pattern.

[0039] In particular, the present invention can produce cell-sized liposomes having a lipid component system using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as the unsaturated phospholipid and dipalmitoylphosphatidylcholine (DPPC) as the saturated phospholipid. Furthermore, in addition to this configuration, cell-sized liposomes can also be produced with a lipid component system that includes cholesterol (Chol), a sterol lipid. The following explanation will mainly focus on cell-sized liposomes having a lipid component system of either DOPC + DPPC or DOPC + DPPC + Chol.

[0040] 3 shows the chemical structures of lipids and ethyl caproate (EC), which are examples of constituent elements of the cell-sized liposomes of the present invention. In the figure, A is DOPC, B is DPPC, C is cholesterol, and D is ethyl caproate.

[0041] When used at room temperature, the lipid component system of the cell-sized liposomes of the present invention can have a molar composition of unsaturated phospholipids and saturated phospholipids in the lipid component system of unsaturated phospholipids:saturated phospholipids = 75-10:25-90. Alternatively, when used at room temperature, the lipid component system may have an equal molar composition of unsaturated phospholipids and saturated phospholipids. Here, room temperature may be the generally understood temperature of about 20°C, or, more preferably, 22±2.0°C.

[0042] As an example of a specific lipid species, the lipid component system of the cell-sized liposome of the present invention, when used at room temperature, has a molar concentration composition in which both DOPC and DPPC are 45 or more, and if the lipid component system also contains cholesterol, the cholesterol can be the remaining amount.

[0043] In the present invention, the most desirable phase-separated domain composition for observation to estimate the ethyl caproate concentration is the So / Ld domain, as described above. The molar concentration composition at which this domain is observed has been reported as follows. DOPC:DPPC:Chol = 37.5~50 : 37.5~50 : 0~15 DOPC:DPPC:Chol = 75~10 : 25~90 : 0~10

[0044] The reason why the composition range differs depending on the reported examples is that the domain formation is affected by temperature. According to the results of the inventor's experiments at room temperature (around 20°C), The ratio is DOPC:DPPC:Chol = 42.5-50: 42.5-50: balance. For example, if Chol is 10, the So / Ld domain structure can be obtained in a molar composition range of DOPC 0-65 and DPPC 25-90. As mentioned above, DOPC and DPPC can also be used in equal amounts. In the examples described below, DOPC and DPPC are used in equal amounts (50:50).

[0045] Phase-separated domains are observed even when the cholesterol content is 15 or more. For example, when the cholesterol content is 20 to 40 and the phospholipid content is 60 to 80, Lo / Ld (liquid-ordered / liquid-disordered) domains are observed.

[0046] As shown in Figures 1, 1-3, and 2(b), the cell-sized liposomes of the present invention may be configured to contain a fluorescent reagent 9 for detecting phase-separated domains. The presence or absence of a phase-separated domain structure is determined by fluorescent observation of the cell-sized liposomes, and the fluorescent reagent 9 is added for this purpose and does not affect the formation of phase separation. The fluorescent reagent 9 may be one or more of the following: a liquid-ordered phase (Lo phase) staining reagent, a liquid-disordered phase (Ld phase) staining reagent, or a sterol-based lipid staining reagent.

[0047] In the examples described below, the fluorescent reagent used is rhodamine B (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt) (rhodamine DHPE), which is added at 1% molar concentration of lipid. This is a relatively common fluorescent reagent for staining the DOPC-rich Ld phase. On the other hand, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4yl)(ammonium salt) (NBD-PE) is used to stain the DPPC-rich So or Lo phases. Because these have different fluorescent wavelengths, they can be used separately or simultaneously.

[0048] In addition, NBD-Cholesterol, for example, is used to fluorescently stain the cholesterol portion. In the present invention, which uses the So / Ld domain structure, the DPPC-rich So domain is stained. When using NBD-Cholesterol, it is thought that the degree of fluorescent staining will be better if the lipid component system contains cholesterol. Of course, fluorescent reagents other than those described above can also be used in the present invention.

