JP4336914B2 - Dietary fiber determination method and kit - Google Patents

Description

【００６６】
に当てはめることによって、小麦粉中の食物繊維の定量を行った。結果を表１に示す。
【００６７】
実施例１ 本発明の方法による小麦粉中の食物繊維の定量
市販の小麦粉（商品名：日清の小麦粉フラワー、日清製粉株式会社製）を、１ｇずつ（0.1mg単位まで）500mL容のトールビーカーに２点量り採った。その値をＷ1及びＷ2とし、その差は20mg以内とした。
【００６８】
採取した各試料に0.05mol/L MES/Tris緩衝液(pH6.3)を50mL加え、耐酸耐熱性α−アミラーゼ（クライスターゼＹ（大和化成株式会社製）を精製して結晶化したもの）水溶液（13,400 LJ/mL）0.1mLを加えてよく振り混ぜ、アルミ箔で覆い、5分間隔でビーカーを振り混ぜつつ、沸騰水浴中で30分間加熱した。
【００６９】
60℃まで冷却後、中性プロテアーゼ（サーモリシン(大和化成株式会社製)）水溶液（443,000 PU/mL）0.1mL及びアミログルコシダーゼ（コクゲンＧ（大和化成株式会社製）を精製したもの)水溶液（438 JGU/mL）3mLを加えて、アルミ箔で覆い、60℃の水浴中で振り混ぜながら30分間反応させた。
【００７０】
得られた酵素処理液の４倍量（約213mL）の95vol％エタノール（あらかじめ60℃に加温しておく）を加え、室温で１時間放置して食物繊維を沈殿させた。あらかじめ、るつぼ型ガラス濾過器（孔径2G2、（株）相互理化学硝子製作所製）にセライト（食物繊維定量用試薬キットTDF-100、シグマ社製）0.5ｇを加えて130℃で１時間加熱後デシケーター中で放冷し、恒量を0.1mg単位まで定めて、使用直前に78vol％エタノールを用いて均一なセライト層を作成しておき、これを用いて吸引濾過を行った。ガラス濾過器上に捕集された残渣を78vol％エタノール（20mL×3回）、95vol％エタノール（10mL×2回）及びアセトン(10mL×2回)で順次洗浄した。残渣をガラス濾過器ごと105℃で一晩乾燥し、シリカゲルを入れたデシケーター中で約１時間放冷した後秤量した。ここで、一方の蛋白質測定用残渣の重量をＲ1、もう一方の灰分測定用残渣の重量をＲ2とした。
【００７１】
残渣Ｒ1の全量をセライトと共にかき取り、比較試験例１と同様にケルダール法によって窒素含量を測定し、得られた窒素含量に係数6.25を乗じて残渣中の蛋白質の質量Ｐ1とした。
【００７２】
また、残渣Ｒ2を濾過器ごと525℃で5時間灰化処理を行い、シリカゲルを入れたデシケーター中で約１時間放冷した後秤量し、残渣中の灰分Ａ1とした。
【００７３】
ブランクについても試料を含まない以外は上記操作と同様に行い、るつぼ型濾過器に捕集されたブランク残渣の各重量ｒ1及びｒ2、ブランク残渣中の蛋白質量ｐ1、及びブランク残渣中の灰分ａ1を求めた。
【００７４】
これらの値を上記数１に当てはめることによって、小麦粉中の食物繊維の定量を行った。結果を表１に示す。
【００７５】
【表１】

【００７６】
実施例１の方法における変動係数は比較試験例１のそれに比べ低い値となり、精度が改良されていることが判る。
【００７７】
比較試験例２ 従来法による大麦中の食物繊維の定量
市販の大麦（押麦）（商品名：押麦、（株）はくばく製）を粉砕し、0.35mm目（42メッシュ）のふるい通過物を試料として、1gずつ（0.1mg単位まで）500mL容のトールビーカーに２点量り採り、比較試験例１と同様の方法によって大麦中の食物繊維の定量を行った。結果を表２に示す。
【００７８】
実施例２ 本発明による大麦中の食物繊維の定量
市販の大麦（押麦）（商品名：押麦、（株）はくばく製）を粉砕し、0.35mm目（42メッシュ）のふるい通過物を試料として、1gずつ（0.1mg単位まで）500mL容のトールビーカーに２点量り採り、実施例１と同様の方法によって大麦中の食物繊維の定量を行った。結果を表２に示す。
【００７９】
【表２】

