Ensuring cleanliness in food factories is essential for product safety. The ATP test provides a quick and effective way to verify cleanliness after cleaning and sterilization, overcoming the time-consuming and complex nature of microbial testing. This article explains the biological significance and chemical structure of ATP, why it is an excellent energy carrier, and the methods and equipment used in ATP testing. By understanding the ATP test and its practical applications, food factories can manage cleanliness more comprehensively and efficiently.
Introduction
After cleaning and sterilization, it's crucial to measure the efficiency of these processes and ensure that cleanliness management is properly implemented. From the perspective of food microbiology, microbial testing is considered the most direct method to prevent secondary contamination of products. However, microbial testing has disadvantages, such as complicated procedures and time consumption, making it not always suitable for daily, quick, and simple execution. On the other hand, the ATP test overcomes these disadvantages, allowing for quick and simple cleanliness measurement. By measuring the amount of ATP in samples taken from surfaces of the environment and equipment, cleanliness can be assessed quickly and easily.
Below, we explain the basic concept of the ATP test, considering the differences from microbial testing, and how it can be practically used for cleanliness management in food factories.
The Biological Significance of ATP
Let's start with the basics.
Revisit the process where solar energy is fixed into organic matter through photosynthesis. In photosynthesis, plants use light energy to combine carbon dioxide and water to synthesize sugars (organic matter). These sugars are like chemical energy stored, which can be compared to glue that binds building blocks together.
When plants "burn" (metabolize), sugars and other organic matters react with oxygen in the air, converting chemical energy into physical energy like heat and light.
On the other hand, heterotrophic organisms such as animals and microbes generate ATP, the currency of energy, during the breakdown of organic matter (sugars, etc.). The produced ATP then functions as chemical energy used for various life activities like muscle contraction and biosynthesis of biomolecules.
In essence, when plants burn, their chemical energy is released as physical energy. Meanwhile, when animals consume plants, their chemical energy is converted into ATP, a chemical energy currency within cells, used for life activities.
The Chemical Structure of ATP
Let's touch briefly on the chemical structure of ATP.
ATP consists of adenosine and three phosphate molecules. Adenosine is a nucleic acid base made up of adenine and ribose. The three phosphate molecules are attached to the ribose part of adenosine. The structure of an ATP molecule is represented as follows.
Why is ATP an Excellent Energy Carrier?
Among many compounds in the body, why is ATP considered an excellent energy carrier?
The reason ATP is an outstanding energy carrier is because the phosphate bonds in the ATP molecule are high-energy. The three phosphate molecules are bonded together while repelling each other, making the phosphate bonds high-energy.
Phosphate molecules carry a negative charge, and the close proximity of these molecules causes them to repel each other, requiring significant force to bind them.
This repulsion and the need for a strong force to maintain the bonds result in a high-energy state in the phosphate bonds. When the ATP phosphate bonds are broken, this high-energy state is released, liberating a large amount of energy.
Thus, ATP is an excellent energy carrier because, despite being a low molecular weight molecule, it can store a significant amount of energy relative to its molecular weight. This allows ATP to be widely used within the body, playing a crucial role as an energy supply source in cells.
ATP Measurement Methods
ATP measurement methods are based on the enzyme luciferase, involved in the bioluminescence phenomenon of fireflies. Why is firefly luciferase used? First, let's understand what luciferase is.
Bioluminescence in Fireflies and Marine Organisms
The reason fireflies use luciferase to produce light is mainly considered to be an ecologically advantageous communication method. Fireflies have unique bioluminescence patterns by species, allowing different species to distinguish each other. Through bioluminescence, male and female fireflies can recognize each other and find mating partners.
Moreover, various types of bioluminescent organisms exist in the ocean that use luciferase. These marine organisms, often living in deep-sea or dark environments, use bioluminescence for functions necessary for survival, such as capturing prey, defending against predators, and communication.
The meaning of bioluminescence in marine organisms that glow with luciferase varies, but one example is bioluminescent plankton, which glows inside the digestive tract of predators when eaten, making the predator visible to larger predators. This defense strategy leads fish to expel the plankton, allowing the plankton to escape predation risk.
The Bioluminescent Reaction with Luciferase
Firefly bioluminescence occurs through a chemical reaction catalyzed by the enzyme luciferase. The reaction proceeds as follows:
First, the carboxyl group of luciferin reacts with ATP to form a luciferyl AMP intermediate.
