Determination of Insoluble, Soluble, and Total Dietary Fiber in Foods Using a Rapid Integrated Procedure of Enzymatic-Gravimetric-Liquid Chromatography: First Action 2022.01

Abstract Background A simple, accurate, and reliable method for the measurement of total dietary fiber (TDF) according to the Codex definition (2009) was developed and successfully validated as AOAC Official Method of Analysis (OMA) 2017.16. Subsequently, OMA 2017.16 was modified to allow separate measurement of soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) fractions. Objective To perform a collaborative study to evaluate the repeatability and reproducibility of OMA 2017.16 modification for the measurement of total dietary fiber (TDF) as IDF and SDF measured as (1) water SDF that precipitates in 78% aqueous ethanol (SDFP), and (2) water SDF that remains soluble in 78% aqueous ethanol (SDFS) of degree of polymerization ≥3. Methods Duplicate test portions are incubated with pancreatic α-amylase (PAA), amyloglucosidase (AMG), and protease under the conditions employed in OMA 2017.16. For the measurement of IDF, the digestate is filtered and the IDF determined gravimetrically. SDFP in the IDF filtrate is precipitated with alcohol and captured by filtration and determined. SDFS in the SDFP filtrate is recovered and quantitated by LC. The matrixes included cereal products and flours, vegetables, health food snacks, soup, chocolate, and beans. Additional materials were analyzed by collaborators as “practice samples”. Results With the diethylene glycol internal standard, all multi-laboratotu (MLV) matrixes resulted in repeatability relative standard deviations (RSDr) for TDF analyses of <3.60% and RSDR ranging from 4.55 to 9.26%. For the practice samples, the RSDR for TDF ranged from 6.69 to 11.68%. Conclusion OMA 2022.01 meets the AOAC requirements for repeatability and reproducibility and the data support First Action status. Highlights OMA 2022.01 is a robust and reproducible method for the analysis of insoluble, soluble (SDFP and SDFS), and TDF in a wide range of matrixes.

definition, a method was developed (2) and validated as AOAC Method 2009.01 (AACC Method 32-45.01) and AOAC Method 2011.25 (AACC Method 32-50.01;3-7). Subsequently, limitations of this method were identified, including the time of incubation with pancreatic a-amylase (PAA) plus amyloglucosidase ([AMG] 16 h; 8,9) not being physiologically relevant, excessive hydrolysis and thus underestimation of phosphate cross-linked starch (RS 4 ), production of resistant oligosaccharides (10,11) from non-RS, underestimation of fructo-oligosaccharides (FOS) (11), and the use of sodium azide (a toxic chemical) as a preservative. This led to the development of an improved method for measurement of total dietary fiber (TDF) in which all limitations identified were addressed, namely, the rapid integrated total dietary fiber method (RINTDF; 8), which was successfully validated in an AOAC/AACCI/ICC multilaboratory study, to become AOAC Official Method of Analysis (OMA) 2017.16 (12), AACCI recommended method 32-60.01 (13), and ICC method 185.
AOAC Method 2017.16 was then modified (according to the steps described in AOAC Method 2011.25) to allow the measurement of insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) [as SDF that precipitates in 78% aqueous ethanol (SDFP) plus SDF that remains soluble in 78% aqueous ethanol (SDFS)] summed as TDF. In the study described here, this modified method has been subjected to interlaboratory validation under the auspices of AOAC INTERNATIONAL.