【００８０】
実施例２の方法における変動係数は比較試験例２のそれに比べ低い値となり、精度が非常に改良されていることが判る。
【００８１】
実施例３ 本発明の方法による大豆中の食物繊維の定量
市販の大豆（きな粉）（商品名：きな粉、日本流通産業株式会社製）を、1gずつ（0.1mg単位まで）500mL容のトールビーカーに２点量り採り、実施例１と同様の方法によって大豆中の食物繊維の定量を行った。結果を表３に示す。
【００８２】
【表３】

【００８３】
実施例４ 本発明の方法によるにんじん中の食物繊維の定量
市販のにんじんを粗く粉砕し凍結乾燥後、更に均一に粉砕し、0.35mm目（42メッシュ）のふるい通過物を試料として、1gずつ（0.1mg単位まで）500mL容のトールビーカーに２点量り採り、実施例３と同様の方法によってにんじん中の食物繊維の定量を行った。結果を表４に示す。但し、乾燥試料から乾燥前試料への換算は下記式にて行った。
【００８４】
【数２】

【００８５】
【表４】

【００８６】
実施例５ 本発明の方法によるキャベツ中の食物繊維の定量
市販のキャベツを粗く粉砕し凍結乾燥後、更に均一に粉砕し、0.35mm目（42メッシュ）のふるい通過物を試料として、1gずつ（0.1mg単位まで）500mL容のトールビーカーに２点量り採り、実施例３と同様の方法によってキャベツ中の食物繊維の定量を行った。結果を表５に示す。但し、乾燥試料から乾燥前試料への換算は上記数２に記載の式にあてはめて行った。
【００８７】
【表５】

