Cell-size liposome, ethyl caproate relative concentration estimation method using cell-size liposome, and ethyl caproate concentration estimation method

Abstract

To provide a simple ethyl caproate analysis method that can be performed at low cost, and is in the form of preliminary analysis for selecting a sample for precision analysis in gas chromatography (GC) such as screening.SOLUTION: A cell-size liposome 110 comprises: a lipid component system formed by using, in addition to one or two or more types of unsaturated phospholipids 12, one or two or more types of saturated phospholipids 13; and a sample or ethyl caproate 18 to be subjected to ethyl caproate concentration estimation. The cell-size liposome generates different phase separation domain generation patterns on the basis of the concentration of ethyl caproate 18, and is used for ethyl caproate concentration estimation from the phase separation domain generation patterns. The lipid component system of the cell-size liposome 110 may include sterol-based lipid such as cholesterol.SELECTED DRAWING: Figure 1-3

Description

[Technical field]

[0001] The present invention relates to cell-sized liposomes, a method for estimating the relative concentration of ethyl caproate using cell-sized liposomes, and a method for measuring the concentration of ethyl caproate, and more particularly to a technique that can estimate the relative concentration of ethyl caproate in sake and the like at low cost in a relatively short period of 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 short lipid chain. Ethyl caproate produced from caproic acid is a major component of ginjo aroma in sake along with isoamyl acetate. Since the ginjo aroma components of sake increase its commercial value, there has long been a demand for yeast that produces a high amount of ethyl caproate. Even if the same yeast is used, the concentration of ethyl caproate in the sake produced will vary depending on the temperature conditions during fermentation.

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

[0004] Gas chromatography (GC) is used in the ethyl caproate concentration measurement test, but this equipment is not only very expensive to purchase, but also has the disadvantage of high running costs because high purity gas is used for each measurement. In addition, it takes several hours to start up the equipment, and a calibration curve is created by running multiple standard samples, and then the concentration measurement is finally performed, 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 technique for accurately measuring ethyl caproate in sake, in which a free fatty acid coloring agent using an acyl-activating enzyme is used to quantify the color intensity according to the concentration by absorptiometry. Furthermore, Non-Patent Document 1, written by the present inventor, discloses a technique for estimating the concentration by adding sake at the stage of preparing a lipid film when preparing cell-sized liposomes and observing the state of the phase-separated domains. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2012-157349 A "Simple method for measuring the concentration of ethyl caproate 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 the commercial value of sake, so if the measurement method were simple, it would be possible to study the production conditions quickly and at low cost. However, the GC used to measure ethyl caproate has problems in that it is expensive in both initial and running costs, and requires considerable time and effort. In addition, although the technology disclosed in the above Patent Document 1 uses a relatively inexpensive absorptiometer, it has the disadvantage that it requires the preparation of multiple reagents such as enzymes and colorants at the time of measurement. Therefore, it would be convenient to have a preliminary analysis for selecting samples to be subjected to precise analysis by GC, that is, a simple analysis method in the form of a so-called screening, which can be performed at the lowest possible cost.

[0009] Liposomes, which are used in many experiments and research as the most simplified biomembrane model, are formed when phospholipids, which have both hydrophilic and hydrophobic fatty chains, self-assemble with the hydrophilic part facing outward to form a lipid bilayer membrane, which then closes into a bag shape. Liposomes with diameters of several μm to several tens of μm are roughly the same size as living cells and are called cell-sized liposomes (giant liposomes). Cell-sized liposomes are useful in research into the elucidation of the control mechanisms of biomembranes, due to the advantages that they can be directly observed with an optical microscope and can be used to create experimental models that mimic cells.

[0010] It has also been revealed that there are phase-separated domains (raft domains) on cell membranes that are rich in saturated lipids and cholesterol and that play a role in signal transmission, etc., and that these raft domains move by sticking together and separating in association with signal transmission between cells. It has been found that similar domain structures are observed in liposomes artificially made from lipids, and research in this field is also being actively conducted. 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) accumulation dynamics of membranes are related to signal transmission using intracellular calcium ions as an indicator, and that changing the structure of lipids and sterols changes the temperature response of the domain structure.

