WO2009136432A1 - 糖質定量方法および糖質定量キット - Google Patents
糖質定量方法および糖質定量キット Download PDFInfo
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- WO2009136432A1 WO2009136432A1 PCT/JP2008/058440 JP2008058440W WO2009136432A1 WO 2009136432 A1 WO2009136432 A1 WO 2009136432A1 JP 2008058440 W JP2008058440 W JP 2008058440W WO 2009136432 A1 WO2009136432 A1 WO 2009136432A1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- G01N2333/934—Glucoamylase
Definitions
- the present invention relates to a carbohydrate quantification method using a low-molecular-weight carbohydrate digestive enzyme and a carbohydrate quantification kit equipped with a low-molecular-weight carbohydrate digestive enzyme. More specifically, the present invention comprises a method for quantifying oligosaccharides (digestible oligosaccharides, indigestible oligosaccharides) using animal-derived low molecular sugar digestive enzymes, and animal-derived low molecular sugar digestive enzymes. The present invention relates to a quantification kit for oligosaccharides (digestible oligosaccharides, indigestible oligosaccharides).
- Oligosaccharides are those whose number of polymerized saccharides is generally 2 or more and less than 10; those digested by digestive enzymes (digestible oligosaccharides), those that cannot be digested or those that are extremely difficult to digest ( Indigestible oligosaccharides). Many oligosaccharides that have already been developed and have added value to health are those that are not digested by digestive enzymes, or those that are difficult to digest.
- Indigestible oligosaccharides are used in health-oriented foods such as foods for specified health use, but these indigestible oligosaccharides are metabolized by different pathways from digestible carbohydrates such as sucrose and starch. . Indigestible oligosaccharides taken orally escape the digestion by ⁇ -amylase and small intestinal mucosal disaccharide hydrolase and reach the large intestine. Therefore, it undergoes fermentation by resident intestinal bacteria and is metabolized to carbon dioxide gas, hydrogen gas, methane gas, bacterial cell components, etc. in addition to short-chain fatty acids such as acetic acid, propionic acid and n-butyric acid. Among these, short-chain fatty acids are absorbed from the large intestine and used as the host's only energy source. In other words, even carbohydrates that are not digested or absorbed supply energy to the living body through fermentation and absorption in the large intestine.
- indigestible oligosaccharides are used in foods for specified insurance as the components involved, but their quantification is carried out by a method unique to the developer, and a common quantification method has not yet been established. Not. The reason is that indigestible oligosaccharides are not only those that are very difficult to digest in the small intestine, but also those that are partially digested, and because the constituent sugars and structures differ depending on the indigestible oligosaccharides, This is because there is a difference in the stability of each oligosaccharide and the action mechanism of the hydrolase.
- Prosky's enzyme-weight method which is a dietary fiber quantification method, is a method in which digestive carbohydrates and proteins are completely digested by hydrolase and the weight of the remaining undigested material having a large molecular weight is measured.
- Dietary fiber which is a polymer, can be quantified by established quantitative methods such as Prosky's enzyme-gravimetric method, but low molecular weight indigestible oligosaccharides and sugar alcohols cannot be quantified by this enzyme-gravimetric method.
- indigestible oligosaccharides and sugar alcohols have come to be used in various health-oriented foods, and a common quantitative method for these has been studied.
- One of the approved methods for quantification of indigestible oligosaccharides by AOAC is the enzyme-HPLC method, which is a partial modification of Prosky's enzyme-gravimetric dietary fiber quantification method (Fig. 6).
- Fig. 6 Prosky's enzyme-gravimetric dietary fiber quantification method
- a digestible component is hydrolyzed with a hydrolase and then an indigestible low molecular weight substance that does not precipitate with ethanol is used in a specific column. This is a method of analyzing and quantifying by HPLC.
- thermostable ⁇ -amylase and amyloglucosidase which are carbohydrate digestive enzymes used in the enzyme-HPLC method, can digest high molecular polysaccharides, but digest low molecular carbohydrates such as oligosaccharides according to the nature of the enzyme. It can be difficult.
- fructooligosaccharides and isomaltoligosaccharides which are said to be indigestible oligosaccharides
- hydrogen gas produced only by fermentation by intestinal bacteria is discharged into the breath from about 2 hours after ingestion. Initially, it reaches a peak at 3 to 4 hours, whereas almost no exhaled hydrogen gas is observed with isomaltoligosaccharides.