Practice Materials
Prior to the collaborative study, all participating laboratories were provided practice samples to familiarize themselves with the method and to ensure adequate method performance. Laboratories were shipped four matrix samples along with OMA 2022.01, required enzymes, a control sample, data reporting sheets, and an Excel calculator. Each laboratory was asked to perform a single analysis of each sample, to ask questions regarding the method, and to provide feedback to the method author. The samples included a health snack bar, cookies containing FOS, cauliflower, and whole meal pita bread.
(a) Health snack bar and cookies containing FOS.-Health snack (Nature Valley Peanut Bar) with high fiber content and cookies containing fiber were purchased from a local supermarket and homogenized with a high-speed blender (e.g., Nutri Bullet). Portions of approximately 100 g were transferred to 2 L beakers and approximately 800 mL petroleum ether (or hexane) was added to each and stirred intermittently with a spatula, in a well-ventilated fume hood over 15 min. The solids were allowed to settle over approximately 4 h and the supernatant solution was carefully decanted and discarded. This process was repeated a further two times. The solids were transferred to a flat polypropylene tray and allowed to dry in a well-ventilated fume hood over approximately 3 h and then weighed. The content of fat remaining in the samples was determined using the ANKOM XT15 extractor. The dry material was ground until 100% passed a 0.5 mm sieve and then thoroughly mixed in a plastic bag by inversion and transferred and stored in well-sealed Duran glass bottles at room temperature away from direct sunlight. The health snack bar, defatted as described above, had an original fat content of 25.4%. Subsequent analysis of the dried product using the ANKOM XT15 extractor gave a residual fat content of 6.3%. Cookies containing FOS, extracted as described above, had an original fat content of 21.2%, and subsequent analysis of the dried product using the ANKOM defatting equipment gave a residual fat content of 3.6%. (b) Cauliflower.-Fresh produce was procured from a local supermarket. The florets were removed and steamed until tender, drained, and cooled to room temperature. The material was chopped finely, weighed, and lyophilized, with wet and dry weights recorded. The recovered dry weight of steamed cauliflower was 11.1%. A sample (approximately 300 g) was ground in a Nutri Bullet homogenizer followed by further grinding in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was thoroughly mixed in a plastic bag by inversion and transferred and stored in well-sealed Duran glass bottles at room temperature away from direct sunlight. (c) Whole meal pita bread.-Product was ground to crumbs in a kitchen blender and lyophilized over 2 days and then further ground in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was thoroughly mixed in a plastic bag by inversion and transferred and stored in wellsealed Duran glass bottles at room temperature away from direct sunlight. Test portions of approximately 5 g of each sample type were transferred to pre-labelled glass vials which were sealed with rubber grommets and screw caps. Upon receipt, the collaborators were instructed to store all test portions at room temperature away from direct sunlight until use. (d) Moisture content of practice samples.-Moisture content of the products used in the study were determined using an Ohaus MB45 moisture analyzer. Values obtained were: health snack bar 9.8%; cookies containing FOS 6.5%; cauliflower 6.1%; whole meal pita bread 2.3%.

Collaborative Study Materials
Eight pre-prepared foods were selected for the collaborative study to cover a broad range of food categories. These included kidney beans, carrots, dark rye crispbread, barley flour, oat bran, chocolate, soup powder containing dietary fiber, and a health food nutrition bar.
(a) Kidney beans (canned and freeze dried).-Product was purchased from a local supermarket. Canned beans were transferred to a sieve and washed with distilled water to remove all viscous solution. One kg washed beans was lyophilized over 2 days and then ground in a kitchen blender and then a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was collected in a plastic bag, mixed thoroughly by inversion, and then transferred to 1 L Duran bottles, well-sealed and stored at room temperature away from direct sunlight. (b) Carrots (steamed and freeze dried).-Products were purchased from a local supermarket, peeled, and steamed until tender (approximately15 min), homogenized in a kitchen blender, weighed, transferred to lyophilizer trays and dried over 2 days. The dry material was ground in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was collected in a plastic bag, mixed thoroughly by inversion, weighed, and then transferred to 1 L Duran bottles, well-sealed and stored at room temperature away from direct sunlight.
(c) Dark rye crispbread (Ryvita).-This product was purchased from a local supermarket and ground in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was collected in a plastic bag, mixed thoroughly by inversion, and then transferred to 1 L Duran bottles, wellsealed and stored at room temperature away from direct sunlight. (d) Barley MAX flour (high fiber variety).-Product was obtained from The Healthy Grain Pty Limited, South Yarra, Victoria, Australia. The flour was ground in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was collected in a plastic bag, mixed thoroughly by inversion, and then transferred to 1 L Duran bottles, well-sealed and stored at room temperature away from direct sunlight. (e) Oat bran.-Product was purchased from a local supplier, ground in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was collected in a plastic bag, mixed thoroughly by inversion, and then transferred to 1 L Duran bottles, well-sealed and stored at room temperature away from direct sunlight. (f) Miso soup powder (containing resistant maltodextrins).-Miso soup powder, containing soluble resistant maltodextrins and seaweed polysaccharide, was obtained from a Japanese supermarket and ground in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was collected in a plastic bag, mixed thoroughly by inversion, and then transferred to 1 L Duran bottles, well-sealed and stored at room temperature away from direct sunlight. (g) Chocolate and health food nutrition bar (Fiber 1).-A commercial chocolate product containing Fibersol-2 was obtained from a supermarket in Japan. A health food nutrition bar (Fiber 1 Salted Caramel Bar) was obtained from a local supermarket. Products were homogenized with a high-speed blender (e.g., Nutri Bullet). Portions of approximately 100 g were transferred to 2 L beakers and approximately 800 mL petroleum ether (or hexane) was added to each and stirred intermittently with a spatula, in a well-ventilated fume hood over 15 min. The solids were allowed to settle over approximately 4 h and the supernatant solution was carefully decanted and discarded. This process was repeated a further two times. The solids were transferred to a flat polypropylene tray and allowed to dry in a well-ventilated fume hood over approximately 3 h and then weighed. The content of fat remaining in the samples was determined using the ANKOM XT15 extractor. The dry material was ground until 100% passed a 0.5 mm sieve and then thoroughly mixed in a plastic bag and transferred and stored in well-sealed Duran glass bottles at room temperature away from direct sunlight. Chocolate, defatted as described above, had an original fat content of 36.2%. Subsequent analysis of the dried product using the ANKOM XT15 extractor gave a residual fat content of 6.5%. The health food bar had an original fat content of 15.4%. Subsequent analysis of the dried product using the ANKOM XT15 extractor gave a residual fat content of 0%.  , sample storage instructions, and an adequate supply of enzymes in the Rapid Integrated TDF assay kit (K-RINTDF), and details on how to prepare and store these, were distributed to collaborating laboratories by express overnight shipment. Upon receipt, the collaborators were instructed to store all test portions at room temperature away from direct sunlight until the start of the study and to store kit components as described on the individual bottle labels.