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

[0012] The inventors also clarified that domain structures are generated when membranes containing antioxidants are subjected to oxidative stress. In other words, when a solution of polyphenols such as resveratrol and theaflavin, which are known as antioxidant functional components, is mixed with cell-sized liposomes, membrane dynamics such as membrane contraction and oscillation are induced, and phase-separated domains appear during observation (HHT. Phan, T. Yoda et al., Biochim. Biophys. Acta, Biomembr. 2014). In addition, it has been revealed by fluorescence microscopy that concanavalin A, a functional component contained in sword beans that activates the immune system, causes 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 undergo phase separation into a DOPC-rich liquid disordered phase domain (Ld domain) and a DPPC- and cholesterol-rich liquid ordered phase domain (Lo domain).

[0014] 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 in Non-Patent Document 1. However, these methods involve a procedure for vacuum drying the organic solvent after film preparation, which may result in volatilization and evaporation of ethyl caproate.

[0015] The problem to be solved by the present invention is to provide a simple analytical method for ethyl caproate concentration in the form of a preliminary analysis for selecting samples for precise analysis by GC, that is, a so-called screening method, based on the progress and results of research using liposomes and the state of the prior art related to measurement, which does not need to be as accurate as GC, but can be performed at the lowest possible cost and in a short time. In other words, to provide a method for easily distinguishing a simple measurement method using a clear difference characterized by observation due to the interaction between ethyl caproate and a membrane, using cell-sized liposomes. Furthermore, the present application also provides a method for suppressing the volatilization and evaporation of ethyl caproate by adding an object to be measured for the concentration of ethyl caproate, such as sake, at a time other than when the film is made. [Means for solving the problem]

[0016] The inventors have carried out research with the above problem in mind. In preparing cell-sized liposomes, we attempted to add sake at the hydration stage. First, we attempted to prepare cell-sized liposomes using only DOPC, and found that when sake was added during hydration, micrometer-scale liposomes were observed. We also found that when sake was added at the hydration stage when preparing cell-sized liposomes using only DOPC, the size was significantly smaller than when hydrated with pure water, and that the size was further reduced when a solution in which ethyl caproate had been added to sake in advance was added at the hydration stage. These findings were consistent with a report (T. Yoda et al., Biomimetics 2020) that showed that when ethyl caproate was added to the film in advance, the size was reduced depending on the concentration of the ethyl caproate (Fig. 1).

[0017] From the above, it was speculated that ethyl caproate in sake could be incorporated into cell-sized liposomes in the formation stage by the method of adding sake during hydration, similar to the method disclosed in the above-mentioned Non-Patent Document 1, i.e., the method of adding sake during film production. Therefore, 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 replaced with ethyl caproate was added was observed, and it was found that the ratio was different. From this, it was found that the content of ethyl caproate in a sample can be determined by the ratio of solid ordered phase / liquid disordered phase, i.e., the ratio of phase separation.

[0018] In addition to the above findings obtained by the experiment using sake, the cost reduction effect was also confirmed by trial calculation, and it was confirmed that the cell-sized liposomes can be used as an ethyl caproate concentration analysis tool. That is, in order to analyze ethyl caproate contained in sake, etc., the relative concentration of ethyl caproate can be estimated in a short time by observing the ratio of domain structures of cell-sized liposomes containing unsaturated lipids, more preferably cell-sized liposomes containing unsaturated lipids and saturated lipids, which are prepared by adding sake during hydration (Note that the reason why "unsaturated lipids" and "saturated lipids" are used here, and not "unsaturated lipids such as DOPC" or "saturated lipids such as DPPC", is that the cell-sized liposomes according to the present invention can also be prepared by using lipids other than the phospholipids that are the main subject of the explanation of this application, such as glycolipids. In other words, these are also within the scope of the present invention). The present invention thus completed, that is, the invention claimed in this application as a means for solving the above 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 produces a phase-separation domain generation pattern that differs depending on the concentration of ethyl caproate, and is thus used to estimate the concentration of ethyl caproate from the phase-separation domain generation pattern. [2] The cell-sized liposome according to [1], characterized in that the lipid component system contains one or more types of saturated phospholipids. [3] The cell-sized liposome according to [2], characterized in that the lipid component system contains one or more types of sterol 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 phospholipid:saturated phospholipid=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], further comprising a fluorescent reagent added thereto for detecting phase-separated domains. [9] The cell-sized liposome according to [8], characterized in that the fluorescent reagent used is one or more of a solid ordered phase (So phase) staining reagent, a liquid disordered phase (Ld phase) staining reagent, and a sterol lipid staining reagent.

