FAO/WHO expert consultation carbohydrates in human nutrition

69

DIETARY FIBRE AND RESISTANT STARCH ANALYSIS

Dietary fibre

There is no single analytical method which meets all the requirements for the nutritional or chemical components of dietary fibre in foods. Analytical methods and techniques continue to be improved, whether this results from changes in the purposes of the analysis or improvements in the accuracy, precision, rapidity, ruggedness and cost effectiveness of the method. This subject has been recently reviewed (43,44).

Currently, dietary fibre methodology can be classified into three major categories as follows:

1.Nonenzymatic-gravimetric

2.Enzymatic-gravimetric

3.Enzymatic-chemical methods, which include

a.Enzymatic-colorimetric

b.Enzymatic-GLC/HPLC

For most foods, the older nonenzymatic-gravimetric methods do not recover a significant portion of what is considered to be total dietary fibre. Among those, the crude fibre method measures fibre as the sum of lignin and cellulose, the acid detergent method measures fibre as the sum of lignin, cellulose, and acid insoluble hemicellulose, and the neutral detergent method measures fibre as the sum of lignin, cellulose and neutral detergent insoluble hemicellulose.

The realization that neither the crude fibre nor the neutral and acid detergent fibre methods were satisfactory for measuring any of the soluble dietary fibre and some of the insoluble dietary fibre led researchers to explore enzymatic approaches for the determination of dietary fibre.

Enzymatic-gravimetric methods

In the early 1980s, a enzymatic-gravimetric method was developed in which the sum of soluble and insoluble polysaccharides and lignin were measured as a unit and considered to be total dietary fibre (TDF). That method is detailed in section 45.4.07 of the AOAC International Official Methods of Analysis (45). The procedure was later extended to the determination of insoluble dietary fibre (IDF) (32.1.16 AOAC) (45) and soluble dietary fibre (SDF) (45.4.08 AOAC) (45). All three methods use the same basic enzymatic-gravimetric procedure with phosphate buffer. An additional method to determine TDF, IDF and SDF was developed in the early 1990s and is detailed in 32.1.17 AOAC (45). It is similar to the first method, in that it uses the same three enzymes (heat stable α-amylase, protease, and amyloglucosidase) and similar incubation conditions but substitutes 2-(N-morpholino) ethanesulfonic acid-tris (hydroxymethyl) aminomethane (MES-TRIS) buffer for the phosphate buffer. The results using the MES-TRIS buffer for determination of dietary fibre are similar to those obtained using the phosphate buffer. The analytical scheme for this procedure is summarized in Figure 4.

70		Annex 2. Background information

Figure 4

Analysis of Total Dietary Fibre (TDF), Insoluble Dietary Fibre (TDF) and Soluble Dietary Fibre (SDF) by AOAC method 32.1.17 (45)

1 g defatted, dried sample

Gelatinization and Termamyl incubation (pH 6.0, 30 min., 100°C)

Protease incubation (pH 7.5, 30 min., 60°C)

Amyloglucosidase incubation (pH 4.5, 30 min., 60°C)

Precpitation with 4 volumes ethanol

Filtration

Washing with ethanol and acetone

Drying (TDF)

(Correction for undigested protein, ash and blank)

Filtration

(Through fritted crucible containing Celite)

	Residue	Filtrate
	(wash with ethanol	(add 4 vol. of ethanol)
	and acetone)
	(IDF)
		Filtration
		Residue
		(SDF)

Source: Adapted from AOAC International 1995 (45)

FAO/WHO expert consultation carbohydrates in human nutrition

71

Enzymatic-chemical methods

Another method recently accepted for official action by the AOAC for the determination of TDF is one based on assays for components of TDF - neutral sugars, uronic acid residues and Klason lignin (45.4.11 AOAC) (45). This procedure is often referred to as the Uppsala Method. Starch is removed enzymatically and soluble polymers are precipitated with ethanol. Precipitated and insoluble polysaccharides are hydrolyzed using sulfuric acid and the released neutral sugars are quantitated as alditol acetates using gas-liquid chromatography. Uronic acids in the acid hydrolysate are determined by colorimetry. Klason lignin is determined gravimetrically. The three values are added together to obtain the TDF figure.

Plant cell wall material comprises much of what is considered to be fibre in the diet. About 90% of endogenous plant cell wall material consists of non-starch polysaccharides (NSP) (46). The analytical procedure known as the Englyst method determines NSP after enzymatic removal of starch, precipitation of NSP, followed by acid hydrolysis and measurement of the released constituent sugars. In an early method the sugars were assayed colourimetrically (47) and in a more recent procedure, they are quantitated by any of three instrumental techniques, i.e. gas-liquid chromatography, high performance liquid chromatography and colourimetry (48).

Resistant starch

Of the many analytical procedures employed for resistant starch (RS), two have emerged as the leading candidates for approval. These are the methods described by Englyst (49) and Champ (50). They both provide similar results. The first step is removal of digestible starch from the food sample using pancreatic I-amylase (in cases where there may be inhibition of the pancreatic enzyme by products of digestion, amyloglucosidase is added). Sometimes the amylolysis is preceded by a proteolysis step with pepsin and trypsin to mimic the action of the stomach and intestine. The RS is quantitated either directly in the residue (50) or by difference between total starch and digestible starch, which are determined separately (49).

A new procedure has been proposed which is derived from several RS analysis systems (51). Its principle is that in-vitro RS is defined as that starch which is not hydrolyzed by incubation with I-amylase. Amyloglucosidase is added to avoid inhibition by by-products of amylase digestion. Hydrolysis products are extracted with 80% ethanol and discarded. The RS is then solubilized with 2N potassium hydroxide and hydrolyzed with amyloglucosidase. The procedure is relatively simple with no particular training required, and is summarized in Figure 5.

72		Annex 2. Background information

Figure 5

Method for the determination of resistant starch

100 mg sample*

Add 10 ml enzyme solution in buffer (pH 6.9); pancreatic α-amylase (500U); 0.1M tris-maleate buffer solution (calcium chloride 4mM)

Mix 16 hours at 37°C; then add 40 ml ethanol

Leave 1 hour; then centrifuge

Wash residue twice with 80% EtOH, dry at 60°C

Add 1.56 ml H2O, then add 1.5 ml 4M KOH

Mix 0.5 hours at room temperature

Add 12 ml H2O

To 1.5 ml dispersion, add approximately 0.65 ml 2M acetic acid (to obtain pH 4.5) and 0.1 ml amyloglucosidase (20U/0.1 ml 0.1M Na acetate buffer pH 4.5)

Shake 90 min. at 65°C

Determine glucose using glucose oxidase assay

The result is resistant starch

*Ground in mincer. The sample must be weighed to contain 50 mg starch

* Ground in a mincer. The sample must be weighed to contain 50 mg starch

Source: Adapted from Champ, M., Noah, L., Loizeau, G., Kozlowski, F. (51)