JP2013072726A - Method for quantifying triacylglycerol in brown rice using near-infrared spectroscopy - Google Patents
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Abstract
Description
本発明は、近赤外分光法を用いた玄米中のトリアシルグリセロールの定量方法に関するものである。 The present invention relates to a method for quantifying triacylglycerol in brown rice using near infrared spectroscopy.
米糠から抽出して得られる米油（米糠油）は、栄養・生理機能に富み、味質、食感にも優れた食用油として注目され、利用が拡大している。昨今、米油の増産のため、油脂を豊富に含んだ高含油米の育種開発が進められている。高含油米を育種開発するためには、膨大な量の育種資源から有用形質遺伝資源を探索する必要があるため、迅速性に富んだ油（トリアシルグリセロール、以下「ＴＡＧ」と記す。）含量分析方法が望まれる。また、交配時の選抜においては、穂の米粒の間にも遺伝的な差があるため、米一粒で分析できることが好ましく、非破壊（そのままの状態）で分析することができれば、分析後の種子を作付けできるため育種開発の効率を飛躍的に高めることができる。しかし、今まで玄米一粒に適用できる迅速性に富んだ脂質含量分析方法や非破壊分析法は公表されていない。 Rice oil obtained by extracting from rice bran (rice bran oil) is attracting attention as an edible oil that is rich in nutrition and physiological functions, and excellent in taste and texture, and its use is expanding. Recently, in order to increase the production of rice oil, breeding development of highly oil-rich rice containing abundant fats and oils has been promoted. In order to breed and develop highly oil-rich rice, it is necessary to search for useful genetic resources from a huge amount of breeding resources, so the content of oil (triacylglycerol, hereinafter referred to as “TAG”) rich in rapidity is high. An analysis method is desired. In addition, in selection at the time of mating, since there is a genetic difference between the rice grains of the ear, it is preferable that analysis can be performed with a single grain of rice, and if analysis can be performed non-destructively (as it is), Since seeds can be planted, the efficiency of breeding development can be dramatically increased. However, a lipid content analysis method and a nondestructive analysis method that are applicable to a single grain of brown rice have not been published so far.
食品の脂質含量の分析方法として一般的な手法は、脂質の抽出を行い、その重量を測定する重量法である。しかし一般に玄米から抽出された脂質中にはＴＡＧ以外にリン脂質、糖脂質などが含まれており、ＴＡＧだけを測定する事は困難である。よって、ＴＡＧを特異的に検出し、かつ微量分析に適用できる迅速分析法の開発が必要となる。 A general method for analyzing the lipid content of food is a gravimetric method in which lipid is extracted and its weight is measured. However, in general, lipids extracted from brown rice contain phospholipids, glycolipids and the like in addition to TAG, and it is difficult to measure only TAG. Therefore, it is necessary to develop a rapid analysis method that can specifically detect TAG and can be applied to microanalysis.
ＴＡＧは１分子のグリセリンに３分子の脂肪酸がエステル結合したアシルグリセロールで、中性脂肪の１つである。中性脂肪は動物の体内脂肪組織に蓄えられる脂肪や、食品中の油脂、植物油（種子）などを構成する脂質の８〜９割を占める。その中でもＴＡＧが圧倒的に多い。
試料中の微量のＴＡＧを測定する方法として、酵素による比色定量法が知られている。血中ＴＡＧ測定用に市販されている試薬キットの多くは、測定が簡便かつ迅速に行える利点から酵素による比色定量法を採用している。しかし酵素による比色定量法を用いた試薬キットは水系試料を適用対象としており、油脂試料や植物体試料には適用されていない。
TAG is an acylglycerol in which three molecules of fatty acid are ester-bonded to one molecule of glycerin, and is one of the neutral fats. Neutral fat occupies 80 to 90% of the fat that is stored in the adipose tissue of animals, the fats and oils in food, and the vegetable oil (seed). Among them, TAG is overwhelmingly large.
As a method for measuring a small amount of TAG in a sample, an enzymatic colorimetric determination method is known. Many of the reagent kits marketed for blood TAG measurement employ an enzyme colorimetric determination method because of the advantage that the measurement can be performed easily and rapidly. However, reagent kits using enzymatic colorimetric determination are applied to aqueous samples, and are not applied to oil and fat samples and plant samples.
従来、油脂試料や植物体試料に含まれるＴＡＧを測定する場合は、試料を破砕し、溶媒にてＴＡＧを抽出、ガスクロマトグラフィー（ＧＣ）または高速液体クロマトグラフィー（ＨＰＬＣ）により定量するのが一般的である。しかし、ＴＡＧは多数の分子種から構成されるため、上記のようなクロマトグラム法ではピークが複数に分かれる事が多く、これらを全て積算する必要がある。また、ＴＡＧを特異的に検出するような検出器は現状存在しないので、ＴＡＧ以外の成分がＴＡＧのピークに重ならないようにカラムや条件を選定する必要もある。その結果、試料ごとに独自の工夫が必要となり、プロトコルを統一できないので、手法としては非常に煩雑である。 Conventionally, when measuring TAG contained in oil and fat samples and plant samples, it is common to crush the sample, extract the TAG with a solvent, and quantify it by gas chromatography (GC) or high performance liquid chromatography (HPLC). Is. However, since TAG is composed of a large number of molecular species, the chromatogram method as described above often has a plurality of peaks, and it is necessary to integrate them all. In addition, since there is currently no detector that specifically detects TAG, it is necessary to select columns and conditions so that components other than TAG do not overlap with the TAG peak. As a result, a unique device is required for each sample, and the protocol cannot be unified, so that the method is very complicated.
上記測定方法は全て原試料を破壊して分析する言わば破壊分析法であり、測定した試料を再利用することはできない。一方、非破壊分析法のメリットとしては、迅速に分析できること、分析操作が簡単なこと、化学薬品を使用しないこと、さらに、分析した試料が、そのままの形で残っていること、などが挙げられる。非破壊分析法には、光を使う方法や電気・磁気を使う方法などがあるが、食品の分析に最も多く使われているのは近赤外分光法である。
近赤外分光法は、物質分子による近赤外光（一般的に波長域８００〜２５００ｎｍを指す）の吸収に基づく分光法である。近赤外光はエネルギーの低い電磁波でありながら、物質を透過しやすい性質を持っている。それゆえ、近赤外分光法では、紫外・可視分光法などで通常用いられる透過測定に加え、透過反射、拡散反射法などの測定方法が多く用いられる。そのため近赤外スペクトルには、それぞれの測定方法のスペクトルが存在する。近赤外分光法は食品の分野で果実、野菜、魚類等の成分測定や加工食品の品質管理などに用いられている。近赤外分光法の利点は第一に、試料を損傷しないことであり、あるがままの状態で分析ができ、多成分の同時定量が可能であるという特徴がある。近赤外分光法は、特に青果物の分析方法として一般的なものである。青果物の非破壊分析法としては糖類の定量方法（例えば特許文献１）、水分の定量方法（例えば特許文献２）、硝酸イオン定量方法（例えば特許文献３）などが報告されている。また、非特許文献１には、近赤外分光法を用いる非破壊分析法が、米のでんぷん、タンパク質、水分、灰分、アミノ酸、食味値に応用されていることが記載されている。しかし、玄米のＴＡＧを非破壊分析法により定量したことは報告されていない。
The above measurement methods are all so-called destructive analysis methods in which the original sample is destroyed and analyzed, and the measured sample cannot be reused. On the other hand, merits of non-destructive analysis include that it can be analyzed quickly, that the analysis operation is simple, that no chemicals are used, and that the analyzed sample remains as it is. . Nondestructive analysis methods include methods using light and methods using electricity and magnetism. Near-infrared spectroscopy is most frequently used for food analysis.
Near-infrared spectroscopy is spectroscopy based on absorption of near-infrared light (generally indicating a wavelength range of 800 to 2500 nm) by substance molecules. Near-infrared light is an electromagnetic wave with low energy, but has the property of being easily transmitted through a substance. Therefore, in near-infrared spectroscopy, in addition to transmission measurement normally used in ultraviolet / visible spectroscopy, measurement methods such as transmission reflection and diffuse reflection are often used. Therefore, the near-infrared spectrum has a spectrum for each measurement method. Near-infrared spectroscopy is used in the field of food for measuring ingredients of fruits, vegetables, fish, etc., and for quality control of processed foods. The advantage of near-infrared spectroscopy is that it does not damage the sample. It can be analyzed as it is, and can be quantified simultaneously with multiple components. Near-infrared spectroscopy is a common method for analyzing fruits and vegetables. As non-destructive analysis methods for fruits and vegetables, a saccharide determination method (for example, Patent Document 1), a moisture determination method (for example, Patent Document 2), and a nitrate ion determination method (for example, Patent Document 3) have been reported. Non-Patent Document 1 describes that a nondestructive analysis method using near-infrared spectroscopy is applied to rice starch, protein, moisture, ash, amino acids, and taste values. However, it has not been reported that TAG of brown rice was quantified by nondestructive analysis.
本発明は、玄米、特に玄米一粒中に含まれるＴＡＧの非破壊分析法を提供することを課題とする。 An object of the present invention is to provide a non-destructive analysis method for TAG contained in brown rice, particularly brown rice.
