JP6805095B2 - Method of determining the strength of heat treatment received by substances in food - Google Patents
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- 238000010438 heat treatment Methods 0.000 title claims description 110
- 239000000126 substance Substances 0.000 title claims description 49
- 235000013305 food Nutrition 0.000 title claims description 44
- 238000000034 method Methods 0.000 title claims description 35
- 238000004587 chromatography analysis Methods 0.000 claims description 21
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 16
- 238000004458 analytical method Methods 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 5
- 238000011088 calibration curve Methods 0.000 claims description 4
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 3
- 239000013076 target substance Substances 0.000 description 19
- 238000004519 manufacturing process Methods 0.000 description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 241000972773 Aulopiformes Species 0.000 description 10
- 235000019515 salmon Nutrition 0.000 description 10
- 230000014759 maintenance of location Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 4
- 241000209140 Triticum Species 0.000 description 4
- 235000021307 Triticum Nutrition 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 238000010411 cooking Methods 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 235000021067 refined food Nutrition 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
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- 241000238631 Hexapoda Species 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
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- 238000005259 measurement Methods 0.000 description 2
- 238000003303 reheating Methods 0.000 description 2
- 238000010025 steaming Methods 0.000 description 2
- BYEAHWXPCBROCE-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoropropan-2-ol Chemical compound FC(F)(F)C(O)C(F)(F)F BYEAHWXPCBROCE-UHFFFAOYSA-N 0.000 description 1
- YEDDVXZFXSHDIB-UHFFFAOYSA-N 1,1,2,2,3,3-hexafluoropropan-1-ol Chemical compound OC(F)(F)C(F)(F)C(F)F YEDDVXZFXSHDIB-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
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- 238000009826 distribution Methods 0.000 description 1
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- 238000005194 fractionation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 235000013324 preserved food Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
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Description
本発明は、食品中の物質が受けた加熱処理の強度を判定する方法に関する。 The present invention relates to a method for determining the strength of heat treatment received by a substance in food.
食品製造分野において、食品への異物の混入は重大な問題である。食品製造工場の衛生管理の観点から、食品への異物の混入時期を調べ、異物が工場の製造ラインで混入したのか、又はその後の流通や保存の過程で混入したのかを判定することは重要である。 In the field of food manufacturing, contamination of food with foreign substances is a serious problem. From the viewpoint of hygiene management in food manufacturing factories, it is important to investigate when foreign substances are mixed in food and determine whether foreign substances are mixed in the manufacturing line of the factory or in the subsequent distribution and storage process. is there.
製造過程で加熱処理された加熱処理食品の場合、食品中の異物が加熱処理を受けているか否かは異物混入時期の判定のための重要な判断材料である。例えば、食品が加工されるに従って、熱、ｐＨ変化、物理的な力等によって、それに含まれるＤＮＡが断片化することが知られている。特許文献１には、食品から採取したＤＮＡを特定のプライマーを用いて増幅させ、増幅産物の生成効率を算出することで該食品の加工の程度を評価する方法が記載されている。 In the case of a heat-treated food that has been heat-treated in the manufacturing process, whether or not the foreign matter in the food has been heat-treated is an important judgment material for determining the time when the foreign matter is mixed. For example, it is known that as food is processed, the DNA contained therein is fragmented by heat, pH change, physical force, and the like. Patent Document 1 describes a method of evaluating the degree of processing of a food by amplifying DNA collected from the food with a specific primer and calculating the production efficiency of the amplified product.
本発明は、食品中におけるＤＮＡを含む物質が受けた加熱処理の強度を判定する方法、及び食品に混入した異物の混入時期を推定する方法を提供する。 The present invention provides a method for determining the strength of heat treatment received by a substance containing DNA in food, and a method for estimating the time when a foreign substance mixed in food is mixed.
本発明者らは、工場での加工食品の製造工程で施されるような強い加熱を受けたＤＮＡと、家庭での加工食品の再加熱で施されるようなより強くない加熱を受けたＤＮＡとが、クロマトグラフィーにより異なるピークとして分離されて検出されることを見出した。さらに本発明者らは、当該加熱強度に依存したＤＮＡのクロマトグラムの違いを利用すれば、加熱処理食品に混入した、ＤＮＡを含む異物が受けた加熱処理の強度を判定することができ、さらにはその結果に基づいて該異物の混入時期を推定することができることを見出した。 The inventors of the present invention have DNA that has been subjected to strong heating, such as that applied in the manufacturing process of processed foods in factories, and DNA that has been subjected to less intense heating, such as that applied to reheating processed foods at home. It was found that was separated and detected as different peaks by chromatography. Furthermore, the present inventors can determine the intensity of the heat treatment received by the foreign substance containing DNA mixed in the heat-treated food by utilizing the difference in the chromatogram of the DNA depending on the heating intensity. Found that the timing of mixing the foreign matter can be estimated based on the result.
したがって、本発明は、加熱処理食品に含まれるＤＮＡを含有する物質が受けた加熱処理の強度を判定する方法であって、
該物質に含まれるＤＮＡをクロマトグラフィー分析すること、
該分析で得られたクロマトグラムに基づいて、該物質が受けた加熱処理の強度を判定すること、
を含む、方法を提供する。
また本発明は、加熱処理食品中に含まれるＤＮＡを含有する異物の混入時期を推定する方法であって、
該異物に含まれるＤＮＡをクロマトグラフィー分析すること、
該分析で得られたクロマトグラムに基づいて、該異物が受けた加熱処理の強度を判定すること、
判定された加熱処理の強度に基づいて、該異物の混入時期を推定すること、
を含む、方法を提供する。
Therefore, the present invention is a method for determining the intensity of heat treatment received by a substance containing DNA contained in a heat-treated food.