[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 a relative concentration of ethyl caproate in an object to be estimated using a cell-sized liposome for obtaining phase-separation domain information according to any one of [1], [2], [3], [4], and [5], which is constituted using an ethyl caproate standard having a known concentration, and using the liposome for obtaining phase-separation domain information, the method comprises: observing the cell-sized liposome for concentration estimation, which is the cell-sized liposome for concentration estimation according to any one of [1], [2], [3], [4], and [5], which is constituted using a sample for which ethyl caproate concentration is to be estimated, under a fluorescent microscope; reading a phase-separation domain generation pattern, i.e., whether or not a phase-separation domain is generated or a pattern when a phase-separation domain is generated; and estimating the relative concentration of ethyl caproate contained in the object to be estimated by referring to information on the ethyl caproate concentration dependency of the phase-separation domain generation pattern in the liposome for obtaining phase-separation domain information.

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

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

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

[12] ; and then precisely measuring the ethyl caproate concentration by gas chromatography for some of the samples that have had their ethyl caproate concentrations estimated.

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

[14] , wherein the subject of concentration measurement is sake or a processed sake product. Effect of the Invention

[0022] The cell-sized liposomes, the method for estimating the relative concentration of ethyl caproate using the cell-sized liposomes, and the method for measuring the concentration of ethyl caproate are configured as described above, and therefore, they can provide a simple measurement method utilizing a clear difference characterized by observation due to the interaction between ethyl caproate and a membrane, and a simple discrimination method using cell-sized liposomes. Specifically, the concentration of ethyl caproate can be analyzed at a significantly lower cost and in a shorter time than in the past.

[0023] Conventional measurement methods, which require expensive GC, have the problem of taking a long time to start up the equipment, etc., but 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 concentration of ethyl caproate in sake, etc., and enabling measurements to be performed quickly.

[0024] In particular, the present invention involves a method in which sake containing the target ethyl caproate is added during hydration to prepare cell-sized liposomes, which are then observed under a fluorescent microscope. With this method, once an optimal condition for the combination of lipids constituting the cell-sized liposomes has been determined, the concentration of ethyl caproate contained can be estimated by simply observing for a short period of time.

[0025] As an effective example of use, the present invention can be used for preliminary analysis to select samples for precise analysis by GC, i.e., for simple determination of the concentration of ethyl caproate in a screening manner, and is highly useful. Therefore, by combining the method of the present invention with GC, a rapid, low-cost, and precise analysis system can be realized. [Brief description 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 phase-separated domain generation pattern in the cell-sized liposome of the present invention. [Figure 1-3] FIG. 2 is a conceptual diagram showing another basic structure of the cell-sized liposome of the present invention, which also contains saturated phospholipids. [Diagram 2] FIG. 2 is a conceptual diagram showing another configuration of the cell-sized liposome of the present invention, which also contains a sterol lipid. [Diagram 3] 1 shows the chemical structural formulae of lipids, which are examples of components of the cell-sized liposome of the present invention, and ethyl caproate. [Figure 4] FIG. 1 is an explanatory diagram showing an example of a method for producing the cell-sized liposome of the present invention. [Diagram 5] FIG. 1 is an explanatory diagram conceptually showing a configuration of a method for estimating a relative concentration of ethyl caproate according to the present invention. [Figure 6] 1 is a flow chart showing the configuration of a method for measuring the concentration of ethyl caproate according to the present invention (the following figures relate to the examples). [Figure 7] 1 is a graph showing the phase-separated domain structural composition in each cell-sized liposome according to the examples (Cases 1, 2, and 3). [Figure 8] 1 is a graph showing the phase transition temperature, i.e., domain disappearance temperature, measurement results for each cell-sized liposome according to the examples (Cases 1, 2, and 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention will now be described in detail 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 of the present invention is a cell-sized liposome composed of a lipid component system using one or more kinds of unsaturated phospholipids 2 (the figure shows an example in which only one kind of unsaturated phospholipid 2 is used, but even with one kind, the desired effect of the present invention can be sufficiently obtained) and a sample for estimating the concentration of ethyl caproate or ethyl caproate 8, and the main structure is that different phase-separation domain generation patterns are generated depending on the concentration of ethyl caproate 8, and thus the concentration of ethyl caproate can be estimated from the phase-separation domain generation patterns. Note that (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 liposome 10 produces and exhibits different phase-separation domain generation patterns depending on the concentration of ethyl caproate 8 contained therein, and the concentration of ethyl caproate contained can be estimated using the 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.