本発明は、上記課題を解決するために、以下の各発明を包含する。
［１］玄米に含まれるトリアシルグリセロールを定量するための回帰式を得る方法であって、以下の（１）〜（３）の工程を含むことを特徴とする方法。
（１）玄米の近赤外光スペクトルを測定する工程
（２）当該玄米のトリアシルグリセロール含量を定量する工程
（３）近赤外光スペクトルを測定した波長範囲の全部または一部の波長領域で得られたスペクトルデータと、定量したトリアシルグリセロール含量とをＰＬＳ（partial least squares）回帰分析により解析し、トリアシルグリセロール含量と関係する因子を決定する工程
［２］近赤外光スペクトルが拡散反射スペクトルであることを特徴とする前記［１］に記載の方法。
［３］前記（３）の工程における波長領域が、１０００〜２５００ｎｍまたはその一部であることを特徴とする前記［１］または［２］に記載の方法。
［４］前記（３）の工程における波長領域が、１６３８〜２３５５ｎｍまたはその一部であることを特徴とする前記［３］に記載の方法。
［５］前記（３）の工程における波長領域が、１７２５±１０、１７６４±１０、２３１７±１０ｎｍの少なくとも１つの領域を含むことを特徴とする前記［３］または［４］に記載の方法。
［６］前記（３）の工程における波長領域が、１８６０±１０、１９６１±１０、２２７６±１０ｎｍの少なくとも１つの領域を含むことを特徴とする前記［５］に記載の方法。
［７］前記（２）の工程において、乾燥処理した玄米から粗脂質を抽出し、粗脂質中のトリアシルグリセロールを定量することを特徴とする前記［１］〜［６］のいずれかに記載の方法。
［８］前記（２）の工程において、酵素を利用した比色定量法を用いることを特徴とする前記［１］〜［７］のいずれかに記載の方法。
［９］前記（２）の工程において、玄米から抽出した粗脂質をｔｅｒｔ−ブチルアルコールまたはヘキサンに溶解させて得られた試料を、酵素を利用した比色定量法に供することを特徴とする前記［８］に記載の方法。
［１０］前記（２）の工程において、玄米から抽出した粗脂質を乾固させた試料を、そのまま酵素を利用した比色定量法に供することを特徴とする前記［８］に記載の方法。
［１１］前記［１］〜［１０］のいずれかに記載の方法により得られた回帰式と、当該回帰式の作成に使用した波長領域における被験玄米の近赤外光スペクトルデータとを用いることを特徴とする玄米に含まれるトリアシルグリセロールの定量方法。
［１２］被験玄米の水分含量に基づいて被験玄米のトリアシルグリセロール含量を補正する工程をさらに含むことを特徴とする前記［１１］に記載の定量方法。
［１３］被験玄米の水分含量が、以下の（ａ）〜（ｃ）の工程により得られた回帰式と、当該回帰式の作成に使用した波長領域における被験玄米の近赤外光スペクトルデータとを用いることにより得られることを特徴とする前記［１２］に記載の定量方法。
（ａ）玄米の近赤外光スペクトルを測定する工程
（ｂ）当該玄米の水分含量を測定する工程
（ｃ）近赤外光スペクトルを測定した波長範囲の全部または一部の波長領域で得られたスペクトルデータと、測定した水分含量とをＰＬＳ回帰分析により解析し、水分含量と関係する因子を決定する工程
The present invention includes the following inventions in order to solve the above problems.
[1] A method for obtaining a regression equation for quantifying triacylglycerol contained in brown rice, which comprises the following steps (1) to (3).
(1) The step of measuring the near-infrared light spectrum of brown rice (2) The step of quantifying the triacylglycerol content of the brown rice (3) In all or part of the wavelength range in which the near-infrared light spectrum was measured The obtained spectral data and the quantified triacylglycerol content are analyzed by PLS (partial least squares) regression analysis to determine factors related to the triacylglycerol content [2] Near-infrared spectrum is diffusely reflected The method according to [1], wherein the method is a spectrum.
[3] The method according to [1] or [2], wherein the wavelength region in the step (3) is 1000 to 2500 nm or a part thereof.
[4] The method according to [3], wherein the wavelength region in the step (3) is 1638 to 2355 nm or a part thereof.
[5] The method according to [3] or [4], wherein the wavelength region in the step (3) includes at least one region of 1725 ± 10, 1764 ± 10, 2317 ± 10 nm.
[6] The method according to [5], wherein the wavelength region in the step (3) includes at least one region of 1860 ± 10, 1961 ± 10, and 2276 ± 10 nm.
[7] The method according to any one of [1] to [6], wherein in the step (2), crude lipid is extracted from the dried brown rice, and triacylglycerol in the crude lipid is quantified. the method of.
[8] The method according to any one of [1] to [7], wherein a colorimetric method using an enzyme is used in the step (2).
[9] In the step (2), the sample obtained by dissolving the crude lipid extracted from brown rice in tert-butyl alcohol or hexane is subjected to a colorimetric determination method using an enzyme. The method according to [8].
[10] The method according to [8], wherein in the step (2), the sample obtained by drying the crude lipid extracted from brown rice is directly subjected to a colorimetric determination method using an enzyme.
[11] Use the regression equation obtained by the method according to any one of [1] to [10] above and the near-infrared light spectrum data of the test brown rice in the wavelength region used for the creation of the regression equation. A method for quantifying triacylglycerol contained in brown rice.
[12] The method according to [11], further comprising a step of correcting the triacylglycerol content of the test brown rice based on the water content of the test brown rice.
[13] The regression formula obtained by the following steps (a) to (c), and the near-infrared light spectrum data of the test brown rice in the wavelength region used to create the regression formula, The quantification method according to [12], which is obtained by using
(A) The step of measuring the near-infrared light spectrum of brown rice (b) The step of measuring the water content of the brown rice (c) Obtained in all or part of the wavelength range in which the near-infrared light spectrum was measured Analyzing the measured spectral data and the measured water content by PLS regression analysis to determine factors related to the water content
本発明によれば、玄米に含まれるＴＡＧの非破壊分析法を提供することができる。特に本発明は、玄米一粒中のＴＡＧ含量を非破壊的に定量できる点で、非常に有用である。 ADVANTAGE OF THE INVENTION According to this invention, the nondestructive analysis method of TAG contained in brown rice can be provided. In particular, the present invention is very useful in that the TAG content in one grain of brown rice can be quantified nondestructively.
〔玄米に含まれるＴＡＧを定量するための回帰式を得る方法〕
本発明の玄米に含まれるＴＡＧを定量するための回帰式を得る方法（以下、「本発明の回帰式を得る方法」という。）は、以下の（１）〜（３）の工程を含むものであればよい。
（１）玄米の近赤外光（以下「ＮＩＲ」と記す。）スペクトルを測定する工程
（２）当該玄米のトリアシルグリセロール含量を定量する工程
（３）ＮＩＲスペクトルを測定した波長範囲の全部または一部の波長領域で得られたスペクトルデータと、定量したＴＡＧ含量とをＰＬＳ（partial least squares）回帰分析により解析し、ＴＡＧ含量と関係する因子を決定する工程
本発明の回帰式を得る方法は、（１）〜（３）以外の工程を含んでいてもよく、その内容は問わない。
[Method of obtaining regression equation for quantifying TAG contained in brown rice]
The method for obtaining a regression equation for quantifying TAG contained in brown rice of the present invention (hereinafter referred to as “method for obtaining the regression equation of the present invention”) includes the following steps (1) to (3). If it is.
(1) Step of measuring the near-infrared light (hereinafter referred to as “NIR”) spectrum of brown rice (2) Step of quantifying the triacylglycerol content of the brown rice (3) All of the wavelength range in which the NIR spectrum was measured or The step of analyzing the spectral data obtained in a part of the wavelength region and the quantified TAG content by PLS (partial least squares) regression analysis and determining the factor related to the TAG content The method for obtaining the regression equation of the present invention is as follows. Steps other than (1) to (3) may be included, and the contents thereof are not limited.
工程（１）では、玄米のＮＩＲスペクトルを測定する。ＮＩＲスペクトルの測定方法は特に限定されないが、拡散反射法が好ましい。測定には、市販の近赤外分光分析計を用いることができるが、固体測定モジュールを備え、拡散反射光を測定できるものが好ましい。具体的にはＭＰＡ（ブルカー・オプティクス）、ＮＩＲ−Ｆｌｅｘ Ｎ−５００（ビュッヒ）、ＩＲＡｆｆｉｎｉｔｙ−１（島津製作所）、ＬＡＭＢＤＡ７５０／９５０／１０５０およびＦｒｏｎｔｉｅｒ ＮＩＲ（パーキンエルマー）、ＦＴ−ＩＲ４０００シリーズおよびＦＴ−ＩＲ ６０００シリーズ（日本分光）、ＦＴ−ＮＩＲ ＭＢ３６００（ＡＢＢ ＢＯＭＥＭ）などが挙げられ、ＭＰＡ（ブルカー・オプティクス）が特に好ましい。得られるスペクトルは、フーリエ変換スペクトルであることが好ましい。 In step (1), the NIR spectrum of brown rice is measured. The method for measuring the NIR spectrum is not particularly limited, but the diffuse reflection method is preferred. For the measurement, a commercially available near-infrared spectrometer can be used, but those equipped with a solid measurement module and capable of measuring diffuse reflected light are preferred. Specifically, MPA (Bruker Optics), NIR-Flex N-500 (Buch), IRAffinity-1 (Shimadzu Corporation), LAMBDA 750/950/1050 and Frontier NIR (Perkin Elmer), FT-IR4000 series and FT-IR 6000 series (JASCO), FT-NIR MB3600 (ABB BOMEM), etc. are mentioned, MPA (Bruker Optics) is particularly preferable. The obtained spectrum is preferably a Fourier transform spectrum.
回帰式を得るための試料および被験試料は、籾殻が除去された玄米であることが好ましい。籾殻が除去されていれば、白米あるいは精米途中の玄米であっても構わない。試料玄米の品種や形状、大きさ、重量は特に限定されないが、ひびや欠損が無いものが好ましい。玄米一粒中のＴＡＧ含量を定量するための回帰式を得るには、一粒の玄米を１試料としてＮＩＲ拡散反射光スペクトルを測定することが好ましい。精度の高い回帰式を得るための試料は、被験試料と類似の品種、形態の玄米であって、ＴＡＧ含量の異なる数種類の玄米を用意することが好ましい。また回帰式を得るための試料数は多いほどよく、好ましくは３０試料以上、より好ましくは５０試料以上である。
試料は原則的に玄米一粒であることが好ましいが、玄米の重量あたりのＴＡＧ含量を測定する目的であれば、複数の玄米を１試料としてもよく、また、玄米を均質に粉砕した粉（玄米粉）を試料としても良い。
The sample for obtaining the regression equation and the test sample are preferably brown rice from which rice husks have been removed. As long as the rice husk is removed, it may be white rice or brown rice in the middle of milling. The variety, shape, size and weight of the sample brown rice are not particularly limited, but those having no cracks or defects are preferred. In order to obtain a regression formula for quantifying the TAG content in one grain of brown rice, it is preferable to measure the NIR diffuse reflection spectrum using one grain of brown rice as one sample. The sample for obtaining a highly accurate regression equation is preferably brown rice having a variety and form similar to that of the test sample and having different TAG contents. Further, the larger the number of samples for obtaining the regression equation, the better, preferably 30 samples or more, more preferably 50 samples or more.
In principle, the sample is preferably a single grain of brown rice, but for the purpose of measuring the TAG content per weight of brown rice, a plurality of brown rice may be used as one sample, or a powder obtained by uniformly grinding brown rice ( Brown rice flour) may be used as a sample.
工程（２）では、工程（１）でＮＩＲスペクトルを測定した玄米のＴＡＧ含量を測定する。工程（１）に供した試料単位でＴＡＧ含量を測定すればよい。玄米のＴＡＧ含量は公知の方法、例えばソックスレー抽出やクロロホルム／メタノール抽出などにて粗脂質を抽出後、ガスクロマトグラフィー（ＧＣ）、高速液体クロマトグラフィー（ＨＰＬＣ）などを用いて測定することができる。なお、酵素による比色定量法を用いて玄米中のＴＡＧ含量を定量する方法については、後述する。 At a process (2), the TAG content of the brown rice which measured the NIR spectrum at the process (1) is measured. What is necessary is just to measure a TAG content in the sample unit provided to the process (1). The TAG content of brown rice can be measured using gas chromatography (GC), high performance liquid chromatography (HPLC), etc. after extracting the crude lipid by a known method such as Soxhlet extraction or chloroform / methanol extraction. A method for quantifying the TAG content in brown rice using an enzyme colorimetric method will be described later.
