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AOAC RESEARCH INSTITUTE AOAC Official Methods of Analysis SM (OMA)

AOAC EXPERT REVIEW PANEL (ERP) FOR DIETARY STARCHES AND DIETARY FIBERS

TUESDAY, SEPTEMBER 26, 2017 10:30 AM – 12:30 PM Meeting Room: M105

AOAC RESEARCH INSTITUTE 2275 RESEARCH BLVD, SUITE 300 ROCKVILLE, MARYLAND 20850 WWW.AOAC.ORG

AOAC Official Methods of Analysis SM (OMA) Expert Review Panel for Dietary Starches and Dietary Fibers

TABLE OF CONTENTS

I. ABOUT AOAC OFFICIAL METHODS OF ANALYSIS SM .......................................................................3 II. AGENDA ......................................................................................................................................7 III. EXPERT REVIEW PANEL ROSTER....................................................................................................9 IV. AOAC INTERNATIONAL VOLUNTEER CONFLICT OF INTEREST, STATEMENT OF POLICY...................11 V. AOAC INTERNATIONAL ANTITRUST POLICY STATEMENT AND GUIDELINES...................................13 VI. AOAC INTERNATIONAL POLICY ON THE USE OF THE ASSOCIATION NAME, INITIALS, IDENTIFYING INSIGNIA, LETTERHEAD, AND BUSINESS CARDS...........................................................................17 VII. MEETING AND METHOD REVIEW INFORMATION ........................................................................19 VIII. AOAC EXPERT REVIEW PANEL ORIENTATION PRESENTATION ........................................................... 21 IX. REVIEW OF METHODS FOR AOAC FIRST ACTION OFFICIAL METHODS OMAMAN-38: TOTAL DIETARY FIBER IN FOODS ENZYMATIC-GRAVIMETRIC-HIGH PRESSURE LIQUID CHROMATOGRAPHY METHOD A. OMAMAN-38 A: COLLABORATIVE STUDY MANUSCRIPT......................................................... 65 B. OMAMAN-38 B: PROTOCOL …................................................................................................. 83 C. OMAMAN-38 C: METHOD USER GUIDE/INSTRUCTIONS FOR USE ........................................ 123 D. OMAMAN-38 D: MATERIAL SAFETY DATA SHEET.................................................................. 147 E. OMAMAN-38 E: METHOD SAFETY CHECKLIST ....................................................................... 159 X. DISCUSS FINAL ACTION REQUIREMENTS FOR FIRST ACTION OFFICIAL METHODS AOAC OFFICIAL METHOD 2014.10, DIETARY STARCH IN ANIMAL FEEDS AND PET FOOD ENZYMATIC- COLORIMETRIC METHOD, FIRST ACTION 2014 A. OMA 2014.10 A: METHOD..................................................................................................... 163 B. OMA 2014.10 B: ARTICLE: DETERMINATION OF DIETARY STARCH IN ANIMAL FEEDS AND PET FOOD BY AN ENZYMATIC-COLORIMETRIC METHOD: COLLABORATIVE STUDY..................... 169 C. OMA 2014.10 C: COLLABORATIVE STUDY PROTOCOL........................................................... 183 D. OMA 2014.10 D: ARTICLE: DETERMINATION OF STARCH, INCLUDING MALTOOLIGOSACCHARIDES, IN ANIMAL FEEDS: COMPARISON OF METHODS AND A METHOD RECOMMENDED FOR AOAC COLLABORATIVE STUDY .......................................................... 217 E. OMA 2014.10 E: CORNELL NET CARBOHYDRATE AND PROTEIN SYSTEM (CNCPS) ............... 225 F. OMA 2014.10 F: FEEDBACK ................................................................................................... 247 G. OMA 2014.10 G: EXPERT REVIEW PANEL REPORT (SEPTEMBER, 2014)................................ 265

ATLANTA MARRIOTT MARQUIS ● 265 PEACHTREE CENTER AVENUE ● ATLANTA, GEORGIA 30303 USA

EXPERT REVIEW PANEL (ERP) FOR DIETARY STARCHES AND DIETARY FIBERS TUESDAY, SEPTEMBER 26, 2017

10:30 AM – 12:30 PM Meeting Room: M105

EXPERT REVIEW PANEL CHAIR: LARS REIMANN, EUROFINS

I.