【００８８】
【図面の簡単な説明】
【図１】本発明の好ましい方法を示す図である。
【図２】酵素処理液を用いて食物繊維を定量する方法を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a dietary fiber quantitative method and a dietary fiber quantitative kit.
[0002]
[Prior art]
As a method for quantifying dietary fiber, the method proposed by Prosky et al. Was adopted as an official method by the Association of Official Analytical Chemists (AOAC Method 985.29, 991.42, 991.43, 993.19) and included the United States and Japan. Adopted by standards in many countries. This method is also adopted by the American Association of Cereal Chemists (AACC) (AACC Method 32-05, 32-07, 32-21). In this method, components other than dietary fiber in the sample are decomposed by enzymatic treatment, and the total amount of dietary fiber is calculated by subtracting the weight of non-digestible protein, ash, and reagent blank from the decomposition residue obtained by ethanol precipitation. It is based on that.
[0003]
However, in this method, the complexity of the enzyme treatment operation is regarded as a problem. Specifically, in the enzyme treatment, three kinds of enzymes, thermostable α-amylase, protease and amyloglucosidase, are used. In order to carry out the enzyme reaction, the sample solution is adjusted to a different pH. In addition, since the sample solution is processed at different temperatures, the enzyme treatment has three stages, and further, the complexity of pH adjustment is required at each stage.
[0004]
As an improvement of this method, there is also known a method of decomposing starch by heat treatment using a pressure cooker instead of thermostable α-amylase (Yano et al., Journal of Japanese Society of Nutrition and Food Science, vol.43, No. 3, pp. 209 to 214, 1990), but workability has not been improved. In addition, in AOAC Method 991.43 and AACC Method 32-07, MES / Tris buffer (pH 8.2) is used, and the pH adjustment between heat-resistant α-amylase treatment and protease treatment is omitted. There has not been a significant improvement in workability (ie, it is necessary to adjust the pH again when amyloglucosidase is allowed to act), especially when handling fruits, it has a high acidity to adjust the pH to a weak alkali. In addition, there are problems in quantifying substances such as pectin that are easily degraded under weak alkaline conditions.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a dietary fiber quantification method and kit with improved complexity and processing time so that dietary fiber can be quantified more easily.
[0006]
[Means for Solving the Problems]
As the conditions for a simpler quantitative method, intensive studies were conducted by (1) improving the complexity of the quantitative operation and (2) completing the entire reaction in a short time.
[0007]
As a result, the present inventors have found that the above object can be achieved for the first time by using a neutral protease instead of the alkaline protease used so far, and have completed the present invention.
[0008]
That is, the present inventors can use three types of enzymes in the same pH range by using neutral protease instead of alkaline protease, and further, treatment with neutral protease and amyloglucosidase. It is also possible to perform the above process simultaneously, and the quantitative operation can be simplified. Furthermore, since the quantitative operation can be simplified, the total amount of the enzyme treatment solution can be reduced, so that the amount of ethanol required for the precipitation operation is reduced, and in addition, the time required for filtration is shortened. It was.
[0009]
That is, the present invention provides a method for quantifying dietary fiber using thermostable α-amylase, protease and amyloglucosidase, and provides a method for quantifying dietary fiber in which the protease is a neutral protease, and a quantification kit according to the method.
[0010]
Specifically, (1) heat-resistant α-amylase is allowed to act on the sample,
(2) It provides a quantitative method in which neutral protease and amyloglucosidase are allowed to act simultaneously.
[0011]
Moreover, as a kit, this invention provides the kit for dietary fiber quantification containing thermostable alpha-amylase, neutral protease, and amyloglucosidase.
[0012]
Furthermore, when acid-stable heat-resistant α-amylase is used as the heat-resistant α-amylase, even if the sample is acidic, the adverse effect on the enzyme due to a slight shift to acidity due to the influence of the sample is reduced, and pH adjustment is possible. In addition to being easy, adverse effects on the enzyme reaction due to pH drop of the buffer during high-temperature treatment were also suppressed, and dietary fiber in the sample could be measured more accurately.
[0013]
As a result, the reaction operation is simplified, the processing solution is reduced, the reaction time and the work time are shortened, and the workability is greatly improved.
[0014]
These quantification methods can quantitate dietary fiber in various samples.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
The neutral protease used in the present invention is not particularly limited as long as it is a known neutral protease. For example, an endo-type neutral protease that can be used at about 60 ° C. can be mentioned. The origin of the enzyme is not particularly a problem, and examples thereof include those derived from animals and microorganisms (bacteria and the like, for example, Bacillus thermoproteolyticus and the like). Further, it may be modified by genetic engineering or manufactured by chemical modification. For example, a modified neutral protease including its production method is disclosed in BIO INDUSTRY vol. 15, No. 5, p39-46 (1998).
[0016]
More specifically, commercially available Samoa C160 (manufactured by Yamato Kasei Co., Ltd.), protease TD “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.), pronase (manufactured by Boehringer Mannheim Co., Ltd.), thermolysin (manufactured by Yamato Kasei Co., Ltd., Bacillus) neutral end-type metalloprotease produced by thermoproteolyticus Rokko).
[0017]
However, the enzyme is preferably a neutral protease containing no contaminating enzyme. More preferably, thermolysin (manufactured by Daiwa Kasei Co., Ltd.), which is a protease crystal product, is preferable.
[0018]
Any known thermostable α-amylase can be used in the present invention. For example, a thermostable α-amylase that can be sufficiently used in an end-type boiling bath (95 ° C.) can be mentioned. Examples of the origin of the enzyme include those derived from microorganisms, and examples include thermostable α-amylase derived from Bacillus subtilis, Bacillus stearothermophilus and Bacillus licheniformis. In addition, it may be modified by genetic engineering or chemically modified.