[0049] FIG. 4 is an explanatory diagram showing an example of a method for preparing the cell-sized liposomes of the present invention. As shown in the figure, the cell-sized liposomes can be prepared by the static hydration method. Alternatively, after preparing a lipid film, a sample whose concentration is to be estimated can be added during hydration of the cell-sized liposomes. Specifically, the liposomes can be prepared using the following basic method: dissolving the lipids constituting the liposomes in a solvent (chloroform CH(Cl)3 in the example shown in the figure) in a container such as a test tube, evaporating the solvent to form a lipid film on the inner wall of the container, and then hydrating the lipid film with sake, which is the sample of interest.

[0050] In the examples described below, the lipid membrane and the sake to be measured were heated to approximately 50°C during hydration, and cell-sized liposomes were prepared using the static hydration method. This is a process to ensure that the lipids and ethyl caproate are uniform in the lipid membrane by heating the temperature above 41°C, the melting point of DPPC. Note that approximately 50°C is merely an example, and it goes without saying that the present invention is not limited to this.

[0051] The phase-separated domain structure is determined not by the production method but by the components of the membrane, such as the constituent lipids. Therefore, the specific method for producing the cell-sized liposomes of the present invention is not limited, and any conventionally known method may be used as appropriate. Examples include the electroformation method, the droplet method, and a new method thereof (Patent No. 6031711). In the present invention, the static hydration method is particularly suitable. The static hydration method is a generally well-known method, and has the advantage that it can be easily produced without the need for special equipment, as long as a test tube, gas, and the specified solution are available, making it ideal for the present invention.

[0052] Figure 5 is an explanatory diagram conceptually illustrating the configuration of the method for estimating ethyl caproate concentration of the present invention. Any of the cell-sized liposomes of the present invention having the configurations described above can be used as liposomes used in estimating the concentration of an object to be estimated, the concentration of which is unknown, i.e., as "liposomes for obtaining phase-separation domain information." In this figure, "liposomes for obtaining phase-separation domain information" are abbreviated as "reference liposomes," and this abbreviation will also be used in the following description.

[0053] In Figure 5, the center column of the table on the left conceptually shows reference liposomes 10a, 10b, ..., and the right column conceptually shows "estimated target liposomes" 10x, 10y, ..., which are liposomes of cell size whose relative concentrations are to be estimated, and the left column shows the concentration n of ethyl caproate contained in each liposome 10a, 10x, etc. The ethyl caproate concentration n=a, etc. of reference liposomes 10a, etc. is known, while the ethyl caproate concentration n=x, etc. of estimated target liposomes 10x, etc. is unknown.

[0054] As shown in the figure, the method for estimating the relative concentration of ethyl caproate of the present invention is a method for estimating the relative concentration n=x, etc. of ethyl caproate in a concentration estimation target using the above-mentioned liposome for obtaining phase-separation domain information (reference liposome) 10a, etc. Reference liposome 10a, etc. is constructed by adding an ethyl caproate standard having a known concentration, such as n=a, to the concentration estimation target. Furthermore, estimation target liposome 10x, etc. is a cell-sized liposome constructed using the concentration estimation target, for example, sake or its processed products, whose relative concentration is to be estimated.

[0055] In the central table of Figure 5, the domain structure generation patterns (domain generation patterns) of each liposome in the left table are shown as columns, and the differences in concentration are shown as rows. That is, reference liposome 10a containing ethyl caproate 8a at a concentration n = a has domain generation pattern Da, and reference liposome 10b containing ethyl caproate 8b at a concentration n = b has domain generation pattern Db. Furthermore, putative target liposome 10x containing ethyl caproate 8x at an unknown concentration n = x has domain generation pattern Dx, and putative target liposome 10y containing ethyl caproate 8y at an unknown concentration n = y has domain generation pattern Dy.