工程（３）では、ＮＩＲスペクトルを測定した波長範囲の全部または一部の波長領域で得られたスペクトルデータと、定量したＴＡＧ含量とをＰＬＳ回帰分析により解析し、ＴＡＧ含量と関係する因子を決定する。ＮＩＲスペクトルを測定した波長範囲の一部とはＮＩＲスペクトルを測定した波長範囲に含まれる任意の部分領域である。
ＮＩＲスペクトルを測定した波長範囲の一部は特に限定されないが、１０００〜２５００ｎｍまたはその一部であることが好ましく、１６３８〜２３５５ｎｍまたはその一部であることがより好ましい。また、ＴＡＧに関連するＡ群（１７２５±１０、１７６４±１０、２３１７±１０ｎｍ）の好ましくは１つ以上、より好ましくは２つ以上の領域を含む領域であり、さらにＴＡＧ以外の成分に関連するＢ群（１８６０±１０、１９６１±１０、２２７６±１０ｎｍ）の少なくとも１つの領域を含むものが好ましい。特に好ましくは、ＴＡＧに関連するＡ群（１７２５±１０、１７６４±１０、２３１７±１０ｎｍ）の２つ以上の領域を含み、かつ、ＴＡＧ以外の成分に関連するＢ群（１８６０±１０、１９６１±１０、２２７６±１０ｎｍ）の１つ以上の領域を含む領域である。また、この１つの波長領域は最大波長と最小波長の差が２０ｎｍ以上の連続した領域であることが好ましく、１００ｎｍ以上の連続した領域である事がより好ましく、２００ｎｍ以上であればさらに好ましい。
In step (3), spectral data obtained in all or a part of the wavelength range in which the NIR spectrum is measured and the quantified TAG content are analyzed by PLS regression analysis to determine factors related to the TAG content. To do. The part of the wavelength range in which the NIR spectrum is measured is an arbitrary partial region included in the wavelength range in which the NIR spectrum is measured.
Although a part of wavelength range which measured the NIR spectrum is not specifically limited, it is preferable that it is 1000-2500 nm or its part, and it is more preferable that it is 1638-2355 nm or its part. Moreover, it is a region including preferably one or more, more preferably two or more regions of Group A (1725 ± 10, 1764 ± 10, 2317 ± 10 nm) related to TAG, and further related to components other than TAG. Those including at least one region of group B (1860 ± 10, 1961 ± 10, 2276 ± 10 nm) are preferred. Particularly preferably, group B (1860 ± 10, 1961 ±) includes two or more regions of group A related to TAG (1725 ± 10, 1764 ± 10, 2317 ± 10 nm) and related to components other than TAG. 10, 2276 ± 10 nm). Further, this one wavelength region is preferably a continuous region where the difference between the maximum wavelength and the minimum wavelength is 20 nm or more, more preferably a continuous region of 100 nm or more, and even more preferably 200 nm or more.
ＰＬＳ回帰分析は市販のソフトウェアを使用して行うことができる。使用するソフトウェアは例えばＴｈｅ Ｕｎｓｃｒａｍｂｌｅｒ（株式会社カモソフトウェアジャパン)、ＯＰＵＳ（ブルカー・オプティクス社）、ＮＩＲＣａｌ（日本ビュッヒ社）、Ｐｉｒｏｕｅｔｔｅ（ジーエルサイエンス社）などが例示できる。これらのソフトウェアを利用して、ＮＩＲ拡散反射光スペクトルを解析し、その結果を本発明の定量法で用いる。使用するスペクトルデータまたはフーリエ変換スペクトルデータは、原スペクトルデータでもよいが、原スペクトルデータを加工したものを使用することが好ましい。データ加工の方法としては、例えば、一次微分、二次微分、三次微分などの多次微分（Ｄｅｒｉｖａｔｉｖｅ）、平滑化（Ｓｍｏｏｔｈｉｎｇ）、スペクトルの減算（Ｓｕｂｔｒａｃｔｉｏｎ）、正規化（Ｎｏｒｍａｌｉｚｅ）、ＭＳＣ補正（ＭｕｌｔｉｐｌｉｃａｔｉｖｅＳｃａｔｔｅｒ Ｃｏｒｒｅｃｔｉｏｎ）、ＳＮＶ補正（Ｓｔａｎｄａｒｄ Ｎｏｒｍａｌ Ｖａｒｉａｔｅ Ｃｏｒｒｅｃｔｉｏｎ）などが挙げられる。 PLS regression analysis can be performed using commercially available software. Examples of the software to be used include The Unscrambler (Camo Software Japan Co., Ltd.), OPUS (Bruker Optics), NIRCal (Nippon Büch), and Pirouette (GL Science). Using these software, the NIR diffuse reflected light spectrum is analyzed, and the result is used in the quantification method of the present invention. The spectrum data or the Fourier transform spectrum data to be used may be original spectrum data, but it is preferable to use data obtained by processing the original spectrum data. As a method of data processing, for example, first-order differentiation, second-order differentiation, third-order differentiation such as third-order differentiation (Derivative), smoothing (Smoothing), spectrum subtraction (Normalization), normalization (Normalize), MSC correction (MultiplicativeScatter) Correction), SNV correction (Standard Normal Variate Correction), and the like.
解析に用いる波長領域のスペクトルデータと定量したＴＡＧ含量とをＰＬＳ回帰分析に供することにより、ＴＡＧ含量と関係する因子を決定して回帰式を得ることができる。ＴＡＧ含量と関係する因子とは、スペクトルデータの中に内在するＴＡＧ含量の変化と相関の高い仮想的な変量を指す。この因子の回帰係数を用いてスペクトルデータからＴＡＧ含量を予測する回帰式を作成する。近赤外分光法では、通常回帰式作成後、検量線作成用試料以外の試料（評価用試料）を用いて、作成された回帰式の測定精度を評価する。回帰式の測定精度は評価用試料の実測値と作成した回帰式によって算出された予測値による分析値との残差（測定誤差）の標準偏差や平均値によって評価する。この予測値（定量値）と実測値の散布図が図３である。検量線回帰式はこの実測値と予測値（定量値）の相関が最大となるように導出された回帰式である。 By using the spectral data of the wavelength region used for analysis and the quantified TAG content for PLS regression analysis, a factor relating to the TAG content can be determined to obtain a regression equation. The factor related to the TAG content refers to a virtual variable having a high correlation with the change in the TAG content inherent in the spectral data. Using the regression coefficient of this factor, a regression formula for predicting the TAG content is created from the spectrum data. In near-infrared spectroscopy, after preparing a regression equation, the measurement accuracy of the created regression equation is evaluated using a sample (evaluation sample) other than the calibration curve creation sample. The measurement accuracy of the regression equation is evaluated by the standard deviation or average value of the residual (measurement error) between the actual measurement value of the evaluation sample and the analysis value based on the predicted value calculated by the created regression equation. A scatter diagram of the predicted value (quantitative value) and the actual measurement value is shown in FIG. The calibration curve regression equation is a regression equation derived so that the correlation between the actual measurement value and the predicted value (quantitative value) is maximized.
〔ＮＩＲ分光法を利用したＴＡＧの定量方法〕
本発明の玄米に含まれる、ＮＩＲ分光法を利用したＴＡＧの定量方法（以下、「本発明の定量方法」という。）は、上記本発明の回帰式を得る方法により得られた回帰式と、当該回帰式の作成に使用した波長領域における被験玄米のＮＩＲスペクトルデータとを用いる定量方法である。具体的には、まず、被験玄米のＮＩＲスペクトルを測定する。ＮＩＲスペクトルを測定する波長範囲は、回帰式の作成に使用した波長領域を含むものであればよく、１０００〜２５００ｎｍであることが好ましい。
[Method of quantifying TAG using NIR spectroscopy]
The method for quantifying TAG using NIR spectroscopy contained in the brown rice of the present invention (hereinafter referred to as “quantitative method of the present invention”) is a regression equation obtained by the method of obtaining the regression equation of the present invention, It is a quantification method using the NIR spectrum data of the test brown rice in the wavelength region used for preparation of the regression equation. Specifically, first, the NIR spectrum of the test brown rice is measured. The wavelength range for measuring the NIR spectrum is not particularly limited as long as it includes the wavelength region used for creating the regression equation, and is preferably 1000 to 2500 nm.
被験玄米のＮＩＲスペクトルデータは、上記本発明の回帰式を得る方法においてＮＩＲスペクトルデータを取得する場合と同様にして取得することができる。すなわち、市販の近赤外分光分析計を用いてＮＩＲスペクトルを測定しスペクトルデータ、好ましくはフーリエ変換スペクトルデータを取得する。スペクトルデータまたはフーリエ変換スペクトルデータは、原スペクトルデータでもよいが、原スペクトルデータを加工したものを使用することが好ましい。データ加工の方法としては、例えば、一次微分、二次微分、三次微分などの多次微分（Ｄｅｒｉｖａｔｉｖｅ）、平滑化（Ｓｍｏｏｔｈｉｎｇ）、スペクトルの減算（Ｓｕｂｔｒａｃｔｉｏｎ）、正規化（Ｎｏｒｍａｌｉｚｅ）、ＭＳＣ補正（Ｍｕｌｔｉｐｌｉｃａｔｉｖｅ Ｓｃａｔｔｅｒ Ｃｏｒｒｅｃｔｉｏｎ）、ＳＮＶ補正（Ｓｔａｎｄａｒｄ Ｎｏｒｍａｌ Ｖａｒｉａｔｅ Ｃｏｒｒｅｃｔｉｏｎ）などが挙げられる。 The NIR spectrum data of the test brown rice can be obtained in the same manner as in the case of obtaining the NIR spectrum data in the method for obtaining the regression equation of the present invention. That is, a NIR spectrum is measured using a commercially available near-infrared spectrometer and spectrum data, preferably Fourier transform spectrum data, is acquired. The spectrum data or the Fourier transform spectrum data may be the original spectrum data, but it is preferable to use the original spectrum data. Examples of data processing methods include, for example, first-order differentiation, second-order differentiation, third-order differentiation and the like (Derivative), smoothing (Smoothing), spectrum subtraction (Normalization), normalization (Normalization), MSC correction (Multiplicative). Scatter correction), SNV correction (standard normal variant correction), and the like.