[0019]
More specifically, as commercially available products, for example, Christase T (manufactured by Daiwa Kasei Co., Ltd.), Termamyl 120L (manufactured by Novo Nordisk), A-3306 (manufactured by Sigma), E-BLAAM (manufactured by Megazyme) Etc.
[0020]
However, thermostable α-amylase free from contaminating enzymes is preferred.
[0021]
Particularly preferred is an acid-resistant and heat-resistant α-amylase that also has acid resistance. Examples of the origin of the enzyme include those described above. Examples of commercially available acid-resistant and heat-resistant α-amylase include Christase Y (manufactured by Daiwa Kasei Co., Ltd.), GC521 (manufactured by Genencor Corp.), Termamyl LC ( Novo Nordisk).
[0022]
However, acid-resistant and heat-resistant α-amylase without contaminating enzymes is preferred.
[0023]
Any known amyloglucosidase can be used in the present invention. The enzyme is a glucosidase that acts on both α1,4 and α1,6 glucosyl bonds, and is not particularly limited as long as it can be used at 60 ° C. The origin is not particularly limited as long as it is normally used, but those derived from mold (for example, Aspergillus) are industrially used. In addition, it may be modified by genetic engineering or chemically modified.
[0024]
Examples of commercially available enzymes include Kokugen G (manufactured by Daiwa Kasei Co., Ltd.), A-9913 (manufactured by Sigma), E-AMGDF (manufactured by Megazyme), and amyloglucosidase (manufactured by Boehringer Mannheim).
[0025]
However, amyloglucosidase without contaminating enzymes is preferred.
[0026]
Each of the enzymes is preferably as pure as possible. The amount and concentration of each enzyme used are appropriately selected according to the type and amount of the sample, the titer of the enzyme, the reaction pH, the reaction temperature, and the like, and are not particularly limited. The concentration of each component in the final enzyme-treated solution to which the enzyme is added can be used within the following range.
[0027]
Thermostable α-amylase: 17,400-122,000 JLU / L
(Acid-resistant heat-resistant α-amylase: 13,000-54,000 LJ / L)
Neutral protease: 443,000-1,770,000 PU / L
Amiloglucosidase: 13,100-52,600 JGU / L.
[0028]
Preferred ranges of the above components are as follows.
[0029]
Thermostable α-amylase: 31,400-66,900 JLU / L
(Acid-resistant heat-resistant α-amylase: 24,000-30,000 LJ / L)
Neutral protease: 797,000-974,000 PU / L
Amiloglucosidase: 23,700-28,900 JGU / L.
[0030]
The meaning of each unit mentioned as the concentration is as follows.
JLU: Based on the bacterial α-amylase activity measurement method defined in JIS K7001-1990.
LJ: According to the bacterial α-amylase activity measurement method defined in the old JIS K7001-1972.
PU: Measured by bacterial protease activity measurement method. When protease is allowed to act on dairy casein at 35 ° C, a non-protein Folin reagent coloring material equivalent to 1 µg of tyrosin in the first minute of the reaction. The amount of enzyme that brings about an increase of 1 PU.
JGU: According to the glucoamylase activity measurement method defined in JIS K7001-1990.
[0031]
The sample used in the method of the present invention is not particularly limited, and various foods such as various cereals, vegetables and fruits can be applied. The preparation of the sample is not particularly limited as long as it is a conventional method, and it is preferable to prepare according to the characteristics of the sample. The amount of specimen used for quantification can be appropriately selected.
[0032]
In the quantification method of the present invention, it is essential to use the above three kinds of enzymes. The enzyme treatment using the above enzyme may be performed in the order of (a) thermostable α-amylase, (b) neutral protease and (c) amyloglucosidase, or (a) after treatment with thermostable α-amylase. (B) You may process by neutral protease and amyloglucosidase.
[0033]
The present invention will be described in detail with reference to FIG.
[0034]
In FIG. 1, first, an appropriate buffer solution such as phosphate buffer solution, acetate buffer solution, MES / Tris buffer solution or the like is added to the prepared sample to adjust pH. Preferably, 0.05 to 0.1 mol / L MES / Tris buffer is good. A preferred pH range is about 5.5 to 6.5.
[0035]
Next, after heat-stable α-amylase is added to the sample suspension and mixed, the mixture is reacted in a boiling bath (90-100 ° C.) for a predetermined time to decompose starch in the sample. The time required is about 15 to 30 minutes.
[0036]
Subsequently, after cooling the temperature to 60 to 65 ° C., neutral protease and amyloglucosidase are added and reacted for a predetermined time to decompose the protein and dextrin contained in the sample. The required time is about 30 minutes.
[0037]
The obtained enzyme treatment liquid is processed according to a conventional method to calculate total dietary fiber. For example, there are the Prosky method (AOAC Method 985.29) and the Prosky modified method (AOAC Method 991.43). When the Prosky method is followed, the details are as follows (see FIG. 2). When the treatment is performed according to the Prosky method, it is necessary to quantify the amount of non-digestible protein and ash after the enzyme treatment. Therefore, at least two points are taken for one analysis, and the sample is subjected to the above operation at the same time.
[0038]
95 vol% ethanol is added to the enzyme treatment solution to precipitate dietary fiber. The amount added is preferably a sufficient amount for precipitation, and specifically about 4 times the amount of the enzyme treatment solution. The required time is about overnight at room temperature, but it is preferable to use 95 vol% ethanol preheated to about 60 ° C. because the required time can be shortened. In that case, specifically, it precipitates in about 1 hour at room temperature.
[0039]
Next, suction filtration is performed using a crucible type filter (for example, made of glass or the like) in which a uniform celite layer is formed on the surface in advance. The collected residue on the filter is washed (for example with ethanol or acetone) and then dried.
[0040]
The protein in the residue is quantified by scraping the entire amount of the residue collected in one crucible filter together with celite and measuring the nitrogen content in the residue by, for example, the Kjeldahl method. The obtained nitrogen content value is multiplied by a coefficient of 6.25 to obtain the protein mass.
[0041]
The amount of ash in the residue is determined by ashing each other crucible type filter (for example, 525 ° C. ± 5 ° C. for 5 hours), allowed to cool and dry, and then the amount of ash is determined. taking measurement.
