Method for detecting spore-forming bacteria

Abstract

This invention provides a simple, highly accurate, and low-cost method for detecting spore-forming bacteria. [Solution] A method for detecting spore-forming bacteria, comprising: a CTC addition step A in which a CTC solution containing 5-cyano-2,3-ditril-2H-tetrazolium salt (CTC) is added to a sample that may contain spore-forming bacteria; an incubation step B in which the sample to which the CTC solution has been added is incubated for a predetermined time after the CTC addition step; and a fluorescence detection step C in which the generated fluorescent formazan (CTF) is detected.

Description

Technical Field

[0001] The present invention relates to a method for detecting spore-forming bacteria.

Background Art

[0002] Among microorganisms, there are bacteria that form spores. Spore-forming bacteria actively divide and grow in a favorable environment for survival, but form spores when the environment becomes unfavorable for growth, such as when the nutrients in the environment decrease or it becomes dry. Spores have extremely high durability and can survive even when the environment deteriorates to a situation where normal bacteria die. However, if there is no change in the growth environment, spores will neither germinate nor grow, and their metabolism is extremely limited.

[0003] Spores are durable cells formed inside vegetative cells, have a very low water content, hardly metabolize, and are in a dormant state so to speak. Spore cells show strong resistance to various stresses and also have strong resistance to heating. Depending on the bacterial species, heating at 100°C or higher for a predetermined time or longer is required to kill them. When dormant spores are placed in an environment suitable for the growth of the bacteria again, the spores germinate and produce bacterial cells with normal growth and metabolic abilities.

[0004] Because spores have high heat resistance, their presence becomes a problem in container-packed foods where microorganisms are controlled by heat sterilization. Spores with particularly high heat resistance are the most important biological hazard factors for container-packed foods and are used as an index for heat sterilization.

[0005] As typical bacteria that form spores, bacteria such as aerobic bacteria of the genus Paenibacillus and Sporosarcina, and anaerobic bacteria of the genus Clostridium are known.

[0006] Advances in food manufacturing technology have significantly improved food quality. However, spoilage, particularly by spore-forming bacteria, remains a major challenge. Spore-forming bacteria are resistant to drying, chemicals, and heat treatment, causing off-odors and changes in flavor in the final product (Non-Patent Documents 1, 2). Spoilage refers to the breakdown of carbohydrates and fats in food ingredients, resulting in a loss of flavor and making the food unsuitable for consumption. Cases of spoilage have been observed in packaged foods (e.g., canned beverages). These spoilage-causing bacteria (hereinafter referred to as spoilage-causing bacteria) are widely distributed in nature, and the risk of spoilage exists at every stage of food production, from raw materials to the processing environment (Non-Patent Document 3).

[0007] Conventionally, methods for detecting spore-forming bacteria have included techniques that utilize microbial culture on agar plates (Non-Patent Documents 4, 5) and multiplex PCR technology that simultaneously detects and amplifies multiple target genes (Non-Patent Document 6).

[0008] In recent years, the market has seen a surge in the launch of various chilled foods, and efforts to develop long-life food products have attracted attention within the food industry. Furthermore, with food waste becoming a major social issue, detection methods for these bacterial species are considered crucial for proper microbiological hygiene management in the food industry. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] N. Kalchayanand, Bibek Ray, Ray A. Field and MC Johnson, “Spoilage of Vacuum-Packaged Refrigerated Beef by Clostridium”, Journal of Food Protection, Vol. 52, No. 6, Pages 424-426 (June 1989) [Non-Patent Document 2] Stephane Andre, Tatiana Vallaeys, Stella Planchon, “Spore-forming bacteria responsible for food spoilage”, Research in Microbiology 168 (2017) 379-387 Non-Patent Document 3 Nidhi Gopal, Colin Hill, Paul R. Ross, Tom P. Beresford, Mark A. Fenelon and Paul D. Cotter, “The Prevalence and Control of Bacillus and Related Spore-Forming Bacteria in the Dairy Industry”, Front. Microbiol., 21 December 2015, Volume 6, Article 1418 Non-Patent Document 4 R. E. Levin, “Detection and Incidence of Specific Species of Spoilage Bacteria on Fish”, Applied Microbiology, Nov. 1968, p. 1734-1737 Non-Patent Document 5 Chandrasekaran M, Lakshmanaperumalsamy P, Chandramohan D., “Fish flesh agar medium--a suitable experimental medium for the detection of spoilage bacteria.”, Antonie Van Leeuwenhoek., 1985;51(2):219-225 Non-Patent Document 6 Ying Chen, Zixuan Wang, Qiaozhen Shi, Shengxiong Huang, Taotao Yu, Linyan Zhang, Huan Yang, “Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases”, Biotech (2021) 11:219 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] In the aforementioned method for detecting spore-forming bacteria, culturing spore-forming bacteria on agar plates takes several days, and many of the procedures depend on the experience of the examiner, which could lead to delays or failures in detection.