Statistical Treatment
Results were submitted by collaborators using supplied Excel-based spreadsheets and evaluated according to AOAC guidelines using an AOAC statistical workbook. Outlier results identified by the Cochran's test for extremes of repeatability and the Grubb's test for extremes of reproducibility were omitted from further calculations. Also determined were repeatability (s r ) and reproducibility (s R ) standard deviations, relative standard deviations of repeatability (RSD r ) and reproducibility (RSD R ), and measurement uncertainty (MU) values. [Applicable to plant material, foods, and food ingredients consistent with CAC Definition adopted in 2009 and modified slightly in 2010 (ALINORM 09/32/REP and ALINORM 10/33/REP, respectively) including naturally occurring, isolated, modified, and synthetic carbohydrate polymers and oligosaccharides meeting that definition. This method is specifically designed for the analysis of foods "as eaten".] Caution: Solvents employed are common-use solvents and reagents. Refer to appropriate manuals or safety data sheets to ensure that the safety guidelines are applied before using chemicals. Store in a flammable liquid storage cabinet. Harmful if inhaled, swallowed, or absorbed through the skin. Use appropriate personal protective equipment such as a lab coat, safety glasses, rubber gloves, and fume hood. Dispose of all materials according to federal, state, and local regulations.

AOAC
See Tables 2022.01A-C for S r , S R , RSD r , and RSD R for insoluble, soluble, and TDF with glycerol internal standard.
See Tables 2022.01D-F for diethylene glycol (DEG) internal standard.

