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Measurement of Dietary Fiber

Overview of Total Dietary Fiber Measurement

Enzymes mimicking human digestion - enzymatic digest - precipitation - non-digestible fractionIn its simplest terms, dietary fiber content in a sample is measured in the laboratory by what is called an enzymatic-gravimetric method.

After defatting, a food sample is treated with enzymes that mimic the digestive process in the human small intestine.

Digestible carbohydrates are broken down into simple sugars and removed from the sample by precipitation and filtration. This mimics absorption of these sugars in the body.

The non-digestible precipitate contains the dietary fiber but also contains protein and inorganic material. These should not be included in dietary fiber so protein and inorganic material must be measured separately and subtracted from the weight. 

Commonly Used Total Dietary Fiber Methods

Megazyme supply an enzyme assay kit , Catalogue Number K-TDFR for the measurement of dietary fiber according to AOAC 985.29, AOAC 991.43.1)     Prosky Method (AOAC 985.29)1 
Introduced in 1985. Uses bacterial α-amylase and harsh   conditions (pH 8.2, 100ºC) for the enzymatic incubation step.
This method does not measure all   components of dietary fiber as currently defined by CODEX Alimentarius (international regulatory body for food ingredients). Most resistant starch and all non-digestible oligosaccharides are not included which results in an underestimation of dietary fiber.

Megazyme supply an enzyme assay kit , Catalogue Number K-INTDF for the measurement of dietary fiber according to AOAC 2009.01, AOAC 2011.25.2)    McCleary Method (AOAC 2009.01)2-4 
Introduced in 2009. Uses pancreatic α-amylase and conditions much closer to physiological (pH 6, 37ºC) for the enzymatic incubation step. This method measures all components of dietary fiber as currently defined by CODEX Alimentarius.

Rapid Integrated Dietary Fiber3)    Rapid Integrated Total Dietary Fiber
Introduced in 2015. Closely resembles AOAC 2009.01. This method addresses the minor limitations that have been identified in the McCleary Method (AOAC 2009.01) and it is envisaged that an interlaboratory evaluation will be conducted in 2016. 

Prosky method - AOAC 985.29 - McCleary Method AOAC 2009.01 - NDOs - resistant starch -bacterial alpha amylase -pancreatic alpha amylase - physiological conditions -

Overview of Soluble and Insoluble Dietary Fiber measurement

In certain cases, it is desirable to know what type of dietary fiber is present in a sample. Modifications to the standard methods (an additional filtration step) exist and these allow the dietary fiber content to be divided into soluble and insoluble dietary fiber (SDF and IDF). In both cases, all other steps in the method remain unchanged.

               AOAC 985.29 AOAC 991.43 AOAC 2009.01 AOAC 2011.25  Prosky Lee McCleary  K-INTDF K-TDFR soluble and insoluble dietary fiber

Lee Method AOAC 991.43 McCleary method AOAC 2011.25 NDOs enzymatic digest insoluble dietary fiber non digestible fraction resistant starch

Dietary Fiber Measurement – more detail

There are many different chemical entities that are classed as dietary fiber and there are distinct analytical methods to measure each of these. The methods approved by CODEX Alimentarius for the measurement of dietary fiber are shown below. Methods highlighted in green were developed by Megazyme (5 out of the 15 CODEX approved methods).

dietary fiber measurement
                high molecular weight dietary fiber HMWDF=IDF+SDFP Dietary fiber insoluble in water and soluble in 78% ethanol resistant starch fructo-oligosaccharides  HMWDF IDF SDFS SDFP FOS RS

'Double Counting of Dietary Fiber Components'

An inherent problem exists with the measurement of dietary fiber by the Prosky/Lee methods (AOAC 985.29/991.43). These methods measure some components of dietary fiber in an incomplete fashion.

The diagram below attempts to highlight the issue.

AOAC Method 2001.02 2001.03 2000.01 997.08 999.03 2002.02 995.16

If Prosky/Lee Methods are used to measure dietary fiber, Galacto-oligosaccharides (GOS), Raffinose and Stachyose are not measured at all while Polydextrose, Resistant Maltodextrins, Inulin, FOS, Pectin, Arabinogalactan and Resistant Starch are partially measured. This poses a challenge for food science analysts because if, for example, Resistant Starch is measured using AOAC 2002.02 and the value obtained is added to the value for Total Dietary Fiber measured using AOAC 985.29, this results in a quantity of resistant starch being ‘double counted’ leading to an artificially high value for dietary fiber.