Chromatographic analysis of the DNA contained in the substance,
Determining the intensity of the heat treatment received by the substance based on the chromatogram obtained by the analysis.
Provide methods, including.
Further, the present invention is a method for estimating the mixing time of a foreign substance containing DNA contained in a heat-treated food.
Chromatographic analysis of the DNA contained in the foreign substance,
Determining the strength of the heat treatment received by the foreign matter based on the chromatogram obtained by the analysis.
To estimate the time when the foreign matter is mixed based on the determined heat treatment intensity.
Provide methods, including.
本発明によれば、食品中の物質が受けた加熱処理の強度を判定し、かつ該加熱処理が家庭等で行われたものか、又は工場での食品の製造過程で行われたものかを評価することができる。本発明は、加熱処理食品への異物の混入時期の推定に有用である。 According to the present invention, the strength of the heat treatment received by the substance in the food is determined, and whether the heat treatment is performed at home or in the process of manufacturing the food in the factory. Can be evaluated. The present invention is useful for estimating the timing of contamination of heat-treated foods with foreign substances.
本発明は、加熱処理食品に含まれるＤＮＡを含有する物質が受けた加熱処理の強度を判定する方法を提供する。また本発明は、加熱処理食品中に含まれるＤＮＡを含有する異物の混入時期を推定する方法を提供する。 The present invention provides a method for determining the intensity of heat treatment received by a substance containing DNA contained in a heat-treated food. The present invention also provides a method for estimating the time of contamination of a foreign substance containing DNA contained in a heat-treated food.
本発明において、加熱処理食品とは、製造過程で加熱処理を施されて製造された食品である。該加熱処理食品の例としては、焼き、揚げ、茹で、蒸し、煮込み等の加熱調理をされた食品、ならびにレトルトパウチ食品、缶詰食品、瓶詰食品等の加圧加熱殺菌処理食品などが挙げられる。 In the present invention, the heat-treated food is a food produced by being heat-treated in the manufacturing process. Examples of the heat-treated food include foods that have been cooked by heating such as baking, fried, boiled, steamed, and stewed, and pressure heat-sterilized foods such as retort pouch foods, canned foods, and bottled foods.
本発明において加熱処理の強度の判定の対象とする物質は、該加熱処理食品に含まれるＤＮＡを含有する物質であれば、その種類は特に限定されない。例えば、該対象物質は、該加熱処理食品に含まれる食品成分（例えば、肉類、魚介類、野菜類、穀物類、それらの加工物等の細胞を含む成分）であってもよく、又は該加熱処理食品に混入したＤＮＡを含有する異物であってもよい。本発明が対象とする異物としては、ＤＮＡを含有する物質であればその種類は特に限定されないが、例えば、ヒトやヒト以外の動物の毛、爪もしくは組織片、植物やその破片、ならびに昆虫等の虫の体やその破片などを挙げることができる。好ましくは、本発明による加熱処理強度の判定における対象物質は、加熱処理食品に混入したＤＮＡを含有する異物である。 In the present invention, the substance to be determined for the strength of the heat treatment is not particularly limited as long as it is a substance containing DNA contained in the heat-treated food. For example, the target substance may be a food component contained in the heat-treated food (for example, a component containing cells such as meat, seafood, vegetables, grains, and processed products thereof), or the heating. It may be a foreign substance containing DNA mixed in the processed food. The type of foreign substance targeted by the present invention is not particularly limited as long as it is a substance containing DNA, but for example, hair, nails or tissue fragments of humans and animals other than humans, plants and fragments thereof, insects and the like. The body of the insect and its fragments can be mentioned. Preferably, the target substance in the determination of the heat treatment strength according to the present invention is a foreign substance containing DNA mixed in the heat treatment food.
当該食品成分や異物からのＤＮＡの抽出は、組織や細胞中のＤＮＡの抽出又は精製に通常用いられる方法に従って行うことができる。例えば、フェノールクロロホルム法、市販のＤＮＡ抽出キット（例えば、Ｑｉａｇｅｎ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ、Ｐｒｏｍｅｇａ、和光純薬工業、ＴＯＹＯＢＯ、タカラバイオ等から入手可能）等を用いることができる。 Extraction of DNA from the food component or foreign substance can be performed according to a method usually used for extraction or purification of DNA in tissues or cells. For example, a phenol-chloroform method, a commercially available DNA extraction kit (for example, available from Qiagen, Sigma-Aldrich, Promega, Wako Pure Chemical Industries, TOYOBO, Takara Bio, etc.) can be used.
本発明においては、当該対象物質から抽出されたＤＮＡをクロマトグラフィーで分析する。好ましくは、該クロマトグラフィーは高速液体クロマトグラフィー（ＨＰＬＣ）である。該クロマトグラフィー用のカラムとしては、ＤＮＡ分離に使用できる種類のものであればよく、好ましくは逆相カラムである。よって好ましくは、該クロマトグラフィーは逆相クロマトグラフィーであり、さらに好ましくは逆相ＨＰＬＣである。逆相カラムの例としては、例えばＤＮＡＰａｃTM ＲＰ（Ｔｈｅｒｍｏｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）などが挙げられるが、これらに限定されない。 In the present invention, the DNA extracted from the target substance is analyzed by chromatography. Preferably, the chromatography is high performance liquid chromatography (HPLC). The column for chromatography may be any type that can be used for DNA separation, and is preferably a reverse phase column. Therefore, the chromatography is preferably reverse phase chromatography, and more preferably reverse phase HPLC. Examples of the reversed-phase column include, but are not limited to, DNAPac TM RP (Thermorphish Scientific) and the like.
