WO2018147443A1 - Kit for measuring degree of contamination of cooking instruments and cooking instrument cleaning program evaluation method - Google Patents

Abstract

In order to provide a method whereby a cleaning program for cooking instruments can be evaluated or improved, a cleaning program evaluation method and improvement method are provided that use an ATP and ADP measurement kit. This method includes: a step (i) in which cooking instruments are cleaned in accordance with a program; a step (ii) in which the kit is used and the degree of contamination is measured for the cleaned cooking instruments; a step (iii) in which the degree of contamination measured in step (ii) is compared with a reference prescribed by the program; a step (iv) in which an evaluation is made that the contamination was not adequately removed in cleaning step (i) or the cleaning step (i) is corrected, if the measured degree of contamination exceeds the reference prescribed by the program; and a step (v) in which steps (i)–(iv) are repeated until the degree of contamination is no more than the reference prescribed ty the program. The kit that is used may include: an enzyme that catalyzes a reaction that generates ATP from ADP; luciferin; luciferase; and metal salts. The kit that is used may include: an enzyme that catalyzes a reaction that generates ATP from AMP; an enzyme that catalyzes a reaction that generates AMP from ADP; luciferin; luciferase; and metal salts.

Description

Cooking-related appliance contamination degree measuring kit and cooking-related appliance cleaning program evaluation method

The present invention relates to a kit for measuring the degree of contamination of cooking-related utensils and a method for measuring the degree of contamination using the kit. Moreover, this invention relates to the evaluation method of the cooking related utensil washing | cleaning program using this kit.

In order to provide food in a clean and hygienic manner, it is necessary to manage the hygiene of the food production environment and the processing environment. This is usually called a general hygiene program. To determine the maintenance management procedures for equipment, cooking-related utensils and handling utensils used in the production or processing of food in general hygiene management programs, and to operate normally and prevent microbial contamination and contamination of food Usually, a standard operating procedure (SOP) is created. Among the SOPs, procedures for preventing contamination and mixing of hazards from the food handling environment, such as cleaning and sterilization of used equipment and fingers, and hygiene management of equipment, especially Sanitation Standard Operating Procedures Procedure, SSOP).

In formulating SOPs and SSOPs for food processing, it is necessary to assess the degree of contamination of cooking-related utensils and confirm cleaning. Examples of SSOP include (A) cleaning and sterilization of machinery and equipment and (B) cleaning and sterilization of workers' hands.

In conventional SSOP, for example, (A) In the cleaning and sterilization of machinery and equipment, the line supervisor visually inspects the condition of the workbench after the determined cleaning and sterilization work, and there are some places where the visual results are not sufficiently cleaned. There are things that are going to start over. In addition, there is one in which a quality control person periodically carries out a wiping inspection of the line and verifies the result of cleaning and sterilization. In addition, in conventional SSOP, for example, (B) In the cleaning and sterilization of workers 'hands, after deciding the cleaning and sterilization method, the line supervisor will check the workers' finger cleaning and sterilization at the start of work, after a break, etc. There are those that observe at the timing, record them in the daily hygiene checklist, and give guidance or training if there are workers who do not follow the cleaning and sterilization procedures. In the case of such a program, whether or not a hygienic state can be realized depends on the visual judgment of the supervisor or the supervisor must observe the employee. Even if regular inspection is performed, lots for which periodic inspection is not performed cannot cope with sudden changes.

In contrast to conventional ATP measurement using luciferase, Patent Document 1 (Japanese Patent Laid-Open No. 9-234099, Japanese Patent No. 34099962) describes an ATP + AMP measurement method that combines pyruvate orthophosphate dikinase (PPDK), which converts AMP to ATP. It is described.
Patent Document 2 (Japanese Patent Laid-Open No. 11-069997) describes a cleanliness inspection reagent and a cleanliness inspection method using the reagent.
Non-Patent Document 1 (Suzuki et al., Japanese Society of Food Microbiology, 2007, Abstract), describes the development of a high-sensitivity cleanliness test method using adenine nucleotides as an index.

With conventional cooking-related utensil cleaning methods, even if qualitative evaluation can be made visually, how much cleaning can be performed at what detergent concentration can remove contamination derived from food such as meat and fish to safety standards, It was not easy to grasp quantitatively. The use of excess synthetic detergent is an environmental burden. Moreover, it has not been easy to optimize the cleaning process to necessary and sufficient conditions only by qualitative judgment. Especially in food processing facilities and large-scale cooking sites, it takes time and labor to evaluate whether the established hygiene management program and SSOP are functioning. It is costly and economical to evaluate individual facilities and sites. It may not be realistic from the aspect, or it may not be fully implemented. In addition, if the program is not functioning or is judged to be inadequate even after evaluation, determine how much the number of steps can be increased or modified to achieve sufficient cleaning and a sanitary environment. It was not easy.

Japanese Patent Laid-Open No. 9-234099 (Japanese Patent No. 3409996) JP 11-069997 A

In conventional hygiene management methods, it was necessary to rely on the visual judgment of the person performing the inspection or to observe the washing and sterilization of the hands of employees by the supervisor. Moreover, even if the periodic inspection was performed, there was a problem that lots that were not subjected to the periodic inspection could not cope with sudden changes.

Enzyme reagents that measure nucleotides such as ATP, ADP, and AMP are known, and these were available in laboratories and laboratories, but samples from individual cooking facilities were transported to laboratory facilities and research facilities one by one. It is expensive and time consuming to make measurements and communicate the results to the cooking site to modify the cleaning program. Moreover, the dishwasher is evaluated by the manufacturer at the time of development, but the evaluation method is not quantitative, such as qualitative or visual. In addition, there has never been a means for accurately and simply evaluating the degree of contamination when it is used at each business office or cooking site. In addition, for contaminated samples that contain a large amount of ATP-degrading enzyme, is the cleaning program executed by ATP degrading into ADP by the ATP-degrading enzyme contained in the sample during sample measurement immediately after washing? It was difficult to accurately evaluate, set an appropriate reference value, and correct the cleaning program accurately.

This invention makes it a subject to provide the cooking related appliance contamination degree measuring kit and cooking related appliance washing program evaluation method which solve the conventional problem.

In view of the above problems, the present invention provides an ATP and ADP measurement kit or an ATP, ADP and AMP measurement kit that can quickly detect contamination of cooking-related utensils, and a cleaning program evaluation using this kit. Provide a method.