The solution to this problem is to replace the Prosky/Lee methods (AOAC 985.29/AOAC 991.43) with the McCleary methods (AOAC 2009.01/2011.25). These methods correctly measure all components of dietary fiber.

Galacto-oligosaccharides Rafinose stachyose Inulin FOS Polydextrose resistant maltodextrins pectin arabinogalactan cellulose beta glucan galactomannan arabinoxylan resistant starch

Dietary Fiber Methods: Prosky (985.29) versus McCleary (2009.01) in detail

By examining the figure below we can identify the major similarities and differences between the Prosky and McCleary methods for total dietary fiber. Both methods involve the use of a 1 g sample and following an enzymatic digestion step, the non-digested material is precipitated and the protein and ash contents are determined separately and subtracted to calculate HMWDF.

The differences between the enzymatic treatment steps are evident. The starch content in a sample is gelatinised during the Prosky amylase incubation while this is avoided under the McCleary amylase incubation. This difference is crucial as gelatinisation removes the ‘resistant’ quality of resistant starch – an important component in dietary fiber. This leads to underestimation of total dietary fiber in certain samples using the Prosky method.

The other major difference between the Prosky and McCleary methods is the inclusion of the quantification of non-digestible oligosaccharides (NDOs) by HPLC. Non-digestible oligosaccharides are not measured in the Prosky method and this leads to underestimation of total dietary fiber in certain samples. 

985.29 versus 2009.01
What is measured by each method

Practicalities of performing AOAC 2009.01/2011.25 in the laboratory

As a routine procedure, AOAC 2009.01 is not difficult to perform but analysts unfamiliar with the topic may find the prospect somewhat daunting. Megazyme provides an individual data booklet for every assay kit sold that outlines clear step-by-step guidelines on how to conduct the relevant experimental protocol. Useful tips and warnings on potential ‘stumbling blocks’ are provided at key points throughout.

K-TDFR can be used for the measurement of total dietary fiber according to AOAC methods 985.29, 991.43, 991.42 and 993.19. The data sheet for this product can also be found here.

K-INTDF can be used for the measurement of total dietary fiber according to AOAC methods 2009.01 and 2011.25. The data sheet for this product can also be found here. Furthermore, an instructional video has been prepared that clearly demonstrates every step in the process.

K-RINTDF is a new product launched in 2015 to enable customers to perform the Rapid Integrated Dietary Fiber method. This method addresses the minor limitations that have emerged in the use of AOAC 2009.901/2011.25. Interlaboratory evaluation of this method will be carried out in 2016. It is discussed in detail below the instructional video.

DVDs can be requested free of charge by simply adding a note to your online order.

To choose a chapter, play the video and select the required chapter from the options on the video display.

Chapter 1: Kit Details
Chapter 2: Principle of the Assay
Chapter 3: Kit Contents
Chapter 4: Reagent Preparation
Chapter 5: Weighing of Samples, Addition of Buffer plus Amylase & AMG
Chapter 6: Addition of Tris Base and Inactivation of Amylase & AMG
Chapter 7: Incubation with Protease
Chapter 8A: Determination of HMWDF & LMWSDF
            (i): pH Adjustment & Addition of D-Sorbitol & Ethanol
            (ii): Filtration & Recovery of HMWDF & LMWSDF
            (iii): Ash & Protein Determination
            (iv): Evaporation of Aqueous Ethanol & Recovery of LMWSDF
            (vi): Desalting of Samples with Ion Exchange Resin
            (vii): Liquid Chromatography of Desalted Samples
Chapter 8B: Determination of IDF, HMWDF & LMWSDF
            (i): pH Adjustment of the Incubation Mixture and Addition of D-Sorbitol
            (ii): Filtration and Recovery of IDF, HMWDF & LMWSDF
Chapter 9: Calculation of Dietary Fiber Content

The development of AOAC 2009.01 was originally reported in 20075. It is unsurprising that since that time, through the application of this method to the entire range of food and beverage samples available, a number of limitations have been identified, including:

