Multilaboratory Validation of an

“Integrated Procedure for the Measurement of CODEX Dietary Fibre”

Barry V. McClearyMegazyme International Ireland Limited

What is Dietary Fibre ?

How can we measure Dietary Fibre ?

Why are we interested in 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.”

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.

Dietary Fibre (CODEX) generally has properties such as:

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.

Trowell (1972) The dietary fibre hypothesis - an inverse relationship between dietary fibre consumption and the incidence of colon cancer and heart disease.

Burkitt (1972) 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.

Trowell (1976) Broadened the dietary fiber definition to become primarily a physiological definition (based on edibility and resistance to digestion).

Definition of Dietary Fibre

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 1972-76Prosky Survey 1979-80

“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 1972-76Prosky Survey 1979-80

Definition of Dietary Fibre(why it is of interest)

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) + 50µL α-AmylaseIncubate at ~ 95oC for 30 min

Adjust pH to 7.5 + 100µL ProteaseIncubate at 60oC for 30 min

Adjust pH to 4.5 + 200µL AmyloglucosidaseIncubate at 60oC for 30 min

Ethanol Precipitation

Filtration + Alcohol and Acetone WashesDry

2 ResiduesProtein Ash

Total Dietary Fibre(TDF)

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

Total Dietary Fibre(AOAC Method 985.29)(AOAC Method 991.43)

β-Galacto-oligosaccharides

Raffinose/Stachyose

CelluloseBeta-Glucan

GalactomannanArabinoxylan

Inulin FOS

Resistant Starch

PectinArabinogalactan

Polydextrose

Fibersol 2

Analysis of Fibre Components

Total Dietary Fibre(AOAC Method 985.29)(AOAC Method 991.43)

β-Galacto-oligosaccharides

Raffinose/Stachyose

CelluloseBeta-Glucan

GalactomannanArabinoxylan

Inulin FOS

Resistant Starch

PectinArabinogalactan

Polydextrose

Fibersol 2

Analysis of Fibre Components

(AOAC Method 2002.02)

(AOAC Method 2001.03)

(AOAC Method 2000.01)

(AOAC Method 2001.02)

(AOAC Method 995.16)

(AOAC Method 997.08 / 999.03)

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

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: 291-308.

Total Dietary FibreCODEX Definition

Method

Galacto-oligosaccharidesRaffinose/Stachyose

PectinArabinogalactan

PolydextroseFibersol 2

Inulin FOS

CelluloseBeta-Glucan

GalactomannanArabinoxylan

Resistant Starch

Analysis of Fibre Components

Resistant Starch (AOAC method 2002.02)

Residue

Glucose

Resistant Starch

Non-Resistant Starch

2 M KOHAcetate BufferAmyloglucosidase

Glucose determination

Sample

Glucose determination

50 % EtOH Solubles(Free Glucose + Maltose)

Pancreatic α-Amylase/Amyloglucosidase

Source of Starch RS (in vivo) RS (in vitro methods/results)

(AOAC 2002.02) Champ Englyst

Native potato 78.8 77.0 77.7 66.5Native Amylomaize 50.3 51.7 52.8 71.4Retrog. Amylomaize 30.1 42.0 29.6 30.5Bean Flakes 9-10.9 14.3 11.2 10.6Corn Flakes 3.1-5.0 4.0 4.3 3.9Canned Beans 16.5 16.5 17.1 17.1ActiStar 59 58 57.0 63

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

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)].

Sample Labs Mean Resistant Starch RSDr RSDR%(“as is” basis) % %

Hylon VII (HAMS) 37 46.29 4.12 8.37

Green banana 36 43.56 3.18 8.47

Native potato starch 35 63.39 4.20 5.94

CrystaLean 34 39.04 1.97 5.13 (Retrograded HAMS)

ActiStarR RS 36 48.28 2.32 5.83

Kidney Beans (canned) 35 4.66 2.42 4.58

Corn Flakes 34 2.20 3.43 10.9

Regular Maize Starch 36 0.67 21.4 44.8

Method Performance for the Measurement of Resistant Starch

(AOAC Method 2002.02; AACC Method 32-40.)

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)

Add 40 mL of 50 mM Na Maleate buffer, pH 6.0 (+ CaCl2)containing purified pancreatic α-amylase + amyloglucosidase.

Remove 1 mL for Available CHO Determination

HMWDF/RS Determination

Add 2.5 mL 10 % Trizma Base to adjust pH to ~ 8.0.

