JP7553034B2 - Analysis method for fat-soluble components - Google Patents

Description

The present invention relates to a method for analyzing fat-soluble components.

Technologies for analyzing the fat-soluble components of a target substance contained in a composition containing various components are important in industries that utilize such compositions.

Conventionally, a method for recovering a specific component from a composition using an organic solvent is known.For example, as a method for recovering a high amount of a component from a composition containing a hydrophilic component, Patent Document 1 describes a method for separating and recovering a water-soluble organic substance at a high concentration and with a high recovery rate, which comprises a process of adding at least one of hydroxides, oxides, carbonates, or hydrogen carbonates to water to be treated that contains water-soluble organic matter and inorganic acids to neutralize the water to be treated, and then adding at least one of salts that have a solubility in water of 30 w/v% or more and a solubility in the water-soluble organic matter to be recovered of 1 w/v% or less to separate the water-soluble organic matter from the water.
Furthermore, in order to extract and measure fat-soluble components from a composition, a method is usually used in which an organic solvent capable of dissolving the fat-soluble components and causing phase separation from water, such as chloroform, is added, and the fat-soluble components are extracted and separated into the chloroform phase and then measured.

JP 2006-175331 A

However, the method described in Patent Document 1 cannot be used for general analysis of fat-soluble components. In addition, the method using chloroform has problems in that the aqueous phase and the chloroform phase are not well separated and the peak of high performance liquid chromatography is not stable, resulting in poor analytical accuracy in quantitative analysis and the like.
Therefore, an object of the present invention is to provide a method for accurately analyzing a fat-soluble component from a composition containing the fat-soluble component by treating the fat-soluble component with a hydrophilic solvent and a hydrophobic solvent having a specific density ratio.

As a result of extensive research, the inventors have discovered that by preparing or adjusting a composition containing a predetermined amount of a hydrophilic solvent and the fat-soluble component to be measured, using an organic solvent having a specific solubility in water and a specific density as an extraction solvent for the fat-soluble component to be measured, and adjusting the density ratio of the hydrophilic solvent to the organic solvent, phase separation can be achieved extremely well and the fat-soluble component to be measured can be analyzed efficiently.
That is, the present invention includes the following aspects.

[Item 1] A method for analyzing a fat-soluble component to be measured in a sample A, comprising the following steps (i) to (iv):
(i) preparing or adjusting sample A containing a fat-soluble component to be measured and 50 w/v% or more of a hydrophilic solvent; (ii) adding medium a to sample A to obtain sample B so as to satisfy the following requirements (X), (Y), and (Z); Requirement (X): the density of medium a at 20 ° C. is less than 0.998 g/ml; (Y): medium a contains at least one organic solvent having an octanol/water partition coefficient of 0.50 to 4.0; (Z): the density ratio of the hydrophilic solvent to medium a at 20 ° C. (density of medium a/density of hydrophilic solvent) is 0.6000 to 0.8500; (iii) separating sample B into upper layer sample C1 and lower layer sample C2, which are analytical specimens; (iv) measuring the content of the fat-soluble component to be measured in sample C1; [Item 2] In step (i), sample A contains cells or tissues containing the fat-soluble component to be measured. The method according to item 1.
[Item 3] The method according to Item 2, wherein in step (i), 40 w/w% or more of the fat-soluble components to be measured in sample A are contained in cells or tissues.
[Item 4] The method according to item 2 or 3, wherein in step (i), the cells or tissues containing the lipid-soluble component to be measured contain acetic acid bacteria.
[Item 5] The method according to any one of Items 1 to 4, wherein in step (i), sample A contains soluble sugars.
[Item 6] The method according to Item 5, wherein in step (i), the content of soluble sugars in the hydrophilic solvent of sample A is 0.1 to 30 w/v %.
[Item 7] The method according to any one of Items 1 to 6, wherein in step (i), sample A contains ceramide as the fat-soluble component to be measured.
[Item 8] The method according to any one of Items 1 to 7, wherein in step (i), sample A contains an acetic acid bacteria product.
[Item 9] The method according to any one of items 1 to 8, wherein in step (i), sample A contains acetic acid.
[Item 10] The method according to Item 9, wherein in step (i), the content of acetic acid relative to the hydrophilic solvent in Sample A is 0.02 to 15 w/v %. [Item 11] The method according to any one of Items 1 to 10, wherein in step (i), the density of the hydrophilic solvent at 20°C is 1.003 g/ml or more.
[Item 12] The method according to any one of Items 1 to 11, wherein in step (i), the pH of the hydrophilic solvent is 4.5 or less.
[Item 13] The method according to any one of Items 1 to 12, wherein in step (i), the total protein content of sample A is 15 w/v% or less.
[Item 14] The method according to any one of Items 1 to 13, wherein in step (i), the turbidity of sample A at a wavelength of 660 nm is 1.100 or more.
[Item 15] The method according to any one of Items 1 to 14, wherein in step (i), sample A is a food or drink.
[Item 16] The method according to any one of items 1 to 15, wherein in step (ii), the medium a contains t-butyl methyl ether.
[Item 17] The method according to any one of items 1 to 16, wherein in step (ii), the medium a contains methanol.
[Item 18] The method according to any one of items 1 to 17, comprising the following step (ii') after step (ii).
(ii') The method according to any one of items 1 to 18, wherein in the step (iii) of mixing sample B for 10 to 1440 minutes [item 19], a precipitate is present in the lower layer sample C2.
[Item 20] The method according to any one of items 1 to 19, comprising the following step (iii') after step (iii).
(iii') The method according to any one of items 1 to 20, wherein the step (iv) of further concentrating the upper layer sample C1 [item 21] is a step of subjecting the sample C1 to high performance liquid chromatography.
[Item 22] The method according to any one of Items 1 to 21, wherein in the step (iv), at least three organic solvents are used in the mobile phase of the high performance liquid chromatography.
[Item 23]
A method for analyzing the change in the content of a fat-soluble component to be measured in sample A over time, comprising the following steps (i) to (iv): (i) preparing or adjusting sample A containing a fat-soluble component to be measured and 50 w/v% or more of a hydrophilic solvent; (ii) adding medium a to sample A to obtain sample B so as to satisfy the following requirements (X), (Y), and (Z); Requirement (X): the density of medium a at 20 ° C. is less than 0.998 g/ml; (Y): medium a contains at least one organic solvent having an octanol/water partition coefficient of 0.50 to 4.0; (Z): the density ratio of the hydrophilic solvent to medium a at 20 ° C. (density of medium a/density of hydrophilic solvent) is 0.6000 to 0.8500; (iii) separating sample B into upper layer sample C1 and lower layer sample C2, which are analytical specimens; (iv) measuring the content of the fat-soluble component to be measured in sample C1;

[Item 24] A method for producing an analytical sample containing a fat-soluble component to be measured, comprising the following steps (i) to (iii):
(i) preparing or adjusting sample A containing a fat-soluble component to be measured and 50 w/v% or more of a hydrophilic solvent; (ii) adding medium a to sample A to obtain sample B so as to satisfy the following requirements (X), (Y), and (Z); Requirement (X): the density of medium a at 20 ° C. is less than 0.998 g/ml; (Y): medium a contains at least one organic solvent having an octanol/water partition coefficient of 0.50 to 4.0; (Z): the density ratio of the hydrophilic solvent to medium a at 20 ° C. (density of medium a/density of hydrophilic solvent) is 0.6000 to 0.8500; (iii) separating sample B into phases and separating upper layer sample C1 and lower layer sample C2, which are analytical specimens; [Item 25] In step (i), sample A contains cells or tissues containing the fat-soluble component to be measured. The method according to item 24.
[Item 26] The method according to Item 25, wherein in step (i), 40 w/w% or more of the fat-soluble components to be measured in sample A are contained in cells or tissues.
[Item 27] The method according to Item 25 or 26, wherein in step (i), the cells or tissues containing the lipid-soluble component to be measured contain acetic acid bacteria.
[Item 28] The method according to any one of Items 24 to 27, wherein in step (i), sample A contains soluble sugars.
[Item 29] The method according to Item 28, wherein in step (i), the content of soluble sugars in the hydrophilic solvent of sample A is 0.1 to 30 w/v %.
[Item 30] The method according to any one of Items 24 to 29, wherein in step (i), sample A contains ceramide as the fat-soluble component to be measured.
[Item 31] The method according to any one of Items 24 to 30, wherein in step (i), sample A contains an acetic acid bacteria product.
[Item 32] The method according to any one of Items 24 to 31, wherein in step (i), sample A contains acetic acid.
[Item 33] The method according to Item 32, wherein in step (i), the content of acetic acid relative to the hydrophilic solvent in Sample A is 0.02 to 15 w/v%. [Item 34] The method according to any one of Items 24 to 33, wherein in step (i), the density of the hydrophilic solvent at 20°C is 1.003 g/ml or more.
[Item 35] The method according to any one of Items 24 to 34, wherein in step (i), the pH of the hydrophilic solvent is 4.5 or less.
[Item 36] The method according to any one of Items 24 to 35, wherein in step (i), the total protein content of sample A is 15 w/v% or less.
[Item 37] The method according to any one of Items 24 to 36, wherein in step (i), the turbidity of sample A at a wavelength of 660 nm is 1.100 or more.
[Item 38] The method according to any one of Items 24 to 37, wherein in step (i), sample A is a food or drink.
[Item 39] The method according to any one of items 24 to 38, wherein in step (ii), the medium a contains t-butyl methyl ether.
[Item 40] The method according to any one of Items 24 to 39, wherein in step (ii), the medium a contains methanol.
[Item 41] The method according to any one of Items 24 to 40, comprising the following step (ii') after step (ii).
(ii') The method according to any one of items 24 to 41, wherein in the step (iii) of mixing sample B for 10 to 1440 minutes [item 42], a precipitate is present in the lower layer sample C2.
[Item 43] The method according to any one of Items 24 to 42, comprising the following step (iii') after step (iii).
(iii') Further concentrating the upper layer sample C1

The present invention provides a method for accurately analyzing fat-soluble components from a composition containing the fat-soluble components using an operationally simple means.

1 shows the change over time in the gradient of the high performance liquid chromatography mobile phase of a comparative sample. 1 shows the change over time in the gradient of the high performance liquid chromatography mobile phase of a test sample. An example of good peak evaluation in high performance liquid chromatography is shown below. An example of poor peak evaluation in high performance liquid chromatography will be described.

In this specification, when multiple upper limits and/or multiple lower limits are indicated for the specification of a numerical range, even if not specifically stated, it is assumed that the specification of the numerical range combining at least the maximum value of the upper limit specification and the minimum value of the lower limit specification is directly stated, and furthermore, all numerical ranges obtained by combining any upper limit value among the upper limits and any lower limit value among the lower limits are included in one embodiment of the present invention. Furthermore, in this specification, a numerical range connected with "~" means a numerical range including the numbers before and after "~" as the lower limit and upper limit. When multiple lower limits and multiple upper limits are indicated separately, it is assumed that any lower limit value and upper limit value can be selected and connected with "~".

In the present invention, when "w/w%" is used, it indicates a percentage "converted to wet mass." "Converted to wet mass" indicates the content ratio of the target component in the sample, calculated using the wet mass of the sample, including water, as the denominator and the mass of the target component in the sample as the numerator. In addition, when "w/v%" is used in the present invention, it indicates the mass (g) of the target component in the sample per volume of the sample (100 ml).

One aspect of the present invention is a method for analyzing a fat-soluble component to be measured in a sample A, comprising the following steps (i) to (iv):
(i) A step of preparing or adjusting sample A containing a fat-soluble component to be measured and 50 w/v% or more of a hydrophilic solvent; (ii) A step of obtaining sample B by adding medium a to sample A so as to satisfy the following requirements (X), (Y), and (Z): Requirement (X): The density of medium a at 20°C is less than 0.998 g/ml; Requirement (Y): Medium a contains at least one organic solvent having an octanol/water partition coefficient of 0.50 to 4.0; Requirement (Z): The density ratio of the hydrophilic solvent and medium a at 20°C (density of medium a/density of hydrophilic solvent) is 0.6000 to 0.8500; (iii) A step of separating sample B into phases, and separating upper layer sample C1, which is an analytical specimen, and lower layer sample C2; (iv) A step of measuring the content of the fat-soluble component to be measured in sample C1.

<Step (i)>
Step (i) is a step of preparing or adjusting a sample A containing a fat-soluble component to be measured and 50 w/v % or more of a hydrophilic solvent.
Step (i) is described in detail below.

<Composition>
The term "composition" is not particularly limited to the commonly used meaning, but may be, for example, a combination of two or more components. Thus, for example, Sample A and Sample B described below are also compositions.

<Sample A>
Sample A is a composition containing a fat-soluble component and 50 w/v % or more of a hydrophilic solvent. Sample A of the present invention may be prepared artificially or may be a composition present in nature.