[0029] The cell-sized liposome of the present invention having the structure shown in FIG. 1 will be described below. The fact that phase-separated domains can be observed under fluorescence observation with a single type of unsaturated lipid, such as DOPC, is believed to be due to active oxygen generated during fluorescence observation. That is, in fluorescence observation, excitation light is irradiated and the fluorescence emitted from the fluorescent dye is observed, and active oxygen is generated at that time, which oxidizes and breaks the unsaturated bond of the unsaturated lipid, i.e., part of the double bond. As a result, two types (or several types) of lipids exist in the system: unoxidized unsaturated lipids and oxidized saturated lipids, which are observed as phase separation in the lipid membrane.

[0030] In fact, unsaturated lipids such as DOPC have a low phase transition temperature, and liposomes can 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 cannot be affected by oxidation by active oxygen during fluorescence observation, so it is thought that the desired formation of phase-separated domains cannot be obtained with liposomes whose lipid component system is composed only of saturated phospholipids.

[0031] FIG. 1-2 is a photograph showing an example of a phase-separated domain generation pattern in the cell-sized liposome of the present invention. These are micrographs showing typical patterns, among which A is an So (solid ordered phase) / Ld (liquid disordered phase) domain structure. B is a uniform membrane vesicle. As a result of observation using a fluorescent microscope, in the present invention, the pattern of the So / Ld domain structure shown in A can be particularly preferably used for concentration estimation. Observation using a fluorescent microscope will be described further below.

[0032] 1-3 is a conceptual diagram 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 a cell-sized liposome composed of a lipid component system using one or more kinds of unsaturated phospholipids 12 (the figure shows an example of only one kind of unsaturated phospholipid 12), as well as one or more kinds of saturated phospholipids 13 (the figure shows an example of only one kind of saturated phospholipid 13. The desired effect of the present invention can be sufficiently obtained even with one kind), and a sample for estimating the concentration of ethyl caproate or ethyl caproate 18, and the main structure is that different phase-separation domain generation patterns are generated depending on the concentration of ethyl caproate 18, and thus the cell-sized liposome is used to estimate the concentration of ethyl caproate from the phase-separation domain generation patterns.

[0033] The cell-sized liposome 110 of this configuration also produces and exhibits 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 the phase-separation domain generation pattern. 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. In order to more fully achieve the intended object of the present invention, the cell-sized liposome 110 of this invention, which is composed of unsaturated lipids and saturated lipids, is more desirable than the cell-sized liposome 10 shown in FIG. 1.

[0034] 2 is a conceptual diagram showing another structure of the cell-sized liposome of the present invention, which also contains a sterol lipid. As shown in (a) of the figure, the cell-sized liposome 210 is a cell-sized liposome composed of a lipid component system including one or more kinds of unsaturated phospholipids 22 (the figure shows an example of only one kind of unsaturated phospholipid 22), one or more kinds of saturated phospholipids 23 (the figure shows an example of only one kind of saturated phospholipid 23), one or more kinds of sterol lipids 25 (the figure shows an example of only one kind of sterol lipid 25. The desired effect of the present invention can be sufficiently obtained with only one kind), and a sample to be subjected to ethyl caproate concentration estimation or ethyl caproate 28, which generates different phase-separation domain generation patterns depending on the concentration of ethyl caproate 28, and is mainly used for estimating the concentration of ethyl caproate from the phase-separation domain generation 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, and therefore 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.

