Multilaboratory Validation of an Integrated Procedure for the Measurement of CODEX Dietary Fibre Barry V.

McCleary Megazyme International Ireland Limited

What is Dietary Fibre ? Why are we interested in Dietary Fibre ?

How can we measure Dietary Fibre ?

CODEX COMMITTEE ON FOODS FOR SPECIAL NUTRITION AND DIETARY USES (CCFSNDU) - Dietary Fibre Definition -

Effort started in 1993 Member Driven Bottom Up Definition Development Driven in Recent Years by
Sweden France

By 2005/2006 reached Stage 7 of an 8 Stage process.

27th Session of the Codex Committee on Nutrition and Foods for Special Dietary Uses (CCNFSD; ALINORM 06/29/26), Bonn Germany, 21-25th November, 2005 (proposed): Dietary fibre means carbohydrate polymers with a degree of polymerization (DP) not lower than 3 which are neither digested nor absorbed in the small intestine. A degree of polymerization not lower than 3 is intended to exclude mono- and disaccharides. It is not intended to reflect the average DP of the mixture. Dietary fibre consists of one or more of: 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.

Dietary Fibre (CODEX) generally has properties such as:

Decreases intestinal transit time and increases stool bulk Fermentable by colonic bacteria Reduce blood total and/or LDL cholesterol levels Reduce post-prandial blood glucose and/or insulin levels.. Material considered as dietary fibre should have at least one of these properties.

Definition of Dietary Fibre

Hipsley (1953)

Coined the term dietary fibre as a shorthand term for the non-digestible constituents of plants that make up the cell wall, e.g. cellulose, hemicellulose and lignin. The dietary fibre hypothesis - an inverse relationship between dietary fibre consumption and the incidence of colon cancer and heart disease. Dietary Fibre - the remnants of plant components that are resistant to hydrolysis by human alimentary enzymes - a physiological / botanical description. Components covered included cellulose, hemicellulose, lignin and associated minor substances such as waxes, cutin and suberin. Edibility was implied. Broadened the dietary fiber definition to become primarily a physiological definition (based on edibility and resistance to digestion).

Trowell (1972)

Burkitt (1972)

Trowell (1976)

Definition of Dietary Fibre (why it is of interest)

Dietary fibre consists of the remnants of edible plant cells, polysaccharides, lignin, and associated substances resistant to (hydrolysis) digestion by the alimentary enzymes of humans.
Trowell 1972-76 Prosky Survey 1979-80

Definition of Dietary Fibre (why it is of interest)

This definition defines a macro constituent of foods which includes cellulose, hemicellulose, lignin, gums, modified celluloses, mucilages, oligosaccharides, and pectins and associated minor substances such as waxes, cutin, and suberin. "
Trowell 1972-76 Prosky Survey 1979-80

From a Working Definition to Practical Application

An AOAC Meeting in Ottawa, Ontario in 1981. Method proposed and an International collaborative study was set up (within months); this involved: 43 Laboratories 29 Countries By 1982 the method approved and used worldwide. The method became AOAC Official Method 985.29 Thus the Definition became practical and workable.

AOAC Official Method 985.29

Sample + Phosphate buffer(pH 8.2) + 50L -Amylase Incubate at ~ 95oC for 30 min Adjust pH to 7.5 + 100L Protease Incubate at 60oC for 30 min Adjust pH to 4.5 + 200L Amyloglucosidase Incubate at 60oC for 30 min Ethanol Precipitation Filtration + Alcohol and Acetone Washes Dry 2 Residues Protein Total Dietary Fibre(TDF) Ash

Limitations of AOAC/AACC Dietary Fibre Methodology

AOAC Method 985.29: - does not measure non-digestible oligosaccharides (NDO). - resistant starch (RS) is not completely measured. An international survey of scientists in 1993 showed that: - 65% of the respondents favoured the inclusion of NDO, and - 80% favoured inclusion of RS. This led to the development of methods for the measurement of : 1. Specific NDO such as: Fructo-oligosaccharides (FOS), Galacto-oligosaccharides, and Starch/glucose derived resistant oligosaccharides such as Fibersol 2 and Polydextrose. 2. Resistant starch

-Galacto-oligosaccharides Raffinose/Stachyose Polydextrose Fibersol 2

Analysis of Fibre Components

Inulin

FOS

Total Dietary Fibre

Pectin Arabinogalactan (AOAC Method 985.29) (AOAC Method 991.43) Cellulose Beta-Glucan Galactomannan Arabinoxylan

Resistant Starch

-Galacto-oligosaccharides
(AOAC Method 2001.02)

Analysis of Fibre Components

Raffinose/Stachyose Polydextrose
(AOAC Method 2000.01)

Inulin

FOS

(AOAC Method 997.08 / 999.03)

Fibersol 2
(AOAC Method 2001.03)

Total Dietary Fibre

(AOAC Method 985.29) (AOAC Method 991.43) Cellulose Beta-Glucan (AOAC Method 995.16) Galactomannan Arabinoxylan

Pectin Arabinogalactan

Resistant Starch
(AOAC Method 2002.02)

Dietary fibre (CODEX 2006-08) - need for an applicable method:

for the measurement of total dietary fibre (including resistant starch), non-digestible oligosaccharides and available carbohydrates.
Anal. Bioanal. Chem. 389: 291-308.