[0011] Furthermore, when performing multiplex PCR, it is necessary to extract template nucleic acids (DNA) from spore-forming bacteria prior to PCR. However, the nucleic acids located inside the core of the spore cell are protected by a rigid core, resulting in poor extraction efficiency. In addition, the process was cumbersome because the conditions for nucleic acid extraction had to be optimized for each type of spore-forming bacterium.

[0012] Multiplex PCR has challenges such as primer design and competition between polymerase and substrate, which can lead to lower sensitivity compared to conventional PCR. Furthermore, food-derived substances such as DNA, sugars, salts, and amino acids can interfere with PCR results, potentially making it difficult to accurately determine the presence or absence of spore-forming bacteria. Additionally, because multiplex PCR uses the DNA of spore-forming bacteria, it cannot distinguish between live and dead spores from the PCR results, making it impossible to detect only living spores (spores that have the potential to germinate and proliferate in the future). Moreover, multiplex PCR involves higher costs for primers, reagents, and PCR equipment.

[0013] Therefore, the object of the present invention is to provide a simple, highly accurate, and low-cost method for detecting spore-forming bacteria. [Means for solving the problem]

[0014] In other words, to achieve the above objective, the inventions shown in [1] to [5] below are provided.

[0015] [1] A method for detecting spore-forming bacteria, comprising: a CTC addition step of adding a CTC solution containing 5-cyano-2,3-ditril-2H-tetrazolium salt (CTC) to a sample of a subject that may contain spore-forming bacteria; an incubation step of incubating the sample to which the CTC solution has been added for a predetermined time after the CTC addition step; and a fluorescence detection step of detecting the generated fluorescent formazan (CTF). [2] A method for detecting spore-forming bacteria as described in [1], wherein the fluorescence detection step is performed by flow cytometry analysis. [3] A method for detecting spore-forming bacteria according to [1] or [2], wherein the spore-forming bacteria are the causative agents of spoilage. [4] A method for detecting spore-forming bacteria according to [3], wherein the causative agent of spoilage is at least one of the genera Paenibacillus, Sporosarcina, and Clostridium. [5] A method for detecting spore-forming bacteria according to [4], wherein the Paenibacillus species is Paenibacillus polymixa, the Sporosarcina species is Sporosarcina cyclophylla, and the Clostridium species is Clostridium putrefasiens.

[0016] According to the nucleic acid extraction method for spore-forming bacteria of the present invention, by performing a CTC addition step, a CTC solution containing CTC can be added to a subject sample that may contain spore-forming bacteria, and CTC staining can be performed on the spore-forming bacteria as an indicator of their viability.

[0017] That is, CTC is reduced by NAD(P)H produced along with the metabolic activity of cells to generate fluorescent formazan (CTF). CTF emits red fluorescence in a highly viscous solution or in a solid state. CTF is lipophilic (insoluble in water) and accumulates as a fluorescent precipitate inside cells. After the CTC addition step, an incubation step is performed, and by performing a fluorescence detection step for detecting the generated CTF, the CTF (fluorescent precipitate) accumulated inside the cells can be detected. By analyzing the CTF detected in this way, living cells and dead cells can be accurately distinguished, and bacteria (living cells) having respiratory activity can be easily detected.