[0036] Sterol lipids are not essential elements for forming 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, so the hydration temperature can be lowered. In the present invention, heating is performed during hydration to prepare cell-sized liposomes, but by performing hydration at a lower temperature, the volatilization or evaporation of the aroma component ethyl caproate can be suppressed.

[0037] In addition, sterol lipids such as cholesterol have the effect of increasing the fluidity of phospholipids, which allows for better mixing of lipids. This reduces the bias in the composition of each liposome produced, and increases the uniformity of the liposomes produced. As a result, the reproducibility of the implementation of the ethyl caproate relative concentration estimation method is improved. There are also other advantages when using a specific fluorescent reagent, which will be described later (see below).

[0038] On the other hand, there is also an advantage in not intentionally including cholesterol in the lipid component system. This is the case when observing mainly the change in the So / Ld phase separation structure, as in the ethyl caproate relative concentration estimation method of the present invention described later with reference to FIG. 5 and the like. By not including cholesterol in the lipid component system, almost all of the obtained cell-sized liposomes are So / Ld, so when observing mainly the change in the So / Ld phase separation structure, the phase separation domain generation pattern can be easily 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 read the discrimination pattern.

[0039] In particular, the present invention can provide 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. In addition to the above configuration, cell-sized liposomes can also be provided having a lipid component system containing cholesterol (Chol), a sterol lipid. In the following description, cell-sized liposomes having either a lipid component system of DOPC+DPPC or a lipid component system of DOPC+DPPC+Chol will be mainly described.

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

[0041] When the lipid component system of the cell-sized liposome of the present invention is used at room temperature, the molar composition of the unsaturated phospholipid and the saturated phospholipid in the lipid component system can be unsaturated phospholipid:saturated phospholipid = 75 to 10: 25 to 90. Alternatively, when the lipid component system is used at room temperature, the molar composition of the unsaturated phospholipid and the saturated phospholipid in the lipid component system can be configured to be equal. Here, room temperature may be about 20°C, which is generally understood, or may be 22±2.0°C, which is more preferable.

[0042] As an example of a specific lipid species, the lipid component system of the cell-sized liposome of the present invention has a molar composition when used at room temperature 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 concentration of ethyl caproate is the So / Ld domain, as described above. The molar concentration composition at which this domain is observed has been reported in the following examples. 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 in 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), DOPC:DPPC:Chol=42.5~50 : 42.5~50 : remaining amount For example, when Chol is 10, the So / Ld domain structure can be obtained in a molar composition range of DOPC 0 to 65 and DPPC 25 to 90. As described above, DOPC and DPPC may be used in equal amounts. In the examples described later, DOPC and DPPC are used in equal amounts (50:50).

[0045] The phase-separated domains themselves are observed even when the cholesterol is 15 or more. For example, when the cholesterol is 20 to 40 and the phospholipid is 60 to 80, Lo / Ld (liquid-ordered phase / liquid-disordered phase) domains are observed. However, no correlation was found between the domain formation rate and the ethyl caproate concentration in this domain pattern.

[0046] The cell-sized liposome of the present invention may be configured to have a fluorescent reagent 9 or the like added thereto for detecting phase-separated domains, such as the cell-sized liposome 10' in Fig. 1, Fig. 1-3, and Fig. 2(b). The presence or absence of a phase-separated domain structure is determined by fluorescent observation of the cell-sized liposome, and the fluorescent reagent 9 is added for that purpose and does not affect the formation of phase separation. The fluorescent reagent 9 may be one or more of the following for staining a liquid ordered phase (Lo phase), a liquid disordered phase (Ld phase), or a sterol lipid stain to constitute the cell-sized liposome 10'.

