Quantitative method for bacteria containing diaminopimelic acid

Abstract

The object of the present invention is to provide a novel method for quantifying bacteria containing diaminopimelic acid in a test sample. The present invention relates to a method for quantifying bacteria, which is a method for quantifying bacteria containing diaminopimelic acid in a test sample, and is characterized by including a step of quantifying the bacteria containing diaminopimelic acid using the amount of diaminopimelic acid derived from the bacteria containing diaminopimelic acid in the test sample as an index.

Description

Technical Field

[0001] The present invention relates to a method for quantifying bacteria containing diaminopimelic acid in a sample to be tested. Background Art

[0002] Due to the improvement of health awareness, etc., lactic acid bacteria and the like are added to foods. Such bacteria are sometimes added as live bacteria (viable bacteria) and sometimes added as dead bacteria (non-viable bacteria). For example, when formulating lactic acid bacteria and other bacteria as functional-related components in functional labeled foods and the like, it is important to determine the number of viable bacteria and non-viable bacteria contained in the product.

[0003] As a method for quantifying viable bacteria contained in a sample, methods such as a method of culturing using an agar medium and then measuring colonies (culture method) are used. For example, Patent Document 1 describes a viable bacteria count method characterized in that, as a method for measuring the viable bacteria count of microorganisms contained in a sample, it has a step of suspending the sample using a dilution liquid for suspension, and a step of measuring the viable bacteria count of the microorganisms contained in the above suspension, and it is described that the viable bacteria count of the microorganisms contained in the suspension can be measured by the culture method of culturing on an agar medium.

[0004] On the other hand, in the case of a powder of dead bacteria or a product to which it is added, since the added bacteria are dead bacteria, the number of bacteria cannot be measured by the culture method. When measuring the number of bacteria in a sample that cannot be measured by the culture method, the total number of bacteria is measured. The measurement of the total number of bacteria mainly uses a method using a microscope, and representative methods such as the DAPI (fluorescence) staining method are known. In this method, for a sample solution obtained by suspending a sample in dilution water, the morphology of cells is observed and measured under a microscope to obtain the number of bacteria. In the case of the DAPI staining method, since dead bacteria and viable bacteria cannot be distinguished, the measurement result is treated as the total number of bacteria (total number of all bacteria).

[0005] Patent Documents

[0006] Patent Document 1: International Publication No. 2009 / 157316 Summary of the Invention

[0007] Among lactic acid bacteria, there are bacteria whose cell walls contain diaminopimelic acid (hereinafter also referred to as DAP). Such lactic acid bacteria containing diaminopimelic acid are added to foods and the like. However, a method for quantifying bacteria containing diaminopimelic acid in a sample to be tested has not been studied.

[0008] In addition, when the test sample contains bacteria that contain DAP as well as bacteria that do not contain DAP, for the quantification of bacteria that contain DAP, for example, if the bacteria that contain DAP are viable bacteria and the bacteria that do not contain DAP are dead bacteria, it can be carried out by the culture method as described above. When in the test sample containing bacteria that contain DAP and bacteria that do not contain DAP, both the bacteria that contain DAP and the bacteria that do not contain DAP are viable bacteria, it is considered that as long as the bacteria that contain DAP can be selectively cultured, the sample is cultured under this condition, and by measuring the formed colonies, the bacteria that contain DAP can be quantified.

[0009] On the other hand, when the bacteria that contain DAP are viable bacteria but the bacteria that do not contain DAP cannot be selectively cultured, or when the bacteria that contain DAP are dead bacteria, the number of bacteria that contain DAP in the test sample containing bacteria that contain DAP and bacteria that do not contain DAP cannot be measured by the culture method. For example, when the bacteria that contain DAP are dead bacteria and the bacteria that do not contain DAP are viable bacteria, it is considered to obtain the number of dead bacteria that contain DAP by subtracting the number of viable bacteria from the total number of bacteria in the test sample, but there is a problem that the measurement methods of the number of viable bacteria and the total number of bacteria are different. A method that can selectively quantify the bacteria that contain DAP regardless of whether they are dead bacteria or viable bacteria in the test sample containing bacteria that contain DAP and bacteria that do not contain DAP has not been studied yet.

[0010] An object of the present invention is to provide a novel method that can quantify bacteria that contain diaminopimelic acid contained in a test sample.

[0011] The inventors of the present invention conducted in-depth research to solve the above problems and found that there is a high correlation between the amount of diaminopimelic acid from lactic acid bacteria that contain diaminopimelic acid and the number of lactic acid bacteria. In the quantification of bacteria that contain diaminopimelic acid, diaminopimelic acid can be used as an index.

[0012] That is, the present invention relates to the following analysis methods.

[0013] [1] A method for quantifying bacteria, which is a method for quantifying bacteria that contain diaminopimelic acid contained in a test sample, characterized by comprising a step of quantifying the bacteria that contain diaminopimelic acid using the amount of diaminopimelic acid from the bacteria that contain diaminopimelic acid in the test sample as an index.

[0014] [2] The method for quantifying bacteria according to [1], characterized in that the test sample contains bacteria that do not contain diaminopimelic acid.

[0015] [3] The quantification method according to [1] or [2], characterized in that the bacteria that contain diaminopimelic acid are dead bacteria.