The present invention includes the following embodiments:
[1] Cleaning program evaluation method using ATP and ADP measurement kit, including the following steps: (i) Cleaning cooking-related utensils according to the program;
(ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
(iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, Evaluate that the cleaning process of i) does not sufficiently remove contamination.
Here, the ATP and ADP measurement kit is an ATP and ADP measurement kit for improving a cooking-related utensil washing program, which includes an enzyme that catalyzes a reaction for generating ATP from ADP, luciferin, luciferase, and a metal salt.
[2] The method according to 1, wherein when it is evaluated that the contamination is not sufficiently removed in step (iv), the cooking-related utensils are further washed by the same or different method as in step (i).
[3] Cleaning program improvement method using ATP and ADP measurement kit, including the following steps, (i) Cleaning cooking-related utensils according to the program,
(ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
(iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
(v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program,
Here, the ATP and ADP measurement kit is an ATP and ADP measurement kit for improving a cooking-related utensil washing program, which includes an enzyme that catalyzes a reaction for generating ATP from ADP, luciferin, luciferase, and a metal salt.
[4] Further before step (i),
(o) creating a cleaning program for cooking-related utensils;
The method according to any one of 1 to 3, comprising
[5] Enzymes that catalyze the reaction of generating ATP from ADP are pyruvate kinase (PK), acetate kinase (AK), creatine kinase (CK), polyphosphate kinase (PPK), hexokinase, glucokinase, glycerol kinase, 5. The method according to any one of 1 to 4, which is selected from the group consisting of fructokinase, phosphofructokinase, riboflavin kinase, and fructose bisphosphatase.
[6] The method according to any one of 1 to 5, wherein the ATP and ADP measurement kit further contains an AMP measurement reagent.
[7] The method according to 6, wherein the AMP measurement reagent comprises an enzyme that catalyzes a reaction for producing ADP or ATP from AMP.
[8] The enzyme according to 7, wherein the enzyme that catalyzes a reaction for generating ADP or ATP from AMP comprises pyruvate orthophosphate dikinase (PPDK), adenylate kinase (ADK), or pyruvate water dikinase (PWDK). Method.
[9] A cleaning program evaluation method using an ATP, ADP and AMP measurement kit, including the following steps:
(i) cleaning cooking-related utensils according to the program;
(ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
(iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, Evaluate that the cleaning process of i) does not sufficiently remove contamination.
Here, the measurement kit includes an enzyme that catalyzes a reaction that generates ATP from AMP, an enzyme that catalyzes a reaction that generates AMP from ADP, luciferin, luciferase, and a metal salt. Said method.
[10] The method according to 9, wherein when it is evaluated that the contamination is not sufficiently removed in step (iv), the cooking-related utensils are further washed by the same or different method as in step (i).
[11] A cleaning program improvement method using an ATP, ADP and AMP measurement kit, including the following steps:
(i) cleaning cooking-related utensils according to the program;
(ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
(iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
(v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program,
Here, the measurement kit includes an enzyme that catalyzes a reaction that generates ATP from AMP, an enzyme that catalyzes a reaction that generates AMP from ADP, luciferin, luciferase, and a metal salt. Said method.
[12] Further before step (i),
(o) creating a cleaning program for cooking-related utensils;
The method according to any one of 9 to 11, comprising:
[13] The enzyme that catalyzes the reaction that generates ATP from AMP is pyruvate orthophosphate dikinase (PPDK) or pyruvate water dikinase (PWDK), and the enzyme that catalyzes the reaction that generates AMP from ADP. The method according to any one of 9 to 12, which is ADP-dependent hexokinase or apyrase.
[14] ATP and ADP measurement kit for use in a cooking-related utensil washing program evaluation method, comprising an enzyme that catalyzes a reaction for generating ATP from ADP, luciferin, luciferase, and a metal salt, wherein the cooking-related utensil washing program is evaluated The method is
(i) cleaning cooking-related utensils according to the program;
(ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
(iii) compare the degree of contamination measured in step (ii) with the references specified in the program; and
(iv) If the measured degree of contamination exceeds the reference specified in the program, evaluate that the cleaning process in (i) does not remove the contamination sufficiently.
A kit comprising the kit.
[15] The kit according to 14, wherein when it is evaluated that the contamination is not sufficiently removed in step (iv), the cooking-related utensils are further washed by the same or different method as in step (i).
[16] ATP and ADP measurement kit for use in a cooking-related utensil washing program improvement method, comprising an enzyme that catalyzes a reaction for generating ATP from ADP, luciferin, luciferase and a metal salt, wherein the cooking-related utensil washing program is improved The method is
(i) cleaning cooking-related utensils according to the program;
(ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
(iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
(v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program,
A kit comprising the kit.
[17] Further before step (i),
(o) creating a cleaning program for cooking-related utensils;
The kit according to any one of 14 to 16, comprising:
[18] Enzymes that catalyze the reaction of generating ATP from ADP are pyruvate kinase (PK), acetate kinase (AK), creatine kinase (CK), polyphosphate kinase (PPK), hexokinase, glucokinase, glycerol kinase, The kit according to any one of 14 to 17, which is selected from the group consisting of fructokinase, phosphofructokinase, riboflavin kinase, and fructose bisphosphatase.
[19] The kit according to any one of 14 to 18, wherein the kit further contains an AMP measurement reagent.
[20] The kit according to 19, wherein the AMP measurement reagent comprises an enzyme that catalyzes a reaction for producing ADP or ATP from AMP.
[21] The enzyme according to 20, wherein the enzyme that catalyzes a reaction for generating ADP or ATP from AMP comprises pyruvate orthophosphate dikinase (PPDK), adenylate kinase (ADK), or pyruvate water dikinase (PWDK). kit.
[22] ATP for use in cooking-related utensil washing program evaluation method, including enzyme that catalyzes reaction to produce ATP from AMP, enzyme that catalyzes reaction to produce AMP from ADP, luciferin, luciferase and metal salt, ADP And the AMP measurement kit, wherein the cooking-related utensil cleaning program evaluation method is:
(i) cleaning cooking-related utensils according to the program;
(ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
(iii) compare the degree of contamination measured in step (ii) with the references specified in the program; and
(iv) If the measured degree of contamination exceeds the reference specified in the program, evaluate that the cleaning process in (i) does not remove the contamination sufficiently.
A kit comprising the kit.
[23] The kit according to 22, wherein when it is evaluated that the contamination is not sufficiently removed in step (iv), the cooking-related utensils are further washed by the same or different method as in step (i).
[24] ATP, ADP for use in cooking-related utensil cleaning program improvement method, including enzyme that catalyzes reaction for generating ATP from AMP, enzyme that catalyzes reaction for generating AMP from ADP, luciferin, luciferase, and metal salt And an AMP measurement kit, wherein the cooking-related utensil cleaning program improvement method is:
(i) cleaning cooking-related utensils according to the program;
(ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
(iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
(v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program,
A kit comprising the kit.
[25] Further before step (i),
(o) creating a cleaning program for cooking-related utensils;
The kit according to any one of 22 to 24, comprising:
[26] The enzyme that catalyzes the reaction that generates ATP from AMP is pyruvate orthophosphate dikinase (PPDK) or pyruvate water dikinase (PWDK), and the enzyme that catalyzes the reaction that generates AMP from ADP. The kit according to any of 22 to 25, which is ADP-dependent hexokinase or apyrase.

This specification includes the disclosure of Japanese Patent Application Nos. 2017-021952, 2017-058940, and 2017-192606, which are the basis of the priority of this application.

As an effect of the present invention, the degree of contamination of cooking-related utensils can be measured without depending on visual observation. In addition to periodic inspections, special inspections can be performed easily and quickly especially for lots that require safety confirmation. Furthermore, by the method of the present invention, the problems of the conventional hygiene management program can be identified more accurately, and the program evaluation can be performed effectively. Further, according to the present invention, ATP and ADP, or ATP is decomposed by heat, pH, time or ATP-degrading enzyme, or ATP and ADP, ATP, AMP, and ADP can be accurately measured, and the cleaning program can be effectively evaluated, a reference value can be set, and the cleaning program can be modified.

Moreover, since the reagents of the present invention are in a kit, it is not necessary to transport samples to a research facility one by one, and contamination can be detected quickly at each cooking site or cooking facility (in situ). And can quickly evaluate cleaning programs.

It is a division figure of the cutting board used for the washing | cleaning evaluation test. It is the figure of the plate which culture | cultivated the wipe sample. It is a figure of a calibration curve. It is a figure of a calibration curve. It is a figure of a calibration curve. It is a figure of a calibration curve. It is a figure of a calibration curve. It is a figure of a calibration curve.

In one embodiment, the present invention provides a cooking-related appliance contamination degree measuring kit and a cooking-related appliance contamination degree measuring method using the same. In certain embodiments, the method uses an enzyme, luciferin, luciferase and a metal salt that catalyses the reaction of producing ATP from ADP, or a kit comprising these. Enzymes that catalyze the reaction of generating ATP from the ADP include pyruvate kinase (PK), acetate kinase (AK), creatine kinase (CK), polyphosphate kinase (PPK), hexokinase, glucokinase, glycerol kinase, fructose It can be selected from the group consisting of kinase, phosphofructokinase, riboflavin kinase, and fructose bisphosphatase. In another embodiment, the method further uses or uses a kit comprising pyruvate orthophosphate dikinase (PPDK), adenylate kinase or pyruvate water dikinase (PWDK). In another embodiment, an enzyme that catalyzes a reaction that produces ATP from AMP, an enzyme that catalyzes a reaction that produces AMP from ADP, luciferin, luciferase, and a metal salt are used, or a kit containing these is used. The enzyme that catalyzes the reaction of generating ATP from the AMP can be selected from the group consisting of pyruvate orthophosphate dikinase (PPDK) and pyruvate water dikinase (PWDK), and generates AMP from the ADP. The enzyme that catalyzes the reaction may be selected from the group consisting of ADP-dependent hexokinase and apyrase.

When the sample contains ATP, it is converted into AMP by luciferase and luminescence occurs. When ADP is contained in the sample, it is converted to ATP by an enzyme that catalyzes a reaction that generates ATP from ADP, and then ATP is subjected to a luminescence reaction. As a result, the total amount of ATP and ADP present in the system can be measured. Furthermore, in a system where PPDK is present, if AMP is contained in the sample, this is converted to ATP by PPDK, PEP, and PPi. Alternatively, in a system where PWDK is present, if AMP is contained in the sample, this is converted to ATP by PWDK, PEP, and phosphoric acid. The generated ATP again emits light by luciferase. Luminescence is maintained stably, and the amount of luminescence correlates with the total amount of ATP and AMP present in the system, so that ATP and AMP can be quantified. In the presence of an enzyme that catalyzes the reaction of generating ATP from ADP and PPDK, ADK, or PWDK, the total amount of ATP, ADP, and AMP can be measured. The advantage of the method using PPDK or the like is that, even in a low-sensitivity apparatus, AMP produced by luciferase is also converted to ATP, so that luminescence can be measured stably without attenuation of luminescence.

If the sample contains AMP and ATP, it is converted to 2 molecules of ADP by adenylate kinase. The resulting ADP molecule can then be converted to ATP by an enzyme that catalyzes a reaction that produces ATP from ADP. The resulting ATP can then be detected by luciferase.