  • the incubation time with pancreatic α-amylase (PAA) plus amyloglucosidase (AMG) is 16 h, in line with published methods for measurement of RS. However a more likely time of residence of food in the small intestine is ~ 4 h.
  • the production of small amounts of resistant maltodextrins on hydrolysis of certain starches.  These oligosaccharides are resistant to further hydrolysis in the analytical method, but are readily hydrolysed by the mucosal α-glucosidase complex of the small intestine – thus they are an artefact generated by the analytical method and should not be included in DF.
  • suggested underestimation of Resistance Starch 4, phosphate cross-linked starch.
  • underestimation of fructo-oligosaccharides (FOS) using the HPLC system chosen (Waters Sugar-Pak® column).  Fructotriose coelutes in this method with disaccharides and thus is not measured.
  • use of sodium azide in the incubation buffer to prevent microbial contamination over the 16 h incubation period. This is undesirable given that sodium azide is notably toxic.

In ongoing research within Megazyme, each of these challenges has been addressed leading to the development of a Rapid Integrated Total Dietary Fiber Method (K-RINTDF). In this method, the incubation time with PAA + AMG was reduced to 4 h in line with likely time of residence of food in the human small intestine. To obtain the same degree of hydrolysis for a range of control starches/resistant starches as obtained with AOAC Method 2009.01 in 16 h, the levels of both PAA and AMG needed to be increased substantially (PAA from 2 to 5 KU/assay and AMG from 0.14 to 1.7 KU/assay).  

Under these conditions the DF values obtained for most samples were very similar to those obtained with AOAC Method 2009.01. However, higher (more physiologically relevant) values were obtained for certain phosphate cross-linked starch samples such as Fibersym® and for native high amylose maize starch (e.g. Hylon VII®).  In addition, under these revised incubation conditions, the problematic resistant maltodextrins were not formed on hydrolysis of starch.  To achieve complete separation of fructotriose (in FOS) from disaccharides, HPLC was performed on a TSKgel G2500PWxL® gel permeation column from Tosoh Biosciences LLC (as recommended by Matsutani Chemical Company) instead of on a Waters Corporation Sugar-Pak® column.  Glycerol was used as the internal standard in place of sorbitol, because on the TSKgel G2500PWxL® column, sorbitol coelutes with glucose. This resulted in a requirement to exclude glycerol from enzyme preparations, which has been accomplished for the Rapid Integrated Total Dietary Fiber assay kit (K-RINTDF). Finally, with the shorter incubation time, microbial infection of the incubation solutions is not a concern, which means that sodium azide can be excluded from the incubation buffer. These results were published recently.5

In conclusion, the new Rapid Integrated Total Dietary Fiber method (K-RINTDF) removes all of the limitations that have been identified with AOAC Method 2009.01. It is expected that this method will undergo interlaboratory evaluation in 2016.


1Prosky, L., Asp, N-G., Furda, I., DeVries, J. W., Schweizer, T. F. & Harland, B. F. (1985). Determination of total dietary fiber in foods and food products: Collaborative study. J. AOAC Chem., 68(4), 677-679. Link to article

2McCleary, B. V., DeVries, J. W., Rader, J. I., Cohen, G., Prosky, L., Mugford, D. C. & Okuma, K. (2010). Determination of total dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid chromatography: Collaborative study. J. AOAC Int., (93)1, 221-233. Link to article

3McCleary, B. V., Sloane, N., Draga, A. & Lazewska, I. (2013). Measurement of Total Dietary Fiber Using AOAC Method 2009.01 (AACC International Approved Method 32-45.01): Evaluation and Updates. Cereal Chem., 90, 396-414. Link to article

McCleary, B. V. (2007) An integrated procedure for the measurement of total dietary fibre (including resistant starch), non-digestible oligosaccharides and available carbohydrates. Anal. Bioanal. Chem.  389(1), 291-308. Link to article

5McCleary, B. V., Sloane, N., & Draga, A (2015). Determination of total dietary fibre and available carbohydrates: A rapid integrated procedure that simulates in vivo digestion. Starch‐Stärke  67 (9-10), 860–883. Link to article