[0016] [4] The quantification method according to any one of [1] to [3], characterized in that the bacterium containing diaminopimelic acid is at least one lactic acid bacterium selected from Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus fabifermentans, Lactobacillus vaccinostercus, Lactobacillus hokkaidonensis, Lactobacillus oligofermentans, Lactobacillus suebicus, Lactobacillus mali, and Lactobacillus divergens.

[0017] [5] The quantification method according to any one of [1] to [4], characterized in that the bacterium containing diaminopimelic acid is Lactobacillus pentosus.

[0018] According to the present invention, a novel method can be provided, which can quantify the bacterium containing diaminopimelic acid contained in a test sample. When using the quantification method of the present invention, for example, when the test sample contains, in addition to the bacterium containing diaminopimelic acid, a bacterium not containing diaminopimelic acid, the bacterium containing diaminopimelic acid in the sample can be selectively quantified. The quantification method of the present invention is useful for the quantification of the bacterium containing diaminopimelic acid contained in foods, pharmaceuticals, etc., and the quality control of products containing the bacterium. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A is a graph showing the time-dependent change in the number of bacteria of Lactobacillus S-PT84 based on the culture time. Figure 1 B is a graph showing the time-dependent change in the amount of diaminopimelic acid based on the culture time.

[0020] Figure 2 To show Figure 1 The number of bacteria of Lactobacillus S-PT84 (number / g) on the vertical axis of A and Figure 1 A graph showing the correlation between the amount of diaminopimelic acid (mg / 100 g) on the vertical axis of B.

[0021] Figure 3A chart showing the correlation between the weight of the Lactobacillus S-PT84 raw material and the amount of diaminopimelic acid contained in the raw material. Detailed implementation mode

[0022] The method for quantifying the bacteria of the present invention is a method for quantifying the bacteria containing diaminopimelic acid contained in a test sample, characterized by comprising a step of quantifying the bacteria containing diaminopimelic acid using the amount of diaminopimelic acid of the bacteria containing diaminopimelic acid from the test sample as an index.

[0023] The test sample of the present invention is usually a sample containing bacteria containing diaminopimelic acid.

[0024] In this specification, diaminopimelic acid (DAP) refers to 2,6-diaminopimelic acid.

[0025] Examples of the bacteria containing diaminopimelic acid include lactic acid bacteria whose cell wall components contain diaminopimelic acid, such as Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus fabifermentans, Lactobacillus vaccinostercus, Lactobacillus hokkaidonensis, Lactobacillus oligofermentans, Lactobacillus suebicus, Lactobacillus mali, Lactobacillus divergens and other lactic acid bacteria.

[0026] The bacteria containing diaminopimelic acid may be one kind or two or more kinds, but preferably one kind. In one mode of the present invention, the bacteria containing diaminopimelic acid is preferably Lactobacillus pentosus.

[0027] The bacteria containing diaminopimelic acid can be viable bacteria or dead bacteria. Since the amount of diaminopimelic acid derived from the bacteria containing diaminopimelic acid in the test sample is related to the number of the bacteria, the number of the bacteria containing diaminopimelic acid (quantifying the number of bacteria) can be determined using the amount of diaminopimelic acid as an index, and the bacteria can be quantified. The quantification method of the present invention is useful for determining the number of bacteria containing diaminopimelic acid. Since the bacteria quantification method of the present invention quantifies the bacteria containing diaminopimelic acid using the amount of diaminopimelic acid derived from the bacteria containing diaminopimelic acid in the test sample as an index, either viable bacteria or dead bacteria can be used, and the bacteria can be quantified.

[0028] The above test sample may also contain bacteria that do not contain diaminopimelic acid. The method of the present invention is useful for quantifying the bacteria containing diaminopimelic acid in a test sample containing both bacteria containing diaminopimelic acid and bacteria that do not contain diaminopimelic acid.

[0029] The bacteria without diaminopimelic acid are not particularly limited. Examples of the bacteria without diaminopimelic acid include, for example, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus coryniformis, Lactobacillus sakei, Lactobacillus brevis, Lactobacillus buchneri and other lactic acid bacteria; Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium adolescentis and other bifidobacteria. The bacteria without diaminopimelic acid can be one kind or two or more kinds, but preferably one kind. In one mode, as the bacteria without diaminopimelic acid, lactic acid bacteria, bifidobacteria, etc. without diaminopimelic acid are preferred, and bifidobacteria are more preferred. In one mode, the test sample can also be a sample containing bacteria with diaminopimelic acid and bifidobacteria and / or lactic acid bacteria (preferably bifidobacteria or lactic acid bacteria) without diaminopimelic acid. The bacteria without diaminopimelic acid can be live bacteria or dead bacteria. When the method of the present invention is used for the quantification of bacteria in a test sample containing bacteria with diaminopimelic acid and bacteria without diaminopimelic acid, either live bacteria or dead bacteria of the bacteria without diaminopimelic acid can be used, and the bacteria with diaminopimelic acid can be selectively quantified using the amount of diaminopimelic acid from the bacteria with diaminopimelic acid in the test sample as an index.

[0030] In one mode, the bacteria containing diaminopimelic acid are preferably dead bacteria.