被験玄米のＮＩＲスペクトルデータ（好ましくは前項の加工を施されたもの）を解析して、先に得られた回帰式を適用することにより、被験玄米のＴＡＧ含量の予測値を算出する。この予測値がＮＩＲによる被験玄米のＴＡＧの定量値となる。ここでのスペクトルデータの解析と予測値（定量値）の算出は市販のソフトウェアを使用して行うことができる。使用するソフトウェアは例えばＴｈｅ Ｕｎｓｃｒａｍｂｌｅｒ(株式会社カモソフトウェアジャパン)、ＯＰＵＳ（ブルカー・オプティクス社）、ＮＩＲＣａｌ（日本ビュッヒ社）、Ｐｉｒｏｕｅｔｔｅ（ジーエルサイエンス社）などが例示できる。 The predicted value of the TAG content of the test brown rice is calculated by analyzing the NIR spectrum data of the test brown rice (preferably those subjected to the processing of the previous item) and applying the regression equation obtained earlier. This predicted value becomes the quantitative value of the TAG of the test brown rice by NIR. The analysis of the spectrum data here and the calculation of the predicted value (quantitative value) can be performed using commercially available software. Examples of the software to be used include The Unscrambler (Camo Software Japan Co., Ltd.), OPUS (Bruker Optics), NIRCal (Nippon Büch), Pirouette (GL Science).
本発明の定量方法において、被験玄米の水分含量に基づいて被験玄米のＴＡＧ含量を補正する工程をさらに含むことが好ましい。被験玄米の水分含量は、玄米の水分含量を定量するための回帰式と、当該回帰式の作成に使用した波長領域における被験玄米のＮＩＲスペクトルデータとを用いることにより定量することができる。 The quantitative method of the present invention preferably further includes a step of correcting the TAG content of the test brown rice based on the water content of the test brown rice. The moisture content of the test brown rice can be quantified by using a regression equation for quantifying the moisture content of the brown rice and the NIR spectrum data of the test brown rice in the wavelength region used to create the regression equation.
玄米の水分含量を定量するための回帰式は、以下の（ａ）〜（ｃ）の工程により得ることができる。
（ａ）玄米のＮＩＲスペクトルを測定する工程
（ｂ）当該玄米の水分含量を測定する工程
（ｃ）ＮＩＲスペクトルを測定した波長範囲の全部または一部の波長領域で得られたスペクトルデータと、測定した水分含量とをＰＬＳ回帰分析により解析し、水分含量と関係する因子を決定する工程
すなわち、上記本発明の回帰式を得る方法において、玄米の水分含量を定量し、ＰＬＳ回帰分析に供することで玄米の水分含量を定量するための回帰式を得ることができる。
玄米の水分含量を定量する方法は特に限定されないが、例えば、乾燥減量法などを好適に用いることができる。
The regression equation for quantifying the moisture content of brown rice can be obtained by the following steps (a) to (c).
(A) Step of measuring the NIR spectrum of brown rice (b) Step of measuring the water content of the brown rice (c) Spectral data obtained in all or part of the wavelength range in which the NIR spectrum was measured, and measurement Step of analyzing the water content by PLS regression analysis and determining a factor related to the water content. That is, in the method of obtaining the regression equation of the present invention, the water content of brown rice is quantified and subjected to PLS regression analysis. A regression equation for quantifying the moisture content of brown rice can be obtained.
The method for quantifying the moisture content of brown rice is not particularly limited, but for example, a loss on drying method can be suitably used.
〔酵素による比色定量法を用いる玄米中のＴＡＧ含量の定量方法〕
本発明者らは、本発明の回帰式を得る方法および本発明の定量方法を完成させる過程において、玄米中のＴＡＧ含量を酵素による比色定量法を用いて定量する方法を新たに考案した。前述のとおり、酵素による比色定量法は、測定が簡便かつ迅速に行えることから、血中ＴＡＧ測定用の試薬キットに多く採用されているが、これらのキットは水系試料を適用対象としており、油脂試料や植物体試料には適用されていない。
本発明者らは、玄米中のＴＡＧ含量を酵素による比色定量法を用いて測定するために試行錯誤を繰り返した結果、比色定量法の油脂試料への適用が難しい原因が、油脂試料をそのまま酵素液と混合した場合は油脂試料が酵素液中に均質に分散しないために反応効率が悪くなることにあることに至った。また、油脂試料を溶媒に溶解して添加する場合には溶媒が酵素活性に影響を及ぼす結果、再現性が得られないことに至った。そこで、油脂試料を酵素液中に均質に分散させること、溶媒を用いない、あるいは酵素活性に影響を及ぼさない溶媒を用いることにより、酵素による比色定量法を用いて玄米中のＴＡＧ含量を定量する方法（以下「玄米ＴＡＧ比色定量法」という。）を完成させた。
[Method for quantifying TAG content in brown rice using enzyme colorimetric method]
The present inventors have devised a new method for quantifying the TAG content in brown rice using an enzyme colorimetric method in the process of obtaining the regression equation of the present invention and in the process of completing the quantification method of the present invention. As described above, the enzyme colorimetric determination method can be easily and quickly measured, and thus is widely used in reagent kits for blood TAG measurement, but these kits are applied to aqueous samples, It is not applied to oil and fat samples and plant samples.
As a result of repeated trial and error in order to measure the TAG content in brown rice using an enzyme colorimetric method, the inventors have found that the reason why the colorimetric method is difficult to apply to an oil sample is When mixed with the enzyme solution as it is, the oil and fat sample is not uniformly dispersed in the enzyme solution, leading to poor reaction efficiency. In addition, when an oil or fat sample is added after being dissolved in a solvent, reproducibility cannot be obtained as a result of the solvent affecting the enzyme activity. Therefore, the TAG content in brown rice is quantified using an enzymatic colorimetric method by uniformly dispersing an oil and fat sample in the enzyme solution, using no solvent, or using a solvent that does not affect the enzyme activity. (Hereinafter referred to as “brown rice TAG colorimetric method”) was completed.
玄米ＴＡＧ比色定量法に用いられる酵素反応としては、例えば以下に示す酵素反応が挙げられる。以下の酵素反応において、第一反応（Ｒ１反応）を行わずに（遊離グリセロールを除去せずに）第二反応（Ｒ２反応）のみを行ってもよい。玄米ＴＡＧ比色定量法に用いることができる市販のキットとしては、例えば、トリグリセライドＥ−テストワコー、ＬタイプワコーＴＧ・Ｍ（和光純薬）、デタミナーＬ ＴＧＩＩ（協和メデックス）、アクアオートカイノスＴＧＩＩ、ＴＧ−ＥＮカイノス（カイノス）、血清トリグリセリド測定キット（シグマ）、Ｔｒｉｇｌｙｃｅｒｉｄｅ Ｑｕａｎｉｔｉｆｉｃａｔｉｏｎ Ｋｉｔ（Ｂｉｏ Ｖｉｓｉｏｎ）、セロテックＴＧ−Ｌ、セロテックＴＧ−Ｓ、セロテックＴＧ−ＣＬ（セロテック）、ネスコートＴＧ−ＧＮ（アズウェル）、セラテスタムＴＧ（日立化成）、コレステストＴＧ（積水メディカル）、富士ドライケムスライドＴＧ−ＩＩＩ（富士フイルム）、ネスコートＶＬＩＩ ＴＧ（アルフレッサファーマ）、スポットケムＩＩトリグリセライド（アークレイ）などが挙げられる。 Examples of the enzyme reaction used in the brown rice TAG colorimetric determination method include the following enzyme reactions. In the following enzyme reaction, only the second reaction (R2 reaction) may be performed without performing the first reaction (R1 reaction) (without removing free glycerol). Examples of commercially available kits that can be used for the brown rice TAG colorimetric determination method include triglyceride E-test Wako, L type Wako TG / M (Wako Pure Chemicals), Determiner L TGII (Kyowa Medex), Aqua Autokinos TGII, TG-EN kainos (Kinos), serum triglyceride measurement kit (Sigma), Triglyceride Quantification Kit (BioVision), Serotech TG-L, Serotech TG-S, Serotech TG-CL (Serotech), Nescoat TG-GN (Aswell) Seratestum TG (Hitachi Chemical), Colles Test TG (Sekisui Medical), Fuji Dry Chem Slide TG-III (Fuji Film), Nescourt VLII TG (Alfresa Pharma), Spot Chem II Glycerides (Akureyri), and the like.
玄米ＴＡＧ比色定量法では、玄米を乾燥処理し、乾燥玄米から粗脂質を抽出して乾固させ、この粗脂質を酵素反応に供してＴＡＧを定量することが好ましい。玄米の乾燥方法は、凍結乾燥、常圧下での加熱、減圧条件下での加熱、乾燥助剤の利用等が挙げられるが、凍結乾燥がより好ましい。乾燥玄米からの粗脂質の抽出は、公知の方法を用いて行うことができる。例えば、乾燥玄米から適当な溶媒を用いて抽出する方法が挙げられる。そのような抽出法としては、ソックスレー抽出法、クロロホルム−メタノール抽出法、酸分解法などが挙げられ、なかでもソックスレー抽出法が好ましい。乾燥玄米は、そのまま溶媒で抽出してもよいが、圧迫により押しつぶす、あるいはすり鉢などですりつぶす、あるいは何らかの器具を用いて粉砕するなどして抽出効率を上げることが好ましく、圧迫により押しつぶすのが回収率と利便性の点でより好ましい。 In the brown rice TAG colorimetric determination method, it is preferable to dry the brown rice, extract the crude lipid from the dried brown rice and dry it, and subject this crude lipid to an enzyme reaction to quantify TAG. Examples of the method for drying brown rice include lyophilization, heating under normal pressure, heating under reduced pressure conditions, use of a drying aid, and the like, and lyophilization is more preferable. Extraction of the crude lipid from the dried brown rice can be performed using a known method. For example, the method of extracting from dry brown rice using a suitable solvent is mentioned. Examples of such an extraction method include a Soxhlet extraction method, a chloroform-methanol extraction method, an acid decomposition method, and the like, and the Soxhlet extraction method is particularly preferable. Dried brown rice may be extracted with a solvent as it is, but it is preferable to increase the extraction efficiency by crushing by pressing, crushing with a mortar, or crushing with some instrument, and crushing by pressing is the recovery rate And more preferable in terms of convenience.