[0042]
The above procedure is similarly performed using at least two points for the sample-free system (blank), that is, only the buffer and the enzyme. From the above results, the dietary fiber content in the sample is calculated according to the Prosky method.
[0043]
The measurement principle of the dietary fiber of the present invention is as described above, but the present invention uses a neutral protease in place of the alkaline protease that has been used so far, and as a result, the pH in enzyme treatment, which has been complicated until now, is as follows. It was found that the adjustment was simplified for the first time, the enzyme treatment with neutral protease and the enzyme treatment with amyloglucosidase could be performed simultaneously, and the time required for the enzyme reaction could be shortened.
[0044]
The method of the present invention can be preferably carried out using a kit, and the present invention also includes such a kit.
[0045]
The kit of the present invention basically contains thermostable α-amylase, neutral protease and amyloglucosidase. The heat resistant α-amylase is preferably an acid resistant heat resistant α-amylase.
[0046]
In addition to the above components, a salt such as NaCl or CaCl ₂ can be contained in the reagent containing each enzyme in order to improve the thermal stability of the enzyme.
[0047]
Moreover, in order to stabilize said each enzyme, you may mix | blend sugar, sugar alcohol, glycerol, etc. in the reagent containing each enzyme.
[0048]
Each enzyme included in the kit may be prepared by adding a buffer solution or water at the time of use in a powder (lyophilized product) state, but preferably in a solution state. Water or a buffer solution to be added may be included in the kit as a reagent.
[0049]
A reagent containing celite may be used as a reagent in the kit.
[0050]
In addition, reagents generally used in the kit may be included in the kit of the present invention.
[0051]
Each component in these reagents is contained in an effective amount in the kit, and those skilled in the art can set an effective amount of each component.
[0052]
In the enzyme reaction, as described above, an appropriate buffer such as a phosphate buffer, an acetate buffer, a MES / Tris buffer, or the like is added to the prepared sample to adjust the pH. These may be included as reagents in the kit.
[0053]
【The invention's effect】
According to the present invention, three different enzymes can be allowed to act in the same pH range, and further, the neutral protease treatment and the amyloglucosidase treatment can be performed at the same time. Simplified. Furthermore, since the quantitative operation can be simplified, the total amount of the enzyme treatment solution can be reduced, so that the amount of ethanol required for the precipitation operation is reduced, and in addition, the time required for filtration is shortened. It was.
[0054]
Furthermore, when acid-resistant heat-resistant α-amylase is used as the heat-resistant α-amylase, the reaction can be performed without being affected by a slight acid shift of the reaction solution even when the sample is acidic. It became possible to measure dietary fiber in a sample more accurately.
[0055]
【Example】
Hereinafter, although it demonstrates in detail using an Example, this invention is not restrict | limited to these.
[0056]
Comparative Test Example 1 Determination of dietary fiber in flour by conventional method Commercial flour (trade name: Nisshin Flour Flour, Nisshin Flour Milling Co., Ltd.) 1g each (up to 0.1mg unit) in a 500mL tall beaker Two points were taken. The values were designated as W1 and W2, and the difference was within 20 mg.
[0057]
50 mL of 0.08 mol / L phosphate buffer (pH 6.0) was added to each sample collected, and heat-resistant α-amylase solution (dietary fiber quantification reagent kit TDF-100, manufactured by Sigma) (30,400 JLU / g) 0.1 Add mL, shake well, cover with aluminum foil and heat in a boiling water bath for 30 minutes while shaking the beaker at 5 minute intervals.
[0058]
After cooling to room temperature, about 10 mL of 0.275 mol / L sodium hydroxide solution was added to adjust to pH 7.5. To this was added 0.1 mL of protease solution (dietary fiber quantification reagent kit TDF-100, Sigma) (72,500 PU / mL), covered with aluminum foil, and allowed to react for 30 minutes while shaking in a 60 ° C water bath. .
[0059]
After cooling to room temperature, about 10 mL of 0.325 mol / L hydrochloric acid was added to adjust to pH 4.5. Add 0.3 mL of amyloglucosidase solution (dietary fiber quantification reagent kit TDF-100, manufactured by Sigma) (3,240 JGU / g), cover with aluminum foil, and react for 30 minutes while shaking in an aqueous solution at 60 ° C. I let you.
[0060]
Four volumes (about 280 mL) of 95 vol% ethanol of the obtained enzyme treatment solution was added, and the mixture was allowed to stand overnight at room temperature to precipitate dietary fiber. Add 0.5g of Celite (reagent kit TDF-100 for dietary fiber determination, manufactured by Sigma Co., Ltd.) to a crucible glass filter (pore size 2G2, manufactured by Reciprocal Chemical Glass Co., Ltd.) in advance and heat at 130 ° C for 1 hour, then desiccator The mixture was allowed to cool, and a constant weight was determined up to 0.1 mg, and a uniform celite layer was prepared using 78 vol% ethanol immediately before use, and suction filtration was performed using this. The residue collected on the glass filter was washed successively with 78 vol% ethanol (20 mL × 3 times), 95 vol% ethanol (10 mL × 2 times) and acetone (10 mL × 2 times). The residue was dried overnight at 105 ° C. together with a glass filter, allowed to cool for about 1 hour in a desiccator containing silica gel, and then weighed. Here, the weight of one protein measurement residue was R1, and the other ash measurement residue weight was R2.
[0061]
The entire amount of residue R1 is scraped with celite, the nitrogen content is measured by the Kjeldahl method (Kjeldahl nitrogen decomposition-distillation apparatus (made by Mitamura Riken Kogyo Co., Ltd.)), and the obtained nitrogen content is multiplied by a factor of 6.25. The mass of protein in the residue was P1.
[0062]
The residue R2 was ashed with the filter at 525 ° C. for 5 hours, allowed to cool in a desiccator containing silica gel for about 1 hour, and weighed to obtain the ash content A1 in the residue.
[0063]
The blank was also processed in the same manner as above except that no sample was contained, and the weights r1 and r2 of the blank residue collected in the crucible filter, the protein mass p1 in the blank residue, and the ash content a1 in the blank residue were determined. Asked.
[0064]
These values are expressed by the following formula:
[Expression 1]

