Method for determining time of death of insect

Abstract

A purpose of the present invention is to provide a novel means for determining the timing of contamination for a biological organism that has contaminated a food / beverage as foreign matter. The present invention provides: a method for determining the time of death of an insect, the method using, as an indicator, the amount of two or more components selected from the group consisting of the ATP, ADP, AMP, IMP, HxR, and Hx contained in the insect's carcass; and a method that uses said determination method to estimate the timing of contamination for the insect that has contaminated a food / beverage.

Description

Method for determining the death time of insects

[0001] The present invention relates to a method for determining the death time of an insect from the corpse of the insect based on the amounts of adenosine triphosphate and its decomposition products in the corpse of the insect.

[0002] In particular, the present invention makes it possible to estimate the time of contamination of an insect mixed in food or drink by determining the death time of the insect based on the amounts of adenosine triphosphate and its decomposition products in the corpse of the insect.

[0003] Food and drink are things that are taken into the body, and of course, their safety must be ensured. Therefore, in the quality control of food and drink production, for example, matters that can cause foreign matter contamination must be thoroughly eliminated.

[0004] In order to thoroughly implement such quality control, it is necessary to accurately grasp the actual situation of foreign matter contamination. Therefore, first, it is important to verify whether the foreign matter mixed in food or drink was mixed during the manufacturing process or after the product was opened, with respect to the time of foreign matter contamination.

[0005] Therefore, methods for determining the time of contamination of the corpse of a living organism mixed as a foreign matter in food or drink have been developed and reported. For example, Patent Document 1 discloses that the degree of fragmentation of nucleic acid derived from the corpse of a living organism can be evaluated, and based on the degree of fragmentation, the death time of the living organism can be determined, and also, by evaluating the degree of fragmentation of nucleic acid derived from the corpse of the living organism, the time of contamination of the living organism or its corpse into food or drink can also be determined.

[0006] However, there are various living organisms and situations that can be mixed as foreign matters in food or drink, and there are various needs in determining the time of contamination. Therefore, in this field, there is still an urgent need for a new method for determining the death time and the time of contamination of a living organism mixed in food or drink.

[0007] Patent Document 2 discloses a method for evaluating the freshness of aquatic animals using the following FI value as an indicator: FI value = {ATP amount - (HxR amount + Hx amount)} / (ATP amount + ADP amount + AMP amount + IMP amount + AdR amount + HxR amount + Hx amount). However, Patent Document 2 does not disclose anything about determining the timing of contamination of food and beverages with organisms that have been mixed in as foreign matter.

[0008] Japanese Patent Publication No. 2020-162586 Japanese Patent Publication No. 2018-100935

[0009] The present invention aims to provide a novel means for determining the time of death of an organism. The present invention also aims to provide a novel means for determining the time of contamination of food or beverages with an organism that was found as a foreign object.

[0010] As a result of diligent research to solve the above problems, the inventors have found that the time of death of an insect can be determined by using as an indicator the amount of two or more components selected from the group consisting of ATP, ADP, AMP, IMP, HxR, and Hx contained in the insect's carcass. Furthermore, they have found that if the insect's carcass was mixed into food or beverage as a foreign object, the time of death of the insect can be estimated by similarly determining the time of death of the insect by using as an indicator the amount of two or more components selected from the group consisting of ATP, ADP, AMP, IMP, HxR, and Hx contained in the insect's carcass, i.e., the time of contamination of the food or beverage.