抽出に用いる溶媒は、ＴＡＧを溶解できるものならばどんなものでもよく、例えばエーテル類、低級アルコール類、アセトン、アセトニトリル、クロロホルム、ヘキサン、シクロヘキサン、ヘプタン、酢酸エチル、ジオキサン、テトラヒドロフラン、トリフルオロ酢酸、ベンゼン、ジメチルスルホキシド（ＤＭＳＯ）、石油エーテル、これらの混合溶媒（均一に混合されたもの）などを好適に用いることができる。これらの中でも、ＴＡＧの溶解性に優れ、低毒性かつ低沸点で加温により容易に留去できる点で、エーテル類、ヘキサン、アセトンがより好ましく、ジエチルエーテルがさらに好ましい。脂質の抽出条件については特に限定されないが、加温部の温度は抽出溶媒の沸点を超える温度に設定することが好ましい。例えばジエチルエーテルを抽出溶媒に用いる場合、３０〜１５０℃の範囲に設定することが好ましく、６０〜１２０℃がさらに好ましい。抽出時間は特に限定されないが、１〜２４時間が好ましく、１〜１２時間がより好ましく、１〜６時間がさらに好ましい。試料の乾燥方法は特に限定されないが、空気酸化を避けるため減圧条件下または不活性ガス雰囲気下で加温することにより、溶媒を除去することが好ましく、減圧条件下での加温がさらに好ましい。減圧条件下での加温により乾燥を行う場合は、試料のロスを避けるため、遠心エバポレーターを使用することが好ましい。加温条件は特に限定されないが、温度は２０〜１００℃が好ましく、３０〜８０℃がより好ましい。また乾燥時間は０．５〜２４時間が好ましく、６〜２４時間がより好ましく、１２〜２４時間がさらに好ましい。減圧条件は特に限定されないが、２０ｈＰａ以下が好ましく、１０ｈＰａ以下がより好ましい。本発明者らは、粉砕した凍結乾燥玄米からエーテルを用いてソックスレー抽出し、ロータリーエバポレーターで乾固させ、クロロホルムに溶解して遠心エバポレーターにより濃縮乾固することで粗脂質を得ているが（実施例１参照）、これに限定されるものではない。 Any solvent can be used for extraction as long as it can dissolve TAG. For example, ethers, lower alcohols, acetone, acetonitrile, chloroform, hexane, cyclohexane, heptane, ethyl acetate, dioxane, tetrahydrofuran, trifluoroacetic acid, benzene , Dimethyl sulfoxide (DMSO), petroleum ether, a mixed solvent thereof (mixed uniformly), and the like can be preferably used. Among these, ethers, hexane, and acetone are more preferable, and diethyl ether is more preferable in that it has excellent TAG solubility, low toxicity, low boiling point, and can be easily distilled off by heating. The lipid extraction conditions are not particularly limited, but the temperature of the heating part is preferably set to a temperature exceeding the boiling point of the extraction solvent. For example, when diethyl ether is used as the extraction solvent, it is preferably set in the range of 30 to 150 ° C, more preferably 60 to 120 ° C. Although extraction time is not specifically limited, 1 to 24 hours are preferable, 1 to 12 hours are more preferable, and 1 to 6 hours are more preferable. The method for drying the sample is not particularly limited, but it is preferable to remove the solvent by heating under reduced pressure or under an inert gas atmosphere to avoid air oxidation, and heating under reduced pressure is more preferable. When drying by heating under reduced pressure conditions, it is preferable to use a centrifugal evaporator to avoid sample loss. Although heating conditions are not specifically limited, 20-100 degreeC is preferable and 30-80 degreeC is more preferable. The drying time is preferably 0.5 to 24 hours, more preferably 6 to 24 hours, and further preferably 12 to 24 hours. The decompression conditions are not particularly limited, but are preferably 20 hPa or less, and more preferably 10 hPa or less. The present inventors obtained crude lipids by extraction from ground lyophilized brown rice using Soxhlet extraction using ether, drying to dryness with a rotary evaporator, dissolving in chloroform and concentrating to dryness with a centrifugal evaporator. However, the present invention is not limited to this.
酵素反応に供する試料として、乾固させた状態の粗脂質をそのまま試料としてもよく、溶媒に溶解させた粗脂質を試料としてもよい。以下、乾固させた状態の粗脂質をそのまま試料とする場合を「直接法」と称し、溶媒に溶解させた粗脂質を試料とする場合を「溶媒法」と称する。 As a sample to be subjected to the enzyme reaction, the crude lipid in a dried state may be used as it is, or a crude lipid dissolved in a solvent may be used as a sample. Hereinafter, the case where the crude lipid in a dried state is used as a sample is referred to as “direct method”, and the case where the crude lipid dissolved in a solvent is used as a sample is referred to as “solvent method”.
直接法では、乾固した粗脂質が入った反応容器に酵素を含む試薬を直接添加し、均質な溶液とするための処理を行う。この際、既に乾固した粗脂質を反応容器に投入してもよいが、粗脂質を適当な溶媒に溶解して所望の分量を反応容器に入れた後に反応容器中で乾固させるのがより好ましい。この時用いる溶媒についてはＴＡＧを溶解できるものならばどのようなものでもよく、例えばエーテル類、低級アルコール類、アセトン、アセトニトリル、クロロホルム、ヘキサン、シクロヘキサン、ヘプタン、酢酸エチル、ジオキサン、テトラヒドロフラン、トリフルオロ酢酸、ベンゼン、ジメチルスルホキシド（ＤＭＳＯ）、石油エーテル、これらの混合溶媒（均一に混合されたもの）などを好適に用いることができる。反応容器中で粗脂質を乾固させる際には、空気酸化を避けるため減圧条件下または不活性ガス雰囲気下で加温することにより乾固することが好ましく、減圧条件下で乾固することがさらに好ましい。減圧条件での加温により乾燥を行う場合は、試料のロスを避けるため、遠心エバポレーターを使用することが好ましい。加温条件は特に限定されないが、温度は２０〜１００℃が好ましく、３０〜８０℃がより好ましい。また乾燥時間は特に限定されないが、１０〜１２０分が好ましく、３０〜８０分がさらに好ましい。減圧条件は特に限定されないが、２０ｈＰａ以下が好ましく、１０ｈＰａ以下がより好ましい。 In the direct method, a reagent containing an enzyme is directly added to a reaction vessel containing dried and dried crude lipid, and a process for obtaining a homogeneous solution is performed. At this time, the crude lipid already dried may be charged into the reaction vessel, but it is more preferable to dissolve the crude lipid in an appropriate solvent and put the desired amount in the reaction vessel, and then dry it in the reaction vessel. preferable. Any solvent may be used as long as it can dissolve TAG. For example, ethers, lower alcohols, acetone, acetonitrile, chloroform, hexane, cyclohexane, heptane, ethyl acetate, dioxane, tetrahydrofuran, trifluoroacetic acid. , Benzene, dimethyl sulfoxide (DMSO), petroleum ether, a mixed solvent thereof (mixed uniformly), and the like can be preferably used. When the crude lipid is dried in the reaction vessel, it is preferable to dry by heating under reduced pressure or inert gas atmosphere in order to avoid air oxidation, and it is preferable to dry under reduced pressure. Further preferred. When drying by heating under reduced pressure conditions, it is preferable to use a centrifugal evaporator to avoid sample loss. Although heating conditions are not specifically limited, 20-100 degreeC is preferable and 30-80 degreeC is more preferable. Moreover, although drying time is not specifically limited, 10 to 120 minutes are preferable and 30 to 80 minutes are more preferable. The decompression conditions are not particularly limited, but are preferably 20 hPa or less, and more preferably 10 hPa or less.
均質な溶液とするための処理としては、例えば超音波処理、転倒混和、攪拌処理、界面活性剤の添加などが挙げられ、これらの方法を必要に応じて併用することが好ましい。中でも、適宜攪拌処理を行いながら、超音波処理を行うことが好ましい。超音波処理を行う場合、温度は特に限定されないが、３０〜６０℃の範囲に加温して行うことが好ましい。超音波処理の時間は特に限定されないが、５秒〜３０分が好ましく、１０秒〜１分がより好ましく、２０秒〜４０秒がさらに好ましい。攪拌処理の方法は特に限定されないが、ボルテックスミキサーを利用して５〜６０秒程度行うのが好ましく、反応容器の天地を入れ替えながら行うのがさらに好ましい。また、これらの均質化処理の後には５〜６０秒間の遠心分離（スピンダウン）により、容器の蓋面や側面に付着した液を容器の底面に落とすことが好ましい。反応容器として用いる容器の材質は使用する溶剤に耐性を持つものであればよく、例えば耐久性を重視してガラス製容器、あるいは利便性を重視してプラスチック製容器などを適宜選択して用いることが好ましい。これらの反応容器は１０ｈＰａ以下の減圧条件での使用に適したものが好ましく、１ｈＰａ以下の減圧条件での使用に適したものがより好ましい。また２００Ｇ以上の遠心分離に適したものが好ましく、２０００Ｇ以上の遠心分離に適したものがより好ましい。反応容器の形状については、蓋が可能で対称な形状のものであれば特に問わないが、底の構造が尖形なものよりは平底ないし丸底のものの方が好ましい。反応容器の容量については、特に限定されないが、０．５ｍＬ〜１０ｍＬが好ましく、１ｍＬ〜５ｍＬがさらに好ましい。酵素を含む試薬が複数ある場合は、酵素を含む試薬を添加するごとに、均質な溶液とするための処理を行うことが好ましい。測定操作は試薬キットの取扱説明書に従って行えばよいが、反応容器の容量に合わせて、試料量と試薬量を調整するのが好ましい。具体的には、最終的な反応溶液の体積が反応容器の容量の１／２〜１／４０であることが好ましく、１／４〜１／２０であることがさらに好ましい。 Examples of the treatment for obtaining a homogeneous solution include ultrasonic treatment, inversion mixing, stirring treatment, addition of a surfactant and the like, and these methods are preferably used in combination as necessary. Among these, it is preferable to perform ultrasonic treatment while appropriately performing stirring treatment. When performing ultrasonic treatment, the temperature is not particularly limited, but it is preferably performed by heating in the range of 30 to 60 ° C. The sonication time is not particularly limited, but is preferably 5 seconds to 30 minutes, more preferably 10 seconds to 1 minute, and further preferably 20 seconds to 40 seconds. The method for the stirring treatment is not particularly limited, but it is preferably performed for about 5 to 60 seconds using a vortex mixer, and more preferably performed while changing the top and bottom of the reaction vessel. Moreover, after these homogenization processes, it is preferable to drop the liquid adhering to the cover surface or side surface of the container to the bottom surface of the container by centrifugation (spin down) for 5 to 60 seconds. The material of the container used as the reaction container only needs to be resistant to the solvent to be used. For example, a glass container that emphasizes durability, or a plastic container that emphasizes convenience is appropriately selected and used. Is preferred. These reaction vessels are preferably suitable for use under reduced pressure conditions of 10 hPa or less, and more preferably suitable for use under reduced pressure conditions of 1 hPa or less. In addition, those suitable for centrifugation of 200 G or more are preferable, and those suitable for centrifugation of 2000 G or more are more preferable. The shape of the reaction vessel is not particularly limited as long as it can be covered and has a symmetrical shape, but a flat bottom or a round bottom is preferable to a pointed bottom structure. Although it does not specifically limit about the capacity | capacitance of reaction container, 0.5 mL-10 mL are preferable and 1 mL-5 mL are more preferable. When there are a plurality of reagents containing an enzyme, it is preferable to perform a treatment for obtaining a homogeneous solution each time the reagent containing an enzyme is added. The measurement operation may be performed according to the instruction manual of the reagent kit, but it is preferable to adjust the sample amount and the reagent amount in accordance with the capacity of the reaction container. Specifically, the volume of the final reaction solution is preferably 1/2 to 1/40 of the capacity of the reaction vessel, and more preferably 1/4 to 1/20.
