A concentration of the guanidinium thiocyanate of the component group (B) blended in the sample preservation solution is preferably 0.01 M to 5 M, more preferably 0.1 M to 5 mM, and particularly preferably 0.5 M to 4 M. Besides, a concentration of the Tris-HCl blended in the sample preservation solution is preferably 10 mM to 300 mM, more preferably 40 mM to 150 mM, and particularly preferably 50 mM to 100 mM. The pH of the Tris-HCL is preferably in a range of 7 to 9, and pH of 7.5 to 8.5 is more preferred, and pH of 8.0 is particularly preferred. A concentration of the EDTA blended in the sample preservation solution is preferably 1 mM to 50 mM, more preferably 10 mM to 50 mM, and particularly preferably 30 mM to 50 mM.

The sample preservation solution of the present invention may contain another component in addition to the above-described components as long as the actions and effects of the present invention are not impaired. Examples of another component include a colorant, a perfume, a dispersant, and a moisturizer.

The sample preserved in the sample preservation solution of the present invention is a biological or environmental sample. Examples of the biological sample include feces, rectal swab, urine, nasal discharge, sputum, saliva, tissue, blood and serum. Among these, although feces contains a large amount of microbes and organic substances, it can be stably preserved in the sample preservation solution of the present invention. Examples of the environmental sample include environmental water from of rivers, lakes and marshes, seas and the like, soil, wastewater, and water and sludge of a biological treatment tank obtained in sewage treatment.

In the present invention, components that are inhibited from changing in the amounts and contents in a sample by preserving the sample in the sample preservation solution are bile acids, phenols, indoles and organic acids. These components will now be described in detail.

Bile acids are steroid compounds biosynthesized in a living body from cholesterol. Bile acids stably preserved in the sample preservation solution of the present invention include primary bile acids, secondary bile acids, reaction intermediates thereof, and conjugated bile acids bound to amino acids. Specific examples of bile acids are not especially limited and include cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid, dehydrocholic acid, isodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycoursodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid, tauroursodeoxycholic acid, α muricholic acid, β muricholic acid, ω muricholic acid, tauro-α muricholic acid, tauro-β muricholic acid, 7-oxodeoxycholic acid, and 7-oxolithocholic acid.

Phenols stably preserved in the sample preservation solution of the present invention include phenols generated in a living body from tyrosine through the action of intestinal bacteria, and examples include, but are not limited to, phenol, p-cresol and 4-ethylphenol. Similarly, indoles stably preserved in the sample preservation solution of the present invention include indoles generated in a living body from tryptophan through the action of intestinal bacterial, and examples include, but are not limited to, indole and scatole.

Organic acids stably preserved in the sample preservation solution of the present invention mainly include short chain fatty acids generated in a living body through the action of intestinal bacteria, and widely include other carboxylic acids in addition to the short chain fatty acids. Examples include acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, isovaleric acid, caproic acid, formic acid, succinic acid, and lactic acid.

The sample preservation solution of the present invention is used with a sample to be preserved immersed or suspended therein for preservation. Thus, a metabolite can be stably preserved under a room temperature environment of 30° C. or less over at least 2 weeks. A volume ratio of an amount of the sample preservation solution to be used is preferably the sample:the sample preservation solution=1:3 or more, more preferably the sample the sample preservation solution=1:4 or more, and particularly preferably the sample:the sample preservation solution=1:5 or more.

The sample preservation solution of the present invention can stably preserve a metabolite such as bile acids described above and also suppress change of a microbial community structure contained in the sample. Therefore, the sample preservation solution can be used in various molecular biological methods using a DNA, such as real time PCR analysis, metagenomic analysis using a next generation sequencer, microbial community structure analysis such as T-RFLP analysis and DGGE analysis, DNA nucleotide sequence analysis of a biological cultured cell strain, RAPD analysis that is a method of DNA polymorphism analysis for identifying a strain, and microsatellite analysis. In this manner, since the analysis of a metabolite and the analysis of a microbial community structure can be performed using the same sample preserved in the sample preservation solution, the sample can be preserved/transported easily and simply, and in addition, these analysis can be definitely performed on the same sample, and thus, comprehensive evaluations from various aspects can be performed.

A storage temperature for preserving a sample using the sample preservation solution of the present invention is under a room temperature environment of 1 to 30° C., but the sample preservation solution of the present invention can stably preserve a metabolite and a microbial community structure also in a temperature range of 10 to 30° C., a temperature range of 15 to 30° C., a temperature range of 20 to 30° C., or a temperature range of 25 to 30° C.

Now, referring to FIGS. 1 to 3, an analysis device including the sample preservation solution, and a preservation container for preserving a sample will be described in detail.

First, the analysis device 1 according to a first embodiment illustrated in FIG. 1 will be described. The analysis device 1 of the present embodiment includes a bottomed cylindrical preservation container 3 having an opening at one end, the sample preservation solution S of the present invention contained in the preservation container 3, a cover member 2 for sealing the opening of the preservation container 3, and a sample collecting member 4 used for collecting a sample. The sample collecting member 4 has a spoon-shaped collecting portion 42, at a tip thereof, formed so that a prescribed amount of a sample can be scooped and held therein, and an axial portion 41 extending toward the opposite end. The axial portion 41 is connected on a tip side thereof to a rear surface of the cover member 2, and is formed so that the sample collecting member 4 can be manipulated with the cover member 2 grasped. The collecting portion 42 of the sample collecting member 4 is provided with two holes 43 penetrating a bottom thereof. The holes 43 provided in the collecting portion 42 are provided so as to cause the sample preservation solution S to come into contact with a sample C held in the collecting portion 42 through the holes 43 in the preservation container 3, and to cause the sample preservation solution S to flow into the bottom of the collecting portion 42. Thus, the sample C held in the collecting portion 42 is moved away from the bottom of the collecting portion 42 into the sample preservation solution S, and the sample C is dispersed and suspended in the sample preservation solution S, and therefore, the sample C can be preserved in a state of being definitely in contact with the sample preservation solution S of the present invention. Although the two holes 43 are provided on the bottom of the collecting portion 42 in the present embodiment, the number thereof is not especially limited, and a structure having no holes 43 may be employed.

