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Total, Insoluble, and Soluble fiber / Total Dietary Fiber

AOAC Method 991.43

Introduction

This method represents a slow evolution of methodologies that combined crude fiber,
detergent fiber and Southgate methodologies.

Principle:

Dry, fat extracted samples are enzymatically digested with α-amylase, amyloglucosidase, and
protease to remove starch and protein. Insoluble fiber is collected by filtration. Soluble fiber
is precipitated by bringing the filtrate to 78% ethanol and collected by filtration. The filtered
fiber residues are washed with ethanol and acetone, oven dried and weighed. The fiber
residues are analysed for protein and ash content [Fiber = weight residue – (weight of protein
and weight of ash)]

Procedure:

Samples are mixed with buffer, a heat stable α-amylase is added, and the pH is adjusted.
Starch is gelatinized and digested by heating the digestion mixture in a boiling water bath.
After cooling, a protein enzyme is added to digest protein. After protein digestion, pH is
adjusted and starch digestion is completed with amyloglucosidase.

Next few steps differ depending on whether total, insoluble, or soluble fiber is being
determined. If fiber into soluble and insoluble fractions, proceed as described in the note at
the bottom figure 1.

To determine insoluble and soluble fiber fractions, the digestion mixture following
amyloglucosidase treatment is filtered through fritted crucibles containing Celite. The
insoluble fiber retained by the filter is washed with water. The soluble fiber is in the filtrate.
Four volumes (vol/vol) of 95% ethanol are added to the filtrate plus water washes to
precipitate the soluble fiber. The precipitate is allowed to form and mixture is vacuum filtered
through fritted crucibles containing Celite. The soluble fiber residue is washed successfully
three times with 78% ethanol.

The fiber residue (total, soluble or insoluble fiber) in the crucible then washed with 95%
ethanol and acetone. The crucibles are oven dried, cooled and weighed. Since some protein
and minerals are complexed with plant cell wall constituents, fiber values must be corrected
for these contaminants. If resistant starch is to be determined, triplicate samples should be
analysed. If resistant starch is not determined, duplicate samples are analysed. One sample is
used to determine nitrogen content by Kjeldahl procedure, and another sample to determine
ash content.

Duplicate reagents blanks must be run through the entire procedure for each types of fiber
determination.
Food sample (1g)

Add 40 mL of buffer (pH 8.2)


Add heat stable α-amylase

Incubate 15 min at 95-100ºC

Cool to 60ºC

Add protease

Incubate 30min at 60ºC

Adjust pH to 4.0-4.7
Add amyloglucosidase

Incubate 30min at 60ºC

Filter the digest

Wash filtered residue with 10 mL of water (2times)

Filtrate + water washes are used for soluble fiber determination *

Insoluble fiber
Wash residue with 10mL of 95% ethanol (2times)

Wash residue with 10mL acetone (2times)

Oven dry

(1a) weight crucible

(1b) Ash one of the duplicates and reweight


(525ºC for at least 5hours)
(1c) Determine residual protein on the other duplicate (Kjeldahl N x 6.25)
(1d) Calculate insoluble fiber content
Soluble fiber
Bring filtrate and water washes to 80g with water

Add 320 mL of 95% ethanol preheated to 60ºC

Precipitate formation (1hr at room temperature)

Filter the digest

Wash filtered residue with 20mL of 78% ethanol (3times)

Wash residue with 10mL of 95% ethanol (2times)

Wash residue with 10mL acetone (2times)

Oven dry

(2a) Weight crucible

(2b) Ash one of the duplicates and reweight


(2c) determine residual protein on the other duplicate (Kjeldahl N x 6.25)
(2d) Calculate soluble fiber content
Total dietary fiber = Insoluble + Soluble fiber

*Total dietary fiber can be determined directly by weighing digest following


amyloglucosidase digestion and either

1) adding four volumes (vol/vol) 95% ethanol preheated to 60ºC or


2) adjusting the volume to 80g with water and then adding 320 mL of 95% ethanol preheated
to 60ºC

After (1) or (2), follow the procedure for determining soluble fiber starting at the precipitate
formation step. Calculation of (2d) would produce total dietary fiber.

Equipments

1. Water bath