- fructooligosaccharide against transient diarrhea is 0.3 to 0.4 g per kg of body weight, whereas isomaltoligosaccharide is 1.2 g or more per kg of body weight. The feature is that it is very high.
- isomaltooligosaccharides are quantified by the AOAC official method of enzyme-HPLC, as described above, it is considered that they are digested because they are difficult to digest low-molecular sugars such as oligosaccharides due to the nature of the enzyme. Isomaltooligosaccharides are detected as indigestible oligosaccharides. This clearly shows that there are problems with the enzyme-HPLC method. In addition, since the digestive enzyme used in the enzyme-HPLC method lacks ⁇ -galactosidase, lactose that should be digested may be quantified as an indigestible oligosaccharide.
- the enzyme-HPLC method which is the official method of AOAC, has a problem that any of the indigestible oligosaccharide and the digestible oligosaccharide is quantified as the indigestible oligosaccharide.
- an object of the present invention is to provide a carbohydrate quantification method capable of more accurately quantifying carbohydrates.
- Another object of the present invention is to provide a carbohydrate quantification kit capable of more accurately quantifying carbohydrates.
- the present invention has been made to solve the above-described problems, and is a carbohydrate quantification method using a digestive enzyme, wherein the digestive enzyme is an animal-derived low-molecular-weight carbohydrate digestive enzyme. Yes.
- the present invention has been made to solve the above-mentioned problems, and is a carbohydrate quantification method using a digestive enzyme, including a first reaction step using a thermostable ⁇ -amylase, a protease, and an amyloglucosidase. And a third reaction step using an animal-derived low-molecular-weight carbohydrate digestive enzyme.
- an animal-derived small intestinal mucosal hydrolase is used as the low-molecular-weight carbohydrate digestive enzyme.
- the present invention has been made in order to solve the above problems, and is a carbohydrate quantification kit having a digestive enzyme, comprising an animal-derived low-molecular-weight carbohydrate digestive enzyme as the digestive enzyme. It is a feature.
- the carbohydrate quantification kit according to the present invention preferably has a structure provided with an animal-derived small intestinal mucosal hydrolase as the digestive enzyme. More specifically, the carbohydrate quantification kit according to the present invention is preferably configured to include thermostable ⁇ -amylase, protease, amyloglucosidase, and animal-derived small intestinal mucosal hydrolase as the digestive enzyme.
- a carbohydrate quantification method capable of more accurately quantifying carbohydrates can be obtained.
- a carbohydrate quantification kit capable of more accurately quantifying carbohydrates can be obtained.
- movement procedure figure of the carbohydrate determination method concerning the 1st Example of this invention is shown.
- the chromatograph of fructooligosaccharide, lactulose, and galactosyl sucrose used as standard substances is shown.
- the chromatographs of isomaltoligosaccharide, sucrose, and lactose used as standard substances are shown. It is the graph which showed the fixed_quantity
- FOS fructooligosaccharide
- IMO isomaltoligosaccharide
- FIG. 3 is a diagram showing an operation procedure of an enzyme-HPLC method in which the dietary fiber determination method by Prosky's enzyme-gravimetric method according to the prior art is partially modified.
- the carbohydrate quantification method according to the first embodiment of the present invention is a carbohydrate quantification method using a digestive enzyme, wherein an animal-derived low-molecular-weight carbohydrate digestive enzyme is used as the digestive enzyme. .
- the carbohydrate quantification method according to the second embodiment of the present invention is a carbohydrate quantification method using a digestive enzyme, and includes a first reaction step using a thermostable ⁇ -amylase, and a first reaction step using a protease and amyloglucosidase. It has two reaction steps and a third reaction step using an animal-derived low-molecular-weight carbohydrate digestive enzyme.
- an animal-derived small intestinal mucosal hydrolase is used as the low-molecular-weight carbohydrate digestive enzyme in the carbohydrate quantification method according to the first embodiment or the second embodiment. It is characterized by.
- the carbohydrate quantification kit according to the fourth embodiment of the present invention is a carbohydrate quantification kit having a digestive enzyme, and is characterized by comprising an animal-derived low-molecular-weight carbohydrate digestive enzyme as the digestive enzyme.
