Journal of Animal and Feed Sciences, 6, 1997, 185-206

Methods for analysis of dietary fibre - advantage

and limitations*

Bach Knudsen K . E , Helle Nygaard Johansen and Vibe Glitse

Danish Institute of Animal Sciences, Department of Nutrition,

Research Centre Foulum, P.O. Box 50
DK-8830 Tjele, Denmark

(Received 25 April 1997; accepted 15 May 1997)

ABSTRACT

The two main approaches for the determination of dietary fibre (DF) in food and feedstuffs are
the enzymatic- and nonenzymatic-gravimetric AOAC (Association of Official Analytical Chemists)
procedures and the enzymatic-chemical Englyst and Uppsala procedures. The main analytical
problems which have been shown to influence the performance of the enzymatic-gravimetric AOAC
procedures are: variable starch removal, too high final phosphate buffer concentration, and hence
variable ash content of gravimetric residues, problems with residual nitrogen determination,
filtration problems of viscous and desugared fruit samples, and variable blank values. Standar-
disation and improvements of the methods have gradually improved the performance of the
gravimetric procedures to acceptable levels. The enzymatic-chemical methods with gas-liquid
chromatography (GLC), high performance liquid chromatography (HPLC) or colorimetry as end-
point determination are generally more complex, involve more steps and require, for the GLC and
HPLC methods, more advance equipment than the gravimetric procedures. The efficiency of starch
removal, the hydrolytic conditions for acid hydrolysis of D F polysaccharides, factorial corrections
for hydrolytic losses of sugars and laboratory expertise are identified as factors influencing the
reproducibility of the methods. Modifications and optimisation of the various steps and the build-up
of expertise in laboratories participating in the collaborative trials have improved the reproducibility
of the enzymatic-chemical methods to a level similar to that of the enzymatic-gravimetric AOAC
methods. Technical problems which may introduce errors in the determination of D F with all
methods are incomplete precipitation in 80% (v/v) ethanol, impurities in bacterial amyloglucosidases
resulting in depolymerisation and potential losses of D F polysaccharides. A recent BCR Reference
Material Study indicate that the various DF methods give comparable results for cereal based

Part of this paper was presented at the Symposium: Dietary fibre - chemical composition and
biological action, 24-25 April 1997, Radzików, Poland
186 BACH-KNUDSEN K.E. ET A L .

materials when appropriate corrections are made for lignin and resistant starch. The same was the
case with full fat soya. It is concluded that the techniques for measuring D F are as reproducible as
other analytical methods for feed and food labelling and for research. The choice of analytical
method depends therefore primarily on the purpose of the analytical work.

K E Y WORDS: dietary fibre, analysis

INTRODUCTION

The need for appropriate analytical methods for the determination of dietary
fibre (DF) in food- and feedstuffs is apparent. It is well recognised that D F is an
important component of the diets for humans and animals and numerous studies
have documented important gastrointestinal and metabolic effects of fibre-rich
diets. Furthermore, dietary guidelines in human nutrition advocate increasing
the consumption of plant foods. This have led to the demand for reproducible
analytical D F methods for control and labelling as well as for nutritional
research. Unfortunately, however, the lack of consensus on the definition of D F
and the wide range of methods, which do not measure the same dietary entities,
have led to inconsistent and confusing data.
The crude fibre method, invented in the middle of the last century, and the
neutral detergent fibre (NDF) method, based on the pionering work of Van
Soest, have been used earlier to determine the D F value in food- and feedstuffs
(Henneberg and Stohmann, 1859; Van Soest, 1963; Van Soest and Wine, 1967).
Both methods, however, have their limitations. In the crude fibre method only
a small and variable fraction of the total D F is measured, while the water-soluble
NSP and water-insoluble pectic substances are lost in the N D F procedure (Bailey
and Ulyatt, 1970; Reichert, 1981; Carre and Brillouet, 1986). Moreover, starch
and protein may contaminate the N D F residue (Theander and Aman, 1980).
Over the last two decades there has been a rapid growth in the development of
robust and reproducible D F methods. The two main approaches are the
enzymatic or non-enzymatic gravimetric AO AC (Association of Official Analy-
tical Chemists) procedures and the enzymatic-chemical Englyst and Uppsala
procedures. In the first approach, all non-fibre components are removed from
the sample by selective extraction and enzymatic degradation and the residue
weighed. The second approach is to measure the dietary fibre constituents
directly after extraction of low-molecular weight sugars, enzymatic removal of
starch, acid hydrolysis of D F polysaccharides and determination of their
monosaccharide residues by gas-liquid chromatography (GLC), high-perfor-
mance liquid chromatography (HPLC) or colorimetry. These methods were in
most cases developed for the analysis of foodstuffs but they can also be applied to
feedstuffs.
ANALYSIS OF D I E T A R Y FIBRE 187

The present summary will primarily deal with advantage and limitations of the
enzymatic-gravimetric and enzymatic-chemical DF methods commonly in use.