[0031] The quantitative method for bacteria of the present invention can be used as a method for quantifying dead bacteria containing diaminopimelic acid in a test sample containing dead bacteria containing diaminopimelic acid and bacteria not containing diaminopimelic acid. The quantitative method of the present invention is useful for measuring the number of dead bacteria containing diaminopimelic acid in a test sample containing dead bacteria containing diaminopimelic acid and bacteria not containing diaminopimelic acid. In addition, the quantitative method for bacteria of the present invention is useful as a method for quantifying dead bacteria containing diaminopimelic acid in a test sample containing dead bacteria containing diaminopimelic acid and live bacteria not containing diaminopimelic acid. When the bacteria containing diaminopimelic acid are dead bacteria, they cannot be quantified by the cultivation method as described above. In addition, in the DAPI (fluorescence) staining method, the total number of bacteria in a test sample containing bacteria containing diaminopimelic acid and bacteria not containing diaminopimelic acid can be determined, but it is difficult to quantify the bacteria containing diaminopimelic acid. The quantitative method for bacteria of the present invention can selectively quantify the bacteria containing diaminopimelic acid contained in the test sample even when the bacteria containing diaminopimelic acid contained in the test sample are dead bacteria and the sample contains bacteria not containing diaminopimelic acid.

[0032] In the quantitative method of the present invention, the bacteria containing diaminopimelic acid are quantified using the amount of diaminopimelic acid from the bacteria containing diaminopimelic acid in the test sample as an index.

[0033] The quantitative method of the present invention may also include a step of quantifying the amount of diaminopimelic acid in the test sample. The quantitative method of the present invention may also be a quantitative method for bacteria, which includes a step of quantifying the amount of diaminopimelic acid in a test sample containing bacteria containing diaminopimelic acid, and a step of quantifying the bacteria containing diaminopimelic acid using the amount of diaminopimelic acid from the bacteria containing diaminopimelic acid in the test sample as an index. The method for quantifying the amount of diaminopimelic acid in the test sample is not particularly limited. The measurement of the amount of diaminopimelic acid in the test sample can be carried out, for example, by subjecting the test sample to acid hydrolysis and quantifying the amount of diaminopimelic acid contained in the decomposition solution. The quantitative method of the present invention may also include a step of subjecting the test sample to acid hydrolysis and quantifying the amount of diaminopimelic acid contained in the decomposition solution. The amount of diaminopimelic acid from the bacteria containing diaminopimelic acid can be determined from the amount of diaminopimelic acid contained in the decomposition solution. Using the obtained amount of diaminopimelic acid from the bacteria containing diaminopimelic acid as an index, the bacteria containing diaminopimelic acid can be quantified.

[0034] The conditions for acid hydrolysis may be those that can decompose the cell wall of bacteria containing diaminopimelic acid. Acid hydrolysis can be carried out in an aqueous solution of an acid. Acids such as hydrochloric acid can be used in acid hydrolysis. Hydrochloric acid is preferred from the perspective of easy operation. The acid concentration in the aqueous solution of the acid is preferably 0.5 to 12 N, more preferably 3 to 9 N, and further preferably 5 to 7 N. In one mode, the temperature of acid hydrolysis is preferably 70 to 150 °C, more preferably 90 to 130 °C, and further preferably 100 to 120 °C. The time of acid hydrolysis can be set to, for example, 0.5 hours or more, preferably 1 hour or more, more preferably 6 hours or more, and further preferably 8 hours or more. In addition, it can be set to 40 hours or less. In one mode, the time of acid hydrolysis can be set to, for example, 0.5 to 40 hours, preferably 1 to 40 hours, more preferably 6 to 40 hours, and further preferably 8 to 40 hours. The method for quantifying the amount of diaminopimelic acid is not particularly limited, and can be carried out by amino acid analysis using an amino acid analyzer, a method using high performance liquid chromatography (HPLC), a method using LC-MS, etc. In one mode, it is preferred to quantify diaminopimelic acid by amino acid analysis.

[0035] The test sample preferably does not contain diaminopimelic acid in components other than bacteria containing diaminopimelic acid. When the test sample does not contain components containing diaminopimelic acid other than bacteria containing diaminopimelic acid, all of the diaminopimelic acid contained in the above decomposition solution comes from bacteria containing diaminopimelic acid. At this time, the amount of diaminopimelic acid in the test sample can be used as the amount of diaminopimelic acid from bacteria containing diaminopimelic acid.

[0036] When components other than bacteria containing diaminopimelic acid contain diaminopimelic acid, the amount of diaminopimelic acid from bacteria containing diaminopimelic acid can be obtained by separately measuring the amount of diaminopimelic acid in components other than bacteria containing diaminopimelic acid in the test sample and subtracting the amount of diaminopimelic acid from components other than bacteria containing diaminopimelic acid from the amount of diaminopimelic acid in the test sample. The quantification method of the present invention may also include a step of quantifying the amount of diaminopimelic acid in components other than bacteria containing diaminopimelic acid in the test sample.