The sample preservation solution S is, in a state of being used as illustrated in FIG. 1, contained in the preservation container 3 up to a position for completely immersing the collecting portion 42 of the sample collecting member 4. The sample preservation solution S is contained in the preservation container 3 in an amount with a volume ratio of preferably the sample C:the sample preservation solution S=1:3 or more, more preferably the sample C:the sample preservation solution S=1:4 or more, and particularly preferably the sample C:the sample preservation solution S=1:5 or more. In the present embodiment, for example, the volume of the collecting portion 42 of the sample collecting member 4 is 1 mL, while 5 mL of the sample preservation solution S is contained.

The analysis device 1 of FIG. 1 is used as follows: First, the cover member 2 of the analysis device 1 is opened, and the sample collecting member 4 is manipulated with the cover member 2 grasped to take the sample C in the collecting portion 42. The resultant sample collecting member 4 is put in the preservation container 3, the collecting portion 42 holding the sample C is immersed in the sample preservation solution S in the preservation container 3, and the preservation container 3 is sealed with the cover member 2. Thus, the sample C is stored in the analysis device 1 in a state of being immersed and suspended in the sample preservation solution S. After the storage, the sample can be preserved and transported at room temperature together with the analysis device 1.

Next, an analysis device 10 according to a second embodiment illustrated in FIGS. 2(A) to 3 will be described. The analysis device 10 of the present embodiment includes a bottomed cylindrical preservation container 3 having an opening at one end, a cover member 20 for sealing the opening of the preservation container 3, the sample preservation solution S of the present invention contained in a containment space within the cover member 20, a pressing member 5 capable of communicating the containment space within the cover member 20 with the preservation container 3 to move the sample preservation solution S into the preservation container 3, and a sample collecting member 4 used for collecting a sample. The sample collecting member 4 has a spoon-shaped collecting portion 42, at a tip thereof, formed so that a prescribed amount of a sample can be scooped and held therein, and an axial portion 41 extending toward the opposite end. A tip side of the axial portion 41 is connected to the inside of the pressing member 5, and the sample collecting member 4 is formed to be able to be manipulated with the outer circumferential surface of the pressing member 5 grasped. Besides, the collecting portion 42 of the sample collecting member 4 is provided with two holes 43 penetrating a bottom thereof. The function and action of the holes 43 are the same as those described in the first embodiment above.

Furthermore, as illustrated in FIGS. 2(A) and 2(B), the pressing member 5 incorporated between the cover member 20 and the preservation container 3 is provided with a pressing pin Sa on a side opposing the cover member 20. On the other hand, a bottom of the containment space within the cover member 20 is provided with a thin portion (not shown) formed to be breakable when pressed by the pressing pin Sa, so that when the preservation container 3 is sealed with the cover member 20, the pressing pin 5a of the pressing member 5 is formed to press the bottom of the cover member 20 to break the bottom to cause the sample preservation solution S in the containment space to leak out through the broken portion. The sample preservation solution S leaked out from the containment space within the cover member 20 flows through a gap of the pressing member 5 into the preservation container 3. In this manner, since a structure in which the sample preservation solution S is not precedently contained in the preservation container 3 is employed, there may be no spill or loss of the sample preservation solution S by mistake in putting the sample collecting member 4 in or out of the preservation container 3, and hence, the collected sample C can be definitely preserved in a prescribed amount of the sample preservation solution S. In addition, since the amount of the sample preservation solution S can be thus made constant, a weight of the sample C preserved in the preservation container 3 can be accurately calculated by measuring the weight of the analysis device 10, and thus, a concentration of a metabolite to be analyzed can be more accurately obtained.

The sample preservation solution S is, in a state of being used as illustrated in FIG. 3, contained in the containment space within the cover member 20 in an amount corresponding to a position for completely immersing the collecting portion 42 of the sample collecting member 4. The sample preservation solution S is contained in the cover member 20 in an amount with a volume ratio of preferably the sample C:the sample preservation solution S=1:3 or more, more preferably the sample C:the sample preservation solution S=1:4 or more, and particularly preferably the sample C:the sample preservation solution S=1:5 or more. In the present embodiment, for example, the volume of the collecting portion 42 of the sample collecting member 4 is 1 mL, while 5 mL of the sample preservation solution S is contained in the cover member 20.

The analysis device 10 of FIGS. 2(A) to 3 are used as follows: First, the cover member 20 of the analysis device 10 is opened, and the sample collecting member 4 is manipulated with the outer circumferential surface of the pressing member 5 grasped to take the sample C in the collecting portion 42. The resultant sample collecting member 4 is put in the preservation container 3 (with the sample preservation solution S not put in the preservation container 3 at this time point), and the pressing member 5 is fit toward the opening of the preservation container 3. Furthermore, the cover member 20 is caused to cover the pressing member 5, and the preservation container 3 is sealed with the cover member 20. At this point, since the pressing pin Sa of the pressing member 5 presses the bottom of the cover member 20, the bottom of the cover member 20 is broken, and the sample preservation solution S is leaked out through the bottom of the cover member 20 to flow through the pressing member 5 into the preservation container 3. In this manner, the collecting portion 42 holding the sample C is immersed in the sample preservation solution S within the preservation container 3, and the sample C is stored in a state of being immersed and suspended in the sample preservation solution S within the analysis device 10. After the storage, the sample can be preserved and transported at room temperature together with the analysis device 10.

Next, the present invention will be described in more detail with reference to examples; however, the present invention is not limited to these examples.