In an embodiment using an enzyme that catalyzes a reaction that generates ATP from AMP, an enzyme that catalyzes a reaction that generates AMP from ADP, luciferin, luciferase, and a metal salt, when ADP is contained in a sample, ADP converts AMP from ADP. It can be converted to AMP by an enzyme that catalyzes the resulting reaction. The resulting AMP can then be converted to ATP by an enzyme that catalyzes the reaction that produces ATP from AMP. ATP is converted to AMP by luciferase and luminescence is produced and can be detected. The sample may contain ATP and / or AMP.

In this specification, the program means a cleaning program. Cleaning programs include general hygiene management programs, SOPs, and SSOPs for managing the hygiene of food manufacturing and processing environments. The cleaning program is sometimes referred to as a cleaning protocol.

In certain embodiments, the present invention provides a method for evaluating a cooking related utensil cleaning program. This method includes the following steps:
(i) cleaning cooking-related utensils according to the program;
(ii) measuring the degree of contamination of the cleaned cooking-related utensils using the kit of the present invention;
(iii) compare the degree of contamination measured in step (ii) with the references specified in the program; and
(iv) If the measured degree of contamination exceeds the reference specified in the program, evaluate that the contamination in (i) does not remove the contamination sufficiently.

In an embodiment, when it is evaluated that the contamination is not sufficiently removed in the step (iv), a cleaning method that is the same as or different from the cleaning method in the step (i) can be further performed.

In certain embodiments, the present invention provides a method for improving a cooking related utensil cleaning program. This method includes the following steps:
(i) cleaning cooking-related utensils according to the program;
(ii) Use the kit of the present invention to measure the degree of contamination of the washed cooking-related utensils.
(iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
(v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program.

In these embodiments, a cooking program for cooking-related utensils can be created as step (o) before step (i).

In this specification, cooking-related utensils refer to cooking utensils and the environment of the cooking site or those in the environment that may be contaminated. As cooking utensils, for example, cutting boards, pans, frying pans, pressure cookers, iron plates, dishes, kitchen knives, chopsticks, chopsticks, spoons, forks, knives, etc., bowl characters, nets, colander, racks, cutting board stands, cooking utensils Examples include, but are not limited to, utensils for cooking such as containers for storing food, packaging containers, and packaging sheets, and utensils related thereto. The environment at the cooking site refers to the environment at the cooking site, food processing plant, food provision facility, etc., which includes equipment, containers such as preparation tanks, piping, filling nozzles, belt conveyors, etc. in the food processing plant Examples include, but are not limited to, a part or place that touches a human hand such as a door knob, a handle of an appliance such as a refrigerator or an oven, a switch, or a receiver. Contamination may include not only ATP, ADP, or AMP but also ATP-degrading enzyme and the like (for example, ATP-degrading enzyme, ADP-degrading enzyme, and enzymes that degrade ATP and ADP). Such contamination may cause ATP to be decomposed into ADP or ADP by the action of ATP degrading enzyme or the like even immediately after washing or during cleanliness measurement. The kit and method of the present invention are also applicable to such contaminated cooking utensils.

Reference can be determined in advance based on standards required for food processing safety management. In this specification, “reference” refers to various indicators that can be associated with the degree of contamination or cleanliness. For example, the reference may be a threshold for a certain index that is found in the sample to be examined, with a threshold or higher being an indicator of contamination, while a value below the threshold is an absence of contamination or a clean state indicator. An example of the indicator is the number of microorganisms. The number of microorganisms is usually expressed as a number per mL (cells / mL). The number of microorganisms can be evaluated by the number of colonies (cfu) formed when cultured under appropriate conditions per mL (cfu / mL).

For example, if the sample to be inspected is tableware or a cooking utensil, the reference is a unit (cfu / mL) in which a colony of microorganisms is formed under a certain condition with respect to a test sample obtained by wiping off the tableware or cooking utensil. It can be expressed as a degree of contamination corresponding to. For example, if the acceptable cfu / mL is 1 cfu / mL, it can be judged that the sample in which 1 cfu / mL is detected is contaminated, and the sample below 1 cfu / mL can be judged to be clean. . The reference for the contamination corresponding to 1 cfu / mL can be determined by examining the luminescence amount of the contaminated sample using the kit of the present invention.

For example, the inspection standard of the coliform group inspection guideline for milk in Europe and the US is 5-10 cfu / mL, and the inspection standard of Japanese Food Sanitation Inspection Law is 0.45 cfu / mL. A facility's cleaning program related to milk production can make references based on these criteria. The method of the present invention does not measure food itself having a specific cfu / mL standard, but measures the presence or absence of contamination after washing cooking-related utensils that handle food. For cooking-related utensils, if a level of contamination is detected, it is checked in advance whether it corresponds to a specific standard (for example, cfu / mL), and the degree of contamination corresponding to the standard is determined. Then, the contamination degree can be used as a threshold value as a reference. The index for deriving the reference is not limited to cfu / mL, and may be various indices depending on the purpose.

Cleaning of cooking-related utensils includes washing with water, running water, standing washing, washing with detergent, ultrasonic washing, boil sterilization, heat sterilization, washing with washing utensils, washing with a dishwasher, drying with a dryer It may include steps such as drying, CIP (fixed cleaning) and COP (decomposition cleaning) in the production line of the food factory. The detergent may be a soap, a synthetic detergent, or a surfactant, and examples of the surfactant include a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and a nonionic surfactant. For cleaning, a commonly used instrument such as a sponge, a brush, a scrubber, a cloth, or a paper towel can be used.

Measure the degree of contamination by quantifying the amount of ATP and ADP or the amount of ATP, ADP and AMP in the sample. The sample may be a wipe of cooking-related utensils or water that has been washed with cooking-related utensils. Samples may be concentrated for evaluation testing.

The comparison between the measured contamination level and the reference may be performed manually or may be automated by the device. The device may comprise a recording medium in which the reference is recorded in advance.

• If the measured degree of contamination exceeds the reference specified in the program, the cleaning program may not be sufficient and therefore the program can be improved. Improvement includes modifying the cleaning process. As used herein, modifying the cleaning process means changing the type of detergent used for cleaning, changing the concentration or amount of detergent used, changing the cleaning time or number of times, allowing the supervisor to supervise the cleaning operation, or Examples include, but are not limited to, strengthening supervision and ensuring that cleaning is performed as per the program.

∙ The modification of the program may be repeated multiple times, and it can be repeated until the pollution level is below the reference specified in the program. The correction of the program multiple times may be a correction for the same process or the same item, or may be a correction for a different process or a different item.

The cleaning program may include, for example, a step of washing or washing detergent-related utensils such as chopping boards and dishes used at the cooking site. At this time, if the washing is not sufficient, the cooking-related utensils remain contaminated. By the method of the present invention, it is possible to evaluate whether the cleaned cooking-related utensils are contaminated or whether the cleaning is sufficient, and the cleaning program can be modified appropriately.

The cleaning program may include, for example, a step of dipping a used dish or cooking utensil in a cleaning tank containing a detergent for a certain period of time at a cooking site. At this time, when the dirty dishes are added and immersed, the cleaning liquid in the cleaning tank is gradually contaminated. And if it exceeds a certain level, the tableware and cooking utensils taken out from the washing tank are not sufficiently washed, and contamination remains. By the method of the present invention, it can be evaluated how often the contamination can be prevented by replacing the cleaning liquid in the cleaning tank.

The method of the present invention can also be used for evaluating the cleaning power of a dishwasher. Put the contaminated dishes and the specified detergent into the dishwasher for cleaning. After washing, measure the degree of contamination of the dishes and compare with the reference. If the contamination exceeds the reference, the detergent used for the dishwasher (type, concentration) and operating conditions (temperature, time, water pressure, process repetition, etc.) may be modified.

The method of the present invention can also be used to evaluate the cleaning power of detergents. Wash with detergent and then measure the degree of contamination of the dishes and compare with the reference. If the contamination exceeds the reference, the usage of the detergent (concentration, amount, time, frequency, temperature, process repetition, etc.) may be modified.

The method of the present invention can be used not only to improve the cooking-related appliance cleaning program, but also to verify whether or not a correctly designed cleaning program is properly executed. For example, a facility where food poisoning has occurred can be subjected to a contamination test under cleaning conditions according to the program of the facility. If contamination is not detected by cleaning according to the program, it can be specified that there is no problem in the program itself, but there may be a problem in its execution. And the execution side can be improved. Such verification and improvement in execution are also widely included in the program improvement method of the present invention.

[Luciferase]
In certain embodiments, the kit of the invention comprises luciferase and luciferin. In this case, metal ions such as magnesium, manganese, and calcium may also be included. A person skilled in the art can determine the concentration of metal ions depending on the enzyme used. The necessary luciferase converts ATP, O ₂ and luciferin to AMP, pyrophosphate, CO ₂ and oxyluciferin, which results in luminescence. The luciferase may be a natural luciferase or a genetically engineered recombinant luciferase variant. The luciferase variant may be one that has been site-directed or randomly mutagenized. It may be a fusion protein with a protein having another function. The luciferase mutant may have a desired property such as one having improved heat resistance and one having improved surfactant resistance.