[0011] The present invention is based on these novel findings and includes the following inventions: [1] A method for determining the time of death of an insect, using as an indicator the amount of two or more components selected from the group consisting of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosinic acid (IMP), inosine (HxR), and hypoxanthine (Hx) contained in the insect's carcass. [2] The method of [1], which includes using the difference between the amounts of group A and group H as an indicator, group A = amount of ATP + amount of ADP + amount of AMP, group H = amount of HxR + amount of Hx, and determining that when the amount of group A is equal to or greater than the amount of group H, it is determined that 30 minutes or less have passed since the insect died, and when the amount of group A is less than the amount of group H, it is determined that more than 30 minutes have passed since the insect died. [3] The method of [2], wherein the amount of group A further includes the amount of IMP. [4] Any method from [1] to [3], which includes determining that if the amount of HxR is equal to or greater than the amount of Hx, less than eight hours have passed since the insect died, and if the amount of HxR is less than the amount of Hx, eight hours or more have passed since the insect died. [5] Any method from [1] to [4], which includes determining the time elapsed since the insect died using the following G value: G value = (AMP amount + Hx amount) / (IMP amount + HxR amount) Time elapsed since death (hours) = (G value - 0.7957) / 0.0632 However, the time elapsed since death (hours) is seven hours or more. [6] Any method from [1] to [5], wherein the insect carcass was mixed into food or beverage. [7] Any method from [1] to [6], wherein the insect carcass has not been subjected to heat treatment.

[0012] [8] A method for estimating the time of contamination of food or beverages with insects, comprising the step of determining the time of death of the insect using as an indicator the amount of two or more components selected from the group consisting of ATP, ADP, AMP, IMP, HxR, and Hx contained in the insect's carcass. [9] The method of [8], wherein the step of determining the time of death of the insect uses as an indicator the difference between the following amounts of Group A and Group H: Amount of Group A = Amount of ATP + Amount of ADP + Amount of AMP Amount of Group H = Amount of HxR + Amount of H When the amount of Group A is equal to or greater than the amount of Group H, it is determined that less than 30 minutes have passed since the insect died, and when the amount of Group A is less than the amount of Group H, it is determined that more than 30 minutes have passed since the insect died.

[10] The method of [9], wherein the amount of Group A further includes the amount of IMP.

[11] Any method from [8] to

[10] , wherein the step of determining the time of death of the insect is to determine that if the amount of HxR is equal to or greater than the amount of Hx, less than eight hours have passed since the insect died, and if the amount of HxR is less than the amount of Hx, eight hours or more have passed since the insect died.

[12] Any method from [8] to

[11] , wherein the step of determining the time of death of the insect is to determine the elapsed time since the insect died using the following G value: G value = (AMP amount + Hx amount) / (IMP amount + HxR amount) Elapsed time after death (hours) = (G value - 0.7957) / 0.0632 However, the elapsed time after death (hours) is seven hours or more.

[13] Any method from [8] to

[12] , wherein the insect carcass has not been subjected to heat treatment.

[14] Any method of [8] to

[13] further comprising the step of estimating the time when the insect was introduced into food or beverages based on the determination of the time of death of the insect. This specification includes the contents described in the specification of Japanese Patent Application No. 2025-11919, filed on 28 January 2025, which is the basis of the priority claim of this application. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

[0013] The present invention provides a novel means for determining the time of death of insects. Furthermore, the present invention provides a novel means for determining the time of contamination of food and beverages with insects that have been found as foreign matter.

[0014] Figure 1 shows a calibration curve created from the G value calculated by dividing the sum of AMP and Hx amounts by the sum of IMP and HxR amounts 7 hours after the insect's death (G value = 0.0632 × elapsed time since insect's death + 0.7957 (R) ２ This indicates that = 0.962.

[0015] In this invention, "insect carcass" means the carcass of an insect, a part of an insect, or a part thereof. The term "insects" is not particularly limited, but includes, for example, cockroaches (e.g., Oriental cockroach, American cockroach, Common cockroach, Japanese cockroach, Brown cockroach, Brown-banded cockroach, Japanese brown cockroach, Small brown cockroach, Turkestan cockroach, Kyoto cockroach, Yellow-striped cockroach, Giant cockroach, Satsuma cockroach, Yaeyama spotted cockroach, Ogasawara cockroach, Gray cockroach, etc.), moths (e.g., Spodoptera litura, Helicopterus litura, Tea leafroller, Apple leafroller, Diamondback moth, etc.), stink bugs (e.g., Brown marmorated stink bug, Brown marmorated stink bug, etc.), aphids (e.g., Peach aphid, Cotton aphid, etc.), planthoppers and leafhoppers (e.g., Brown planthopper, Black-tipped leafhopper, etc.) (e.g., small flies), whiteflies (e.g., tobacco whiteflies), thrips (e.g., citrus thrips), leafminers (e.g., tomato leafminers), spider mites (e.g., two-spotted spider mites), mosquitoes (e.g., Culex pipiens, Aedes albopictus), flies (e.g., houseflies, gnats, moth flies, Drosophila melanogaster, and pygmy flies) Examples include adults and larvae of various insects such as clothes moths (e.g., the common clothes moth, the striped rice moth, etc.), rice weevils (e.g., the rice weevil, etc.), confused flour beetles (e.g., the flat confused flour beetle, etc.), cigarette beetles (e.g., the cigarette beetle, etc.), carpet beetles (e.g., the small carpet beetle, etc.), and termites (e.g., the Formosan subterranean termite, etc.).