[0018] Moreover, according to the method for detecting spore-forming bacteria of the present invention, by performing the CTC addition step, the incubation step, and the fluorescence detection step, the time required until the analysis of CTF is completed can be significantly shortened compared to the time required for culturing spore-forming bacteria on an agar medium. In addition, the above-mentioned CTC addition step, incubation step, and fluorescence detection step can be easily carried out compared to the case of culturing spore-forming bacteria on an agar medium, which has many procedures depending on the experience of the examiner.

[0019] Furthermore, according to the method for detecting spore-forming bacteria of the present invention, the cost required for detection can be significantly reduced compared to the method for detecting spore-forming bacteria to which the multiplex PCR technique that requires primers, reagents, PCR equipment, etc. is applied.

[0020] Therefore, according to the method for detecting spore-forming bacteria of the present invention, a method for detecting spore-forming bacteria that is simple, has high detection accuracy, and is low in cost can be provided.

[0021] After the CTC addition step, an incubation step is performed in which the test sample to which the CTC solution has been added is incubated for a predetermined time. By adjusting the incubation time, the staining efficiency of spore-forming bacteria by CTC can be adjusted.

[0022] By performing the fluorescence detection step, for example, by flow cytometry analysis, living cells of spore-forming bacteria can be detected quickly and easily.

[0023] Many spoilage-causing bacteria are included among spore-forming bacteria, and if the spoilage-causing bacteria are at least one of the genera Paenibacillus, Sporosarcina, and Clostridium, then at least one of Paenibacillus polymixa, Sporosarcina cyclophylla, and Clostridium putrefasiens, which are thought to cause spoilage of packaged foods (e.g., canned beverages), can be detected by the spore-forming bacteria detection method of the present invention. [Brief explanation of the drawing]

[0024] [Figure 1] This is a flowchart illustrating the method for detecting spore-forming bacteria according to the present invention. [Figure 2] This is a photographic diagram showing the results of Wiltz staining. [Figure 3] This graph shows the growth curves of vegetative cells for each bacterial species. [Figure 4] This figure shows the results of examining the metabolic activity of vegetative cells of each bacterial species. [Figure 5] This is a photographic diagram showing the results of confirming purified spores using Wiltz staining. [Figure 6] This figure shows the results of monitoring the metabolic activity of purified spores after they were added to a liquid culture medium. [Figure 7] This graph shows the growth curve of purified spores added to a liquid culture medium. [Figure 8] This figure shows the results of monitoring the metabolic activity of purified spores after they were added to a liquid culture medium. [Figure 9] This graph shows the growth curves of spores added to each food sample. [Figure 10] This figure shows the results of monitoring bacterial metabolic activity based on growth curves in each food sample. [Modes for carrying out the invention]

[0025] Embodiments of the present invention will be described below with reference to the drawings. The present invention provides a method for detecting spore-forming bacteria, comprising: a CTC addition step A, in which a CTC solution containing 5-cyano-2,3-ditril-2H-tetrazolium salt (CTC) is added to a sample that may contain spore-forming bacteria; an incubation step B, in which the sample to which the CTC solution has been added is incubated for a predetermined time after the CTC addition step; and a fluorescence detection step C, in which the generated fluorescent formazan (CTF) is detected (Figure 1).

[0026] The term "spore-forming bacteria" is not particularly limited to any bacteria that form spore cells. In this embodiment, the spoilage-causing bacteria described below are given as examples of spore-forming bacteria, but the definition is not limited to these.

[0027] "Spoilage-causing bacteria" are not particularly limited to bacteria that are thought to cause spoilage, which leads to a deterioration in the flavor of packaged foods (e.g., canned beverages) and makes them unsuitable for consumption, or to bacterial genera to which such bacteria belong (e.g., Paenibacillus, Sporosarcina, Clostridium, Bacillus, Sporolactobacillus, Morellella, Diobacillus, and Thermoanaerobacterium). Of these spoilage-causing bacteria, it is preferable to be able to detect Paenibacillus, Sporosarcina, and Clostridium.