[0037] From the amount of diaminopimelic acid obtained from bacteria containing diaminopimelic acid, the number of bacteria containing diaminopimelic acid can be determined. The method for determining the number of bacteria containing diaminopimelic acid from the above-mentioned amount of diaminopimelic acid is not particularly limited. For example, for the bacteria containing diaminopimelic acid contained in the test sample, a method preferably implemented using a calibration curve prepared in advance with a sample having a known number of bacteria is used. Specifically, for the calibration curve, a sample having a known number of bacteria containing diaminopimelic acid contained in the sample is used, and as described above, the sample is subjected to acid hydrolysis, and a calibration curve is prepared from the measured value obtained by quantifying the amount of diaminopimelic acid from the bacteria containing diaminopimelic acid contained in the decomposition solution. The calibration curve is usually prepared using a sample containing the same type of bacteria as the bacteria containing diaminopimelic acid contained in the test sample and having a known number of bacteria. In the quantification method of the present invention, the total number of bacteria containing diaminopimelic acid (the total number of viable and dead bacteria) contained in the test sample can be measured. In the quantification method of the present invention, when all the bacteria containing diaminopimelic acid contained in the test sample are viable bacteria, the number of viable bacteria can be measured, and when all the bacteria are dead bacteria, the number of dead bacteria can be measured.

[0038] The quantification method of the present invention can be a quantification method for bacteria containing diaminopimelic acid and bacteria not containing diaminopimelic acid in the test sample. The quantification method of the present invention may also include a step of quantifying bacteria not containing diaminopimelic acid contained in the test sample. For example, when the bacteria not containing diaminopimelic acid are viable bacteria, the quantification method of the present invention may also include a step of quantifying the viable bacteria not containing the above-mentioned diaminopimelic acid in the above-mentioned test sample. At this time, the implementation order of the quantification step for bacteria containing diaminopimelic acid and the quantification step for viable bacteria not containing diaminopimelic acid is not particularly limited.

[0039] The method for quantifying viable bacteria is not particularly limited. For example, a cultivation method or the like can be used in the measurement of the number of viable bacteria. For example, in the quantification step for viable bacteria not containing diaminopimelic acid, the above-mentioned test sample is cultivated, and the viable bacteria can be quantified by measuring the colonies of the viable bacteria not containing diaminopimelic acid. The cultivation can be carried out by a cultivation method (solid cultivation method) carried out on an agar medium.

[0040] The cultivation and the measurement of the colonies of viable bacteria can be carried out by a known method according to the type of bacteria. For example, a dilution solution of the test sample is prepared, the dilution solution is cultivated on an agar medium, and the number of colonies formed is measured. The number of colonies formed at this time is the number of viable bacteria of the bacteria contained in the dilution solution cultivated in the agar medium. Therefore, the number of viable bacteria of the bacteria contained in the test sample can be determined from the number of colonies and the dilution ratio.

[0041] Regarding the cultivation conditions, when the bacteria containing diaminopimelic acid are dead bacteria, the growth conditions for live bacteria without diaminopimelic acid are sufficient. When the test sample contains two or more types of live bacteria, selective media or the like can also be used for cultivation to measure the viable cell count of each bacterium. For example, when the bacteria containing diaminopimelic acid are live bacteria, by not allowing the growth of this bacterium and using the growth conditions for live bacteria without diaminopimelic acid, selective media, etc., the live bacteria without diaminopimelic acid can be selectively quantified.

[0042] The test sample used in the present invention is not particularly limited, and examples thereof include foods (including health foods), pharmaceuticals, cosmetics, and their raw materials. The form of the test sample is not particularly limited, and any form such as solid, powder, paste, gel, jelly, tablet, or liquid can be used. If necessary, the test sample can be pulverized for the quantification of diaminopimelic acid and the quantification of viable bacteria.

[0043] According to the quantification method of the present invention, the bacteria containing diaminopimelic acid contained in foods, pharmaceuticals, cosmetics, etc. or their raw materials containing bacteria containing diaminopimelic acid can be simply quantified. The quantification method of the present invention can quantify the bacteria in the above samples whether the bacteria containing diaminopimelic acid are dead bacteria or live bacteria. The quantification method of the present invention is useful, for example, in the quality control of these foods, pharmaceuticals, cosmetics, and their raw materials.

[0044] Examples

[0045] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[0046] <Quantification method of diaminopimelic acid>

[0047] The sample is subjected to acid hydrolysis, and the decomposition solution is analyzed by an automatic amino acid analyzer to quantify diaminopimelic acid.

[0048] (1) Analytical instruments, etc.

[0049] · Fully automatic amino acid analyzer (JLC-500 / V2, manufactured by JEOL Ltd.)

[0050] · Analytical column [LCR-6 (5 μm, 4.0 mm i.d. × 120 mm, manufactured by JEOL Ltd.)]

[0051] (2) Reagents

[0052] · 2,6-Diaminopimelic acid (DAP) (Tokyo Chemical Industry Co., Ltd.)

[0053] · 20% hydrochloric acid (for precision analysis, manufactured by FUJIFILM Wako Pure Chemical Corporation) (6 mol / L hydrochloric acid)

[0054] · 2-Mercaptoethanol (FUJIFILM Wako Pure Chemical Corporation)

[0055] · Hydrochloric acid (for amino acid automatic analysis, FUJIFILM Wako Pure Chemical Corporation)

[0056] · Trisodium citrate dihydrate (for amino acid automatic analysis, FUJIFILM Wako Pure Chemical Corporation)

[0057] · Octanoic acid [n-Octanoic acid] (for amino acid automatic analysis, FUJIFILM Wako Pure Chemical Corporation)

[0058] · Thiodiglycol (for amino acid automatic analysis, FUJIFILM Wako Pure Chemical Corporation)

[0059] · Sodium citrate buffer solution (H-01 to H-04) (JEOL Ltd.)

