JP2885353B2 - Determination of chitin and chitosan in foods - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for determining chitin and chitosan in foods.

[0002]

2. Description of the Related Art Chitin / chitosan is a polysaccharide widely present in crustaceans, insects and fungi, and chitin is a partially deacetylated N-acetyl-β-D-glucosamine polymer. On the other hand, chitosan, which has advanced deacetylation of chitin and is soluble in dilute acid, is expected to have the effect of so-called dietary fiber due to its indigestibility.In particular, since chitosan has many amino groups, it has a large number of amino groups. By adsorbing bile acids, it exhibits an excellent serum cholesterol lowering effect by inhibiting cholesterol absorption and bile acid reabsorption.

[0003] Although chitin and chitosan, which are excellent dietary fiber materials, occur naturally, foods designed to be added to food and exert their effects are important in the present day when dietary fiber intake is insufficient. It seems to be. Therefore it is important from a nutritional point of view to accurately determine its content.

[0004]

Generally, in a method for quantifying chitin / chitosan, the amount of β-D-glucosamine produced by hydrolysis with hydrochloric acid is measured. However, since the food is directly hydrolyzed, the components in the food, In particular, glucose and amino acids produced by hydrolysis of starch and protein affect the subsequent determination of glucosamine, and it is difficult to obtain an accurate quantitative value. In addition, in the hydrolysis with hydrochloric acid, it is difficult to set the conditions for the decomposition of chitosan, and the decomposition is incomplete or glucosamine is decomposed, so that it is difficult to obtain a stable quantitative value.

[0005]

Means for Solving the Problems As a result of various studies, the present inventors have found that after removing starch and proteins from foods with a hydrolytic enzyme, the remaining chitin and chitosan are hydrolyzed using sulfuric acid. As a result, an accurate and highly reproducible quantitative value of chitin / chitosan was obtained.

The present invention has been completed based on the above findings. That is, the present invention is characterized in that after decomposing starch and protein in a food containing chitin or chitosan by a decomposing enzyme, the remaining chitin and chitosan are hydrolyzed using sulfuric acid, and the amount of glucosamine produced is determined. It relates to a method for determining chitin and chitosan.

[0007] The enzymes used in the present invention to degrade starch and proteins in foods are hydrolytic enzymes, and they have no chitinase or chitosanase activity and can be derived from animals or microorganisms. For example, enzymes that degrade starch include α-amylase, amyloglucosidase, pancreatin and the like, and enzymes that degrade proteins include protease and pepsin. The decomposition conditions are not particularly limited as long as they are optimal conditions for the enzyme to be used. For example, the temperature is 4 to 110 ° C., and the decomposition time is about 0.5 to 12 hours. The amount of enzyme to be used is preferably not less than the amount at which starch and protein in food are completely degraded.For example, when amyloglucosidase is used as a starch-degrading enzyme and protease is used as a proteolytic enzyme, the enzyme activity of amyloglucosidase is reduced. Assuming that the amount of an enzyme that produces 1 mg of glucose in 3 minutes using starch as a substrate is 1 unit, 50-10000 units, preferably 1 unit, per gram of food is used.
About 00-2000 units are good. When a protease is used as a protease, the enzyme activity of the protease can be determined by using the casein as a substrate and the Folin-Ci
The amount of the enzyme that produces a hydrolysis product showing a color development corresponding to 1.0 μmol of tyrosine with the ocateu reagent is defined as 1 to 10 to 500 units, and preferably about 20 to 100 units. The hydrolysis may be performed in any order of starch degradation and protein degradation. After the decomposition, it is preferable to recover chitin or chitosan by centrifugation or filtration.

[0008] The hydrolysis of chitin / chitosan after the enzymatic treatment uses sulfuric acid, preferably dilute sulfuric acid.
C, preferably 30-70 C for 0.5-12 hours,
Preferably, it is performed for 0.5-3 hours. The concentration of sulfuric acid is not particularly limited, but is 0.1-3 molar, preferably 0.2-3 molar.
-0.5 molar is preferred. The amount used is 0.005 to 1 g of the weight of the enzyme-treated product after removing the starch and protein.
-0.03 mol, preferably about 0.01-0.2 mol. Before the hydrolysis, the enzyme-treated product after removing starch and protein from sulfuric acid having a concentration of 10-14 mol, preferably 11-13 mol, is subjected to 4-30 ° C, preferably 10-3 ° C.
Good results are obtained by immersion at 0 ° C. for 0.5-6 hours, preferably 1-3 hours. After the hydrolysis, the undegraded product is separated by centrifugation or filtration to obtain an analysis sample.

The quantification of glucosamine from the hydrolyzed sample can be performed by a known method, for example, a colorimetric method such as an indole hydrochloride method, a pyrrole hydrochloride method, an Elson-Morgan method, a Morgan-Elson method involving acetylation, or high performance liquid chromatography. Any method such as a gas chromatography method or a gas chromatography method may be used, but the indole hydrochloric acid method, which is simple in operation and high in sensitivity, is preferred.