E N Z Y M A T I C - G R A V I M E T R I C METHODS

The original AOAC enzymatic-gravimetric method for the determination of

total dietary fibre (TDF) was developed on basis of the joint experience of Asp et
al. (1992). The origins of these methods can be tracked back to the biochemical
approach of measuring the indigestible residues in human foods introduced by
Williams and Olmsted in the 1930s, (1935) and to the work of Hellendrom and
collegues (1975). These methods use enzymes to remove the digestible com-
ponents, which was considered more physiological than extraction with chemical
reagents as used in the detergent method.
The main steps in the AOAC procedure (Prosky et al., 1988) and the recent
modification proposed by Lee et al. (1992) are outlined in Table 1. Briefly, the
procedure consists of the following steps: (a) weighing of -1 g in a beaker, (b)
incubation with enzymes to remove starch and protein, (c) precipitation of
soluble polymers with four volumes of ethanol, (d) quantitative transfer of
sample to a pre-weighed crucible, (e) filtration, (f) weighing of the dry crucible
and (g) correction for ash and protein in residues. Blanks are carried through the
procedure in parallel. In the modification of Lee et al. (1992) the phosphate

TABLE 1
Main steps of the original and modified enzymatic-gravimetric AOAC methods

AOAC original AOAC modified

Prosky et al. (1988) Lee et al. (1992)

Sample ~1 g ~1 g
Buffer Na-phosphate p H 6 MES/TRIS p H 8.2
Enzyme step 1 Termamyl 100°C, 15-30 min Termamyl 95-100°C, 35 min
p H adjustment pH 7.5
Enzyme step 2 Protease 60°C, 30 min Protease 60°C, 30 min
pH adjustment pH 4.0-4.6 pH 4.1-4.7
Enzyme step 3 Amyloglucosidase 60°C, Amyloglucosidase 60°C,
30 min 30 min
Volume of 96% EtOH
required to precipitage
soluble D F 280 ml 225 ml
Filtering aid Celite 545 Celite
Protein correction N x 6.25 N x 6.25
Ash correction Incineration 525°C Incineration 525°C
188 BACH-KNUDSEN K.E. ET A L .

buffer is replaced with a MES/TRIS (2(N-morpholino) ethanesulphonic acid/tris

hydroxymethyl amino-methane) buffer, one of the p H adjustment is eliminated
and the total volume is reduced.

Starch removal

Starch is removed by simultaneous gelatinisation in a water bath at 100°C and

incubation with heat-stable cc-amylase (Termamyl). This step is followed by
a amyloglucosidase treatment to completely degrade the malto-oligosaccharides
to glucose. Although the incubation with heat-stable a-amylase in combination
with gelatinisation is very efficient, retrograded amylose, formed during food
processing, will not be removed by Termamyl but requires solubilisation with
either 2 mol/1 of potassium hydroxide or dimethylsulphoxide (DMSO). This step
is not included in the official AOAC procedure, hence resistant starch is
measured as D F . Because of the relatively short incubation time, it is important
to grind the samples to particles <0.5 mm. Too large particles have been found
to result in variable and insufficient starch removal (Prosky et al., 1984).

Protein correction

The samples can not be completely depleted for protein by the protease
treatment and it is therefore necessary to correct for residual protein by the factor
N x 6.25. The error introduced by applying the arbitrary conversion factor of
N x 6.25 are insignificant in most cases as residual nitrogen represent only
a minor component of most foods. However, exceptions may include e.g.
sorghum based foods, where a relatively big proportion (40-90%) of the protein
is found as residual nitrogen as compared to other cereals (10-20%) (Bach
Knudsen and Munck, 1985; Bach Knudsen et al., 1988). The determination of
nitrogen in the residues is not an easy task as demonstrated in the first
collaborative trial with the AOAC enzymatic-gravimetric method. The enor-
mous variation in the T D F value for soya isolate could be attributed to variable
residual nitrogen determination (Prosky et al., 1984). The same conclusion was
drawn in the recent BCR Detailed Method Study for the determination of
protein in blanks (Predlington and Brookes, 1995).

Buffer

The first intercollaborative trial with the AOAC method also identified too
high final phosphate concentration, and hence variable ash content of the
gravimetric residues as one of the underlying causes for the relatively poor
reproducibility obtained in this study (Prosky et al., 1984). Reducing the
ANALYSIS OF D I E T A R Y FIBRE 189

phosphate concentration in the following study improved the performance of the

method (Prosky et al., 1985). The replacement of the phosphate buffer with
MES/TRIS buffer in the modified method of Lee et al. (1992) has further reduced
this problem.

Filtration and filter aid

Filtration difficulties, in particular with viscous and desugared fruit samples,

have frequently been encountered as a major problem influencing the general
performance of the method. To avoid some of these difficulties, it is recommen-
ded to reduce portion size from 1.0 to 0.5-0.25 g, which will reduce the filtration
time to reasonable levels (Lee et al., 1992).
Improved performance of the enzymatic-gravimetric procedures can be
obtained by standardising the type of celite used. In a survey among the
participants in the AOAC/AACC 92 collaborative trial, it was reported that
collaborators in research laboraties preferred to use Celite Analytical Filter Aid
(CAFA), instead of Celite 545 AW, because CAFA allowed a minimum loss
during filtration (Lee et al., 1992). However, most laboratories, in which
a multitude of test samples were handled daily, supported the use of Celite 545
AW because of faster filtration rate as compared to that of CAFA. Filtration loss
and incineration loss of crucibles are the most likely cause for the negative
blank-ash values reported for I D F (19 out of 26) and in some cases for T D F ,
while only 3 out of 27 ash values were negative for the SDF blank determination
(Prosky et al., 1992).

Blanks and technical limitations of the gravimetric techniques

All gravimetric techniques, because of the unspecificity, have limitations when

applied to samples with low levels of dietary constituents (Faulks and Boenke,
1994). For the D F methods this is even more pronounced because a correction
for blanks has to be applied. Moreover, the quantitatively smallest D F
subfraction - soluble D F (SDF) - is the one with the largest and most variable
blank value; the 5-95% SDF-blank fraction varies from 0.1 to 11.2 with a mean
value of 5.95 (Table 2) (Prosky et al., 1992). Given these limitation, it is not
surprising that all collaborative trials performed so far have shown far the biggest
relative standard deviations for the SDF fraction.

Collaborative trials

The results obtained in the main collaborative trails with the enzymatic-
gravimetric AOAC methods are shown in Table 3. The method for T D F
190 BACH-KNUDSEN K.E. ET A L .

