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32 Fiber Methods​

32-01.01 Guideline on Method Selection for Dietary Fiber Analysis​​

The introduction of new fiber sources, along with traditional ingredients, raises methodology challenges when measuring fiber levels within food products. This guideline will help identify the proper methods to use, depending on the ingredients in the formulation. Referencing this guideline is a good starting point, but consulting with an expert in this area will lead to the best possible results. 

32-05.01 Total Dietary Fiber

Total dietary fiber (TDF) is determined by gelatinizing duplicate samples (previously fat-extracted if fat content is greater than 10%) with heat-stable alpha-amylase, digesting with protease and amyloglucosidase to remove protein and starch, and diluting the aqueous digest with four volumes ethanol to precipitate soluble dietary fiber. The residue is filtered; washed with 78% ethanol, 95% ethanol, and acetone; dried; and weighed. One duplicate is analyzed for protein, the other incinerated at 525° to determine ash. The TDF collected is corrected for method blanks, which include protein and ash determinations. This method is applicable to cereal grains and cereal-based products for nutritional labeling purposes.

32-06.01 Total Dietary Fiber—Rapid Gravimetric Method

This method determines the dietary fiber of food or feed products by parallel determinations of water-soluble and neutral detergent-insoluble fractions.

32-07.01 Soluble, Insoluble, and Total Dietary Fiber in Foods and Food Products

This method is a modification of Methods 32-05 and 32-21. The MES-TRIS buffer substitutes for phosphate buffer in this method. See Note 1. Duplicate 1-g dried food samples are briefly subjected to sequential enzymatic digestion by heat-stable alpha-amylase, protease, and amyloglucosidase. This method determines soluble, insoluble, and total dietary fiber (TDF) content in processed foods and raw materials, such as cereal products, fruits, and vegetables.

Insoluble dietary fiber (IDF) is filtered, and the residue is washed with warm distilled water. A solution combining filtrate and water washings is precipitated with 4 volumes 95% EtOH for soluble dietary fiber (SDF) determination. The precipitate is then filtered and dried. Both SDF and IDF residues are corrected for protein, ash, and blank for final calculation of SDF and IDF values.

SDF is precipitated with EtOH, and the residue is then filtered, dried, and weighed. TDF value is corrected for protein and ash.

32-10.01 Crude Fiber in Flours, Feeds and Feedstuffs

To determine crude fiber in flours, feeds, and feedstuffs. Crude fiber is the loss on ignition of the dried residue remaining after digestion of the sample with 1.25% H2SO4 and 1.25% NaOH solutions under specific conditions. This method utilizes the Oklahoma State filter screen or the Modified California State Buchner funnel for filtration and uses prepared ceramic fiber as a filtration aid instead of asbestos. This method is applicable to grains, meals, flours, feeds, and fiber-bearing material from which fat can be extracted to leave workable residue.

32-20.01 Insoluble Dietary Fiber

To determine the neutral detergent-insoluble dietary fiber of food, food products, and feedstuffs.

32-21.01 Insoluble and Soluble Dietary Fiber in Oat Products—Enzymatic-Gravimetric Method

This method describes the determination of insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) fractions in oats and oat products. The procedures are an extension of AACC Method 32-05 and AOAC International Method 985.29 for total dietary fiber determination. Duplicate samples are gelatinized and then enzymatically digested with protease and amyloglucosidase to remove protein and starch.

Insoluble Dietary Fiber
Residue is filtered and washed with distilled water. Filtrate and wash are saved. Residue (IDF) is washed with 95% ethanol and acetone. After drying, residue is weighed. One of duplicates is analyzed for protein; the other is incinerated at 525° and ash is determined. IDF is weight of residue minus weight of protein and ash.

Soluble Dietary Fiber
Four volumes of 95% ethanol are added to combined filtrate and water washing to precipitate SDF. Precipitate is filtered and washed with 78% EtOH, 95% EtOH, and acetone. After drying, residue is weighed. One of duplicates is analyzed for protein; the other is incinerated at 525° and ash is determined. SDF is weight of residue minus weight of protein and ash.

32-22.01 beta-Glucan in Oat Fractions and Unsweetened Oat Cereals

This is a method to determine β-glucan content in oat fractions and unsweetened oat cereals via an enzymatic procedure using lichenase and β-glucosidase enzymes for two stage hydrolysis. The (1→3)(1→4)-β-D-glucan is converted to glucose, which is subsequently measured using the glucose oxidase/peroxidase procedure with visible spectrophotometry.