The amount of luminescence of the luciferase can be determined by a suitable luminescence measuring device such as a luminometer (Berthold, Centro LB960 or Lumat 3 LB9508, Kikkoman Biochemifa, Lumitester C-110, Lumitester C-100, Lumitester PD-30. The relative light emission intensity (RLU) obtained using a Lumitester PD-20 or the like can be evaluated as an index. Usually, luminescence generated upon conversion from luciferin to oxyluciferin is measured. As a luminescence measuring device, it is possible to measure with high sensitivity, and a device equipped with a photomultiplier tube (manufactured by 3M, etc.) or a device equipped with a photodiode (manufactured by Hygina, Neogen, etc.) may be used. it can.

The luciferase is not particularly limited as long as it uses ATP as a substrate, and those derived from bacteria, protozoa, animals, mollusks, and insects can be used. Insects include beetle luciferases, such as the genus Photinus, such as the North American firefly (Photinus pyralis), the genus Photuris, such as the Photouris lucicrescens, the Shoturis pennsylvanica, the genus Luciola, such as the Luciola crucia , Firefly (Luciola lateralis), Japanese firefly (Luciola parvula), genus firefly (Pyrocoelia) genus, firefly (Lucidina biplagiata) firefly and Pyrophorus (Pyrophorus) genus beetle derived. Numerous luciferase genes have been reported, and their nucleotide sequences and amino acid sequences can be obtained from known databases such as GeneBank.

The luciferase gene may be a wild type or may have a mutation. The mutation may be a site-specific introduction or a random mutation. Known mutations include those that improve the amount of luminescence as described in JP-A-2011-188787, mutations that increase the persistence of luminescence as described in JP-A-2000-197484, and Japanese Patent No. 2666561. Mutations that change the emission wavelength as described in JP-A No. 2003-512071, mutations that increase the resistance to surfactants as described in JP-A No. 11-239493, and International Publication No. 99/02697 Mutations that increase the substrate affinity as described in the pamphlet, JP 10-512750 A or JP 2001-518799 A, Japanese Patent No. 3048466, JP 2000-197487 A, JP 9-9 Mutations that increase stability, as described in JP 510610 and JP 2003-518912 Including but not limited to this.

The luciferase gene and its recombinant DNA can be prepared by conventional methods. For example, Japanese Patent Publication No. 7-112434 describes a Heike firefly luciferase gene. JP-A-1-51086 describes a genji firefly luciferase gene.

The luciferase gene can be incorporated into a vector such as a plasmid, bacteriophage, cosmid, etc., to transform or transduce an appropriate host. The host can be a microorganism, a bacterium such as E. coli, or a yeast. The transformed host capable of producing luciferase can be cultured by various known methods.

As the medium, tryptone, yeast extract, meat extract, peptone, corn steep liquor, or one or more nitrogen sources such as soybean or wheat bran leachate, sodium chloride, monopotassium phosphate, dipotassium phosphate, chloride One or more inorganic salts such as magnesium, ferric chloride, magnesium sulfate, or manganese sulfate are added, and if necessary, saccharide raw materials, vitamins, and the like are added.

The initial pH of the medium can be 7-9, for example. The culture can be performed, for example, at 30 to 40 ° C. for 2 to 24 hours by aeration and agitation culture, shaking culture, stationary culture, or the like. After the culture, luciferase is recovered from the culture by a known method.

Specifically, the cells are subjected to ultrasonic crushing treatment, grinding treatment or the like by a conventional method, or luciferase is extracted using a lytic enzyme such as lysozyme. The obtained extract is filtered, centrifuged, etc., nucleic acid is removed with streptomycin sulfate, if necessary, and ammonium sulfate, alcohol, acetone, etc. are added thereto and fractionated to obtain a crude enzyme.

The crude enzyme may be further purified by various gel filtration and chromatographic techniques. A commercially available luciferase can also be used, for example, a luciferase of Kikkoman Biochemifa, catalog number 61314 can be used. This luciferase is described in Japanese Patent Application Laid-Open No. 11-239493 (Patent No. 3794628) (SEQ ID NO: 1 in this document). Also, commercially available luciferases of molecular probes (registered trademark) from Sigma-Aldrich, Promega, and Life Technology can be used.

[Luciferin]
The luciferin may be any luciferin that is recognized as a substrate by the luciferase used, and may be natural or chemically synthesized. Any known luciferin derivative can also be used. The basic skeleton of luciferin is imidazopyrazinone, and there are many tautomers. Examples of luciferin include firefly luciferin. Firefly luciferin is a substrate for firefly luciferase (EC 1.13.12.7). Luciferin derivatives can be those described in JP-A-2007-91695, JP-T 2010-523149 (International Publication No. 2008/127777) and the like.

In one embodiment, the final concentration in the luciferase measurement system is 0.001 μg protein / mL or more, 0.01 μg protein / mL or more, 0.02 μg protein / mL or more when the absorbance at 280 nm is luciferase concentration (mg protein / mL), 0.05 μg protein / mL or more, 0.10 μg protein / mL or more, 0.20 μg protein / mL or more, or 0.25 μg protein / mL or more. In one embodiment, the final concentration in the luciferase measurement system is 1 μg protein / mL or less, 0.5 μg protein / mL or less, or 0.3 μg protein / mL or less when the absorbance at 280 nm is luciferase concentration (mg protein / mL). be able to. In certain embodiments, the final concentration of the luciferin or luciferin derivative in the measurement system may be 0.01 mM to 20 mM, 0.05 mM to 20 mM, 0.1 mM to 20 mM, 0.5 mM to 10 mM, such as 0.75 mM to 5 mM.

Enzymes that catalyze reactions that produce ATP from ADP In certain embodiments, the methods of the invention employ enzymes that catalyze reactions that produce ATP from ADP. ADP present in the system is converted to ATP by an enzyme that catalyzes a reaction that generates ATP from ADP. Next, ATP is converted to AMP by luciferase and light is emitted.

As the enzyme that catalyzes the reaction of generating ATP from ADP, any known enzyme can be used, for example, a kinase having ATP generating ability. Examples of the kinase having ATP generation ability include pyruvate kinase, acetate kinase, creatine kinase, polyphosphate kinase, hexokinase, glucokinase, glycerol kinase, fructokinase, phosphofructokinase, riboflavin kinase, fructose bisphosphatase and combinations thereof However, it is not limited to this.

[Pyruvate kinase (PK)]
Pyruvate kinase (EC 2.7.1.40) converts phosphoenolpyruvate to pyruvate in the glycolytic system, where ADP is converted to ATP. This reaction is an Ergon reaction with negative Gibbs energy and is irreversible under natural conditions:
PEP + ADP → Pyruvate + ATP
The reverse reaction is catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase in gluconeogenesis to produce PEP and ADP from ATP and pyruvate. When cell extraction is performed, various enzymes are mixed in the system, and the above reaction can proceed in both directions. At that time, if phosphoenolpyruvate is present at a high concentration, ADP can be converted to ATP. Further, if not only phosphoenolpyruvate but also pyruvate kinase is present in the system, it is considered that ADP is more converted to ATP. Although it does not specifically limit, For example, animals derived from microorganisms, such as animals, such as a rabbit, a rat, a chicken, yeast, Bacillus stearothermophilus (Bacillus stearothermophilus), can be used.

[Acetate kinase (AK)]
Acetate kinase (EC 2.7.2.1) catalyzes the conversion between ATP and acetic acid and ADP and acetylated phosphate in the presence of cations:
ATP + acetic acid ← → ADP + acetylated phosphate Acetate kinase (AK) is also called ATP: acetate phosphotransferase, acetyl kinase. As used herein, these terms can be interchanged. In vivo, ATP and acetic acid are used to generate ADP and acetylated phosphate, and ultimately promote the reaction to produce acetyl CoA. If the system contains acetylated phosphate and ADP generated from acetyl-CoA, it can be converted to acetic acid and ATP. Although it does not specifically limit, For example, Escherichia coli (Escherichia coli), Bacillus stearothermophilus (Bacillus stearothermophilus), Costridium pasteurianum (Costridium pasteurianum), Lactobacillus delbrucki (Lactobacillus delbruckii) The one derived from Veillonella alcalescence can be used.

[Creatine kinase (CK)]
Creatine kinase (EC 2.7.3.2) mediates the conversion reaction between creatine and ATP and creatine phosphate and ADP:
Creatine + ATP ← → Creatine phosphate + ADP
Creatine kinase (CK) is also called creatine phosphokinase (CPK) or phosphocreatine kinase. As used herein, these terms can be interchanged. Usually, creatine phosphate and ADP are produced from creatine and ATP in animal muscles and the like. However, this reaction is a reversible reaction, and if creatine phosphate and ADP are present in a high concentration in the system, the reaction proceeds in the reverse direction, and creatine and ATP can be generated. In vivo, cytoplasmic creatine kinase is composed of two subunits B or M. Therefore, three isozymes, CK-MM, CK-BB and CK-MB may exist depending on the combination of subunits. The isozyme pattern varies depending on the tissue, but any combination can be used in the present invention. Although it does not specifically limit, the thing derived from an animal can be used, For example, the thing derived from a rabbit, a chicken, a cow, a pig, a carp, a catfish, a frog is mentioned.