Incubate in shaking water bath at 37oC for 16 h.

Incubate at 100oC for 20 min. Cool to ~ 60oC.

Add 0.1 mL protease

Incubate at 60oC for 30 min. Cool to room temp.

NDO Determination

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

Add 2.5 mL of 1 M HCl (to adjust pH to ~ 4.5).

Codex DefinitionMethod

AOAC 2002.02 New TDF/RS MethodNative Potato Starch 64.9 56.8

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

Resistant Starch Determined by AOAC 2002.02 and theCodex Definition Method (in Bottles)

Sample Details Total Dietary Fibre, % w/w (dwb)

AOAC 991.43 New TDF/RS Methodβ-Glucan 98.0 96.0Casein 0 0Pectin 86.5 87.0Wheat Starch 0.1 0.1Larch Arabinogalactan 83.5 84.0High Amylose Maize Starch 29.3 46.5

TDF Determined by AOAC 2002.02 and the Codex Definition Method (in Bottles)

Sample Details Total Dietary Fibre, % w/w (as is)

AOAC 991.43 New TDF/RS MethodHylon VII 25.6 49.3

Novelose 240 47.1 44.3Novelose 330 35.0 39.9ActistarR 0.5 47.3Green Banana 7.5 37.6

Native Potato Starch 0.9 64.6Red Kidney Beans 20.4 21.8Cooked/Cooled Potato 7.1 9.6Red Lentils 11.3 14.8Pinto Beans (dry milled) 17.3 54.9Haricot Beans (dry milled) 23.3 51.9Regular Maize Starch 0.1 0.72

TDF Determined by AOAC 2002.02 and theCodex Definition Method (in Bottles)

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

Add 40 mL of 50 mM Na Maleate buffer, pH 6.0 (+ CaCl2)containing purified pancreatic α-amylase + amyloglucosidase.

Remove 1 mL for Available CHO Determination

HMWDF/RS Determination

Add 2.5 mL 10 % Trizma Base to adjust pH to ~ 8.0.

Incubate in shaking water bath at 37oC for 16 h.

Incubate at 100oC for 20 min. Cool to ~ 60oC.

Add 0.1 mL protease

Incubate at 60oC for 30 min. Cool to room temp.

NDO Determination

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

Add 2.5 mL of 1 M HCl (to adjust pH to ~ 4.5).

Codex DefinitionMethod

+ Sorbitol

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

Aqueous incubation solution following enzyme incubations:

Measurement of NDO

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

Glycerol (from enzyme preparations)

b. Full enzyme incubation

a. No enzyme incubation

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

SorbitolGlycerol

Glucose

Fructose

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

Coordinators:

Barry V. McCleary, Megazyme International IrelandJon DeVries, Medallion Laboratories/General MillsGerry Cohen, KRAFT FoodsJeanne Rader, US Food and Drug AdministrationLeon Prosky, Retired-US Food and Drug AdministrationDavid Mugford, Retired-Australian Bread Research Inst.Kazuhiro Okuma-MatsutaniMartine Champ-University of Nantes

Funding of Interlaboratory Study:

Kellogg CompanyKRAFT FoodsTate & LyleCollaborating laboratories

Collaborative Study: CODEX Definition Method(AOAC International/AACC International)

Collaborative Study: CODEX Definition Method

AOACI/AACCI Interlaboratory Study: Collaborators:Mike Marshak / Jonathan De Vries; General Mills, USAJerry Cohen; Kraft Foods, USAGrace Lai / Julie Lorenz; Kellogg Company, USAAnnette Evans; Tate and Lyle, USAMary Ellen Camire; University of Maine, USAToyohide Nishibata; Matsutani Chemical Company, JapanErik Konings; VWA, The NetherlandsNancy Ames; Agriculture and AgriFood Canada; CanadaSonja Ehrhardt; Sudzucker AG, GermanyAndrea Rizzo; Neotron S.P.A. ItalyJean Michel Roturier; Roquette, FranceRichard Walker / Luzmila Ludena, BRI, AustraliaSneh D. Bhandari; Silliker, Ilinois, USAMonique Steegmans; Benio Group, BelgiumKenichiro Kanaya; OCN, JapanGraeme Richardson; DTS, AustraliaBarry McCleary; Megazyme, IrelandAnders Pagh Thomsen; Eurofins, USAMeinolf Lindhauer; Max Rubner Institute, GermanySteve 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).