Measurement of Dietary Fiber

AOAC Method 2017.16

Barry V. McCleary, CEO

Megazyme

Trowel Definition of Dietary Fibre (1976)

Trowell definition:

- primarily a physiological definition, based on edibility and

resistance to digestion in the human small intestine;

the definition includes indigestible polysaccharides such as

gums, modified celluloses, mucilages and pectin, and non-

digestible oligosaccharides (NDO).

Prosky method (introduced to serve the Trowell definition):

Thermostable a-amylase at ~98oC plus AMG at 60oC to hydrolyse starch to

glucose.

Protease at 60oC to hydrolyse protein to peptides.

Higher molecular weight DF recovered by alcohol precipitation, which also

removed fragments derived from starch and protein.

However:

- Protein is not completely hydrolysed - so the protein remaining in the

residue is measured and subtracted (as also is ash).

- starch is not completely hydrolysed (concept of resistant starch).

Measurement of Dietary Fiber (HMWDF; AOAC Method 985.29 - Prosky)

Sample (1 g)

Starch hydrolysis

Protein hydrolysis

+ Thermostable a-Amylase

(30 min, 95-98oC, pH 8.2)

Change pH to ~ 7.5, ~ 60oC

Alcohol precipitation, solvent washing, drying,

filtration; (2 residues)

Ash

Determination

Protein

Determination

HMWDF (IDF + SDFP)

+ Protease

+ AmyloglucosidaseStarch hydrolysis

Change pH to ~ 4.5, ~ 60oC

(AOAC Method 985.29)

The aim was simply to remove

protein and starch so that the

residue could be measured

gravimetrically and ash and

residual protein subtracted to

give dietary fiber values.

Measurement of Dietary Fibre

Sample (1 g)

Starch hydrolysis

Protein hydrolysis

+ Thermostable a-Amylase

(30 min, 95oC, pH 8.2)

Change pH to ~ 7.5, ~ 60oC

Alcohol precipitation, solvent washing, drying,

filtration; (2 residues)

Ash

Determination

Protein

Determination

HMWDF (IDF + SDFP)

+ Protease

+ AmyloglucosidaseStarch hydrolysis

Change pH to ~ 4.5, ~ 60oC

(AOAC Method 985.29)

AOAC

985.29

991.43

991.42

993.19

994.13

2001.03

AOAC

HMWDF (IDF + SDFP) - Prosky

IDF and SDFP - Lee

IDF

SDFP (Prosky addition)

HMWDF (sugar composition) (Uppsala)

HMWDF (Prosky) + SDFS (Matsutani)

Concentrate ethanolic

filtrate. Desalt, HPLC

SDFS (NDO)

Codex Definition of Dietary Fibre

In 2008 the Codex Committee on Nutrition and Foods for Special Dietary Use

(CCNFSDU) established a definition of Dietary Fibre.

In June, 2009, this definition was accepted by the Codex Alimentarius

Commission (CAC).

Dietary fibre: Carbohydrate not hydrolysed by the endogenous enzymes in the human small intestine; - (which include resistant starch)

• Edible carbohydrate polymers naturally occurring in the food as consumed.

• Carbohydrate polymers which have been obtained from raw materials by physical, enzymatic or chemical means

• Synthetic carbohydrate polymers

The latter two groups must be shown to have a physiological effect of benefit to health.

Footnotes: Dietary fibre: • Includes fractions of lignin and the other compounds “associated” with plant

polysaccharides

• The decision on whether to include carbohydrates from 3 to 9 monomeric units is left to national authorities

Dietary Fiber(AOAC Method 985.29)

(AOAC Method 991.43)

Galacto-oligosaccharides

Raffinose/Stachyose

Cellulose

Beta-Glucan

Galactomannan

Arabinoxylan

Inulin FOS

Resistant Starch

Pectin

Arabinogalactan

Polydextrose

Fibersol 2

Analysis of Fiber Components – AOAC Method 985.29 (Prosky)

Resistant Starch Values: in vitro Vs in vivo Data

Source of

Starch

RS (in vivo) McCleary

(AOAC

2002.02)

Champ Englyst

Native

potato

78.8 77.0 77.7 66.5

Native

amylomaize

50.3 51.7 52.8 71.4

Retrograded

Amylomaize

30.1 42.0 29.6 30.5

Bean Flakes 9 – 10.9 14.3 11.2 10.6

Corn Flakes 3.1 – 5.0 4.0 4.3 3.9

Canned

Beans

16.5 16.5 17.1 17.1

ActistarR 59 58 57.0 63

RS, % (in vitro Methods/Results)