[0016] In this invention, insect carcasses may be those that were mixed into food or beverages. In this invention, "food or beverages" means dishes served in restaurants, etc., and various processed foods that can be distributed frozen, chilled, or at room temperature. Examples of processed foods include, but are not limited to, retort products such as curry, stew, soup, and sauce; roux products such as curry and stew; frozen foods; various spice products such as wasabi paste, mustard paste, and mustard; seasoning products such as mayonnaise and dressing; dairy products such as yogurt, butter, cheese, and ice cream; dessert products such as jelly and pudding; confectionery products such as chocolate and cookies; and beverage products such as tea, coffee, fruit drinks, and soft drinks.

[0017] In the present invention, it is preferable that the insect carcasses have not been subjected to heat treatment. Examples of heat treatment include treatment equivalent to 65°C for 15 minutes or more, 90°C for 5 minutes or more, and preferably 100°C for 5 minutes or more. Typical examples of such heat treatment include, but are not limited to, cooking and heat sterilization in the manufacturing process of food and beverages. More preferably, in the present invention, the insect carcasses have been at room temperature, for example, 4°C to 35°C, preferably 10°C to 30°C, more preferably 20°C to 30°C, and even more preferably 23°C to 26°C.

[0018] The present invention relates to a method for determining the time of death of an insect from its carcass (hereinafter sometimes simply referred to as "the present invention method"). The present invention method includes a step of determining the time of death of the insect by using as an indicator the amount of two or more components selected from the group consisting of ATP, ADP, AMP, IMP, HxR, and Hx contained in the insect's carcass.

[0019] "ATP" stands for adenosine triphosphate, a compound involved in muscle contraction activity in insects. It is a type of nucleotide consisting of a base, a pentose sugar, and a phosphate group. Energy is generated when a phosphate group is removed from ATP.

[0020] "ADP" stands for adenosine diphosphate, a nucleotide with one less phosphate group than ATP.

[0021] "AMP" stands for adenosine monophosphate (adenylic acid), which is a nucleotide with one phosphate group.

[0022] "IMP" represents inosinic acid (inosine monophosphate), a nucleotide that has been deaminated from the base of AMP.

[0023] "HxR" indicates inosine, which is an N-riboside composed of hypoxanthine and D-ribose.

[0024] "Hx" indicates hypoxanthine, a component of the nucleic acid bases found in inosinic acid and inosine.

[0025] The method of the present invention generally comprises a measurement step and a determination step.

[0026] In the measurement process, the amount of substance (so-called mol amount) of two or more components selected from the group consisting of ATP, ADP, AMP, IMP, HxR, and Hx contained in the insect carcass is measured. The insect carcass used for measurement is not particularly limited as long as it contains parts that may contain each component, but it is preferable that it contains muscle tissue. The amount of substance of each component can be measured by any means and is not particularly limited, but for example, it can be done by high-performance liquid chromatography (HPLC), thin-layer chromatography, etc.

[0027] As detailed in the following examples, the HPLC method can measure the amounts of ATP, ADP, AMP, IMP, HxR, and Hx contained in insect carcasses. Specifically, first, perchloric acid solution is added to the insect carcasses, which are then pulverized and homogenized. After that, NaOH is added to adjust the pH to 2.5-3.5, and the proteins are precipitated while cooling. After that, the supernatant is collected after returning to room temperature, mixed with phosphate buffer, and filtered through a cellulose filter (φ0.45) to prepare a sample for HPLC.