A. Principle
The method measures IDF, SDF and TDF as defined by the CAC (1). The method quantitates IDF and SDF which precipitates in 78% aqueous ethanol (SDFP) by gravimetric procedures, SDF which remains soluble in 78% aqueous ethanol (SDFS) by HPLC, and TDF by gravimetric and HPLC procedures (Figure 2022.01A). SDF is calculated by combining the weights of SDFP and SDFS. RS is captured in the IDF fraction. The method combines key attributes of OMA 985.29, 2001OMA 985.29, .03, 2011OMA 985.29, .25, and 2017. Duplicate test portions are incubated with PAA and AMG for 4 h at 37 C in sealed 250 mL bottles in a shaking water bath while mixing in orbital motion or stirring with a magnetic stirrer, during which time non-RS is solubilized and hydrolyzed to glucose and maltose by the combined action of the two enzymes. The reaction is terminated by pH adjustment followed by temporary heating. Protein in the sample is digested with protease. For the separate measurement of Table 2022.01C. Interlaboratory study results for TDF in foods (RINTDF Method glycerol internal standard) in which outlier data from laboratories 4, 9, 11, and 12 (see Table 3) were excluded; statistical evaluation according to AOAC statistics format   IDF and SDF, the sample suspension is filtered and the filtrate recovered. IDF is captured on a sintered glass crucible, washed with ethanol (EtOH) or industrial methylated spirits (IMS) and acetone, dried and weighed. With all gravimetric determinations, one of the duplicate residues is analyzed for protein, the other for ash and these weights are subtracted from the residue weights. To the filtrate, ethanol is added to a concentration of 78%, and the precipitated SDFP is captured on a sintered glass crucible, washed with ethanol and acetone, dried, and weighed. SDFS in the filtrate is concentrated, deionized with resins, and quantitated by HPLC.   Laboratories 5,8,9,and 11 (see   Improved deionization and HPLC separation of SDFS is incorporated, glycerol or DEG is used as the internal standard, and potentially hazardous sodium azide is removed from the incubation buffer. One unit AMG activity is the amount of enzyme required to release one mmole D-glucose from soluble starch per minute at 40 C and pH 4.5; one unit PAA activity is the amount of enzyme required to release one mmole p-nitrophenyl from Ceralpha reagent per minute at 40 C and pH 6.9; OMA 2002.01. PAA/AMG preparations should be essentially devoid of b-glucanase, b-xylanase, and detectable levels of free D-glucose. Stable for $4 years at À20 C. (e) PAA (4 KU/5 mL)/AMG (1.7 KU/5 mL).-Immediately before use, dissolve 1 g PAA/AMG powder, B(d), in 50 mL sodium maleate buffer (50 mM, pH 6.0 plus 2 mM CaCl 2 ) and stir for approximately 5 min. Store on ice during use. Use on the day of preparation. Alternatively: Some individuals are allergic to powdered PAA and/or AMG. In this instance, engage an analyst who is not allergic to prepare the powdered enzymes as an ammonium sulphate suspension as follows: Gradually add 5 g PAA/AMG powder mix [PAA 40 KU/ g plus AMG 17 KU/g; B(d)] to 70 mL cold, distilled water in a 200 mL beaker on a magnetic stirrer in a laboratory hood and stir until the enzymes are completely dissolved (approximately 5 min). Add 35 g granular ammonium sulphate and dissolve by stirring. Adjust the volume to 100 mL with ammonium sulphate solution (50 g/100 mL) and store at 4 C. This preparation contains PAA at 2 KU/mL and AMG at 0.85 KU/mL. Stable at 4 C for 3 months.  (h) Diethyleneglycol (DEG) internal standard (100 mg/mL).-Carefully weigh 10.00 g of diethylene glycol into a 100 mL beaker on an analytical balance. Remove the beaker from the balance and add $30 mL of aqueous sodium azide solution (0.02%, w/v), B(p). Transfer the solution to a 100 mL volumetric flask (using a funnel). Wash the beaker with approximately 2 Â 20 mL of the aqueous sodium azide solution to remove all DEG and transfer this to the volumetric flask. Adjust the volume to 100 mL with aqueous sodium azide solution, B(p). Transfer the solution to a 100 mL Duran bottle and store at room temperature in the dark. Stable for $6 months stored in the dark at room temperature. diethylene glycol into a 100 mL beaker on an analytical balance. Remove the beaker from the balance and add $30 mL aqueous sodium azide (0.02% w/v), B(p), and mix well. Separately, weigh 1.00 g dried glucose into a separate 100 mL beaker on an analytical balance. Remove the beaker from the balance and add $30 mL aqueous sodium azide (0.02% w/v) and mix well. Transfer both solutions to a 100 mL volumetric flask and use aqueous sodium azide solution (0.02% wv) B(p) to completely rinse the beakers and transfer the washings into the volumetric flask (using a funnel). Adjust the final volume to 100 mL and mix the contents well. Transfer the solution to a 100 mL Duran bottle and store at room temperature in the dark. Stable for $6 months stored in the dark at room temperature. (l) Sodium maleate buffer (50 mM, pH 6.0, with 2 mM CaCl 2 ).-Dissolve 11.6 g maleic acid in 1600 mL deionized water and adjust the pH to 6.0 with 4 M (160 g/L) NaOH solution. Add 0.6 g calcium chloride (CaCl 2 Á2H 2 O) and adjust volume to 2 L. Stable for approximately 2 weeks at 4 C.     Thermometer.-Capable of measuring to 100 C.

