JP6449136B2 - Method for analyzing periodontal disease-causing bacteria - Google Patents

Description

  This invention is a method for analyzing periodontal disease-causing bacteria. Specifically, it is possible to analyze periodontal disease-causing bacteria measured at high temperature at room temperature, and the periodontal disease-causing bacteria that do not require special equipment. It relates to the analysis method.

Periodontal disease is said to affect about 80% of adults in Japan. Periodontal disease has been shown to cause tooth loss, resulting in abnormal taste and salivary secretion as well as abnormalities in the central nervous system and autonomic nervous system. Furthermore, in recent years, it has been pointed out that periodontal disease is a risk factor for various systemic diseases such as coronary heart disease and cerebral infarction.
Among the test items for periodontal disease, as a method for examining bacterial infection and inflammation, a method of visually judging a stained tooth surface using a plaque staining solution, a periodontal probe and a dental probe without staining, etc. Teeth that scrape the tooth surface at the tip of the tooth to determine the presence or absence of plaque adhesion, subgingival plaque is collected at a paper point, and the number of bacteria is measured by a DNA quantification method, etc. by requesting an inspection organization There are peripathogenic bacteria tests, antibody test methods for measuring IgG antibody titers in serum against periodontal pathogenic bacteria, and the like. These methods are not only time consuming, labor intensive and expensive, but also require special facilities and techniques, so it is not possible to examine multiple patients simultaneously and simply, and the burden on the patients Is also big.

Among the periodontal disease-causing bacteria, three species of Porphyromonas gingivalis (P. g), Treponema denticola (T. d), and Tannerella forsythesis (T. f) are called Red Complex. It is said to be the most dangerous group of bacteria causing periodontal disease. These three bacterial species have arginine-specific peptidase activity (trypsin-like enzyme activity), and methods for detecting trypsin-like enzyme activity using a synthetic substrate are reported in Patent Documents 1 to 3.
It is also known that this enzyme activity is activated by a reducing agent (Patent Documents 3 to 4, Non-Patent Document 1).
If the trypsin-like enzyme activity can be easily analyzed and determined in a short time for the presence of three important species of periodontal disease-causing bacteria, it is a useful method for screening for periodontal disease.

Japanese National Patent Publication No. 8-500241 JP-A-5-317095 International Publication No. 2004/106541 Wendy E. Kaman et al. "Highly Specific Protease-Based Approach for Detection of Porphyromonys gingivalis in Diagnosis of Periodontitis, Journal of Clinicolol. 50, Number 1, p. 104-112, 2012

  However, trypsin-like enzyme activity cannot obtain sufficient sensitivity unless its optimum temperature is 50 to 60 ° C., and a special instrument capable of maintaining the optimum temperature is required for measurement. Periodontal disease It becomes an obstacle to the spread of the inspection of causative bacteria. Therefore, among the periodontal disease-causing bacteria, g, T. d. When analyzing trypsin-like enzyme activity peculiar to the three species of bacterium f, it has been anxious to enable analysis at room temperature without the need for special equipment.

  Therefore, the present inventors have described P.I. g, T. d. Focusing on the chemical characteristics of fungus f, the presence of an antioxidative compound or a compound capable of protecting the SH group (mercapto group) and cleaving disulfide bonds when analyzing trypsin-like enzyme activity Thus, it was found that trypsin-like enzyme activity can be analyzed at room temperature, and the present invention was completed.

  The present invention relates to P. albundum among periodontal disease-causing bacteria measured under high temperature conditions. g, T. d. It is an object of the present invention to provide a method for analyzing periodontal disease-causing bacteria that can analyze trypsin-like enzyme activities peculiar to the three species of fungus f at room temperature and does not require special equipment.

The invention according to claim 1 is an analysis method for periodontal disease-causing bacteria, which analyzes the presence or absence of periodontal disease-causing bacteria by analyzing trypsin-like protease activity peculiar to periodontal disease-causing bacteria. A first compound having an action and / or a second compound having an action of protecting a SH group and cleaving a disulfide bond are combined with N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride in the above trypsin-like manner. This is a method for analyzing periodontal disease-causing bacteria, which is present for 10 minutes during the analysis of protease activity, and the trypsin-like protease activity is analyzed at room temperature using a coloring reagent .