[0056] The ethyl caproate concentration estimation method of the present invention involves fluorescent microscopic observation of the reference liposomes 10a, 10b, etc., to read the phase-separation domain generation patterns Da, Db, etc., i.e., whether or not a phase-separation domain is generated or the pattern that occurs when a phase-separation domain is generated; fluorescent microscopic observation of the cell-sized liposomes 10x, 10y, etc., whose concentration is to be estimated, to read the phase-separation domain generation patterns Dx, Dy, etc., i.e., whether or not a phase-separation domain is generated or the pattern that occurs when a phase-separation domain is generated; and estimating the relative concentration of ethyl caproate contained in the object whose concentration is to be estimated by referring to ethyl caproate concentration-dependence information such as the phase-separation domain generation pattern Da in the reference liposomes 10a, etc.

[0057] That is, in this method for estimating the concentration of ethyl caproate, the phase-separated domain formation patterns Da, Db, ... in the reference liposome 10a, etc., are dependent on the concentration of ethyl caproate. Therefore, this pattern information is compared to estimate the relative concentrations in the estimation target liposome 10x, etc., i.e., the relative ranking of the ethyl caproate concentrations contained in the target substance, and the concentration range is estimated. In Figure 5, an example estimation result is shown on the right side of the figure, in which the concentrations are highest in the order of a, x, y, and b. At the same time, the estimation result is also obtained that the unknown concentrations x and y are both lower than the known concentration a and higher than b.

[0058] 6 is a flow diagram showing the configuration of the method for measuring the concentration of ethyl caproate of the present invention. As shown in the figure, this method for measuring the concentration of ethyl caproate comprises a relative concentration estimation process P10 in which the concentrations of ethyl caproate are estimated for all samples N to be subjected to concentration estimation using the above-mentioned ethyl caproate concentration estimation method, and a precise measurement process P20 in which the concentrations of ethyl caproate are precisely measured by GC for a portion of samples n after the ethyl caproate concentration estimation process.

[0059] This ethyl caproate concentration measurement method involves a preliminary analysis in the relative concentration estimation process P10, a simple screening-like determination of the ethyl caproate concentration, to select samples for precision analysis by GC in the precision measurement process P20. After the relative concentration estimation process P10 determines whether the ethyl caproate content in the sample is relatively high or low, precision measurement process P20 is performed on a portion of the sample n. This allows for a rapid, low-cost, and precise analysis system.

[0060] The ethyl caproate concentration estimation method and ethyl caproate concentration measurement method of the present invention described above can be used for all estimation / measurement targets that are expected to contain ethyl caproate, and it goes without saying that these methods of the present invention can also be applied to sake containing ethyl caproate or products processed from it.

[0061] Here, the method for estimating the concentration of ethyl caproate according to the present invention, which has already been described, will be explained again. 6-2 is a flow diagram showing the configuration of the method for estimating the concentration of ethyl caproate of the present invention. As shown in (i) in the figure, the method for estimating the concentration of ethyl caproate includes a pattern reading process Q510 in which cell-sized liposome 510x (liposome to be estimated) is observed under a fluorescent microscope to determine whether or not a phase-separated domain is formed, or to read a phase-separated domain generation pattern 5Dx, which is a pattern that would be observed if a phase-separated domain is formed, and a concentration estimation process Q520 in which ethyl caproate concentration-dependent information Ref of the phase-separated domain generation pattern in liposome 510a (reference liposome) for obtaining phase-separated domain information is referenced to estimate the concentration of ethyl caproate contained in the object to be estimated.

[0062] In this ethyl caproate concentration estimation method having such a configuration, in the pattern reading process Q510, the cell-sized liposome 510x to be subjected to concentration estimation is observed under a fluorescent microscope to read the phase-separation domain generation pattern 5Dx, and then in the concentration estimation process Q520, the ethyl caproate concentration-dependent information Ref of the phase-separation domain generation pattern in the liposome 510a for obtaining phase-separation domain information is referenced to estimate the concentration of ethyl caproate contained in the object to be subjected to concentration estimation, and an estimated concentration Est is obtained.