TABLE 2
Blank values (mg) obtained for total dietary fibre (TDF), soluble dietary fibre (SDF) and insoluble
dietary fibre (IDF)
Mean Range* Reference

TDF-blanks Faulks and Boenke (1994)

Protein 19.67 3.8 - 28.6
Ash 1.65 -5.7 - 13.8
Mean blank 9.30 7.0 - 13.0
SDF-blanks Prosky et al. (1992)
Protein 5.95 0.4 - 9.3
Ash 3.34 -4.7 - 7.9
Mean blank 5.95 0.1 -11.2
IDF-blanks Prosky et al. (1992)
Protein 1.75 0.4 - 6.0
Ash -2.08 -11.3 - 2.1
Mean blank 1.44 -1.2 - 3.9

*values for SDF- and IDF-blanks are 5-95% intervals

determination was accepted as official AO AC method after two (AOAC 84 and

AOAC 85) international collaborative trials (Prosky et al., 1984, 1985). The first
inter-laboratory study, however, revealed big variations and poor repeatability
(r ) and reproducibility (R ); the R was in the range 1.7 to 21.2 giving a relative
95 95 95

reproducibility standard deviation (RSD ) in the range 2.9 to 100.9 %. As

R

indicated previously, three main problems were identified: too high final
phosphate concentration and hence variable ash content of the gravimetric
residues, problems with residual nitrogen determination and variable starch
removal. Addressing these limitations in the second collaborative trial resulted in
a significant improvement in reproducibility (R 0.8-3.8) and repeatability (r
95 95

0.4-2.8) (Prosky et al., 1985). The collaborative study performed locally in

Switzerland (Swiss 87) demonstrated even better reproducibilities with values in
the range of 1.0 to 1.1 (Schweizer et al., 1988). The method for T D F is now
approved the legal or recommended procedure for food analysis in the USA and
several continental European countries.
The wish to validate a method for separate determinations of soluble and
insoluble D F was studied in three AOAC and one Ministry of Agriculture,
Fisheries and Food (MAFF) organised collaborative studies (Prosky et al., 1988,
1992, 1994; Wood et al., 1993). The result of the first AOAC collaborative trial
(AOAC 88) was satisfactory with regard to the determination of TDF, and there
was also a good agreement between the sum of I D F and SDF and the separate
T D F determination (Prosky et al., 1988). The reproducibility of SDF, however,
was unacceptably high. In a follow-up study (AOAC 92) comprising 22 samples,
the R of I D F ranged from 1.7 to 22.2 for the samples analysed; almost half the
95
ANALYSIS OF DIETARY FIBRE 191

samples tested had a R S D < 10% (Prosky et al., 1992). For SDF it was higher
R

and approximately 50% of the samples analysed had an R S D > 20% and 45%
R

had an RSD between 10 and 20%. In the most recent AOAC collaborative trial
R

(AOAC 94) comprising six samples, the average R of SDF was 2.52 (Prosky et
95

al., 1994). However, in 4 of the 6 samples RSD was lower than 10%, while two
R

samples with 1.21 and 4.13% SDF had RSD of 28.28 and 24.12% RSDR
R

respectively. Based on the last AOAC collaborative trial, the enzymatic-

gravimetric method for the determination of SDF was adopted first action of
AOAC International. In the M A F F IV study the R was higher with average
95

values for SDF of 2.33; for I D F of 3.11 and of T D F of 5.34 (Wood et al., 1993).
Supplementation of the well known enzyme-neutral detergent fibre (NDF)
procedure with an separate determination of SDF has been proposed to provide
a practical method for rapid determination of T D F (Mongeau and Brassard,
1986, 1990). This method was tested in a collaborative trial in Canada (Canada
90), and claims to give values for N D F and soluble (SOL) D F in agreement with
T D F obtained with the original enzymatic-gravimetric AOAC method. As
shown in Table 3, the R and r are satisfactory and comparable with the
95 95

enzymatic-gravimetric AOAC procedure (Mongeau and Brassard, 1990). A ma-

jor drawback with the method, however, is that the analysis of insoluble and
soluble D F are performed on two different samples and under different
solubilisation conditions. The risk is that some D F components are analysed
twice or not at all.
Recently, a nonenzymatic-gravimetric method for determination of D F in
products with little ( < 2 % ) or no starch such as fruits, and vegetables and many
purified polysaccharides were adopted first action by AOAC. The method gives
comparable results to the enzymatic-gravimetric AOAC method (Li and
Cardozo, 1994).

E N Z Y M A T I C - C H E M I C A L METHODS

The main characteristics of the most recent modifications of the enzymatic-

chemical Englyst and Uppsala methods are summarised in Table 4. In short the
methods consist of the following steps: (a) weighing into a centrifuge tube, (b)
solubilisation and removal of starch by specific enzymes, (c) precipitation of
soluble polymers by 80 % (v/v) ethanol, (d) swelling of polysaccharides with 12
mol/I of H S 0 and hydrolysis of polysaccharides to monosaccharides with
2 4