溶媒法では、溶媒に溶解した粗脂質を試料とする。ここでの溶媒としては、ＴＡＧを溶解できるものならどの様なものでもよく、例えばエーテル類、低級アルコール類、アセトン、アセトニトリル、クロロホルム、ヘキサン、シクロヘキサン、ヘプタン、酢酸エチル、ジオキサン、テトラヒドロフラン、トリフルオロ酢酸、ベンゼン、ジメチルスルホキシド（ＤＭＳＯ）、石油エーテルおよびこれらの混合溶媒（ただし均一に混合されたもの）などを好適に用いることができるが、酵素活性への影響が小さい低級アルコール類およびヘキサンが好ましく、ｔ−ＢｔＯＨとヘキサンが再現性や精度の点からさらに好ましい。溶液状の試料と酵素を含む試薬とを混合し、均質な溶液とするための処理を行う。均質な溶液とするための処理としては特に限定されないが、例えば超音波処理、転倒混和、攪拌処理、界面活性剤の添加などが挙げられ、これらの方法を必要に応じて併用することが好ましい。中でも、適宜攪拌処理を行いながら、超音波処理を行うことが好ましい。超音波処理を行う場合、温度は特に限定されないが、３０〜８０℃の範囲に加温して行うことが好ましい。超音波処理の時間は特に限定されないが、５秒〜３０分が好ましく、１０秒〜１分がより好ましく、２０秒〜４０秒がさらに好ましい。攪拌処理の方法は特に限定されないが、ボルテックスミキサーを利用して１０秒〜１分程度行うのが好ましく、反応容器の天地を入れ替えながら行うのがさらに好ましい。また、これらの均質化処理の後には５〜６０秒間の遠心分離（スピンダウン）により、容器の蓋面や側面に付着した液を容器の底面に落とすことが好ましい。反応容器として用いる容器の材質は使用する溶剤に耐性を持つものであればよく、例えば耐久性を重視してガラス製容器、あるいは利便性を重視してプラスチック製容器などを適宜選択して用いることが好ましい。これらの反応容器は２００Ｇ以上の遠心分離に適したものが好ましく、２０００Ｇ以上の遠心分離に適したものがより好ましい。反応容器の形状については、蓋が可能で対称な形状のものであれば特に問わないが、底の構造が尖形なものよりは平底ないし丸底のものの方が好ましい。反応容器の大きさについては特に限定されないが、０．５〜１０ｍＬが好ましく、１〜５ｍＬがさらに好ましい。酵素を含む試薬が複数ある場合は、酵素を含む試薬を添加するごとに、均質な溶液とするための処理を行うことが好ましい。測定操作は試薬キットの取扱説明書に従って行えばよいが、反応容器の容量に合わせて、試料量と試薬量を調整するのが好ましい。具体的には、最終的な反応溶液の体積が反応容器の容量の１／２〜１／４０であることが好ましく、１／４〜１／２０であることがさらに好ましい。 In the solvent method, a crude lipid dissolved in a solvent is used as a sample. The solvent here may be any solvent that can dissolve TAG, such as ethers, lower alcohols, acetone, acetonitrile, chloroform, hexane, cyclohexane, heptane, ethyl acetate, dioxane, tetrahydrofuran, trifluoroacetic acid. , Benzene, dimethyl sulfoxide (DMSO), petroleum ether, and mixed solvents thereof (but uniformly mixed) can be suitably used, but lower alcohols and hexane having a small influence on enzyme activity are preferable, t-BtOH and hexane are more preferable from the viewpoint of reproducibility and accuracy. A solution-like sample and an enzyme-containing reagent are mixed to perform a process for obtaining a homogeneous solution. Although it does not specifically limit as a process for setting it as a homogeneous solution, For example, an ultrasonic process, inversion mixing, stirring process, addition of surfactant, etc. are mentioned, It is preferable to use these methods together as needed. Among these, it is preferable to perform ultrasonic treatment while appropriately performing stirring treatment. When performing ultrasonic treatment, the temperature is not particularly limited, but it is preferably performed by heating in the range of 30 to 80 ° C. The sonication time is not particularly limited, but is preferably 5 seconds to 30 minutes, more preferably 10 seconds to 1 minute, and further preferably 20 seconds to 40 seconds. The method of the stirring treatment is not particularly limited, but is preferably performed for about 10 seconds to 1 minute using a vortex mixer, and more preferably performed while changing the top and bottom of the reaction vessel. Moreover, after these homogenization processes, it is preferable to drop the liquid adhering to the cover surface or side surface of the container to the bottom surface of the container by centrifugation (spin down) for 5 to 60 seconds. The material of the container used as the reaction container only needs to be resistant to the solvent to be used. For example, a glass container that emphasizes durability, or a plastic container that emphasizes convenience is appropriately selected and used. Is preferred. These reaction vessels are preferably those suitable for centrifugation at 200 G or more, and more preferably those suitable for centrifugation at 2000 G or more. The shape of the reaction vessel is not particularly limited as long as it can be covered and has a symmetrical shape, but a flat bottom or a round bottom is preferable to a pointed bottom structure. Although it does not specifically limit about the magnitude | size of a reaction container, 0.5-10 mL is preferable and 1-5 mL is more preferable. When there are a plurality of reagents containing an enzyme, it is preferable to perform a treatment for obtaining a homogeneous solution each time the reagent containing an enzyme is added. The measurement operation may be performed according to the instruction manual of the reagent kit, but it is preferable to adjust the sample amount and the reagent amount in accordance with the capacity of the reaction container. Specifically, the volume of the final reaction solution is preferably 1/2 to 1/40 of the capacity of the reaction vessel, and more preferably 1/4 to 1/20.
直接法および溶媒法のいずれの場合も、標準試料（例えば、段階希釈した米油を含む試料）を同時に測定して検量線を作成し、この検量線を用いて被験試料のＴＡＧ含量を定量することができる。標準試料は、ＴＡＧを主成分とし、その濃度が既知であるものならば特に限定されない。例えばトリオレイン、トリリノレインなどのＴＡＧの市販試薬、あるいは市販のサラダ油などが利用可能であるが、ＴＡＧ濃度が既知の植物油であることが好ましく、米油であることがさらに好ましい。この米油は原油でも、製油後のサラダ油でも、精製途中の油であっても構わない。また、米油の原料となる米の品種については特に限定されない。
本玄米ＴＡＧ比色定量法は、直接法および溶媒法のいずれを用いても、玄米一粒中のＴＡＧ含量を、簡便かつ再現性よく定量できる点で非常に有用である。
In both the direct method and the solvent method, a standard sample (for example, a sample containing serially diluted rice oil) is simultaneously measured to prepare a calibration curve, and the TAG content of the test sample is quantified using this calibration curve. be able to. The standard sample is not particularly limited as long as TAG is the main component and the concentration is known. For example, commercially available TAG reagents such as triolein and trilinolein, or commercially available salad oils can be used, but vegetable oils having a known TAG concentration are preferable, and rice oil is more preferable. This rice oil may be crude oil, refined salad oil, or refined oil. Moreover, it is not specifically limited about the rice varieties used as the raw material of rice oil.
This brown rice TAG colorimetric determination method is very useful in that the TAG content in a brown rice grain can be determined easily and with good reproducibility using either the direct method or the solvent method.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.
〔実施例１：玄米一粒中のＴＡＧ含量の定量〕
１．粗脂質の抽出
(1) 玄米一粒を秤量した後凍結乾燥処理し、ろ紙に包んで、薬さじにて押しつぶした。
(2) 押しつぶした試料をろ紙ごと抽出用円筒ガラスフィルターに入れ、セミ・ミクロ脂肪抽出器セットを用いて、エーテル２５ｍＬを用いて、加温機を１００℃に設定し、６時間ソックスレー抽出を行った。
(3) 抽出液をナシ型フラスコに移し、ロータリーエバポレーターにより乾固した。
(4) ナシ型フラスコの粗脂質をクロロホルム１．２ｍＬに溶解し、脂質のクロロホルム溶液を２ｍＬバイアル瓶に移し、遠心エバポレーターにより濃縮乾固することで玄米一粒の粗脂質を得た。
[Example 1: Determination of TAG content in one grain of brown rice]
1. Extraction of crude lipid
(1) One grain of brown rice was weighed, freeze-dried, wrapped in filter paper, and crushed with a spoon.
(2) Put the crushed sample together with the filter paper into a cylindrical glass filter for extraction, use a semi-micro fat extractor set, use 25 mL of ether, set the heater to 100 ° C, and perform Soxhlet extraction for 6 hours It was.
(3) The extract was transferred to a pear-shaped flask and dried with a rotary evaporator.
(4) The crude lipid of the pear-shaped flask was dissolved in 1.2 mL of chloroform, and the chloroform solution of the lipid was transferred to a 2 mL vial, and concentrated and dried by a centrifugal evaporator to obtain one grain of crude rice.
２．ＴＡＧ含量の測定
（２−ａ）直接法によるＴＡＧ含量の測定
(1)米サラダ油（築野食品工業（株））のクロロホルム溶液（０〜２．０ｍｇ／ｍＬ）を調製し、標準溶液とした。
(2) 上記１．(4)で濃縮乾固した玄米粗脂質に５００μＬのクロロホルムを加えて攪拌し、次いで超音波処理（５０℃、３０秒）を行って完全に溶解させ、試料溶液とした。
(3) 反応容器に標準溶液または試料溶液を２５μＬずつ分注し、遠心エバポレーターを用いて乾固した。
(4) ＴＡＧ含量の測定には、デタミナーＬ ＴＧＩＩ（協和メデックス）を用いた。デタミナーＬ ＴＧＩＩは以下の成分を持つ二つの試薬（試薬Ｒ１、Ｒ２）から構成される。
［試薬Ｒ１］
・グリセロールキナーゼ
・グリセロール−３−リン酸オキシダーゼ
・カタラーゼ
・Ｎ−（３、５−ジメトキシフェニル）−Ｎ’−サクシニルエチレンジアミンＮａ塩（ＤＯＳＥ）
・アデノシン−５’−三リン酸二ナトリウム（ＡＴＰ）
［試薬Ｒ２］
・リポプロテインリパーゼ
・パーオキシダーゼ
・４−アミノアンチピリン（４−ＡＡ）
(5) 乾固した粗脂質が入った反応容器に上記試薬Ｒ１を２４０μＬ加えて混和し、素早く超音波処理（３７℃、３０秒）を行って均質な溶液とし、３７℃の水浴にて５分間静置した。
(6) 続いて、上記試薬Ｒ２を８０μＬ加えて混和し、素早く超音波処理（３７℃、３０秒）を行って均質な溶液とし、３７℃の水浴にて１０分間静置した。
(7) 反応溶液を遠心分離し、上清をシリンジフィルターでろ過した後、分光光度計にて５８５ｎｍの吸光度を測定した。
(8) 各濃度の標準溶液の吸光度から検量線（図１（Ａ））を作成し、本検量線を用いて玄米一粒中のＴＡＧ含量を米サラダ油換算で定量した。
2. Measurement of TAG content (2-a) Measurement of TAG content by direct method
(1) A chloroform solution (0 to 2.0 mg / mL) of rice salad oil (Tsukino Food Industry Co., Ltd.) was prepared and used as a standard solution.