The first compound is not particularly limited as long as it is a chemical substance having an antioxidant action, and a known substance can be used. Examples include ascorbic acid, α-tocopherol, glutathione, cysteine, N-acetylcysteine, and the like. The first compound may be one kind or a combination of plural kinds.
The second compound is not particularly limited as long as it is a chemical substance that protects the SH group and cleaves the disulfide bond, and a known substance can be used. For example, sodium tetrahydroborate, dithiothreitol (DTT), mercaptoacetic acid (thioglycolic acid), 3-mercapto-1,2-propanediol (thioglycerol), 2-mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride Examples include salts (TCEP), tributylphosphine (TBP), iodoacetamide, and the like. The second compound may be one type or a combination of a plurality of types.
In the analysis of trypsin-like protease activity, either the first compound or the second compound may be present, or both the first compound and the second compound may be mixed.

  Room temperature refers to a state in which neither an external system is heated nor cooled. It is approximately 20 ° C to 30 ° C.

  Specimens are samples obtained by diluting samples to be analyzed such as oral wipe samples, plaque samples, tongue coating samples, saliva, etc. with a suitable medium, or extracts obtained by extracting periodontal disease-causing bacteria from a sample to be analyzed using a suitable medium. It is.

Invention of Claim 2 WHEREIN: The said 1st compound was included in the absorptive substance with the said N- (alpha) -benzoyl-DL-arginine-2-naphthylamide hydrochloride, The periodontal disease of Claim 1 This is an analysis method for causative bacteria.
In the invention described in claim 3, the second compound is contained in an absorbent material together with the N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride. This is an analysis method for periodontal disease-causing bacteria.

The first compound and the second compound can be included in the water-absorbing substance together with the substrate (dry holding method). As the substrate, N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride, which is a substance that undergoes a chemical reaction under the action of the enzyme of the periodontal disease-causing bacteria contained in the sample, is used . This reagent is contained in a water-absorbing substance (test piece, carrier), the analyte to be analyzed and the water-absorbing substance are brought into direct contact, and the presence or absence of a coloring substance that is released by the enzyme activity of periodontal disease-causing bacteria is analyzed. In this case, a coloring reagent can be used as necessary.
Various known reagents can be used as the coloring reagent. For example, sodium 4-hydroxy-3-[(2,4-dihydroxy-3-quinolyl) azo] benzenesulfonate, 4-benzoylamino-2,5-diethoxybenzenediazonium, 4- (dimethylamino) cinnamaldehyde, etc. Can be used.

  The water-absorbing substance is not particularly limited as long as it has a water-absorbing property and can contain the first compound and the second compound. Examples thereof include paper, filter paper, cellulose, non-woven fabric, glass fiber, and cotton. The water-absorbing substance can contain a substrate. This water-absorbing substance can be used as it is, or can be provided on an appropriate member such as waterproof paper, glass, plastic, wood, metal, or the like to facilitate handling. The shape, length, and thickness of the water-absorbing substance are not particularly limited as long as the specimen can be contacted.

As the dry retention method, the first compound and the second compound can be dissolved or dispersed in an appropriate solvent together with the substrate or separately, and the solution (or dispersion) can be contained in the water-absorbing substance. As the solvent, for example, tris-HCl buffer, trismaleic acid buffer, phosphate buffer, or purified water can be used. Furthermore, those obtained by adding a suitable surfactant to these solvents, for example, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, octylphenol ethoxylate and the like can be used.
A solution (dispersion) of 1 mM to 100 mM of the first compound and the second compound is impregnated with 10 μL to 500 μL of the water-absorbing substance, and then dried by natural drying, air drying, vacuum drying under vacuum or freeze drying. Can be used.

The invention according to claim 4 includes the first compound in a sample, and the sample containing the first compound is converted into the N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride. The method for analyzing periodontal disease-causing bacteria according to claim 1, which is added to the salt.
The invention according to claim 5, said second compound contained in the specimen, the second compound contains analyte, the N-alpha-benzoyl--DL- arginine-2-naphthylamide hydrochloride The method for analyzing periodontal disease-causing bacteria according to claim 1 or 4, which is added to salt.