当該クロマトグラフィー分析の手順は、使用するカラムや機器のマニュアルに従って行うことができる。例えば、カラムからのＤＮＡの溶出のための溶離液としては、水、メタノール、アセトニトリル、イソプロピルアルコール、トリエチルアミン（ＴＥＡ）、ヘキサフルオロプロパノール（ＨＦＩＰ）、ＴＥＡ−ＨＦＩＰ、それらの混合液などを用いることができる。好ましくは、該溶出はグラジエント溶出である。グラジエントの例としては、ＴＥＡ−ＨＦＩＰと、ＴＥＡ−ＨＦＩＰ／メタノール（５０／５０）との０：１００〜１００：０グラジエントが挙げられる。クロマトグラフィー検出シグナルの測定波長は、好ましくは２００〜３００ｎｍである。当該クロマトグラフィー分析により、対象物質に含まれるＤＮＡについてのクロマトグラムが得られる。 The chromatographic analysis procedure can be performed according to the manual of the column or instrument used. For example, as the eluent for elution of DNA from the column, water, methanol, acetonitrile, isopropyl alcohol, triethylamine (TEA), hexafluoropropanol (HFIP), TEA-HFIP, a mixture thereof, or the like can be used. it can. Preferably, the elution is a gradient elution. Examples of gradients include TEA-HFIP and TEA-HFIP / methanol (50/50) 0: 100-100: 0 gradients. The measurement wavelength of the chromatography detection signal is preferably 200 to 300 nm. By the chromatographic analysis, a chromatogram of DNA contained in the target substance can be obtained.
上記分析で得られたクロマトグラムには、断片化されたＤＮＡの画分のピークと、断片化が進んでいないＤＮＡの画分のピークが含まれる。該断片化されたＤＮＡの画分のピークは、１つのピークからなるものであっても、２つ以上のピークからなるものであってもよい。また、該断片化が進んでいないＤＮＡの画分のピークも、１つのピークからなるものであっても、２つ以上のピークからなるものであってもよい。いずれの場合も、該断片化されたＤＮＡの画分と断片化が進んでいないＤＮＡの画分は、クロマトグラム上で保持時間の異なる互いに分離された２つの画分として現れる。また通常、これら２つの画分は、クロマトグラム上で最も顕著な２つの画分として現れる。したがって当業者は、クロマトグラム上のピークの中から、該断片化されたＤＮＡの画分のピークと断片化が進んでいないＤＮＡの画分のピークとをそれぞれ選別することができる。画分の選別は、クロマトグラフィー分析の条件等を考慮して、保持時間に基づいて行えばよい。あるいは、対象物質由来の加熱処理強度が既知であるＤＮＡ（例えば、非加熱のＤＮＡや、ＤＮＡが十分に断片化する程度に加熱したＤＮＡ）のクロマトグラムを基準として、断片化されたＤＮＡの画分と断片化が進んでいないＤＮＡの画分をそれぞれ選別することができる。 The chromatogram obtained by the above analysis includes a peak of a fragmented DNA fraction and a peak of a non-fragmented DNA fraction. The peak of the fragmented DNA fraction may consist of one peak or two or more peaks. Further, the peak of the DNA fraction in which the fragmentation has not progressed may also consist of one peak or two or more peaks. In either case, the fragmented DNA fraction and the unfragmented DNA fraction appear on the chromatogram as two separate fractions with different retention times. Also, these two fractions usually appear as the two most prominent fractions on the chromatogram. Therefore, those skilled in the art can select the peak of the fragmented DNA fraction and the peak of the unfragmented DNA fraction from the peaks on the chromatogram. The fraction may be selected based on the retention time in consideration of the conditions of chromatographic analysis and the like. Alternatively, a picture of the fragmented DNA is based on a chromatogram of DNA derived from the target substance and whose heat treatment intensity is known (for example, unheated DNA or DNA heated to the extent that the DNA is sufficiently fragmented). Fractions and DNA fractions that have not been fragmented can be selected.
当該クロマトグラムにおける断片化されたＤＮＡの画分のピークと断片化が進んでいないＤＮＡの画分のピークは、対象物質中のＤＮＡの受けた加熱の強さを反映する指標となる。より詳細には、後述の実施例に示すとおり、断片化されたＤＮＡの画分のピークの高さ及び面積は、対象物質中のＤＮＡが受けた加熱強度の強さに従って増大する。逆に、断片化が進んでいないＤＮＡの画分のピークの高さ及び面積は、対象物質中のＤＮＡが受けた加熱強度の強さに従って減少する。したがって、対象物質に含まれるＤＮＡのクロマトグラフィー分析で得られたクロマトグラムにおける断片化されたＤＮＡの画分のピーク又は断片化が進んでいないＤＮＡの画分のピークに基づいて、該ＤＮＡ（又はそれを含む物質）が受けた加熱処理の強度を判定することができる。 The peak of the fragmented DNA fraction and the peak of the unfragmented DNA fraction in the chromatogram serve as an index reflecting the intensity of heating received by the DNA in the target substance. More specifically, as shown in Examples described later, the height and area of the peak of the fragmented DNA fraction increase according to the strength of the heating intensity received by the DNA in the target substance. On the contrary, the height and area of the peak of the unfragmented DNA fraction decrease according to the strength of the heating intensity received by the DNA in the target substance. Therefore, based on the peak of the fragmented DNA fraction or the peak of the unfragmented DNA fraction in the chromatogram obtained by chromatographic analysis of the DNA contained in the target substance, the DNA (or The intensity of the heat treatment received by the substance containing it) can be determined.