EXAMPLES
Example 1

1. Analysis (1) of Phenols Contained in Feces Sample

A feces sample cryopreserved in an ultra low temperature freezer at −80° C. immediately after collection was used, and feces samples A to C respectively obtained from 3 subjects were used. 1 g each of thawed feces samples was taken to be suspended in 5 mL of each of three sample preservation solutions, and the resultant was stored in a constant temperature bath at 30° C. for 14 days. As the three sample preservation solutions, a “0.5% Tween 20/4 M GTC solution” containing 0.5% by weight of Tween 20 (polyoxyethylene sorbitan monolaurate), 4 M guanidinium thiocyanate, 100 mM Tris-HCl, 40 mM EDTA (pH 8.0) and water; a “10 mM Na pyrophosphate/4 M GTC solution” containing 10 mM sodium pyrophosphate, 4 M guanidinium thiocyanate, 100 mM Tris-HCl, 40 mM EDTA (pH 8.0) and water; and a “4 M GTC solution” containing 4 M guanidinium thiocyanate, 100 mM Tris-HCl, 40 mM EDTA (pH 8.0) and water were used. In each of a sample suspension immediately before the storage at 30° C. (day 0) and a sample suspension after the storage (day 14), concentrations of phenol, p-cresol and 4-ethylphenol were measured by the following method. A prescribed amount of the sample suspension was precisely weighed in a centrifuge tube, and a phosphate buffer containing an internal standard substance (4-isopropylphenol) was added thereto to be mixed. The resultant was heated at 85° C. for 15 minutes, then cooled, and subjected to solvent extraction. The thus obtained crude extract solution was purified with a solid phase cartridge, and the resultant extract solution was used as a measurement specimen. The measurement specimen was used for measuring concentrations of phenol, p-cresol and 4-ethylphenol by a gas chromatography-mass spectrometry-selected ion monitoring method (GC-MS-SIM). Results are shown in Table 1 below and FIG. 4. It is noted that 4-ethylphenol was not detected in the feces sample B.

TABLE 1

Phenol
p-Cresol
4-Ethylphenol

Sample
(μg/g)
(μg/g)
(μg/g)

No.
day 0
day 14
day 0
day 14
day 0
day 14

0.5% Tween20
A
0.96
0.66
120.45
108.72
1.33
1.28

4M GTC solution

100%
69%
100%
90%
100%
96%

B
45.48
40.62
60.96
57.42
—
—

100%
89%
100%
94%

C
4.98
3.86
130.22
112.04
1.54
145

100%
78%
100%
86%
100%
94%

10 mM Na
A
0.96
0.77
120.45
113.03
1.33
0.95

pyrophosphate

100%
81%
100%
94%
160%
72%

4M GTC solution
B
45.48
45.73
60.96
63.92
—
—

100%
101%
100%
105%

C
4.98
4.12
130.22
113.23
1.54
1.57

100%
69%
100%
90%
100%
96%

4M GTC solution
A
0.96
0.56
120.45
88.02
1.33
0.94

100%
58%
100%
73%
100%
70%

B
45.48
37.41
60.96
50.32
—
—

100%
82%
100%
83%

C
4.98
3.67
130.22
111.95
1.54
1.58

100%
74%
100%
86%
100%
103%

In Table 1, a concentration of each substance to be measured is shown in an upper column, and a ratio (%) to a concentration before the storage (concentration on day 0) is shown in a lower column. It was revealed based on these results that phenol compounds in each feces sample can be stably retained after elapse of 14 days at 30° C. when the sample preservation solution in which Na pyrophosphate or Tween 20 is blended in addition to the 4 M GTC solution is used. Besides, FIG. 4 illustrates regression lines of the respective sample preservation solutions with the concentration of phenol in the feces sample before the storage (concentration on day 0) plotted on the abscissa, and with the concentration of phenol in the feces sample obtained on day 14 plotted on the ordinate. According to this graph, it was found that the sample preservation solution in which Na pyrophosphate or Tween 20 is blended in addition to the 4 M GTC solution has a larger slope of regression equation (close to 1), that even after the storage at 30° C. for 14 days, the concentrations of the phenols in the feces sample are not largely changed, and that the phenols can be thus quantitatively determined.

Example 2

2. Analysis (2) of Phenols Contained in Feces Sample

A feces sample cryopreserved in an ultra low temperature freezer at −80° C. immediately after collection was used, and this test was performed on a feces sample within 1 week after the cryopreservation at −80° C. Feces samples No. 1-1 to No. 1-6 obtained from 6 subjects were used. 1 g each of thawed feces samples was taken to be suspended in 5 mL of a sample preservation solution containing 100 mM sodium pyrophosphate, 4 M guanidinium thiocyanate, 100 mM Tris-HCl, 40 mM EDTA (pH 8.0) and water, and the resultant was stored in a constant temperature bath at 30° C. In each of a sample suspension immediately before the storage at 30° C. (day 0 raw feces) and sample suspensions after storage for 7 days (day 7), after storage for 14 days (day 14), and after storage for 28 days (day 28), concentrations of phenol, p-cresol and 4-ethylphenol were measured by a method similar to that of Example 1. A remaining portion of each feces sample was frozen at −80° C. again to be stored at −80° C. for 28 days, and then thawed to be suspended in the sample preservation solution in the same manner as described above. In this sample suspension (day 28 frozen feces), the concentrations of phenol, p-cresol and 4-ethylphenol were measured. Results of phenol are shown in Table 2 and FIG. 5, results of p-cresol are shown in Table 3 and FIG. 6, and results of 4-ethylphenol are shown in Table 4 and FIG. 7. It is noted that 4-ethylphenol was not detected in the feces samples No. 1-3, No. 1-5 and No. 1-6.