<Fat-soluble components>
The fat-soluble component refers to a component that has a hydrophobic group and is soluble in fats and oils. The fat-soluble component may be an amphipathic component that has both a hydrophobic group and a hydrophilic group, but it is useful in the present invention that the fat-soluble component has a higher solubility in fats and oils than in water. The fat-soluble component to be measured is literally a fat-soluble component contained in sample A and is a fat-soluble component to be measured. Examples of the fat-soluble component of the present invention include fat-soluble components contained in animal cells, plant cells, microorganisms, etc., and specific examples thereof include fatty acyls, glycerolipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, polyketides, etc., and more specific examples thereof include fatty acids, neutral fats, phospholipids, glycolipids, sterols, sphingosine, diglycerides, interesterified oils, etc., but are not particularly limited thereto. More specific examples of the fat-soluble component of the present invention include saturated fatty acids, unsaturated fatty acids, terpenes, fat-soluble vitamins, sphingolipids, and the like. More specific examples include carotenoids, steroids, vitamin A, vitamin D, vitamin E, vitamin K, EPA (eicosapentaenoic acid), DPA (docosapentaenoic acid), DHA (docosahexaenoic acid), α-linolenic acid, linoleic acid, arachidonic acid, oleic acid, lauric acid, myristic acid, palmitic acid, stearic acid, limonene, α-terpinene, β-terpinene, γ-terpinene, β-terpinene, β-terpinene, α-terpinene, β ... -terpinene, δ-terpinene, p-cymene, murolol, cadalene, tetrahydroxybacteriohopane (C35-pentacyclic terpene alcohol), hopane-22-ol, hop-22(29)-ene, ornityl tau phospholipid 3-(palmitoyl)-hydroxypalmitoyl-ornityl-taurine, ornithine lipid 3-(palmitoyl)-hydroxypalmitoyl-ornithine, lysoornithine lipid 3-hydroxypalmitoyl-ornithine, cis-vaccenic acid, lecithin, ceramide, and the like. These ingredients include, for example, sesame oil, olive oil, avocado oil, rapeseed oil, perilla oil, camellia oil, rice oil, peanut oil, palm oil, coconut oil, corn oil, rapeseed oil, egg yolk oil, soybean oil, lemon essential oil, lard, chicken oil, beef tallow, fish oil (liver oil, herring oil, sardine oil, mackerel oil, salmon oil, etc.), butter, high oleic rapeseed oil, palm stearin, palm olein, palm kernel oil, palm fractionated oil (PMF), cottonseed oil, sunflower oil, high oleic sunflower oil, safflower oil, and linseed oil. The oils and fats may be those contained in naturally derived oils and fats such as grape seed oil, almond oil, salad oil, canola oil, milk fat, ghee, cocoa butter, acai oil, amaranth oil, apple seed oil, mustard oil, nutmeg butter, okra seed oil, papaya seed oil, apricot kernel oil, pequy oil, shiso oil, prune kernel oil, quinoa oil, rambutan oil, rice bran oil, thistle oil, tomato seed oil, and wheat germ oil, or may be those refined from these or chemically synthesized.

Since the present invention relates to a method for analyzing fat-soluble components, it is preferable that the content of the fat-soluble component to be measured in sample A is a predetermined amount. The content of the fat-soluble component to be measured in sample A of the present invention may be preferably 0.0000001 to 20 w/v%, more preferably 0.0000005 to 15.0 w/v%, even more preferably 0.000001 to 6.0 w/v%, even more preferably 0.00001 to 5.0 w/v%, particularly preferably 0.00005 to 3.0 w/v%, particularly preferably 0.0001 to 0.5 w/v%, 0.0002 to 0.005 w/v%, or 0.0002 to 0.002 w/v%. Furthermore, the upper limit is not particularly limited, and can be, for example, 19.0 w/v% or less, 18.0 w/v% or less, 16.0 w/v% or less, 15.0 w/v% or less, 14.0 w/v% or less, 13.0 w/v% or less, 11.0 w/v% or less, 9.0 w/v% or less, 7.0 w/v% or less, 5.0 w/v% or less, 4.5 w/v% or less, 4.0 w/v% or less, 3.5 w/v% or less, 3.0 w/v% or less, 2.5 w/v% or less, 2.0 w/v% or less, 1.5 w/v% or less, 1.0 w/v% or less, 0.7 w/v% or less, 0.5 w/v% or less, or 0.3 w/v% or less. The lower limit is not particularly limited, but can be, for example, 0.0000001 w/v% or more, 0.0000005 w/v% or more, 0.000001 w/v% or more, 0.000005 w/v% or more, 0.00001 w/v% or more, 0.00005 w/v% or more, 0.0001 w/v% or more, 0.00005 w/v% or more, 0.0001 w/v% or more, 0.0002 w/v% or more, 0.0003 w/v% or more, 0.0004 w/v% or more, 0.00045 w/v% or more, or 0.0005 w/v% or more.

In view of the fact that the conventional technology acts as an impediment to the separation of the hydrophilic solvent and the organic solvent, which makes the problem of the present invention more serious, it is preferable that sample A contains sphingolipids as the fat-soluble component to be measured, more preferably ceramide, even more preferably human ceramide, and even more preferably acetic acid bacteria ceramide. Although the principle behind this is unclear, the present invention is useful because conventional technology was particularly difficult to apply to human ceramides such as acetic acid bacteria ceramide that do not have a glucosyl group.

<Ceramide>
Ceramide is a general term for a compound in which a fatty acid is amide-bonded to the amino group of sphingosine, which is a long-chain base, and specifically includes natural ceramide and synthetic ceramide. Natural ceramides are further divided into plant-type ceramide and human-type ceramide (without glucosyl group) based on the difference in their structure. Specific examples of natural ceramides include sphingosine, dihydrosphingosine, N-acylsphinganine, etc. Specific examples of synthetic ceramides include (N-hexadecyloxyhydroxypropyl)-N-hydroxyhexadecanamide, (N-hexadecyloxyhydroxypropyl)-N-hydroxydecanamide, etc. Acetobacter ceramide is a natural ceramide produced by acetic bacteria, and is a type of human ceramide. Acetobacter ceramide has a structure in which sphinganine, a precursor of sphingosine in the sphingoid base portion of human ceramide, is amide-bonded to a fatty acid. Examples of acetic acid bacteria ceramides include N-acylsphinganines (more specifically, N-2'-hydroxypalmitoyl-sphinganine, N-palmitoyl-sphinganine, O-1-glucuronyl-N-2'-hydroxypalmitoyl-sphinganine, N-cis-vaccenoyl-sphinganine, O-1-glucuronyl-N-palmitoyl-sphinganine, etc.).

When sample A of the present invention contains sphingolipids, more preferably ceramides, and even more preferably acetic acid bacteria ceramides, as the fat-soluble components to be measured, preferably 40 w/w% or more, more preferably 50 w/w% or more, even more preferably 60 w/w% or more, even more preferably 70 w/w% or more, particularly preferably 80 w/w% or more, particularly preferably 90 w/w% or more, 95 w/w% or more, or the entire amount of the fat-soluble components to be measured in sample A may be sphingolipids, more preferably ceramides, and even more preferably acetic acid bacteria ceramides. Also, when sample A of the present invention contains sphingolipids, more preferably ceramides, and even more preferably acetic acid bacteria ceramides as the fat-soluble components to be measured, the content preferably satisfies the regulations regarding the range and upper and lower limits of the content of the fat-soluble components to be measured.

<Cells or tissues containing lipid-soluble components to be measured>
The sample A of the present invention preferably contains cells or tissues containing the fat-soluble component to be measured. In this embodiment, the present invention is useful because, in the prior art, the cells or tissues act like an emulsifier when separating the organic solvent from the hydrophilic solvent, and the organic solvent and the hydrophilic solvent cannot be sufficiently separated from each other.

The term "cell" is not particularly limited to the commonly used meaning, and may be derived from any of animal cells, plant cells, bacteria, eukaryotes, and archaea, and there is no limitation as to whether the cell is alive or dead. Examples of such cells include animal cells, plant cells, Escherichia coli, acetic acid bacteria, yeast, Bacillus subtilis natto, and lactic acid bacteria. Furthermore, "tissue" refers to an aggregate of such cells, a part of such cells, or a mixture thereof.

In the present invention, it is particularly preferable that the fat-soluble component to be measured in sample A is contained in a predetermined amount in the cells or tissues, from the viewpoint that the conventional technology is difficult to apply and the problem is of great importance. Although the principle is unclear, in this embodiment, in the conventional technology, the cells or tissues act like an emulsifier when separating the organic solvent from the hydrophilic solvent, and the fat-soluble component to be measured remains in the cells or tissues. In the present invention, the density ratio of the hydrophilic solvent and the organic solvent is adjusted to make it easier to separate the hydrophilic solvent and the organic solvent by gravity, osmotic pressure, etc., and it is possible that the extraction of the fat-soluble component to be measured into the organic solvent is promoted. In other words, preferred embodiments of the present invention include an embodiment in which sample A contains cells or tissues containing the fat-soluble component to be measured, more preferably an embodiment in which sample A contains cells or tissues of bacteria, fungi, and archaea containing the fat-soluble component to be measured, even more preferably an embodiment in which sample A contains acetic acid bacteria cells, and particularly preferably an embodiment in which sample A contains acetic acid bacteria cells containing acetic acid bacteria ceramide. Furthermore, of the fat-soluble components to be measured contained in sample A, preferably 40 w/w% or more, more preferably 50 w/w% or more, even more preferably 60 w/w% or more, even more preferably 70 w/w% or more, particularly preferably 80 w/w% or more, especially preferably 85 w/w% or more, 88 w/w% or more, 90 w/w% or more, 91 w/w% or more, 92 w/w% or more, 93 w/w% or more, 94 w/w% or more, 95 w/w% or more, 96 w/w% or more, 98 w/w% or more, 99 w/w% or more, or the entire amount may be derived from the cells or tissues described above. In addition, in an embodiment in which sample A contains cells or tissues containing the fat-soluble component to be measured, more preferably an embodiment in which sample A contains cells or tissues of bacteria, fungi, or archaea containing the fat-soluble component to be measured, even more preferably an embodiment in which sample A contains acetic acid bacteria cells, and particularly preferably an embodiment in which sample A contains acetic acid bacteria cells containing acetic acid bacteria ceramide, the content of the fat-soluble component to be measured in sample A may satisfy the regulations regarding the range and upper and lower limits of the content of the fat-soluble component to be measured.

In the present invention, the content of cells or tissues containing the fat-soluble component to be measured in sample A is preferably a predetermined amount, since conventional techniques are difficult to apply and the problem to be solved by the present invention becomes large. The content may be, for example, 10 to 300 mg/100 ml, preferably 15 to 280 mg/100 ml, more preferably 20 to 260 mg/100 ml, even more preferably 22 to 240 mg/100 ml, even more preferably 25 to 220 mg/100 ml, particularly preferably 28 to 210 mg/100 ml, and particularly preferably 30 to 200 mg/100 ml, in terms of mass (dry mass) relative to the volume of sample A. In addition, the upper limit is not particularly limited, and may be, for example, 290 mg/100 ml or less, 270 mg/100 ml or less, 250 mg/100 ml or less, 230 mg/100 ml or less, 210 mg/100 ml or less, 190 mg/100 ml or less, 170 mg/100 ml or less, 150 mg/100 ml or less, 130 mg/100 ml or less, 110 mg/100 ml or less, 100 mg/100 ml or less, 90 mg/100 ml or less, 80 mg/100 ml or less, or 70 mg/100 ml or less. The lower limit is not particularly limited, but may be, for example, 0.1 mg/100 ml or more, 1.0 mg/100 ml or more, 3.0 mg/100 ml or more, 4.0 mg/100 ml or more, 5.0 mg/100 ml or more, 10 mg/100 ml or more, 15 mg/100 ml or more, 18 mg/100 ml or more, 21 mg/100 ml or more, 25 mg/100 ml or more, 32 mg/100 ml or more, 40 mg/100 ml or more, or 50 mg/100 ml or more. In addition, the content (dry mass) of cells or tissues in sample A can be calculated as the mass excluding ash from the dry weight of the precipitate obtained by centrifuging sample A (3000 G for 10 minutes) in an embodiment in which sample A is prepared by adding cells or tissues containing the fat-soluble component to be measured to a composition not containing cells or tissues containing the fat-soluble component to be measured.

In view of the difficulty of applying conventional techniques and the importance of the problem, sample A of the present invention particularly preferably contains acetic acid bacteria cells (acetic acid bacteria cells refer to acetic acid bacteria cells, aggregates of acetic acid bacteria cells, parts of acetic acid bacteria cells, or mixtures thereof, which are one aspect of the cells or tissues described above). Although the principle is unclear, acetic acid bacteria have properties such as high acid resistance and alcohol resistance, so there is a possibility that their membrane structure has some characteristics that inhibit the separation of fat-soluble components from the hydrophilic solvent, and this may be eliminated by adjusting the density ratio of the hydrophilic solvent to the organic solvent. When sample A of the present invention contains acetic acid bacteria cells, the cells or tissues contained in sample A may be preferably 40 w/w% or more, more preferably 50 w/w% or more, even more preferably 60 w/w% or more, even more preferably 70 w/w% or more, particularly preferably 80 w/w% or more, particularly preferably 90 w/w% or more, 95 w/w% or more, or the entire amount may be acetic acid bacteria cells. In addition, the content may satisfy the regulations regarding the range and upper and lower limits of the content of the cells or tissues described above.

Here, in the present invention, unless otherwise specified, the "dry mass" refers to the mass of the remainder obtained by subtracting the moisture content calculated from the "moisture content" from the mass of the entire composition. The moisture content can be measured as the dry moisture content by heating to 90°C using the vacuum heating drying method in accordance with the 2015 edition (7th edition) of the Standard Tables of Food Composition in Japan. Specifically, an appropriate amount of sample is collected in a weighing container (W0) that has been brought to a constant weight in advance, weighed (W1), and placed in a vacuum electric constant temperature dryer adjusted to a predetermined temperature (more specifically, 90°C) at normal pressure with the lid of the weighing container removed or with the mouth open, the door is closed, the vacuum pump is operated, and the sample is dried at a predetermined reduced pressure for a certain period of time, the vacuum pump is stopped, dry air is pumped in to return the sample to normal pressure, the weighing container is removed, the lid is put on, and the sample is allowed to cool in a desiccator, and weighed (W2). This process is repeated, and the moisture content (mass%) is calculated using the following formula.