[Polyphosphate kinase (PPK)]
Polyphosphate kinase (EC 2.7.4.1) catalyzes the reaction of converting polyphosphate (PolyP _n ) and ADP to polyphosphate (PolyP _n-1 ) and ATP:
ADP + PolyP _n ← → ATP + PolyP _n-1
Polyphosphate kinase (PPK) is also called ATP: polyphosphate phosphotransferase. As used herein, these terms can be interchanged. PPK is involved in oxidative phosphorylation in vivo. If polyphosphoric acid (n) and ADP are present in the system, they can be converted to polyphosphoric acid (n-1) and ATP. Although it does not specifically limit, For example, microorganism-derived things, such as Escherichia coli (Escherichia coli), yeast, Corynebacterium xerosis (Corynebacterium xerosis), can be used.

[Riboflavin kinase (FMNK)]
Riboflavin kinase (EC 2.7.1.26), also described as FMNK, catalyzes the reaction of converting riboflavin and ATP to riboflavin phosphate (FMN) and ADP:
ATP + riboflavin ← → ADP + FMN
Riboflavin kinase belongs to ATP: riboflavin 5′-phosphotransferase (also referred to as flavokinase). Although not particularly limited, for example, those derived from microorganisms and animals can be used, and examples include those derived from yeast, rat, and legume (Phaseolus radiatus).

[Phosphofructokinase 1 (PFK1)]
Phosphofructokinase 1 (EC 2.7.1.11), also described as PFK1, converts fructose-6-phosphate (Fru6P) and ATP to fructose-1,6-bisphosphate (Fru1,6-BP) and ADP Catalyze the reaction to:
Fru6P + ATP ← → Fru1,6-BP + ADP
Phosphofructokinase 1 belongs to phosphofructokinase. In the present specification, phosphofructokinase 1 is sometimes referred to as Fru-1,6BPK. Although not particularly limited, those derived from animals and microorganisms can be used, for example, those derived from microorganisms include baker's yeast, beer yeast, Clostridium pasteurianum, Escherichia coli, Bacillus Examples are those derived from Bacillus licheniformis.

[Fructose bisphosphatase (FBPase)]
Fructose bisphosphatase (EC 3.1.3.11), also described as FBPase, is a reaction that converts fructose-1,6-bisphosphate (Fru1,6-BP) and ADP to fructose-6-phosphate (Fru6P) and ATP. Catalyze:
Fru1,6-BP + ADP ← → Fru6P + ATP
Fructose bisphosphatase may be described as FBP or FBP1. Although not particularly limited, those derived from animals, plants, and microorganisms can be used, and examples include those derived from rabbits and chickens.

[Pyruvate-phosphate dikinase (PPDK)]
Pyruvate-phosphate dikinase (EC 2.7.9.1) is a reaction between ATP, pyruvate and orthophosphate and adenosine monophosphate (AMP), phosphoenolpyruvate (PEP) and pyrophosphate (PPi). Catalyze:
ATP + pyruvic acid + phosphoric acid ← → AMP + PEP + PPi
Pyruvate-phosphate dikinase (PPDK) is also called ATP: pyruvate, phosphate phosphotransferase, pyruvate orthophosphate dikinase, pyruvate phosphate ligase. As used herein, these terms can be interchanged. PPDK usually converts pyruvic acid to PEP, and in the process, one ATP molecule is consumed and converted to AMP. The reaction is divided into the following three reversible reactions.
1. The enzyme PPDK binds to ATP and results in conversion to AMP and diphosphorylated PPDK.
2. Diphosphorylated PPDK binds to inorganic phosphoric acid, resulting in diphosphoric acid and monophosphorylated PPDK.
3. Monophosphorylated PPDK binds to pyruvic acid, yielding PEP and PPDK again.
At this time, if the PEP concentration present in the system is high, the reaction proceeds in the reverse direction as follows.

For convenience, reaction steps are described with the same numbers as above.
3. PEP binds to PPDK, producing monophosphorylated PPDK and pyruvate.
2. Diphosphorylated PPDK and inorganic phosphoric acid are produced from diphosphoric acid and monophosphorylated PPDK.
1. PPDK and ATP are produced from biphosphorylated PPDK and AMP.
The PPDK is not particularly limited, and examples thereof include, for example, Microbispora thermorosea, Propionibacterium shremanii, Bacteroides symbiosus, Entamoeba histolytica, Acetobacter xylinum described in JP-A-8-168375 (Patent No. 3181801). And those derived from microorganisms such as Propionibacter shermanii, and those derived from plants such as corn and sugarcane.

[Adenylate kinase (ADK)]
Adenylate kinase (EC 2.7.4.3), also called adenylate kinase, catalyzes the following reaction in the presence of metal ions:
ATP + AMP ← → 2ADP
This reaction is reversible. ADK is an example of an enzyme that catalyzes a reaction that generates ADP from AMP. When ADK is combined with PK or the like, ADP is converted to ATP, and as a result, ATP, ADP, and AMP can be measured. ADK is not particularly limited, and examples thereof include those derived from microorganisms such as yeast and those derived from animals such as rabbits, pigs, cows, rats, and pigs.

[Pyruvate water dikinase (PWDK)]
Pyruvate water dikinase (EC 2.7.9.2) catalyzes the following reaction:
ATP + pyruvate + H ₂ O ← → AMP + phosphoenolpyruvate (PEP) + phosphate (P)
Pyruvate water dikinase is also called phosphoenolpyruvate synthase; pyruvate water dikinase (phosphorylation); PEP synthetase; phosphoenolpyruvate synthetase; phosphoenolpyruvic synthetase; phosphopyruvate synthetase. As used herein, these terms can be interchanged.

∙ ATP generation from AMP and PEP can be promoted by using PWDK together with PEP. When PWDK is combined with PK or the like, ADP is converted to ATP, and as a result, ATP, ADP, and AMP can be measured. The PWDK is not particularly limited. For example, E. coli, Pseudomonas fluorescens, Pyrococcus furiosus, Staphylothermus marinus, Sulfolobus solfataric ), Thermococcus kodakarensis, Thermoproteus tenax, and corn (Zea mays).

[RNA-degrading enzyme]
In certain embodiments, the kit of the present invention may comprise an RNase. In one embodiment, the method of the present invention may use an RNase. Here, the RNase means an RNase that is not derived from a sample.

In the present specification, RNase means an enzyme that catalyzes a reaction that generates 5′-mononucleotides (AMP, GMP, CMP, and UMP) from RNA, and examples thereof include the following: (1) Endonuclease S1 (EC3.1.30.1), (2) Venom exonuclease (EC3.1.15.1), (3) Phosphodiesterase One ( Phospho diesterase 1) (EC3.1.4.1). The endonuclease s-one includes nuclease pi-one (Nuclease P1), mung bean nuclease (Mung beans nuclease), and neurospora crassa nuclease.

In another embodiment, the kit of the invention does not contain RNase or does not contain a substantial amount of RNase. Also, in certain embodiments, the methods of the invention do not use RNases or do not use substantial amounts of RNases. In this embodiment, RNase derived from the sample may be included in the reaction system. In the present specification, the “substantial amount of RNase” refers to the effect of the kit or method of the present invention (for example, the effect of providing an accurate contamination detection method that is hardly affected by the ATP degradation activity). An amount of RNase that has no effect. For example, the final concentration in the reaction system is 0.3 U / mL or less, 0.15 U / mL or less, 0.1 U / mL or less, 0.05 U / mL or less, 0.01 U / mL as an example not including the substantial amount of RNase Hereinafter, a kit containing an RNase of 0.001 U / mL or less, or a method using such an amount of RNase is mentioned. In this specification, the enzyme unit of RNase focuses on the RNA resolution of the enzyme, and the activity unit (U) of the enzyme having RNA resolution is 37 ° C., and 1.0 μmol of substrate per minute is acid-soluble. Defined as the amount of enzyme converted to nucleotides. For example, the enzyme unit of Nuclease P1 is defined as the amount of enzyme that converts 1.0 μmol of substrate into acid-soluble nucleotides per minute at 37 ° C., pH 5.3 (as defined in the definition of the enzyme activity of Nuclease P1). For details, see the Merck catalog (http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/nuclease_p1.pdf).

In certain embodiments where the kit or method of the invention comprises, uses, or substantially comprises or uses an RNase, the RNase may or may not contribute to the luminescence reaction by luciferase. You don't have to contribute.