TABLE 2

Day 0

Day 28

Phenol
(raw

(frozen

(μg/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 1-1
5.00
5.31
4.04
5.23
4.44

No. 1-2
11.01
11.69
8.21
10.33
11.24

No. 1-3
7.65
9.17
6.31
7.49
8.13

No. 1-4
7.30
5.23
5.45
5.01
6.92

No. 1-5
1.76
1.53
2.03
1.78
1.37

No. 1-6
11.43
7.86
9.80
7.01
8.74

TABLE 3

Day 0

Day 28

p-Cresol
(raw

(frozen

(μg/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 1-1
18.69
16.17
12.31
13.53
20.84

No. 1-2
32.51
29.90
24.35
28.10
34.51

No. 1-3
88.70
92.41
66.93
63.38
75.14

No. 1-4
139.71
106.11
112.28
104.85
120.03

No. 1-5
106.36
87.89
100.20
77.13
99.65

No. 1-6
1.38
0.87
1.03
1.17
1.51

TABLE 4

Day 0

Day 28

4-Ethylphenol
(raw

(frozen

(μg/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 1-1
20.93
18.62
16.23
16.63
16.92

No. 1-2
0.48
0.45
0.41
0.50
0.48

No. 1-4
6.37
4.77
4.62
4.84
6.96

Tables 2 to 4 show the concentrations of each substance to be measured obtained at the respective measurement times. FIGS. 5 to 7 illustrate graphs of regression lines obtained based on these results with the concentration of each substance to be measured in the feces sample immediately before the storage at 30° C. (day 0 raw feces) plotted on the abscissa, and with the concentration of each substance to be measured in the feces sample obtained in each measurement time plotted on the ordinate. According to these graphs, the feces sample stored at 30° C. in the sample preservation solution of the present invention (100 mM sodium pyrophosphate+4 M GTC solution) was not largely changed in the concentrations as compared with the feces sample continuously stored at −80° C. for 28 days (day 28 (frozen feces)), and thus it was found that the feces sample can be stably stored at 30° C. for a long period of 28 days. Specifically, FIG. 5 reveals that similar analysis values are obtained for phenol through storage at 30° C. for 7 days (slope of regression equation: 0.84) and storage at −80° C. for 28 days (slope of regression equation: 0.90). It was also revealed that the analysis value tended to be slightly decreased through storage for 14 days or 28 days, but the slope of the regression equation remained at about 0.7. Besides, FIG. 6 reveals that similar analysis values were obtained for p-cresol through storage at 30° C. for 14 days (slope of regression equation: 0.80 to 0.85) and storage at −80° C. for 28 days (slope of regression equation: 0.85). It was also revealed that the analysis value tended to be slightly decreased through storage for 28 days, but the slope of the regression equation remained at about 0.7. Furthermore, FIG. 7 reveals that similar analysis values were obtained for 4-ethylphenol through storage at 30° C. for 28 days (slope of regression equation: 0.78 to 0.90) and storage at −80° C. for 28 days (slope of regression equation: 0.78).

Comparative Example 1

3. Analysis (3) of Phenols Contained in Feces Sample

A feces sample cryopreserved in an ultra low temperature freezer at −80° C. immediately after collection was used, and feces samples 1 to 3 respectively obtained from 3 subjects were used. 1 g each of thawed feces samples was taken to be suspended in 5 mL of each of four sample preservation solutions, and the resultant was stored in a constant temperature bath at 30° C. for 7 days. As the four sample preservation solutions, “20 mM Na pyrophosphate” containing 20 mM sodium pyrophosphate and water, “50 mM Na pyrophosphate” containing 50 mM sodium pyrophosphate and water, “100 mM Na pyrophosphate” containing 100 mM sodium pyrophosphate and water, and a “4 M GTC solution” containing 4 M guanidinium thiocyanate, 100 mM Tris-HCl, 40 mM EDTA (pH 8.0) and water were used. In each of a sample suspension immediately before the storage at 30° C. (day 0) and a sample suspension after the storage (day 7), concentrations of phenol, p-cresol and 4-ethylphenol were measured by a method similar to that of Example 1. Since 4-ethylphenol was not detected in the feces samples 1 to 3, measurement results of phenol and p-cresol are shown in Table 5 below.

TABLE 5

Sample
Phenol (μg/g)
p-Cresol (μg/g)

No.
day 0
day 7
day 0
day 7

20 mM
1
0.85
6.30
38.67
467.87

Na

100%
741%
100%
1210%

pyrophosphate
2
1.89
5.42
88.17
236.80

100%
287%
100%
269%

3
0.33
22.18
78.93
233.68

100%
6686%
100%
296%

50 mM
1
0.84
13.39
37.83
480.31

Na

100%
1586%
100%
1270%

pyrophosphate
2
1.98
6.20
90.93
334.43

100%
314%
100%
368%

3
0.42
71.55
66.83
296.47

100%
16848%
100%
444%

100 mM
1
0.86
374.90
43.10
226.35

Na

100%
43609%
100%
525%

pyrophosphate
2
2.10
7.93
97.50
344.91

100%
377%
100%
354%

3
0.41
75.71
66.28
270.30

100%
18384%
100%
408%

4M
1
2.72
1.88
44.18
41.63

GTC

100%
69%
100%
94%

solution
2
5.05
2.12
103.12
85.34

100%
42%
100%
83%

3
2.49
4.16
80.59
97.07

100%
167%
100%
120%

In Table 5, a concentration of each substance to be measured is shown in an upper column, and a ratio (%) to a concentration before the storage (concentration on day 0) is shown in a lower column. It was revealed based on these results that the phenol compounds in each feces sample were largely changed in the concentrations as compared with that before the storage when the sample preservation solution singly containing sodium pyrophosphate was used. As the concentration of sodium pyrophosphate blended was higher, the degree of the concentration change tended to be larger, and therefore, it was presumed that sodium pyrophosphate may possibly improve the growth of microbes contained in a feces sample. Examples 1 and 2 reveal, however, that a sample preservation solution capable of stably preserving phenols under a condition of 30° C. can be obtained by combining sodium pyrophosphate with the “4 M GTC solution” containing 4 M guanidinium thiocyanate, 100 mM Tris-HCl and 40 mM EDTA (pH 8.0).