0.4-2.0 mol/1 H S 0 and (e) end-point determination of neutral non-starch

2 4

polysaccharide (NSP) residues by either GLC, HPLC or colorimetry and

measurements of uronic acids by colorimetry.
The Uppsala method was published in 1979 and the Englyst method in 1982
(Theander and Aman, 1979; Englyst et al., 1982). The Uppsala method was
h-) TABLE 3
< Summary of main collaborative studies of methods for total dietary fibre (TDF) insoluble dietary fibre (IDF) and soluble dietary fibre (SDF) using
gravimetric procedures
H
PJ Number of Number of DF DF content Regeatability r 95 Regroducibility R 95
W
results labs samples component mean range mean range mean range Reference
ú
z
w
AOAC methods
AOAC 84 750 (32) 13 TDF 17.5 (3-89) 2.39 (1.3-3.8) 5.43 (1.7-8.8) c
Prosky et al. (1984)
W5 AOAC 85 160 (9) 9 TDF 19.1 (1-87) 1.26 (0.4-2.8) 2.00 (0.8-3.8) Prosky et al. (1985)
Q Swiss 87 90 (15) 3(P) TDF 11.0 (2-20) 0.73 (0.3-1.1) 1.07 (1.0-1.1) Schweizer et al. (1989)
D AOAC 88 172 (9) 10 TDF 21.0 (1-87) 1.78 (1.0-2.7) 2.68 (1.3-4.0) d
Prosky et al. (1988)
Z SDF 2.4 (0-9) 1.05 (0.5-2.0) 1.47 (0.7-4.5) c
í¿
i IDF 19.3 (1-87) 1.30 (0.1-3.6) 2.20 (0.9-4.0/
EC AOAC/AACC 92 98 (10) 8(P) TDF 39.5 (13-72) 2.58 (1.1-4.0) 3.64 (2.2-6.7) Lee et al. (1992)
U SDF 10.6 (3-31) 1.70 (1.0-2.6) 3.30 (1.6-6.7)
< AOAC 92 821 (10) 22
IDF
SDF
29.3
10.8
(11-36)
(2-33)
2.30
2.44
(1.3-3.6)
(1.1-6.1)
3.36
4.80
(1.8-7.3)
(1.7-12.1) 6
Prosky et al. (1992)
h
IDF 30.1 (4-65) 3.22 (2.0-7.9) 7.53 (1.7-22.2)
AOAC 94 340 (10) 6 TDF 29.1 (14-66) 2.32 (1.4-3.2) 3.47 (2.2-5.7) Prosky et al. (1994)
SDF 9.5 (1-21) 1.65 (0.6-2.5) 2.52 (1.0-3.8)
IDF 20.4 (13-46) 2.27 (1.7-3.6) 3.64 (2.5-6.1)
MAFF IV 282 (16) 12 TDF" 10.8 (3-27) 2.11 (1.1-3.4) 5.34 (2.0-6.8)' Wood et al. (1993)
SDF 2.5 (0-6) 1.11 (0.4-2.1) 2.33 (0.7-4.9)
IDF 8.1 (3-22) 1.50 (0.7-3.1) 3.11 (1.2-6.7)
Neutral detergent fibre plus soluble fibre estimate
Canada 90 98 (10) 5(P) TDF 19.6 (1-46) 1.99 (0.5-2.9) 3.60 (0.6-5.8) Mangenau and
Brassard (1990)
SOL 5.4 (0-10) 1.19 (0.3-2.2) 2.34 (0.7-4.5)
NDF 14.2 (1-41) 1.44 (0.3-2.3) 2.34 (0.8-4.4)
AOAC simplified method
AOAC 94 108 (9) 6 TDF 31.6 (13-77) 1.69 (0.6-3.8) 3.83 (2.0-7.7) Li and Cardozo (1994)
a (P) denotes trials in which pre-trial samples were given f without fabulous fibre (R = 12.60) and soy isolate (R
95 95 = 5.10)
b TDF is calculated as the sum of IDF and SDF g without prunes (R = 26.68) and raisins (R, = 16.68)
95 5
c without soy isolate (R = 21.2)
95 h without prunes (R = 25.14) and raisins (R = 26.57)
95 95
d without fabulous fibre(R = 5.33) and soy isolate (R =3.89)
95 95
i without coconut (R = 17.00)
95
OS e without fabulous Fibre(R = 1 1.32)
95
 TABLE 4
Comparison of main steps in the Englyst and Uppsala methods for the determination of dietary fibre by GLC
Procedure Englyst method (Englyst, 1994) Uppsala method (Theander et al., 1994)
Sample size ( D M ) 50-300 mg 250-500 mg
Fat removal Extraction with acetone i f fat > 5 % Extraction with petrolium ether i f fat > 6%
Starch removal (1) DMSO (0.5 h, 50°C + 10 min at 100°C) (1) Termamyl (0.5 h, 96°C) in acetate buffer
(2) Termamyl (10 min, 100°C) in acetate buffer (0.1 M , pH 5)
(0.08 M , pH 5.2) (2) Amyloglucosidase (16 h, 60°C)
(3) Pancreatin and pullulanase
(0.5 h, 50°C + 10 at 100°C)
Precipitation of soluble fibre 80% (v/v) acidified ethanol (30 min, 0°C) 80% (v/v) ethanol (1 h, 4°C)
Drying of the fibre Acetone washing, 80°C until dry 45°C overnight
Analysis of neutral sugars (1) 12 mol/1 H S 0 (1 h, 35°C)
2 4 (1) 12 mol/1 H S 0 (1 h, 35°C)
2 4

(2) 2 mol/1 H S 0 (1 h, 100°C)

2 2 (2) Addition of inernal standard (myo-inositol)
(3) Addition of internal standard (allose) (3) 0.4 mol/1 H,S0 (1 h, 125°C)
4

(4) Reduction of neutral sugars to alcohols with (4) Reduction of neutral sugars to alcohols
sodium borohydrate with sodium boryhydrate
(5) Alditole acetate formation of alcohols with (5) Alditole acetate formation of alcohols for
1-rnethylimidazole/acetic acid anhydride with 1-methylimidazole/acetic acid
anhydride
(6) Individual correction factors for acid (6) Individual correction factors determined for
hydrolysis losses each sugars
Analysis of uronic acids (1) Scott procedure with 3,5-dimethylphenol (1) Scott preocedure with 3,5-dimethylphenol
(2) Calibration based on galacturonic acid with (2) Calibration on galacturonic acid with
a fixed correction for hydrolysis losses corretion of acid hydrolysis losses
Lignin Not determined Gravimetrically as Klason lignin
194 BACH-KNUDSEN K.E. ET A L .