EXPERIMENTAL EXAMPLE [0010] Breads (chitosan added: 2.48%), chimpanzees (chitosan added: 2.19%), and hot cakes (chitosan added: 0.18%) added with chitin powder, chitosan powder and chitosan.
91%), using the quantification method shown below. 1. Removal of starch and protein by enzymatic decomposition of chitin / chitosan-containing food Enzyme used: α-amylase Termamyl 120L Nobo Laboratories Inc. Manufactured by: Protease Protease P-5380 Sigma Chemical Co. Manufactured by: Amyloglucosidase
case A-9913 Sigma Chemical Co. Enzyme digestion method 1 g of an analysis sample is precisely weighed, and 400 ml
After adding 50 ml of 0.05M phosphate buffer (pH 6.0), add 0.2 ml of α-amylase, cover with an aluminum hoist, incubate in a boiling water bath for 30 minutes, and paste starch And decomposition. After cooling in running water, add 10 ml of 0.171 N sodium hydroxide to adjust the pH to 0.1 ± 0.1.
Adjust with sodium hydroxide N and 0.205 M phosphoric acid and add 5 mg of alkaline protease. Cover with aluminum foil and incubate at 60 ° C for 30 minutes while infiltrating to degrade proteins. 10 ml of 0.205 M phosphoric acid was added, and 0.171 N sodium hydroxide and pH 4.5 ± 0.2 were added.
After adjustment with 0.205 M phosphoric acid, amyloglucosidase
Add 0.3 ml, cover with aluminum foil, incubate at 60 ° C for 30 minutes while infiltrating to decompose dextrin. In this way, the obtained enzyme-treated solution was added at 60 ° C.
280 ml of the above 95% ethanol was added, and the mixture was allowed to stand for 60 minutes to aggregate the chitosan dissolved in the solution, followed by suction filtration using a glass filter-Prosity-2 to obtain a residue. afterwards,
3 times with about 20 ml of 78% ethanol, 10 m of 95% ethanol
After washing the residue on the filter twice with 1 l and twice with 10 ml of acetone, the residue is dried at 105 ° C. to obtain an enzymatic decomposition residue.

2. Hydrolysis of chitin and chitosan in enzymatic decomposition residue Take the enzyme-treated residue obtained in the above, add 40 ml of 0.358 mol sulfuric acid per 0.1 g of the residue, attach a condenser tube and hydrolyze in a boiling water bath for 6 hours. It is collected by centrifuging for 5 minutes, and glucosamine is 20-400 μg /
Make up to a volume of ml.

3. 1. Determination of glucosamine by indole hydrochloric acid method 0.5% 5% sodium nitrite in 0.5 ml of sample
Add 5 ml and 0.5 ml of 33% acetic acid and mix well.
A deamination reaction is performed by leaving the mixture to stand for 0 minutes. 1
0.5% aqueous solution of 2.5% ammonium sulfamate
And add 30 minutes with occasional mixing to remove excess nitrous acid. 2 ml of 5% hydrochloric acid and 1% indole-ethano
Solution in a boiling water bath for 5 minutes.
Of ethanol and stirred, and the absorbance at 490 nm is measured. At this time, the residue containing a large amount of other dietary fiber and non-digestible protein shows a high value. Therefore, when a large amount of dietary fiber is contained, the color is formed without performing the deamination reaction, and the difference is determined. Further, when a large amount of non-digestible protein is contained, a different absorption spectrum is shown. Therefore, the effect is reduced by subtracting the value of 490 nm from 490 nm.

The results are shown in Table 1. The results obtained by the conventional method (hydrochloric acid decomposition method: decomposition of 6N hydrochloric acid at 100 ° C. for 24 hours) are shown as a control. The numerical values in the table are numerical values calculated as acetylglucosamine polymer (chitin) or glucosamine polymer (chitosan).

[0014]

[Table 1]

In the case of chitosan powder, the recovery rate was higher than that obtained by hydrolyzing with hydrochloric acid in the present method, and stable quantitative results were obtained. In the case of chitin powder, the same results as those obtained by the conventional method were obtained.
In addition, even when chitosan was added to each food, a stable recovery rate was exhibited. In addition, as for food, blank food without chitosan was also quantified, but no chitosan was detected.

[0016]

From the above results, it is clear that this method is an excellent method for quantitatively determining chitin and chitosan from foods.

Claims (1)

(57) [Claims] The present invention is characterized in that starch and protein in a food containing chitin or chitosan are decomposed by a decomposing enzyme, and then the remaining chitin or chitosan is hydrolyzed using sulfuric acid, and glucosamine produced is quantified. Determination of chitin and chitosan.