Oxygen then reacts with the intermediate, producing an excited state of oxyluciferin.
The excited state of oxyluciferin returns to its ground state by emitting a photon, resulting in light emission.
Application of Firefly Luciferase for ATP Quantification
The amount of light produced in the luciferase bioluminescent reaction is proportional to the amount of ATP.
Therefore, the amount of ATP in biological samples can be measured by utilizing the amount of light produced by luciferase.
The first attempt to use firefly luciferase for ATP concentration measurement was believed to be in 1966 by Brewer and Knutsen, in a study measuring ATP concentration in blood.
Brewer, G.J., Knutsen, C.A.
A technique for the processing of blood samples for subsequent assay of ATP, and an investigation of the method of standardization of the firefly-luciferase ATP assay
Clinica Chimica Acta 14(6), pp. 836-839（ 1966 ）
Since the publication of Brewer and Knutsen's paper, this method has been used in various fields of biochemical research.
Mass Production of Luciferase through Genetically Modified E. coli
As mentioned, the principles and methods for measuring ATP concentration using firefly luciferase have been clear since the 1960s. However, extracting and purifying luciferase from fireflies was costly and had issues with stability, limiting its widespread adoption.
The breakthrough for applying luciferase to ATP concentration measurement in the food industry came in the late 1980s. At that time, cloning techniques and recombinant expression were in the dawn of molecular biology. In 1988, using these new technologies, Dr. Shigehiro Homma and his team at Kikkoman Corporation in Japan successfully incorporated the firefly luciferase gene into E. coli for mass expression.
Dr. Homma's development became the starting point for the broad application of ATP in the food industry. For more details on these developments, the following reference is recommended:
ATP Testing for Cleanliness Management in Food Factories
The ATP measurement method has become widely used in the food industry and other fields, thanks to the possibility of mass-producing luciferase.
Why is the ATP test important in food factories?
The reason is that the ATP test allows for rapid and simple cleanliness checks. Traditionally, methods for measuring microbial counts after cleaning were time-consuming and labor-intensive. However, with the advent of the ATP test, it's now possible to quickly verify the cleaning effectiveness in food factories.
Initially, the ATP test was expected to serve as an alternative to traditional microbial measurements. However, since ATP can also be found in food-derived organic materials, it's not a perfect indicator of microbial counts.
Currently, the ATP test is utilized in food factories to determine the amount of organic material remaining after cleaning operations. This enables food factories to quickly check the amount of organic material left on production lines after cleaning and ensure proper cleaning has been conducted.
Measurement Procedure
Here, we'll briefly outline the general procedure for the ATP test. However, please note that this blog is not intended to provide an experimental manual, but rather to help understand the experiment's overview, focusing on that aspect.
❶Sample Collection
First, collect samples from the surfaces of the environment and equipment within the food factory. Swabs, which are included with the ATP measurement equipment, are commercially available for sample collection. Typically, the first stage involves collecting samples across the entire factory area, followed by selecting areas or points on the production line with a high potential for contamination in the second stage.
❷Sample Extraction
Soak the collected samples in an appropriate extraction liquid to leach the ATP from the samples. The process is completed simply by inserting the swab used for sample collection into the extraction liquid, and kits for this process are commercially available.
❸ATP Measurement
To measure the amount of ATP in the extraction liquid, use the chemiluminescent reagents luciferin and luciferase. Mix the extraction liquid and reagent, detecting the light emitted as a result of the reaction with ATP. The intensity of the light emission is proportional to the amount of ATP in the sample. Kits that allow for simultaneous operation of sample extraction and mixing with the chemiluminescent reagent are also available commercially.
❹Measurement Equipment
ATP measurements are conducted using a specialized device called a luminometer. The luminometer detects the light emitted from the mixture of reagent and extraction liquid and calculates the amount of ATP based on the intensity of the light. Some luminometers also have the capability to record and analyze the measurement results as data.
❺Result Interpretation
The measurement results obtained from the luminometer are compared with pre-set standard values to evaluate cleanliness. The standard values should be set according to the conditions of each food factory.
Conclusion
By properly implementing the ATP test and understanding its limitations, it becomes possible to manage the cleanliness of food factories more comprehensively when combined with other testing methods. For example, identifying areas prone to organic material residue through the ATP test and focusing on cleaning and sterilization in those areas, and ultimately combining it with microbial tests as necessary, can enable more effective utilization.