WELCOME AND INTRODUCTIONS Expert Review Panel Co-Chairs

II. REVIEW OF AOAC VOLUNTEER POLICIES & EXPERT REVIEW PANEL PROCESS OVERVIEW AND GUIDELINES Deborah McKenzie, Senior Director, Standards Development and Method Approval Processes, AOAC INTERNATIONAL and AOAC Research Institute REVIEW OF METHODS (DIETARY FIBERS) For each method, the assigned ERP members will present a review of the proposed collaborative study manuscript, after which the ERP will discuss the method and render a decision on the status for each method. A. OMAMAN-38: Total Dietary Fiber in Foods Enzymatic-Gravimetric-High Pressure Liquid Chromatography Method Study Director: Barry McCleary, Megazyme, Bray Business Park, Southern Cross Road, Bray, Ireland ERP will discuss, review and track First Action methods for 2 years after adoption, review any additional information (i.e., additional collaborative study data, proficiency testing, and other feedback) and make recommendations to the Official Methods Board regarding Final Action status. A. AOAC Official Method 2014.10, Dietary Starch in Animal Feeds and Pet Food Enzymatic-Colorimetric Method, First Action 2014 Original Study Director: Mary Beth Hall, USDA-ARS, U.S. Dairy Forage Research Center, 1925 Linden Drive West Madison, WI 53706 IV. DISCUSS FINAL ACTION REQUIREMENTS FOR FIRST ACTION OFFICIAL METHODS (DIETARY STARCHES )

III.

V.

ADJOURNMENT

*Agenda is subject to change. V1

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Official Methods of Analysis SM (OMA) Expert Review Panel MEETING AND METHOD REVIEW GUIDANCE

The AOAC Research Institute administers AOAC INTERNATIONAL's premier methods program, the AOAC Official Methods of Analysis SM (OMA). The program evaluates chemistry, microbiology, and molecular biology methods. It also evaluates traditional benchtop methods, instrumental methods, and proprietary, commercial, and/or alternative methods and relies on gathering the experts to develop voluntary consensus standards, followed by collective expert judgment of methods using the adopted standards. The Official Methods of Analysis of AOAC INTERNATIONAL is deemed to be highly credible and defensible. All Expert Review Panel (ERP) members are vetted by the AOAC Official Methods Board (OMB) and serve at the pleasure of the President of AOAC INTERNATIONAL. In accordance to the AOAC Expert Review Panel Member and Chair Volunteer Role Description all Expert Review Panel members are expected to 1) serve with the highest integrity, 2) perform duties and method reviews, and 3) adhere to review timelines and deadlines.

To assist the ERP Chair and its members, please note the following in preparation for Expert Review Panel meetings and method reviews.

Pre-Meeting Requirements 1. Confirm availability and plan to be present to ensure a quorum of the ERP.

(Please refer to page 25, Quorum Guidelines, Expert Review Panel Information Packet ) 2. Ensure that your laptop, CPU or mobile device can access online web documentation. 3. Be prepared for the meeting by reviewing all relevant meeting materials and method documentation.

In-Person Meeting and Teleconference Conduct 1. Arrive on time.

2. Advise the Chair and ERP members of any potential Conflicts of Interest at the beginning of the meeting. 3. Participation is required from all members of the ERP. All members have been deemed experts in the specific subject matter areas. 4. The ERP Chair will moderate the meeting to ensure that decisions can be made in a timely manner. 5. Follow Robert’s Rules of Order for Motions. 6. Speak loud, clear, and concise so that all members may hear and understand your point of view. 7. Due to the openness of our meetings, it is imperative that all members communicate in a respectful manner and tone. 8. Refrain from disruptive behavior. Always allow one member to speak at a time. Please do not interrupt. 9. Please note that all methods reviewed and decisions made during the Expert Review Panel process are considered confidential and should not be discussed unless during an Expert Review Panel meeting to ensure transparency. Reviewing Methods Prior to the Expert Review Panel meeting, ERP members are required to conduct method reviews. All methods are reviewed under the following criteria, technical evaluation, general comments, editorial criteria, and recommendation status. These methods are being reviewed against their collaborative study protocols as provided in the supplemental documentation. Note: The method author(s) will be present during the Expert Review Panel session to answer any questions.

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Version 1 – OMA ERP Meeting Conduct

Official Methods of Analysis SM (OMA) Expert Review Panel MEETING AND METHOD REVIEW GUIDANCE

Reviewing Methods (Cont’d)

Reviewers shall conduct in-depth review of method and any supporting information. In-depth reviews are completed electronically via the method review form. The method review form must be completed and submitted by the deadline date as provided. All reviews will be discussed during the Expert Review Panel meeting. Any ERP member can make the motion to adopt or not to adopt the method. If the method is adopted for AOAC First Action status, Expert Review Panel members must track and present feedback on assigned First Action Official Methods . Recommend additional feedback or information for Final Action consideratio n. Here are some questions to consider during your review based on your scientific judgment: 1. Does the method sufficiently follow the collaborative study protocol? 2. Is the method scientifically sound and can be followed? 3. What are the strengths and weaknesses of the method? 4. How do the weaknesses weigh in your recommendation for the method? 5. Will the method serve the community that will use the method? 6. What additional information may be needed to further support the method? 7. Can this method be considered for AOAC First Action OMA status? Reaching Consensus during Expert Review Panel Meeting 1. Make your Motion. 2. Allow another member to Second the Motion. 3. The Chair will state the motion and offer the ERP an option to discuss the motion. 4. The Chair will call a vote once deliberations are complete. 5. Methods must be adopted by unanimous decision of ERP on first ballot, if not unanimous, negative votes must delineate scientific reasons. Negative voter(s) can be overridden by 2/3 of voting ERP members after due consideration. 6. All other motions will require 2/3 majority for vote to carry.