The first compound and the second compound can be dissolved in an appropriate solvent and then added to the specimen (solution method). Also, the first compound and the second compound can be added directly to the specimen.
As the solvent, for example, tris-HCl buffer, trismaleic acid buffer, phosphate buffer, or purified water can be used. Furthermore, those obtained by adding a suitable surfactant to these solvents, for example, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, octylphenol ethoxylate and the like can be used.
1/10 volume of the volume of the specimen can be added to the specimen from a 10 mM to 1000 mM lysate of the first compound and the second compound. Moreover, when adding a 1st compound and a 2nd compound directly to a test substance, the addition amount is about 1 mM-100 mM.

  The invention according to claim 6 is characterized in that the analysis of the trypsin-like protease activity is carried out by the presence or absence of N-benzoyl-DL-arginine peptidase, according to any one of claims 1 to 5. This is an analysis method.

  According to the present invention, the first compound having an antioxidant action and / or the second compound having the action of protecting the SH group and cleaving the disulfide bond are included in the specimen, thereby being measured under a conventional high temperature condition. P. g, T. d. It becomes possible to analyze the trypsin-like enzyme activity peculiar to three strains of the bacterium f at room temperature. Thereby, trypsin-like enzyme activity can be easily analyzed and determined in a short time, and is useful as a screening for periodontal disease.

In the method for analyzing periodontal disease-causing bacteria according to Example 1 of the present invention (dry retention method using the first compound and the second compound alone), It is a graph which shows the detection sensitivity of g microbe. In the method for analyzing periodontal disease-causing bacteria according to Example 2 of the present invention (solution method using the first compound and the second compound alone), It is a graph which shows the detection sensitivity of g microbe. In the method for analyzing periodontal disease-causing bacteria according to Example 3 of the present invention (dry retention method by mixing the first compound and the second compound). It is a graph which shows the detection sensitivity of g microbe. In the method for analyzing periodontal disease-causing bacteria according to Example 4 (solution method by mixing the first compound and the second compound) of the present invention, It is a graph which shows the detection sensitivity of g microbe.

  Examples of the present invention will be specifically described below, but they do not limit the scope of the present invention.

(Preparation of the first compound and the second compound)
As an anti-oxidant compound (first compound), L-ascorbic acid, L-cysteine hydrochloride, and glutathione were collected at 62.5 mM, 125 mM, 250 mM, 500 mM, and 1000 mM, respectively, and the collected first compound was 50 mM. After dissolving in trismaleic acid buffer pH 8.5, pH adjustment was performed so that the pH of the solution was 8.5.
DTT, thioglycol, thioglycerol, mercaptoethanol, and TCEP in 62.6 mM, 125 mM, 250 mM, 500 mM, and 1000 mM concentrations as compounds having a function of protecting the SH group and cleaving disulfide bonds (second compound) After being dissolved in 50 mM trismaleic acid buffer pH 8.5, pH adjustment was performed so that the pH of the solution was 8.5.

(Preparation of substrate)
As a substrate, N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride (obtained from Sigma-Aldrich Japan LLC) was dissolved in 50 mM Trismaleic acid buffer pH 8.5. The substrate concentration is 0.11% by weight.

(Preparation of coloring solution)
4- (Dimethylamino) cinnamaldehyde (DMAC) (obtained from Sigma Aldrich Japan LLC) was dissolved in 1 mol / L hydrochloric acid. The concentration of DMAC is 0.1% by weight.

(Pg culture (specimen) culture)
Prepared Porphyromonas gingivalis (P. g. Bacterium) JCM12257 was inoculated into bouillon medium and anaerobically cultured at 37 ° C. for 24 hours to obtain a bacterial culture solution of 1.1 × 10 ⁶ cfu / mL.
The bouillon medium was prepared and prepared with 3.0 g of tryply case soy broth, 0.5 g of yeast extract, 0.05 g of L-cysteine hydrochloride, 0.1 mL of hemin solution, 0.02 mL of vitamin K1 solution, and 100 mL of distilled water. Later, sterilization was performed at 121 ° C. for 15 minutes.
The hemin solution is obtained by dissolving 0.005 g of hemin and 0.0174 g of potassium hydrogen phosphate in 1 mL of distilled water. The vitamin K1 solution is obtained by dissolving 5 mg of vitamin K1 in 1 mL of ethanol.
The method for measuring the number of bacteria is as follows: a broth culture solution after anaerobic culture is diluted 10-fold with sterile physiological saline, inoculated on a sheep blood agar medium for CDC anaerobic bacteria (obtained from Becton Dickinson), and then 37 ° C. The number of bacteria was measured by culturing for 48 hours and measuring the colonies grown on the medium with the naked eye.