D. Preparation of Test Samples
Collect and prepare samples as intended to be eaten, i.e., baking mixes should be prepared and baked, pasta should be cooked, etc. Defat as per AOAC OMA 985.29 if >10% fat. For highmoisture samples (>25%), it may be desirable to freeze-dry. Grind ca 50 g in a grinding mill, C(a), to pass a 0.5 mm sieve. Transfer all material to a wide-mouthed plastic jar, seal, and mix well by shaking and inversion. Store in the presence of a desiccant.

E. Enzyme Purity
To ensure absence of undesirable enzymatic activities and effectiveness of desirable enzymatic activities, analyze the standards listed in     for 10 min to equilibrate to 37 C. Alternatively, transfer the bottles (without stirrer bar) to a Grant OLS 200 shaking incubation bath, C(g), or similar, and secure in place with the shaker frame springs or a polypropylene holder, Figure 2107.16B, and shake at 150 rpm in orbital motion for 10 min.

I. Determination of SDFS
Proper deionization of the filtrate is an essential part of obtaining quality chromatographic data on SDFS. See   Note: For samples containing significant levels of lactose or isomaltose, clear delineation of SDFS from disaccharides on chromatography on TSKgel G2500PW XL columns is difficult. This problem can be resolved by hydrolyzing lactose and/or isomaltose to glucose and galactose as follows: To a 5 mL aliquot of the SDFS fraction add 1 mL 1 M sodium acetate buffer (pH 4.0) to give a final pH of $4.5. Then add 0.1 mL of a suspension of b-galactosidase (EC 3.2.1.23; 2000 U/mL) and oligo-a-1, 6-glucosidase (EC 3.2.1.10;2000 U/mL, Megazyme Cat. No. E-BGOG) and incubate at 40 C for 30 min. Heat the solution in a boiling water bath for 3 min, cool to room temperature, and deionize by adding add 1.5 g of both Amberlite FPA53 (OH À ) and Ambersep 200 (H þ ) resins and mixing over 5 min. Filter solutions through a 47 mm, 0.45 mm syringe filter and apply to TSKgel G2500PW XL HPLC columns with in-line deionization. and (c) a sample (b) was analyzed with a Bio-Rad deionization pre-cartridges in place. Deionization with resins in a polypropylene tube, as described here, removes >95% of the salt from the sample, thus ensuring more efficient use of the expensive Bio-Rad deionization pre-cartridges. This deionization step increases the effectiveness of the deionization cartridges and allows up to 10 times more samples to be chromatographed before the need to regenerate or replace the deionization cartridges.
Under these conditions essentially all of the galactooligosaccharides (GOS) in the sample will also be hydrolyzed to galactose and glucose. Hydrolysis of lactose and isomaltose by a mixture of oligo-a-1,6-glucosidase and b-galactosidase (E-BGOG) is shown in Figure 2022.01H.

J. Calculation of IDF (by Gravimetry)
(a) Blank (B, mg) determination.- where BR 1 and BR 2 ¼ residue mass, in mg, for duplicate IDF blank determinations, respectively; and P B and A B ¼ mass, in mg, of protein and ash, respectively, determined on first and second blank residues.
where R 1 ¼ IDF residue mass 1 from M 1 in mg; R 2 ¼ IDF residue mass 2 from M 2 in mg; M 1 ¼ test portion mass 1 in g; M 2 ¼ test portion mass 2 in g; P ¼ protein mass in mg from R 1 ; A ¼ ash mass in mg from R 2 ; and B ¼ IDF blank from J(a).

K. Calculation of SDFP (by Gravimetry)
where BR 1 and BR 2 ¼ residue mass, in mg, for duplicate SDFP blank determinations, respectively; and P B and A B ¼ mass, in mg, of protein and ash, respectively, determined on the first and second blank residues.
where R 1 ¼ SDFP residue mass 1 from M 1 in mg; R 2 ¼ SDFP residue mass 2 from M 2 in mg; M 1 ¼ test portion mass 1 in g; M 2 ¼ test portion mass 2 in g; P ¼ protein mass, in mg, from R 1 ; A¼ ash mass, in mg, from R 2 ; and B ¼ SDFP blank from K(a).  where PA Glu ¼ peak area of D-glucose; PA IS ¼ peak area of glycerol or DEG internal standard; Wt Glu ¼ mass of D-glucose/mL in standard, B(j) and Wt IS ¼ mass of glycerol/mL B(j); or Wt Glu ¼ mass of D-glucose/mL in standard, B(k) and Wt IS ¼ mass of DEG/mL in standard B(k).