- the fifth embodiment of the present invention is characterized in that the carbohydrate quantification kit according to the fourth embodiment is provided with an animal-derived small intestinal mucosal hydrolyzing enzyme as the digestive enzyme.
- the carbohydrate quantification method according to this embodiment described above is a carbohydrate quantification method using a digestive enzyme, and is characterized by using an animal-derived low-molecular-weight carbohydrate digestive enzyme as the digestive enzyme. More specifically, animal-derived low-molecular-weight carbohydrate digestive enzymes are added to the enzyme treatment reaction system in the carbohydrate quantification method according to the present embodiment.
- the carbohydrate quantification kit according to the present embodiment may have any configuration as long as the above-described carbohydrate quantification method can be performed, and as a digestive enzyme, low-molecular-weight carbohydrate digestion derived from animals is used. It is characterized by having an enzyme.
- animal is a concept excluding microorganisms. That is, “animal-derived low-molecular-weight carbohydrate digestive enzyme” is a concept excluding “microorganism-derived low-molecular-weight carbohydrate digestive enzyme”.
- a pig small intestine mucosa brush border membrane suspension is used as an animal-derived low-molecular-weight carbohydrate digestive enzyme.
- a method for preparing a pig small intestine mucosa brush border membrane suspension will be described.
- Porcine small intestinal mucosa brush border membrane suspension was prepared by the method of Kessler et al. (Kessler M, Acuto O, Storelli C, Murer H, Muller M, Semenza G (1978) A modified procedure for the rapid preparation of efficiently transporting vesicles small intestinal brush border membranes. Biochim Biophys Acta 506: 136-54.).
- the porcine small intestine provided by the Nagasaki Prefectural Meat Hygiene Inspection Station carefully cuts off fats and the like adhering to the small intestine on an ice-cold vat, and the upper half of the small intestine with high disaccharide hydrolase activity is spaced at 10 cm intervals. Carved.
- the cut small intestine is cut vertically on an ice-cooled glass plate, the inside of the small intestine lumen is washed twice or three times with physiological saline, water drops are removed with a paper towel, and the small intestine is spread and stacked. Stored at ⁇ 80 ° C. until preparation of brush border membrane suspension.
- the suspension was crushed for 20 minutes with an ultrasonic device (US-4, manufactured by SND Co., Ltd.). And powdery calcium chloride was added to this suspension so that final concentration might be 10 mM, and it left still for 20 minutes under ice-cooling.
- the mixture was centrifuged at 3000 ⁇ g and 4 ° C. for 15 minutes, and the obtained supernatant was further centrifuged at 27000 ⁇ g and 4 ° C. for 30 minutes.
- the precipitate was suspended in 2 mM Tris-Cl buffer (pH 7.1) containing 50 mM mannitol, and centrifuged at 27000 ⁇ g, 4 ° C. for 30 minutes. This operation is repeated once more, and the resulting precipitate is suspended in an appropriate amount of 0.05 M phosphate buffer (pH 7.0), centrifuged at 27000 ⁇ g for 30 minutes at 4 ° C.
- 0.05 M phosphate buffer pH 7.0
- the resulting precipitate (purified brush border membrane) is suspended in an appropriate amount of 0.05M phosphate buffer (pH 7.0) and then dispensed into a 10 mL plastic tube for actual carbohydrate determination. It was stored frozen at ⁇ 80 ° C. until used.
- FIG. 1 is an operation procedure diagram of the carbohydrate quantification method according to the first embodiment of the present invention. More specifically, FIG. 1 shows an operation procedure diagram of carbohydrate determination by the enzyme-HPLC method according to this example.
- FOS fructto-oligosaccharide
- IMO isomalto-oligosaccharide
- sucrose and lactose were used as the digestible sugar (carbohydrate). That is, the carbohydrate quantification method according to the present embodiment, which can more accurately quantify the saccharide using these hardly digestible saccharide and digestible saccharide, will be described.
- each indigestible saccharide and 1 g of each digestible saccharide were weighed and placed in a tall beaker (capacity 500 mL), and 0.05 M MES / TRIS buffer (pH 6. 3) It melt
- an ⁇ -amylase treatment is performed. Specifically, 200 ⁇ L of heat-resistant ⁇ -amylase (derived from Bacillus licheniformis), a digestive enzyme, is added, the tall beaker is covered with aluminum foil, and DIGITAL HOT PLATATE / STIRRE DP-1M (manufactured by AS ONE Co., Ltd.) is used. After the liquid temperature in the tall beaker reached 95 ° C., the reaction was carried out with stirring for 30 minutes (first reaction step).