(Example 1) Execution of dry holding method by adding the first compound and the second compound alone (A) Dry holding of the substrate, the first compound and the second compound The adjusted first compound or the second compound The compound and the substrate were mixed at a ratio of 1: 9, and 80 μL was soaked per 10 mm paper disc (obtained from Advantech). Thereafter, the paper disc was dried at 25 ° C. overnight.

(B) P.I. Preparation of g bacteria The bacterial culture after 24 hours of culture was diluted 10-fold with 50 mM Trismaleic acid buffer pH 8.5, 1.1 × 10 ⁶ cfu / mL, 1.1 × 10 ⁵ cfu / mL, Bacteria solutions of 1.1 × 10 ⁴ cfu / mL and 1.1 × 10 ³ cfu / mL were prepared.

(C) Test method P. C. after preparation on a paper disc impregnated with each concentration of the first compound or the second compound and the substrate. 2 g of the bacterial solution soaked in 80 μL were prepared, and one was allowed to stand at room temperature and the other at 50 ° C. for 10 minutes. The former is called room temperature analysis and the latter is called 50 ° C. analysis. Thereafter, 30 μL of a coloring solution was dropped on the paper disk, and the color of the paper disk was observed and judged with the naked eye.

(D) Results P. of the first compound and the second compound. FIG. 1 shows the detection sensitivity of bacteria g. As shown in FIG. 1, only 1.1 × 10 ⁶ cfu / mL was detected in the room temperature analysis in which the first compound and the second compound were not present, but 1.1 × 10 ⁴ cfu was detected in the 50 ° C. analysis. / 50 mL can be detected, and it can be said that 50 ° C. analysis is 100 times more sensitive than room temperature. In the presence of the first compound and the second compound, it has been found that the sensitivity obtained by the 50 ° C. analysis can be equivalent to that at room temperature. That is, when the first compound and the second compound are not present, a sensitivity difference of 100 times is recognized between the room temperature analysis and the 50 ° C. analysis, but the kind of the compound can be obtained by the presence of the first compound and the second compound. Although there was a difference in detection sensitivity, it was found that the sensitivity of room temperature analysis and 50 ° C. analysis was almost the same for all compounds. Table 1 shows the minimum detection sensitivity obtained by the presence of the first compound and the second compound and the optimum temperatures of the first compound and the second compound. As shown in Table 1, it was confirmed that the effects of the first compound and the second compound had a wide optimum concentration.

(Example 2) Implementation of the solution method by adding the first compound and the second compound alone (A) Dry substrate retention The prepared substrate is 9: 1 in volume ratio with 50 mM Trismaleic acid buffer pH 8.5. 80 μL per 10 mm paper disc (obtained from Advantech). Thereafter, the paper disc was dried at 25 ° C. overnight.

(B) P.I. Preparation of g-bacterium The adjusted first compound or second compound is diluted 9-fold with 50 mM trismaleic acid buffer pH 8.5 to prepare a diluted compound solution.
The bacterial culture after 24 hours of culturing was diluted in 10 stages using a compound diluent, 1.1 × 10 ⁶ cfu / mL, 1.1 × 10 ⁵ cfu / mL, 1.1 × 10 ⁴ cfu / mL. 1.1 × 10 ³ cfu / mL bacterial solution was prepared.

(C) Test method The P.P. 2 g of the bacterial solution soaked in 80 μL were prepared, and one was allowed to stand at room temperature and the other at 50 ° C. for 10 minutes. The former is called room temperature analysis and the latter is called 50 ° C. analysis. Thereafter, 30 μL of a coloring solution was dropped on the paper disk, and the color of the paper disk was observed and judged with the naked eye.