L. Calculation of SDFS (by HPLC)
(a) SDFS determination.- where Rf ¼ response factor from L(a); Wt IS ¼ mass in mg of internal standard contained in 1 mL internal standard solution (100 mg), B(g) or B(h), pipetted into sample before filtration; PA SDFS ¼ peak area of the SDFS; PA IS ¼ peak area of the internal standard; and M ¼ test portion mass (g), M 1 or M 2 , of the sample whose filtrate was concentrated and analyzed by LC.

Results and Discussion
Samples were selected to cover a wide range of foods, feeds, and ingredients for which insoluble, soluble, and TDF values would be useful. The samples chosen contained various levels of IDF, SDFP, and SDFS to ensure accurate measurement of all dietary fiber components. All materials were prepared, dried, dispensed into sealed tubes, and blind coded before dispatch to avoid possible deterioration during shipping.

Practice Sample Testing
To ensure adequate method performance and a good understanding of the method by collaborators, practice samples were analyzed by all the participating laboratories. Collaborators were sent four samples, labelled P1-P4, along with copies of the method, an Excel-based data calculator, and the required enzymes, control solutions, and ion exchange resins. Each laboratory was asked to analyze a single test portion of each sample, to ask questions regarding the procedures, and to provide feedback to the method author. The results of the analysis on the practice samples are shown in Table 1. In the gravimetric determinations of IDF and SDFP, no specific problems were identified by the collaborators. Some problems and misunderstandings occurred in the measurement of the SDFS fraction by HPLC. The need for use of the stated HPLC columns, with ongoing maintenance, and correct deionization of samples using resin in tubes together with in-line deionization was again reiterated. The importance of maintaining the sample in suspension during the incubation with PAA/AMG was again highlighted. The results of the practice samples were typical for dietary fiber methods. Repeatability values were within the range of performance limits typically found for dietary fiber methods, wherein a significant number of manual steps are necessary to perform the assay. Samples were analyzed for IDF and SDFP gravimetrically and SDFS by HPLC. The reproducibility SD (S R ) for TDF ranged from 0.79 to 1.91 g/100 g, and the RSD R ranged from 6.69 to 11.68%, values consistent with those reported for analyses of similar samples with other dietary fiber assay formats ( Table 2). A similar range of RSD R values were obtained for IDF i.e., 5.16 to 13.45%. The higher RSD R values for the SDF fraction are in line with a similar range obtained in other studies (AOAC 2011.25) and were thought to be due mainly to the unfamiliarity of collaborators with the requirements of this assay.
It was concluded that the method was ready for a collaborative study. It is generally known that some food samples contain glycerol either as a natural component or as an added ingredient, thus glycerol is not an ideal internal standard for use in SDFS determinations by HPLC. Some laboratories routinely use DEG as internal standard as it elutes as a discrete peak separate from all other components in the samples. To gain further insight into the relative value of these two compounds as internal standards, collaborators were requested to employ both compounds in the MLV study.