[Equation 1]
Moisture (mass%) = (W1-W2)/(W2-W0) x 100

"Ash" refers to non-volatile inorganic matter contained in the composition, and the content in a sample can be analyzed using the dry ashing method adopted for the analysis of inorganic matter in the Analysis Manual for the 2015 Edition (7th revision) of the Standard Tables of Food Composition in Japan.

<Acetic acid bacteria>
The acetic acid bacteria contained in sample A of the present invention are not particularly limited as long as they are bacteria belonging to the Acetobacteraceae family and capable of producing acetic acid. Examples of acetic acid bacteria include bacteria belonging to the genus Acetobacter, bacteria belonging to the genus Gluconobacter, bacteria belonging to the genus Gluconacetobacter, bacteria belonging to the genus Komagataeibacter, bacteria belonging to the genus Asaia, and bacteria belonging to the genus Acidmonas. , bacteria belonging to the genus Acidiphyllum (Acidiphilium), bacteria belonging to the genus Acidisphaera (Acidisphaera), bacteria belonging to the genus Acidocella (Acidocella), bacteria belonging to the genus Acidomonas (Acidomonas), bacteria belonging to the genus Asaia (Asaia), bacteria belonging to the genus Belnapia (Belnapia), bacteria belonging to the genus Claurococcus (Craurococcus), Kozakia Bacteria belonging to the genus Kozakia, bacteria belonging to the genus Leahibacter, bacteria belonging to the genus Muricoccus, bacteria belonging to the genus Neoasiaia, bacteria belonging to the genus Oleomonas, bacteria belonging to the genus Paracraurococcus, bacteria belonging to the genus Rhodopila, bacteria belonging to the genus Roseococcus Examples of bacteria that can cause malaria include bacteria belonging to the genus Roseococcus, bacteria belonging to the genus Rubritepida, bacteria belonging to the genus Saccharibacter, bacteria belonging to the genus Stella, bacteria belonging to the genus Swaminathania, bacteria belonging to the genus Teichococcus, and bacteria belonging to the genus Zavarzinia.

Acetobacter genus acetic acid bacteria include Acetobacter polyoxogenes, Acetobacter tropicalis, Acetobacter indonesia, Acetobacter syzygii, Acetobacter cibinongensis, Acetobacter orientalis, Acetobacter pasteurianus, Acetobacter orleanensis, and Acetobacter polyoxogenes. orleanensis, Acetobacter lovaniensis, Acetobacter aceti, Acetobacter pomorum, Acetobacter malorum, etc. are examples.

Examples of acetic acid bacteria of the genus Gluconobacter include Gluconobacter frateurii and Gluconobacter cerinus.

The acetic acid bacteria of the genus Gluconacetobacter include Gluconacetobacter swingsii, Gluconacetobacter xylinus, Gluconacetobacter diazotrophicus, Gluconacetobacter intermedius, Gluconacetobacter sacchari, and Gluconacetobacter maltaceti. Examples include Gluconacetobacter maltaceti, Gluconacetobacter kombuchae, and Gluconacetobacter liquefaciens.

Examples of acetic acid bacteria of the genus Komagataebacter include Komagataebacter hansenii, Komagataebacter xylinus, Komagataebacter europaeus, and Komagataebacter oboediens.

Examples of acetic acid bacteria in the Asaia genus include Asaia bogorensis and Asaia siamensis.

An example of an acetic acid bacterium of the genus Acidomonas is Acidomonas methanolicus.

Preferred examples of acetic acid bacteria include bacteria belonging to the genus Acetobacter, bacteria belonging to the genus Gluconobacter, bacteria belonging to the genus Gluconacetobacter, bacteria belonging to the genus Asaia, bacteria belonging to the genus Acidomonas, and the like. From the viewpoint of bacterial body odor, flavor and/or texture adjusting action, etc., more preferred examples of acetic acid bacteria include bacteria belonging to the genus Acetobacter, Gluconacetobacter, and Komagataebacter, and even more preferred examples include Acetobacter malorum, Komagataebacter xylinus NBRC 15237, and Komagataebacter europaeus NBRC 3261, and particularly preferred examples include Acetobacter malorum.

As an example of Acetobacter malorum, the Acetobacter malorum NCI1683 strain (FERM BP-10595) is preferably used. The Acetobacter malorum NCI1683 strain (FERM BP-10595) is an acetic acid bacterium isolated from smetana, a Russian fermented food. The ubiquinone type is Q9, and the 16S rRNA sequence is completely identical to that of the Acetobacter malorum strain (see, for example, International Journal of Systematic and Evolutionary Microbiology, Vol. 52, p. 1551-1558, 2002). It was deposited at the National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) on April 7, 2006 under the accession number FERM BP-10595.

<Acetic acid bacteria products>
The acetic acid bacteria product refers to all components produced by acetic acid bacteria. Examples of the acetic acid bacteria product of the present invention include acetic acid, cellulose, polysaccharides, acetic acid bacteria-type ceramide, isoamyl alcohol, isobutanol, butanol, propanol, n-amyl alcohol, isovaleric acid, isobutyric acid, butyric acid, propionic acid, and valeric acid.

<Hydrophilic Solvent>
The "hydrophilic solvent" refers to a solvent constituting the liquid portion of sample A, and is preferably a substantially aqueous solution. Components contained other than water include water-soluble proteins, water-soluble amino acids, water-soluble dietary fibers (pectin, glucomannan, etc.), soluble sugars, water-soluble vitamins, water-soluble alcohols, polyphenols, minerals, mineral salts, pH adjusters, etc.

The water content in the hydrophilic solvent is usually 50 w/w% or more, preferably 55 w/w% or more, more preferably 60 w/w% or more, even more preferably 65 w/w% or more, even more preferably 70 w/w% or more, particularly preferably 80 w/w% or more, and particularly preferably 90 w/w% or more. The upper limit is not particularly limited, but may be, for example, 99.99 w/w% or less, 99.9 w/w% or less, 99.0 w/w% or less, 98.0 w/w% or less, 97.0 w/w% or less, 96.0 w/w% or less, 95.0 w/w% or less, 94.0 w/w% or less, 93.0 w/w% or less, 92.0 w/w% or less, 91.0 w/w% or less, or 90 w/w% or less. The lower limit may be, for example, 51 w/w% or more, 54 w/w% or more, 59 w/w% or more, 65 w/w% or more, 70 w/w% or more, 73 w/w% or more, 75 w/w% or more, 78 w/w% or more, 80 w/w% or more, 83 w/w% or more, 85 w/w% or more, or 88 w/w% or more.

The hydrophilic solvent content in sample A is preferably a predetermined value or more from the viewpoint of the importance of the problem of the present invention. The hydrophilic solvent content in sample A may be usually 50 w/v% or more, preferably 55 w/v% or more, more preferably 60 w/v% or more, even more preferably 65 w/v% or more, even more preferably 70 w/v% or more, particularly preferably 80 w/v% or more, and particularly preferably 90 w/v% or more. In addition, the upper limit is not particularly limited, but may be, for example, 99.99 w/v% or less, 99.9 w/v% or less, 99.0 w/v% or less, 98.0 w/v% or less, 97.0 w/v% or less, 96.0 w/v% or less, 95.0 w/v% or less, 94.0 w/v% or less, 93.0 w/v% or less, 92.0 w/v% or less, 91.0 w/v% or less, or 90 w/v% or less. The lower limit may be, for example, 51 w/v% or more, 54 w/v% or more, 59 w/v% or more, 65 w/v% or more, 70 w/v% or more, 73 w/v% or more, 75 w/v% or more, 78 w/v% or more, 80 w/v% or more, 83 w/v% or more, 85 w/v% or more, or 88 w/v% or more. The content of the hydrophilic solvent in sample A of the present invention may be preferably 50 to 99.999 w/v%, more preferably 60 to 99.999 w/v%, even more preferably 70 to 99.999 w/v%, particularly preferably 80 to 99.999 w/v%, 83 to 99.999 w/v%, 85 to 99.999 w/v%, 88 to 99.999 w/v%, or 90 to 99.999 w/v%. By adjusting the content of the hydrophilic solvent within the above range, components other than the fat-soluble components to be measured can be efficiently separated into the lower layer sample C2 described below, resulting in improved analytical accuracy of the fat-soluble components to be measured.

<Soluble sugars>
It is preferable that sample A of the present invention contains "soluble sugars" in the hydrophilic solvent. By containing soluble sugars in sample A of the present invention, it becomes easy to set the density of the hydrophilic solvent described later within a predetermined range. In addition, particularly in the case where sample A of the present invention contains the cells or tissues, in the prior art, the presence of soluble sugars and the cells or tissues acts as an inhibitor on the separation of the organic solvent phase and the aqueous phase. Although the principle is unclear, it is presumed that, since soluble sugars generally have high water retention, the adsorption of soluble sugar molecules to the cells or tissues inhibits the separation of the aqueous phase and the organic solvent phase. The present invention can solve these problems by adjusting the density of the hydrophilic solvent and the medium a containing an organic solvent, and therefore, particularly in the case where sample A contains the cells or tissues and contains soluble sugars, the effects of the invention can be enhanced while solving the problems of the prior art. Thus, examples of preferred aspects of the present invention include, for example, an aspect in which sample A contains cells or tissues containing the fat-soluble component to be measured and also contains soluble sugars, an aspect in which sample A contains cells or tissues of bacteria, fungi or archaea containing the fat-soluble component to be measured and also contains soluble sugars, an aspect in which sample A contains acetic acid bacteria cells containing the fat-soluble component to be measured and also contains soluble sugars, and an aspect in which sample A contains acetic acid bacteria cells which contain ceramide as the fat-soluble component to be measured and also contains soluble sugars.

In the present invention, "soluble sugars" refers to sugars that are soluble in water, and is a general term for monosaccharides and oligosaccharides (sugars in which 2 to 10 monosaccharides are bonded). Therefore, it does not include starch, which has far more sugars bonded to it.

The soluble sugar content in sample A of the present invention is preferably a predetermined content, from the viewpoint that the conventional technology is difficult to apply, the problem to be solved is important, and the effect of the present invention is great. The content can be, for example, in the range of 0.01 to 25 w/v% as a concentration in a hydrophilic solvent, and may be preferably 0.02 to 24 w/v%, more preferably 0.05 to 23 w/v%, even more preferably 0.08 to 22 w/v%, even more preferably 0.1 to 21.5 w/v%, particularly preferably 0.15 to 20.5 w/v%, and particularly preferably 0.20 to 16.0 w/v%. The upper limit is preferably 25 w/v% or less, but may be 20 w/v% or less, 18 w/v% or less, 17 w/v% or less, 15 w/v% or less, 14 w/v% or less, 13 w/v% or less, 12 w/v% or less, 11 w/v% or less, 10 w/v% or less, 9 w/v% or less, 8 w/v% or less, 7 w/v% or less, or 6 w/v% or less. On the other hand, the lower limit is not limited, but may be 0.01 w/v% or more, 0.05 w/v% or more, 0.1 w/v% or more, 0.2 w/v% or more, 0.3 w/v% or more, 0.5 w/v% or more, 0.8 w/v% or more, 1.0 w/v% or more, 1.5 w/v% or more, or 2.0 w/v% or more.

The soluble sugar content in Sample A of the present invention is measured using high performance liquid chromatography in accordance with the method for measuring "available carbohydrates (glucose, fructose, galactose, sucrose, maltose, lactose and trehalose)" in the Standard Tables of Food Composition in Japan, 2015 Edition (7th Edition), and each measured value is compared with the content of a standard sample of monosaccharides or oligosaccharides (2-10 sugars) of known concentration, and the obtained values are calculated by summing up.

As described above, it is preferable that sample A of the present invention contains cells or tissues containing fat-soluble components and contains soluble sugars, but in this embodiment, for the same reasons as above, it is difficult to apply conventional technology, the importance of the problem to be solved increases, and the effect of the present invention is great, so it is preferable that the content ratio is within a predetermined range. Specifically, it is more preferable that the value obtained by dividing the value of the content (mg/100 ml) of cells or tissues in sample A by the value of the content (g) of soluble sugars per 100 ml of hydrophilic solvent in sample A (referred to as value D) is within a predetermined range. The range of value D in sample A of the present invention is preferably 1 to 1000, more preferably 2 to 700, even more preferably 3 to 500, even more preferably 4 to 400, particularly preferably 6 to 300, and particularly preferably 8 to 250. That is, examples of preferred aspects of the present invention include an aspect in which sample A contains cells or tissues containing a fat-soluble component, contains soluble sugars, and value D satisfies the above range, an aspect in which sample A contains cells or tissues of bacteria, fungi, or archaea containing the fat-soluble component to be measured, contains soluble sugars, and value D satisfies the above range, an aspect in which sample A contains acetic acid bacteria cells, contains soluble sugars, and value D satisfies the above range, and an aspect in which sample A contains acetic acid bacteria cells containing ceramide, contains soluble sugars, and value D satisfies the above range.

