JP-2026075412-A - Method for detecting spore-forming bacteria

Abstract

[Problem] To provide 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. [Selection Diagram] Figure 1

Inventors

• 河合 総一郎

Assignees

• 公益財団法人東洋食品研究所

Dates

Publication Date

20260508

Application Date

20241022

Claims (5)

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 potentially containing 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. The 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.

Description

This invention relates to a method for detecting spore-forming bacteria. Among microorganisms, there are bacteria that form spores. Spore-forming bacteria actively divide and multiply in favorable environments, but form spores when the environment becomes unfavorable for growth, such as when nutrients decrease or the environment dries up. Spores possess extremely high durability and can survive even in situations where the environment deteriorates to the point where normal bacteria die. However, if the growth environment does not change, spores will not germinate or multiply, and their metabolism is extremely limited. Spores are durable cells formed within vegetative cells. They have a very low water content, metabolize very little, and are essentially in a dormant state. Spore cells exhibit strong resistance to various stresses and are highly resistant to heat. Depending on the bacterial species, killing them requires heating at over 100°C for a specified period of time. When surviving dormant spores are placed again in an environment suitable for bacterial growth, these spores germinate, producing bacterial cells with normal growth and metabolic capabilities. Because spores are highly heat-resistant, their presence poses a problem in packaged foods where microbial control is achieved through heat sterilization. Spores with particularly high heat resistance are the most important biological hazards for packaged foods and serve as an indicator for heat sterilization. Representative bacteria that form spores include aerobic bacteria such as Paenibacillus and Sporosarcina species, and anaerobic bacteria such as Clostridium species. 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 and 2). Spoilage, where carbohydrates and fats in food ingredients decompose, resulting in a loss of flavor and making the food unsuitable for consumption, has 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). Conventionally, methods for detecting spore-forming bacteria have included techniques using microbial culture on agar plates (Non-Patent Documents 4 and 5) and multiplex PCR technology for simultaneously detecting and amplifying multiple target genes (Non-Patent Document 6). 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 garnered significant 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. N. Kalchayanand, Bibek Ray, Ray A. Field and M. C. Johnson, “Spoilage of Vacuum-Packaged Refrigerated Beef by Clostridium”, Journal of Food Protection, Vol. 52, No. 6, Pages 424-426 (June 1989)Stephane Andre, Tatiana Vallaeys, Stella Planchon, “Spore-forming bacteria responsible for food spoilage”, Research in Microbiology 168 (2017) 379-387Nidhi 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 1418R. E. Levin, “Detection and Incidence of Specific Species of Spoilage Bacteria on Fish”, Applied Microbiology, Nov. 1968, p. 1734-1737Chandrasekaran 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):219-225Ying 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 This is a flowchart illustrating the method for detecting spore-forming bacteria according to the present invention.This is a photographic diagram showing the results of Wiltz staining.This graph shows the growth curves of vegetative cells for each bacterial species.This figure shows the results of examining the metabolic activity of vegetative cells of each bacterial species.This is a photographic diagram showing the results of confirming purified spores using Wiltz staining.This figure shows the results of monitoring the metabolic activity of purified spores after they were added to a liquid culture medium.This graph shows the growth curve of purified spores added to a liquid culture medium.Thi