[0047] In the examples described below, Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Triethylammonium Salt (Rhodamine DHPE) is added as a fluorescent reagent at 1% molar concentration of lipids. This is a relatively commonly used 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 a fluorescent reagent for staining the DPPC-rich So or Lo phases. These have different fluorescent wavelengths, so they can be used separately or two types can be used at the same time.

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

[0049] FIG. 4 is an explanatory diagram showing an example of a method for producing the cell-sized liposomes of the present invention. As shown in the figure, the cell-sized liposomes can be produced by the static hydration method. Alternatively, after producing a lipid film, a sample whose concentration is to be estimated can be added when hydrating the cell-sized liposomes. That is, the lipids constituting the liposomes are dissolved in a solvent (chloroform CH(Cl)3 in the example shown in the figure) in a container such as a test tube, the solvent is evaporated to form a lipid film on the inner wall of the container, and the lipid film is hydrated with sake, which is the target sample, to produce the liposomes.

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

[0051] The phase separation domain structure is determined by the components in the membrane, such as the constituent lipids, and not by the production method. Therefore, the specific production method of the cell-sized liposome of the present invention is not limited, and a conventionally known method may be used as appropriate. For example, there are electroformation method, droplet method, and its new method (Patent No. 6031711), but in the present invention, the static hydration method can be particularly preferably used. 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 there is a test tube, gas, and a specified solution, and is optimal for the present invention.

[0052] 5 is an explanatory diagram conceptually showing the configuration of the method for estimating the relative concentration of ethyl caproate of the present invention. The cell-sized liposome of the present invention having any of the configurations described above can be used as a liposome used in estimating the concentration of an estimation target having an unknown concentration of ethyl caproate, that is, as a "liposome for obtaining phase-separation domain information." In this figure, the "liposome for obtaining phase-separation domain information" is abbreviated to "reference liposome," and this abbreviation will also be used in the following description.

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

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

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

[0056] The ethyl caproate concentration estimation method of the present invention comprises observing the reference liposomes 10a, 10b, ...under a fluorescent microscope to read the phase-separation domain formation patterns Da, Db, ..., i.e., whether or not a phase-separation domain is formed or the pattern that would occur if a phase-separation domain is formed; observing the cell-sized liposomes 10x, 10y, ...under a fluorescent microscope to read the phase-separation domain formation patterns Dx, Dy, ..., i.e., whether or not a phase-separation domain is formed or the pattern that would occur if a phase-separation domain is formed; and estimating the relative concentration of ethyl caproate contained in the object to be estimated by referring to ethyl caproate concentration-dependence information such as the phase-separation domain formation pattern Da in the reference liposome 10a, etc.

[0057] That is, in this ethyl caproate concentration estimation method, the phase-separated domain generation patterns Da, Db, ... in the reference liposome 10a, etc., have ethyl caproate concentration dependency, so this pattern information is compared, and the relative concentration in the estimation subject liposome 10x, etc., that is, the relative ranking of the concentration of ethyl caproate contained in the concentration estimation subject, and the concentration range is estimated. In FIG. 5, an estimation result in which the concentrations were highest in the order of a, x, y, and b is shown on the right side of the figure as an example. At the same time, the estimation result that the unknown concentrations x and y are both lower than the known concentration a and higher than b is also obtained.

[0058] 6 is a flow diagram showing the configuration of the ethyl caproate concentration measurement method of the present invention. As shown in the figure, the ethyl caproate concentration measurement method is composed of a relative concentration estimation process P10 in which the ethyl caproate concentrations of all samples N to be subjected to concentration estimation are estimated by the above-mentioned ethyl caproate relative concentration estimation method, and then a precision measurement process P20 in which the ethyl caproate concentrations of some samples n that have already been subjected to the ethyl caproate concentration estimation process are precisely measured by GC.

[0059] This ethyl caproate concentration measurement method is a method in which a simple ethyl caproate concentration judgment is performed in a relative concentration estimation process P10 for preliminary analysis to select samples for precision analysis by GC in a precision measurement process P20, in the form of a sort of screening, and after the relative concentration estimation process P10 has determined whether the ethyl caproate content in the samples is relatively high or low, the precision measurement process P20 is performed on a portion of the samples n. Therefore, a rapid, low-cost, and precise analysis system can be realized.