[0063] As shown in (ii) in the figure, in the concentration estimation step Q52C of the present ethyl caproate concentration estimation method, when estimating the ethyl caproate concentration, a calibration curve Ccv based on the observation results of the phase-separation domain information acquisition liposome 510a can be used as the ethyl caproate concentration-dependent information Ref of the phase-separation domain generation pattern.

[0064] As a result, in the concentration estimation process Q52C, the ethyl caproate concentration is estimated using the calibration curve Ccv to obtain the estimated concentration EsC, which further improves the accuracy and precision of concentration estimation and measurement, enabling concentration measurement with a consistent level of precision. The calibration curve Ccv can be created by focusing on the proportion of cell-sized liposomes that have not phase-separated, and can also be created using multiple lipid species of cell-sized liposomes. The actual use of the calibration curve as information on the ethyl caproate concentration dependence of the phase-separated domain generation pattern will be described later in the Examples section [5. Concentration Estimation Experiments and Results Using the Calibration Curve]. [Example]

[0065] Examples of the present invention will be described below, but the present invention is not limited to these examples. Note that the examples will be explained by outlining some of the experimental results that led to the completion of the present invention. [Research topic] Development of a simple method for detecting ethyl caproate using observation of the phase-separated domain structure of cell-sized liposomes [Research purpose] To create a phase-separated domain structure that depends on the concentration of ethyl caproate, the main component of ginjo aroma in sake, and to establish a technique for estimating the concentration of ethyl caproate using this, i.e., a screening technique for precise measurement.

[0066] [Experimental details] [1. Basics of cell-sized liposome production] The method for preparing cell-sized liposomes was as shown in Figure 4 above. The lipid component system was composed as follows: The fluorescent reagent added was one that stains the DOPC-rich Ld phase. DOPC / DPPC=50:50 +Lissamine Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Triethylammonium Salt(Rhodamine DHPE) 1% (stained liquid disordered domain) This results in a liposome having an So / Ld domain structure as shown in A in Figure 1-2 above.

[0067] 2. Fluorescence Microscopic Observation of Phase-Separated Domain Structure The conditions for fluorescence microscopic observation of the phase-separated domain structure were as follows. Microscope used: Olympus BX51 Observation conditions: WIG (excitation wavelength 530-550 nm, fluorescence wavelength 575 nm) It can also be observed with WIY (excitation wavelength 545-580 nm, fluorescence wavelength 610 nm).

[0068] [3. Experiments using sake] The EC concentration in sake with a pronounced ginjo aroma has been reported to be approximately 7 mg / L ("Breeding of High-Ester-Producing Yeast and Development of Fruity Sake Without the Need for Extensive Polishing," Takahashi Toshinari, Journal of the Brewing Society of Japan, Vol. 115, 2020, hereafter referred to as "Academic Reference 1"). Furthermore, previous analyses conducted by the inventors have yielded values ​​of 1.6 mg / L for standard ginjo sake and 11 mg / L for ginjo sake fermented with yeast that produces a high level of ethyl caproate (T. Yoda and T. Saito 2020 Membranes). In this study, the samples used were a commercially available sake that claims to contain a pronounced ginjo aroma, ethyl caproate (referred to as "Sake 1"), a sake in which 10% of the volume of Sake 1 was replaced with pure EC (referred to as "Sake 1 + EC"), and pure water. During the hydration process of the cell-sized liposome preparation, each sample was added to a lipid film to produce cell-sized liposomes. We then observed the phase-separated domain structure and attempted to estimate the ethyl caproate concentration.