(2) Above 1. 500 μL of chloroform was added to the brown rice crude lipid concentrated and dried in (4) and stirred, and then subjected to ultrasonic treatment (50 ° C., 30 seconds) to completely dissolve it to obtain a sample solution.
(3) 25 μL of the standard solution or sample solution was dispensed into the reaction vessel and dried using a centrifugal evaporator.
(4) Determiner L TGII (Kyowa Medex) was used to measure the TAG content. The determiner L TGII is composed of two reagents (reagents R1, R2) having the following components.
[Reagent R1]
・ Glycerol kinase ・ Glycerol-3-phosphate oxidase
Catalase N- (3,5-dimethoxyphenyl) -N′-succinylethylenediamine Na salt (DOSE)
Adenosine-5′-triphosphate disodium (ATP)
[Reagent R2]
・ Lipoprotein lipase ・ Peroxidase ・ 4-Aminoantipyrine (4-AA)
(5) Add 240 μL of the above-mentioned reagent R1 to the reaction vessel containing the dried crude lipid and mix, rapidly sonicate (37 ° C., 30 seconds) to make a homogeneous solution, and add 5% in a 37 ° C. water bath. Let sit for a minute.
(6) Subsequently, 80 μL of the reagent R2 was added and mixed. The mixture was rapidly sonicated (37 ° C., 30 seconds) to obtain a homogeneous solution, and left in a 37 ° C. water bath for 10 minutes.
(7) The reaction solution was centrifuged, the supernatant was filtered with a syringe filter, and the absorbance at 585 nm was measured with a spectrophotometer.
(8) A calibration curve (FIG. 1 (A)) was prepared from the absorbance of the standard solution at each concentration, and the TAG content in one grain of brown rice was quantified in terms of rice salad oil using this calibration curve.
（２−ｂ）溶媒法（ヘキサン）によるＴＡＧ含量の測定
(1) 米サラダ油のヘキサン溶液（０〜５．０ｍｇ／ｍＬ）を調製し、標準溶液とした。
(2) 上記１．(4)で濃縮乾固した玄米粗脂質に５００μＬのヘキサンを加えて攪拌し、次いで超音波処理（５０℃、３０秒）を行って完全に溶解させ、試料溶液とした。
(3) デタミナーＬ ＴＧＩＩ（協和メデックス）を用いて、ＴＡＧ含量を測定した。
(4)反応容器に上記試薬Ｒ１を２４０μＬ入れ、試料溶液１０μＬを加えて混和し、素早く超音波処理（３７℃、３０秒）を行って均質な溶液とし、３７℃の水浴にて５分間静置した。
(5) 続いて、上記試薬Ｒ２を８０μＬ加えて混和し、素早く超音波処理（３７℃、３０秒）を行って均質な溶液とし、３７℃の水浴にて１０分間静置した。
(6) 反応溶液を遠心分離し、上清をシリンジフィルターでろ過した後、分光光度計にて５８５ｎｍの吸光度を測定した。
(7) 各濃度の標準溶液の吸光度から検量線（図１（Ｂ））を作成し、本検量線を用いて玄米一粒中のＴＡＧ含量を米サラダ油換算で定量した。
(2-b) Measurement of TAG content by solvent method (hexane)
(1) A hexane solution (0-5.0 mg / mL) of rice salad oil was prepared and used as a standard solution.
(2) Above 1. 500 μL of hexane was added to the brown rice crude lipid concentrated and dried in (4) and stirred, and then subjected to ultrasonic treatment (50 ° C., 30 seconds) to completely dissolve it to obtain a sample solution.
(3) TAG content was measured using Determiner L TGII (Kyowa Medex).
(4) Add 240 μL of the above-mentioned reagent R1 to the reaction vessel, add 10 μL of the sample solution, mix, quickly sonicate (37 ° C., 30 seconds) to make a homogeneous solution, and leave it in a 37 ° C. water bath for 5 minutes. I put it.
(5) Subsequently, 80 μL of the above-mentioned reagent R2 was added and mixed. The mixture was rapidly sonicated (37 ° C., 30 seconds) to obtain a homogeneous solution, and left in a 37 ° C. water bath for 10 minutes.
(6) The reaction solution was centrifuged, the supernatant was filtered with a syringe filter, and the absorbance at 585 nm was measured with a spectrophotometer.
(7) A calibration curve (FIG. 1 (B)) was prepared from the absorbance of the standard solution at each concentration, and the TAG content in one grain of brown rice was quantified in terms of rice salad oil using this calibration curve.
（２−ｃ）溶媒法（ｔ-ＢｔＯＨ）によるＴＡＧ含量の測定
上記（２−ｂ）のヘキサンをｔ-ＢｔＯＨに変えて同様の処理を行い、検量線（図１（Ｃ））を作成し、本検量線を用いて玄米一粒中のＴＡＧ含量を定量した。
(2-c) Measurement of TAG content by solvent method (t-BtOH) The hexane of the above (2-b) was changed to t-BtOH, the same treatment was performed, and a calibration curve (Fig. 1 (C)) was prepared. The TAG content in one grain of brown rice was quantified using this calibration curve.
（比較例１）溶媒法（クロロホルム）によるＴＡＧ含量の測定
上記（２−ｂ）のヘキサンをクロロホルムに変えて同様の処理を行い、検量線（図１（Ｄ））を作成し、本検量線を用いて玄米一粒中のＴＡＧ含量を定量した。
(Comparative Example 1) Measurement of TAG content by solvent method (chloroform) The same treatment was performed by changing hexane in (2-b) above to chloroform, and a calibration curve (Fig. 1 (D)) was prepared. Was used to quantify the TAG content in one grain of brown rice.
図１（Ａ）〜（Ｄ）において、縦軸は５８５ｎｍの吸光度、横軸はＴＡＧ量（米サラダ油の量、μｇ）を表す。ここでのＴＡＧ量は標品の米サラダ油の重量で示している。図１（Ａ）、（Ｂ）、（Ｃ）からわかるように、直接法、溶媒法（ヘキサン）および溶媒法（ｔ-ＢｔＯＨ）で得られたＴＡＧの検量線の傾きに有意な差はなかった。一方、図１（Ｄ）から明らかなように、比較例１の溶媒法（クロロホルム）で得られたＴＡＧの検量線の傾きは顕著に小さくなった。この原因はクロロホルムが試薬中の酵素の活性を低下させているためと推測された。
表１に、直接法、溶媒法（ヘキサン）、溶媒法（ｔ-ＢｔＯＨ）および溶媒法（クロロホルム、比較例１）によりキヌヒカリ玄米一粒中のＴＡＧ含量の測定結果を示した。表１から、直接法、溶媒法（ヘキサン）および溶媒法（ｔ-ＢｔＯＨ）の定量結果には有意な差はなかったが、比較例１の溶媒法（クロロホルム）では測定誤差が大きくなっていることが示された。これらの結果から、クロロホルムのように試薬中の酵素の活性に影響を及ぼす溶媒は、本発明に適していないことが明らかとなった。
1A to 1D, the vertical axis represents absorbance at 585 nm, and the horizontal axis represents the amount of TAG (the amount of rice salad oil, μg). The amount of TAG here is indicated by the weight of the standard rice salad oil. As can be seen from FIGS. 1 (A), (B), and (C), there is no significant difference in the slope of the calibration curve of TAG obtained by the direct method, solvent method (hexane), and solvent method (t-BtOH). It was. On the other hand, as is clear from FIG. 1D, the slope of the calibration curve of TAG obtained by the solvent method (chloroform) of Comparative Example 1 was significantly reduced. This was presumed to be due to the fact that chloroform reduced the activity of the enzyme in the reagent.
Table 1 shows the measurement results of the TAG content in one grain of Kinuhikari brown rice by the direct method, the solvent method (hexane), the solvent method (t-BtOH) and the solvent method (chloroform, Comparative Example 1). From Table 1, there was no significant difference in the quantitative results of the direct method, the solvent method (hexane), and the solvent method (t-BtOH), but the measurement error was large in the solvent method (chloroform) of Comparative Example 1. It was shown that. From these results, it became clear that a solvent that affects the activity of the enzyme in the reagent, such as chloroform, is not suitable for the present invention.
〔実施例２：水分含量検量線回帰式の作成〕
(1) １９品種のイネの玄米を各１５粒用意した。
(2) 水分含量の範囲を広げるため、乾燥器を用いて４０℃にて０〜３６０分間乾燥した。
(3) 分析装置としてＦＴ型近赤外分析計ＭＰＡ（ブルカー・オプティクス社）を用いた。玄米試料のＮＩＲ拡散反射光を１０００ｎｍから２５００ｎｍ（１００００〜４０００ｃｍ−１）の範囲で測定した。
(4) スペクトルを取得した各玄米の水分含量を、乾燥減量法（１３５℃、１５時間）で測定した。
(5) 得られた原スペクトルの前処理（ＳＮＶ補正）を行い、前処理スペクトル（１３３３〜１８３６ｎｍ）の波長データを説明変数とし、測定した水分含量を目的変数とするＰＬＳ回帰分析により、玄米一粒の水分検量モデルを作成した。ＮＩＲによる予測値（定量値）と化学分析値（実測値）の相関を図２に示した。ここでのＮＩＲによる予測値（％、定量値）は水分の検量線回帰式とＮＩＲスペクトルのデータから測定装置によって導出される計算値である。
[Example 2: Preparation of moisture content calibration curve regression equation]
(1) Fifteen rice varieties of 19 varieties of rice were prepared.
(2) In order to widen the range of moisture content, drying was performed at 40 ° C. for 0 to 360 minutes using a dryer.
(3) An FT type near-infrared analyzer MPA (Bruker Optics) was used as an analyzer. The NIR diffuse reflected light of the brown rice sample was measured in the range of 1000 nm to 2500 nm (10000 to 4000 cm −1 ).
(4) The moisture content of each brown rice from which spectra were acquired was measured by the loss on drying method (135 ° C., 15 hours).