例えば、当該断片化されたＤＮＡの画分のピークと断片化が進んでいないＤＮＡの画分のピークのいずれか１つ以上のピークの高さ又は面積に基づいて、該ＤＮＡ又はそれを含む物質が受けた加熱処理の強度を判定することができる。好ましくは、本発明で測定されるピークの高さとは、各画分の１つもしくは２つ以上のピークのうちの最も高いピークの高さであるか、又は該１つもしくは２つ以上のピークの高さの合計もしくはその平均値である。また好ましくは、本発明で測定されるピーク面積とは、各画分の１つもしくは２つ以上のピークの面積の合計もしくはその平均値である。 For example, the DNA or a substance containing the DNA based on the height or area of one or more of the peaks of the fragmented DNA fraction and the peaks of the unfragmented DNA fraction. Can determine the strength of the heat treatment received by. Preferably, the peak height measured in the present invention is the height of the highest peak of one or more peaks of each fraction, or the one or more peaks. The total height of or the average value thereof. Further, preferably, the peak area measured in the present invention is the total or average value of the areas of one or two or more peaks of each fraction.
好ましくは、当該断片化されたＤＮＡの画分と断片化が進んでいないＤＮＡの画分のいずれか一方又は両方のピークの面積に基づいて、該ＤＮＡ又はそれを含む物質が受けた加熱処理の強度が判定される。後述の実施例に示すとおり、該２つの画分のピークの面積はいずれも、ＤＮＡが受けた加熱処理の強度と相関する。 Preferably, the heat treatment received by the DNA or a substance containing it is based on the peak area of either or both of the fragmented DNA fraction and the unfragmented DNA fraction. The strength is determined. As shown in Examples described later, the peak areas of the two fractions correlate with the intensity of the heat treatment received by the DNA.
本発明における加熱処理強度の判定は、検量線を用いて、又は対照との比較によって行うことができる。例えば、分析したＤＮＡについての当該２つの画分のうちのいずれかのピークの高さ又は面積、あるいは該２つの画分のピーク高さ又はピーク面積の比率を、予め作成した各画分のピークの高さもしくは面積、又はそれらの画分間での比率と加熱処理強度に関する検量線にあてはめることによって、該ＤＮＡ又はそれを含む物質が受けた加熱処理の強度を判定することができる。また例えば、分析したＤＮＡについての各画分のピーク高さもしくは面積、又はそれらの画分間での比率を、対照（加熱処理強度が既知の同種の物質由来のＤＮＡ）から得られたデータと比較することによって、該ＤＮＡ又はそれを含む物質が受けた加熱処理の強度を判定することができる。当該加熱処理強度の判定は、定量的であっても定性的であってもよい。本発明において、加熱処理強度の定量的評価とは、例えば、該加熱処理で負荷された熱量（例えば、加熱温度と時間の推定、例えば８０℃で３０分間又は９０℃で１０分間に相当する熱量が加えられたか、など）を測定することであり得る。また本発明において、加熱処理強度の定性的評価とは、所定の強度以上（もしくは以下）の加熱処理、又は所定の条件の加熱処理を受けたか否かを判定することであり得る。 The determination of the heat treatment intensity in the present invention can be performed using a calibration curve or by comparison with a control. For example, the height or area of the peak of either of the two fractions of the analyzed DNA, or the ratio of the peak height or peak area of the two fractions is the peak of each fraction prepared in advance. The intensity of the heat treatment received by the DNA or a substance containing the DNA can be determined by applying it to the calibration curve relating to the height or area of the DNA, or the ratio of these fractions and the heat treatment intensity. Also, for example, the peak height or area of each fraction of the analyzed DNA, or the ratio of those fractions, is compared with the data obtained from a control (DNA derived from the same substance having a known heat treatment intensity). By doing so, the intensity of the heat treatment received by the DNA or a substance containing the DNA can be determined. The determination of the heat treatment strength may be quantitative or qualitative. In the present invention, the quantitative evaluation of the heat treatment intensity means, for example, the amount of heat loaded in the heat treatment (for example, the estimation of the heating temperature and time, for example, the amount of heat corresponding to 30 minutes at 80 ° C. or 10 minutes at 90 ° C. Was added, etc.) could be measured. Further, in the present invention, the qualitative evaluation of the heat treatment strength may be to determine whether or not the heat treatment has received a heat treatment of a predetermined strength or more (or less) or a heat treatment of a predetermined condition.