- heat-resistant ⁇ -amylase derived from Bacillus licheniformis
- STIRRE DP-1M manufactured by AS ONE Co., Ltd.
- a pig small intestine disaccharide hydrolase treatment is performed. Specifically, in order to digest the oligosaccharide, an animal-derived low-molecular-weight carbohydrate digestive enzyme was added and reacted at 37 ° C. for 1 hour (third reaction step).
- an animal-derived small intestinal mucosal hydrolase was used as the “animal-derived low-molecular-weight carbohydrate digestive enzyme”. More specifically, a pig small intestine mucosa brush border membrane suspension (pH 7.0) prepared in-house as described above was used.
- the protease was inactivated so that the animal-derived low-molecular-weight carbohydrate digestive enzyme was not degraded by the protease (inactivation step).
- the supernatant obtained by ethanol precipitation was reacted while sucking with an ASPIRATOR A-3S (manufactured by Tokyo Science Instruments Co., Ltd.) into a glass filter in which a filter layer was previously formed with Celite.
- the solution was injected and fractionated into a residue and a supernatant (filtration fractionation step).
- the residue on the inner wall of the tall beaker and the glass filter is washed 3 times with 20 mL of 78% ethanol, twice with 10 mL of 95% ethanol, and further twice with 10 mL of acetone, and the washing solution is added to the Erlenmeyer flask. Collected (washing process).
- the evaporated solution was dissolved in a small amount of water and desalted with an amphoteric ion exchange resin (Amberlite MB-3, manufactured by Organo Corp.) (desalting step).
- an amphoteric ion exchange resin Amberlite MB-3, manufactured by Organo Corp.
- the collected solution is fixed to 50 mL using a graduated cylinder, and then impurities such as proteins are filtered using a membrane filter (Millex-GV Non-Sterile 0.22 ⁇ m ⁇ 13 mm, Millipore Co., USA). It was set as the analytical sample of HPLC.
- Measuring instrument LC-20AD (manufactured by Shimadzu Corporation) Column Shodex SUGAR KS-802 (8.0 ⁇ ⁇ 300mm, Showa Denko K.K.) Column temperature 70 ° C Mobile phase H 2 O Flow rate 0.5mL / min Sample flow rate 10 ⁇ L Detector Differential refractometer RID-10A (manufactured by Shimadzu Corporation)
- the indigestible saccharides and digestible saccharides that were not decomposed by a series of enzyme treatments of the saccharide quantification method (enzyme-HPLC method improvement method) according to this example were analyzed using a Shodex SUGAR KS-802 column. Analysis was performed at a column temperature of 70 ° C. As the standard solution, each indigestible saccharide solution and digestible saccharide solution (5.0 mg / mL each) dissolved in distilled water were used.
- FIG. 2 and FIG. 3 show elution profiles of standard substance solutions used for quantification of each indigestible saccharide and digestible saccharide by HPLC.
- Recovery rate of undigested saccharides after the enzyme treatment was calculated by the following formula.
- Recovery rate of undigested material (%) (((A / B) ⁇ dilution rate) / sample weight (g)) ⁇ 100
- A Peak area of carbohydrate after enzyme treatment
- B Peak area of standard substance solution
- the carbohydrate determination method according to the present example can obtain the following results by having the above-described configuration.
- FIG. 4 and FIG. 5 show the results of the carbohydrate quantification method according to the above-described example.
- FIG. 4 is a graph showing the quantification results of the indigestible saccharide fraction concerning fructooligosaccharide (FOS) and isomaltoligosaccharide (IMO) when the saccharide quantification method according to this example is used.
- FIG. 5 is a graph showing the quantification results of the indigestible saccharide fraction relating to sucrose and lactose when the saccharide quantification method according to this example is used.
- IMO which is regarded as an indigestible carbohydrate, is almost completely digested by adding disaccharide hydrolase derived from porcine small intestine to the enzyme treatment reaction system, and only glucose, which is the minimum constituent unit of IMO, is detected. (See FIG. 4).