McCleary B. V. (2007) An integrated procedure

Analysis of Fibre Components

Galacto-oligosaccharides Raffinose/Stachyose Polydextrose Fibersol 2

Inulin

FOS

Total Dietary Fibre

CODEX Definition Method Cellulose Beta-Glucan Galactomannan Arabinoxylan

Pectin Arabinogalactan

Resistant Starch

Resistant Starch (AOAC method 2002.02)

Sample Pancreatic -Amylase/Amyloglucosidase

Residue 2 M KOH Acetate Buffer Amyloglucosidase

50 % EtOH Solubles (Free Glucose + Maltose)

Glucose
Glucose determination

Glucose determination

Non-Resistant Starch

Resistant Starch

Comparison of Resistant Starch Values: in vivo Vs in vitro Data

Source of Starch

RS (in vivo)

RS (in vitro methods/results) (AOAC 2002.02) Champ 77.7 52.8 29.6 11.2 4.3 17.1 57.0 Englyst 66.5 71.4 30.5 10.6 3.9 17.1 63

Native potato Native Amylomaize Retrog. Amylomaize Bean Flakes Corn Flakes Canned Beans ActiStar

78.8 50.3 30.1 9-10.9 3.1-5.0 16.5 59

77.0 51.7 42.0 14.3 4.0 16.5 58

Values are presented as a percentage of the total starch content of the sample. All data except that of McCleary and values for ActiStarR, are from Champ et al. [Advanced Dietary Fibre Technology, Blackwell Scientific Press (2000)].

Method Performance for the Measurement of Resistant Starch (AOAC Method 2002.02; AACC Method 32-40.)
Sample Hylon VII (HAMS) Green banana Native potato starch CrystaLean (Retrograded HAMS) ActiStarR RS Kidney Beans (canned) Corn Flakes Regular Maize Starch Labs 37 36 35 34 36 35 34 36 Mean Resistant Starch %(as is basis) 46.29 43.56 63.39 39.04 48.28 4.66 2.20 0.67 RSDr % 4.12 3.18 4.20 1.97 2.32 2.42 3.43 21.4 RSDR % 8.37 8.47 5.94 5.13 5.83 4.58 10.9 44.8

1.00 g sample + 40 mL of buffered enzyme (pH 6.0)

Orbital motion (150 rpm) 16 h, at 37oC.

Sample (1.00 g) in sealed 250 mL Duran bottle (in duplicate)

Codex Definition Method

Add 40 mL of 50 mM Na Maleate buffer, pH 6.0 (+ CaCl2) containing purified pancreatic -amylase + amyloglucosidase. Incubate in shaking water bath at 37oC for 16 h. Add 2.5 mL 10 % Trizma Base to adjust pH to ~ 8.0. Incubate at 100oC for 20 min. Cool to ~ 60oC. Add 0.1 mL protease Incubate at 60oC for 30 min. Cool to room temp. Add 2.5 mL of 1 M HCl (to adjust pH to ~ 4.5). Remove 1 mL for Available CHO Determination Add 4 volumes of ethanol, stir, store at room temp for 1 h, then filter.

HMWDF/RS Determination

NDO Determination

Resistant Starch Determined by AOAC 2002.02 and the Codex Definition Method (in Bottles)
AOAC 2002.02
Native Potato Starch Hylon VII Novelose 240 Hi Maize 1043 CrystaLean Pinto Beans (dry milled) Novelose 330 Amylose (potato) Haricot Beans (dry milled) Red Kidney Beans Red Lentils Flagelot beans (freeze-dried) Cooked/Cooled Potato Corn flakes Regular maize starch 64.9 50.0 48.4 41.0 39.8 39.4 38.8 38.2 36.9 5.0 7.6 5.3 4.0 2.2 0.5

New TDF/RS Method

56.8 48.6 44.2 41.7 37.9 35.6 38.7 36.6 31.2 5.3 6.1 4.5 3.2 2.4 0.8

TDF Determined by AOAC 2002.02 and the Codex Definition Method (in Bottles)
Sample Details
-Glucan Casein Pectin Wheat Starch Larch Arabinogalactan High Amylose Maize Starch