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Version 1 – OMA ERP Meeting Conduct

OMAMAN-38 A: Collaborative Study Manuscript Expert Review Panel Use Only September, 2017

AOAC Official Method 20 17 .xx Total Dietary Fiber in Foods Enzymatic-Gravimetric-High Pressure Liquid Chromatography Method

(Applicable to plant material, foods, and food ingredients).

A. Principle A method is described for the measurement of total dietary fiber as defined by Codex Alimentarius Commission (CAC). The method quantitates high molecular weight dietary fiber (HMWDF), which includes insoluble dietary fiber (IDF) and fiber which precipitates in the presence of 78% ethanol (SDFP); and fiber which is soluble in 78% ethanol (SDFS). Resistant starch (RS) is captured in the IDF fraction ( Figure 2017.xxA ). This method combines the key attributes of AOAC Official Methods of Analysis 991.43, 2001.03, and 2002.02 and is an update of AOAC Method 2009.01. Duplicate test portions are incubated with pancreatic α -amylase and amyloglucosidase (AMG) for 4 hr at 37 o 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-resistant starch is solubilised and hydrolysed to glucose and maltose by the combined action of the two enzymes. The reaction is terminated by pH adjustment and temporary heating. Protein in the sample is digested with protease. For the measurement of total dietary fiber, ethanol or industrial methylated spirits (IMS) are added and the IDF and SFDP are captured, on a scintered glass crucible, washed with ethanol and acetone, dried and weighed. One of the duplicate residues is analysed for protein, the other for ash. SDFS in the filtrate is concentrated, desalted with resins and quantitating by HPLC. This method differs from AOAC Method 2009.01 in that incubation time with pancreatic α -amylase and amyloglucosidase is reduced from 16 h to 4 h (with higher concentrations of enzymes used) to better simulate human intestinal residence time; improved desalting and HPLC separation of SDFS is incorporated; glycerol is used as the internal standard; and sodium azide is deleted from the incubation buffer. (a) Grinding mill.— Centrifugal, with 12-tooth rotor and 0.5 mm sieve, or similar device. Alternatively, cyclone mill can be used for small test laboratory samples provided they have sufficient air flow or other cooling to avoid overheating samples. AOAC Research Institute ERP Use Only B. Apparatus

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(b) Digestion Bottles.— 250 mL Fisherbrand ® soda glass, wide mouth bottles with polyvinyl lined cap (cat. no. 11798859) ( Figures 2017.xxB&C ) . (https://se.fishersci.com/se/index.php?option=com_insight2&task=getproduct&prod uctCode=11798859) (accessed 8 th May, 2016). (c) Fritted crucible.— Corning Büchner, fritted disk, Pyrex ® 50 mL, pore size, coarse, ASTM 40-60 µm, or equivalent. (product no. 32940-50C). Prepare as follows: i. Ash overnight at 525°C in muffle furnace, cool furnace to 130°C before removing crucibles to minimize breakage.

Remove any residual Celite and ash material by using a vacuum Soak in 2 % Micro cleaning solution, [C(o)] at room temperature for 1 hr.

ii.

iii. iv.

Rinse crucibles with water and deionized water. For final rinse, use 15 mL acetone and air dry.

v.

Add approximately 1.0 g Celite to dried crucibles and dry at 130°C to constant weight. Cool crucible in desiccators for approximately 1 hr and record weight of crucible containing Celite.

vi.

vii.

(d) (e)

Filtering flask. — heavy-walled, 1-L with side arm.

Rubber ring adaptors .— for use to join crucibles with filtering flasks.

(f) Vacuum source .— vacuum pump or aspirator with regulator capable of regulating vacuum. (g) Water bath(s) .— rotary motion, shaking, large-capacity (20-24 L) with covers; capable of maintaining temperature of 37+ 1°C and 60 + 1°C. Ensure that shaking action/sample agitation in water bath is sufficient to maintain sample solids in suspension and that no residue buildup or rings of sample material form in the digestion bottle during the enzymatic digestions (i.e. at 150 rev/min) ( Figure 2017.xxB ). If the water bath is used in linear motion (not preferred motion), then the bottles must be placed at an angle of 45 o to ensure continual suspension of the sample during the 4 h incubation period with PAA/AMG. Alternatively, mixing can be achieved with a 2mag Mixdrive 15 ® submersible magnetic stirrer with a 30 × 7 mm stirrer bar, set at 170 rpm ( Figure 2017.xxC ). AOAC Research Institute ERP Use Only

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Balance .— 0.1 mg readability, accuracy, and precision .

(h)

Ovens .— two, mechanical convection, set at 103 ± 2°C and 130 ± 3°C.

(i) (j)

Timer.