Example 3

4. Analysis (1) of Bile Acids Contained in Feces Sample

Experimental Method

A feces sample cryopreserved in an ultra low temperature freezer at −80° C. immediately after collection was used, and feces samples A to D respectively obtained from 4 subjects were used. 1 g each of thawed feces samples was taken to be suspended in 5 mL of each of three sample preservation solutions, and the resultant was stored in a constant temperature bath at 30° C. for 14 days. As the three sample preservation solutions, the same solutions as those described in Example 1 were used. In each of a sample suspension immediately before the storage at 30° C. (day 0) and a sample suspension after the storage (day 14), concentrations of cholic acid, chenodeoxycholic acid and ursodeoxycholic acid were measured based on an analysis method of Kakiyama et al., (Kakiyama G, Muto A, Takei H, Nittono H, Murai T et al., A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS, J Lipid Res., 2014, Vol. 55, pp. 978-990). Incidentally, a pretreatment method for each specimen was modified as follows. A prescribed amount of the sample suspension was precisely weighed in a bead tube, and a 9-fold amount of a sodium acetate buffer/ethanol mixed solution was added thereto to pulverize the sample suspension. The resultant was heated at 85° C. for 30 minutes, cooled, and then centrifuged at 18,400×g for 10 minutes. The thus obtained supernatant was collected, diluted by 4 times with ultrapure water, and supplied to a solid phase extraction column (Bond Elut C18 cartridge, Agilent Technologies Japan, Ltd.) for solid phase extraction. The thus obtained extract was solidified by drying, the resultant was dissolved in 50% ethanol to be filtered through a hydrophilic PTFE filter having a hole size of 0.2 μm, and an internal standard solution was added to the resultant filtrate to obtain a measurement specimen solution. The concentrations of cholic acid, chenodeoxycholic acid and ursodeoxycholic acid in the measurement specimen solution were measured with a liquid chromatography-quadrupole time-of-flight mass spectrometer (LC-QTOF/MS). Results are shown in Table 6 below and FIGS. 8 and 9. It is noted that ursodeoxycholic acid was not detected in the feces samples A and C.

TABLE 6

Cholic acid
Chenodeoxycholic acid
Ursodeoxycholic acid

Sample
(μmol/g)
(μmol/g)
(μmol/g)

No.
day 0
day 14
day 0
day 14
day 0
day 14

0.5% Tween20
A
0.05
0.03
0.07
0.05
—
—

4M GTC solution

100%
54%
100%
68%

B
5.03
4.37
2.90
2.54
0.93
0.79

100%
87%
100%
88%
100%
85%

C
0.06
0.03
0.08
0.05
—
—

100%
53%
100%
65%

D
0.84
0.66
0.45
0.39
0.64
0.55

100%
79%
100%
86%
100%
85%

10 mM Na
A
0.05
0.03
0.07
0.05
—
—

pyrophosphate

100%
52%
100%
67%

4M GTC solution
B
5.03
4.84
2.90
2.88
0.93
0.67

100%
96%
100%
99%
100%
94%

C
0.06
0.03
0.08
0.05
—
—

100%
52%
100%
63%

D
0.84
0.73
0.45
0.43
0.64
0.57

100%
87%
100%
84%
100%
89%

4M GTC solution
A
0.05
0.03
0.07
0.05
—
—

100%
51%
100%
68%

B
5.03
3.71
2.90
2.26
0.93
0.71

100%
74%
100%
78%
100%
76%

C
0.06
0.03
0.08
0.05
—
—

100%
52%
180%
62%

D
0.84
0.61
0.45
0.36
0.64
0.51

100%
73%
100%
80%
100%
79%

In Table 6, a concentration of each substance to be measured is shown in an upper column, and a ratio (%) to a concentration before the storage (concentration on day 0) is shown in a lower column. It was revealed based on these results that bile acid compounds such as cholic acid, chenodeoxycholic acid and ursodeoxycholic acid in the feces sample can be stably retained after elapse of 14 days at 30° C. when the sample preservation solution in which Na pyrophosphate or Tween 20 is blended in addition to a 4 M GTC solution is used. Besides, FIGS. 8 and 9 illustrate regression lines of the respective sample preservation solutions with the concentration of cholic acid or chenodeoxycholic acid in the feces sample before the storage (concentration on day 0) plotted on the abscissa, and with the concentration of cholic acid or chenodeoxycholic acid in the feces sample obtained on day 14 plotted on the ordinate. According to these graphs, it was found that the sample preservation solution in which Na pyrophosphate or Tween 20 is blended in addition to the 4 M GTC solution has a larger slope of regression equation (close to 1), that even after the storage at 30° C. for 14 days, the concentrations of the bile acids in the feces sample are not largely changed, and that the bile acids can be thus quantitatively determined.