(5) Pretreatment (SNV correction) of the obtained original spectrum was performed, and PLS regression analysis using the wavelength data of the preprocessed spectrum (1333-1386 nm) as an explanatory variable and the measured water content as a target variable A moisture calibration model of the grain was created. The correlation between the predicted value (quantitative value) by NIR and the chemical analysis value (actual measurement value) is shown in FIG. The predicted value (%, quantitative value) by NIR here is a calculated value derived by the measuring device from the moisture calibration curve regression equation and NIR spectrum data.
〔実施例３：ＴＡＧ含量検量線回帰式の作成〕
(1) ２０品種のイネの玄米を各三粒用意した。
(2) 実施例２の(3)と同様に、玄米試料のＮＩＲ拡散反射光を測定した。
(3) スペクトルを取得した玄米について、実施例１のＴＡＧ含量の測定法（直接法）を用いて、各玄米一粒中のＴＡＧ含量を定量した。
(4) 得られた原スペクトルの前処理（二次微分処理）を行い、種々の波長範囲の前処理スペクトルの波長データを説明変数とし、測定したＴＡＧ含量を目的変数とするＰＬＳ回帰分析により、玄米一粒のＴＡＧ検量モデルを作成した。
[Example 3: Preparation of TAG content calibration curve regression formula]
(1) Three kinds of 20 rice varieties of brown rice were prepared.
(2) NIR diffuse reflected light of the brown rice sample was measured in the same manner as in Example 2 (3).
(3) About the brown rice which acquired the spectrum, using the measuring method (direct method) of the TAG content of Example 1, the TAG content in each grain of brown rice was quantified.
(4) Pre-processing (second-order differential processing) of the obtained original spectrum, using wavelength data of pre-processed spectra in various wavelength ranges as explanatory variables, and PLS regression analysis using the measured TAG content as an objective variable, A TAG calibration model of one grain of brown rice was created.
得られた検量モデルの全条件の決定係数Ｒ２を表２に示した。波長は用いた波長範囲を示しており、最小波長と最大波長で区切られた１区間（条件Ｎｏ．５、８以外）ないし２区間（条件Ｎｏ．５、８）のデータを用いて解析を行った。表２からわかるように各条件でＮＩＲ予測値（定量値）と化学分析値には高い相関関係が見られた。中でも、条件Ｎｏ．１、２、３、４、５、６、７）のような、ＴＡＧに関連するＡ群（１７２５±１０、１７６４±１０、２３１７±１０ｎｍ）の領域を２つ以上含み、かつ、ＴＡＧ以外の成分に関連するＢ群（１８６０±１０、１９６１±１０、２２７６±１０ｎｍ）の領域を１つ以上含む検量線にて相関係数が高くなり、最も高い相関（Ｒ２＝０．９２６）が得られた波長範囲は、条件Ｎｏ．１の１６３８．８〜２３５４．８ｎｍであった。 Table 2 shows the determination coefficient R2 of all conditions of the calibration model obtained. The wavelength indicates the wavelength range used, and analysis is performed using data from one section (other than condition Nos. 5 and 8) or two sections (conditions No. 5 and 8) divided by the minimum wavelength and the maximum wavelength. It was. As can be seen from Table 2, there was a high correlation between the predicted NIR value (quantitative value) and the chemical analysis value under each condition. Among these, condition no. Including two or more regions of group A (1725 ± 10, 1764 ± 10, 2317 ± 10 nm) related to TAG, such as 1, 2, 3, 4, 5, 6, 7), and other than TAG The correlation coefficient increases in a calibration curve including one or more regions of group B (1860 ± 10, 1961 ± 10, 2276 ± 10 nm) related to the component, and the highest correlation (R2 = 0.926) is obtained. The wavelength range indicated is condition No. 1 of 1638.8 to 2354.8 nm.
至適条件（条件Ｎｏ．１）および最大範囲の１０００〜２５００ｎｍ（条件Ｎｏ．６）におけるＮＩＲによる予測値（定量値）と化学分析値（実測値）の相関を、それぞれ図３（Ａ）および（Ｂ）に示した。なお、ここでの実測値は、
実測値＝玄米粗脂質の中のＴＡＧ重量（ｍｇ）／玄米一粒の重量（ｍｇ）×１００（％）
として算出している。また、ＮＩＲによる予測値（％、定量値）はＴＡＧの検量線回帰式とＮＩＲスペクトルのデータから測定装置によって導出される計算値である。
The correlation between the predicted value (quantitative value) by NIR and the chemical analysis value (actual measurement value) in the optimum condition (condition No. 1) and the maximum range of 1000 to 2500 nm (condition No. 6) is shown in FIG. Shown in (B). The actual measured value here is
Measured value = TAG weight in brown rice crude lipid (mg) / weight of brown rice grain (mg) × 100 (%)
It is calculated as Moreover, the predicted value (%, quantitative value) by NIR is a calculated value derived by the measuring device from the TAG calibration curve regression equation and NIR spectrum data.
〔実施例４：玄米一粒中のＴＡＧ含量の定量〕
(1) ＦＴ型近赤外分析計ＭＰＡ（ブルカー・オプティクス社）を用いて、玄米試料のＮＩＲ拡散反射光を１０００ｎｍから２５００ｎｍ（１００００〜４０００ｃｍ−１）の範囲で測定した。
(2) 実施例２で作成した水分検量モデルに(1)で測定したＮＩＲスペクトルデータを当てはめて、水分含量を定量した。
(3) 実施例３で作成したＴＡＧ検量モデルに(1)で測定したＮＩＲスペクトルデータを当てはめて、ＴＡＧ含量を定量した。
(4) (2)で定量した水分含量および(3)で定量したＴＡＧ含量に基づいて、玄米試料の乾物重量当たりのＴＡＧ含量を得た。
[Example 4: Determination of TAG content in one grain of brown rice]
(1) Using a FT type near-infrared analyzer MPA (Bruker Optics), NIR diffuse reflected light of a brown rice sample was measured in the range of 1000 nm to 2500 nm (10000 to 4000 cm −1 ).
(2) The NIR spectrum data measured in (1) was applied to the moisture calibration model created in Example 2 to quantify the moisture content.
(3) The TAG content was quantified by applying the NIR spectral data measured in (1) to the TAG calibration model created in Example 3.
(4) Based on the water content determined in (2) and the TAG content determined in (3), the TAG content per dry matter weight of the brown rice sample was obtained.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。 The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.
Claims (13)
（１）玄米の近赤外光スペクトルを測定する工程
（２）当該玄米のトリアシルグリセロール含量を定量する工程
（３）近赤外光スペクトルを測定した波長範囲の全部または一部の波長領域で得られたスペクトルデータと、定量したトリアシルグリセロール含量とをＰＬＳ（partial least squares）回帰分析により解析し、トリアシルグリセロール含量と関係する因子を決定する工程 A method for obtaining a regression equation for quantifying triacylglycerol contained in brown rice, comprising the following steps (1) to (3).
(1) The step of measuring the near-infrared light spectrum of brown rice (2) The step of quantifying the triacylglycerol content of the brown rice (3) In all or part of the wavelength range in which the near-infrared light spectrum was measured A step of analyzing the obtained spectral data and the quantified triacylglycerol content by PLS (partial least squares) regression analysis to determine factors related to the triacylglycerol content
（ａ）玄米の近赤外光スペクトルを測定する工程
（ｂ）当該玄米の水分含量を測定する工程
（ｃ）近赤外光スペクトルを測定した波長範囲の全部または一部の波長領域で得られたスペクトルデータと、測定した水分含量とをＰＬＳ回帰分析により解析し、水分含量と関係する因子を決定する工程 Use the regression formula obtained by the following steps (a) to (c) and the near-infrared light spectrum data of the test brown rice in the wavelength region used for the creation of the regression formula for the moisture content of the test brown rice. The quantification method according to claim 12, which is obtained by:
(A) The step of measuring the near-infrared light spectrum of brown rice (b) The step of measuring the water content of the brown rice (c) Obtained in all or part of the wavelength range in which the near-infrared light spectrum was measured Analyzing the measured spectral data and the measured water content by PLS regression analysis to determine factors related to the water content
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| CN103411913A (en) * | 2013-05-22 | 2013-11-27 | 温州大学 | Infrared spectroscopy PLS (Partial Least Square) modeling method based on genetic algorithm self-adaptive selection segment point |
| JP2015135323A (en) * | 2013-12-20 | 2015-07-27 | 築野食品工業株式会社 | Quantifying method of free fatty acid in vegetable oil or in raw material thereof using near infrared spectroscopy method |
| CN109239105A (en) * | 2018-09-19 | 2019-01-18 | 天津大学 | It is a kind of for identifying the millimeter wave method of glyceryl monooleate phase |
| CN109374548A (en) * | 2018-11-14 | 2019-02-22 | 深圳职业技术学院 | A method of quickly measuring nutritional ingredient in rice using near-infrared |
| CN109799207A (en) * | 2019-01-15 | 2019-05-24 | 上海交通大学 | The quantitative detecting method of talcum powder is mixed in the root of Dahurain angelica based on near-infrared spectrum analysis |
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| CN103411913A (en) * | 2013-05-22 | 2013-11-27 | 温州大学 | Infrared spectroscopy PLS (Partial Least Square) modeling method based on genetic algorithm self-adaptive selection segment point |
| JP2015135323A (en) * | 2013-12-20 | 2015-07-27 | 築野食品工業株式会社 | Quantifying method of free fatty acid in vegetable oil or in raw material thereof using near infrared spectroscopy method |
| CN109239105A (en) * | 2018-09-19 | 2019-01-18 | 天津大学 | It is a kind of for identifying the millimeter wave method of glyceryl monooleate phase |
| CN109239105B (en) * | 2018-09-19 | 2020-12-25 | 天津大学 | Millimeter wave method for identifying phase of glycerol monooleate |
| CN109374548A (en) * | 2018-11-14 | 2019-02-22 | 深圳职业技术学院 | A method of quickly measuring nutritional ingredient in rice using near-infrared |
| CN109799207A (en) * | 2019-01-15 | 2019-05-24 | 上海交通大学 | The quantitative detecting method of talcum powder is mixed in the root of Dahurain angelica based on near-infrared spectrum analysis |
| CN110044842A (en) * | 2019-05-22 | 2019-07-23 | 江南大学 | A kind of method of the odd sub- seed moisture content of quick measurement |
| CN111272695A (en) * | 2020-02-26 | 2020-06-12 | 北京市农林科学院 | Nondestructive testing method for content of antitumor component precursor 3-methylindolyl thioglycoside in broccoli powdery raw material |
| CN115201144A (en) * | 2022-09-14 | 2022-10-18 | 武汉工程大学 | Quantitative detection method, system and medium for amino acid and protein of rapeseed |
| CN115201144B (en) * | 2022-09-14 | 2022-12-09 | 武汉工程大学 | Quantitative detection method, system and medium for amino acid and protein of rapeseed |
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