developed by workers whose primary interest was plant cell wall chemistry
(Theander and Aman, 1979a), while the method of Englyst evolved within
a nutritional research environment focussed on the measurement and charac-
terisation of the carbohydrates in foods (Englyst et al., 1988, 1989) as well as
understanding how the polysaccharides of plant cell walls in the food influence
the physiology of the human gastrointestinal tract (Macfarlane and Cummings,
1991). Although a number of modifications have converged the methods, there
are still differences of which the most important are: (a) the inclusion of the
DMSO solubilisation step for "resistant starch" in the Englyst procedures, (b)
the acid hydrolytic conditions, (c) the type of internal standard applied, (d) the
compensation for hydrolytic losses and (e) the measurement of Klason lignin in
the Uppsala method (Englyst et al., 1994; Theander et al., 1994).

Starch removal

The Uppsala and the Englyst methods use heat stable a-amylase (Termamyl)
to hydrolyse starch to malto-oligosaccharides but differ in the use of pancreatin
and pullulanase (Englyst) and amyloglucosidase (Uppsala) to complete the
degradation to glucose. The method of Englyst further includes a DMSO step to
solubilise "resistant starch". It has been questioned i f the DMSO treatment
increases the solubility of D F components, since this reagent is used in structural
analysis to solubilise hemicellulose polysaccharides. However, Englyst and
Cummings (1984) did not found any differences in either cellulose or non-
cellulosic polysaccharides in samples treated with or without DMSO. It is also
clear that DMSO treatment did not cause any loss of NSP as documented in the
BCR Detailed Method Study and the BCR Reference Materials Study (Faulks
and Boenke, 1994; Pendlington et al., 1996).

Acid hydrolysis

Acid hydrolysis of insoluble D F polysaccharides requires a two-step procedu-

re involving dispersion of cellulose with 12 mol/1 of H S 0 and hydrolysis with
2 4

either 2 mol/1 of H S 0 for 1 h at 100°C (Englyst) or 0.4 mol/1 of H S 0 for 1 h at

2 4 2 4

125°C (Uppsala). The conditions chosen for the acid hydrolysis will inevitably be
a compromise between the wish of obtaining maximal yield of sugars as well as
minimal destruction of the released monosaccharides. The typical losses during
acid hydrolysis (directly related to the treatment with acid or incomplete
desulphation) are in the range of 4-11% (Englyst et al., 1992). I t is possible to
account for hydrolysis losses by using corrections factors derived either from
concomitant hydrolysis of reference sugar mixtures (Uppsala) or using fixed
corrections factors (Englyst) (Theander and Westerlund, 1986; Englyst et al.,
ANALYSIS OF DIETARY FIBRE 195

1992). In the Uppsala procedure, the internal standard - myo-inositol - is added

before the acid hydrolysis step, while the Englyst method adds the internal
standard - D-allose - after completing the acid hydrolysis. Individual corrections
for each sugars are used in both methods. However, when converting from
mono- to polysaccharides the Englyst methods use a mean conversion factor of
0.89, while the Uppsala method use 0.88 for pentoses and deoxyhexoses and 0.90
for other hexoses.
The uronic acids determination is a challenge in itself. In terms of specificity
the decarboxylation method (Theander and Aman, 1979), originally used in the
Uppsala procedure, has several advantages over the colorimetric procedures
(Quigley and Englyst, 1994). However, because the equipment for performing
the decarboxylation determination is not available in all laboratories, the
colorimetric method of Scott, in spite of its limitations, is the procedure
employed for uronic acid determination in both the Englyst and the Uppsala
procedures (Englyst et al., 1994; Theander et al., 1995). The compensation for
hydrolysis losses of uronic acids are done in different ways in the two methods.
While the Uppsala method carry the uronic acid standards through the
autoclaving procedure and further compensate for the lower degradation of
oligomers than of monomers by a factor of 0.88, the Englyst method use a fixed
correction of the standard to compensate for the hydrolysis losses of uronic
acids. The BCR Reference Material Study did not show any significant
differences between the two methods when analysing for uronic acids in cereal
samples with low levels of uronic acids (Pendlington and Brookes, 1995).

End-point determination

The end-point determination of sugars can optionally be performed by

HPLC. The advantage of the HPLC procedure is avoiding the derivatisation step
and the possibility of detecting any oligosaccharides deriving from incomplete
hydrolysis of NSP. Recent progress in column technology and the introduction
of the pulsed amperometric detection have made the HPLC technique more
sensitive and specific. However, although HPLC has several theoretical advan-
tages over GLC for the determination of sugar residues, HPLC is less frequently
used as compared to GLC for end-point determination of sugar residues in D F
analysis (Englyst et al., 1994). This is presumably due to the successful
introduction of the 1-methylimidazole to catalyse the acetylation of sugar
alcohols in D F analysis (Englyst and Cummings, 1984). Compared with previous
techniques, the derivatisation has become much faster and easier and can be
performed in the presence of borate without interference. Moreover, the GLC
technique has the advantage over the HPLC technique that a chromatographic
run is completed within 8-10 min as compared to ~25 min for a HPLC run.
196 BACH-KNUDSEN K.E. ET A L .

Colorimetric assays are an alternative to GLC and HPLC determinations if

only values for total NSP (or insoluble and soluble) are required. However,
colorimetric methods have limitations. The method for measuring total reducing
sugars by dinitrosalicyclic acid used by Englyst and Hudson (1987) exhibit
different colour yields for pentoses, hexoses and uronic acids. The same is the
case with para-hydroxy benzoic acid hydraside used by Faulks and Timms
(1985). This may explain why some foods show differences in NSP values
obtained by GLC or colorimetry in spite of an overall good correlation. Recent
modifications of the dinitrosalicyclic acid assay have addressed some of these
problems and corrected for the interferences from other food components of
processed foods, and thereby made the procedure more robust (Englyst et al.,
1994).