[0060] · Ninhydrin color developing solution kit-II for JEOL (FUJIFILM Wako Pure Chemical Corporation)

[0061] (3) Preparation of test solution

[0062] · 20% Hydrochloric acid (containing 0.04% 2-mercaptoethanol)

[0063] Add 0.2 mL of 2-mercaptoethanol to 500 mL of 20% hydrochloric acid and mix.

[0064] · Sodium citrate buffer solution (pH 2.2)

[0065] Weigh 980 g of trisodium citrate dihydrate, add about 3500 mL of ion-exchanged water to dissolve it. Add about 700 mL of 36% hydrochloric acid and 5 mL of octanoic acid and stir. Adjust to pH 2.2 with 20% hydrochloric acid, and then adjust to 5000 mL with ion-exchanged water (stock solution). Add 500 mL of the stock solution, 100 mL of thiodiglycol and 4000 mL of ion-exchanged water and stir. Adjust to pH 2.2 with 20% hydrochloric acid, and then adjust to 5000 mL with ion-exchanged water.

[0066] · 0.01 mol / L hydrochloric acid

[0067] Add 80 mL of 20% hydrochloric acid to 400 mL of ion-exchanged water and stir (1 mol / L hydrochloric acid). Dilute 1 mol / L hydrochloric acid 100 times with water.

[0068] (4) Preparation of sample for analysis

[0069] 1) Take the sample to be analyzed into a test tube for hydrolysis, add 20 mL of 20% hydrochloric acid (containing 0.04% 2-mercaptoethanol), and seal the tube under reduced pressure.

[0070] 2) Hydrolyze the test tube after sealing at 110 °C for 24 hours.

[0071] 3) After cooling, open the tube and make up the volume of the hydrolyzate to 100 mL with ion-exchanged water.

[0072] 4) Take 10 mL of this solution and concentrate it under reduced pressure using an evaporator to make it dry and solidify.

[0073] 5) Dissolve the residue with 5 mL of sodium citrate buffer (pH 2.2), and use the filtered substance through a membrane filter (0.45 μm) as the test solution.

[0074] 6) Weigh 0.0475 g of DAP precisely, dissolve it in 0.01 mol / L hydrochloric acid, and then make up the volume to 100 mL as the standard stock solution (concentration: 2.5 μmol / mL).

[0075] 7) Dilute the standard stock solution 25-fold with sodium citrate buffer (pH 2.2) as the standard solution (concentration: 0.1 μmol / mL).

[0076] 8) Supply the test solution and the standard solution to the conditions of the amino acid autoanalyzer shown below, and quantify DAP based on the peak height.

[0077] (5) Analysis based on the amino acid autoanalyzer

[0078] Analyze the sample prepared by the above method under the analysis conditions shown below to perform the quantification of DAP.

[0079] (Analysis conditions)

[0080] Column: LCR-6 (5 μm, 4.0 mm i.d. × 120 mm, manufactured by JEOL Ltd.)

[0081] Flow rate: Mobile phase 0.42 mL / min, reaction solution 0.22 mL / min

[0082] Mobile phase: Sodium citrate buffer (H-01 to 04) (manufactured by JEOL Ltd.)

[0083] Reaction solution: Ninhydrin color developing solution kit-II for JEOL (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0084] Injection volume: 30 μL

[0085] The proportion (vol%) of sodium citrate buffer H-01 to H-04 in the mobile phase and the column temperature are shown in Table 1.

[0086] (Amino acid autoanalyzer time program)

[0087] [Table 1]

[0088]

[0089] (6) Calculation method

[0090] The DAP content (mg / 100 g) is calculated by the following formula.

[0091] [Mathematical formula 1]

[0092]

[0093] A: Peak height of the test solution

[0094] B: Peak height of the standard solution

[0095] 50: Dilution factor of the test solution during the operations 1) to 5) in (4) above

[0096] W: Sample collection amount (g)

[0097] 0.1: Concentration of the standard solution (μmol / mL)

[0098] 190.20: Molecular weight of DAP

[0099] The peaks in A and B of the above formula are the peaks of DAP.

[0100] (7) Qualitative confirmation

[0101] The test solution and the standard solution are analyzed by the method described in (5), and the peak with the same elution retention time as the DAP standard solution is regarded as DAP.

[0102] <Test example 1>

[0103] The correlation between the cell count of Lactobacillus pentosus SAM2336 (hereinafter referred to as lactic acid bacterium S-PT84) and the amount of diaminopimelic acid (DAP) was investigated. Lactobacillus pentosus SAM2336 is a lactic acid bacterium stored in the Patent Biological Depositary, National Institute of Technology and Evaluation (NITE-IPOD) (Deposit number: FERM BP-10028) and identified as Lactobacillus pentosus SAM 2336.