- sucrose digested with sucrase was digested almost completely by adding disaccharide hydrolase derived from porcine small intestine to the enzyme treatment reaction system as in IMO (see FIG. 5).
- lactose digested with lactase was digested only 56.8% due to the low lactase activity of the porcine small intestinal mucosa brush border membrane suspension. It was also confirmed that a disaccharide other than lactose was newly produced by reacting with the porcine small intestinal mucosa brush border membrane suspension (see FIG. 5).
- the carbohydrate digestive enzyme used in the enzyme-HPLC method according to the prior art can digest high molecular polysaccharides, but low molecular oligosaccharides are difficult to digest. . Therefore, although IMO is easily digested by human and rat small intestinal mucosal enzymes, it is only slightly hydrolyzed by the hydrolyzing enzymes for dietary fiber determination according to the prior art. This indicates that the enzyme-HPLC method, which is an official method of AOAC, has a defect that is quantified as an indigestible oligosaccharide regardless of whether it is digestible or indigestible oligosaccharide.
- the carbohydrate-digesting enzymes currently used in the enzyme-HPLC method lack ⁇ -glucosidase and ⁇ -galactosidase that digest low-molecular-weight carbohydrates, so by adding both to the reaction system, Sugar (oligosaccharide) can be quantified more accurately.
- a porcine small intestine mucosa brush border membrane suspension pig small intestinal mucosal digestive enzyme
- this example is a carbohydrate quantification method using a digestive enzyme, wherein the digestive enzyme is an animal-derived low-molecular-weight carbohydrate digestive enzyme.
- the present example is a carbohydrate determination method using a digestive enzyme, which is a first reaction step using a thermostable ⁇ -amylase, a second reaction step using a protease and amyloglucosidase, and an animal-derived method. And a third reaction step using a low-molecular-weight carbohydrate digestive enzyme.
- an animal-derived small intestinal mucosal hydrolase is used as a low-molecular-weight carbohydrate digestive enzyme.
- any kit can be used as long as it has this feature. It belongs to the technical scope of the present invention.
- the present invention is a carbohydrate quantification kit having a digestive enzyme, and is characterized by comprising an animal-derived low-molecular-weight carbohydrate digestive enzyme as the digestive enzyme.
- the carbohydrate quantification kit according to the present invention is characterized by comprising thermostable ⁇ -amylase, protease, amyloglucosidase, and animal-derived small intestinal mucosal hydrolase as digestive enzymes.
- the carbohydrate quantification kit according to the present invention preferably has a constitution provided with an animal-derived small intestinal mucosal hydrolase as a digestive enzyme.
- low molecular carbohydrate digestive enzyme derived from porcine pig small intestinal mucosa brush border membrane suspension
- animal derived low molecular carbohydrate digestive enzyme animal derived low molecular carbohydrate digestive enzyme
- the present invention is not limited to this configuration, and low molecular carbohydrate digestive enzymes derived from “other animals” can be used as long as their activities are similar to those of “human”. is there. Therefore, for example, the small intestinal mucosal hydrolase of other animals such as rats and cows may be used.
- Dietary fiber and indigestible oligosaccharides / sugar alcohols have been shown to have special physiological effects on health that are different from conventional carbohydrates and are deeply involved in the prevention of lifestyle-related diseases. .
- health-consciousness has become stronger due to changes in national values, and functional foods such as foods for specified health use that emphasize various physiological functions by food manufacturers and pharmaceutical manufacturers are also actively developed.
- the dietary fiber quantification method and the indigestible oligosaccharide quantification method which are the current official methods (according to the prior art), are considered to be overestimated, and may be false. Consumers have the right to know the right information based on scientific evidence.