[0028] Next, for example, using a Shimadzu HPLC analysis system (Class VP), the amount of ATP, ADP, AMP, IMP, HxR, and Hx is analyzed under the following conditions: column: CAPCELL PAK ADME-HR (S5), 4.6 mm ID x 250 mm (manufactured by OSAKA SODA), mobile phase: 0.05 mol / L phosphate buffer (pH 7.0), column temperature: 40°C, flow rate: 1.0 mL / min, injection volume: 10 μL, detection wavelength: 260 nm. In high-performance liquid chromatography, by selecting an appropriate column through solvent selection, analysis in different modes such as ion exchange and reversed phase becomes possible, and by selecting the conditions, the separation ability of the substance to be measured can be improved and the analysis time can be shortened. Furthermore, it is possible to simplify the pretreatment method for extracting the substance to be measured, and the optimal analytical method for the amount of ATP, ADP, AMP, IMP, HxR, and Hx can be selected.

[0029] In thin-layer chromatography, for example, the substance to be measured is coated onto the lower end of a thin-layer plate made of a carrier suitable for nucleic acid-related substances such as ecteolar cellulose or polyethyleneimine (PEI) cellulose. The sample is then developed in a sealed container using an elevating method or similar technique with a solvent composition optimized for spot separation, such as a mixture of distilled water or organic solvents of different polarities. The separated sample spots can then be quantitatively analyzed using a densitometer to measure absorbance.

[0030] Furthermore, the amount of ATP, ADP, AMP, IMP, HxR, and Hx contained in insect carcasses may be measured by a combination of any means or methods capable of measuring these amounts. Such means or methods include, but are not limited to, commercially available measuring devices (e.g., QS-SOLUTION's "Freshness Checker"), colorimetric methods, and enzymatic methods such as enzyme sensor methods.

[0031] In living insects, ATP is consumed by activity and converted to ADP, but it is regenerated back into ATP through respiration. However, once death occurs and respiration stops, ATP regeneration ceases, and it is thought that ATP is irreversibly broken down through the following main pathway: ATP → ADP → AMP → IMP → HxR → Hx

[0032] Therefore, by comparing two or more components selected from the group consisting of ATP, ADP, AMP, IMP, HxR, and Hx contained in the insect carcass, it is possible to determine how far the irreversible degradation of ATP has progressed, that is, how much time has passed since the insect died.

[0033] In one embodiment of the present invention, the determination step calculates the amount of group A and group H from the following formulas based on the amounts of substance of ATP, ADP, AMP, HxR, and Hx, and optionally IMP, measured in the measurement step, and uses the difference between the amount of group A and the amount of group H as an indicator to determine the time of death of the insect. Amount of group A = Sum of the amount of substance of ATP (referred to as "amount of ATP") + Aum of substance of ADP (referred to as "amount of ADP") + Aum of substance of AMP (referred to as "amount of AMP"). The amount of group A may further include the amount of substance of IMP contained in the insect carcass (referred to as "amount of IMP"). Including the amount of IMP in the amount of group A makes the difference between the amount of group A and the amount of group H clearer, which is preferable. Amount of group H = Sum of the amount of substance of HxR (referred to as "amount of HxR") + Amount of substance of Hx (referred to as "amount of Hx").

[0034] In the determination step of this embodiment, the difference between the calculated amounts of group A and group H is used as an indicator to determine the time of death of the insect in question according to (1) or (2) below.

[0035] (1) When the amount of group A is equal to or greater than the amount of group H, preferably when the amount of group A exceeds the amount of group H, it means that the insect has died relatively recently. In this case, it can be determined that approximately 30 minutes have passed since the insect died (for example, within 25 minutes, within 20 minutes, within 15 minutes, within 10 minutes, within 5 minutes, 0 minutes or more, etc.).

[0036] (2) When the amount of group A falls below the amount of group H, it means that a longer time has passed since the death of the insect than in the case of (1) above. In this case, it can be determined that approximately more than 30 minutes has passed since the death of the insect (for example, 40 minutes or more, 50 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 20 hours or more, 24 hours or more, 36 hours or more, 40 hours or more, 48 hours or more, 60 hours or more, 80 hours or more, 100 hours or more, 120 hours or more, 140 hours or more, 160 hours or more, 180 hours or more, and the upper limit is not particularly limited but preferably 720 hours or less).