例として、当該断片化されたＤＮＡの画分と断片化が進んでいないＤＮＡの画分との間でのピークの大きさの比較に基づいた、本発明による加熱処理強度の判定について詳述する。ＤＮＡがより強い強度の加熱を受けているほど、熱によるＤＮＡ断片化が進むため、該断片化が進んでいないＤＮＡの画分のピークの高さ又は面積に対する、断片化されたＤＮＡの画分のピークの高さ又は面積の比は大きくなる。したがって、対象物質のＤＮＡについて測定した当該比の値を、同じ種類の物質について予め分析した結果をもとに設定した基準値と比較することによって、該対象物質やそのＤＮＡの受けた加熱処理の強度を判定することができる。例えば、工場での加熱処理食品の製造工程で施されるような相対的に強い強度の加熱処理（以下「強い加熱処理」という）を受けた物質や、家庭での加熱処理食品の再加熱で施されるような相対的により強くない強度の加熱処理（以下「弱い加熱処理」という）を受けた物質について、上述したクロマトグラフィー分析を行って、該断片化されたＤＮＡの画分と断片化が進んでいないＤＮＡの画分とのピークの高さ又は面積の比を算出し、それに基づいて比の基準値を設定しておく。対象物質についての同じ分析から得られた比を該基準値と比較すれば、対象物質又はそれに含まれるＤＮＡが、当該強い加熱処理を受けているか否かを判定することができる。より具体的には、対象物質について算出された比（[断片化されたＤＮＡの画分のピークの高さ又は面積]／[断片化が進んでいないＤＮＡの画分のピークの高さ又は面積]）が該基準値よりも大きければ、該対象物質又はそれに含まれるＤＮＡは、当該強い加熱処理を受けていると判定され得、一方、当該算出された比が該基準値よりも小さければ、該対象物質又はそれに含まれるＤＮＡは、当該強い加熱処理を受けていないと判定され得る。該[断片化されたＤＮＡの画分のピークの高さ又は面積]／[断片化が進んでいないＤＮＡの画分のピークの高さ又は面積]比の代わりに、（[断片化が進んでいないＤＮＡの画分のピークの高さ又は面積]／[断片化されたＤＮＡの画分のピークの高さ又は面積]）比を用いることもできる。この場合、ＤＮＡがより強い強度の加熱を受けているほど、比の値は小さくなる。 As an example, the determination of the heat treatment intensity according to the present invention based on the comparison of the peak size between the fragmented DNA fraction and the unfragmented DNA fraction will be described in detail. .. The stronger the heat of the DNA, the more fragmented the DNA is due to the heat. Therefore, the fragmented DNA fraction is relative to the peak height or area of the unfragmented DNA fraction. The ratio of peak height or area of is large. Therefore, by comparing the value of the ratio measured for the DNA of the target substance with the reference value set based on the result of pre-analysis of the same type of substance, the heat treatment of the target substance and its DNA is performed. The strength can be determined. For example, a substance that has undergone a relatively strong heat treatment (hereinafter referred to as "strong heat treatment"), which is performed in the manufacturing process of heat-treated food at a factory, or a reheat of a heat-treated food at home. The above-mentioned chromatographic analysis is performed on a substance that has undergone a relatively less intense heat treatment (hereinafter referred to as "weak heat treatment") as applied, and the fragmented DNA fraction and fragmentation are performed. The ratio of the peak height or area to the fraction of the DNA in which the amount is not advanced is calculated, and the reference value of the ratio is set based on the ratio. By comparing the ratio obtained from the same analysis on the target substance with the reference value, it can be determined whether or not the target substance or the DNA contained therein has undergone the strong heat treatment. More specifically, the ratio calculated for the target substance ([Peak height or area of fragmented DNA fraction] / [Peak height or area of unfragmented DNA fraction] ]) Is greater than the reference value, the target substance or the DNA contained therein can be determined to have undergone the strong heat treatment, while the calculated ratio is less than the reference value. It can be determined that the target substance or the DNA contained therein has not been subjected to the strong heat treatment. Instead of the [Peak height or area of fragmented DNA fraction] / [Peak height or area of unfragmented DNA fraction] ratio, ([Fragmentation progresses The peak height or area of the non-fragmented DNA fraction] / [peak height or area of the fragmented DNA fraction]) ratio can also be used. In this case, the stronger the heat of the DNA, the smaller the ratio value.
またあるいは、当該２つの画分のピークを比較したときに、当該断片化されたＤＮＡの画分のピークの高さ又は面積が、当該断片化が進んでいないＤＮＡの画分のピークと比べて所定の割合以上（例えば、断片化が進んでいないＤＮＡの画分のピークの高さ又は面積の２５％以上、５０％以上、７５％以上、又は１００％以上）であれば、対象物質又はそれに含まれるＤＮＡは当該強い加熱処理を受けていると判定され得る。一方、該断片化されたＤＮＡの画分のピークの高さ又は面積が、該断片化が進んでいないＤＮＡの画分ピークと比べて当該所定の割合以下であれば、対象物質又はそれに含まれるＤＮＡは当該強い加熱処理を受けていないと判定され得る。 Alternatively, when comparing the peaks of the two fractions, the height or area of the peak of the fragmented DNA fraction is compared to the peak of the unfragmented DNA fraction. If it is a predetermined ratio or more (for example, 25% or more, 50% or more, 75% or more, or 100% or more of the peak height or area of the unfragmented DNA fraction), the target substance or it. It can be determined that the contained DNA has undergone the strong heat treatment. On the other hand, if the height or area of the peak of the fragmented DNA fraction is equal to or less than the predetermined ratio of the fraction peak of the unfragmented DNA, it is included in the target substance or it. It can be determined that the DNA has not undergone the strong heat treatment.