[0069] The materials used to prepare the samples used in the experiment are as follows: Sake 1 Manufacturer: Kikumasamune Sake Brewery Co., Ltd. Brand name: Kikumasamune Freshly Squeezed Gin Pack EC pure product Manufacturer: Kanto Chemical Co., Ltd. Product name: Ethyl n-hexanoate 500mL, Deer Special Grade

[0070] The lipid film formation for cell-sized liposomes was carried out as follows. The lipid was prepared as a chloroform solution with a concentration of 2 mM, and a total of 40 μL of solution was added. The organic solvent was evaporated to dryness, after which hydration was carried out. Below, we list the experimental cases using each sample and describe the conditions. Case 1 (Sake 1) Case 2 (Sake 1 + EC) Case 3 (pure water)

[0071] Case 1 Sake 1 Based on the value in Non-Patent Document 1, the EC concentration in sake 1, whose concentration is unknown, was assumed to be 7.2 mg / L. Since the amount of sample added when preparing cell-sized liposomes was 400 μL, the assumed molar weight was as follows: 7.2 x 400 / 1000 / 1000 / 144 x 1000 x 1000 = 20 μM (convert to 400 μL, then divide by molecular weight and adjust units)

[0072] Case 2 Sake 1 + EC First, 10% of sake 1 was replaced with pure EC, and 400 μL was added during hydration. The concentration of the pure product is stated as "at least 98% or more," but we assumed that 100% was EC. Also, since the specific gravity is 0.87, we assumed that the EC concentration of the pure product was 870 g / L. The amount added was 40 μL, or 10% of the total 400 μL. Therefore, the molar weight of the sample is as follows: 7.2 x 400 / 1000 / 1000 / 144 x 1000 x 1000 x 0.9 + 870 x 40 / 1000 / 1000 / 144 x 1000 x 1000 = 260 μM (After converting to 400 μL, divide by molecular weight, adjust units and round off.)

[0073] Case 3 Pure water Pure water is used, so the molar weight of EC in the cell-sized liposome solution is 0 μM.

[0074] 4. Experimental Results Figure 7 is a graph showing the structural composition of the phase-separated domains in each cell-sized liposome according to the examples (Cases 1, 2, and 3). As a result of fluorescence microscopic observation, as shown in the figure, the So / Ld domain was approximately 80% in the cell-sized liposomes using pure water in Case 3. According to Non-Patent Document 1, the EC concentration in pure water is naturally 0 mol%, so the obtained estimation results were confirmed to be valid.

[0075] In the putative target liposomes from Case 1 Sake 1, domains were difficult to observe, but approximately 39% of the So / Ld domains were still confirmed by fluorescence microscopy.

[0076] In the case of Case 2, the estimated target liposomes made from sake 1 + EC, the So / Ld domains confirmed by fluorescence microscopy were approximately 31%.

[0077] In Non-Patent Document 1, the inventors reported a technique for estimating the concentration of cell-sized liposomes by adding sake during the lipid film preparation stage and observing the state of phase-separated domains. The same document also reported that the higher the concentration of ethyl caproate in the lipid membrane, the more difficult it is to observe the So / Ld domains.

[0078] In this study, the order of increasing ethyl caproate concentration was Case 2, Case 1, and Case 3. Furthermore, the order of decreasing So / Ld domain proportions observed in this example was Case 2, Case 1, and Case 3, showing the relative concentration order. Furthermore, because the ethyl caproate concentration was lower in Case 1 Sake 1 than in Case 2 Sake 1 + EC, it was found that the ethyl caproate concentration in Case 1 sake was lower than the concentration of the ethyl caproate standard added in Case 2.

[0079] From the above, it was confirmed that the method of the present invention can be used to simply rank the relative concentrations of ethyl caproate. Furthermore, it was confirmed that the method of the present invention can simply estimate the concentration range of ethyl caproate.