[0037] In another embodiment of the present invention, the determination step determines the time of death of the insect in question, using the HxR amount and Hx amount measured in the measurement step as indicators, according to (3) or (4) below.

[0038] (3) When the amount of HxR is equal to or greater than the amount of Hx, it can be determined that approximately less than 8 hours have elapsed since the insect died, for example, within approximately 7.5 hours, within 7.4 hours, within 7.2 hours, preferably within approximately 7 hours (for example, 0 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, etc.).

[0039] (4) When the amount of HxR is less than the amount of Hx, it means that a longer time has passed since the insect died than in the case of (3) above, and in this case it can be determined that approximately 8 hours or more have passed since the insect died (for example, 10 hours or more, 12 hours or more, 20 hours or more, 24 hours or more, 36 hours or more, 40 hours or more, 48 hours or more, 60 hours or more, 80 hours or more, 100 hours or more, 120 hours or more, 140 hours or more, 160 hours or more, 180 hours or more, and the upper limit is not particularly limited but preferably 720 hours or less).

[0040] In another aspect of the method of the present invention, the determination step determines the death time of the insect according to the following (5) using the amounts of AMP, IMP, HxR, and Hx measured in the measurement step as indices.

[0041] (5) Calculate the G value from the following formula, and using the G value, the elapsed time (hours) after the death of the insect can be obtained: G value = (amount of AMP + amount of Hx) / (amount of IMP + amount of HxR) Elapsed time after death (hours) = (G value - 0.7957) / 0.0632 However, the elapsed time after death (hours) is 7 hours or more (for example, 8 hours or more, 10 hours or more, 12 hours or more, 20 hours or more, 24 hours or more, 36 hours or more, 40 hours or more, 48 hours or more, 60 hours or more, 80 hours or more, 100 hours or more, 120 hours or more, 140 hours or more, 160 hours or more), and the upper limit is not particularly limited but is preferably 720 hours or less, more preferably 168 hours or less).

[0042] Here, the elapsed time (hours) after the death of the insect calculated using the G value means a value within a range of approximately ±19 hours, preferably ±17 hours, more preferably ±12 hours, still more preferably ±8 hours, even more preferably ±4 hours, and particularly preferably ±2 hours with respect to the actual elapsed time after the death of the insect.

[0043] In the method of the present invention, the determination step can be performed according to any one or a combination of the above (1) to (5).

[0044] The present invention also relates to a method for estimating the contamination time of an insect mixed in a food or drink (hereinafter, may be simply referred to as "this estimation method").

[0045] This estimation method includes a step of determining the time of death of an insect that has been mixed into food or drink, more specifically, the time of death of the dead insect that has been mixed into food or drink. In this estimation method, according to the above-described method of the present invention, the amount of substances of two or more components selected from the group consisting of ATP, ADP, AMP, IMP, HxR, and Hx contained in the dead body of the insect is used as an index to determine the time of death of the insect. Thereby, when it is determined that the time of death of the insect mixed into the food or drink is relatively recent, it can be estimated that the time of mixing of the insect into the food or drink is relatively recent (for example, after the product is opened, after the food or drink is provided, etc.). On the other hand, when it is determined that the time of death of the insect mixed into the food or drink is relatively long after death, it can be estimated that the time of mixing of the insect into the food or drink is relatively early (for example, during the manufacturing process or distribution process of the food or drink). Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples.

[0046] Time-dependent changes in the amounts of substances of ATP and its decomposition products in the dead body of an insect (black cockroach) (1) Preparation of samples The purchased black cockroaches (Sumika Technoservice) were frozen to death at -80°C. The frozen black cockroaches were taken out, put into a tube for a crusher, weighed, and then left at room temperature (25°C) for a predetermined time (0 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 24 hours, 30 hours, 168 hours), which was taken as the elapsed time since the insects died.