Example 4

5. Analysis (2) of Bile Acids Contained in Feces Sample

A feces sample cryopreserved in an ultra low temperature freezer at −80° C. immediately after collection was used, and this test was performed on a feces sample within 1 week after the cryopreservation at −80° C. Feces samples No. 2-1 to No. 2-5 respectively obtained from different 5 subjects were used. 1 g each of thawed feces samples was taken to be suspended in 5 mL of a sample preservation solution containing 100 mM sodium pyrophosphate, 4 M guanidinium thiocyanate, 100 mM Tris-HCl, 40 mM EDTA (pH 8.0) and water, and the resultant was stored in a constant temperature bath at 30° C. In each of a sample suspension immediately before the storage at 30° C. (day 0 raw feces) and sample suspensions after storage for 7 days (day 7), after storage for 14 days (day 14), and after storage for 28 days (day 28), concentrations of cholic acid, chenodeoxycholic acid and ursodeoxycholic acid were measured by a method similar to that of Example 3. A remaining portion of each feces sample was frozen at −80° C. again to be stored at −80° C. for 28 days, and then thawed to be suspended in the sample preservation solution in the same manner as described above. In this sample suspension (day 28 frozen feces), the concentrations of cholic acid, chenodeoxycholic acid and ursodeoxycholic acid were measured. Results of cholic acid are shown in Table 7 and FIG. 10, results of chenodeoxycholic acid are shown in Table 8 and FIG. 11, and results of ursodeoxycholic acid are shown in Table 9 and FIG. 12. It is noted that ursodeoxycholic acid was not detected in the feces sample No. 2-5, and hence a feces sample No. 2-6 obtained from a different subject was used to perform a similar test.

TABLE 7

Cholic
Day 0

Day 28

acid
(raw

(frozen

(μmol/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 2-1
0.70
0.57
0.64
0.59
0.64

No. 2-2
2.07
2.06
2.04
1.71
2.01

No. 2-3
1.90
1.89
1.79
1.62
1.74

No. 2-4
0.51
0.41
0.38
0.42
0.46

No. 2-5
0.11
0.10
0.18
0.15
0.10

TABLE 8

Chenodeoxycholic
Day 0

Day 28

acid
(raw

(frozen

(μmol/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 2-1
0.53
0.45
0.52
0.42
0.55

No. 2-2
0.88
0.67
0.76
0.56
0.81

No. 2-3
0.92
0.70
0.71
0.58
0.70

No. 2-4
0.41
0.38
0.41
0.32
0.38

No. 2-5
0.11
0.15
0.16
0.14
0.10

TABLE 9

Ursodeoxycholic
Day 0

Day 28

acid
(raw

(frozen

(μmol/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 2-1
0.18
0.15
0.16
0.19
0.14

No. 2-2
1.18
1.05
1.07
0.82
1.07

No. 2-3
0.28
0.26
0.25
0.25
0.23

No. 2-4
0.32
0.32
0.31
0.28
0.29

No. 2-6
0.19
0.19
0.16
0.19
0.17

Tables 7 to 9 show the concentrations of each substance to be measured obtained at the respective measurement times. FIGS. 10 to 12 illustrate graphs of regression lines obtained based on these results with the concentration of each substance to be measured in the feces sample immediately before the storage at 30° C. (day 0 raw feces) plotted on the abscissa, and with the concentration of each substance to be measured in the feces sample obtained in each measurement time plotted on the ordinate. According to these graphs, the feces sample stored at 30° C. in the sample preservation solution of the present invention (100 mM sodium pyrophosphate+4 M GTC solution) was not largely changed in the concentrations as compared with the feces sample continuously stored at −80° C. for 28 days (day 28 (frozen feces)), and thus it was found that the feces sample can be stably stored at 30° C. for a long period of 28 days. Specifically, FIG. 10 reveals that similar analysis values were obtained for cholic acid through storage at 30° C. for 14 days (slope of regression equation: 0.99) and storage at −80° C. for 28 days (slope of regression equation: 0.96), and that the slope of the regression equation remained at 0.82 also through the storage at 30° C. for 28 days. Besides, FIG. 11 reveals that similar analysis values were obtained for chenodeoxycholic acid through storage at 30° C. for 14 days (slope of regression equation: 0.67 to 0.71) and storage at −80° C. for 28 days (slope of regression equation: 0.8). Furthermore, FIG. 12 reveals that similar analysis values were obtained for ursodeoxycholic acid through storage at 30° C. for 14 days (slope of regression equation: 0.88 to 0.91) and storage at −80° C. for 28 days (slope of regression equation: 0.93).

Example 5

6. Analysis (1) of Organic Acids Contained in Feces Sample

A feces sample cryopreserved in an ultra low temperature freezer at −80° C. immediately after collection was used, and feces samples A to C respectively obtained from 3 subjects were used. 1 g each of thawed feces samples was taken to be suspended in 5 mL of each of three sample preservation solutions, and the resultant was stored in a constant temperature bath at 30° C. for 14 days. As the three sample preservation solutions, the same solutions as those described in Example 1 were used. In each of a sample suspension immediately before the storage at 30° C. (day 0) and a sample suspension after the storage (day 14), concentrations of acetic acid, propionic acid and n-butyric acid were measured by the following method. A prescribed amount of the sample suspension was precisely weighed in a bead tube, and an extract solution was added thereto to be mixed. The resultant was heated at 85° C. for 15 minutes, and after cooling, the sample suspension was pulverized with beads and then centrifuged at 18,400×g for 10 minutes. The thus obtained supernatant was collected to be filtered through a membrane filter having a hole size of 0.20 μm to obtain a measurement specimen solution. The concentrations of acetic acid, propionic acid and n-butyric acid in the measurement specimen solution were measured with a high performance liquid chromatograph (product of Shimadzu Corporation, organic acid analysis system).