Collaborative Study Results
All of the 17 laboratories that analyzed the practice samples completed the study and 16 reported a full set of results with the glycerol internal standard; 14 laboratories reported a full set of results with the DEG internal standard; and a fifteenth laboratory reported a partial set of results with the DEG internal standard. Collaborating laboratory data were evaluated statistically according to AOAC guidelines using an AOAC workbook (version 4.8).
In measurement of IDF with the glycerol internal standard, of the eight valid pairs of assay results reported from 16 collaborators, Laboratories 1, 5, and 8 reported a single duplicate pair outlier and Laboratories 9 and 11 reported two duplicate pairs of outliers (see Table 3a). With the DEG internal standard employed, Laboratories 5 and 8 reported a single duplicate pair outlier and Laboratories 9 and 11 reported two duplicate pairs of outliers (see Table 4a). For SDF with glycerol internal standard (IS), B(g), Laboratory 5 reported a single statistical outlier pair (samples K and N) and Laboratories 4 and 9 reported two statistical outliers (see Table 3b); with DEG IS, single outliers were reported by Laboratories 9 and 13. For TDF with glycerol IS, Laboratories 4,9,11,and 12 reported single outliers (see Table 3c); with DEG IS, single outliers were reported for Laboratories 9, 11 and 13 (see Table 4c). IDF levels ranged from 5.64 to 23.00% (glycerol IS;  Tables 3 and 4. The S r , S R , RSD r , and RSD R for IDF, SDF, and TDF with glycerol IS are shown in Tables 2022.01A-C and with DEG IS in  Tables 2022.01D-F. For IDF, the values with the glycerol and DEG IS are very similar as would be expected. The small difference in values is due to the fact that not all laboratories included both IS in the samples being analyzed; Laboratory 2 did not include the glycerol IS and Laboratories 1 and 3 did not include the DEG IS. Laboratory 11 included the DEG IS for just half of the samples analyzed. Values for S r , S R , RSD r , and RSD R for these fractions are in a similar range as those reported for AOAC Method 2011.25 [Reference (4), Table 1A]. With the glycerol IS, expanded measurement uncertainty (MU) in measurement of IDF ranged from 0.79 to 3.99%, whereas with the DEG IS expanded MU ranged from 0.70 to 3.86%. For SDF, there is a significant difference in SDFS values obtained with either the glycerol or the DEG IS. This difference is a result of the presence of glycerol in the sample, and the effect this has on the determined value of SDFS (Table 5). However, for this effect to be of any significance, the sample must have a glycerol content of >0.6 g/100 g. For example, with sample G/H with a glycerol content of 0.6 g/100 g, there is a moderate decrease in the determined SDFS value (approximately 0.6%) over the value obtained with the DEG IS. However, for sample K/N with a glycerol content of 2.23 g/100 g, a dramatic decrease in the measured SDFS content is observed, with a value of 23.44 g/100 g with the DEG IS and a value of 13.36 with the glycerol IS. Sample K/N is a high-fiber nutrition bar into which significant quantities of glycerol have been added, possibly as an anti-staling agent and to help maintain texture. The S r , S R , RSD r , and RSD R for SDF with glycerol IS are shown in Table 2022.01B and with DEG IS in Table 2022.01E. With the DEG IS, s r for SDF ranged from 0.41 to 0.84 g/100 g, and s R ranged from 0.78 to 2.94 g/100 g. RSD R ranged from 7.56 to 12.08%. Expanded MU ranged from 1.56 to 5.88%. Similar values were obtained with the glycerol IS. For TDF analysis with the DEG IS, s r ranged from 0.59 to 1.35 g/100 g, and s R ranged from 1.11 to 3.05 g/100 g. RSD R ranged from 4.55 to 9.26%. Expanded MU ranged from 2.21 to 6.10%. Again, similar values for TDF were obtained with the glycerol IS.
Repeatability, reproducibility, and expanded MU values were within the range of performance characteristics typically found for similar methods or similar analytical formats [ Table 2 and Table 2011.25A-C (5)].
DEG is a better IS than glycerol because on chromatography on TSK columns, there is a clear delineation of it from glycerol, oligosaccharides, and apparently all other components in the sample extract. Previously, glycerol had been chosen over DEG because of a perceived lesser stability of aqueous solutions of DEG over time. However, our recent studies have shown that aqueous solutions of DEG, in the presence or absence of glucose, are stable for at least 6 months at room temperature (in the dark), which is an acceptable period of stability to be of value in an analytical laboratory. Consequently, DEG is recommended as the preferred IS.