<Density of hydrophilic solvent>
Since the present invention includes a step of setting the density of the hydrophilic solvent in sample A and the density of the medium a described later within a predetermined range, it is preferable to set the density of the hydrophilic solvent within a predetermined range. Although the principle is unclear, it is considered that the density of the hydrophilic solvent within a predetermined range facilitates phase separation between the organic solvent and the hydrophilic solvent, and facilitates the migration of the fat-soluble component to be measured from sample A to the organic solvent. For example, the density may be preferably 1.003 to 1.250 g/ml, more preferably 1.0035 to 1.230 g/ml, even more preferably 1.004 to 1.200 g/ml, even more preferably 1.005 to 1.180 g/ml, particularly preferably 1.005 to 1.155 g/ml, and particularly preferably 1.005 to 1.145 g/ml at 20°C. In addition, the upper limit is not particularly limited, and may be, for example, 1.230 g/ml or less, 1.200 g/ml or less, 1.190 g/ml or less, 1.160 g/ml or less, 1.150 g/ml or less, 1.140 g/ml or less, 1.130 g/ml or less, 1,110 g/ml or less, 1.100 g/ml or less, 1.090 g/ml or less, 1.070 g/ml or less, 1.060 g/ml or less, 1.040 g/ml or less, 1.030 g/ml or less, or 1.010 g/ml or less at 20°C. The lower limit is not particularly limited, but may be, for example, 1.001 g/ml or more, 1.0015 g/ml or more, 1.002 g/ml or more, 1.0025 g/ml or more, 1.003 g/ml or more, 1.0035 g/ml or more, 1.004 g/ml or more, 1.0045 g/ml or more, 1.005 g/ml or more, 1.0055 g/ml or more, 1.006 g/ml or more, 1.0065 g/ml or more, 1.007 g/ml or more, 1.0075 g/ml or more, 1.0 08 g/ml or more, 1.0085 g/ml or more, 1.009 g/ml or more, 1.0095 g/ml or more, 1.010 g/ml or more, 1.0150 g/ml or more, 1.020 g/ml or more, 1.025 g/ml or more, 1.030 g/ml or more, 1.035 g/ml or more, 1.040 g/ml or more, 1.045 g/ml or more, 1.050 g/ml or more, 1.055 g/ml or more, 1.060 g/ml or more, or 1.070 g/ml or more.

<Step (ii)>
Step (ii) is a step of obtaining sample B by adding medium a to sample A so as to satisfy the following requirements (X), (Y), and (Z).
Step (ii) is described in detail below.

<Sample B>
"Sample B" is a composition obtained by adding medium a to sample A. In step (ii), sample B is obtained so as to satisfy the following requirements (X), (Y), and (Z).
Requirement (X): The density of medium a at 20°C is less than 0.998 g/ml. Requirement (Y): Medium a contains at least one organic solvent with an octanol/water partition coefficient of 0.50 to 4.0. Requirement (Z): The density ratio of the hydrophilic solvent to medium a at 20°C (density of medium a/density of hydrophilic solvent) is 0.6000 to 0.85000.

<Medium A>
"Medium a" refers to an organic solvent or a liquid composition containing at least one organic solvent. The amount of medium a added to sample A may be a predetermined amount, for example, 0.1 to 100 ml, 0.5 to 70 ml, 1.0 to 50 ml, 1.5 to 30 ml, or 2.0 to 10 ml per 1 ml of sample A. The upper limit is not particularly limited, but may be, for example, 100 ml or less, 90 ml or less, 80 ml or less, 70 ml or less, 60 ml or less, 50 ml or less, 40 ml or less, 30 ml or less, 20 ml or less, 10 ml or less, or 5 ml or less per 1 ml of sample A. The lower limit is not particularly limited, but may be, for example, 0.1 ml or more, 0.3 ml or more, 0.7 ml or more, 1.0 ml or more, 1.5 ml or more, or 2.0 ml or more per 1 ml of sample A.

<Organic solvent>
The term "organic solvent" refers to a general term for an organic compound that dissolves other substances. The organic solvent contained in the medium a of the present invention is not particularly limited as long as it satisfies the requirements X and Y, and examples thereof include alcohols, aliphatic carboxylates, ketones, ethers, and the like. Examples of the alcohols include methanol, ethanol, propanol, isopropanol, butanol, and the like. Examples of the aliphatic carboxylates include branched or straight-chain saturated fatty acids such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and caproic acid, as well as alcohol esters of various unsaturated fatty acids, such as the esters of the above alcohols. Examples of the ketones include acetone, ethyl methyl ketone, methyl propyl ketone, isopropyl methyl ketone, butyl methyl ketone, and isobutyl ketone. Examples of the ethers include dimethyl ether, ethyl methyl ether, diethyl ether, diphenyl ether, ethylene oxide, tetrahydrofuran (THF), furan, 1,4-dioxane, anisole, benzofuran, dibenzofuran, crown ether, and t-butyl methyl ether. Other examples of the solvent include dichloromethane, chloroform, ethyl acetate, toluene, hexane, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). Medium a of the present invention preferably contains two or more organic solvents. Medium a of the present invention preferably contains t-butyl methyl ether. Furthermore, medium a of the present invention particularly preferably contains t-butyl methyl ether and one or more organic solvents, and particularly preferably contains at least t-butyl methyl ether and methanol.

<t-Butyl methyl ether>
The medium a of the present invention preferably contains t-butyl methyl ether. t-Butyl methyl ether (also referred to as methyl t-butyl ether, methyl tert-butyl ether, MTBE, or tBME) is an organic solvent represented by "CAS: 1634-04-4". The content ratio of t-butyl methyl ether in the medium a of the present invention (volume of t-butyl methyl ether/volume of medium a) is preferably 0.100 to 0.970, more preferably 0.200 to 0.930, even more preferably 0.300 to 0.900, even more preferably 0.400 to 0.870, particularly preferably 0.450 to 0.850, and particularly preferably 0.500 to 0.800. In addition, the upper limit value is not particularly limited, but is, for example, 0.980 or less, 0.960 or less, 0.940 or less, 0.920 or less, 0.900 or less, 0.880 or less, 0.860 or less, 0.840 or less, 0.820 or less, 0.800 or less, 0.780 or less, 0.760 or less, 0.740 or less, 0.720 or less, 0.700 or less, 0.680 or less, 0.660 or less, 0.640 or less, 0.620 or less, and 0.600 or less. Furthermore, the lower limit is not particularly limited, but is, for example, 0.100 or more, 0.140 or more, 0.180 or more, 0.220 or more, 0.260 or more, 0.280 or more, 0.320 or more, 0.360 or more, 0.400 or more, 0.440 or more, 0.480 or more, or 0.520 or more.

<Methanol>
The medium a of the present invention preferably contains methanol (CAS: 67-56-1). The content ratio of methanol in the medium a of the present invention (volume of methanol/volume of medium a) is preferably 0.060 to 0.900, more preferably 0.090 to 0.800, even more preferably 0.120 to 0.700, even more preferably 0.150 to 0.600, particularly preferably 0.180 to 0.500, and particularly preferably 0.210 to 0.400. The upper limit is not particularly limited, but may be, for example, 0.950 or less, 0.0850 or less, 0.750 or less, 0.650 or less, 0.550 or less, 0.450 or less, 0.350 or less, 0.300 or less, or 0.280 or less. The lower limit is not particularly limited, but is, for example, 0.050 or more, 0.0800 or more, 0.110 or more, 0.120 or more, 0.140 or more, 0.160 or more, 0.180 or more, 0.200 or more, 0.220 or more, or 0.240 or more.

It is preferable that the medium a of the present invention contains both t-butyl methyl ether and methanol. The content ratio (volume of t-butyl methyl ether/volume of methanol) is not particularly limited, but is preferably 0.300 to 0.950, more preferably 0.350 to 0.930, even more preferably 0.400 to 0.900, even more preferably 0.450 to 0.870, particularly preferably 0.500 to 0.850, and particularly preferably 0.550 to 0.830. The upper limit is not particularly limited, but may be, for example, 0.950 or less, 0.920 or less, 0.890 or less, 0.860 or less, 0.830 or less, 0.800 or less, 0.770 or less, 0.760 or less, 0.755 or less, 0.750 or less, 0.730 or less, 0.700 or less, 0.680 or less, 0.660 or less, 0.640 or less, 0.620 or less, or 0.600 or less. The lower limit is not particularly limited, but may be, for example, 0.120 or more, 0.160 or more, 0.200 or more, 0.250 or more, 0.300 or more, 0.350 or more, 0.400 or more, 0.440 or more, 0.480 or more, or 0.520 or more. Furthermore, the lower limit of the ratio of the total content of t-butyl methyl ether and methanol in medium a (total volume of t-butyl methyl ether and methanol/volume of medium a) may be, for example, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, 0.70 or more, 0.75 or more, 0.80 or more, 0.85 or more, 0.90 or more, 0.95 or more, or 1. The upper limit may be, for example, 1 or less, 0.99 or less, 0.95 or less, 0.90 or less, 0.85 or less, or 0.80 or less. It is preferable that t-butyl methyl ether and methanol are contained so that the octanol/water partition coefficient of medium a is within a predetermined range, and specifically, may be 0.50 or more and 4.0 or less. More specifically, the lower limit may be 0.50 or more, or 0.60 or more, or 0.70 or more, or 0.80 or more, or 0.90 or more, and the upper limit is not particularly limited, but may usually be 4.0 or less, or 3.9 or less, or 3.6 or 3.5 or less, or 3.0 or less, or 2.5 or less, or 2.0 or less, or 1.9 or less, or 1.8 or less, or 1.7 or less, or 1.6 or less, or 1.5 or less, or 1.4 or less, or 1.3 or less, or 1.2 or less, or 1.15 or less, or 1.10 or less.

<Requirement X>
The medium a of the present invention usually has a density of less than 0.998 g/ml at 20° C. The density may be preferably 0.700 to 0.995 g/ml, more preferably 0.720 to 0.900 g/ml, even more preferably 0.730 to 0.890 g/ml, still more preferably 0.735 to 0.880 g/ml, particularly preferably 0.736 to 0.850 g/ml, and particularly preferably 0.739 to 0.785 g/ml at 20° C. The upper limit is not particularly limited, but may be, for example, 0.995 g/ml or less, 0.992 g/ml or less, 0.990 g/ml or less, 0.985 g/ml or less, 0.980 g/ml or less, 0.975 g/ml or less, 0.970 g/ml or less, 0.965 g/ml or less, 0.963 g/ml or less, 0.960 g/ml or less, 0.955 g/ml or less, 0.950 g/ml or less, 0.945 g/ml or less, 0.94 0g/ml or less, 0.935g/ml or less, 0.930g/ml or less, 0.925g/ml or less, 0.920g/ml or less, 0.915g/ml or less, 0.910g/ml or less, 0.905g/ml or less, 0.900g/ml or less, 0.895g/ml or less, 0.890g/ml or less, 0.88 5g/ml or less, 0.880g/ml or less, 0.875g/ml or less, 0.870g/ml or less, 0.865g/ml or less, 0 ．． 860g/ml or less, 0.855g/ml or less, 0.850g/ml or less, 0.845g/ml or less, 0.840g/ml or less, 0.835g/ml or less, 0.830g/ml or less, 0.825g/ml or less, 0.820g/ml or less, 0.817g/ml or less, 0.814g/ml or less, 0. 811g/ml or less, 0.809g/ml or less, 0.806g/ml or less, 0.803g/ml or less, 0.800g/ml or less or less, 0.797 g/ml or less, 0.794 g/ml or less, 0.791 g/ml or less, 0.788 g/ml or less, 0.785 g/ml or less, 0.782 g/ml or less, 0.779 g/ml or less, 0.776 g/ml or less, 0.773 g/ml or less, 0.770 g/ml or less, 0.767 g/ml or less, 0.764 g/ml or less, 0.761 g/ml or less, 0.758 g/ml or less, or 0.755 g/ml or less. The lower limit is not particularly limited, but may be, for example, 0.660 g/ml or more, 0.663 g/ml or more, 0.665 g/ml or more, 0.668 g/ml or more, 0.670 g/ml or more, 0.673 g/ml or more, 0.675 g/ml or more, 0.677 g/ml or more, 0.680 g/ml or more, 0.683 g/ml or more, 0.685 g/ml or more, 0.687 g/ml or more, 0.690 g/ml or more, 0.693 g/ml or more, 0.696 g/ml or more, 0.700 g/ml or more, 0.703 g/ml or more, 0.705 g/ml or more, 0.707 ... 710 g/ml or more, 0.712 g/ml or more, 0.715 g/ml or more, 0.718 g/ml or more, 0.720 g/ml or more, 0.723 g/ml or more, 0.725 g/ml or more, 0.727 g/ml or more, 0.730 g/ml or more, 0.733 g/ml or more, 0.735 g/ml or more, 0.738 g/ml or more, 0.740 g/ml or more, 0.741 g/ml or more, 0.742 g/ml or more, 0.743 g/ml or more, 0.744 g/ml or more, 0.745 g/ml or more, 0.746 g/ml or more, 0.750 g/ml or more, or 0.751 g/ml or more.