32-23.01 beta-Glucan Content of Barley and Oats—Rapid Enzymatic Procedure

This is a method to determine β-glucan content in barley and oats. Lichenase is used to hydrolyze the (1→3)-4 link in (1→3)(1→4)-β-D-glucan. The resulting mixed-linkage β-oligosaccharides are separated from the insoluble material by centrifugation. An aliquot is removed to determine free glucose (sample blank), and duplicate aliquots are further hydrolyzed to glucose using β-glucosidase. Subsequent colorimetric measurement of the derived glucose is performed using glucose oxidase/peroxidase reagent. This method is suitable for flours from whole oat, barley, and rye grains, milling fractions, and unsweetened cereal products. With minor modifications (described in Reference 1), the method may be used for cereal products containing high levels of simple sugars (glucose, sucrose, fructose, and corn sweeteners).

32-24.01 beta-Glucan Viscosity in Cereal Products with the Rapid Visco Analyzer

The objective of this method is to determine the viscosity of cereal products containing β-glucan without the need for pretreatments. This measurement is obtained by mixing an aqueous suspension of ground samples with digestive enzymes in a disposable canister in the Rapid Visco Analyzer (RVA). The method can be used in industrial and research laboratories as a screening tool to identify products that may have positive physiological effects. It can also be used as a quality-assurance method to ensure consistency of β-glucan characteristics during production.

32-25.01 Total Dietary Fiber—Determined as Neutral Sugar Residues, Uronic Acid Residues, and Klason Lignin (Uppsala Method)

The principle of the method is illustrated in Fig. 1. The method includes selective and enzymatic removal of starch, using a thermostable alpha-amylase and an amyloglucosidase; precipitation of soluble polysaccharides with 80% ethanol; hydrolysis of amylase-resistant polysaccharides (precipitated and insoluble) with sulfuric acid; and subsequent quantification by gas-liquid chromatography (GLC) of released neutral sugars as alditol acetates. Uronic acids in acid hydrolysate are determined by colorimetry, and Klason lignin (sulfuric acid lignin) is calculated gravimetrically as acid-insoluble residue after correction for ash. The method determines total dietary fiber as the sum of the neutral sugar residues, uronic acid residues, and Klason lignin in food raw material and products. This method is applicable to cereal and vegetable products.

32-28.02 Polydextrose in Foods by Ion Chromatography

This method measures the polydextrose content of foods. Polydextrose is a 1 kcal/g randomly bonded polysaccharide used as a food ingredient, and has physiological benefits consistent with dietary fiber. The value obtained from this polydextrose assay may be added to the values from the enzyme-gravimetric methods without concern for double counting. Polydextrose is extracted from food with hot water and centrifuged. The supernatant then passes through a centrifugal ultrafilter to remove high molecular weight interferences. The filtrate is treated with an enzyme mix (isoamylase, amyloglucosidase, and fructanase) to remove any oligosaccharide interferences, mainly maltooligomers and fructans. Polydextrose standards undergo the same treatment. High-pressure anion-exchange chromatography with electrochemical detection (HPAEC-ED) is used to detect and quantitate the high molecular weight fraction of polydextrose.

32-31.01 Fructans in Foods and Food Products—Ion Exchange Chromatographic Method

Fructans are polydisperse mixtures of molecules that consist of fructose moieties linked to each other by beta(2-1) bonds. A glucose moiety may be linked to the end of the chain by an alpha(1-2) bond, as in sucrose. The degree of polymerization (DP) may vary from 2 to several hundreds. The main components of fructans are inulin (mainly DP 2–60) and oligofructose (DP 2–10). This method allows the quantitative determination of fructans in processed foods and raw materials, such as cereal products, fruits, and vegetables.

The method relies on the enzymatic treatment of the sample with an inulinase enzyme, followed by determination of the released sugars. The fructans are extracted from the sample with boiling water. An aliquot of the extract is hydrolyzed using a lyophilized amyloglucosidase enzyme. Subsequently, a part of that hydrolysate is treated with Fructozym.