In one embodiment, the kit of the present invention substantially contains an RNase, the kit of the present invention may or may not contain an enzyme that generates ATP from AMP. In certain embodiments where the methods of the invention substantially use RNases, the methods of the invention may or may not use enzymes that produce ATP from AMP.

In certain embodiments, where the kit or method of the present invention includes or uses an RNase, the present invention provides for the ATP, ADP, And the amount of luminescence is measured before it affects the measurement of AMP. For example, the amount of luminescence when RNase is included is 2 times or less, 1.8 times or less, 1.5 times or less, 1.2 times or less, 1.1 times or less, or equivalent to the amount of luminescence without RNase. Measurements can be made at such times. The measurement time can be appropriately set according to the amount of RNase, for example, within 10 minutes, within 5 minutes, within 4 minutes, preferably within 3 minutes, within 2 minutes, or within 1 minute, within 30 seconds, or Can be within 10 seconds. Even if a large amount of RNase is contained, the action of RNase can be reduced by shortening the reaction time.

In certain embodiments where the kit or method of the invention includes or uses an RNase, the present invention may be used on samples that are free of RNA or substantially free of RNA. As such an example, the amount of luminescence when RNase is included is 2 times or less, 1.8 times or less, 1.5 times or less, 1.2 times or less, 1.1 times or less than the amount of luminescence when RNAase is not included, Or the sample which becomes equivalent is mentioned.

Based on the disclosure of this specification, various modifications are possible. In certain embodiments, the present invention provides an enzyme that generates AMP from ADP, an enzyme that generates ATP from AMP (eg, PPDK), a kit including luciferin, luciferase, and a metal salt, and a measurement method using the kit. When combining an enzyme that generates AMP from ADP and an enzyme that generates ATP from AMP (eg PPDK), ADP is converted to AMP, and AMP is converted to ATP. As a result, ATP, ADP, and AMP should be measured. Can do. The kit can further include PEP and PPi. The enzyme that generates ATP from AMP is as described above, and examples of the enzyme that generates AMP from ADP include ADP-dependent hexokinase and apyrase.

[ADP-dependent hexokinase]
ADP-dependent hexokinase (EC 2.7.1.147), also called ADP-specific hexokinase, catalyzes the following reaction:
D-glucose + ADP ← → D-glucose-6-phosphate + AMP

[Apyrase]
Apyrase (EC 3.6.1.5), also called adenosine diphosphatase, ADPase, ATP diphosphatase, or ATP diphosphohydrolase, catalyzes the following two reactions:
ATP + H ₂ O ← → ADP + phosphoric acid (P)
ADP + H ₂ O ← → AMP + phosphoric acid (P)

In the present specification, the enzymes that catalyze the reaction of generating ATP from ADP, PPDK, PWDK, and ADK, and the enzyme that generates AMP from ADP may be collectively referred to as an enzyme having ATP generating ability.

As the enzyme having ATP generating ability, any known ones such as those derived from microorganisms, bacteria, eukaryotes, protists, plants, animals, etc. can be used, for example, commercially available ones. Can be used. The addition amount of the enzyme can be appropriately set according to the target concentration and reaction system.

Various enzymes are known that have ATP-producing ability. In this specification, focusing on the ATP-producing ability of the enzyme, the enzyme activity unit (U) having the ATP-producing ability is 37 ° C., pH 7.8, and the amount of enzyme that converts 1.0 μmol of substrate into ATP per minute Define (1U = 1μmol ATP / min, pH7.8, 37 ℃). In one embodiment, the enzyme having ATP generation ability is added so that the activity unit in the measurement system is 0.001 U or more, 0.01 U or more, 0.1 U or more, 1 U or more, 2 U or more, 3 U or more, 4 U or more, or 5 U or more. be able to. In one embodiment, the enzyme having ATP generating ability is added so that the activity unit in the measurement system is 10,000 U or less, 1000 U or less, 100 U or less, 50 U or less, 10 U or less, 9 U or less, 8 U or less, 7 U or less, or 6 U or less. be able to. A person skilled in the art can appropriately determine the amount of the enzyme added.

When using an enzyme having ATP-producing ability, a substrate for each enzyme can be added, and is not particularly limited. For example, phosphoenolpyruvate and pyrophosphate for PPDK, For PK, AK, CK, PPK, FMNK, PFK1, and FBPase, use phosphoenolpyruvate, acetyl phosphate, creatine phosphate, polyphosphate, riboflavin phosphate, fructose-1,6-bisphosphate, respectively. it can. For example, for PWDK, phosphoenolpyruvate and phosphate can be used. For example, glucose can be used for ADP-dependent hexokinase. In certain embodiments, the kits of the invention further comprise these substrates. In certain embodiments, the methods of the invention may further use these substrates.

[Phosphoenolpyruvate (PEP)]
The method of the present invention may use phosphoenolpyruvate (PEP). In some cases, the addition of an excess amount of PEP to the system can facilitate the measurement of ATP and AMP present in the system. The concentration of PEP to be used includes 0.001 mM to 4500 mM, for example, 2.1 mM as the final concentration.

[Pyrophosphate (PPi)]
The method of the present invention may use pyrophosphate (PPi). In some cases, by adding an excessive amount of PPi to the system, measurement of ATP and AMP present in the system can be promoted. The concentration of PPi used is 0.001 mM to 2000 mM, for example, 0.2 mM as the final concentration.

The reaction reagent can also include an enzyme stabilizer such as bovine serum albumin or gelatin that protects reporter molecules such as luciferase from degradation. The reaction reagent may also be added with a substance that improves pH adjustment and storage stability. For example, suitable pH buffer (HEPES, Tricine, Tris, phosphate buffer, acetate buffer, etc.), reducing agent (dithiothreitol (DTT), 2-mercaptoethanol, etc.), sugar (glucose, sucrose, trehalose, etc.) Etc.

In certain embodiments, the kit of the invention may include instructions for use. The instructions for use may be one in which the procedure of the cleaning program evaluation method of the present invention, the procedure of the cleaning program improvement method and the method of using the kit of the present invention are described.

[ATP assay method using luciferase]
The assay method using luciferase is described below. The conditions are exemplary. Prepare an ATP measurement reagent containing:
MES 1 mM
Magnesium acetate 5.1 mM
Potassium pyrophosphate 0.15mM
Potassium phosphoenolpyruvate 2.1 mM
Luciferin 0.8 mM
Tricine 25mM
Luciferase 12.5μg protein / mL (The absorbance at 280nm is the luciferase concentration (mg protein / mL).)
Add 0.1 mL of sample solution containing ATP to 0.1 mL of the above ATP measurement reagent and measure luminescence. The amount of luminescence can be measured using a known luminometer (Berthold Centro LB960 or Lumat3 LB9508, Kikkoman Biochemifa luminometer, etc.).
Luminescence can be described as a relative luminescence unit (RLU) relative to a certain standard. A calibration curve is prepared using a substrate solution with a known ATP concentration. Next, the above ATP measurement reagent is added to a sample solution with an unknown ATP concentration, and luminescence is measured under the same conditions.

[ATP + ADP assay method 1 (luciferase + PK)]
The ATP + ADP assay method is described below. The conditions are exemplary.
Prepare an ATP + ADP measurement reagent containing:
MES 1 mM
Magnesium acetate 5.1 mM
Potassium pyrophosphate 0.15mM
Potassium phosphoenolpyruvate 2.1 mM
Luciferin 0.8 mM
Tricine 25mM
Luciferase 12.5 μg protein / mL (The absorbance at 280 nm is the luciferase concentration (mg protein / mL).)
PK 25U / mL

[ATP + ADP assay method 2 (luciferase + AK)]
The ATP + ADP assay method is described below. The conditions are exemplary.
Prepare an ATP + ADP measurement reagent containing:
MES 1 mM
Magnesium acetate 5.1 mM
Potassium pyrophosphate 0.15mM
Potassium phosphoenolpyruvate 2.1 mM
Luciferin 0.8 mM
Tricine 25mM
Luciferase 12.5 μg protein / mL (The absorbance at 280 nm is the luciferase concentration (mg protein / mL).)
AK 25U / mL

[ATP + AMP assay method (luciferase + PPDK)]
The ATP + AMP assay method will be described below. The conditions are exemplary.
A reagent for measuring ATP + AMP containing the following is prepared.
MES 1 mM
Magnesium acetate 5.1 mM
Potassium pyrophosphate 0.15 mM
Potassium phosphoenolpyruvate 2.1 mM
Luciferin 0.8 mM
Tricine 25 mM
Luciferase 12.5 μg protein / mL (The absorbance at 280 nm is the luciferase concentration (mg protein / mL).)
PPDK 2 U / mL

[ATP + AMP + ADP assay method 1 (luciferase + PK + PPDK)]
The ATP + AMP + ADP assay method will be described below. The conditions are exemplary.
Prepare an ATP + AMP + ADP measurement reagent containing:
MES 1 mM
Magnesium acetate 5.1 mM
Potassium pyrophosphate 0.15mM
Potassium phosphoenolpyruvate 2.1 mM
Luciferin 0.8 mM
Tricine 25mM
Luciferase 12.5 μg protein / mL (The absorbance at 280 nm is the luciferase concentration (mg protein / mL).)
PK 25U / mL
PPDK 2U / mL
As an alternative, in the above, PWDK can be used (2U / mL) instead of PPDK. In this case, phosphoric acid is used instead of potassium pyrophosphate.