<Requirement (Y)>
The medium a of the present invention contains an organic solvent having a certain degree of hydrophobicity, and specifically, the medium a of the present invention may be a medium relatively hydrophobic to the hydrophilic solvent used in step (i). Specifically, the medium a of the present invention contains at least one organic solvent having an octanol/water partition coefficient of a predetermined value or more. The octanol/water partition coefficient can be measured by the flask shake method defined in OECD Test Guideline 107 (Adopted: revised on July 27, 1995). The octanol/water partition coefficient of at least one organic solvent contained in the medium a is preferably within a predetermined range, and specifically may be 0.50 or more and 4.0 or less. More specifically, the lower limit may be 0.50 or more, or 0.60 or more, or 0.70 or more, or 0.80 or more, or 0.90 or more. The upper limit is not particularly limited, but may be usually 4.0 or less, or 3.9 or less, or 3.6 or 3.5 or less, or 3.0 or less, or 2.5 or less, or 2.0 or less, or 1.9 or less, or 1.8 or less, or 1.7 or less, or 1.6 or less, or 1.5 or less, or 1.4 or less, or 1.3 or less, or 1.2 or less, or 1.15 or less, or 1.10 or less. The medium a may also satisfy the above-mentioned provision regarding the octanol/water partition coefficient.
In addition, it is preferable that the organic solvent having the above-mentioned octanol/water partition coefficient is contained in the medium a at a predetermined ratio or more. Specifically, the lower limit may be 10 w/v % or more, 20 w/v % or more, 30 w/v % or more, 40 w/v % or more, 50 w/v % or more, 60 w/v % or more, 70 w/v % or more, 80 w/v % or more, 90 w/v % or more, or 100 w/v %.
Furthermore, the medium a may contain a predetermined ratio of an organic solvent so as to satisfy the above-mentioned requirement regarding the octanol/water partition coefficient.
The medium a of the present invention may also contain at least one organic solvent having a solubility in water of 0.0002 to 10 w/v% at 20°C. The solubility can be calculated by the method described in JIS K8001:2017 General Rules for Testing Methods for Reagents. That is, a certain amount of solute is powdered if the sample is solid, then placed in water, and shaken vigorously for 30 seconds every 5 minutes at 20°C ± 5°C. The volume (ml) of water required to dissolve within 30 minutes can be measured. The solubility in water at 20°C of at least one organic solvent contained in the medium a is preferably within a predetermined range, and the range may be preferably 0.0005 to 9.0 w/v%, more preferably 0.005 to 8.0 w/v%, even more preferably 0.01 to 7.0 w/v%, even more preferably 0.1 to 6.0 w/v%, particularly preferably 0.5 to 5.5 w/v%, and particularly preferably 1.0 to 5.0 w/v%. Furthermore, the upper limit is not particularly limited, and may be, for example, 10.0 w/v% or less, 9.0 w/v% or less, 8.0 w/v% or less, 7.5 w/v% or less, 7.0 w/v% or less, 6.5 w/v% or less, 6.0 w/v% or less, 5.5 w/v% or less, 5.3 w/v% or less, 5.0 w/v% or less, or 4.5 w/v% or less at 20°C. Furthermore, the lower limit is not particularly limited, and may be, for example, 0.002 w/v% or more, 0.004 w/v% or more, 0.008 w/v% or more, 0.012 w/v% or more, 0.015 w/v% or more, 0.020 w/v% or more, 0.080 w/v% or more, 0.100 w/v% or more, 0.200 w/v% or more, 0.400 w/v% or more, 1.0 w/v% or more, 1.3 w/v% or more, or 1.5 w/v% or more at 20°C.
In addition, the organic solvent having the above solubility in medium a may be 10 w/v% or more, or 20 w/v% or more, or 30 w/v% or more, or 40 w/v% or more, or 50 w/v% or more, or 60 w/v% or more, or 70 w/v% or more, or 80 w/v% or more, or 90 w/v% or more, or 100 w/v%.
Furthermore, the medium a may contain an organic solvent so as to satisfy the above-mentioned requirement regarding the solubility in water at 20°C.

<Requirement Z>
In the analytical method of the present invention, it is preferable that the density ratio between the hydrophilic solvent and medium a (density of medium a/density of hydrophilic solvent) is within a predetermined range at 20° C. Although the principle behind this is unclear, it is believed that when the density ratio is within the predetermined range, phase separation between the organic solvent and the hydrophilic solvent is facilitated, and the transfer of the lipid-soluble component to be measured from sample A to the organic solvent is facilitated. The density ratio of the hydrophilic solvent to medium a (density of medium a/density of hydrophilic solvent) at 20°C is usually 0.6000 to 0.8500, preferably 0.6020 to 0.8000, more preferably 0.6040 to 0.7900, even more preferably 0.6060 to 0.7800, still more preferably 0.6100 to 0.7700, particularly preferably 0.6150 to 0.7600, particularly preferably 0.6200 to 0.7550, 0.6300 to 0.7530, 0.6350 to 0.7500, or 0.6400 to 0.7490. In addition, the upper limit is not particularly limited, but is, for example, 0.7950 or less, 0.7850 or less, 0.7750 or less, 0.7650 or less, 0.7600 or less, 0.7550 or less, 0.7530 or less, 0.7510 or less, 0.7480 or less, 0.7450 or less, 0.7430 or less, 0.7420 or less, 0.7410 or less, 0.7400 or less, 0.7380 or less, 0.7360 or less, 0.7320 or less, 0.7300 or less, 0.7210 or less, 0.7110 or less, or 0.7000 or less at 20°C. The lower limit is not particularly limited, but is, for example, 0.6000 or more, 0.6010 or more, 0.6030 or more, 0.6040 or more, 0.6050 or more, 0.6080 or more, 0.6120 or more, 0.6140 or more, 0.6160 or more, 0.6210 or more, 0.6250 or more, 0.6350 or more, 0.6420 or more, 0.6500 or more, 0.6600 or more, 0.6700 or more, 0.6750 or more, 0.6800 or more, 0.6900 or more, 0.6950 or more, or 0.7000 or more at 20°C.

Mixing of Sample B; Step (ii')
In the analysis method of the present invention, the measurement target fat-soluble component can be extracted into an organic solvent by adding medium a to sample A to obtain sample B. Therefore, in the step of obtaining sample B, a step (step (ii')) of mixing sample B for a predetermined time may be included in order to efficiently extract the organic solvent into sample B. The mixing method may include, for example, mixing using a shaking incubator. The mixing time is not particularly limited, but is preferably 10 to 1440 minutes, more preferably 12 to 1080 minutes, even more preferably 15 to 540 minutes, even more preferably 20 to 480 minutes, particularly preferably 25 to 240 minutes, and particularly preferably 30 to 90 minutes. The upper limit of the mixing time is not particularly limited, but may be, for example, 1200 minutes or less, 1100 minutes or less, 1000 minutes or less, 900 minutes or less, 800 minutes or less, 700 minutes or less, 600 minutes or less, 500 minutes or less, 300 minutes or less, 200 minutes or less, 180 minutes or less, 150 minutes or less, 120 minutes or less, or 80 minutes or less. The lower limit of the mixing time is not particularly limited, but may be, for example, 10 minutes or more, 12 minutes or more, 13 minutes or more, 16 minutes or more, 18 minutes or more, 20 minutes or more, 24 minutes or more, 27 minutes or more, 33 minutes or more, 40 minutes or more, 45 minutes or more, or 48 minutes or more.

<Step (iii)>
Step (iii) is a step of subjecting sample B to phase separation to separate an upper layer sample C1, which is an analytical specimen, from a lower layer sample C2.
Step (iii) is described in detail below.
Step (iii) comprises

<Upper layer sample C1>
"Upper layer sample C1" refers to the upper layer of the composition obtained by phase separation of sample B. Since the analytical method of the present invention satisfies the above requirements (X), (Y), and (Z), the fat-soluble component to be measured is extracted into upper layer sample C1. Therefore, by recovering upper layer sample C1, it is possible to obtain an analytical specimen for the fat-soluble component to be measured.

The method for phase-separating sample B is not particularly limited, and may be, for example, centrifuging using a tabletop centrifuge. The centrifugation may be performed, for example, at 20°C for 3 to 30 minutes, 5 to 15 minutes, or 7 to 12 minutes at 3,000 rpm.

<Step (iii')>
The upper layer sample C1 may be used as the analytical specimen as it is, or may be further concentrated (step (iii')), and the concentrated product may be used as the analytical specimen. Alternatively, the concentrated product may be diluted with water, the organic solvent, or a mixture thereof to be used as the analytical specimen. In addition, the mixture of water and the organic solvent may be, for example, a mixture containing water, chloroform, and methanol. By performing the concentration, the analytical accuracy can be improved, for example, even when the amount of the fat-soluble component to be measured is small. In addition, the concentration method is not particularly limited, and examples thereof include a method of removing the solvent with a rotary evaporator, and in this embodiment, the concentrate may be in a solid form.

In particular, when sample A of the present invention contains cells or tissues containing the fat-soluble component to be measured and/or soluble sugars, if the fat-soluble component to be measured is extracted and concentrated using the conventional technology, the cells or tissues or soluble sugars will remain in the organic solvent, making the concentrate sticky and unsuitable for analysis. Therefore, the present invention is useful in that the concentration can be easily performed.

<Lower Layer Sample C2>
"Lower layer sample C2" refers to the lower layer of the composition obtained by phase separation of sample B. Since the analytical method of the present invention satisfies the above requirements (X), (Y), and (Z), a layer of hydrophilic solvent is formed in the lower layer sample C2. When cells or tissues are contained in sample A, a precipitate of cells or tissues may be present in the lower layer sample C2. In the conventional technology, the precipitate may be present at the interface between the aqueous phase and the organic solvent phase, but in the method of the present invention, the majority of the precipitate is usually present in the lower layer sample C2, more preferably 60 w/w% or more, even more preferably 70 w/w% or more, particularly preferably 80 w/w% or more, particularly preferably 90 w/w% or more, 95 w/w% or more, or the entire amount is present in the lower layer sample C2.

In the present invention, depending on the type of fat-soluble component to be measured, the fat-soluble component to be measured may remain in the lower layer sample C2. Therefore, medium a may be added to the collected lower layer sample C2 to extract the fat-soluble component again.

<Step (iv)>
Step (iv) is a step of measuring the fat-soluble component content in sample C1.
Step (iv) is described in detail below.

<Measurement of fat-soluble components>
The method for measuring the content of the fat-soluble component to be measured in sample C1 is not particularly limited, and high performance liquid chromatography (HPLC) or gas chromatography can be applied, but high performance liquid chromatography (HPLC) is preferred.

<High performance liquid chromatography>
The step (iv) of the present invention preferably includes a step of subjecting the sample C1 to high performance liquid chromatography. In the analysis method of the present invention, drift, swell and noise are suppressed in high performance liquid chromatography, and the peak of the target component can be made approximately horizontal before and after the peak. Therefore, it is easy to compare the area ratio of the peak of the target component of known concentration with the peak of the target component in the analysis sample, and the measurement error is reduced. The conditions for subjecting to high performance liquid chromatography are not particularly limited, but since the present invention can be used universally, various fat-soluble components can be extracted into the upper layer sample C1. Therefore, it is preferable to use multiple organic solvents as the mobile phase in high performance liquid chromatography. This makes it easy to separate the peaks of various fat-soluble components in high performance liquid chromatography. As an example of the condition, for example, a method of adjusting the polarity of the mobile phase using at least two or three organic solvents can be mentioned. As an example, for example, it is preferable to adjust the flow rate of a solvent with low polarity and a solvent with high polarity in the mobile phase, and it is particularly preferable to have a step of increasing the polarity of the mobile phase over time. The at least two or three organic solvents can be selected arbitrarily, for example, from methanol, acetonitrile, chloroform, hexane, tetrahydrofuran, isopropanol, isooctane, N-heptane, diethyl ether, cyclohexane, ethyl acetate, toluene, benzene, dichloromethane, ethanol, propanol, dimethylformamide, tetrahydrofuran, t-butyl methyl ether, and the like. As an example, methanol and t-butyl methyl ether can be used. In this embodiment, the method may include a step of adjusting the content ratio of t-butyl methyl ether to the content ratio of methanol in the mobile phase to be 3 times or more and 10 times or less, and then a step of decreasing the content ratio of t-butyl methyl ether to the content ratio of methanol in the mobile phase over time so that the content ratio of t-butyl methyl ether to the content ratio of methanol becomes 0.25 times or more and 0.55 times or less. Furthermore, methanol, t-butyl methyl ether, and hexane (CAS number 110-54-3) may be used as the at least three organic solvents used in the mobile phase. In this embodiment, the step of adjusting the content ratio of t-butyl methyl ether to the content ratio of methanol in the mobile phase to be 3 to 10 times may include a step of adjusting the content ratio of hexane to the total content ratio of methanol and t-butyl methyl ether to be 7 times or more, and then may include a step of adjusting the content ratio of hexane to the total content ratio of methanol and t-butyl methyl ether in the mobile phase to be 0.1 times or less. The polarity of various organic solvents can be determined by comparing the relative dielectric constants (ratio of the dielectric constant of the solvent to the dielectric constant of a vacuum) at the same temperature, and a solvent with a larger relative dielectric constant value can be determined to have a larger polarity.

<Turbidity>
The turbidity of the sample A of the present invention is preferably within a predetermined range. The turbidity can be specified by a value obtained by measuring the absorbance of the stirred sample at a wavelength of 660 nm using a spectrophotometer. Specifically, the turbidity of the sample A of the present invention is preferably 1.100 to 3.600, more preferably 1.100 to 3.400, even more preferably 1.100 to 3.200, even more preferably 1.100 to 3.000, particularly preferably 1.100 to 2.800, and particularly preferably 1.100 to 2.700. The upper limit is not particularly limited, but may be, for example, 3.800 or less, 3.700 or less, 3.600 or less, 3.400 or less, 3.200 or less, 3.000 or less, 2.800 or less, 2.700 or less, 2.500 or less, 2.400 or less, 2.200 or less, or 2.000 or less. The lower limit is not particularly limited, and may be, for example, 1.010 or more, 1.030 or more, 1.060 or more, 1.090 or more, 1.110 or more, 1.130 or more, 1.150 or more, 1.200 or more, or 1.300 or more.