[0080] 5. Concentration estimation experiment using calibration curve and results Next, the concentration of ethyl caproate in the sample to be measured is estimated using the calibration curve. As explained above in [1. Basics of Cell-Sized Liposome Preparation], the method for preparing cell-sized liposomes is shown in Figure 4. The lipid component systems were composed of the following three types. Similarly, the fluorescent reagent used was rhodamine DHPE, which stains the DOPC-rich Ld phase. Lipid composition1. DOPC / DPPC=50:50 +Rhodamine DHPE 1% Lipid composition 2. DOPC / DPPC / Chol=45:45:10 +Rhodamine DHPE 1% Lipid composition 3. DOPC / DPPC / Chol=40:40:20 +Rhodamine DHPE 1%

[0081] Hydration was performed using the three lipid compositions described above to estimate the ethyl caproate concentration of target samples, and reference liposomes were prepared using sample solutions with known ethyl caproate concentrations. Four sample solutions were used for hydration to prepare these reference liposomes: pure water alone, and sample solutions containing ethyl caproate at concentrations of 5 mg / L, 10 mg / L, and 15 mg / L, respectively. A calibration curve was created by observing the reference liposomes encapsulating these four sample solutions with different ethyl caproate concentrations. For the hydration of the reference liposomes prepared for the calibration curve, the pure EC product and pure water described above in [3. Experiments using sake] were used as sample solutions with known concentrations.

[0082] In lipid compositions 2 and 3, Lo / Ld domains were observed in addition to So / Ld domains, but both were considered to be phase-separated cell-sized liposomes. On the other hand, homogenous cell-sized liposomes, which were uniformly stained with fluorescent dye and had not undergone phase separation, were also observed. In this experiment, phase-separated cell-sized liposomes and homogenous cell-sized liposomes without phase separation were counted separately.

[0083] As mentioned above, in Non-Patent Document 1, the inventors reported that when cell-sized liposomes are prepared, the So / Ld domain becomes less observable as the ethyl caproate concentration in the lipid film increases, as observed by adding sake during lipid film preparation and observing the state of the phase-separated domains. The results of this experiment confirmed this. In other words, it was shown that the higher the ethyl caproate concentration in the sample solution used for hydration, the more difficult it becomes to observe the So / Ld domain or the Lo / Ld domain.

[0084] Furthermore, we discovered that the proportion of homogenous cell-sized liposomes in the observed cell-sized liposomes increased linearly in proportion to the ethyl caproate concentration, meaning that it was possible to create a calibration curve plotting the proportion of homogenous cell-sized liposomes on the vertical axis and the ethyl caproate concentration in the sample solution on the horizontal axis. 8, 9, and 10 show graphs of calibration curves prepared under conditions of lipid composition 1, lipid composition 2, and lipid composition 3, respectively.

[0085] To create the calibration curves shown in Figures 8, 9, and 10, cell-sized liposomes were prepared and observed at least three times, with at least 60 samples observed per observation. The average values ​​and standard errors are shown as error bars when creating the calibration curves.

[0086] At the same time, cell-sized liposomes were prepared by hydration using samples for estimating ethyl caproate concentrations. The samples for estimating ethyl caproate concentrations were the three cases described above in [3. Experiments using sake]: Case 1 (Sake 1), Case 2 (Sake 1 + EC), and Case 3 (Pure water). The respective ethyl caproate concentrations were 7.2 mg / L, 93.6 mg / L, and 0 mg / L, as previously described.

[0087] The estimated concentration of ethyl caproate using the calibration curve showed different values ​​depending on the lipid composition, as shown below. Lipid composition 1 Case 1 (Sake 1) 22.18 mg / L Case 2 (Sake 1 + EC) 31.01 mg / L Case 3 (pure water) 0.85 mg / L Lipid composition 2 Case 1 6.05 mg / L Case 2 13.72 mg / L Case 3 -0.06 mg / L Lipid composition 3 Case 1: 40.0 mg / L Case 2: 56.7 mg / L Case 3 1.8 mg / L

[0088] In case 3 (pure water), the error was within ±2.0 mg / L for all lipid compositions, and in case 1 (sake 1), the estimated concentration was within ±2.0 mg / L for lipid composition 2. In the other cases, although the concentrations were significantly different from the actual concentrations, the relative rankings were accurate for all lipid compositions.