[0028] Examples of Paenibacillus species include Paenibacillus polymixa, Paenibacillus odorifer, and Paenibacillus terae; examples of Sporosarcina species include Sporosarcina cyclophylla and Sporosarcina globispora; and examples of Clostridium species include Clostridium putrefaciens, Clostridium perflingens, and Clostridium difficile, but are not limited to these.

[0029] The "subject sample" can be a food sample taken in whole or in part from a containerized food such as retort foods, canned goods, PET bottle beverages, pouched prepared foods, packaged beverages, or other chilled foods containing the target food or beverage in a container. However, it is not limited to this, and any sample that may contain spore-forming bacteria (spoilage-causing bacteria) is acceptable.

[0030] 5-Cyano-2,3-Ditril-2H-tetrazolium salt (CTC) is reduced by NAD(P)H produced in response to cellular metabolic activity, generating fluorescent formazan (CTF). CTF emits red fluorescence in viscous solutions and in solid form. Since CTF is lipid-soluble (insoluble in water) and accumulates as a fluorescent precipitate within cells, the number of respiratory bacteria can be determined by incubating CTC with a sample and then counting them under a fluorescence microscope or analyzing them by flow cytometry.

[0031] CTF is detected at excitation wavelengths of 480 nm and emission wavelengths of 630 nm. Therefore, in this invention, CTC staining is used as an indicator of the viability of spore-forming bacteria in order to distinguish between living cells (spores that have the potential to germinate and proliferate in the future) and dead cells.

[0032] Step A, the addition of CTC, involves adding a CTC-containing CTC solution to a sample that may contain spore-forming bacteria. The CTC solution can be any form containing CTC and can be used together with an enhancing reagent (dimethyl sulfoxide (DMSO) solution). The CTC solution can be prepared, for example, by adding water (preferably ultrapure water) to a commercially available CTC powder.

[0033] While there are no particular limitations to the type of CTC solution described, this embodiment describes the use of Bacstain-CTC Rapid Staining Kit (manufactured by Dojin Chemical Research Institute Co., Ltd.). The CTC solution is preferably used with a CTC concentration of, for example, 0.5 to 250 mmol / L, preferably 0.5 to 50 mmol / L, and more preferably 50 mmol / L, but is not limited to these values. If the concentration exceeds the upper limit (250 mmol / L), it is undesirable because the high concentration of CTC may kill the bacteria or prevent them from performing proper metabolism. Conversely, if the concentration is below the lower limit (0.5 mmol / L), it is undesirable because the fluorescence intensity will be weak and detection will be difficult, or it will be difficult to distinguish it from dirt and background particles, reducing the accuracy of detection.

[0034] After step A, which involves adding CTC, an incubation step B is performed in which the sample to which the CTC solution has been added is incubated for a predetermined time. The conditions for the incubation step can be, for example, 5 minutes to 24 hours, preferably 15 to 30 minutes, at room temperature (20 to 37°C) and in the dark (light-shielding conditions), but the temperature and incubation time are not limited to these. For example, the staining efficiency of spore-forming bacteria by CTC can be adjusted by adjusting the incubation time. A temperature within the above range is preferable because it is within the appropriate temperature range for the survival of each bacterium. A temperature outside the above range is undesirable because it is outside the appropriate temperature range for the survival of each bacterium. An incubation time within the above range is preferable because it allows for efficient detection of the red fluorescence of CTF. An incubation time outside the above range is undesirable because it becomes difficult to efficiently detect the red fluorescence of CTF.

[0035] Fluorescence detection step C is a step in which the generated fluorescent formazan (CTF) is detected. Fluorescence detection is not particularly limited as long as it can measure the fluorescence intensity of the CTF and analyze the fluorescence. For example, fluorescence detection can be performed using flow cytometry, fluorescence microscopy, or a spectrophotometer equipped with an excitation wavelength laser around 450 nm and capable of detecting fluorescence in the 630-640 nm range. These analyses should be performed according to standard procedures.