<Protein>
Although the preferred aspects of sample A of the present invention are as described above, it is preferable that the total protein content of sample A of the present invention is within a predetermined range from the viewpoint of efficiently carrying out phase separation of sample B. The total protein content of sample A of the present invention is preferably 0 to 15 w/v%, more preferably 0 to 12 w/v%, more preferably 0 to 10 w/v%, even more preferably 0 to 7.0 w/v%, still more preferably 0 to 4.0 w/v%, particularly preferably 0 to 2.0 w/v%, and particularly preferably 0 to 0.8 w/v%. Furthermore, the lower limit is not particularly limited, and is, for example, 0.0001 w/v% or more, 0.01 w/v% or more, 0.02 w/v% or more, 0.03 w/v% or more, 0.04 w/v% or more, 0.05 w/v% or more, 0.06 w/v% or more, 0.07 w/v% or more, 0.08 w/v% or more, 0.09 w/v% or more, or 0.10 w/v% or more. The upper limit is, for example, 15 w/v% or less, 13 w/v% or less, 11 w/v% or less, 9.0 w/v% or less, 8.5 w/v% or less, 8.0 w/v% or less, 7.5 w/v% or less, 7.0 w/v% or less, 6.5 w/v% or less, 6.0 w/v% or less, 5.5 w/v% or less, 5.0 w/v% or less, 4.5 w/v% or less, 4.0 w/v% or less, 3.5 w/v% or less, 3.0 w/v% or less, 2.5 w/v% or less, 2.0 w/v% or less, 1.5 w/v% or less, 1.0 w/v% or less, 0.7 w/v or less, 0.6 w/v% or less, 0.4 w/v% or less, or 0.3 w/v% or less. The protein content of sample A can be measured, for example, by the Kjeldahl method.

<pH>
The preferred embodiment of sample A of the present invention is as described above, but in the present invention, from the viewpoint of safely extracting fat-soluble components by imparting a bactericidal effect and denaturing substances such as proteins that inhibit total separation of upper layer sample B, it is preferable that the pH of the hydrophilic solvent is within a predetermined range. The pH can be adjusted, for example, by adding an organic acid or an organic acid salt (e.g., sodium hydroxide, potassium hydroxide, potassium carbonate, calcium carbonate, potassium gluconate, lactic acid, citric acid, tartaric acid, malic acid, acetic acid, etc.) to sample A. The pH of the hydrophilic solvent of the present invention is preferably 7 or less, more preferably 5 or less, even more preferably 4.5 or less, even more preferably 4.4 or less, particularly preferably 4 or less, particularly preferably 3.9 or less, 3.8 or less, 3.6 or less, or 3.3 or less. In addition, the lower limit of the pH is not particularly limited, but is preferably 1.3 or more, 1.5 or more, 1.7 or more, 1.9 or more, or 2.1 or more. The preferred ranges of pH of the hydrophilic solvent of the present invention are 1.2 to 7, 1.5 to 5, 1.7 to 4.5, 1.9 to 3.6, and 2.1 to 3.5. The method for adjusting the pH is not particularly limited, but can be carried out, for example, by adding various organic acids or sodium salts having a buffering effect to sample A. The type of organic acid is not particularly limited, but it is preferable to add acetic acid from the viewpoint of a high bactericidal effect.

<Acetic acid>
The term "acetic acid" refers to acetic acid molecules (CH ₃ COOH) and acetate ions (CH ₃ COO ^- ), and the content of acetic acid refers to the total concentration of these, which can be measured by high performance liquid chromatography. The origin of acetic acid in the vinegar of the present invention is not particularly limited, and may be, for example, commercially available acetic acid, acetic acid contained in vinegar, or acetic acid derived from acetic acid bacteria. However, it is preferable that the majority of acetic acid contained in sample A is derived from vinegar and/or acetic acid bacteria. The content of acetic acid in sample A of the present invention is, as a concentration in a hydrophilic solvent, preferably 0.02 to 15 w/v%, more preferably 0.02 to 10 w/v%, even more preferably 0.02 to 8.0 w/v%, even more preferably 0.02 to 7.0 w/v%, particularly preferably 0.02 to 5 w/v%, and particularly preferably 0.02 to 4.0 w/v%. The lower limit of the content of acetic acid is not particularly limited, and can be, for example, 0.01 w/v% or more, 0.02 w/v% or more, 0.1 w/v% or more, 0.2 w/v% or more, 0.7 w/v% or more, 1.0 w/v% or more, 1.5 w/v% or more, 2.0 w/v% or more, 2.5 w/v% or more, or 4 w/v% or more, and the upper limit can be, for example, 21 w/v% or less, 19 w/v% or less, 17 w/v% or less, 15 w/v% or less, 13 w/v% or less, 11 w/v% or less, 9 w/v% or less, 8 w/v% or less, 7 w/v% or less, or 6 w/v% or less, 5.0 w/v% or less, 4.5 w/v% or less, 4.0 w/v% or less, 3.0 w/v% or less, or 1.0 w/v% or less.

<Vinegar>
Vinegar refers to a sour seasoning containing acetic acid as an active ingredient. The type of vinegar is not particularly limited, but includes synthetic vinegar in which acetic acid or glacial acetic acid is diluted with water and seasoned to have a vinegar flavor, brewed vinegar, grain vinegar, fruit vinegar, rice vinegar, black rice vinegar, apple vinegar, grape vinegar, or sake vinegar produced by acetic acid fermentation using ethanol as a raw material, Chinese vinegar, sherry vinegar, etc., as specified by the Japanese Agricultural Standards. In addition, fruits that can be used for the fruit vinegar include, for example, fruits described below. When sample A of the present invention contains vinegar, preferably 50 w/w% or more, more preferably 60 w/w% or more, even more preferably 70 w/w% or more, particularly preferably 80 w/w% or more, particularly preferably 85 w/w% or more, 90 w/w% or more, 95 w/w% or more, or the entire amount may be derived from vinegar. Furthermore, the proportion (mass %) of brewed vinegar to the total vinegar content is preferably 50 w/w% or more, more preferably 60 w/w% or more, even more preferably 70 w/w% or more, particularly preferably 80 w/w% or more, particularly preferably 90 w/w% or more, 95 w/w% or more, or may be 100 w/w% (total amount).

<Sodium>
"Sodium" refers to sodium ions, and is measured by atomic absorption spectrometry in accordance with "sodium" in the 2015 edition (seventh edition) of the Standard Tables of Food Composition in Japan. The sodium content in sample A of the present invention is not particularly limited, but may be, for example, in the range of 0.01 w/v% to 15 w/v% as a concentration in a hydrophilic solvent. Specifically, the lower limit is usually 0.01 w/v% or more, but may be 0.03 w/v% or more, or 0.05 w/v% or more, or 0.07 w/v% or more, or 0.1 w/v% or more, or 0.3 w/v% or more, or 0.5 w/v% or more, or 0.6 w/v% or more, or 0.7 w/v% or more, or 0.8 w/v% or more, or 1.0 w/v% or more. On the other hand, the upper limit can be, for example, 10 w/v% or less, or 8.5 w/v% or less, or 8.0 w/v% or less, or 7.5 w/v% or less, or 7.0 w/v% or less, or 6.5 w/v% or less, or 6.0 w/v% or less, or 5.5 w/v% or less, or 5.0 w/v% or less, or 3.0 w/v% or less, or 2.0 w/v% or less, or 1.0 w/v% or less, 0.5 w/v% or less, or 0.3 w/v% or less. In order to satisfy the requirement (Z), the analysis method of the present invention may adjust the density of the hydrophilic solvent by adjusting the sodium content, but it is more convenient to use soluble saccharides from the viewpoint of high solubility in water and excellent safety.

Since the present invention relates to a method for analyzing fat-soluble components, it is preferable that the total lipid content in sample A is within a predetermined range. The lipid content is measured by a Soxhlet extraction method using diethyl ether in accordance with the 2015 edition (seventh revision) of the Standard Tables of Food Composition in Japan. From the viewpoint of increasing the technical significance of the present invention, the lipid content of sample A of the present invention is preferably 45 w/v% or less, more preferably 35 w/v% or less, even more preferably 25 w/v% or less, even more preferably 15 w/v% or less, particularly preferably 10 w/v% or less, and particularly preferably 7 w/v% or less, as a concentration in sample A. In addition, the upper limit is preferably 45 w/v% or less, or 40 w/v% or less, or 30 w/v% or less, while the lower limit is not limited, but can be 0.001 w/v% or more, or 0.002 w/v% or more, or 0.003 w/v% or more, or 0.005 w/v% or more.

The present invention is an analytical method that can be used universally, but it is particularly preferable that sample A is a food or beverage, since the conventional techniques are difficult to apply and the problem to be solved is of great importance.

<Food and drink>
The term "food and drink" is not particularly limited to the commonly used meaning, and examples thereof include food and drink that is consumed as is, and compositions for preparing food and drink that is consumed as is (concentrates for beverage preparation, seasonings, etc.). The food and drink preferably contains 50 w/v % or more water. The food and drink may also be used as the aforementioned sample A as is, and it is preferable that the water content of the aforementioned sample A is satisfied. Furthermore, the range of the content of various components in sample A disclosed in this specification may be satisfied.
The food or drink of the present invention may also preferably be a beverage, a concentrate for the preparation of a beverage, or a seasoning.

Foods and beverages that are consumed as is are not particularly limited, but examples include beverages, confectioneries, soups, etc. Among these, beverages are preferred.

Examples of beverages include beverages containing fruit juice (e.g., citrus fruits (mandarin oranges, oranges, lemons, limes, grapefruits, yuzu, kabosu, sudachi, bergamot, pink grapefruit, hassaku, calamansi, shekwasha, etc.), tropical fruits (pineapple, banana, guava, mango, acerola, papaya, passion fruit, etc.), lychee, strawberry, apple, peach, grapes (white grapes, red grapes, etc.), black currant, raspberry, pomegranate, plum, pear, apricot, plum, kiwi, etc.) Fruit juices such as roots, melon, blueberry, acai, etc., ades, near water, beauty drinks, smoothies, etc.), dairy products (such as milk and its processed products such as skim milk powder, whole milk powder, concentrated milk, yogurt, fresh cream, condensed milk, butter, skim milk, cream powder, sweetened milk powder, modified milk powder, whey powder, buttermilk powder, and other dairy products), vegetable drinks (such as tomato, carrot, pumpkin juice, smoothies, green juice, etc.), refreshing drinks Drinking water (e.g. sports drinks, lemonade and other ades, fruit-flavored drinks), carbonated drinks, jelly drinks, grain drinks (e.g. grain drinks made mainly from rice, soy milk, almonds, etc.), tea drinks (e.g. black tea, oolong tea, green tea, black tea, matcha, jasmine tea, rosehip tea, chamomile tea, roasted green tea, blended tea (grains such as barley, barley, brown rice, soybeans, corn, persimmon leaves, loquat leaves, bear bamboo, gynostemma, angelica tree, dokudami leaves, kelp, safflower, Examples of such beverages include shiitake mushrooms, lychees, etc.), coffee beverages, powdered beverages (e.g., cocoa, green juice, etc.), alcoholic beverages (e.g., beer, beer-flavored beverages such as low-malt beer, brewed alcoholic beverages such as fruit wine and sake, distilled alcoholic beverages such as shochu, whiskey, brandy, and spirits, mixed alcoholic beverages such as liqueurs in which distilled alcoholic beverages are mixed with auxiliary ingredients such as sugars, and further cocktails, fizz, chuhai, etc. in which fruit juice, flavorings, emulsified flavorings (flavoring preparations in which oil-soluble flavorings are emulsified so as to be stably dispersed in water), carbon dioxide, etc. are added to such alcoholic beverages). Among these, fruit juice-containing beverages are preferred.

The fruit juice content of the fruit juice-containing beverage of the present invention (calculated as straight fruit juice) is not particularly limited, but can be, for example, in the range of 0.05 w/v% to 100 w/v% in wet mass terms. Specifically, the lower limit is usually 0.05 w/v% or more, but is preferably 0.1 w/v% or more, 0.15 w/v% or more, or 0.2 w/v% or more, while the upper limit can be 100 w/v% or less, or 90 w/v% or less.

In the present invention, "fruit juice" refers to the liquid portion of fruit obtained by squeezing or extracting fruit, or in the case of strained or grated fruit puree or grated fruit, the liquid portion of the puree or grated fruit. Examples of fruit juices used in the liquid seasoning of the present invention include fruit juices derived from citrus fruits (e.g., lemon, Valencia orange, navel orange, grapefruit, lime, shikuwasa, bitter orange, yuzu, kabosu, sudachi, citron, bush citron, natsumikan, hassaku, hyuganatsu, sweetie, dekopon, iyokan, tankan, seminole, pomelo, mandarin orange, unshu mikan, ponkan, Kishu mikan, kumquat, yuko, pomelo, banpeiyu, etc.), apple, pineapple, peach, grape, strawberry, pear, banana, kiwi, black currant, acerola, blueberry, raspberry, persimmon, apricot, guava, plum, mango, papaya, lychee, etc. One or more of these fruit juices can be used. In addition, the above fruit juices can also be used after processing such as freezing, concentration, reduction, etc.

In addition, the above fruit juice preferably has a citric acid content, calculated on a wet mass basis, of 0.1 w/v% or more, or 0.25 w/v% or more, or 0.5 w/v% or more, or 0.75 w/v% or more, or 1.0 w/v% or more, or 1.5 w/v% or more, and is preferably a fruit juice that is standardized as having citric acid in the JAS standard for fruit juice (Japanese Agricultural Standards for Fruit Drinks, Notification No. 3118 of the Ministry of Agriculture, Forestry and Fisheries of December 24, 2013), and particularly preferably lemon juice, lime juice, or kabosu juice. The fruit juice preferably has a sugar content of 1.0 w/v% or more, or 1.5 w/v% or more, or 2.0 w/v% or more, or 2.5 w/v% or more, and is preferably standardized as having a sugar content standard in the JAS standard for fruit juice (Japanese Agricultural Standards for Fruit Drinks, Notification No. 3118 of the Ministry of Agriculture, Forestry and Fisheries, December 24, 2013), with citrus juice (excluding fruit juices without a sugar content standard) or apple juice being particularly preferred.