(k) Desiccator .— airtight, with SiO 2 or equivalent desiccant. Desiccant dried biweekly overnight in 130 °C oven, or more frequently as needed. (l) pH meter. (m) Pipettors and tips.— 50-200 µL and 5 mL capacity. (n) Dispensers.— i. 15 ± 0.5 mL for 78 % EtOH (or IMS), 95 % ethanol (or IMS), and acetone. ii. 35 ± 0.5 mL for buffer. (o) Cylinder , graduated, 100 mL and 500 mL. (p) Magnetic stirrers and stirring bars. (q) Rubber spatulas (r) Muffle furnace .— 525 ± 5°C (s) Polypropylene tube; 40 mL, 84 x 30 mm, flat base with screw cap; 13 mL, 101 x 16.5 mm, flat base, with screw cap. (t) High Performance Liquid chromatograph (HPLC).— With oven to maintain a column temperature of 80°C and a 50 µL injection loop. Column operating conditions are: Temperature, 80°C; mobile phase, distilled water, flow rate, 0.5 mL/min. (u) HPLC columns.— Two TSK-Gel G2500PWXL columns, 30 cm × 7.8 mm, connected in series. Operate at 80°C. Mobile phase: distilled water at 0.5 mL/min. System must be capable of separating maltose from maltotriose ( Figure 2017.xxD ). Run time of 60 min to ensure that all materials from the injection are cleared from the column prior to the next injection. (v) Cation and anion exchange guard column (containing de-ashing/ de-salting cartridges).— Cation and anion exchange guard cartridges, H + and CO 2 3- forms respectively. (Bio-Rad Laboratories, Cat. No. 125-0118, includes one cation and one anion cartridge), with guard column holder (Bio-Rad Labratories, Cat. No. 125-039) to hold the two guard cartridges in series, cation cartridge preceeding anion cartridge). AOAC R search Institute ERP Use Only

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(w) Guard column (or pre-column).— TSK Gel PWx1 guard column (TOSOH). (x) Detector.— Refractive index (RI); maintained at 50°C. (y) Data integrator or computer.— For peak area measurement. (z) Filters for disposable syringe.— 0.45 µm membrane, 13 mm or 25 mm. (aa) Filters for water.— polyvinylidene fluoride, pore size 0.45 µm, 47 mm. (bb) Filter apparatus.— To hold 47 mm, 0.45 µm filter, [B(aa)] ; to filter larger volumes of water. (cc) Syringes.— 10 mL, disposable, plastic. (dd) Syringes.— Hamilton 100 µ L, 710SNR syringe. (ee) Rotary evaporator .—Heidolph Laborota 4000 or equivalent. (ff) Thermometer .—Capable of measuring to 100°C. (a) Ethanol 95 % v/v. or Industrial Methylated Spirits (IMS). Industrial Methylated Spirits made up of: ethanol 84.8333 (w%), 85.952 (v%); water 5.6571 (w%), 4.524 (v%); 2- propanol 4.9118 (w%), 5.0000 (v%); methanol 4.5979 (w%), 4.524 (v%). Can be prepared by mixing 5 volumes of 2 propanol with 95 volumes of denatured ethanol formula SDA- 3A[100 volumes of 95% ethanol combined with 5 volumes of methanol]. (b) Ethanol (or IMS), 78 % .— Place 179 mL water into 1-L volumetric flask. Dilute to volume with 95 % ethanol or IMS. Mix. (c) Acetone , reagent grade. (d) Stock PAA plus AMG powder .— PAA (40 KU/g) plus AMG (17 KU/g) as a freeze-dried powder mixture. Note: One Unit of AMG activity is the amount of enzyme required to release one µ mole of D -glucose from soluble starch per minute at 40°C and pH 4.5; one Unit of PAA activity is the amount of enzyme required to release one µ mole of p -nitrophenyl from Ceralpha reagent per minute at 40°C and pH 6.9; AOAC Method 2002.01). PAA/AMG preparations should be essentially devoid of β -glucanase, β -xylanase and detectable levels of free D -glucose. Stable for > 4 years at −20°C. (e) PAA (4 KU/5 mL)/AMG (1.7 KU/5 mL) .— Immediately before use, dissolve 1 g of PAA/AMG powder in 50 mL of sodium maleate buffer (50 mM, pH 6.0 plus 2 mM CaCl 2 ) and stir for approx. 5 min. Store on ice during use. Use on the day of preparation. AOAC Research Institute ERP Use Only C. Reagents