[0036] By performing fluorescence detection step C using flow cytometry analysis, viable cells of spore-forming bacteria can be detected rapidly and easily.

[0037] By performing fluorescence detection step C to detect the generated CTF, the CTF (fluorescent precipitate) accumulated inside the cells can be detected. By analyzing the CTF detected in this way, living cells and dead cells can be accurately distinguished, and bacteria with respiratory activity (living cells) can be easily detected.

[0038] According to the spore-forming bacteria detection method of the present invention, the time required from the CTC addition step A, incubation step B, and fluorescence detection step C to the completion of CTF analysis is approximately 15 to 30 minutes. Conventional methods, such as culturing spore-forming bacteria on agar plates, require several days. Therefore, the spore-forming bacteria detection method of the present invention can significantly reduce the detection time for spore-forming bacteria.

[0039] Furthermore, the CTC addition step A, incubation step B, and fluorescence detection step C can be performed more easily compared to culturing spore-forming bacteria on agar plates, which involve many procedures that depend on the experience of the inspector.

[0040] Furthermore, the method for detecting spore-forming bacteria according to the present invention can significantly reduce the cost required for detection compared to, for example, a method for detecting spore-forming bacteria using multiplex PCR technology.

[0041] Therefore, the present invention provides a simple, highly accurate, and low-cost method for detecting spore-forming bacteria. [Examples]

[0042] Examples of the spore-forming bacteria detection method of the present invention will be described below.

[0043] [Example 1] (Culturing of bacterial strains) All bacterial strains described below were purchased from the Japan Microbial Strain Preservation Center (JCM). The aerobic bacteria Paenibacillus polymyxa (JCM 2507=American Type Culture Collection [ATCC] 842=Leibniz Institute DSMZ [DSM] 36) and Sporosarcina psychrophila (JCM 9075=ATCC 23304=DSM 3)) were cultured in tryptic soy broth (BD Difco) at 30°C and 25°C, respectively. The anaerobic bacterium Clostridium putrefaciens (JCM 1431=ATCC 25786=DSM 1291) was cultured at 26°C in cooked meat medium (BD Difco) using an AnaeroPack system (Mitsubishi Gas Chemical Company) under conditions of 15% CO2 and <0.1% O2. The cultured bacteria were transferred to a medium containing 1 mM manganese, which has been reported to induce spore formation, and incubated at 20°C for a further 10 days ("Heat tolerance and growth at low temperatures of low-temperature spore-forming bacteria," Aoyama and Enda, Toyo Food Research Institute Research Report, 28, 47-53 (2010)).

[0044] The conditions under which each bacterial strain forms spores were confirmed by culturing them under the optimal conditions listed in the JCM catalog (Paenibacillus polymixa: 30°C, tryptic soy broth; Sporosarcina cyclophylla: 25°C, tryptic soy broth; Clostridium putrefaciens: 26°C, cooked meat medium) and then staining with Wirtz (Figure 2A). For the culture, the optical density (OD600) of the sample (100 μL) was measured at 600 nm using a Synergy HTX spectrophotometer (Bio-Tec). If the OD600 exceeded 1.0, the sample was diluted with fresh medium and measured again until the OD was 1.0.

[0045] Wirtz staining was performed using the Wirtz spore staining kit (Muto Chemical Co., Ltd.) according to the manufacturer's protocol. Specifically, 1 mL of 5% malachite green was added to the sample fixed on a glass slide and incubated for 2 minutes. Next, the slide was heated and washed with ultrapure water (Millipore Sigma) for 30 seconds. Then, 1 mL of 0.5% safranin was added, incubated for 30 seconds, washed with ultrapure water, and observed under a microscope.

[0046] Subsequently, spore formation was confirmed by culturing the samples in a manganese-containing medium for an additional 10 days (Figure 2B). The spore formation rates were approximately 75% for Paenibacillus polymixa, approximately 90% for Sporosarcina cyclophylla, and approximately 12% for Clostridium putrefaciens (see arrows).