[0066]
Quantification of dietary fiber in the flour was performed. The results are shown in Table 1.
[0067]
Example 1 Determination of dietary fiber in wheat flour by the method of the present invention Commercial flour (trade name: Nisshin Flour Flour, Nisshin Flour Milling Co., Ltd.) 1 g each (up to 0.1 mg unit) 500 mL tall beaker 2 points were taken. The values were designated as W1 and W2, and the difference was within 20 mg.
[0068]
50 mL of 0.05 mol / L MES / Tris buffer (pH 6.3) was added to each sample collected, and acid-resistant and heat-resistant α-amylase (Christase Y (manufactured by Daiwa Kasei Co., Ltd.) was purified and crystallized). (13,400 LJ / mL) 0.1 mL was added and shaken well, covered with aluminum foil, and heated in a boiling water bath for 30 minutes while shaking the beaker at intervals of 5 minutes.
[0069]
After cooling to 60 ° C, neutral protease (thermolysin (manufactured by Daiwa Kasei Co., Ltd.) aqueous solution (443,000 PU / mL) 0.1 mL and amyloglucosidase (kokugen G (manufactured by Daiwa Kasei Co., Ltd.) purified) aqueous solution (438 JGU / mL) 3 mL was added, covered with aluminum foil, and reacted for 30 minutes while shaking in a 60 ° C. water bath.
[0070]
Four volumes (about 213 mL) of 95 vol% ethanol (previously heated to 60 ° C.) of the obtained enzyme treatment solution was added, and the mixture was allowed to stand at room temperature for 1 hour to precipitate dietary fiber. Add 0.5g of Celite (reagent kit TDF-100 for dietary fiber determination, manufactured by Sigma Co., Ltd.) to a crucible glass filter (pore size 2G2, manufactured by Reciprocal Chemical Glass Co., Ltd.) in advance and heat at 130 ° C for 1 hour, then desiccator The mixture was allowed to cool, and a constant weight was determined up to 0.1 mg, and a uniform celite layer was prepared using 78 vol% ethanol immediately before use, and suction filtration was performed using this. The residue collected on the glass filter was washed successively with 78 vol% ethanol (20 mL × 3 times), 95 vol% ethanol (10 mL × 2 times) and acetone (10 mL × 2 times). The residue was dried overnight at 105 ° C. together with a glass filter, allowed to cool for about 1 hour in a desiccator containing silica gel, and then weighed. Here, the weight of one protein measurement residue was R1, and the other ash measurement residue weight was R2.
[0071]
The entire amount of the residue R1 was scraped with celite, the nitrogen content was measured by the Kjeldahl method in the same manner as in Comparative Test Example 1, and the obtained nitrogen content was multiplied by a factor of 6.25 to obtain the protein mass P1 in the residue.
[0072]
The residue R2 was ashed with the filter at 525 ° C. for 5 hours, allowed to cool in a desiccator containing silica gel for about 1 hour, and weighed to obtain the ash content A1 in the residue.
[0073]
The blank was also processed in the same manner as above except that no sample was contained, and the weights r1 and r2 of the blank residue collected in the crucible filter, the protein mass p1 in the blank residue, and the ash content a1 in the blank residue were determined. Asked.
[0074]
By applying these values to the above equation 1, dietary fiber in the flour was quantified. The results are shown in Table 1.
[0075]
[Table 1]