本発明における「強い加熱処理」（工場での加熱処理食品の製造工程で施されるような条件での加熱処理）としては、例えば、通常行われる食品の加熱調理（例えば、焼き調理、揚げ調理、又は１０分以上の茹で、蒸しもしくは煮込み調理等）における加熱処理、レトルト殺菌等の通常の加圧加熱殺菌処理における加熱処理（例えば、１２１℃３０分）、あるいはチルド食品（パウチ惣菜）の殺菌で行われる加熱処理（例えば、９０℃３０分）など、又はこれらと同等以上の熱量を負荷する加熱処理が挙げられる。また本発明における「弱い加熱処理」（家庭での加熱処理食品の再加熱で施されるような条件での加熱処理）としては、例えば、電子レンジによる再加熱、数分程度の茹でや蒸し、湯煎等、又はこれらと同等以下の熱量を負荷する加熱処理が挙げられる。 As the "strong heat treatment" (heat treatment under the conditions performed in the manufacturing process of the heat-treated food in the factory) in the present invention, for example, the usual cooking of food (for example, baking cooking, fried cooking) Or, heat treatment in boiling for 10 minutes or more, steaming or boiling cooking, etc., heat treatment in normal pressure heat sterilization treatment such as retort sterilization (for example, 121 ° C. for 30 minutes), or sterilization of chilled foods (pouch side dishes). (For example, 90 ° C. for 30 minutes), or a heat treatment in which an amount of heat equal to or higher than these is applied can be mentioned. Further, as the "weak heat treatment" (heat treatment under the condition that the heat-treated food is reheated at home) in the present invention, for example, reheating with a microwave oven, boiling or steaming for about several minutes, Examples thereof include hot water boiling and heat treatment in which a heat amount equal to or less than these is applied.
本発明において、加熱処理強度の判定の対象物質が加熱処理食品中に混入した異物である場合、その判定の結果は、該異物が、工場での加熱処理食品の製造工程で施されるような強い強度の加熱処理を受けた経験を有するか否かについての情報を与える。すなわち、該異物が当該強い加熱処理を受けていると判定された場合は、該異物は、工場での製造過程における加熱処理を受けているものであるから、該加熱処理食品の製造過程で混入したと推定される。一方で、該異物が当該強い加熱処理を受けていないと判定された場合は、該異物は、該加熱処理食品の製造後、少なくとも該加熱処理よりも後の段階で混入したと推定される。 In the present invention, when the target substance for determining the heat treatment strength is a foreign substance mixed in the heat-treated food, the result of the determination is that the foreign substance is applied in the manufacturing process of the heat-treated food at the factory. Gives information about whether or not they have undergone intense heat treatment. That is, when it is determined that the foreign matter has undergone the strong heat treatment, the foreign matter has been heat-treated in the manufacturing process at the factory and is therefore mixed in the manufacturing process of the heat-treated food. It is estimated that it was done. On the other hand, if it is determined that the foreign matter has not been subjected to the strong heat treatment, it is presumed that the foreign matter was mixed at least after the production of the heat-treated food, at least after the heat treatment.
したがって、本発明はまた、加熱処理食品中に含まれるＤＮＡを含有する異物の混入時期を推定する方法を提供する。当該方法では、上述した異物が受けた加熱処理の強度を判定する方法と同様に、該異物に含まれるＤＮＡのクロマトグラフィー分析により該異物が受けた加熱処理の強度を判定し、次いで判定された加熱処理の強度に基づいて、該異物の混入時期を推定する。混入時期推定の基準は上記のとおりである。 Therefore, the present invention also provides a method for estimating the time of contamination of a foreign substance containing DNA contained in a heat-treated food. In this method, the strength of the heat treatment received by the foreign substance was determined by chromatographic analysis of the DNA contained in the foreign substance, and then determined in the same manner as the method for determining the intensity of the heat treatment received by the foreign substance. The timing of mixing the foreign matter is estimated based on the strength of the heat treatment. The criteria for estimating the mixing time are as described above.
次に本発明をさらに具体的に説明するために実施例を掲げるが、本発明は以下の実施例のみに限定されるものではない。 Next, examples will be given to more specifically explain the present invention, but the present invention is not limited to the following examples.
１）試薬及びサンプルＤＮＡ
・アセトニトリル（ＨＰＬＣ用）
・トリエチルアミン（ＴＥＡ）（特級）
・ヘキサフルオロ−２−プロパノール（ＨＦＩＰ）（ＨＰＬＣ用）
・メタノール（特級）
・サケ白子ＤＮＡ（Ｓａｌｍｏｎ Ｓｐｅｒｍ，Ｓｏｎｉｃａｔｅｄ、ＤＮＡ濃度：１０ｍｇ／ｍＬ）
・小麦ＤＮＡ（小麦種子からＤｎｅａｓｙ Ｐｌａｎｔ Ｍａｘｉ Ｋｉｔ（Ｑｉａｇｅｎ）により抽出、ＤＮＡ濃度：３００ｎｇ／μＬ）
1) Reagents and sample DNA
-Acetonitrile (for HPLC)
・ Triethylamine (TEA) (special grade)
Hexafluoro-2-propanol (HFIP) (for HPLC)
・ Methanol (special grade)
・ Salmon milt DNA (Salmon Sperm, Sonicated, DNA concentration: 10 mg / mL)
-Wheat DNA (extracted from wheat seeds by Dneasy Plant Maxi Kit (Qiagen), DNA concentration: 300 ng / μL)
２）ＨＰＬＣ分析条件
機器：Ｓｈｉｍａｄｚｕ ３０Ａ
カラム：ＴｅｒｍｏＦｉｓｈｅｒ ＤＮＡＰａｃ ２．１ｍｍ×５０ｍｍ×４μｍ
カラム温度：５５℃
カラム流量：０．３０ｍＬ
測定波長：２６０ｎｍ
溶離液：Ａ：１５ｍＭ ＴＥＡ−４００ｍＭ ＨＦＩＰ
Ｂ：（１５ｍＭ ＴＥＡ−４００ｍＭ ＨＦＩＰ）／メタノール（５０／５０）