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[ 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 of PAA/AMG powder mix (PAA 40 KU/g plus AMG 17 KU/g; reagent 4) to 70 mL of cold, distilled water in a 200 mL beaker on a magnetic stirrer in a laboratory hood and stir until the enzymes are completely dissolved (approx. 5 min). Add 35 g of 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 o C. (This preparation contains PAA at 2 KU/mL and AMG at 0.85 KU/mL). Stable at 4 o C for 3 months]. (f) Protease suspension (50 mg/mL, ~ 6 Tyrosine U/mg).— Stabilised suspension in 3.2 M ammonium sulphate. -Swirl gently before use. Dispense using a positive displacement dispenser. Protease must be devoid of α -amylase and essentially devoid of β -glucanase and β -xylanase. Use as supplied. Stable for > 4 years at 4°C. (g) Glycerol internal standard.– 100 mg/mL containing sodium azide (0.02% w/v). Stable for > 4 years at 4°C. Diethyleneglycol (100 mg/mL) in sodium azide (0.02 %) is an alternative internal standard. This is less stable than the glycerol standard, so must be prepared on a weekly basis. (h) LC retention time standard (malto-oligosaccharides).— Dissolve 1.25 g of retention time standard (consisting of corn syrup solids (DP > 3) and maltose in 30 mL of 0.02% sodium azide solution and transfer to 50-mL volumetric flask. Pipette 5 mL of glycerol internal standard (100 mg/mL). Bring to 50 mL with 0.02% sodium azide solution [C(n)]. Transfer solutions to 50-mL Duran bottle. Stable at 4 o C for > 2 years. (i) D -Glucose/glycerol LC standard.— 10 mg/mL of each containing sodium azide (0.02% w/v). Stable for > 4 years at 4°C. (j) Sodium maleate buffer.— 50 mM, pH 6.0 plus 2 mM CaCl 2 and 0.02 % sodium azide. Dissolve 11.6 g of maleic acid in 1600 mL of deionised water and adjust the pH to 6.0 with 4 M (160 g/l) NaOH solution. Add 0.6 g of calcium chloride (CaCl 2 .2H 2 O) and adjust the volume to 2 L. Stable for ~ 2 weeks at 4°C. ( Alternatively , prepare the buffer with the addition of sodium azide. In this case, add 0.4 g of sodium azide to 2 L of final buffer solution and dissolve with stirring. Stable for > 2 years at 4°C). [ NOTE: do not add the sodium azide until the pH has been adjusted. Acidification of sodium azide releases a poisonous gas] and adjust the volume to 2 L. Stable for > 1 year at 4°C. AOAC Research Institute ERP Use Only

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(k) MES buffer.— {this can be used as an alternative to sodium maleate buffer; [c(j)] }. 50 mM, pH 6.0 plus 2 mM CaCl 2 .– Dissolve 19.5 g of MES [2-( N -morpholino) ethanesulfonic acid] (Megazyme cat. no. B-MES250) in 1600 mL of deionised water and adjust the pH to 6.0 with 4 M (160 g/l) NaOH solution. Add 0.6 g of calcium chloride (CaCl 2 .2H 2 O) and adjust the volume to 2 L. Solution is stable for ~ 2 weeks at 4°C. ( Alternatively , prepare the buffer with the addition of sodium azide. In this case, add 0.4 g of sodium azide to 2 L of final buffer solution and dissolve with stirring. Stable for > 2 years at 4°C). (l) Trizma Base (Megazyme cat. no. B-TRIS500), 0.75 M.— Add 90.8 g of Trizma base to approx. 800 mL of distilled water and dissolve. Adjust volume to 1 L. Stable for > 1 year at room temperature . (m)Acetic acid solution, 2 M.— Add 115 mL of glacial acetic acid (Fluka 45731) to a 1-L volumetric flask. Dilute to 1-L with distilled water. Stable for > 1 year at room temperature. (n) Sodium azide solution (0.02 % w/v). — Add 0.2 g of sodium azide to 1 L of deionized water and dissolve by stirring. Stable at room temperature for > 1 year. (o) Cleaning solution.— Micro (International Products Corp., Trenton, NJ). Make a 2 % solution with deionized water. (p) pH standards.— Buffer solutions at pH 4.0, 7.0 and 10.0. (q) Deionized water . (r) Celite.— acid-washed, pre-ashed (Megazyme G-CEL100 or G-CEL500). (s) Amberlite ® FPA53 (OH − ) resin (Megazyme cat. no. G-AMBOH), ion exchange capacity 1.6 meq/mL (minimum) and Ambersep ® 200 (H + ) resin (Megazyme cat. no. G-AMBH), ion exchange capacity: 1.6 meq/mL (minimum).

Items (d), (f), (g), (h) and (i) are supplied in the Rapid Integrated Total Dietary Fiber kit available from Megazyme, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland, but preparations of reagents and buffers which meet the criteria as specified in the method above may also be used. AOAC Research Institute ERP Use Only

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D. Preparation of Test Samples

Collect and prepare samples as intended to be eaten. Defat if >10% fat. For high moisture samples it may be desirable to freeze dry. Grind ca 50 g in a grinding mill [B(a)] to pass a 0.5 mm sieve. Transfer all material to a wide mouthed plastic jar 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, run standards listed in Table 991.43B each time enzyme lot changes or at a maximum 6 month interval.

F. Enzymatic Digestion of Sample

(1) Blanks

With each assay, run two blanks along with samples to measure any contribution from reagents to residue.

(2) Samples

(a) Weigh duplicate 1.000±0.005 g samples accurately into 250 mL polypropylene bottles.