TABLE 10

Acetic acid
Propionic acid
n-Butyric acid

Sample
(mg/g)
(mg/g)
(mg/g)

No.
day 0
day 14
day 0
day 14
day 0
day 14

0.5% Tween20
A
1.00
1.06
0.52
0.50
0.34
0.33

4M GTC solution

100%
108%
100%
96%
100%
98%

B
4.80
4.71
2.02
1.98
1.06
1.05

100%
98%
100%
98%
100%
99%

C
1.37
1.29
0.99
0.90
1.81
1.64

100%
94%
100%
91%
100%
91%

10 mM Na
A
1.00
1.14
0.52
0.47
0.34
0.31

pyrophosphate

100%
114%
100%
91%
190%
91%

4M GTC solution
B
4.80
5.06
2.02
2.10
1.06
1.10

100%
106%
100%
104%
100%
104%

C
1.37
1.34
0.99
0.93
1.81
1.69

100%
98%
100%
94%
100%
94%

4M GTC solution
A
1.00
1.19
0.52
0.53
0.34
0.36

100%
119%
100%
103%
100%
108%

B
4.80
5.20
2.02
2.16
1.06
1.12

100%
108%
100%
107%
100%
105%

C
1.37
1.27
0.99
0.89
1.81
1.62

100%
93%
100%
91%
100%
90%

In Table 10, a concentration of each substance to be measured is shown in an upper column, and a ratio (%) to a concentration before the storage (concentration on day 0) is shown in a lower column. It was revealed based on these results that organic acid compounds in each feces sample can be stably retained after elapse of 14 days at 30° C. when the sample preservation solution in which Na pyrophosphate or Tween 20 is blended in addition to a 4 M GTC solution is used.

Example 6

7. Analysis (2) of Organic Acids Contained in Feces Sample

A feces sample cryopreserved in an ultra low temperature freezer at −80° C. immediately after collection was used, and this test was performed on a feces sample within 1 week after the cryopreservation at −80° C. Feces samples No. 3-1 to No. 3-14 respectively obtained from 14 subjects were used. 1 g each of thawed feces samples was taken to be suspended in 5 mL of a sample preservation solution containing 100 mM sodium pyrophosphate, 4 M guanidinium thiocyanate, 100 mM Tris-HCl, 40 mM EDTA (pH 8.0) and water, and the resultant was stored in a constant temperature bath at 30° C. In each of a sample suspension immediately before the storage at 30° C. (day 0 raw feces) and sample suspensions after storage for 7 days (day 7), after storage for 14 days (day 14), and after storage for 28 days (day 28), concentrations of acetic acid, propionic acid, n-butyric acid, n-valeric acid and iso-valeric acid were measured by HPLC in the same manner as in Example 5. A remaining portion of each feces sample was frozen at −80° C. again to be stored at −80° C. for 28 days, and then thawed to be suspended in the sample preservation solution in the same manner as described above. In this sample suspension (day 28 frozen feces), the concentrations of acetic acid, propionic acid, n-butyric acid, n-valeric acid and iso-valeric acid were measured. Results of acetic acid are shown in Table 11 and FIG. 13, results of propionic acid are shown in Table 12 and FIG. 14, results of n-butyric acid are shown in Table 13 and FIG. 15, and results of n-valeric acid are shown in Table 14 and FIG. 16, and results of iso-valeric acid are shown in Table 15 and FIG. 17. It is noted that n-butyric acid and n-valeric acid were not detected in the feces samples No. 3-10 to 3-14, and iso-valeric acid was not detected in the feces samples No. 3-3, 3-11, 3-13 and 3-14.

TABLE 11

Acetic
Day 0

Day 28

acid
(raw

(frozen

(mg/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 3-1
2.14
2.41
2.60
2.57
2.16

No. 3-2
3.03
3.01
3.09
3.10
2.96

No. 3-3
4.78
4.63
4.69
4.78
5.12

No. 3-4
3.69
3.68
3.82
3.83
3.76

No. 3-5
2.90
3.06
3.23
3.25
3.30

No. 3-6
2.51
2.57
2.67
2.95
2.82

No. 3-7
3.71
3.43
3.45
3.56
3.76

No. 3-8
3.73
3.67
3.94
3.91
3.83

No. 3-9
3.04
2.97
3.12
3.27
2.97

No. 3-10
2.54
2.66
2.65
2.79
2.74

No. 3-11
2.05
2.04
2.32
2.44
1.78

No. 3-12
1.18
1.29
1.46
1.79
1.40

No. 3-13
3.24
3.25
3.41
3.47
3.18

No. 3-14
5.12
5.13
5.57
5.55
5.22

TABLE 12

Day 0

Day 28

Propionic acid
(raw

(frozen

(mg/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 3-1
1.12
1.14
1.20
1.12
1.13

No. 3-2
2.18
2.10
2.12
2.09
2.11

No. 3-3
1.34
1.27
1.27
1.32
1.34

No. 3-4
1.83
1.77
1.86
1.75
1.83

No. 3-5
1.25
1.31
1.32
1.31
1.33

No. 3-6
1.70
1.65
1.66
1.77
1.74

No. 3-7
1.19
1.12
1.14
1.15
1.13

No. 3-8
1.14
1.09
1.19
1.16
1.14

No. 3-9
1.83
1.70
1.71
1.74
1.77

No. 3-10
1.06
1.06
0.95
0.99
1.03

No. 3-11
1.33
1.10
1.15
1.10
1.10

No. 3-12
0.80
0.79
0.87
0.79
0.78

No. 3-13
1.25
1.21
1.26
1.27
1.20

No. 3-14
1.42
1.39
1.56
1.49
1.45

TABLE 13

n-Butyric
Day 0

Day 28

acid
(raw

(frozen

(mg/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 3-1
1.11
1.12
1.18
1.07
1.07

No. 3-2
1.12
1.06
1.07
1.03
1.05

No. 3-3
2.00
1.94
1.93
1.93
2.02

No. 3-4
1.35
1.29
1.23
1.22
1.33

No. 3-5
1.50
1.58
1.59
1.54
1.61

No. 3-6
0.85
0.81
0.82
0.80
0.83

No. 3-7
1.25
1.19
1.16
1.16
1.18

No. 3-8
1.32
1.23
1.36
1.30
1.30

No. 3-9
0.76
0.70
0.69
0.63
0.75

TABLE 14

n-Valeric
Day 0

Day 28

acid
(raw

(frozen

(mg/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 3-1
0.24
0.23
0.24
0.20
0.23