Lignin determination

Klason lignin, together with structural protein the non-carbohydrate part of

the cell walls, is measured gravimetrically as the sulphuric acid resistant residue
in the Uppsala method, but not by the Englyst method (Theander and Aman,
1979b; Theander and Westerlund, 1986; Englyst et al., 1992). In plant materials
used for animal production lignin is an important dietary constituent with values
in the rangeof 1-7% in whole grain cereals. Lower (flour< 1%) and higher values
(bran 7-15%) may be found in cereal flour for human consumption or
by-products for animal feeding (Bach Knudsen, 1997). It is argued that lignin, in
human diets, represent only a small and therefore insignificant fraction. The
physiological importance of lignin lies in the affect on the solubility of D F
polysaccharides which has implications for the degradation of fibre in the large
bowel of man and animals and consequently for the bulking properties and the
energy values of the fibre. It should also be mentioned that Klason lignin, in
addition to true lignin, also contain Maillard reaction products and degradation
products formed as the result of heat processing (Theander et al., 1990).

Collaborative trials

The Englyst GLC and colorimetric procedures have been evaluated in four
collaborative trials organised by the M A F F (Table 5). In the first study, M A F F I ,
five different methods were compared, but only the Englyst GLC method was
selected for further studies (Cummings et al., 1985). In M A F F I I seven breads
were analysed by the GLC procedure, which showed acceptable repeatability but
unsatisfactory reproducibility; the R was in the range of 3.7 to 8.7 correspon-
95

ding to RSD in the range of 29.9 to 59.5 % (Englyst et al., 1987a). In M A F F I I I

R

similar samples were analysed by an improved GLC procedure and by two

 >
z
>
r
<
TABLE 5
Summary of main collaborative studies of methods for total non-starch polysaccharides (T-NSP), soluble non-starch polysaccharides (S-NSP), O
insoluble non-starch polysaccharides 0-NSP) and total dietary fibre (TDF) using enzymatic-chemical methods
O
Number of Number of DF DF content Repeatability r Reproducibility R H-(
95 95
ra
lf
resu s (labs) samples" component mean (range) mean (range) mean (range) Reference H
>
Englyst methods
MAFF I I 238 (17) 7 T-NSP 5.9 (3-10) 1.48 (0.6-2.3) 6.04 (3.7-8.7) Englyst et al. (1987a)
<
HH

MAFF I I I Englyst et al. (1987b)

GLC 266 (19) 7(P) T-NSP 6.5 (1-10) 1.67 (0.7-3.1) 2.62 (1.2-3.8) »
Colorimetry 266 (19) 7(P) T-NSP 7.2 (1-12) 1.77 (0.9-2.7) 3.20 (1.1-5.2) ra
MAFF IV Wood et al. (1993)
GLC 438 (22) 12 T-NSP 8.9 (1-25) 1.24 (0.7-3.2) 2.67 (1.3-8.5)
S-NSP 3.8 (0-12) 1.36 (0.6-4.2) 2.01 (0.6-6.6)
604 (31) 12 I-NSP 5.1 (1-13) 0.95 (0.5-1.8) 1.92 (0.9-4.9)
Colorimetry T-NSP 9.4 (2-24) 1.31 (0.7-2.9) 3.56 (2.0-6.2) Wood et al. (1993)
S-NSP 3.8 (1-10) 1.61 (0.8-3.1) 2.28 (1.0-5.0)
I-NSP 5.6 (2-14) 0.98 (0.7-1.9) 2.75 (1.2-4.6)
Uppsala methods
AOAC 95 Theander et al. (1995)
GLC/gravimetry 112 (7) 8 TDF 32.1 (5-84) 3.23 (0.9-8.1) 6.03 (1.5-13.7)
" (P) denotes trials in which pre-trial samples were given
198 BACH-KNUDSEN K.E. ET A L .

colorimetric procedures of which only the dinitrosalicyclic acid assay was

applicable to all food types (Englyst et al., 1987b). The R95 of the GLC method
was strongly improved in the M A F F I I I study, and it was concluded that the
Englyst method with GLC end-point determination was recommended as
a reference method for the determination of D F in the U K (Englyst et al., 1987).
The results further indicate that the Englyst procedure with colorimetric
end-point was suitable as quality control method. In the most recent M A F F I V
study 12 samples were analysed for total, soluble and insoluble D F by the
enzymatic-gravimetric AOAC and the Englyst GLC and colorimetric methods
(Wood et al., 1993). The r and R of the Englyst GLC and colorimetric
95 95

methods were in line with results obtained in the M A F F I I I study with R95
values for total, soluble and insoluble NSP of the Englyst GLC method of 2.67,
2.01 and 1.92 and of the Englyst colorimetry method of 3.56, 2.28 and 2.75,
respectively. These results are also acceptable compared with results obtained
with the enzymatic-gravimetric AOAC method (Table 2).
Currently, the Uppsala method has only been studied in one AOAC organised
collaborative trial (Table 5). The mean R95 for T D F was 6.03, which is
somewhat higher than obtained for T-NSP in the M A F F IV study by the Englyst
GLC method (Theander et al., 1995). However, taking into account that the
samples used in the AOAC 95 study had a higher D F level, the RSD were in the
R