(b) Wet the sample with 1.0 mL of ethanol (or IMS) and add 35 mL of 50 mM sodium maleate buffer [C(j)] or MES buffer [C(k)] and a 7 x 30 mm stirrer bar to each bottle. Place bottles on a 2mag Mixdrive 15 magnetic stirrer apparatus in a water bath set at 37 o C [B(g)]. Stir the contents at 170 rpm for 10 min to equilibrate to 37 o C. Alternatively, transfer the bottles (without stirrer bar) to a Grant OLS 200 shaking incubation bath (or similar), secure in place with the shaker frame springs and shake at 150 rpm in orbital motion for 10 min. (c) Incubation with pancreatic α -amylase plus AMG.— Add 5.0 mL of PAA/AMG solution (reagent 5) (PAA 4 KU/mL and AMG 1.7 KU/mL) to each bottle, cap the bottles and incubate the reaction solutions at 37C with stirring at 170 rpm for exactly 4 h using a AOAC Research Institute ERP Use Only

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magnetic stirrer bar and a 2mag Mixdrive 15 magnetic stirrer apparatus; alternatively incubate in a shaking water bath maintained at 37C at 150 revolutions/min (orbital motion) for exactly 4 h. (Alternatively, If employing the ammonium sulphate suspension of PAA/AMG [PAA (2 KU/mL) /AMG (0.85 KU/mL)] { [C(e)] , alternative}, gently swirl the suspension before use and add 2.0 mL of this suspension and 3 mL of maleate buffer [C(j)] or MES buffer [C(k)] to each bottle and incubate as indicated. (d) Adjustment of pH to approx. 8.2 (pH 7.9-8.4), Inactivation of α -amylase and AMG.— After 4 h, remove all sample bottles from the stirring or shaking water bath, and immediately add 3.0 mL of 0.75 M Tris base solution (reagent 12) to adjust pH to approximately 8.2 (7.9–8.4), at which pH AMG has no activity. Immediately, slightly loosen the caps of the sample bottles, place the bottles in a boiling water bath (nonshaking; 95-100°C), and incubate for 20 min with occasional agitation (by hand). This inactivates both PAA and AMG. With a thermometer, ensure that the final temperature of the bottle contents is >90°C. Checking just one bottle is adequate. (At the same time, if only one shaker bath is available, increase the temperature of the shaking incubation bath to 60°C in readiness for the protease incubation step). (e) Cool and protease treatment.— Remove all sample bottles from the hot water bath and cool to approx. 60°C. Add 0.1 mL of protease suspension [C(f)] with a positive displacement dispenser (solution is thick) and incubate at 60°C for 30 min.

(f) pH adjustment.— Add 4.0 mL of 2 M acetic acid to each bottle and mix. This gives a final pH of approx 4.3. (g) Proceed to step [G(a)] for determination of HMWDF. AOAC Research Institute ERP Use Only (g) Add internal standard.— To each sample, add 1 mL of 100 mg/mL glycerol (or diethyleneglycol) internal standard solution [C(g)].

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G. Determination of HMWDF (IDF + SDFP)

(a) Precipitation of high molecular weight soluble dietary fiber (HMWSDF).— To each sample, add 207 mL (measured at room temperature) of 95 % (v/v) EtOH or IMS preheated to 60°C and mix thoroughly. Allow the precipitate to form at room temperature for 60 min (overnight precipitation is acceptable). (b) Filtration setup.— Tare crucible containing Celite to nearest 0.1 mg. Wet and redistribute the bed of Celite in the crucible, using 15 mL of 78 % (v/v) EtOH (or IMS) from wash bottle. Apply suction to crucible to draw Celite onto fritted glass as an even mat. Discard these washings. (c) Filtration.— Using vacuum, filter precipitated enzyme digest [G(a)] through crucible. Using a wash bottle with 78 % (v/v) EtOH or IMS, quantitatively transfer all remaining particles to crucible and wash the residue successively with two 15 mL portions of 78% v/v EtOH or IMS. Retain filtrate and washings for determination of SDFS [ H(a) ]. (d) Wash.— Using a vacuum, wash residue successively with two 15 mL portions of the following: 78 % (v/v) EtOH or IMS; 95 % (v/v) EtOH or IMS; Acetone. Discard these washings. Draw air through the crucibles for at least 2 min to ensure all acetone is removed before drying crucibles in an oven.

Dry crucibles containing residue overnight in 103°C oven.

(e)

(f) Cool crucible in desiccators for approximately 1 hr. Weigh crucible containing dietary fiber residue and Celite to nearest 0.1 mg. To obtain residue weight, subtract tare weight, i.e., weight of dried crucible and Celite. of protein. For ash analysis, incinerate the second residue for 5 hr at 525°C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract crucible and Celite weight to determine ash. AOAC Research Institute ERP Use Only (g) Protein and ash determination.— The residue from one crucible is analyzed for protein, and the second residue of the duplicate is analyzed for ash. Perform protein analysis on residue using Kjeldahl or combustion methods. Use 6.25 factor for all cases to calculate g

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Proceed to step [I(a)] for calculations.