グラジエント：Ａ：Ｂ＝４２：５８（０分）→５：９５（１５分）→５：９５（２５分）→４２：５８（２５．１分）→４２：５８（３５分）
2) HPLC analysis condition equipment: Shimadzu 30A
Column: Thermo Fisher DNAPac 2.1 mm x 50 mm x 4 μm
Column temperature: 55 ° C
Column flow rate: 0.30 mL
Measurement wavelength: 260 nm
Eluent: A: 15 mM TEA-400 mM HFIP
B: (15 mM TEA-400 mM HFIP) / methanol (50/50)
Gradient: A: B = 42:58 (0 minutes) → 5:95 (15 minutes) → 5:95 (25 minutes) → 42:58 (25.1 minutes) → 42:58 (35 minutes)
実施例１ ＤＮＡ加熱処理によるクロマトグラムの変化
１）サケ白子ＤＮＡの分析
サケ白子ＤＮＡをサンプルとした。サンプルＤＮＡを水で１０００倍に希釈し、１ｍＬずつにわけ、７０、８０、９０、及び９５℃に設定した湯浴でそれぞれ０、１０、２０、３０、６０又は１２０分加熱した。加熱処理したサンプルＤＮＡをＨＰＬＣで測定した。得られたクロマトグラムから、断片化されたＤＮＡの画分及び断片化が進んでいないＤＮＡの画分（それぞれ、クロマトグラム上で最も顕著な２つの画分のうち、保持時間のより短い画分及びより長い画分）を選択し、選択した各画分のピークの面積を求めた。ピーク面積は、クロマトグラフィーにおけるＵＶ検出値に基づいて算出した。加熱処理条件毎に各ピーク面積の平均値を求めた（Ｎ＝３）。
Example 1 Changes in chromatogram due to DNA heat treatment 1) Analysis of salmon milt DNA Salmon milt DNA was used as a sample. The sample DNA was diluted 1000-fold with water, divided into 1 mL portions, and heated in hot water baths set at 70, 80, 90, and 95 ° C. for 0, 10, 20, 30, 60, or 120 minutes, respectively. The heat-treated sample DNA was measured by HPLC. From the obtained chromatogram, a fraction of fragmented DNA and a fraction of unfragmented DNA (each of the two most prominent fractions on the chromatogram, the one with the shorter retention time). And longer fractions) were selected and the peak area of each selected fraction was determined. The peak area was calculated based on the UV detection value in chromatography. The average value of each peak area was calculated for each heat treatment condition (N = 3).
９０℃で加熱処理したサンプルＤＮＡについてのクロマトグラムを図１に示す。２つの主なピークが検出された（図１中の矢印で示したピーク）。これら２つのピークのうち、断片化が進んでいないＤＮＡの画分のピーク（保持時間がより長いピーク；図１の右矢印）の高さ及び面積は、加熱処理の時間が長くなるに従って減少した。一方、断片化されたＤＮＡの画分のピーク（保持時間がより短いピーク；図１の左矢印）の高さ及び面積は、加熱処理の時間が長くなるに従って増加した。他の温度で加熱処理したサンプルについても、同様の結果が得られた。すなわち、図１と同様に２つの主なピークが検出され、それらのピークの高さ及び面積は加熱処理の時間に従って変化した。 A chromatogram of the sample DNA heat-treated at 90 ° C. is shown in FIG. Two major peaks were detected (peaks indicated by arrows in FIG. 1). Of these two peaks, the height and area of the unfragmented DNA fraction peak (peak with longer retention time; right arrow in FIG. 1) decreased as the heat treatment time increased. .. On the other hand, the height and area of the peaks (peaks with shorter retention time; left arrow in FIG. 1) of the fragmented DNA fraction increased as the heat treatment time increased. Similar results were obtained for samples heat-treated at other temperatures. That is, two main peaks were detected as in FIG. 1, and the height and area of those peaks changed according to the time of heat treatment.
検出された２つの画分のピークの面積と加熱処理条件との関係を調べた。表１〜２は、各加熱処理条件における、該２つの画分のピークそれぞれの面積の平均値を示す。検出された２つの画分のピークのうちの、保持時間がより長い断片化が進んでいないＤＮＡの画分のピーク（以下、第１ピークと称する）の結果を表１に、保持時間がより短い断片化されたＤＮＡの画分のピーク（以下、第２ピークと称する）の結果を表２に示す。第１ピークの面積は、加熱温度の上昇、又は加熱時間の延長とともに減少した。第２ピークの面積は、加熱温度の上昇、又は加熱時間の延長とともに増加した。温度条件ごとの第１及び第２ピークの面積の加熱時間に対するプロットを図２〜５に示す。第１ピークの面積は、特に３０分間以上の加熱に対して、加熱時間による影響を受けて変動した。第２ピークの面積は、短時間の加熱によっても変動した。いずれの温度条件においても、ピークの面積と加熱時間との間に高い相関性（Ｒ2＞０．９９）がみられた。 The relationship between the peak areas of the two detected fractions and the heat treatment conditions was investigated. Tables 1 and 2 show the average value of the areas of the peaks of the two fractions under each heat treatment condition. Table 1 shows the results of the peaks of the unfragmented DNA fractions (hereinafter referred to as the first peak) having a longer retention time among the peaks of the two detected fractions. Table 2 shows the results of the peaks of the short fragmented DNA fractions (hereinafter referred to as the second peak). The area of the first peak decreased as the heating temperature increased or the heating time was extended. The area of the second peak increased with increasing heating temperature or extension of heating time. Plots for the heating time of the areas of the first and second peaks for each temperature condition are shown in FIGS. 2-5. The area of the first peak fluctuated due to the influence of the heating time, especially for heating for 30 minutes or more. The area of the second peak also fluctuated with short heating. A high correlation (R 2 > 0.99) was observed between the peak area and the heating time under all temperature conditions.