[0104] Lactobacillus pentosus SAM2336 was cultured, and the cell count of Lactobacillus pentosus SAM2336 and the amount of DAP at each culture time were measured.

[0105] At the time of evaluation, sampling was carried out every 3 hours from 0 hour to 24 hours. In the determination of the DAP amount, 150 g of the culture solution sampled at the specified time was sterilized and centrifuged, and then the supernatant was removed. The substance prepared by adding water to the precipitate to 1.5 g (100-fold concentrated solution) was used as the DAP amount measurement sample, and the DAP amount was measured by the above-mentioned quantitative method of diaminopimelic acid. In addition, for the determination of the number of Lactobacillus S-PT84 bacteria, Lactobacillus S-PT84 was collected from the culture solution used for the determination of the DAP amount, stained with DAPI, and then the number of bacteria was measured under a microscope. In addition, regarding the sample for the determination of the number of bacteria, since the bacteria aggregated when using the 100-fold concentrated solution and the number of bacteria could not be correctly measured, the value obtained by multiplying the measurement result of the culture solution by 100 was used as the number of bacteria. Based on these results, the correlation between the DAP amount and the number of Lactobacillus S-PT84 bacteria was studied.

[0106] Figure 1 A and Figure 1 B represent the time-dependent changes in the number of Lactobacillus S-PT84 bacteria ( Figure 1 A) and the time-dependent changes in the amount of diaminopimelic acid ( Figure 1 B) based on the culture time. Figure 1 The number of Lactobacillus bacteria on the vertical axis of A is the value obtained by converting the number of Lactobacillus S-PT84 bacteria per 1 g of the culture solution multiplied by 100 to the number of Lactobacillus S-PT84 bacteria per 100-fold concentrated solution. Figure 1 The DAP amount (mg) on the vertical axis of B is the value per 100 g of the 100-fold concentrated solution. Figure 2 To represent Figure 1 the correlation between the number of Lactobacillus S-PT84 bacteria (number / g) on the vertical axis of A and Figure 1 the DAP amount (mg / 100 g) on the vertical axis of B. As Figure 1 shown in A, the number of Lactobacillus S-PT84 bacteria began to increase from 3 hours, and no change in the number of bacteria was observed after 21 hours. In addition, the DAP amount also showed the same tendency ( Figure 1 B). Further, when the results of the number of Lactobacillus S-PT84 bacteria and the DAP amount were plotted and their correlation was confirmed by an approximate straight line, a correlation with an R 2 value as high as 0.984 was confirmed ( Figure 2 ).

[0107] <Test Example 2>

[0108] In order to confirm the correlation between the raw material weight of Lactobacillus S-PT84 and the DAP amount, the quantification of DAP per unit raw material weight of Lactobacillus S-PT84 was carried out by the above-mentioned quantitative method of diaminopimelic acid. This test was repeated once a day for 3 days.

[0109] As the raw material of Lactobacillus S-PT84, dead cell powder of Lactobacillus S-PT84 (manufactured by Taiyo Kagaku Co., Ltd., containing 1.8×10 11 cells / g) was used. The number of dead cells is based on the measured value of the raw material manufacturer.

[0110] Table 2 shows the measured values of DAP per unit weight of the Lactobacillus S-PT84 raw material and the analysis results. The graph drawn from the results shown in Table 2 is shown in Figure 3 (horizontal axis: sample size (Lactobacillus raw material) (g), vertical axis: average analysis result (DAP amount) (mg)). Figure 3 The shown graph represents the correlation between the weight of the Lactobacillus S-PT84 raw material and the amount of DAP contained in the raw material.

[0111] When confirming the linearity between the weight of the Lactobacillus S-PT84 raw material and the DAP amount, a linearity with an R 2 value as high as 0.999 was confirmed (Table 2 and Figure 3 ). The regression formula obtained from the data shown in Figure 3 is y = 3.0331x + 0.0099.

[0112] [Table 2]

[0113]

[0114] In Test Example 1, when evaluating the correlation between the number of Lactobacillus S-PT84 cells and the DAP amount at each culture time, a high correlation with an R 2 value of 0.984 was confirmed in the approximate straight line between the number of Lactobacillus S-PT84 cells and the DAP amount. Since DAP is contained in the cell wall of Lactobacillus S-PT84, it is considered that the amount of DAP also increases with the proliferation of Lactobacillus S-PT84. In Test Example 2, when studying the correlation between the weight of the Lactobacillus S-PT84 raw material and the DAP amount, a linearity with an R 2 value as high as 0.999 was clarified. From this, it can be known that by measuring the DAP amount, the number of bacteria containing DAP or the blending amount of the bacterial raw material can be calculated.

[0115] In addition, in (4) Preparation of the analysis sample of the above method for quantifying diaminopimelic acid, 2), except that the hydrolysis time was set to 8, 16, 32, or 40 hours, the correlation between the number of Lactobacillus S-PT84 cells and the DAP amount was evaluated in the same manner as above. Even when the hydrolysis time was set to 8, 16, 32, or 40 hours, the same results as those obtained when hydrolyzing for 24 hours were obtained, and it was confirmed that the Lactobacillus S-PT84 raw material was sufficiently hydrolyzed.