[0047] (Evaluation of bacterial growth and metabolic responses in optimal media using CTC staining and flow cytometry analysis) CTC staining and flow cytometry analysis were used to confirm bacterial metabolic responses and assess the presence of living vegetative cells and spores. First, growth curves of vegetative cells for each bacterial species were plotted (Figure 3). Paenibacillus polymixa and Sporosarcina cyclophylla entered the logarithmic growth phase approximately 9 hours and 6 hours after inoculation, and both reached the stationary phase at 15 hours. Paenibacillus polymixa entered the late stationary phase or death phase approximately 21 hours later, while Sporosarcina cyclophylla maintained the stationary phase even after 24 hours. The anaerobic bacterium Clostridium putrefasiens entered the logarithmic growth phase approximately 9–12 hours after inoculation and reached the stationary phase at 40 hours later.

[0048] Furthermore, vegetative cells and spores were incubated with 9% PAA (Millipore Sigma) diluted to a final concentration of 0.2% in ultrapure water for 15 minutes at room temperature. After centrifugation at 12,000 × g at 4°C for 2 minutes, the supernatant was removed, and the pellet was washed twice with PBS (0.2% PAA treatment). All bacteria treated with 0.2% PAA to halt metabolic activity did not show growth.

[0049] To confirm metabolic activity, culture samples of Paenibacillus polymixa and Sporosarcina cyclophylla were collected after 18 hours, and samples of Clostridium putrefaciens were collected after 48 hours. After CTC staining (CTC addition step A), incubation step B and flow cytometry analysis (fluorescence detection step C) were performed.

[0050] Step A, the addition of CTC, was carried out using the Bacstain-CTC Rapid Staining Kit (manufactured by Dojin Chemical Laboratories Co., Ltd.) in accordance with the manufacturer's instructions, as follows: That is, cells (1 × 10 6 To 1 mL of cells (cells / mL), add 20 μL of CTC solution (10 mg of CTC dissolved in 750 μL of ultrapure water) and 1 μL of the enclosed enhancing reagent (Enhancing reagent A) to stain the cells. After inversion to relax the cells, the mixture was incubated for 30 minutes at room temperature (37°C) in the dark (Incubation step B).

[0051] The fluorescence detection step C was performed as follows. The samples stained by step A (CTC addition) were diluted 100-1000 times with 1×PBS. 1 mL of the diluted sample was placed in a Guava easyCyte flow cytometer (Cytek Industries) and detected under excitation wavelength 480 nm and emission wavelength 630 nm conditions. The results were analyzed using FlowJo v10.10.0 software (BD Biosciences). The results are shown in Figure 4.

[0052] In Figure 4, the X-axis defines the location of spore appearance, and the Y-axis defines the ranges of positive (metabolic activity present) and negative (metabolic activity absent). The upper part of the horizontal line in the graph represents the spore signal, and the lower part represents the signal from food residue, etc. The right side of the vertical line in the graph represents positive, and the left side represents negative.

[0053] Compared to bacteria treated with 0.2% PAA for 15 minutes, Paenibacillus polymixa, Sporosarcina cyclophylla, and Clostridium putrefasiens maintained metabolic activity in the stationary phase in approximately 43%, 69%, and 25% of cases, respectively, suggesting bacterial viability. Cells treated with 0.2% PAA did not show metabolic activity in flow cytometry analysis.

[0054] (Purification of spores) The spores were purified as follows. Cells were collected by centrifugation at 12,000 × g at 4°C for 2 minutes (Eppendorf Hi-Mac Technologies). The cell pellet was washed twice with phosphate-buffered saline (PBS) (Fujifilm Wako Pure Chemical Industries, Ltd.) and then resuspended in a solution containing 10 mg / mL of lysozyme (Fujifilm Wako Pure Chemical Industries, Ltd.) in 10 mM Tris-HCl pH 7.5 (Nippon Gene Co., Ltd.). The suspension was incubated overnight at 37°C with shaking at 120 rpm. Subsequently, the cell pellet was collected by centrifugation at 12,000 × g at 4°C for 2 minutes. It was washed twice with PBS and treated with 1% sodium lauryl sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.) at 37°C for 1 hour with shaking at 120 rpm. Finally, it was centrifuged at 12,000 × g at 4°C for 2 minutes and washed three times with PBS.