The first and the second hydrolysate and the initial sample are analyzed using high-performance anion-exchange chromatography (HPAEC)-pulsed amperometric detection. In sugar analysis 1, free fructose (Ff) and sucrose (S) are determined in the initial sample. In sugar analysis 2, the sum of the amount of free glucose (Gf) and glucose from maltodextrins and starch (Gm) are determined in the first hydrolysate. In sugar analysis 3, the total amount of glucose (Gt) and the total amount of fructose (Ft) are determined in the second hydrolysate. The concentration of glucose and fructose released from fructan is calculated by difference from these determinations.

Glucose released from fructan (Gi) = Gt – S/1.9 – (Gf + Gm)
Fructose released from fructan (Fi) = Ft – S/1.9 – Ff

The fructan content (i) is the sum of Gi and Fi, corrected for the water loss during hydrolysis: i = k (Gi + Fi). For inulin from chicory, k = 0.91, and for oligofructose, k = 0.925.

32-32.01 Measurement of Total Fructan in Foods by an Enzymatic/Spectrophotometric Method

Fructan and fructooligosaccharides occur in a wide range of plant materials. Interest in measurement of fructans such as inulin and oligofructose has been stimulated by applications made to regulatory authorities that fructans be incorporated into dietary fiber for food-labeling purposes. In this method, samples are extracted with hot water to dissolve the fructan. Aliquots of extract are treated with a specific sucrase to hydrolyze sucrose to glucose and fructose and with a mixture of pure starch-degrading enzymes to hydrolyze starch to glucose. All reducing-sugars are then reduced to the sugar alcohols by treatment with alkaline borohydride. The solution is neutralized and excess borohydride is removed by treatment with dilute acetic acid. The fructan is then hydrolyzed to fructose and glucose with purified fructanase (exoinulinase plus endoinulinase), and these sugars are measured with the p-hydroxybenzoic acid hydrazide (PAHBAH) method for reducing sugars. With this method, color response is the same for fructose and glucose. The procedure can be used to measure fructan in a wide range of plant materials and food materials to which fructan has been added.

A spreadsheet calculator accompanies this method.

32-33.01 Determination of trans-Galactooligosaccharides in Selected Food Products by Ion-Exchange Chromatographic Method

trans-Galactooligosaccharides (TGOS) are fully soluble glycosides produced from lactose by enzymatic transgalactosylation and are used by the food and feed industry as prebiotic ingredients in numerous applications. TGOS are di- to octasaccharides composed of one to seven galactose units linked to a glucose molecule at the reducing end. The bond type is strongly dependent on the enzyme and the conditions used in the reaction. In healthy humans, TGOS are barely digested in the small intestine but are fermented in the colon by the bacterial flora and can thus be classified as dietary fiber (2). Even transgalactosylated disaccharides consisting of galactose and glucose with different beta-glycoside bonds from lactose are considered dietary fiber since they have physiological characteristics similar to those of longer TGOS (5). The prebiotic TGOS affect the host beneficially by selectively stimulating the growth of one or a limited number of health-promoting intracolonic bacteria, such as bifidobacteria.

Having a generally applicable and reliable method for the analysis of TGOS in different types of foods is very important. Classical methods such as HPLC lack the desired sensitivity and selectivity, so a new procedure has been developed, based on the method published by Quemener et al (4). This method relies on the enzymatic treatment of a test solution with a beta-galactosidase enzyme, followed by the quantitative determination of galactose by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

TGOS and lactose are extracted from a test portion with hot phosphate buffer. The extract is treated with beta-galactosidase to hydrolyze TGOS and lactose. Both the initial and the treated solutions are analyzed using HPAEC-PAD. In the first assay, free galactose and lactose are determined in the initial test solution. In the second assay, the total amount of galactose released from TGOS and lactose is determined in the treated solution. TGOS are calculated from the concentrations of lactose and galactose.