[0116] <Example 1>

[0117] In the raw material of Lactobacillus S-PT84, the same dead bacteria powder of Lactobacillus S-PT84 as in Test Example 2 was used (manufactured by Taiyo Kagaku Co., Ltd., containing 1.8×10 11 cfu / g).

[0118] As the DAP-free bacterium, Bifidobacterium was used. The above-mentioned raw material of Lactobacillus S-PT84 and the raw material of Bifidobacterium were mixed to prepare a sample containing 4.1×10 12 cfu of Lactobacillus S-PT84 (dead bacteria) per 100 g.

[0119] The amount of DAP in the sample was quantified by the above-described method for quantifying diaminopimelic acid. In addition, a calibration curve was prepared by the following method, and the number of Lactobacillus S-PT84 bacteria was determined from the amount of DAP in the sample.

[0120] (Preparation of calibration curve)

[0121] 0.1, 0.3, 0.5, 0.8, and 1.0 g of the raw material of Lactobacillus S-PT84 used in the preparation of the sample were collected, and DAP was quantified according to the procedures described in (4) to (7) of the above-described method for quantifying diaminopimelic acid, and a regression line of the amount of DAP and the weight of the raw material of Lactobacillus S-PT84 was prepared. This regression line was used as the calibration curve. In addition, (5) of (4) in the above-described method for quantifying diaminopimelic acid was changed to the following 5').

[0122] 5') The residue was dissolved in 20 mL of sodium citrate buffer (pH 2.2), and the filtered material using a membrane filter (0.45 μm) was used as the test solution.

[0123] (Conversion of the number of Lactobacillus S-PT84 bacteria)

[0124] Using the slope and intercept of the calibration curve (regression line) prepared above, the weight of the raw material of Lactobacillus S-PT84 per 100 g of the sample was calculated by the following formula.

[0125] (Weight of the raw material of Lactobacillus S-PT84 per 100 g of the sample) (g / 100 g) = (E - intercept) / slope

[0126] E: DAP content (mg / 100 g)

[0127] The weight of the raw material of Lactobacillus S-PT84 per 100 g of the sample was converted to the number of Lactobacillus S-PT84 bacteria per 100 g of the sample by the following formula.

[0128] Number of Lactobacillus S-PT84 bacteria per 100 g of the sample (cfu / 100 g) = M × T

[0129] M: Weight of Lactobacillus S-PT84 raw material per 100 g of sample (g / 100 g)

[0130] T: Number of bacteria per unit weight of Lactobacillus S-PT84 raw material (CFU / g)

[0131] [The number of bacteria (CFU / g) is the number of dead bacteria of Lactobacillus S-PT84 (CFU / g), as determined by the raw material manufacturer]

[0132] Using the above method, the number of Lactobacillus S-PT84 bacteria per 100 g of sample was determined to be 4.2×10 12 CFU / 100 g.

[0133] In the sample prepared in Example 1, in addition to Lactobacillus S-PT84, it contained a Bifidobacterium raw material. The DAP content of the Bifidobacterium raw material was confirmed. As a result, no DAP was detected from the Bifidobacterium raw material.

[0134] Regarding the Lactobacillus S-PT84 raw material, the cells were cultured on a medium (containing yeast extract and glucose), then separated by solid-liquid separation, and the precipitate was freeze-dried and pulverized to obtain the raw material. Since it was considered that the medium might be mixed into this raw material, the DAP content of the medium raw material for Lactobacillus S-PT84 was confirmed. As a result, no DAP was detected from any sample. From the above results, it was considered that in Example 1, the DAP detected from the sample all came from Lactobacillus S-PT84.

[0135] From the above results, it can be seen that the number of bacteria containing DAP can be measured using the DAP content as an index.

[0136] <Example 2>

[0137] As the bacteria without DAP, Bifidobacterium bifidum (B. bifidum) NBRC100015 strain (hereinafter referred to as Bifidobacterium NBRC100015) was used. Bifidobacterium NBRC100015 can be obtained from the National Institute of Technology and Evaluation, Biotechnology Center (NBRC No. NBRC 100015). To mix with the Lactobacillus S-PT84 raw material, skim milk powder was added to the culture solution of Bifidobacterium NBRC100015 to prepare a powdered Bifidobacterium NBRC100015 raw material (product containing 3.4×10 10 CFU / g of live bacteria). In addition, the number of live bacteria of the Bifidobacterium NBRC100015 raw material was based on the measurement value of the Japan Food Analysis Center, a general incorporated foundation. The Lactobacillus S-PT84 raw material used was the same dead bacteria powder of Lactobacillus S-PT84 as in Test Example 2 (manufactured by Taiyo Kagaku Co., Ltd., product containing 1.8×10 11 CFU / g of dead bacteria).

[0138] The lactic acid bacteria S-PT84 raw material and the Bifidobacterium NBRC100015 raw material were mixed to prepare a mixture containing 4.1×10 12 lactic acid bacteria S-PT84 (dead bacteria), 2.6×10 12 The samples were of Bifidobacterium NBRC100015 (live bacteria).

[0139] The amount of DAP in the sample was quantified by the above-mentioned diaminopimelate quantification method. A calibration curve was prepared by the DAP amount and the weight of the lactic acid bacteria S-PT84 raw material in the same manner as in Example 1, and the number of lactic acid bacteria S-PT84 was calculated from the DAP amount in the sample.