[0055] The purified spores were observed under a microscope and measured using a hemocytometer (Sunlead Glass Co., Ltd.) at a density of 1.0 × 10⁻⁶. 8 The concentration was adjusted by diluting to cells / mL. The spores were stored at 4°C, with a shelf life of 2 weeks.

[0056] (Monitoring bacterial spore germination in culture media) The purified spores were stained with Wiltz stain using the method described above, and the presence of spores and reduced vegetative cells was confirmed (Figure 5). Purified spores (1.0 × 10⁻⁶) 6 Cells were added to a liquid culture medium, and their proliferation and metabolic activity were monitored (Figures 6-8).

[0057] Initially, no metabolic activity was detected (Figure 6), but compared to vegetative cells, it was confirmed that Paenibacillus polymyxa and Sporosarcina cyclophylla spores entered the logarithmic growth phase with a delay of approximately 6 hours, while Clostridium putrefaciens spores entered the logarithmic growth phase with a delay of approximately 15 hours (Figure 7). Spores treated with 0.2% PAA did not show proliferation (Figure 7).

[0058] Furthermore, metabolic activity during the delayed phase (9 hours after inoculation for Paenibacillus polymixa and Sporosarcina cyclophylla, and 20 hours after inoculation for Clostridium putrefasiens), the logarithmic growth phase (18 hours after inoculation for Paenibacillus polymixa and Sporosarcina cyclophylla, and 36 hours after inoculation for Clostridium putrefasiens), and the early stationary phase (24 hours after inoculation for Paenibacillus polymixa and Sporosarcina cyclophylla, and 48 hours after inoculation for Clostridium putrefasiens) was evaluated by CTC staining using the method described above (Figure 8).

[0059] CTC staining revealed that approximately 18%, 20%, and 12% of Paenibacillus polymixa, Sporosarcina cyclophylla, and Clostridium putrefasiens, respectively, showed metabolic activity before entering the logarithmic growth phase. Furthermore, during the logarithmic growth phase, approximately 90% of Paenibacillus polymixa, 82% of Sporosarcina cyclophylla, and 31% of Clostridium putrefasiens showed metabolic activity at 18 and 36 hours. In the early stationary phase, approximately 48% of Paenibacillus polymixa, 67% of Sporosarcina cyclophylla, and 35% of Clostridium putrefasiens showed metabolic activity.

[0060] (Monitoring the growth and germination of spore-forming bacteria using food samples) As described above, we were able to track spore germination using CTC staining, and we monitored the germination and proliferation trends of vegetative cells and spores using actual food samples.

[0061] Paenibacillus and Sporosarcina species have been reported in soil and processed vegetables (such as bottled white asparagus, canned corn, and kimchi), while Clostridium species have been reported in meat and meat-based soups (McSpadden Gardener, "Ecology of Bacillus and Paenibacillus spp. in Agricultural Systems" Phytopathology. 2004 Nov;94(11):1252-8, 2004; Inatsu et al., "Characterization of Paenibacillus spp. Isolated from Spoiled Corn Paste", Japanese Journal of Food Microbiology 34(2):126-130 2017; Helen E. Ross, "Clostridium putrefaciens: A Neglected Anaerobe", Journal of Applied Bacteriology, Volume 28, Issue 1, 1 April 1965, Pages) 49-51,10). Therefore, bottled white asparagus (imported by Toko Co., Ltd.) and chicken bouillon soup (manufactured by Maruhachi Muramatsu Co., Ltd.) were purchased, and the growth conditions when vegetative cells or spores were added were monitored.