[0089] 5. Effectiveness of Fluorescence Microscopy It should be noted that the implementation of the fluorescence microscopic observation according to the present invention is guaranteed without any problems. 11 is a graph showing the phase transition temperature, i.e., the domain disappearance temperature, of each cell-sized liposome according to the examples (Cases 1, 2, and 3). As shown in the figure, in each case, the temperature range at which the generated domains disappear is generally above 34°C, confirming that there is no problem with fluorescence microscopy observation at room temperature.

[0090] [6. Estimation of cost reduction effects] The costs (expenses and time) were estimated for the conventional measurement method using GC and the method of the present invention. The initial costs include the cost of instruments and reagents that can be reused after purchase, but the cost of items that must be purchased in minimum units was included for the conventional method and the method of the present invention. 1) Existing method (GC) Initial cost GC equipment: 16,880,000 yen (Agilent) Running costs Helium gas (one cylinder) 13,000 yen Adsorption device PDMS Twister 59,700 yen EC (measurement standard) 1,800 yen Methyl caproate 12,730 yen (measurement reference) Total: 87,230 yen Measurement time 1-2 days (including equipment setup and calibration sample measurement)

[0091] 2) The method of the present invention (cell-sized liposomes) Initial cost Microscope 1,146,260 yen Fluorescent unit 1,400,000 yen (All manufactured by Olympus) Total: 2,546,260 yen Running costs Lipid DOPC 8,800 yen DPPC 9,000 yen Chol 5,000 yen Rhodamine DHPE 50,900 yen Solvent: Chloroform 2,850 yen Total: 76,550 yen Measurement time Preparation: 8 hours (liposome preparation (film preparation 1 hour + vacuum drying 3 hours + hydration 4 hours)) Measurement: 1 hour (time required to observe 60 samples) Total: 9 hours

[0092] As described above, the method of the present invention is estimated to be cheaper than the conventional method using GC, with an initial cost of about 14,000,000 yen and a running cost of about 10,000 yen. Furthermore, the time required for measurement is estimated to be shortened to about half a day (note that glassware, pipettes, plastic tubes, filters, and syringes are inexpensive and make almost no difference, so they have not been included in this calculation). [Industrial Applicability]

[0093] The cell-sized liposomes, the method for estimating ethyl caproate concentration using the cell-sized liposomes, the method for estimating ethyl caproate concentration using the observation results of the cell-sized liposomes and a calibration curve, and the method for measuring ethyl caproate concentration of the present invention enable analysis of ethyl caproate concentration at significantly lower cost and in a shorter time than conventional methods. In particular, the method for estimating ethyl caproate concentration using a calibration curve can further improve the accuracy and precision of concentration estimation and measurement. Therefore, this invention has high industrial applicability, particularly in the fields of food component analysis, food quality control, and all related fields. [Explanation of symbols]

[0094] 2, 12, 22...unsaturated phospholipids 8, 8a, 8b, 8x, 8y, 18, 28...Ethyl caproate 9, 19, 29…Fluorescent reagents for detecting phase-separated domains 10, 10', 110, 110', 210, 210'...cell-sized liposomes 10a, 10b, 510a...cell-sized liposomes (reference liposomes) 10x, 10y, 510x...cell-sized liposomes (estimated target liposomes) 13, 23...Saturated phospholipids 25...Sterol lipids Ccv...calibration curve Da, Db, Dx, Dy, 5Dx...phase separation domain generation pattern EsC, Est...Estimated concentration N: All samples for which concentration estimation is required n: A portion of the sample that has been subjected to concentration estimation processing P10: Relative concentration estimation process P20…Precision measurement process Q510...Pattern reading process Q520, Q52C…Concentration estimation process Ref…Ethyl caproate concentration dependence of phase-separated domain formation pattern

Claims

1. A lipid component system comprising one or more unsaturated phospholipids; and The sample to be estimated for ethyl caproate concentration or ethyl caproate A cell-sized liposome comprising: The ethyl caproate concentration resulted in different phase-separated domain formation patterns. This allows cell-sized liposomes to be used to estimate the concentration of ethyl caproate from the phase-separated domain formation pattern.