[0060] The above-described method for estimating the relative concentration of ethyl caproate and the method for measuring the concentration of ethyl caproate of the present invention can be used for all objects to be estimated or measured that are expected to contain ethyl caproate, and it goes without saying that the methods of the present invention can also be applied to sake containing ethyl caproate or its processed products. EXAMPLES

[0061] The following describes examples of the present invention, but the present invention is not limited to these examples. Note that the examples are provided 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 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.

[0062] [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) As a result, a liposome having an So / Ld domain structure as shown in A in FIG. 1-2 above is obtained.

[0063] 2. Fluorescence microscopy 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) In addition, it can also be observed with WIY (excitation wavelength 545-580 nm, fluorescence wavelength 610 nm).

[0064] [3. Experiments using sake] It has been reported that the EC concentration in sake with a pronounced ginjo aroma is about 7 mg / L ("Breeding of yeasts producing high esters and development of fruity sake that does not require extensive polishing", Takahashi Toshinari, Journal of the Brewing Society of Japan, Vol. 115, 2020, hereafter referred to as "Academic Literature 1"). In addition, in a previous analysis conducted by the inventors, values ​​of 1.6 mg / L were obtained for regular ginjo sake and 11 mg / L for ginjo sake fermented with yeasts producing high levels 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 amount of 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 hydration in the cell-sized liposome preparation process, each sample was added to the lipid film to prepare cell-sized liposomes. We then observed the phase-separated domain structure and attempted to estimate the ethyl caproate concentration.

[0065] The materials used to prepare each sample 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

[0066] The lipid film formation for the production of cell-sized liposomes was carried out as follows. The lipid was adjusted to a concentration of 2 mM as a chloroform solution, and a total of 40 μL of solution was added. Hydration was carried out after evaporating and drying the organic solvent. Below, we list the cases in which experiments were carried out using each sample, and describe the conditions. Case 1 (Sake 1) Case 2 (Sake 1 + EC) Case 3 (pure water)

[0067] Case 1 Sake 1 Based on the value in Non-Patent Document 1, the EC concentration in sake 1 of unknown concentration was assumed to be 7.2 mg / L. Since the amount of sample added when preparing cell-sized liposomes was 400 μL, the molar weight based on the assumption is 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)

[0068] Case 2 Sake 1+EC 10% of sake 1 was replaced with pure EC in advance, and 400μL was added during hydration. The concentration of the pure product is stated to be "at least 98% or more," but we assumed that 100% was EC, and since the specific gravity is 0.87, we assumed that the EC concentration of the pure product was 870g / L. The amount added was 40μL, which is 10% of the total amount of 400μL. Therefore, the molar weight in 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.)

[0069] Case 3 Pure water Pure water is used. Therefore, the molar weight of EC in the cell-sized liposome solution is 0 μM.

[0070] 4. Experimental Results 7 is a graph showing the phase-separated domain structural composition in each cell-sized liposome according to the examples (cases 1, 2, and 3). As a result of fluorescence microscopy, as shown in the figure, in the cell-sized liposome using pure water in case 3, the So / Ld domain was about 80%. According to Non-Patent Document 1, the EC concentration in pure water is naturally 0 mol%, so it was confirmed that the obtained estimation result was valid.

[0071] In the putative target liposomes from Case 1, Sake 1, the domains were difficult to observe, but approximately 39% of the So / Ld domains were still confirmed by fluorescence microscopy.

[0072] In the putative target liposomes made from sake 1 + EC in case 2, the So / Ld domains that could be confirmed by fluorescence microscopy accounted for approximately 31%.

[0073] In Non-Patent Document 1, the inventors reported a technique for estimating the concentration of cell-sized liposomes by adding sake during lipid film preparation 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.

[0074] In the present results, the order of highest ethyl caproate concentration was Case 2, Case 1, and Case 3. Also, the order of lowest ratio of So / Ld domain observed in this example was Case 2, Case 1, and Case 3, which were in order of relative concentrations. Also, since 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.