[0076]
The coefficient of variation in the method of Example 1 is lower than that of Comparative Test Example 1, indicating that the accuracy is improved.
[0077]
Comparative Test Example 2 Determination of dietary fiber in barley by the conventional method Commercial barley (Oshigi) (trade name: Oshiba, manufactured by Hakuba Co., Ltd.) was pulverized, and the 0.35 mm (42 mesh) sieve passing material was used as a sample. 1 g each (up to 0.1 mg unit) was weighed in a 500 mL tall beaker, and dietary fiber in barley was quantified in the same manner as in Comparative Test Example 1. The results are shown in Table 2.
[0078]
Example 2 Quantification of dietary fiber in barley according to the present invention Commercial barley (Oshigi) (trade name: Oshiba, Co., Ltd., made by Hakubaku) was pulverized, and the 0.35 mm (42 mesh) sieve passing material was used as a sample. Two points were weighed into a 500 mL tall beaker (1 g each) (up to 0.1 mg unit), and dietary fiber in barley was quantified in the same manner as in Example 1. The results are shown in Table 2.
[0079]
[Table 2]

[0080]
The coefficient of variation in the method of Example 2 is lower than that of Comparative Test Example 2, indicating that the accuracy is greatly improved.
[0081]
Example 3 Determination of dietary fiber in soybean by the method of the present invention Commercial soybean (kina flour) (trade name: Kinako flour, manufactured by Japan Ryutsu Sangyo Co., Ltd.) 1 g each (up to 0.1 mg unit) in a 500 mL tall beaker Two points were weighed and dietary fiber in soybean was quantified by the same method as in Example 1. The results are shown in Table 3.
[0082]
[Table 3]