[0047] After the elapse of the predetermined time, 4 mL of perchloric acid solution (60 w / w%) was added (an amount sufficient to submerge the whole body), and it was crushed with a crusher (constantly ice-cooled during the waiting time). Using a simple pH meter, after adjusting the pH to 2.5 - 3.5 with 4 mol / L NaOH, it was cooled to precipitate proteins and the like. Then, after returning to room temperature, 300 μL of 0.25 mol / L phosphate buffer (pH 7) was added to 1200 μL of the supernatant, mixed, diluted, and filtered through a cellulose filter (φ0.45). This was used as a sample for HPLC analysis and subjected to the following analysis.

[0048] (2) HPLC analysis Each sample was subjected to HPLC analysis using the following apparatus and conditions, and the amounts (moles) of HxR, Hx, ATP, ADP, AMP, and IMP in the sample were measured.

[0049] (3) Results The measurement results of the amounts of HxR, Hx, ATP, ADP, AMP, and IMP in each sample are shown in Table 1 below. In Table 1, the amount of each component is shown as a relative value with the total amount (total mol) of HxR, Hx, ATP, ADP, AMP, and IMP in each sample set to 100%.

[0050] These results confirmed that when the amount of group A was higher than the amount of group H, the time elapsed since the insect's death was less than 30 minutes.

[0051] On the other hand, when the amount of group A was lower than the amount of group H, it was confirmed that the time elapsed since the insect's death was more than one hour.

[0052] Furthermore, it was confirmed that when the HxR amount was higher than the Hx amount, the time elapsed since the insect's death was 7 hours or less.

[0053] Furthermore, it was confirmed that when the HxR amount was lower than the Hx amount, the time elapsed since the insect's death was 8 hours or more.

[0054]

[0055] Furthermore, regarding AMP levels, an increase was observed after 7 hours from the time elapsed since insect death. Therefore, the G value was calculated by dividing the sum of AMP and Hx levels, which were higher after 8 hours compared to 7 hours after insect death, by the sum of IMP and HxR levels, which are the decomposition products after AMP (Table 2). Next, a calibration curve (G value = 0.0632 × time elapsed since insect death + 0.7957) was created from this G value (Figure 1). In Table 2, the amounts of each component are shown as relative values ​​with the total amount (total mol) of HxR, Hx, ATP, ADP, AMP, and IMP in each sample set to 100%.

[0056] These results confirm that, based on the calibration curve, it is possible to determine the elapsed time from 7 hours after the insect's death using the G value.

[0057]

Claims

1. A method for determining the time of death of an insect, the method using the amount of two or more components selected from the group consisting of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosinic acid (IMP), inosine (HxR), and hypoxanthine (Hx) contained in the insect's carcass as an indicator.

2. The method according to claim 1, wherein the difference between the following amounts of A and H is used as an indicator: Amount of A = Amount of ATP + Amount of ADP + Amount of AMP Amount of H = Amount of H x R + Amount of H When the amount of A is equal to or greater than the amount of H, it is determined that 30 minutes have elapsed since the insect died, and when the amount of A is less than the amount of H, it is determined that more than 30 minutes have elapsed since the insect died.

3. The method according to claim 1, wherein the amount of group A further includes the amount of IMP.

4. The method according to claim 1, comprising determining that when the amount of HxR is equal to or greater than the amount of Hx, less than eight hours have passed since the insect died, and when the amount of HxR is less than the amount of Hx, eight hours or more have passed since the insect died.

5. The method according to claim 1, comprising determining the time elapsed since the death of the insect using the following G value: G value = (AMP amount + Hx amount) / (IMP amount + HxR amount) Time elapsed since death (hours) = (G value - 0.7957) / 0.0632 However, the time elapsed since death (hours) is 7 hours or more.

6. The method according to claim 1, wherein the insect carcass was mixed into food or beverage.

7. The method according to claim 1, wherein the insect carcass has not been subjected to heat treatment.

8. A method for estimating the time of contamination of food or beverages with insects, comprising the step of determining the time of death of the insects using the amount of two or more components selected from the group consisting of ATP, ADP, AMP, IMP, HxR, and Hx contained in the insect carcasses as an indicator.

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