[0075] 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 easily estimate the concentration range of ethyl caproate.

[0076] 5. Effectiveness of Fluorescence Microscopy It should be noted that the implementation of the fluorescence microscopy according to the present invention is guaranteed without any problems. 8 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 34° C. or higher, and it has been confirmed that there is no problem at all with fluorescence microscopy observation performed at room temperature.

[0077] [6. Estimation of cost reduction effect] 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 equipment and reagents that can be used repeatedly after purchase, but the prices of items that need to be purchased in minimum units were 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 sample) 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)

[0078] 2) 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 RhodamineDHPE 50,900 yen Solvent: Chloroform 2,850 yen Total: 76,550 yen Measurement time Preparation: 8 hours (liposome preparation (film preparation 1H + vacuum drying 3H + hydration 4H)) Measurement: 1 hour (time required to observe 60 samples) Total: 9 hours

[0079] 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. In addition, 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 do not make much difference, so they are not included in this calculation). [Industrial Applicability]

[0080] According to the cell-sized liposome, the method for estimating ethyl caproate concentration, and the method for measuring ethyl caproate concentration of the present invention, the concentration of ethyl caproate can be analyzed at a significantly lower cost and in a shorter time than in the past. Therefore, the invention is highly industrially applicable, particularly in the fields of food component analysis, food quality control, and all related fields. [Explanation of symbols]

[0081] 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...cell-sized liposomes (reference liposomes) 10x, 10y...cell-sized liposomes (estimated target liposomes) 13, 23...Saturated phospholipids 25...Sterol lipids Da, Db, Dx, Dy...Phase separation domain generation pattern N: All samples for which concentration is estimated n: A portion of the sample that has been subjected to concentration estimation processing P10…Relative concentration estimation process P20…Precision measurement process

Claims

1. A lipid component system comprising one or more unsaturated phospholipids; and Samples for which ethyl caproate concentration is to be estimated or ethyl caproate A cell-sized liposome comprising: Different ethyl caproate concentrations resulted in different phase-separated domain formation patterns. This allows the cell-sized liposome to be used to estimate the concentration of ethyl caproate from the phase-separated domain generation 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, characterized in that the lipid component system contains one or more sterol lipids.

4. The cell-sized liposome according to claim 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, dipalmitoylphosphotidylcholine (DPPC).

5. The cell-sized liposome according to claim 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, dipalmitoylphosphotidylcholine (DPPC), and a sterol lipid, cholesterol.

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, 3, 4 or 5, wherein the molar concentration composition of the unsaturated phospholipid and the saturated phospholipid in the lipid component system is equal when used at room temperature.

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

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 a sterol lipid.

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, characterized in that 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 a relative concentration of ethyl caproate in a concentration estimation subject, comprising using a cell-sized liposome according to any one of claims 1, 2, 3, 4, and 5, which is configured using an ethyl caproate standard having a known concentration, as a liposome for acquiring phase-separation domain information, and using the liposome for acquiring phase-separation domain information, Fluorescence microscopy is performed on the cell-sized liposome as a target for estimating the concentration of ethyl caproate, the cell-sized liposome being the target for estimating the concentration of ethyl caproate, A phase-separation domain generation pattern, i.e., whether or not a phase-separation domain is generated, or a pattern when a phase-separation domain is generated, is read; With reference to the ethyl caproate concentration dependency information of the phase-separation domain generation pattern in the phase-separation domain information acquisition liposome, Estimate the relative concentration of ethyl caproate contained in the target substance. A method for estimating a relative concentration of ethyl caproate, comprising:

13. The method for estimating a relative concentration of ethyl caproate according to claim 12, wherein an object of concentration estimation is sake or a processed sake product.

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 as described in claim 12; and then precisely measuring the concentrations of ethyl caproate by gas chromatography for some of the samples that have already been subjected to the ethyl caproate concentration estimation process.

15. 15. The method for measuring the concentration of ethyl caproate according to claim 14, wherein the subject of concentration measurement is sake or a processed sake product.

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