[0083]
Example 4 Determination of dietary fiber in carrot by the method of the present invention A commercially available carrot was coarsely pulverized, freeze-dried, and then uniformly pulverized, and 1 g each using a 0.35 mm (42 mesh) sieve passing material as a sample ( Two points were weighed into a 500 mL tall beaker (up to 0.1 mg unit), and the dietary fiber in the carrot was quantified in the same manner as in Example 3. The results are shown in Table 4. However, the conversion from the dried sample to the pre-dried sample was performed by the following formula.
[0084]
[Expression 2]

[0085]
[Table 4]

[0086]
Example 5 Determination of dietary fiber in cabbage by the method of the present invention A commercially available cabbage was coarsely pulverized, freeze-dried, and then uniformly pulverized, and 1 g each of samples passing through a 0.35 mm (42 mesh) sieve ( Two points were weighed into a 500 mL tall beaker (up to 0.1 mg unit), and the dietary fiber in the cabbage was quantified in the same manner as in Example 3. The results are shown in Table 5. However, the conversion from the dried sample to the pre-dried sample was performed by applying the equation described in Equation 2 above.
[0087]
[Table 5]

[0088]
[Brief description of the drawings]
FIG. 1 illustrates a preferred method of the present invention.
FIG. 2 is a diagram showing a method for quantifying dietary fiber using an enzyme treatment solution.

Claims (6)

In a method for quantifying dietary fiber using thermostable α-amylase, neutral protease and amyloglucosidase , between pH 5.5 and 6.5
(1) A thermostable α-amylase is allowed to act on the sample,
(2) A method for quantifying dietary fiber, wherein neutral protease and amyloglucosidase are allowed to act simultaneously . The method according to claim 1 , wherein the heat-resistant α-amylase is an acid-resistant heat-resistant α-amylase. A kit for carrying out the method according to claim 1 or 2, comprising a thermostable α-amylase, a neutral protease and an amyloglucosidase. A kit for carrying out the method according to claim 1 or 2, comprising a mixture of a neutral protease and amyloglucosidase and a thermostable α-amylase. The kit for dietary fiber determination according to claim 3 or 4, comprising a buffer for adjusting the pH to 5.5 to 6.5. The kit according to any one of claims 3 to 5 , wherein the heat-resistant α-amylase is an acid-resistant heat-resistant α-amylase.

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