(D) Results P. of the first compound and the second compound. FIG. 2 shows the detection sensitivity of bacteria g. As shown in FIG. 2, only 1.1 × 10 ⁶ cfu / mL was detected in the room temperature analysis in which the first compound and the second compound were not present, but 1.1 × 10 ⁴ cfu was detected in the 50 ° C. analysis. / 50 mL can be detected, and it can be said that 50 ° C. analysis is 100 times more sensitive than room temperature. When the first compound and the second compound were present, it was found that the sensitivity obtained by analysis at 50 ° C. was equivalent to that at room temperature, as in the dry retention method shown in Example 1. That is, when the first compound and the second compound are not present, a sensitivity difference of 100 times is recognized between the room temperature analysis and the 50 ° C. analysis, but the kind of the compound can be obtained by the presence of the first compound and the second compound. Although there was a difference in detection sensitivity, it was found that the sensitivity of room temperature analysis and 50 ° C. analysis was almost the same for all compounds. Table 2 shows the minimum detection sensitivity obtained by the presence of the first compound and the second compound, and the optimum temperatures of the first compound and the second compound. As shown in Table 2, it was confirmed that the effects of the first compound and the second compound had a wide optimum concentration.

  In the present invention, the enzyme activity at an optimal temperature of 50 to 60 ° C., which does not provide sufficient sensitivity by room temperature analysis, protects the first compound or SH group having an antioxidant action and cleaves the disulfide bond. It has been clarified that a compound having an action is kept dry in a water-absorbing substance or added to a specimen, so that even room temperature analysis can be analyzed with the same sensitivity as 50 ° C. analysis.

(Example 3) Execution of dry holding method by mixing addition of first compound and second compound (A) Mixed preparation of first compound and second compound Table 3 shows the first compound and the second compound. The combination shown in FIG. The mixing concentration is such that 500 mM each of the first compound and the second compound is dissolved in 50 mM trismaleic acid buffer pH 8.5, and then equal amounts of the first compound and the second compound are mixed so that each becomes 250 mM. Prepared.

(B) Keeping the substrate and the mixture of the first compound and the second compound dry The solution prepared by mixing the first compound and the second compound and the prepared substrate were mixed at a ratio of 1: 9, and 10 mm paper The disk was soaked with 80 μL per sheet. Thereafter, the paper disc was dried at 25 ° C. overnight. (The mixed first compound and second compound each have a final concentration of 25 mM)

(C) P.I. Preparation of g bacteria The bacterial culture after 24 hours of culture was diluted 10-fold with 50 mM Trismaleic acid buffer pH 8.5, 1.1 × 10 ⁶ cfu / mL, 1.1 × 10 ⁵ cfu / mL, Bacteria solutions of 1.1 × 10 ⁴ cfu / mL and 1.1 × 10 ³ cfu / mL were prepared.

(D) Test method P. C. after adjustment to a paper disk impregnated with a mixture of a first compound and a second compound and a substrate. 2 g of the bacterial solution soaked in 80 μL were prepared, and one was allowed to stand at room temperature and the other at 50 ° C. for 10 minutes. The former is called room temperature analysis and the latter is called 50 ° C. analysis. Thereafter, 30 μL of a coloring solution was dropped on the paper disk, and the color of the paper disk was observed and judged with the naked eye.

(E) Results P.I. mixed with the first compound and the second compound. FIG. 3 shows the detection sensitivity of bacteria g. As shown in FIG. 3, as in the case where the first compound or the second compound was added alone, it was confirmed that the same sensitivity was obtained in the room temperature analysis and the 50 ° C. analysis. Regarding the detection sensitivity, in the combination of L-ascorbic acid and mercaptoethanol, a phenomenon was observed in which the sensitivity was reduced as compared with each addition. In other combinations, the sensitivity was the same as that of single addition.
Therefore, even when the first compound and the second compound were mixed and used, the same sensitivity was obtained in the room temperature analysis and the 50 ° C. analysis as in the case of single use.