[ATP + ADP + AMP assay method 2 (luciferase + PK + ADK)]
The ATP + AMP + ADP assay method will be described below. The conditions are exemplary.
Prepare ATP and AMP + ADP measurement reagents including the following:
Magnesium acetate 5.1 mM
Potassium pyrophosphate 0.15mM
Potassium phosphoenolpyruvate 2.1 mM
Luciferin 0.8 mM
Tricine 25mM
Luciferase 12.5μg protein / mL (The absorbance at 280nm is the luciferase concentration (mg protein / mL).)
PK 25U / mL
ADK 500U / mL

[ATP + ADP + AMP assay method 3 (luciferase + PPDK + ADP-dependent hexokinase or apyrase)]
The ATP + AMP + ADP assay method will be described below. The conditions are exemplary.
Prepare ATP and AMP + ADP measurement reagents including the following:
MES 1 mM
Magnesium acetate 5.1 mM
Potassium pyrophosphate 0.15 mM
Potassium phosphoenolpyruvate 2.1 mM
Luciferin 0.8 mM
Tricine 25 mM
Luciferase 12.5 μg protein / mL (The absorbance at 280 nm is the luciferase concentration (mg protein / mL).)
ADP-dependent hexokinase 30 U / mL + glucose 10 mM or apyrase 1 U / mL
PPDK 2U / mL

[Example 1]
[Measurement reagent]
Nucleotide measurement was performed using the following reagents. These are merely examples and can be replaced with other reagents. ATP (conventional 1 component) includes Lucipak II (manufactured by Kikkoman Biochemifa), ATP + AMP (conventional 2 components) includes Lucipak Pen (manufactured by Kikkoman Biochemifa), ATP + ADP (present invention 2 components) Is a reagent in which 0.1 mg of phosphoenolpyruvate (PEP) and 5 U of pyruvate kinase (Lee Biosolutions Inc. Catalog No. 500-20) are added to Lucipak II, and Lucipak is added to ATP + ADP + AMP (3 components of the present invention). Using a reagent containing 5U of pyruvate kinase in Pen, ATP (conventional one component) and ATP + ADP (two components of the present invention) are ATP + AMP using Lumitester C-110 (Kikkoman Biochemifa). Luminity tester PD-30 (manufactured by Kikkoman Biochemifa) was used to measure the amount of luminescence for (conventional two components) and ATP + ADP + AMP (three components of the present invention). In this reagent, when the same concentration of ATP solution is measured, the same amount of luminescence is displayed.

Lucipak II and Lucipak Pen can be replaced with a composition comprising: Luciferase manufactured by Kikkoman Biochemifa (catalog number: 61314) was used, and PPDK described in JP-A-8-168375 (Japanese Patent No. 3181801) was used.
<Lushi Pack II composition>
Magnesium acetate 7mM
Luciferin 0.5 mM
Tricine 25mM
Luciferase 0.7 μg protein / mL (The absorbance at 280 nm is the luciferase concentration (mg protein / mL).)
<Lusipak Pen composition>
Magnesium acetate 7mM
Luciferin 0.5 mM
Tricine 25mM
Luciferase 0.2 mg protein / mL (The absorbance at 280 nm is the luciferase concentration (mg protein / mL).)
Potassium pyrophosphate 0.2mM
Potassium phosphoenolpyruvate 1.4 mM
PPDK 1.3U / mL

[Evaluation of contamination of cooking-related utensils]
The cleanliness evaluation by the difference of the cleaning method of cooking related utensils was performed by the following method.
As shown in the photograph of FIG. 1, the cutting board was divided into a grid. One square is 6.5cm x 6.8cm. The meat was adhered by rubbing the pork so that it was uniform over the entire top surface of the cutting board.
Next, the cutting board is processed under the conditions 1 to 5 below, and wiped with a cotton swab for each square on the top row divided in a lattice pattern. ATP (conventional 1 component), ATP + AMP (conventional 2 component), ATP + ADP + AMP (3 components of the present invention) and ATP + ADP (2 components of the present invention) were measured:
Washing conditions No sticking of meat, no cleaning process (negative control)
2. Stick meat and do not wash (positive control)
3. Let the meat adhere, and then run for about 10 seconds with running water (washing with running water)
4). Adhere meat, then rub lightly with a sponge and wash with running water for about 10 seconds (sponge + washing with running water)
5). Adhere the meat, then rub it with a sponge with detergent and wash with running water (detergent washing + running water washing)
After the measurement is completed, spray the sterilized ultrapure water on the entire upper surface of the cutting board with a spray, and place the cutting board in a sealed plastic container filled with water, and place the cutting board on the rack so that the cutting board does not come into contact with the stretched water. The sealed plastic container was covered and stored at 30 ° C. for 2 days. After storage, wipe the surface of the lower 4 squares divided in a grid pattern on the upper surface of the cutting board with a cotton swab, dissolve in 1 mL of sterilized ultrapure water, dilute the solution 100 times with sterilized ultrapure water, and add to tryptosy agar medium. 50 μL was seeded, cultured at 30 ° C. for 20 hours (FIG. 2), and the number of colonies was counted. The number of bacteria was calculated as cfu / mL, taking into account the dilution factor (100 times) and the coating amount (50 μL), and multiplying the number of colonies obtained by 2000.

[result]
The results are as follows. The luminescence unit is RLU and the bacterial count unit is cfu / mL.

As shown in Table 1, the cleanliness evaluation results by the cleaning method were divided according to the number and types of components to be measured. When only ATP is measured, When washed with running water, 4. Luminescence was lower than in the case of washing with sponge + running water. This tendency was not consistent with the case where two components of ATP + AMP were measured. Therefore, if only ATP is measured, the cleanliness evaluation may not be stable. In the two-component measurement, the total luminescence when ATP + AMP was measured was lower than that of ATP + ADP. The total amount of luminescence in the three-component measurement was the largest, but when ATP + ADP was measured, the amount of luminescence was the same. If two components of ATP + ADP were measured, the three components of ATP + ADP + AMP were It was shown that almost the same evaluation can be performed as when measured.

In the count of bacteria, “2. Positive control” and “3. Washing with running water” were too numerous to grow and could not be counted. In “4. Sponge + washing with running water”, the number of bacteria was 96000 cfu / mL and it was confirmed that contamination remained, but the amount of luminescence for this sample varied depending on the components to be measured. Regarding “4. Sponge + running water cleaning”, in Table 1, there is a sensitivity difference of about 5 to 10 times depending on the components to be measured. If the surface to be wiped is smaller, the ATP alone or ATP + AMP measurement results in a lower luminescence value, and in fact it is clean even though contamination remains and bacteria may grow. May be judged. Further, regarding residual contamination in the case of “4. Sponge + washing with water”, the contamination can be detected more accurately by measuring ATP + ADP or ATP + ADP + AMP.

This test is a kind of accelerated test using conditions suitable for the growth of bacteria, but it is considered that washing is performed to reduce the number of growing bacteria at actual cooking sites. In that case, contamination cannot be detected by the amount of luminescence when ATP + AMP is measured, but it may be detected by measuring ATP + ADP or ATP + ADP + AMP. Similarly, for samples containing ATP-degrading enzymes, etc., the amount of luminescence when ATP + AMP is measured cannot be detected, but it may be detected by measuring ATP + ADP or ATP + ADP + AMP. It is done.

The above example is only a model system. This cleaning method is not sufficient because contamination remains, and 5. It turns out that it is necessary to modify the program to the cleaning method. A person skilled in the art can evaluate the cleanliness when the cleaning program is executed, measure the presence of contamination, and modify or improve the program as necessary based on such knowledge.

[Example 2]
Construction of an ATP + ADP + AMP measurement system using an enzyme that catalyzes the reaction that generates AMP from ADP. ADP-dependent hexokinase (ADP + AMP) is an enzyme that catalyzes the reaction that generates AMP from ADP. Asahi Kasei Pharma, T-93 ADP-HKTII) and glucose were added to investigate whether ATP + ADP + AMP could be measured. The composition of the luminescent reagent is as follows.

Prepare standard products (1 × 10 ^-9 M to 1 × 10 ^-6 M) of various concentrations of ATP, ADP, or AMP, mix with the luminescent reagent in the following ratio to the above luminescent reagent, and Lumitester C -110 (Kikkoman Biochemifa) (n = 2):
0.1mL Luminescent reagent
0.01 mL ATP, ADP, or AMP solutions at various concentrations The mol amount of ATP, ADP, and AMP in the solution at the time of luminescence was calculated, and a calibration curve was prepared. The results are shown in FIGS. 3-1 to 3-3.