Total Dietary Fiber(AOAC Method 985.29)

(AOAC Method 991.43)

Galacto-oligosaccharides(AOAC Method 2001.02)

Raffinose/Stachyose

Cellulose

Beta-Glucan(AOAC Method 995.16)

Galactomannan

Arabinoxylan

Inulin FOS(AOAC Method 997.08 / 999.03)

Resistant Starch(AOAC Method 2002.02)

Pectin

Arabinogalactan

Polydextrose(AOAC Method 2000.01)

Fibersol 2(AOAC Method 2001.03)

Analysis of Fiber Components; AOAC 985.29 + Extra

Total Dietary Fiber(AOAC Method 985.29)

(AOAC Method 991.43)

Galacto-oligosaccharides

Raffinose/Stachyose

Cellulose

Beta-Glucan

Galactomannan

Arabinoxylan

Inulin FOS

Resistant Starch

Pectin

Arabinogalactan

Polydextrose

Fibersol 2

Analysis of Fiber Components; AOAC Methods 2009.01 / 2011.25

Measurement of TDF (AOAC Method 2009.01)

Sample (1 g)

Alcohol precipitation, solvent washing, drying,

filtration; (2 residues)

Ash DeterminationProtein Determination

Ethanolic filtrate. Concentrate; desalt on column,

re-concentrate;

HPLC – Waters Sugar Pak

Starch hydrolysis

Change pH to ~ 8.2, Heat to ~ 95oC -To denature protein and,

-To inactivate starch hydrolyzing enzymes

+ Protease (60oC, pH 8.2)

AOAC 2009.01: INTDF

Change pH to ~ 4.5, ~ 60oC

PAA (2 KU) / AMG 0.14 KU

(16 h, 37oC, pH 6.0)

SDFS (NDO)

Protein hydrolysis

TDF = HMWDF + SDFS

HMWDF (IDF + SDFP)

AOAC 2002.02 AOAC Method 2009.01

Native Potato Starch 64.9 56.8

ActistarR 58.0 48.8

Hylon VII 50.0 48.6

Novelose 240 48.4 44.2

Hi Maize 1043 41.0 41.7

CrystaLean 39.8 37.9

Pinto Beans (dry milled) 39.4 35.6

Novelose 330 38.8 38.7

Amylose (potato) 38.2 36.6

Haricot Beans (dry milled) 36.9 31.2

Red Kidney Beans 5.0 5.3

Red Lentils 7.6 6.1

Flagelot beans (freeze-dried) 5.3 4.5

Cooked/Cooled Potato 4.0 3.2

Corn flakes 2.2 2.4

Regular maize starch 0.5 0.8

Sample Details Resistant Starch, % w/w (dwb)

Resistant Starch Determined by AOAC 2002.02 and the

Codex Definition Method (in Bottles)

1. The incubation time employed is 16 h. However, the likely time of

residence of food in the small intestine is 3-5 h.

2. Resistant maltodextrins are produced from non-resistant starch

under the incubation conditions used.

3. Fructo-oligosaccharides are underestimated using Sugar-PakR

columns [inulinotriose (F3) elutes with disaccharides; so it is not

included in the DF value].

4. Sample deionisation for HPLC is tedious.

5. Sodium azide preservative is essential with incubation times of 16 h,

but is undesirable.

6. Phosphate cross-linked starches (RS4) were considered to be

underestimated.

7. D-Sorbitol is not considered to be the best internal standard (it is

found in some fruits and food products).

Problems and Challenges in the application of AOAC Method 2009.01

to a range of food samples and ingredients

1. Read et al Gut 1982, 23, 824-828

2. Geboes et al., Aliment. Pharmacol. Ther. 2003, 18, 721–729

3. Geypens et al., J. Nucl. Med. 1999, 40, 1451–1455

4. Sadik et al., Scand J Gastroenterol. 2003, 38, 1039–1044

5. Deiteren et al., Neurogastroenterol. Motil. 2010, 22, 415–423

6. Zarate et al., Am. J. Physiol. 2010, 299, G1276–G1286

7. Camilleri et al., Neurogastroenterol. Motil. 2010, 22, 874–882

Choosing the time of Incubation with PAA/AMG

- Small Intestine Transit Time -

0

1

2

3

4

5

6

1 2 3 4 5 6 7

Smal

l Bo

wel

Tra

nsi

t Ti

me

(ho

urs

)