same order.
The M A F F I I I and I V study and the recent BCR Detailed Method Study
revealed that the Englyst colorimetric assay gave from 5 to 15 % higher values
than the GLC end-point assay (Englyst et al., 1987b; Wood et al., 1993; Faulks
and Boenke, 1994). It has been speculated that this was due to incomplete acid
hydrolysis, which would affect the D F values from the GLC method more than
from the colorimetric method, as the latter measure in part di- and oligosac-
charides (Schweizer, 1989). However, this is presumably not the case as tests in
our laboratory indicate that the acid hydrolytic conditions applied in the Englyst
procedures result in almost complete hydrolysis and limited destruction of
monosaccharides. The BCR Detailed Method Study and the recent BCR
Reference Materials Study support this view (Faulks and Boenke, 1994;
Pendlington and Brookes, 1995). It is therefore more likely that products formed
during heat processing (5-hydroxy-methyl-2-furaldehyde and 2-furaldehyde)
and other non-carbohydrate components of the cell walls interfere in the
colorimetric assay. A recent modification of the colorimetric assay claims to
solve interference from products formed during food processing (Englyst et al.,
1994).
The need for special expertise and experience when performing enzymatic-
chemical GLC procedures was earlier recognised as one of the reasons why these
methods performed less satisfactory as compared with the more simple
ANALYSIS OF D I E T A R Y FIBRE 199

enzymatic-gravimetric procedures (Englyst et al., 1987a). As the laboratories

participating in the collaborative trials have become more experienced, the
reproducibility of the more complex enzymatic-chemical Englyst and the
Uppsala methods has reached acceptable levels (Table 5). Moreover, the results
of the BCR Reference Materials Study show that the coefficient of variation
between laboratories in absolute terms are more or less at the same level for the
AOAC enzymatic-gravimetric methods (original and modified), the Englyst
GLC and colorimetric methods and the Uppsala method (Pendlington and
Brookes, 1995).

T E C H N I C A L PROBLEMS RELATED TO A L L METHODS

Enzymes

A common feature of the enzymatic-gravimetric and enzymatic-chemical D F

methods is the use of heat-stable a-amylase (Termamyl) to degrade starch to
malto-oligosaccharides. It has been documented that the simultaneous gelatini-
sation and hydrolysis of starch at 100°C does not degrade fibre polysaccharides
(Theander and Aman, 1979b). For complete hydrolysis of starch to glucose, the
AOAC and the Uppsala procedures use various types of amyloglucosidases,
while the Englyst methods use a combination of pancreatin and pullulanase.
Unfortunately, many commercial amyloglucosidase preparations at present
have side activities, e.g. /?-glucanase and xylanase, which will result in depolyme-
risation of these fibre polysaccharides and potentially lead to lower values for the
fibre contents in, for example, cereal samples (Theander et al, 1990). Consequen-
tly, it is important to check all enzyme batches for side activities prior to use in
D F analysis.

Alcohol strength

Alcohol strength of around 80% (v/v) is employed in all D F methods for the
recovery of soluble polysaccharides and is generally considered sufficient to
precipitate saccharides with a degree of polymerisation of 10 or more; i.e. the
arbitrary defined borderline between oligo- and polysaccharides. However,
highly branched polysaccharides, e.g. arabinans in sugar beet fibre, may be
soluble even at significantly higher strengths of alcohol (Asp, 1990; Theander et
al., 1990). In the BCR Detail Method Study, 2-4% of the sugar residues in the
polysaccharides mixture and 3-7% of the sugar residues in the sugar beet fibre
were found to be soluble in 80% ethanol (Faulks and Boenke, 1994). These data
are in accordance with studies conducted by Theander and Westerlund (1988),
200 BACH-KNUDSEN K.E. ET A L .

who found that the amount of soluble polysaccharides not recovered in 80%
ethanol corresponded to 1-6% of the total fibre content of the original sample.
The highest values were found in the most severely heat-treated samples (bread
crust).

INTERCOMPARISON STUDIES

The number of controlled comparisons between methods is extremly limited.

In general, however, the available data show that the AOAC and the Uppsala
procedures give higher values for D F than those obtained by the Englyst
procedures. This is seen of Table 6 that list D F values obtained by these methods
in the most recent BCR Reference Materials Study (Pedlington et al, 1996). The
reason for the discrepancies in D F values is primarily that the three procedures
do not measure the same components. The AOAC (Asp et al. 1992) and the
Uppsala procedures (Theander et al., 1994) are designed to measure total D F as
NSP, resistant starch (retrograded amylose) and lignin, while the Englyst
procedures (Englyst et al., 1994) are designed to measure all NSP constituents
excluding any form of resistant starch. When correcting for resistant starch and
lignin the AOAC, Uppsala and Englyst procedures gives identical mean values
for the samples used in the intercomparison study of the recent BCR Reference
Materials Study: white bread, whole wheat bread, a mixture of white and whole
wheat bread and cornflakes (Pedlington and Brookes, 1995). The same factors
also explain the differences between the values obtained for bran breakfast cereal
and harricot beans, while for the pectin containing materials (carrot and apple),
the differences between the methods are more difficult to explain. The very good
agreement between all methods for full fat soya, however, is very encouraging.