No. 3-2
0.15
0.14
0.15
0.11
0.15

No. 3-3
0.16
0.16
0.14
0.15
0.16

No. 3-4
0.34
0.32
0.33
0.27
0.33

No. 3-5
0.22
0.21
0.23
0.19
0.22

No. 3-6
0.20
0.17
0.16
0.18
0.19

No. 3-7
0.32
0.30
0.29
0.29
0.28

No. 3-8
0.13
0.13
0.13
0.13
0.14

No. 3-9
0.23
0.22
0.20
0.20
0.22

TABLE 15

iso-Valeric
Day 0

Day 28

acid
(raw

(frozen

(mg/g)
feces)
Day 7
Day 14
Day 28
feces)

No. 3-1
0.41
0.42
0.47
0.39
0.41

No. 3-2
0.14
0.14
0.12
0.12
0.13

No. 3-4
0.14
0.13
0.14
0.13
0.15

No. 3-5
0.30
0.29
0.32
0.25
0.31

No. 3-6
0.23
0.22
0.24
0.20
0.21

No. 3-7
0.19
0.18
0.17
0.16
0.17

No. 3-8
0.12
0.12
0.13
0.13
0.14

No. 3-9
0.24
0.22
0.19
0.21
0.21

No. 3-10
0.43
0.42
0.36
0.36
0.41

No. 3-12
0.28
0.26
0.26
0.23
0.26

Tables 11 to 15 show the concentrations of each substance to be measured obtained at the respective measurement times. FIGS. 13 to 17 illustrate graphs of regression lines obtained based on these results with the concentration of each substance to be measured in the feces sample immediately before the storage at 30° C. (day 0 raw feces) plotted on the abscissa, and with the concentration of each substance to be measured in the feces sample obtained in each measurement time plotted on the ordinate. According to these graphs, the feces sample stored at 30° C. in the sample preservation solution of the present invention (100 mM sodium pyrophosphate+4 M GTC solution) was not largely changed in the concentrations as compared with the feces sample continuously stored at −80° C. for 28 days (day 28 (frozen feces)), and thus it was found that the feces sample can be stably stored at 30° C. for a long period of 28 days. Specifically, FIG. 13 reveals that similar analysis values were obtained for acetic acid through storage at 30° C. for 28 days (slope of regression equation: 0.89 to 0.95) and storage at −80° C. for 28 days (slope of regression equation: 1.0). Besides, FIG. 14 reveals that similar analysis values were obtained for propionic acid through storage at 30° C. for 28 days (slope of regression equation: 0.92 to 0.95) and storage at −80° C. for 28 days (slope of regression equation: 0.99). Similarly, FIG. 15 reveals that similar analysis values were obtained for n-butyric acid through storage at 30° C. for 28 days (slope of regression equation: 1.00 to 1.04) and storage at −80° C. for 28 days (slope of regression equation: 1.06). FIG. 16 reveals that similar analysis values were obtained for n-valeric acid through storage at 30° C. for 28 days (slope of regression equation: 0.81 to 0.95) and storage at −80° C. for 28 days (slope of regression equation: 0.85). Furthermore, FIG. 17 reveals that similar analysis values were obtained for iso-valeric acid through storage at 30° C. for 14 days (slope of regression equation: 0.86 to 1.02) and storage at −80° C. for 28 days (slope of regression equation: 0.98).

Comparative Example 2

8. Analysis (3) of Organic Acids Contained in Feces Sample

A feces sample cryopreserved in an ultra low temperature freezer at −80° C. immediately after collection was used, and feces samples 1 to 3 respectively obtained from 3 subjects were used. 1 g each of thawed feces samples was taken to be suspended in 5 mL of each of four sample preservation solutions, and the resultant was stored in a constant temperature bath at 30° C. for 7 days. As the four sample preservation solutions, “20 mM Na pyrophosphate” containing 20 mM sodium pyrophosphate and water, “50 mM Na pyrophosphate” containing 50 mM sodium pyrophosphate and water, “100 mM Na pyrophosphate” containing 100 mM sodium pyrophosphate and water, and a “4 M GTC solution” containing 4 M guanidinium thiocyanate, 100 mM Tris-HCl, 40 mM EDTA (pH 8.0) and water were used. In each of a sample suspension immediately before the storage at 30° C. (day 0) and a sample suspension after the storage (day 7), concentrations of acetic acid, propionic acid, n-butyric acid and iso-valeric acid were measured by a method similar to that of Example 5. Results are shown in Table 16 below.

TABLE 16

Acetic acid
Propionic acid
n-Butyric acid
iso-Valeric acid

Sample
(mg/g)
(mg/g)
(mg/g)
(mg/g)

No.
day 0
day 7
day 0
day 7
day 0
day 7
day 0
day 7

20 mM
1
4.06
16.92
1.89
4.70
1.36
12.03
0.16
1.76

Na pyrophosphate

100%
417%
100%
249%
100%
882%
100%
1093%

2
3.86
15.90
1.36
8.47
1.66
5.58
0.33
2.30

100%
412%
100%
621%
100%
336%
100%
697%

3
1.47
10.39
0.77
3.78
0.35
2.81
0.26
0.78

100%
707%
100%
493%
100%
806%
100%
295%

50 mM
1
4.10
19.42
1.91
5.29
1.38
12.97
0.17
2.43

Na pyrophosphate

100%
474%
100%
277%
100%
941%
100%
1425%

2
3.30
20.49
1.23
11.06
1.48
6.79
0.27
2.80

100%
622%
100%
897%
100%
460%
100%
1032%

3
1.46
12.00
0.77
3.87
0.33
2.90
0.28
2.73

100%
820%
100%
506%
100%
869%
100%
987%

100 mM
1
3.90
21.05
1.86
3.98
1.32
10.51
0.17
2.82

Na pyrophosphate

100%
540%