32-40.01 Resistant Starch in Starch Samples and Plant Materials

The procedure described here can be used for the measurement of the resistant starch (RS) content of plant materials, food products, and commercial starch samples. (See Note 1.) The range of applicability of the test is 2-64% RS. The method is not suitable for samples with less than 1% RS (e.g., regular maize starch, which is 0.6% RS), although an indicative result is possible. For such samples, relative standard deviation values for repeatability (RSDr) and for reproducibility (RSDR) are high. In principle, samples are incubated in a shaking water bath with pancreatic alpha-amylase and amyloglucosidase (AMG) for 16 hr at 37°, during which time nonresistant starch is solubili1zed and hydrolyzed to glucose by the combined action of the two enzymes. The reaction is terminated by the addition of an equal volume of aqueous ethanol or industrial methylated spirits (IMS), and the RS is recovered as a pellet on centrifugation. This is then washed twice by suspension in aqueous ethanol or IMS (50% v/v), followed by centrifugation. Free liquid is removed by decantation. RS in the pellet is dissolved in 2M KOH by vigorously stirring in an ice-water bath over a magnetic stirrer. This solution is neutralized with acetate buffer, and the starch is quantitatively hydrolyzed to glucose with AMG. Glucose is measured with glucose oxidase/peroxidase reagent (GOPOD), and this is a direct measure of the RS content of the sample. Nonresistant (solubilized) starch can be determined by pooling the original supernatant and the washings, adjusting the volume to 100 ml, and measuring the glucose content with GOPOD.

32-41.01 Total Dietary Fiber in Foods Containing Resistant Maltodextrin—Enzymatic-Gravimetric Method and Liquid Chromatography Determination

This method determines the total dietary fiber (TDF) value of processed foods containing insoluble dietary fiber (IDF) and high-molecular-weight soluble dietary fiber (HMW-SDF), which are precipitated in ethanol, and low-molecular-weight resistant maltodextrin (LMW-RMD), which is soluble in ethanol. This method defines dietary fiber (DF) as consisting of nondigestible carbohydrates having a degree of polymerization with three sugar moieties (DP3) or higher after enzymatic hydrolysis (ref. 2). All the starches contained in food are converted to glucose after this enzymatic hydrolysis. This method to determine TDF content in processed foods containing resistant maltodextrin (RMD) is a combination of AOAC Official Method 985.29 (ref. 1) for DF and a liquid chromatographic (LC) method for LMW-RMD described in the method. A food is first analyzed for the total quantity of IDF and HMW-SDF, which is precipitated in ethanol, according to the AOAC method (ref. 1). Then an LC determination is conducted on the desalted filtrate to obtain the quantity of LMW-RMD not precipitated in the 78% alcohol preparation. These two values ([IDF + HMW-SDF] and LMW-RMD) are summed to obtain the TDF value in the food.

32-45.01 Total Dietary Fiber (Codex Alimentarius Definition)

This method determines total dietary fiber in foods and food ingredients, as defined by the Codex Alimentarius Commission. The method quantitates high molecular weight dietary fiber (HMWDF), including resistant starch (RS), and low molecular weight soluble dietary fiber (LMWSDF), including nondigestible oligo-saccharides. In this method, enzymatic digestion at 37°C is used to simulate hu-man intestinal digestion, followed by gravimetric isolation to quantitate HMWDF and high-performance liquid chromatography (HPLC) to quantitate LMWSDF. The method measures the complete range of dietary fiber components from RS (by utilizing the digestion conditions of Method 32-41.01) to digestion-resistant oligosaccharides (by incorporating the deionization and HPLC procedures of Method 32-40.01).

32-50.01 Insoluble, Soluble, and Total Dietary Fiber (Codex Definition) by an Enzymatic-Gravimetric Method and Liquid Chromatography

This method determines insoluble, soluble, and total dietary fiber, as defined by the Codex Alimentarius Commission. It is applicable to plant material, foods, and food ingredients consistent with the Codex Alimentarius Commission Definition adopted in 2009 (ALINORM 09/32/REP) and modified slightly in 2010 (ALINORM 10/33/REP), including naturally occurring, isolated, modified, and synthetic polymers meeting the definition.

32-60.01 Rapid Integrated Method for Total Dietary Fiber

This method determines total dietary fiber (TDF) in foods and food ingredients, as defined by Codex Alimentarius. The method measures soluble and insoluble dietary fiber, including resistant starch, as well as nondigestible oligosaccharides. In this method, enzymatic digestion is used to simulate human intestinal digestion. Insoluble dietary fiber (IDF) and soluble dietary fiber that precipitates in 78% ethanol (SDFP) are separated by filtration and quantified gravimetrically. Additionally, highly soluble oligosaccharides (SDFS) are quantified by chromatographic separation. TDF is reported as the sum of the gravimetric and high-performance liquid chromatography (HPLC) results. The digestion and chromatographic conditions of this method have been modified from those of AACC Approved Methods 32-45.01 and 32-50.01 in an attempt to better simulate human digestion and to allow for more exact quantitation.