[0062] Bottled white asparagus (350g) was homogenized, and residue was removed using 100μm and 40μm Corning cell strainers (Corning). The prepared sample (5mL) was placed in sterile 15mL cell tubes (Greiner Bio-One) and stored at room temperature until use. Chicken bouillon soup was prepared by dissolving 15g of soup powder in 300mL of ultrapure water. After sterilization at 121°C for 15 minutes, the prepared sample (5mL) was dispensed into sterile 15mL cell tubes and stored at room temperature in an AnaeroPack system (Mitsubishi Gas Chemical Company).

[0063] cells (1×10 6The cells / mL were added to 5 mL of each food sample prepared above, and Paenibacillus polymixa was cultured at 30°C, Sporosarcina cyclophylla at 25°C, and Clostridium putrefaciens at 26°C. Growth curves were drawn in the same manner as above (Figure 9). It was confirmed that the growth peaks of the bacteria in these food samples were reduced compared to when cultured in culture medium (Figures 3 and 9). Specifically, the doubling time of vegetative cells was extended by approximately 3 times for Paenibacillus polymixa, approximately 6 times for Sporosarcina cyclophylla, and approximately 1.5 times for Clostridium putrefaciens. Similarly, the doubling time of spores in the food samples was extended by approximately 4.5 times for Paenibacillus polymixa, approximately 6.5 times for Sporosarcina cyclophylla, and approximately 2 times for Clostridium putrefaciens.

[0064] [Table 1]

[0065] To monitor bacterial metabolic activity based on growth curves in food samples, spores were added to each food sample as described above. Bacterial samples were collected at 20, 30, and 48 hours for Paenibacillus polymixa and Sporosarcina cyclophylla, and at 20, 36, and 48 hours for Clostridium putrefaciens. CTC staining was performed according to the method described above, and spore germination was tracked (Figure 10).

[0066] Before entering the logarithmic growth phase, approximately 14%, 27%, and 15% of the spores of Paenibacillus polymixa, Sporosarcina cyclophylla, and Clostridium putrefasiens, respectively, showed metabolic activity. During the logarithmic growth phase, approximately 97% of Paenibacillus polymixa, 94% of Sporosarcina cyclophylla, and 30% of Clostridium putrefasiens showed metabolic activity at 30 and 36 hours. In the stationary phase, approximately 47% of Paenibacillus polymixa, 60% of Sporosarcina cyclophylla, and 45% of Clostridium putrefasiens showed metabolic activity at 48 hours.

[0067] Based on the above results, it was confirmed that the spore-forming bacteria detection method of the present invention can detect spore-forming bacteria with high accuracy from a sample containing spore-forming bacteria.

[0068] This indicates that spore-forming bacteria can be detected with high accuracy even in sample materials that may contain them. The spore-forming bacteria detected in this case are thought to be at least one of the following genera: Paenibacillus, Sporosarcina, and Clostridium. [Industrial applicability]

[0069] This invention can be used in a method for detecting spore-forming bacteria.

Claims

1. A method for detecting spore-forming bacteria, comprising: a CTC addition step of adding a CTC solution containing 5-cyano-2,3-ditril-2H-tetrazolium salt (CTC) to a subject sample that may contain spore-forming bacteria; an incubation step of incubating the subject sample to which the CTC solution has been added for a predetermined time after the CTC addition step; and a fluorescence detection step of detecting the generated fluorescent formazan (CTF).

2. A method for detecting spore-forming bacteria according to claim 1, wherein the fluorescence detection step is performed by flow cytometry analysis.

3. A method for detecting spore-forming bacteria according to claim 1 or 2, wherein the spore-forming bacteria are the causative agents of spoilage.

4. The method for detecting spore-forming bacteria according to claim 3, wherein the spoilage-causing bacteria is at least one of the following: Paenibacillus, Sporosarcina, and Clostridium.

5. The method for detecting spore-forming bacteria according to claim 4, wherein the Paenibacillus species is Paenibacillus polymixa, the Sporosarcina species is Sporosarcina cyclophylla, and the Clostridium species is Clostridium putrefasiens.

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