In the present invention, the "fruit juice content (converted to straight fruit juice)" refers to the w/v% concentration when the straight fruit juice obtained by squeezing fruit is taken as 100%, and can be calculated by multiplying the content (w/v%) of the fruit juice blended in the food or drink by the concentration ratio of the fruit juice. For example, when apple juice with a concentration ratio of 5 times is blended in the food or drink at 10 w/v%, the fruit juice content (converted to straight juice) is 50 w/v%. The concentration ratio of each fruit juice can be converted based on the minimum value of the sugar refractometer reading standard or acidity standard for the straight fruit juice of various fruits shown in the JAS standard (Japanese Agricultural Standards for Fruit Drinks, Notification No. 3118 of the Ministry of Agriculture, Forestry and Fisheries, December 24, 2013).

Confectionery is food that is manufactured and prepared to emphasize tastes such as sweetness or saltiness, or to enhance tactile sensations such as texture, and to satisfy taste sensations such as olfactory sensations with various smells. More specific examples include jellies, puddings, chocolates, bars (snack bars), frozen desserts (ice cream, sherbet, etc.), etc., among which jellies and frozen desserts (ice cream, sherbet, etc.) are preferred, and among these, jellies are particularly preferred.

Soup is a food that contains a lot of water and is obtained by cooking ingredients such as meat, seafood, eggs, milk, vegetables, fruits, herbs, and seaweed in an appropriate manner. Specific examples of soup include minestrone, sanratan, baitan soup, and chige soup.

The composition for preparing food and drink to be consumed is not particularly limited, but examples thereof include compositions for preparing beverages, dessert sauces and creams, seasonings, retort foods, etc. Among these, preferred are compositions for preparing beverages, dessert sauces and creams, seasonings, etc., and more preferred are compositions for preparing beverages.

An example of a composition for preparing a beverage is a concentrated type of beverage. This is diluted with an appropriate beverage (e.g., water or one of the beverages exemplified above) before consumption. The recommended dilution ratio is, for example, 1.1 to 50 times, preferably 2 to 20 times, more preferably 3 to 12 times, and even more preferably 4 to 8 times.

Dessert sauces and creams are liquid, powdered, or semi-solid sauces or creams that are poured, placed on, or mixed into beverages or confectioneries (such as jellies, cakes, and ice cream) to add flavor, texture, or color to desserts. Specific examples include caramel sauce, custard sauce, chocolate sauce, and fruit sauces such as raspberry sauce, strawberry sauce, blueberry sauce, apple sauce, and pomegranate sauce.

The seasonings are not particularly limited, but examples thereof include sauces (sesame-containing seasonings such as sesame sauce, yakiniku sauce, etc.), dressings (oil-free dressings, separated dressings, emulsified dressings, etc.), seasoning vinegars (e.g., general-purpose seasoning vinegars, seasoning vinegars for vinegared dishes, seasoning vinegars for sushi rice, seasoning liquids for pickling (e.g., pickles, etc.), sweet vinegar, etc.), seasonings for rice, ponzu seasonings, seasonings containing dashi (e.g., noodle soup, hot pot soup, etc.), seasonings for natto, seasonings for pickles, seasonings for meat, vinegar, Worcestershire sauce, ketchup, oyster sauce, salsa, sambal sauce, chili sauce, seasonings containing hot spices, chutney, mustard, mayonnaise, etc. In addition, the seasonings of the present invention may contain amino acids or nucleic acids.

<Amino acids>
In the present invention, "amino acid" refers to an amino acid and its salt, and the type of amino acid is not particularly limited. The amino acid contained in the food and drink of the present invention may be contained in the food material as a raw material, or may be added from the outside separately from the food material. In addition, when adding an amino acid from the outside, a highly purified preparation that has been extracted and purified may be added, or it may be added in the form of some material containing the amino acid (e.g., fruit juice) or a processed material product (e.g., extract, concentrated fruit juice). However, it is preferable that the majority (more preferably all) of the amino acid contained in the food and drink is derived from some food material. Examples of the amino acid-containing seasoning include amino acid-based seasonings, and examples of amino acid-based seasonings include L-sodium glutamate, DL-alanine, glycine, L- or DL-tryptophan, L-phenylalanine, L- or DL-methionine, L-lysine, L-aspartic acid, L-sodium aspartate, L-arginine, and the like. These amino acids may be used alone in one type, or two or more types may be contained in any combination or in any ratio.

The total amino acid content of sample A of the present invention is not particularly limited, but is, for example, 0.3 to 1500 mg/100 ml, preferably 0.5 to 1200 mg/100 ml, more preferably 0.8 to 1000 mg/100 ml, even more preferably 1.0 to 800 mg/100 ml, particularly preferably 2.0 to 500 mg/100 ml, and particularly preferably 3.0 to 250 mg/100 ml. The upper limit is, for example, 1600 mg/100 ml or less, 1400 mg/100 ml or less, 1100 mg/100 ml or less, 900 mg/100 ml or less, 750 mg/100 ml or less, 650 mg/100 ml or less, 600 mg/100 ml or less, 550 mg/100 ml or less, 450 mg/100 ml or less, 400 mg/100 ml or less, 350 mg/100 ml or less, 300 mg/100 ml or less, 240 mg/100 ml or less, 200 mg/100 ml or less, 180 mg/100 ml or less, 160 mg/100 ml or less, 140 mg/100 ml or less, 120 mg/100 ml or less, 110 mg/100 ml or less, 100 mg/100 ml or less, 90 mg/100 ml or less, 80 mg/100 ml or less, 70 mg/100 ml or less, 60 mg/100 ml or less, 50 mg/100 ml or less, 40 mg/100 ml or less, 30 mg/100 ml or less. The lower limit is, for example, 0.01 mg/100 ml or more, 0.02 mg/100 ml or more, 0.05 mg/100 ml or more, 0.1 mg/100 ml or more, 0.2 mg/100 ml or more, 0.4 mg/100 ml or more, 0.6 mg/100 ml or more, 0.8 mg/100 ml or more, 1.1 mg/100 ml or more, 1.5 mg/100 ml or more, 2.0 mg/100 ml or more, 2.4 mg/100 ml or more, 3.5 mg/100 ml or more, 4.0 mg/100 ml or more, 4.5 mg/100 ml or more, 5.5 mg/100 ml or more, 6.5 mg/100 ml or more, 7.5 mg/100 ml or more, 8.5 mg/100 ml or more, or 10 mg/100 ml or more. Amino acid analysis can be performed by high performance liquid chromatography.

<Nucleic Acid>
In the present invention, "nucleic acid" refers to nucleic acid and its salt. The nucleic acid contained in the food and drink of the present invention may be contained in the raw material food material, or may be added from the outside separately from the food material. When adding nucleic acid from the outside during the production of the food and drink of the present invention, a highly purified and extracted preparation may be added, or it may be added in the form of some processed material product (e.g., an extract) containing nucleic acid. However, it is preferable that the majority (more preferably all) of the nucleic acid contained in the food and drink is derived from some food material. Examples of nucleic acid-containing seasonings include amino acid-based seasonings, and examples of nucleic acid-based seasonings include 5'-disodium inosinate, 5'-disodium guanylate, 5'-disodium uridylate, 5'-disodium cytidylate, 5'-calcium ribonucleotide, and 5'-disodium ribonucleotide. These nucleic acids may be contained alone, or two or more types may be contained in any combination or in any ratio.

There are no particular limitations on the retort food, so long as it is a cooked or semi-cooked food packed in a retort pouch or can. Examples of the food to be packed include the above-mentioned foods themselves, or foods that can be obtained by simple cooking (for example, adding ingredients and cooking with heat).

The present invention also relates to a method for analyzing the change over time in the content of a fat-soluble component to be measured in sample A.

As described above, the present invention is a method capable of accurately analyzing the content of the fat-soluble component to be measured in sample A, and therefore can analyze the change in the content of the fat-soluble component to be measured when sample A is stored for a specified period of time. The period is not particularly limited, but may be, for example, within 10 years, within 5 years, within 4 years, within 3 years, within 2 years, within 1 year and 6 months, or within 1 year.

The present invention also relates to a method for producing an analytical sample containing a fat-soluble component to be measured.

When the present invention is a method for producing an analytical sample containing a fat-soluble component to be measured, as described above, the upper layer sample C1 can be collected to produce an analytical sample of the fat-soluble component to be measured. The upper layer sample C1 may be further concentrated, and the concentrated product may be used as the analytical sample. The concentrated product may be diluted with water, the organic solvent, or a mixture thereof to produce an analytical sample. Examples of the mixture of water and an organic solvent include mixtures containing water, chloroform, and methanol. Therefore, the conditions related to steps (i) to (iii), (ii'), and (iii') disclosed in this specification can be directly applied to the method for producing an analytical sample containing a fat-soluble component to be measured of the present invention.

The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative and are not intended to limit the present invention in any way.

<Preparation of medium containing organic solvent>
A control solvent and a test solvent (medium A) were prepared by mixing t-butyl methyl ether (Kanto Chemical; purity 99.8 w/w%), methanol (Kanto Chemical; purity 99.8 w/w%), and chloroform (Kanto Chemical; purity 99.0 w/w%) according to the composition in Table 1. The t-butyl methyl ether, methanol, and chloroform used were reagents (for HPLC) manufactured by Kanto Chemical.

<Step (i): Preparation of Sample A>
Lemon juice, apple juice, black vinegar, apple vinegar, acetic acid bacteria (ceramide content 0.8 w/w%), white sugar, acetic acid, flavoring, and emulsified flavoring were appropriately mixed to prepare Sample A as shown in Table 2. The ceramide content in the acetic acid bacteria was measured in the same manner as described in JP 2007-306882 A, by extracting total lipids from the freeze-dried acetic acid bacteria with an organic solvent having a composition of chloroform:methanol:water=1:2:0.8 according to the known Bligh-dyer method, followed by subjecting the extract to high performance liquid chromatography.

<Preparation of Analytical Samples in Comparative Examples 1 and 2>
In Comparative Examples 1 and 2, Sample A was prepared as described above, and then the control solvent was added to Sample A in an amount of 3.6 ml per 1 ml of volume, and the mixture was stirred with a vortex (vortex mixer: Pasolina: NS-80), subjected to ultrasonic waves for 20 minutes, and then centrifuged at 3000 rpm for 10 minutes (refrigerated centrifuge 2800; KUBOTA).

Then, the supernatant was filtered through a 0.45 μm PTFE filter and collected in a 100 ml eggplant flask.

Next, water and a control solvent were added to the precipitate as appropriate, and the precipitate was centrifuged and filtered in the same manner as above, and then collected in the same 100 ml eggplant flask from which the supernatant was initially collected.

Next, the sample collected in the recovery flask was subjected to a rotary evaporator to remove the solvent (concentrate the sample), and a specified amount of the control solvent was added to the resulting residue again to prepare a sample for high-performance liquid chromatography. The sample was sealed in a vial and subjected to high-performance liquid chromatography, and detected by ELSD. When quantifying the ceramide content in the sample, a ceramide standard with a known concentration (manufactured by Nagara Science Co., Ltd.; Ceramide from Acetobacter malorum (Ceramide mix)) was subjected to high-performance liquid chromatography in the same manner as above, and a calibration curve was created.

<High performance liquid chromatography>
The following equipment was used for high performance liquid chromatography:
・Liquid chromatograph (Shimadzu Corporation; LC-20AD)
・Autosampler (Shimadzu Corporation; SIL-20AC HT)
・Communication module (Shimadzu Corporation; CBM-20A)
・Column oven (Shimadzu Corporation; CTO-20AC)
・ELSD detector (Shimadzu Corporation; ELSD-LTII)
・HRL column (Shimadzu Corporation; Shim-Pack HRC-SIL 4.6 mm fwai * 25 cm)
Column: (Shimadzu Corporation; SHIM-PACK HRC-SIL (250 mm, keru 4.6 mm))

The high performance liquid chromatography was carried out under the following conditions.
Mobile phase: The flow rate of methanol and chloroform was adjusted over time as shown in Figure 1. Flow rate: 1.0 mL/min
Injection: 4μL
Autosampler temperature: 15°C
Detector ELSD temperature: 40°C
ELSD detection sensitivity: Gain 8
Column temperature: 50 ° C.

<Step (ii): Preparation of Sample B in Test Example>
In the test example, after preparing sample A as described above, a test solvent was added to sample A in an amount of 5.2 ml per 1 ml of sample A to obtain sample B, which was then stirred with a vortex mixer and shaken (120 rpm) at 20° C. for 1 hour with a shaker (EYELA; MULTI SHAKER MMS) (step (ii')). Then, the sample was allowed to stand for 10 minutes.

<Step (iii): Preparation of upper layer sample C1 and lower layer sample C2 in test example>
Next, sample B was centrifuged at 20° C. for 10 minutes at 3000 rpm in a tabletop centrifuge to obtain upper layer sample C1 and lower layer sample C2.

Next, the upper layer sample C1 was collected in a 100 ml eggplant flask.

Additionally, water and test solvent were added to the lower layer sample C2, which was then shaken and centrifuged again, and the upper layer was collected in the same recovery flask as sample C1.

Next, the sample collected in the recovery flask was subjected to a rotary evaporator to remove the solvent (concentrating the sample; step (iii')), and a solvent with a chloroform:methanol:ultrapure water (MilliQ water) ratio of 60:30:4.5 was added to the resulting residue to prepare an analytical sample. The sample was sealed in a vial, subjected to high-performance liquid chromatography, and detected by ELSD. In addition, when quantifying the ceramide content in the sample, a ceramide standard with a known concentration (manufactured by Nagara Science Co., Ltd.; Ceramide from Acetobacter malorum (Ceramide mix)) was subjected to high-performance liquid chromatography in the same manner as above, and a calibration curve was created.