Citation Number

4 hour

Measurement of TDF (AOAC Method 2009.01 cf AOAC Method 2017.16)

Sample (1 g)

Alcohol precipitation, solvent washing, drying,

filtration; (2 residues)

Ash DeterminationProtein Determination

Starch hydrolysis

Change pH to ~ 8.2, Heat to ~ 95oC -To denature protein and,

-To inactivate starch hydrolyzing enzymes

+ Protease (60oC, pH 8.2)

AOAC 2017.16: RINTDFAOAC 2009.01: INTDF

Change pH to ~ 4.5, ~ 60oC

PAA (2 KU) / AMG 0.14 KU

(16 h, 37oC, pH 6.0)

Change pH to ~ 8.2, Heat to ~ 95oC -To denature protein and,

-To inactivate starch hydrolyzing enzymes

Change pH to ~ 4.5, ~ 60oC

SDFS (NDO)SDFS (NDO)

Protein hydrolysis Protein hydrolysis

PAA (4 KU) / AMG 1.7 KU

(4 h, 37oC, pH 6.0)

+ Protease (60oC, pH 8.2)

Starch hydrolysis

TDF = HMWDF + SDFS

HMWDF (IDF + SDFP)

Ethanolic filtrate. Concentrate,

Desalt in tube + In-line

HPLC - TOSOH TSK

Ethanolic filtrate. Concentrate; desalt on column,

re-concentrate;

HPLC – Waters Sugar Pak

Reaction bottles in a shaking water bath in orbital motion at 37oC

Orbital motion

150 rpm, 37oC, 4 h

Resistant Starch is the most difficult DF component to measure.

It is defined as “that portion of starch that is not susceptible to

hydrolysis by the enzymes of the human small intestine”, thus the

aim is to get an in vitro measurement that correlates with in vivo results.

In attempting to develop an in-vitro method for the measurement of

RS, the parameters that need to be considered are:

- Enzyme type, concentration and purity

- Incubation conditions such as stirring/shaking of reaction

containers, pH, temperature and time

Resistant Starch

Time Course of Hydrolysis of Starch under Englyst Incubation Conditions

(Tubes + marbles + guar, but at pH 6.0)

Varying levels of PAA with saturating AMG

Varying levels of AMG with saturating PAA

Time course of hydrolysis of starches by PAA (4 KU) plus AMG (1.7 KU/assay)

- Shaken in tubes and bottles and Stirred in Bottles -

A comparison of dietary fibre values obtained for a range of starch

containing samples using AOAC Method 2009.01

with those obtained using the RINTDF assay format .

Sample HMWDF%

SDFS%

TDF(HMWDF +

SDFS), %

HMWDF%

SDFS%

TDF(HMWDF +

SDFS), %

Wholemeal bread 12.4 1.8 14.2 12.0 1.5 13.5

Oat bran 18.9 0.6 19.5 19.9 1.4 21.3

Weetabix 9.8 2.8 12.6 10.1 1.4 11.6

Kellogg All Bran 26.6 3.9 30.5 28.1 3.6 31.7

Whole wheat pasta 9.9 2.8 12.7 10.1 2.2 12.3

Semi-ripe banana 30.2 0.9 31.1 30.2 1.4 31.6

Sweet corn (tinned) 12.7 0.4 13.1 12.4 0.5 12.9

Garden peas (tinned) 29.1 1.4 30.5 29.1 2.2 31.3

Broccoli 28.1 0.4 28.5 29.7 0.6 30.3

Carrots 21.8 0.6 22.4 22.2 1.2 23.4

Fibersym® 28.6 1.1 29.7 59.2 1.0 60.2

Hylon VII® 48.6 0.5 49.3 58.8 0.0 58.8

Polydextrose® 1.5 83.3 84.8 1.1 83.3 84.4

AOAC Method 2009.01 AOAC Method 2017.16

TDF Values Determined for a Range of Samples Traditionally used

as Standards in TDF Assays

Sample AOAC Method 991.43

AOAC Method 2009.01

AOAC Method2017.16

b-Glucan 98.0 96.0 96.2

Citrus Pectin 86.5 87.0 84.5

Larch galactan 83.5 84.0 86.0

Wheat arabinoxylan 95.0 94.5 96.0

Hylon VII® (high amylose

maize starch)