(h)

H. Determination of SDFS Proper deionization of the filtrate is an essential part of obtaining quality chromatographic data on SDFS. Refer to ( Figure 2017.xxE ) to see patterns of glycerol and D-glucose in the presence and absence of buffer salts. To ensure that the resins being used are of adequate deionizing capacity, add 0.1 mL of protease suspension [C(f)] to 40 mL of either maleate buffer [C(j)] or MES buffer [C(k)] along with 3.0 mL of 0.75 M Tris base solution [C(l)] , 4.0 mL of 2M acetic acid [C(m)] , 1 mL of glycerol internal standard (100 mg/mL) [C(g)] and 1 mL of D-glucose solution (100 mg/mL). Concentrate this solution to dryness on a rotary evaporator and re-dissolve the residue in 32 mL of deionised water. To 5 mL of this solution in a 13 mL polypropylene tube [ B (s)] add 1.5 g of Amberlite ® FPA53 (OH − ) resin and 1.5 g of Ambersep ® 200 (H + ) and swirl the contents regularly over 5 min. Allow the resin to settle and remove the supernatant (1.5-2.0 mL) with a syringe [ B (cc)] and filter through a polyvinylidene fluoride filter, pore size 0.45 μm [ B (z)] . Inject an aliquot (50 L) of this solution onto the TSK columns (Bio-Rad ® de-ashing pre-cartridges in place). No salt peaks should be seen on HPLC. ( a) Filtrate recovery, desalting, and LC analysis.— (Set aside the filtrate from one of the sample duplicates [G(c)] to use in case of spills or if duplicate LMWDF data is desired. Transfer the filtrate [G(c)] into a 500 mL measuring cylinder. Adjust the volume to 300 mL with 78 % v/v aqueous ethanol [ C (b)], transfer to a 1 L beaker and mix thoroughly. Transfer ~ 75 mL (~ 25 %) of this solution to a 500 mL evaporator flask, and concentrate with a rotary evaporator to dryness at 50°C. [Note: it is not essential to quantitatively transfer all solution because SDFS is determined by the ratio of these peaks on HPLC to that of glycerol internal standard]. (b) Desalting of sample.— Dissolve the residue in the evaporator flask in 8 mL of deionized water and transfer most of this solution to a 40 mL polypropylene container (apparatus 19). Transfer 5 mL of this solution to a 13 mL polypropylene tube (apparatus 19) containing 1.5 g of Amberlite ® FPA53 (OH − ) resin and 1.5 g of Ambersep ® 200 (H + ) (Fig. 5). Cap the container and invert the contents regularly over 5 min. Alternatively, if the ammonium AOAC Research Institute ERP Use Only

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sulphate suspension of PAA/AMG is used for starch digestion [see Reagents 5, alternative], then use 2 g of Amberlite ® FPA53 (OH − ) resin and 2 g of Ambersep ® 200 (H + ) to ensure effective removal of most of the salt in the sample. (c) Prepare samples for LC analysis . Remove a sample (approx. 1.5-2.0 mL) of the supernatant solution from the resin slurry ( Figure 2017.xxF ) with a syringe [ B (cc)] and filter through a polyvinylidene fluoride filter, pore size 0.45 μm [ B (z)]. Use this solution as the sample extract in step [H(f)] . HPLC patterns for non-desalted sample, sample desalted with resin in tube, and sample of desalted preparation run onto TSK columns through Bio-Rad ® de- ashing pre-cartridges are shown in ( Figure 2017.xxE ). (d) Determine the response factor for D-glucose. (Since D-glucose provides an LC refractive index response equivalent to the response factor for the nondigestible oligosaccharides that make up SDFS, D-glucose is used to calibrate the LC and the response factor is used for determining the mass of SDFS). Use a 100-μL LC syringe [ B (dd)] to fill the 50μL injection loop for the standard internal standard/D-glucose solution [ C (i)]. Inject in triplicate. Internal standard method. Obtain the values for the peak areas of D-glucose and internal standard (glycerol) from duplicate chromatograms. The ratio of peak area of D-glucose/peak area of glycerol to the ratio of the mass of D-glucose/mass of glycerol is the “response factor.” The average response factor for D-glucose is approximately 0.82 vs. glycerol.

Response factor (Rf) = (PA-IS)/ (PA-Glu) x (Wt-Glu)/(Wt-IS) (e) Calibrate the area of the chromatogram to be measured for SDFS. Use a 100-μL LC syringe [ B (dd)] to fill the 50-μL injection loop with retention time standard [C(h)] Inject in duplicate. Determine the demarcation point between DP2 and DP3 oligosaccharides (disaccharide maltose versus higher oligosaccharides) (Figure 2017.xxD). AOAC Research Institute ERP Use Only where PA-Glu = peak area of D-glucose; PA-IS = peak area of internal standard (glycerol);Wt-Glu = mass of D-glucose in standard; and Wt-IS = mass of internal standard (glycerol) in standard.