<High performance liquid chromatography>
High performance liquid chromatography was carried out under the following conditions.
Column: Diol-HILIC column (YMC: YMC-Triart Diol-HILIC 4.6 x 250 mm, base material: organic silica hybrid, particle size: 1.9 μm, 3 μm, 5 μm, pore size: 12 nm, usable pH range 2 to 10)
Mobile phase: The flow rates of hexane, t-butyl methyl ether, and methanol were adjusted over time as shown in FIG. 2. Flow rate: 1.0 mL/min
・Injection: 10μL
Autosampler temperature: 20°C
Detector ELSD temperature: 40°C
・ELSD detection sensitivity: Gain 6
Column temperature: 30°C

<Peak evaluation>
The peak of the high performance liquid chromatography was observed, and if the drift, swell and noise in the baseline were suppressed and the baseline was approximately horizontal around the peak of the target component as shown in Figure 3, it was rated as ◯, and if not, it was rated as × as shown in Figure 4. Note that Figure 3 shows the results of Test Example 4, and Figure 4 shows the results of Comparative Example 2.

<Error evaluation>
The test was repeated three times for each analytical sample, and the ceramide content (w/w%) relative to the acetic acid bacteria cell content in the analytical sample was calculated. When the calculated values (n=3) based on the measurement results were all within the error range of 0.8 w/w% ±15% (the ceramide content relative to the acetic acid bacteria cell content was 0.68-0.92 w/w%), the error was evaluated as ◯. In other cases, the error was evaluated as ×. The measurement error was calculated by measuring the ceramide content relative to the acetic acid bacteria cell content in the raw material state, and calculating the ceramide content in the composition from the amount of acetic acid bacteria cells as a theoretical value.

<Results>
The results are shown in Table 2. In Comparative Example 2, the sample became wet and sticky when concentrated by the rotary evaporator, and it was presumed that impurities other than fat-soluble components were contained in the analyzed specimen. In Test Examples 1, 3, and 4, emulsified flavoring, skim milk powder, flavoring, etc. were added to Sample A, and excellent results were obtained.

Claims (49)

A method for analyzing a fat-soluble component to be measured in a sample A, comprising the following steps (i) to (iv), wherein the fat-soluble component is a component selected from fatty acyl, glycerolipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, polyketides, and phospholipids :
(i) A step of preparing or adjusting sample A containing a fat-soluble component to be measured and 50 w/v % or more of a hydrophilic solvent that is an aqueous solution having a density of 1.003 g/ml or more at 20 ° C. (ii) A step of obtaining sample B by adding medium a to sample A so as to satisfy the following requirements (X), (Y), and (Z): Requirement (X): The density of medium a at 20 ° C. is less than 0.998 g/ml Requirement (Y): Medium a contains at least one organic solvent having an octanol/water partition coefficient of 0.50 to 4.0 Requirement (Z): The density ratio of the hydrophilic solvent to medium a at 20 ° C. (density of medium a/density of the hydrophilic solvent) is 0.6000 to 0.8500 (iii) A step of separating sample B into phases and separating an upper layer sample C1 and a lower layer sample C2, which are analytical specimens (iv) A step of subjecting sample C1 to high performance liquid chromatography to measure the content of the fat-soluble component to be measured in sample C1 The method according to claim 1 , wherein the fat-soluble component is selected from the group consisting of saturated fatty acids, unsaturated fatty acids, terpenes, fat-soluble vitamins and sphingolipids. The fat-soluble component is selected from the group consisting of carotenoids, steroids, vitamin A, vitamin D, vitamin E, vitamin K, EPA (eicosapentaenoic acid), DPA (docosapentaenoic acid), DHA (docosahexaenoic acid), α-linolenic acid, linoleic acid, arachidonic acid, oleic acid, lauric acid, myristic acid, palmitic acid, stearic acid, limonene, α-terpinene, β-terpinene, γ-terpinene, δ-terpinene, p-cymene, murolol, cadalene, and tetrahydroxybacillin. The method according to claim 1, wherein the component is selected from the group consisting of cteriohopane (C35-pentacyclic terpene alcohol), hopan-22-ol, hop-22(29)-ene, ornityl tau phospholipid 3-(palmitoyl)-hydroxypalmitoyl-ornityl-taurine, ornithine lipid 3-(palmitoyl)-hydroxypalmitoyl-ornithine, lysoornithine lipid 3-hydroxypalmitoyl-ornithine, cis-vaccenic acid, lecithin, and ceramide. The method according to claim 1, wherein in step (i), sample A contains cells or tissues containing the lipid-soluble component to be measured. The method according to claim 4 , wherein in step (i), 40 wt % or more of the fat-soluble components to be measured in sample A are contained in cells or tissues. The method according to claim 4 or 5 , wherein in step (i), the cells or tissues containing the lipid-soluble component to be measured contain acetic acid bacteria. The method according to claim 1 or 4 , wherein in step (i), sample A contains soluble sugars. The method according to claim 7 , wherein in step (i), the content of soluble sugars in the hydrophilic solvent of sample A is 0.1 to 30 w/v %. The method according to claim 1 or 4 , wherein in step (i), sample A contains ceramide as the fat-soluble component to be measured. The method according to claim 1 or 4 , wherein in step (i), sample A contains an acetic acid bacteria product. The method according to claim 1 or 4 , wherein in step (i), sample A contains acetic acid. The method according to claim 11 , wherein in step (i), the content of acetic acid in the hydrophilic solvent of sample A is 0.02 to 15 w/v %. The method according to claim 1 or 4 , wherein in step (i), the density of the hydrophilic solvent at 20° C. is 1.003 g/ml or more and 1.250 g/ml or less . The method according to claim 1 or 4 , wherein in step (i), the pH of the hydrophilic solvent is 4.5 or less. The method according to claim 1 or 4 , wherein in step (i), the total protein content of sample A is 15 w/v% or less. The method according to claim 1 or 4 , wherein in step (i), the turbidity of sample A at a wavelength of 660 nm is 1.100 or more. The method according to claim 1 or 4 , wherein in step (i), sample A is a food or beverage. The method according to claim 1 or 4 , wherein in step (ii), the medium a contains t-butyl methyl ether. The method according to claim 18 , wherein in step (ii), the medium a further contains methanol. The method according to claim 1 or 4 , comprising step (ii) followed by step (ii'):
(ii') mixing sample B for 10 to 1440 minutes The method according to claim 1 or 4 , wherein in step (iii), a precipitate is present in the lower layer sample C2. The method according to claim 1 or 4 , comprising step (iii) followed by step (iii'):
(iii') Further concentrating the upper layer sample C1 The method according to claim 1 or 4 , wherein in step (iv), at least two or three organic solvents are used in the mobile phase of the high performance liquid chromatography. The method according to claim 1 or 4 , wherein in step (iv), at least three organic solvents are used in the mobile phase of the high performance liquid chromatography. A method for analyzing a change in the content of a fat-soluble component to be measured in sample A over time, the method comprising the following steps (i) to (iv), wherein the fat-soluble component is a component selected from fatty acyl, glycerolipids, sphingolipids, sterol lipids, prenol lipids, glycolipids, polyketides, and phospholipids :
(i) A step of preparing or adjusting sample A containing a fat-soluble component to be measured and 50 w/v % or more of a hydrophilic solvent that is an aqueous solution having a density of 1.003 g/ml or more at 20 ° C. (ii) A step of obtaining sample B by adding medium a to sample A so as to satisfy the following requirements (X), (Y), and (Z): Requirement (X): The density of medium a at 20 ° C. is less than 0.998 g/ml Requirement (Y): Medium a contains at least one organic solvent having an octanol/water partition coefficient of 0.50 to 4.0 Requirement (Z): The density ratio of the hydrophilic solvent to medium a at 20 ° C. (density of medium a/density of the hydrophilic solvent) is 0.6000 to 0.8500 (iii) A step of separating sample B into phases and separating an upper layer sample C1 and a lower layer sample C2, which are analytical specimens (iv) A step of subjecting sample C1 to high performance liquid chromatography to measure the content of the fat-soluble component to be measured in sample C1 The method according to claim 25, wherein the fat-soluble component is selected from the group consisting of saturated fatty acids, unsaturated fatty acids, terpenes, fat-soluble vitamins, and sphingolipids. The fat-soluble component is selected from the group consisting of carotenoids, steroids, vitamin A, vitamin D, vitamin E, vitamin K, EPA (eicosapentaenoic acid), DPA (docosapentaenoic acid), DHA (docosahexaenoic acid), α-linolenic acid, linoleic acid, arachidonic acid, oleic acid, lauric acid, myristic acid, palmitic acid, stearic acid, limonene, α-terpinene, β-terpinene, γ-terpinene, δ-terpinene, p-cymene, murolol, cadalene, and tetrahydroxybacillin. The method according to claim 25, wherein the component is selected from the group consisting of cteriohopane (C35-pentacyclic terpene alcohol), hopane-22-ol, hop-22(29)-ene, ornityl tau phospholipid 3-(palmitoyl)-hydroxypalmitoyl-ornityl-taurine, ornithine lipid 3-(palmitoyl)-hydroxypalmitoyl-ornithine, lysoornithine lipid 3-hydroxypalmitoyl-ornithine, cis-vaccenic acid, lecithin, and ceramide. A method for producing an analytical specimen containing a fat-soluble component to be measured, comprising the following steps (i) to (iii), wherein the fat-soluble component is a component selected from fatty acyl, glycerolipid, sphingolipid, sterol lipid, prenol lipid, glycolipid, polyketide, and phospholipid :
(i) A step of preparing or adjusting sample A containing a fat-soluble component to be measured and 50 w/v % or more of a hydrophilic solvent that is an aqueous solution having a density of 1.003 g/ml or more at 20 ° C. (ii) A step of obtaining sample B by adding medium a to sample A so as to satisfy the following requirements (X), (Y), and (Z): Requirement (X): The density of medium a at 20 ° C. is less than 0.998 g/ml; (Y): Medium a contains at least one organic solvent having an octanol/water partition coefficient of 0.50 to 4.0; (Z): The density ratio of the hydrophilic solvent to medium a at 20 ° C. (density of medium a/density of the hydrophilic solvent) is 0.6000 to 0.8500; (iii) A step of separating sample B into upper layer sample C1 and lower layer sample C2, which are analytical specimens, by phase separation. The method according to claim 28, wherein the fat-soluble component is selected from the group consisting of saturated fatty acids, unsaturated fatty acids, terpenes, fat-soluble vitamins, and sphingolipids. The fat-soluble component is selected from the group consisting of carotenoids, steroids, vitamin A, vitamin D, vitamin E, vitamin K, EPA (eicosapentaenoic acid), DPA (docosapentaenoic acid), DHA (docosahexaenoic acid), α-linolenic acid, linoleic acid, arachidonic acid, oleic acid, lauric acid, myristic acid, palmitic acid, stearic acid, limonene, α-terpinene, β-terpinene, γ-terpinene, δ-terpinene, p-cymene, murolol, cadalene, and tetrahydroxybacillin. The method of claim 28, wherein the component is selected from the group consisting of cteriohopane (C35-pentacyclic terpene alcohol), hopane-22-ol, hop-22(29)-ene, ornityl tau phospholipid 3-(palmitoyl)-hydroxypalmitoyl-ornityl-taurine, ornithine lipid 3-(palmitoyl)-hydroxypalmitoyl-ornithine, lysoornithine lipid 3-hydroxypalmitoyl-ornithine, cis-vaccenic acid, lecithin, and ceramide. The method according to claim 28 , wherein in step (i), sample A contains cells or tissues containing the lipid-soluble component to be measured. The method according to claim 31 , wherein in step (i), 40 wt % or more of the fat-soluble components to be measured in sample A are contained in cells or tissues. The method according to claim 28 or 32 , wherein in step (i), the cells or tissues containing the lipid-soluble component to be measured contain acetic acid bacteria. 33. The method of claim 28 or 32 , wherein in step (i), sample A contains soluble sugars. The method according to claim 32 , wherein in step (i), the content of soluble sugars in the hydrophilic solvent of sample A is 0.1 to 30 w/v %. The method according to claim 28 or 32 , wherein in step (i), sample A contains ceramide as the lipid-soluble component to be measured. The method according to claim 28 or 32 , wherein in step (i), sample A contains an acetic acid bacteria product. 33. The method of claim 28 or 32 , wherein in step (i), sample A contains acetic acid. The method according to claim 38 , wherein in step (i), the content of acetic acid relative to the hydrophilic solvent in sample A is 0.02 to 15 w/v %. The method according to claim 28 or 32, wherein in step (i), the density of the hydrophilic solvent at 20° C. is 1.003 g/ml or more and 1.250 g/ml or less . 33. The method of claim 28 or 32 , wherein in step (i), the pH of the hydrophilic solvent is 4.5 or less. 33. The method of claim 28 or 32 , wherein in step (i) the total protein content of sample A is 15 w/v% or less. The method according to claim 28 or 32 , wherein in step (i), the turbidity of sample A at a wavelength of 660 nm is 1.100 or more. The method according to claim 28 or 32 , wherein in step (i), sample A is a food or beverage. The method according to claim 28 or 32 , wherein in step (ii), the medium a contains t-butyl methyl ether. The method according to claim 45 , wherein in step (ii), the medium a further contains methanol. 33. The method of claim 28 or 32 , comprising step (ii) followed by step (ii'):
(ii') mixing sample B for 10 to 1440 minutes The method according to claim 28 or 32 , wherein in step (iii), a precipitate is present in the lower layer sample C2. 33. The method of claim 28 or 32 , comprising step (iii) followed by step (iii'):
(iii') Further concentrating the upper layer sample C1