[0140] When the number of lactic acid bacteria S-PT84 was calculated from the amount of DAP, the number of lactic acid bacteria S-PT84 in the sample was 4.5×10 12 When the amount of DAP in the Bifidobacterium NBRC100015 raw material used was confirmed, no DAP was detected from the raw material.

[0141] The number of viable cells of Bifidobacterium NBRC100015 in the sample was determined by culturing the sample on an agar medium and then measuring the colonies (culturing method). The number of viable cells of Bifidobacterium NBRC100015 in the sample was 2.0×10 12 Pieces / 100g.

[0142] <Example 3>

[0143] As a DAP-free bacterium, lactic acid bacteria Lactobacillus acidophilus (L.acidophilus) NBRC13951 strain (hereinafter referred to as lactic acid bacteria NBRC13951) was used. Lactic acid bacteria NBRC13951 can be obtained from the Biotechnology Center of the Institute of Technology for Product Evaluation (NBRC No. NBRC 13951). In order to mix with the lactic acid bacteria S-PT84 raw material, skim milk powder was added to the culture solution of lactic acid bacteria NBRC13951 to prepare a powdered lactic acid bacteria NBRC13951 raw material (live bacteria 1.0×10 8 The number of viable cells of the lactic acid bacteria NBRC13951 raw material is a value measured by the Japan Food Analysis Center.

[0144] The lactic acid bacteria S-PT84 raw material used was the same lactic acid bacteria S-PT84 dead bacteria powder as in Test Example 2 (manufactured by Taiyo Fragrance Co., Ltd., dead bacteria 1.8×10 11 / g contains products).

[0145] Mix the raw material of Lactobacillus S-PT84 and the raw material of Lactobacillus NBRC13951 to prepare a sample containing 4.1×10 12 Lactobacillus S-PT84 (dead bacteria) per 100 g and 7.7×10 9 Lactobacillus NBRC13951 (live bacteria).

[0146] Quantify the amount of DAP in the sample by the above quantitative method for diaminopimelic acid. In the same manner as in Example 1, prepare a calibration curve from the amount of DAP and the weight of the raw material of Lactobacillus S-PT84, and determine the number of Lactobacillus S-PT84 bacteria from the amount of DAP in the sample.

[0147] When determining the number of Lactobacillus S-PT84 bacteria from the amount of DAP, the number of Lactobacillus S-PT84 bacteria in the sample is 4.7×10 12 per 100 g. When confirming the amount of DAP in the raw material of Lactobacillus NBRC13951 used, DAP was not detected from this raw material.

[0148] Regarding the number of live bacteria of Lactobacillus NBRC13951 in the sample, it was determined by the method of culturing the sample using an agar medium and measuring the colonies (culture method). The number of live bacteria of Lactobacillus NBRC13951 in the sample is 7.4×10 9 per 100 g.

[0149] "Implementation of Management of Inspection and Other Operations for Food Hygiene Inspection Facilities, etc." (April 1, 1997) (Health Food No. 117) records in the "General Guidelines for Precision Management" of "III Precision Management of Microbiological Examinations" the standard that the recovery rate of added known microorganisms should ensure at least 70% to 120% ("Setting of Target Values Required for Precision Management" "(1) Confirmation of Recovery Rate, etc."). In Lactobacillus S-PT84 of Examples 1 to 3 and Bifidobacterium NBRC100015 and Lactobacillus NBRC13951 of Examples 2 and 3, the measured values of the number of bacteria in the sample are within the above range relative to the number of bacteria in the sample (calculated value) calculated from the quantitative values of each raw material. It can be said that the quantitative method of the present invention has sufficient precision in the quantification of bacteria.

Claims

1. A method for quantifying the number of bacteria, which is a method for quantifying the number of bacteria containing diaminopimelic acid in a test sample, characterized in that, it includes a step of quantifying the number of bacteria containing diaminopimelic acid by using the amount of diaminopimelic acid from the bacteria containing diaminopimelic acid in the test sample as an index, the test sample contains bacteria that do not contain diaminopimelic acid and one kind of bacteria that contains diaminopimelic acid, the bacteria containing diaminopimelic acid is Lactobacillus pentosus, the test sample is a food, a pharmaceutical, a cosmetic, a raw material of a food, a raw material of a pharmaceutical or a raw material of a cosmetic, in the step of quantifying the number of bacteria containing diaminopimelic acid, the number of bacteria containing diaminopimelic acid is obtained from the amount of diaminopimelic acid by using a calibration curve prepared with a sample in which the number of bacteria containing diaminopimelic acid contained in the test sample is known.

2. The quantification method according to claim 1, characterized in that, the bacteria containing diaminopimelic acid are dead bacteria.

3. The quantification method according to claim 1 or 2, characterized in that, the bacteria containing diaminopimelic acid is Lactobacillus pentosus SAM 2336.

4. The quantification method according to claim 1 or 2, characterized in that, the bacteria that do not contain diaminopimelic acid are live bacteria.

5. The quantification method according to claim 1 or 2, characterized in that, the bacteria that do not contain diaminopimelic acid are Bifidobacterium.

Images