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Dietary Fiber Assay Kit

A simple and modified version of Prosky's method. Compared to conventional Prosky's method, it assures high measurement accuracy. Furthermore, enzyme treatment time is shortened.

  • Operation Procedure
  • Package Insert (906 KB; 20 page)
  • image
    Kit Contents:
    1. Thermostable alpha-Amylase Solution (20 mL x 1 vial)
    2. Protease Solution (20 mL x 1 vial)
    3. Amyloglucosidase Solution (20 mL x 1 vial)
    4. Diatomaceous Earth (100 g x 1 vial)


    Operation Procedure

    1. Addition of Sample
    2. Digestion with thermostable α-amylase
    3. Wash the Beaker and Cool it
    4. Digestion with protease and amyloglucosidase
    5. Ethanol Precipitation
    6. Filtration
    7. Wash
    8. Drying and Weighing
    9. Protein Determination
    10. Ash Determination
    11. Calculation of Total Dietary Fiber Content

    Perform the below-mentioned procedure without samples and obtain each blank value.
    See the Package Insert (906 KB; 20 page) for detailed procedure.

    1. Addition of Sample

    Accurately weigh 1 g of samples for determination of undigested protein (W1) and for determination of ash content (W2), respectively. Then, add a sample to 40 mL of 50 mmol/L MES-Tris buffer (pH6.3, 24ºC) in a 500 mL tall beaker and disperse it.

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    2. Digestion with thermostable &alpha-amylase

    Add 0.2 mL of thermostable ƒ¿-amylase and cover the tall beaker with aluminum foil. Let the mixture react in a boiling water bath for 30 min. while shaking it every 5 min.

    image

    3. Wash the wall of the tall beaker and cool it

    Add 10 mL of water while washing the wall of the tall beaker, cool to 60ºC.

    image

    4. Digestion with protease and amyloglucosidase

    Add simultaneously 0.2 mL of protease and 0.2 mL of amyloglucosidase. Let the mixture react in a thermostatic bath for 30 min. while continuous shaking.

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    5. Ethanol Precipitation

    Add a quadruple volume of 95% Ethanol (preheated at 60ºC) to the enzyme treated solution obtained, and allow to stand for 1 hour at room temperature to precipitate enzyme undigested macromolecule.

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    6. Filtration

    Conduct a suction filtration of Ethanol Precipitation solution using a glass filter crucible, which has a homogeneous Diatomaceous Earth layer.

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    7. Wash the wall of the tall beaker and cool it

    Under suction, rinse the residue in the tall beaker with 78% Ethanol (20 mL x 3 times), 95% Ethanol (10 mL x 2 times), and acetone (10 mL x 2 times), and add the washings to the residue in the glass filter crucible.

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    8. Drying and Weighing

    Dry the residue with the glass filter crucible at 105±3ºC overnight. Weigh the residue to a tenth of a milligram after allowing to cool in a desiccator. Residues for protein determination and that for ash determination are set at R1 and R2, respectively.

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    9. Protein Determination

    Scrape the total amount of residues for protein determination together with Diatomaceous Earth and determine the nitrogen content in the residue by Kjeldahl protein determination. Multiply the obtained nitrogen content by 6.25 to obtain the protein content (P1).

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    10. Ash Determination

    Conduct incineration treatment of the residues for ash determination with the glass filter crucible at 525ºC for 5 hours, allow to cool in a desiccator, weigh to the tenth of a milligram and obtain the ash content (A1).

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    11. Calculation of Total Dietary Fiber Content

    Obtain total dietary fiber content by subtracting the protein content, the ash content and the blank value from the residue.

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        W1, W2: weighed samples (g)
        R1: Residue for protein determination (g)
        R2: Residue for ash determination (g)
        P1: Protein content (g)
        A1: Ash content (g)
        B: Blank test (g) = (r1+r2)/2 x {1-(p1/r1 + a1/r2)}
        (Note) r1, r2, p1 and a1: blank values of R1, R2, P1 and A1

    References

    1. Kitamoto, T.,Ogomori, K., Tateishi, J., and Prusiner, S. B.: Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids., Lab. Invest., 57, 230-236 (1987)
    2. Okamura, N., et al.: Styrylbenzoxazole derivatives for in vivo imaging of amyloid plaques in the brain, J. Neurosci., 24, 2535-2541 (2004)
    3. Okamura, N., Kudo, Y., et al.: Quinoline and benzimidazole derivatives: candidate probes for in vivo imaging of tau pathology in Alzheimer's disease., J. Neurosci., 25, 10857-10862 (2005)

    Product List

    Description
    Wako Catalog No.
    Package Size
    Dietary Fiber Assay Kit
    291-59701
    100 tests

    Reagents required but not supplied in the kit

    Description
    Wako Catalog No.
    Package Size
    Reagents for enzyme reactions
    2-(N-Morpholino)ethanesulfonic Acid (MES) [Dojindo]
    341-01622
    25 g
    2-Amino-2-hydroxymethyl-1,3-propanediol (TRIS)
    203-06272
    25 g
    Sodium Chloride (NaCl)
    191-01665
    500 g
    Potassium Chloride (KCl)
    169-03542
    25 g
    Ethanol (95)
    051-00476
    500 mL
    Acetone
    016-00346
    500 mL
    Reagents for Kjeldahl method
    Potassium Sulfate (K2SO4)
    169-04485
    500 g
    Copper (II) Sulfate Pentahydrate (CuSO4·5H2O)
    039-04412
    25 g
    Sulfuric Acid
    192-04696
    500 mL
    Hydrogen Peroxide (30) (H2O2)
    081-04215
    500 mL
    Boric Acid (H3BO3)
    027-02192
    25 g
    Bromocresol Green-Methyl Red Ethanol Solution
    020-14571
    100 mL
    Sodium Hydroxide (NaOH)
    199-08621
    100 g
    Note
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