29.3 46.5 58.8

Casein 0 0 0.2

Wheat starch 0.1 0.1 0.1

Recovery of Oligosaccharides of DP > 3 in Original Samples

and on Incubation of the samples

according to AOAC 2009.01 and the RINTDF Method

Sample Original Oligosaccharides

AOAC Method 2009.01

AOAC2017.16

Neosugars® (FOS) 93.0 92.9 92.8

Raftilose P95® (FOS)* 89.0 76.2 88.2

Polydextrose® 84.3 85.1 82.5

Fibersol 2® 88.5 83.4 82.4

Galacto-

oligosaccharides

(GOS)

76.0 70.6 72.0

Xylo-oligosaccharides

(XOS)

78.0 78.6 76.2

Raffinose 99.0 99.0 98.0

AdvantaFibre®

(Isomalto-

oligosaccharides)

65.4 29.0 10.8

* The FOS analysed had a high content of inulinotriose.

Resistant Maltodextrins produced on hydrolysis of non-resistant starch

a-Glc a-Glc

a-Glc-(1-4)-a-Glc-(1-4)-a-Glc-(1-4)-a-Glc-(1-4)-a(b)-Glc

63,65-di-(1-6)-a-D-Glucosyl (1-4)-a-D-maltopentaose

6 6

Resistant Heptasaccharide incubated under RIN-TDF format

Resistant Heptasaccharide (50 mg)

Glc

Glucosyl-maltotriose (50 mg)

Glucosyl-maltotrioseincubated under RIN-TDF format

Glycerol

Glc

G-M3

a-Glc

a-Glc-(1-4)-a-Glc-(1-4)-a-Glc

6

In-line desalting of samples for HPLC

Kestose (trisaccharide)

F3 (trisaccharide)

Sucrose

Glycerol

b) Raftilose P-95 on TSKR gel-permeation columns

Measurement of FOS - Separation of Fructotriose (F3) and Disaccharides

a) Raftilose P-95 on Waters Sugar-PakR column

Desalting of Samples for HPLC

Desalting of samples for HPLC

Desalting of samples in tubes in preparation for HPLC

Desalting of Samples for HPLC

Barry V. McCleary, Naomi Sloane and Anna Draga (2015) “Determination of

total dietary fibre and available carbohydrates: a rapid integrated procedure

that simulates in-vivo digestion”. Starch/Starke, 66, 1-24.

SUMMARY

AOAC Method 2017.16 - Changes to AOAC Method 2009.01

1. Incubation time with PAA/AMG reduced to 4 h (physiologically

significant) by increasing the levels of PAA and AMG per assay.

2. Higher RS and DF values are obtained for some phosphate cross-

linked starches (RS4) samples (e.g. FibersymR).

3. Higher RS values are obtained for native high amylose maize starch

(RS2).

4. Resistant maltodextrins that are produced on hydrolysis of non-RS

in AOAC Method 2009.01 are not produced in the RIN-TDF Method.

5. Complete and accurate measurement of FOS is achieved by using

TSKgelRPWXL HPLC columns.

6. Sodium azide is not required in the incubation buffer.

7. HPLC is simplified by desalting of sample in a tube with resins,

followed by the use of a HPLC desalting pre-column.

9. Glycerol or diethylene glycol are used as the internal standard.

Interlaboratory evaluation of RINTDF Procedure for TDF

Data for individual samples from the 13 laboratories

(and outliers)

International Interlaboratory evaluation

of RINTDF Procedure for TDF

Statistical analysis of Data

NOTE: Laboratories 2 and 12 were deleted from statistical analysis because the final

HPLC step was necessarily performed in the laboratory of the method author.

MethodNo.

Title of method Sr % RSDr SR % RSDR

985.29 TDF: Total dietary fiber in foods

0.15-0.99 0.56-66.25 0.27-1.36 1.58-66.25

991.43 IDF: Insoluble dietary fiber in food and food products

0.36-1.06 1.50-6.62 0.85-2.06 1.58-12.17

2002.02 RS: Resistant starch in starch and plant materials

0.08-2.66 1.97-4.12 0.21-3.87 4.58-10.9

2009.01 TDF: Integrated total dietary fiber in food products (HMWDF + SDFS)

0.41-1.43 1.65-12.34 1.18-5.44 4.70-17.97

2011.25 TDF: Integrated TDF procedure for fiber in food products (IDF + SDFP + SDFS)

0.47-1.41 2.88-8.60 0.95-3.14 6.85-14.48

RINTDF TDF: Rapid integrated TDF procedure(HMWDF + SDFS)

0.27-0.76 1.22-6.52 0.54-3.99 2.14-10.62

Comparison of AOAC/AACC Method Data

RINTDF Interlaboratory study - Collaborators