Subsequently, apyrase (Sigma A6536), an enzyme that catalyzes the reaction to generate AMP from ADP, is added to the luminescent reagent for ATP + AMP measurement of the same composition instead of hexokinase, and ATP + ADP + AMP It was investigated whether measurement was possible. The composition of the luminescent reagent is as follows.

A calibration curve was prepared by calculating the molar amounts of ATP, ADP, and AMP in the solution during luminescence. The results are shown in FIGS. 4-1 to 4-3.
These results indicate that ATP + ADP + AMP can be measured by using PPDK that catalyzes the reaction that generates ATP from AMP and an enzyme that catalyzes the reaction that generates AMP from ADP. Yes. Using these methods, the degree of contamination of the cooking-related utensils can be evaluated.

By using the kit of the present invention, the degree of contamination of cooking-related utensils can be measured. Moreover, the cleaning method and cleaning program for cooking-related utensils can be modified or altered according to the degree of contamination.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (26)

1. Cleaning program evaluation method using ATP and ADP measurement kit, including the following steps,
   (i) cleaning cooking-related utensils according to the program;
   (ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
   (iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, Evaluate that the cleaning process of i) does not sufficiently remove contamination.
   Here, the ATP and ADP measurement kit is an ATP and ADP measurement kit for improving a cooking-related utensil washing program, which includes an enzyme that catalyzes a reaction for generating ATP from ADP, luciferin, luciferase, and a metal salt.
2. The method according to claim 1, wherein if it is evaluated that the contamination is not sufficiently removed in step (iv), the cooking-related utensils are further washed by the same or different method as in step (i).
3. Cleaning program improvement method using ATP and ADP measurement kit, including the following steps:
   (i) cleaning cooking-related utensils according to the program;
   (ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
   (iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
   (v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program,
   Here, the ATP and ADP measurement kit is an ATP and ADP measurement kit for improving a cooking-related utensil washing program, which includes an enzyme that catalyzes a reaction for generating ATP from ADP, luciferin, luciferase, and a metal salt.
4. Furthermore, before step (i),
   (o) creating a cleaning program for cooking-related utensils;
   The method according to any one of claims 1 to 3, comprising:
5. Enzymes that catalyze the reaction to generate ATP from ADP are pyruvate kinase (PK), acetate kinase (AK), creatine kinase (CK), polyphosphate kinase (PPK), hexokinase, glucokinase, glycerol kinase, fructokinase The method according to any one of claims 1 to 4, which is selected from the group consisting of phosphofructokinase, riboflavin kinase, and fructose bisphosphatase.
6. The method according to any one of claims 1 to 5, wherein the ATP and ADP measurement kit further contains an AMP measurement reagent.
7. The method according to claim 6, wherein the AMP measurement reagent contains an enzyme that catalyzes a reaction for producing ADP or ATP from AMP.
8. The method according to claim 7, wherein the enzyme that catalyzes a reaction for generating ADP or ATP from AMP comprises pyruvate orthophosphate dikinase (PPDK), adenylate kinase (ADK), or pyruvate water dikinase (PWDK). .
9. Cleaning program evaluation method using ATP, ADP and AMP measurement kit, including the following steps:
   (i) cleaning cooking-related utensils according to the program;
   (ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
   (iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, Evaluate that the cleaning process of i) does not sufficiently remove contamination.
   Here, the measurement kit includes an enzyme that catalyzes a reaction that generates ATP from AMP, an enzyme that catalyzes a reaction that generates AMP from ADP, luciferin, luciferase, and a metal salt. Said method.
10. 10. The method according to claim 9, wherein if it is evaluated that the contamination is not sufficiently removed in step (iv), the cooking-related utensils are further washed by the same or different method as in step (i).
11. A cleaning program improvement method using an ATP, ADP and AMP measurement kit, including the following steps:
    (i) cleaning cooking-related utensils according to the program;
    (ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
    (iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
    (v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program,
    Here, the measurement kit includes an enzyme that catalyzes a reaction that generates ATP from AMP, an enzyme that catalyzes a reaction that generates AMP from ADP, luciferin, luciferase, and a metal salt. Said method.
12. Furthermore, before step (i),
    (o) creating a cleaning program for cooking-related utensils;
    The method according to any one of claims 9 to 11, comprising:
13. The enzyme that catalyzes the reaction that generates ATP from AMP is pyruvate orthophosphate dikinase (PPDK) or pyruvate water dikinase (PWDK), and the enzyme that catalyzes the reaction that generates AMP from ADP depends on ADP. The method according to any one of claims 9 to 12, which is a sex hexokinase or an apyrase.
14. ATP and ADP measurement kit for use in a cooking-related utensil washing program evaluation method, comprising an enzyme that catalyzes a reaction for producing ATP from ADP, luciferin, luciferase and a metal salt, wherein the cooking-related utensil washing program evaluation method comprises:
    (i) cleaning cooking-related utensils according to the program;
    (ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
    (iii) compare the degree of contamination measured in step (ii) with the references specified in the program; and
    (iv) If the measured degree of contamination exceeds the reference specified in the program, evaluate that the cleaning process in (i) does not remove the contamination sufficiently.
    A kit comprising the kit.
15. The kit according to claim 14, wherein if it is evaluated that the contamination is not sufficiently removed in step (iv), the cooking-related utensils are further washed by the same or different method as in step (i).
16. ATP and ADP measurement kit for use in a cooking-related utensil washing program improvement method, comprising an enzyme that catalyzes a reaction for generating ATP from ADP, luciferin, luciferase, and a metal salt, wherein the cooking-related utensil washing program improvement method comprises:
    (i) cleaning cooking-related utensils according to the program;
    (ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
    (iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
    (v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program,
    A kit comprising the kit.
17. Furthermore, before step (i),
    (o) creating a cleaning program for cooking-related utensils;
    The kit according to any one of claims 14 to 16, comprising:
18. Enzymes that catalyze the reaction to generate ATP from ADP are pyruvate kinase (PK), acetate kinase (AK), creatine kinase (CK), polyphosphate kinase (PPK), hexokinase, glucokinase, glycerol kinase, fructokinase The kit according to any one of claims 14 to 17, which is selected from the group consisting of phosphofructokinase, riboflavin kinase, and fructose bisphosphatase.
19. The kit according to any one of claims 14 to 18, wherein the kit further contains an AMP measurement reagent.
20. The kit according to claim 19, wherein the AMP measurement reagent contains an enzyme that catalyzes a reaction for producing ADP or ATP from AMP.
21. 21. The kit according to claim 20, wherein the enzyme that catalyzes a reaction for generating ADP or ATP from AMP comprises pyruvate orthophosphate dikinase (PPDK), adenylate kinase (ADK), or pyruvate water dikinase (PWDK). .
22. ATP, ADP and AMP measurements for use in cooking-related utensil washing program evaluation methods, including enzymes that catalyze reactions that produce ATP from AMP, enzymes that catalyze reactions that produce AMP from ADP, luciferin, luciferase and metal salts Kit, where the cooking-related utensil cleaning program evaluation method is:
    (i) cleaning cooking-related utensils according to the program;
    (ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
    (iii) compare the degree of contamination measured in step (ii) with the references specified in the program; and
    (iv) If the measured degree of contamination exceeds the reference specified in the program, evaluate that the cleaning process in (i) does not remove the contamination sufficiently.
    A kit comprising the kit.
23. The kit according to claim 22, wherein if it is evaluated that the contamination is not sufficiently removed in step (iv), the cooking-related utensils are further washed by the same or different method as in step (i).
24. ATP, ADP and AMP measurements for use in cooking-related utensil cleaning program improvement methods, including enzymes that catalyze reactions that produce ATP from AMP, enzymes that catalyze reactions that produce AMP from ADP, luciferin, luciferase and metal salts Kit, where the cooking-related utensil washing program improvement method is:
    (i) cleaning cooking-related utensils according to the program;
    (ii) Use the kit to measure the degree of contamination of washed cooking-related utensils.
    (iii) Compare the pollution degree measured in step (ii) with the reference specified in the program. (iv) If the measured pollution degree exceeds the reference specified in the program, modify the cleaning process of i), and
    (v) Repeat steps (i) to (iv) until the pollution level is below the reference specified in the program,
    A kit comprising the kit.
25. Furthermore, before step (i),
    (o) creating a cleaning program for cooking-related utensils;
    The kit according to any one of claims 22 to 24, comprising:
26. The enzyme that catalyzes the reaction that generates ATP from AMP is pyruvate orthophosphate dikinase (PPDK) or pyruvate water dikinase (PWDK), and the enzyme that catalyzes the reaction that generates AMP from ADP depends on ADP. The kit according to any one of claims 22 to 25, which is a sex hexokinase or an apyrase.

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