Total Dietary Fibre, % w/w (dwb) AOAC 991.43

98.0 0 86.5 0.1 83.5 29.3

New TDF/RS Method

96.0 0 87.0 0.1 84.0 46.5

TDF Determined by AOAC 2002.02 and the Codex Definition Method (in Bottles)
Sample Details
Hylon VII Novelose 240 Novelose 330 ActistarR Green Banana Native Potato Starch Red Kidney Beans Cooked/Cooled Potato Red Lentils Pinto Beans (dry milled) Haricot Beans (dry milled) Regular Maize Starch

Total Dietary Fibre, % w/w (as is) AOAC 991.43

25.6 47.1 35.0 0.5 7.5 0.9 20.4 7.1 11.3 17.3 23.3 0.1

New TDF/RS Method

49.3 44.3 39.9 47.3 37.6 64.6 21.8 9.6 14.8 54.9 51.9 0.72

Sample (1.00 g) in sealed 250 mL Duran bottle (in duplicate)

Codex Definition Method

Add 40 mL of 50 mM Na Maleate buffer, pH 6.0 (+ CaCl2) containing purified pancreatic -amylase + amyloglucosidase. Incubate in shaking water bath at 37oC for 16 h. Add 2.5 mL 10 % Trizma Base to adjust pH to ~ 8.0. Incubate at 100oC for 20 min. Cool to ~ 60oC. Add 0.1 mL protease Incubate at 60oC for 30 min. Cool to room temp. Add 2.5 mL of 1 M HCl (to adjust pH to ~ 4.5).

+ Sorbitol

Remove 1 mL for Available CHO Determination

Add 4 volumes of ethanol, stir, store at room temp for 1 h, then filter.

HMWDF/RS Determination

NDO Determination

Measurement of NDO
Aqueous incubation solution following enzyme incubations: 1. Add sorbitol standard solution 2. Add 227 mL of ethanol, mix thoroughly, allow precipitate to form. 3. Filter through celite. 4. Concentrate filtrate by rotary evaporation. 5. Deionise the solution on a mixed bed resin [Amberlite IRA 900 (OH-) plus Amberlite 200 C (H+)]. 7. Concentrate the eluate and washings and adjust the volume to 10 mL. 8. Analyse the solution by HPLC using a Waters Sugar-PakR column

HPLC on Waters Sugar-Pac Column of Raftilose (FOS)

a. No enzyme incubation

b. Full enzyme incubation

Glycerol (from enzyme preparations)

HPLC on Waters Sugar-Pac Column of Glycerol and D-Sorbitol

Glycerol Sorbitol Glucose Fructose

Determination of D-Glucose, D-Fructose and D-Galactose by HPLC: Area Under the Curve Relative to D-Sorbitol Standard

Collaborative Study: CODEX Definition Method (AOAC International/AACC International) Coordinators: Barry V. McCleary, Megazyme International Ireland Jon DeVries, Medallion Laboratories/General Mills Gerry Cohen, KRAFT Foods Jeanne Rader, US Food and Drug Administration Leon Prosky, Retired-US Food and Drug Administration David Mugford, Retired-Australian Bread Research Inst. Kazuhiro Okuma-Matsutani Martine Champ-University of Nantes Funding of Interlaboratory Study:
Kellogg Company KRAFT Foods Tate & Lyle Collaborating laboratories

Collaborative Study: CODEX Definition Method

AOACI/AACCI Interlaboratory Study: Collaborators:
Mike Marshak / Jonathan De Vries; General Mills, USA Jerry Cohen; Kraft Foods, USA Grace Lai / Julie Lorenz; Kellogg Company, USA Annette Evans; Tate and Lyle, USA Mary Ellen Camire; University of Maine, USA Toyohide Nishibata; Matsutani Chemical Company, Japan Erik Konings; VWA, The Netherlands Nancy Ames; Agriculture and AgriFood Canada; Canada Sonja Ehrhardt; Sudzucker AG, Germany Andrea Rizzo; Neotron S.P.A. Italy Jean Michel Roturier; Roquette, France Richard Walker / Luzmila Ludena, BRI, Australia Sneh D. Bhandari; Silliker, Ilinois, USA Monique Steegmans; Benio Group, Belgium Kenichiro Kanaya; OCN, Japan Graeme Richardson; DTS, Australia Barry McCleary; Megazyme, Ireland Anders Pagh Thomsen; Eurofins, USA Meinolf Lindhauer; Max Rubner Institute, Germany Steve Cui; Agriculture Canada, Canada

Study Design
8 Food Matrices Blind Duplicates 20 Laboratories requested/received samples

Matrices
Whole Grains Bread Haricot Beans Carrots (Freeze Dried) Brocolli Resistant Starch Red Kidney Beans All Bran Whole Wheat Pasta

Current Status
Laboratories performed well on the initial ruggedness test (6 samples). Approximately 1/3 of labs have returned the data from the collaborative study. Performance results are comparable to those obtained with the current Official Method(s) of Analysis (e.g. AOAC 985.29).