(Example 4) Implementation of a solution method by mixing and adding a first compound and a second compound (A) Mixed preparation of a first compound and a second compound Table 1 shows the first compound and the second compound. Mixed in the combinations shown. The mixed concentration was obtained by dissolving 55.5 mM of each of the first compound and the second compound in 50 mM trismaleic acid buffer pH 8.5, mixing equal amounts of the first compound and the second compound, and each containing 27.75 mM. It prepared so that it might become.

(B) Substrate dry holding The prepared substrate was mixed with 50 mM Trismaleic acid buffer pH 8.5 to a volume ratio of 9: 1, and 80 μL per 10 mm paper disc (obtained from Advantech) was soaked. . Thereafter, the paper disc was dried at 25 ° C. overnight.

(C) P.I. Preparation of g fungus The bacterial culture solution after 24 hours of culture was diluted 10 stages with the prepared first compound and second compound mixture to obtain 1.1 × 10 ⁶ cfu / mL, 1.1 × 10 ^5. Cfu / mL, 1.1 × 10 ⁴ cfu / mL, and 1.1 × 10 ³ cfu / mL bacterial solutions were prepared.

(D) Test method The P.P. 2 g of the bacterial solution soaked in 80 μL were prepared, and one was allowed to stand at room temperature and the other at 50 ° C. for 10 minutes. The former is called room temperature analysis and the latter is called 50 ° C. analysis. Thereafter, 30 μL of a coloring solution was dropped on the paper disk, and the color of the paper disk was observed and judged with the naked eye.

A mixture of the first compound and the second compound added together. FIG. 4 shows the detection sensitivity of bacteria g. As shown in FIG. 4, as in the case where the first compound or the second compound was added alone, it was confirmed that the same sensitivity was obtained in the room temperature analysis and the 50 ° C. analysis. Regarding the detection sensitivity, in the combination of L-cysteine hydrochloric acid and TCEP and in the combination of glutathione and TCEP, a phenomenon was observed in which the sensitivity decreased compared to each addition alone, but both the first compound and the second compound The sensitivity was 10 times as high as that in the absence. In other combinations, the sensitivity was the same as that of single addition. Therefore, even when the first compound and the second compound were mixed and used, the same sensitivity was obtained in the room temperature analysis and the 50 ° C. analysis as in the case of single use.

  In the present invention, the enzyme activity at an optimum temperature of 50 to 60 ° C., which cannot obtain sufficient sensitivity by room temperature analysis, protects the first compound having an antioxidant action and the SH group and cleaves the disulfide bond. It was clarified that by mixing a compound having an action and keeping it dry in a water-absorbing substance or adding it to a specimen, analysis at room temperature analysis can be performed with sensitivity equivalent to 50 ° C. analysis.

  The present invention is useful for technology for easily grasping periodontal disease risk determination and confirmation of progress, further confirmation of therapeutic effect, and the like in detection and diagnosis of periodontal disease-causing bacteria.

Claims (6)

Analyzing periodontal disease-causing bacteria by analyzing the trypsin-like protease activity specific to periodontal disease-causing bacteria,
The first compound having an antioxidant action and / or the second compound having an action of protecting the SH group and cleaving a disulfide bond are combined with N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride, A method for analyzing periodontal disease-causing bacteria, wherein the trypsin-like protease activity is present for 10 minutes at the time of analysis of trypsin-like protease activity, and the trypsin-like protease activity is analyzed using a coloring reagent at room temperature.   The method for analyzing periodontal disease-causing bacteria according to claim 1, wherein the first compound is contained in an absorptive substance together with the N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride.   The method for analyzing periodontal disease-causing bacteria according to claim 1 or 2, wherein the second compound is contained in an absorptive substance together with the N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride. . Including the first compound in a sample ;
The method for analyzing periodontal disease-causing bacteria according to claim 1, wherein the specimen containing the first compound is added to the N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride. Including the second compound in a sample ;
The method for analyzing periodontal disease-causing bacteria according to claim 1 or 4, wherein a specimen containing the second compound is added to the N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride.   6. The method for analyzing periodontal disease-causing bacteria according to any one of claims 1 to 5, wherein the analysis of the trypsin-like protease activity is performed based on the presence or absence of N-benzoyl-DL-arginine peptidase.

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