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Abstract
Description
図1は、本発明の第一実施例にかかる糖質定量方法の操作手順図を示したものである。より具体的には、図1は、本実施例にかかる酵素-HPLC法による糖質定量の操作手順図を示したものである。
カラム Shodex SUGAR KS-802
(8.0φ×300mm,昭和電工(株)製)
カラム温度 70℃
移動相 H2O
流速 0.5mL/min
試料流入量 10μL
検出器 示差屈折計 RID-10A((株)島津製作所製)
未消化物の回収率(%)=(((A/B)×希釈倍率)/試料重量(g))×100
ここで、A:酵素処理後の糖質のピークエリア
B:標準物質溶液のピークエリア
なお、本発明は、上記実施形態および実施例(以下「実施形態等」という。)に限定されるものではなく、必要に応じて種々の変更を加えて実施することも可能であり、それらはいずれも本発明の技術的範囲に含まれる。
Claims (5)
- 消化酵素を用いた糖質定量方法であって、
前記消化酵素として、動物由来の低分子糖質消化酵素が用いられる
ことを特徴とする糖質定量方法。 - 消化酵素を用いた糖質定量方法であって、
耐熱性α-アミラーゼを用いた第一反応工程と、
プロテアーゼおよびアミログルコシダーゼを用いた第二反応工程と、
動物由来の低分子糖質消化酵素を用いた第三反応工程とを備えた
ことを特徴とする糖質定量方法。 - 前記低分子糖質消化酵素として、動物由来の小腸粘膜水解酵素が用いられる
請求項1または2に記載の糖質定量方法。 - 消化酵素を有する糖質定量キットであって、
前記消化酵素として、動物由来の低分子糖質消化酵素を備えた
ことを特徴とする糖質定量キット。 - 前記消化酵素として、動物由来の小腸粘膜水解酵素を備えた
請求項4に記載の糖質定量キット。
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US12/991,606 US8367365B2 (en) | 2008-05-06 | 2008-05-06 | Method for determining carbohydrate and kit for determining carbohydrate |
PCT/JP2008/058440 WO2009136432A1 (ja) | 2008-05-06 | 2008-05-06 | 糖質定量方法および糖質定量キット |
JP2010510973A JP5532256B2 (ja) | 2008-05-06 | 2008-05-06 | 糖質定量方法および糖質定量キット |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0690130A1 (en) * | 1994-06-27 | 1996-01-03 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Saccharide composition with reduced reducibility, and preparation and uses thereof |
JPH10150934A (ja) * | 1996-11-20 | 1998-06-09 | Matsutani Chem Ind Ltd | 還元難消化性水飴およびこれを用いた食品 |
WO2006035725A1 (ja) * | 2004-09-27 | 2006-04-06 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | イソサイクロマルトオリゴ糖及びイソサイクロマルトオリゴ糖生成酵素とそれらの製造方法並びに用途 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US3249514A (en) * | 1963-11-18 | 1966-05-03 | Bode Harold Eli | Production and use of amyloglucosidase |
GB1446965A (en) * | 1974-02-14 | 1976-08-18 | Agricultural Vegetable Prod | Preparation of food products |
JP3183500B2 (ja) | 1989-04-10 | 2001-07-09 | 日本化薬株式会社 | 糖アルコールの定量方法 |
JP4070250B2 (ja) * | 1994-06-27 | 2008-04-02 | 株式会社林原生物化学研究所 | 還元性を低減させた糖質とその製造方法並びに用途 |
-
2008
- 2008-05-06 JP JP2010510973A patent/JP5532256B2/ja active Active
- 2008-05-06 US US12/991,606 patent/US8367365B2/en not_active Expired - Fee Related
- 2008-05-06 WO PCT/JP2008/058440 patent/WO2009136432A1/ja active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0690130A1 (en) * | 1994-06-27 | 1996-01-03 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Saccharide composition with reduced reducibility, and preparation and uses thereof |
JPH10150934A (ja) * | 1996-11-20 | 1998-06-09 | Matsutani Chem Ind Ltd | 還元難消化性水飴およびこれを用いた食品 |
WO2006035725A1 (ja) * | 2004-09-27 | 2006-04-06 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | イソサイクロマルトオリゴ糖及びイソサイクロマルトオリゴ糖生成酵素とそれらの製造方法並びに用途 |
Non-Patent Citations (2)
Title |
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MCCLEARY, BV. ET AL.: "An integrated procedure for the measurement of total dietary fibre (including resistant starch), non-digestible oligosaccharides and available carbohydrates.", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 389, no. 1, 2007, pages 291 - 308, XP019537567, DOI: doi:10.1007/s00216-007-1389-6 * |
OSAMU CHONAN ET AL.: "Galacto-oligosaccharide no Nanshokasei no Kento", JOURNAL OF THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY, vol. 51, no. 1, 2004, pages 28 - 33 * |
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US20110059474A1 (en) | 2011-03-10 |
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