CHOICE OF A N A L Y T I C A L M E T H O D

The choice of analytical method depends primarily on the purpose of the

analytical work (Southgate, 1995). This involves consideration of the criteria
which the method and the values produced must meet to satisfy the needs of the
analytical work. Since the performance of the D F methods after improvements is
almost comparable (Tables 3, 5, 6) the choice of method has to be set against the
criteria determined by the purpose of the analysis (Table 7). While the methods
are prescribed for analysts involved in analytical work of a regulatory kind,
researcher can choose the methods giving the information that best suit the
purpose of his work. Whereas a physiologist studying mechanisms may want to
use a method providing detailed informations of the D F fractions, total D F may
 TABLE 6
Dietary fibre values (mean standard deviation) of reference materials analysed with the gravimetric procedures and the enzymatic-chemical methods
Whole bread Mix of white and Bran breakfast Harricot Carrot Apple Full fat soya Method
whole-meal bread cereal beans
AOAC 11.8+0.6 8.0 + 0.6 30.2 + 0.8 25.6 + 0.5 31.1+0.6 16.4 + 0.4 12.6 + 0.5 Prosky et al. (1988)
AOAC 12.1 + 0.8 8.1+0.5 30.5 + 0.6 25.9+1.5 29.5 + 0.4 14.9+1.0 12.4+2.1 Lee et al. (1992)
Uppsala 11.5 + 0.6 8.2 + 0.6 27.6+1.8 23.7+1.5 29.8+1.1 16.2 + 0.8 12.8 + 0.9 Theander et al. (1994)
Englyst GC 9.1+0.7 6.3 + 0.5 24.1+0.8 19.8+1.0 27.1 + 0.6 13.7 + 0.5 11.9 + 0.7 Englyst et al. (1994)
Englyst Col. 9.6 + 0.6 6.8 + 0.4 25.0+1.1 20.1 + 0.6 25.2+1.2 13.4+0.5 12.3 + 0.8 Englyst et al. (1994)
reference: Pedlington and Brookes (1995); Pedlington et al. (1996)
202 BACH-KNUDSEN K.E. ET A L .

TABLE 7
Purposes for which dietary fibre is measured and the criteria which the analytical method has to meet
Purpose Characteristics of a suitable method Resource required Time required
Research on Detailed characterisation of dietary Not critical; Not
physiological fibre. Flexible methods amenable outcome most critical
mechanism of to expansion to give even greater important
action detail

Nutritional Nutritionally relevant to biological Critical in relation Critical in relation

composition role and to current nutritional to work load to costs
data bases guidance. Compoartive with
labelling regulations

Food labelling Defined, with good reproducibility. Very critical Very critical
Compatible with nutritional adcive
to consumer

Food regulation Defined precise protocol. Very critical Very critical

Reproducible

Quality control Simple emperical method. Very critical Very critical

Compatible with regulation.
Repeatable, consistens performance
over long period

reference: Southgate (1995)

be enough when studying the variation in chemical composition of cereals as

influenced by e.g. variety, location and year of harvest. In all case, however, the
researcher needs to take into consideration whether resistant starch and lignin
should be included in the D F value or measured separately from the NSP.

CONCLUSIONS

The enzymatic-gravimetric and the enzymatic-chemical techniques for the

determination of D F in foods have progressed to a point where they are as
reproducible as other analytical methods used for food and feed labelling and for
research. The higher values obtained by the enzymatic-gravimetric AO AC
methods and the Uppsala method as compared to the Englyst methods are
primarily due to retrograded amylose (resistant starch) and lignin being included
in the former methods. Accounting for these differences, the analytical methods
give comparable results.
ANALYSIS OF D I E T A R Y FIBRE 203

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STRESZCZENIE

Zalety i ograniczenia metod analizy włókna pokarmowego

Istnieją dwa zasadnicze sposoby oznaczania włókna pokarmowego (DF) w żywności dla ludzi
i paszach dla zwierząt, a mianowicie: enzymatyczna bądź nieenzymatyczna grawimetryczna metoda
AOAC lub enzymatyczno-chemiczna Englysfa i Uppsala. Głównymi czynnikami analitycznymi
wpływającymi na przebieg analizy metodą enzymatyczno-grawimetryczną są: zmienność stopnia
usuwalności skrobi, zbyt wysokie stężenie końcowe buforu fosforanowego i spowodawana tym duża
zmienność w zawartości popiołu w masie końcowego produktu, zagadnienie sączenia lepkich
i odcukrzonych prób owoców i wreszcie zmienna wartość prób ślepych. Standaryzacja i poprawki
wprowadzane do metod grawimetrycznych poprawiły wyniki doprowadzając do zadowalającego
stopnia dokładności. Metody enzymatyczno-chemiczne z zastosowaniem chromatografii cieczowo-
206 BACH-KNUDSEN K.E. ET A L .

-gazowej (GLC), wysokosprawnej chromatografii cieczowej (HPLC) lub kolorymetrii stosowane do

końcowego pomiaru wartości są zazwyczaj bardziej skomplikowane, wielostopniowe i wymagają,
w przypadku metod GLC I HPLC, bardziej złożonej i nowoczesnej aparatury niż metoda
grawimetryczna. Stopień usunięcia skrobi, hydrolityczne warunki kwaśnej hydrolizy polisachary­
dów DF, poprawkowe współczynniki dotyczące strat hydrolitycznych cukrów i postępowanie
laboratoryjne zotały uznane jako główne czynniki wpływające na odtwarzalność wyników stosowa­
nymi metodami. Modyfikacje i optymalizacja poszczególnych etapów oraz ulepszenia techniczne
w laboratoriach uczestniczących we wspólnych badaniach poprawiły powtarzalność metod en-
zymatyczno-chemicznych do poziomu podobnego jaki uzyskiwany jest metodami enzymatyczno-
grawimetrycznymi AOAC. Zagadnieniami technicznymi, które mogą powodować błędy w oznacza­
niu D F wszystkimi metodami są: niezupełne wytrącenie 80%(v/v) etanolem, zanieczyszczenia
bakteryjnymi amyloglukozydazami powodującymi depolimeryzację i potencjalne straty polisachary­
dów DF. Ostatnie badania z zastosowaniem prób referencyjnych (Reference Materiał Study)
wskazują, że różne metody dają porównywalne wyniki dla produktów zbożowych, gdy zastosuje się
poprawki na ligninę i na skrobię oporną na hydrolizę. To samo dotyczy całych nasion soi.
W posumowaniu można stwierdzić, że metody oznaczania D F dają zadowalająco odtwarzalne
wyniki podobnie jak inne metody analityczne stosowane do oceny pasz i żywności oraz do celów
badawczych.