2. 2. The cell-sized liposome of claim 1, wherein the lipid component system contains one or more saturated phospholipids.

3. The cell-sized liposome according to claim 2, wherein the lipid component system contains one or more sterol-based lipids.

4. The cell-sized liposome according to claim 1, characterized in that the lipid component system is composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), which is an unsaturated phospholipid, and Dipalmitoylphosphotidylcholine (DPPC), which is a saturated phospholipid.

5. The cell-sized liposome according to claim 1, characterized in that the lipid component system is composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), which is an unsaturated phospholipid, dipalmitoylphosphotidylcholine (DPPC), which is a saturated phospholipid, and cholesterol, which is a sterol lipid.

6. The cell-sized liposome according to any one of claims 2, 3, 4, and 5, characterized in that, when used at room temperature, the molar composition of unsaturated phospholipids and saturated phospholipids in the lipid component system is unsaturated phospholipid:saturated phospholipid=75-10:25-90.

7. 6. The cell-sized liposome according to claim 2, wherein the unsaturated phospholipid and the saturated phospholipid in the lipid component system have an equal molar concentration when used at room temperature.

8. 6. The cell-sized liposome according to claim 1, wherein a fluorescent reagent for detecting phase-separated domains is added.

9. The cell-sized liposome described in claim 8, characterized in that the fluorescent reagent used is one or more of a reagent for staining a solid ordered phase (So phase), a reagent for staining a liquid disordered phase (Ld phase), or a reagent for staining sterol-based lipids.

10. 6. The cell-sized liposome according to claim 1, 2, 3, 4, or 5, which is prepared by a static hydration method.

11. The cell-sized liposome according to claim 10, wherein after the lipid film is prepared, a sample whose concentration is to be estimated is added during hydration of the cell-sized liposome.

12. A method for estimating the relative concentration of ethyl caproate in a target substance for concentration estimation, comprising using a cell-sized liposome according to any one of claims 1 to 5, which is constructed using an ethyl caproate standard having a known concentration, as a liposome for obtaining phase-separation domain information, and using the liposome for obtaining phase-separation domain information, Fluorescence microscopic observation of the cell-sized liposomes to be used for estimating the concentration of ethyl caproate, which are cell-sized liposomes according to any one of claims 1 to 5, constructed using a sample for which the concentration of ethyl caproate is to be estimated; The phase separation domain generation pattern, i.e., whether or not a phase separation domain is generated, or the pattern when a phase separation domain is generated, is read; Referring to the ethyl caproate concentration dependency information of the phase-separation domain generation pattern in the liposome for obtaining phase-separation domain information, Estimate the relative concentration of ethyl caproate contained in the target substance. A method for estimating the concentration of ethyl caproate, comprising:

13. The method for estimating the concentration of ethyl caproate according to claim 12, wherein the concentration of ethyl caproate is estimated using a calibration curve based on the observation results of the liposome for obtaining phase-separation domain information.

14. 14. The method for estimating the concentration of ethyl caproate according to claim 12, wherein the subject of concentration estimation is sake or a processed sake product.

15. 14. A method for measuring the concentration of ethyl caproate, comprising: first estimating the relative concentrations of ethyl caproate for a plurality of samples by the method for estimating the concentration of ethyl caproate according to claim 12 or 13; and then precisely measuring the concentrations of ethyl caproate by gas chromatography for some of the samples that have been subjected to the ethyl caproate concentration estimation process.

16. 16. The method for measuring the concentration of ethyl caproate according to claim 15, wherein the object of concentration measurement is sake or a processed sake product.

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