Statistical Treatment
Collaborating laboratory data were evaluated statistically according to AOAC protocols using AOAC software. The raw data and statistically paired data from the blind duplicate results for IDF, SDF, and TDF with either the glycerol or DEG IS reported by the collaborating laboratories are shown in  Tables 3a-c, 4a-c, 2022.01A-C and 2022.01D-F. Outliers and the reason for outlier removal are indicated and footnoted in Tables 3a-c, 4a-c. Measurement uncertainty (14) of the insoluble, soluble and TDF procedure was assessed and calculated in accordance with the guidelines specified by Eurachem CITAC Guide CG4 (QUAM:2012.P1). The use of the s R derived from a collaborative study as a measure of the combined standard uncertainty of the method is appropriate on the basis that: this is an empirical method whereby the measurement is dependent on the method used, there is no available certified reference material (CRM), and the method has been subject to a collaborative study whereby laboratory collaborators performed all stages of the method. Expanded MU is calculated using a coverage factor (k) of 2 on the basis that all mean results have a degrees of freedom greater than 25. The coverage factor (k) of 2 provides a level of confidence of approximately 95%.
The method was applied to collaborative study using eight sample matrixes (16 homogeneous test samples as 8 blind duplicates) analyzed by 17 collaborators.
The TDF value of a given sample is the sum of total of SDF (SDFS plus SDFP) and IDF. Raw data for IDF, SDF and TDF reported by the collaborating laboratories are shown in Tables 3a-c (glycerol IS ) and Tables 4a-c (DEG IS). Extremely high or low RSD R values for IDF can be explained either by the fact that the measurement values were low (samples C/M and I/O), or by the fact that the sample contained carbohydrate material that was sparingly soluble in water (sample I/O) and values obtained were influenced by the severity of extraction (shaking/stirring). This is particularly evident with sample I/O (Miso soup) which contains seaweed polysaccharides.

Discussion and Comments from Collaborators
In the procedure employed for the measurement of IDF, SDFS, SDFP, and TDF described in AOAC 2022.01, there are several steps that must be performed as described to obtain correct values. In the incubation of PAA/AMG with samples, the enzyme employed, pH, temperature, and time of incubation are critical. However, there is some flexibility in the method concerning agitation during incubation with the proviso that the sample must be completely suspended during the full incubation period. The recommended procedure is to either use continual orbital shaking in a temperature-controlled bath or to continually stir the samples with an immersible magnetic stirrer. Laboratories 14 and 17 used linear shaking of incubation bottles attached at 45 angle to the direction of shaking while Laboratories 5,6,9,and 16 used ANKOM TDF equipment to achieve sample mixing. In most cases, insoluble fractions were recovered by filtration through Celite or using the ANKOM filtration system. One laboratory (6) used centrifugation to recover residues. In the analysis of the practice samples, glycerol was employed as the IS. However, because glycerol is found in various food samples at varying levels, several collaborating laboratories routinely employ DEG as the IS. The presence of significant levels of glycerol in samples leads to erroneously low values for the SDFS fraction. Consequently, in the analysis of the MLV samples, all collaborating laboratories, except Laboratories 1, 2, and 3 included both glycerol and DEG IS. Laboratories 1 and 3 used only glycerol and Laboratory 2 used only DEG. Because Laboratory 7 had initiated the analysis of the MLV samples before the decision was made to include the DEG as well as the glycerol IS, half of the results reported by them did not include DEG data. Two laboratories (6 and 11) analyzed residual protein using the Dumas method, and one of these labs (laboratory 11) analyzed a single sample and divided the residue for determination of both ash and protein. Laboratory 17 noted that in recovering the SDFP fraction from samples I and O, a sticky residue was present on the bottom of the bottles, and this was difficult to recover quantitatively. This sample (Miso soup powder) contains Wakame seaweed, which in turn contains carrageenan polysaccharides which are gelatinous and sparingly soluble in water. The nature of this polysaccharide most likely explains the very high RSD r and RSD R for the IDF fraction of this sample. One collaborator recommended that the washings in step H(d) be combined with the filtrate H(c) to ensure 100% recovery of the SDFS fraction. In the method described here, this is not important as the amount of SDFS is quantitated using the ratio of the areas of the SDFS fraction and DEG or glycerol IS. To ensure 100% recovery of the SDFS fraction the method was modified to include the washings H(d) with the SDFS fraction H(c). Another collaborator highlighted the fact that several disaccharides and other components, like lactose, elute on TSK HPLC at various points between maltose and the SDFS fraction. These compounds include lactitol, maltitol, and isomalt (6-O-a-D-glucopyranosyl-D-glucitol mixed with 1-O-a-D-glucopyranosyl-D-mannitol). A method has been developed to remove lactose and isomaltose in the sample to allow clearer delineation between disaccharide and SDFS. Clearly, if these other compounds find significant use in food products at levels that might interfere with accurate measurement of SDFS, then procedures, possible enzymatic, could be developed to remove these compounds.