２）小麦ＤＮＡの分析
小麦ＤＮＡをサンプルとした。サンプルＤＮＡを水で１００倍に希釈し、０．５ｍＬずつにわけ、９０℃に設定した湯浴でそれぞれ０、１０、２０、３０、６０又は１２０分加熱した。上記１）と同様の手順で、加熱処理したサンプルＤＮＡをＨＰＬＣで測定し、得られたクロマトグラムから第１ピーク及び第２ピークの面積を求めた。加熱処理条件毎に各ピーク面積の平均値を求めた（Ｎ＝３）。
2) Analysis of wheat DNA Wheat DNA was used as a sample. The sample DNA was diluted 100-fold with water, divided into 0.5 mL portions, and heated in a hot water bath set at 90 ° C. for 0, 10, 20, 30, 60 or 120 minutes, respectively. The heat-treated sample DNA was measured by HPLC in the same procedure as in 1) above, and the areas of the first peak and the second peak were determined from the obtained chromatogram. The average value of each peak area was calculated for each heat treatment condition (N = 3).
サンプルＤＮＡのクロマトグラムを図６に示す。サケ白子ＤＮＡと同様に、２つの主なピークが検出された。これら２つのピークのうち、保持時間がより長い断片化が進んでいないＤＮＡの画分のピーク（第１ピーク；図６の右矢印）の高さ及び面積は、加熱処理の時間が長くなるに従って減少した。一方、保持時間がより短い断片化されたＤＮＡの画分のピーク（第２ピーク；図６の左矢印）の高さ及び面積は、加熱処理の時間が長くなるに従って増加した。表３に示すとおり、加熱時間の延長とともに、第１ピークの面積は減少し、第２ピークの面積は増加した。第１及び第２ピークの面積の加熱時間に対するプロットを図７に示す。第１及び第２ピークともピーク面積と加熱時間との間に高い相関性（Ｒ2＞０．９９）がみられた。 A chromatogram of sample DNA is shown in FIG. Similar to salmon milt DNA, two major peaks were detected. Of these two peaks, the height and area of the unfragmented DNA fraction peak (first peak; right arrow in FIG. 6) having a longer retention time increases as the heat treatment time increases. Diminished. On the other hand, the height and area of the peak (second peak; left arrow in FIG. 6) of the fragmented DNA fraction having a shorter retention time increased as the heat treatment time increased. As shown in Table 3, the area of the first peak decreased and the area of the second peak increased with the extension of the heating time. A plot of the areas of the first and second peaks for the heating time is shown in FIG. A high correlation (R 2 > 0.99) was observed between the peak area and the heating time for both the first and second peaks.
以上の結果から、ＤＮＡのクロマトグラムのピークの高さ及び面積が、該ＤＮＡに負荷された加熱処理の強度を反映することが示された。 From the above results, it was shown that the height and area of the peak of the chromatogram of DNA reflected the intensity of the heat treatment loaded on the DNA.
実施例２ クロマトグラムに基づくＤＮＡ加熱処理条件の定量
サケ白子ＤＮＡをサンプルとし、実施例１と同様の条件で加熱処理した。ただし温度は９０℃、加熱時間は１５、２５、３５及び４５分とした（Ｎ＝１）。実施例１と同様の手順で、加熱処理したサンプルＤＮＡをＨＰＬＣで測定し、得られたクロマトグラムから第１ピークの面積を求めた。求めたピーク面積を図４に記載した第１ピークの近似式にあてはめて加熱時間を推定したところ、実際の加熱時間とほぼ一致した（表４）。したがって、クロマトグラムのピークに基づいて、ＤＮＡに負荷された加熱処理時間を定量可能であることが示された。
Example 2 Quantification of DNA heat treatment conditions based on chromatogram Salmon milt DNA was used as a sample and heat-treated under the same conditions as in Example 1. However, the temperature was 90 ° C. and the heating time was 15, 25, 35 and 45 minutes (N = 1). The heat-treated sample DNA was measured by HPLC in the same procedure as in Example 1, and the area of the first peak was determined from the obtained chromatogram. When the heating time was estimated by applying the obtained peak area to the approximate expression of the first peak shown in FIG. 4, it was almost the same as the actual heating time (Table 4). Therefore, it was shown that the heat treatment time loaded on the DNA can be quantified based on the peak of the chromatogram.
Claims (13)
該物質に含まれるＤＮＡをクロマトグラフィー分析すること、
該分析で得られたクロマトグラムに基づいて、該物質が受けた加熱処理の強度を判定すること、
を含む、方法。 A method for determining the strength of heat treatment received by a substance containing DNA contained in a heat-treated food.
Chromatographic analysis of the DNA contained in the substance,
Determining the intensity of the heat treatment received by the substance based on the chromatogram obtained by the analysis.
Including methods.
該異物に含まれるＤＮＡをクロマトグラフィー分析すること、
該分析で得られたクロマトグラムに基づいて、該異物が受けた加熱処理の強度を判定すること、
判定された加熱処理の強度に基づいて、該異物の混入時期を推定すること、
を含む、方法。 It is a method of estimating the mixing time of foreign substances containing DNA contained in heat-treated foods.
Chromatographic analysis of the DNA contained in the foreign substance,
Determining the strength of the heat treatment received by the foreign matter based on the chromatogram obtained by the analysis.
To estimate the time when the foreign matter is mixed based on the determined heat treatment intensity.
Including methods.
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