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(f) Determine peak areas of SDFS (PA-SDFS) and internal standard (PA-IS) in chromatograms of sample extracts. Inject sample extracts [ H (c)] on LC. Record areas of all

peaks of DP greater than the DP2/DP3 demarcation point as PA-SDFS. Record the peak area of internal standard as PA-IS.

I. Calculations for HMWDF (IDF + SDFP) Blank (B, mg) determination .

B = [(BR 1 + BR 2 )]/2 – P B – P A

Where: BR 1 and BR 2 = residue mass, in milligrams, for duplicate blank determinations, respectively, and P B and P A = mass, in milligrams, of protein and ash, respectively, determined on first and second blank residues.

HMWDF (mg/100 g) = [(R 1 + R 2 )/2 – P B – P A – B]/(M 1 + M 2 )/2] x 100

Where: R 1 = residue mass 1 from M 1 in milligrams; R 2 = residue mass 2 from M 2 in milligrams; M 1 = test portion mass 1 in grams; M 2 = test portion mass 2 in grams; P A = ash mass in milligrams from R 1 ; P B = protein mass in milligrams from R 2 .

J. Calculations for SDFS

Internal Standard Method SDFS(mg/100 g) = Rf ×Wt-IS × (PA-SDFS)]/(PA-IS) x 100/M AOAC Research Institute ERP Use Only where Rf = the response factor; Wt-IS = milligrams of internal standard contained in 1 mL of internal standard solution (100 mg/mL) pipetted into sample before filtration; PA-SDFS = the peak area of the SDFS; PA-IS = the peak area of the internal standard; M = the test portion mass M 1 or M 2 of the sample whose filtrate was concentrated and analyzed by LC.

L. Calculation of Total Dietary Fiber

Total Dietary Fiber (%) = (IDF + HMWSDF + LMWSDF)/1000

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Calculations can be simplified by using an Excel ® based calculator (Supporting Information)

M.

References

Figures: Figure 2017.xxA. Rapid integrated total dietary fiber assay procedure showing the key steps in the procedure. Figure 2017.xxB. Incubation of samples in Fisherbrand ® incubation bottles in a shaking water bath, showing custom made polypropylene bottle holder. Figure 2017.xxC. 2mag Mixdrive 15 ® submersible magnetic stirrer in custom built bath. With Fisherbrand ® incubation bottles. Figure 2017.xxD. Chromatograms of a mixture of maltodextrins, glucose and glycerol on two TSK gel filtration columns (G2500PWXL) in series. Solvent: distilled water; flow rate: 0.5 mL/min; temperature: 80°C. The arrows show demarcation between DP2 (maltose) and DP 3 (higher maltodextrins). The fraction shown as SDFS denotes the fraction that would be collected as SDFS, however, in this case these are maltodextrins that would be hydrolysed by the PAA/AMG mixture. Figure 2017.xxE. Chromatograms on TSK-Gel G2500PWXL columns of glucose/glycerol mixtures. A mixture of glycerol (100 mg) and glucose (100 mg) was analysed according to the RINTDF procedure. The ethanolic filtrate (for SDFS determination) was concentrated to dryness and re-dissolved in 32 mL of deionised water. A sample of this was analysed by HPLC; a) directly with no desalting and no Bio-Rad ® de-ashing pre-cartridges in place; b) a sample (5 mL) was desalted by mixing with 1.5 g of Amberlite ® FPA53 (OH − ) and 1.5 g of Ambersep ® 200 (H + ) resins over 5 min and the supernatant was analysed by HPLC with no Bio-Rad ® de-ashing pre-cartridges in place; c) Sample b) was analysed with a Bio-Rad ® de-ashing pre-cartridges in place. Desalting 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 ® de-ashing pre-cartridges. This desalting step increases the effectiveness of the de-ashing cartridges and allows up to 10-times more samples to be chromatographed before the need to regenerate or replace the de-ashing cartridges. Figure 2017.xxF. Desalting of samples for HPLC. Five (5) mL of concentrated eluate mixed with 1.5 g of Amberlite ® FPA53 (OH − ) and 1.5 g of Ambersep ® 200 (H + ) resins in a polypropylene tube. AOAC R search Institute ERP Use Only

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Figure 2017.xxA. Rapid integrated total dietary fiber assay procedure showing the key steps in the procedure.

Figure 2017.xxB. Incubation of samples in Fisherbrand ® incubation bottles in a shaking water bath, showing custom made polypropylene bottle holder. AOAC Research Institute ERP Use Only

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Figure 2017.xxC. 2mag Mixdrive 15 ® submersible magnetic stirrer in custom built bath. With Fisherbrand ® incubation bottles.

Figure 2017.xxD. Chromatograms of a mixture of maltodextrins, glucose and glycerol on two TSK gel filtration columns (G2500PWXL) in series. Solvent: distilled water; flow rate: 0.5 mL/min; temperature: 80°C. The arrows show demarcation between DP2 (maltose) and DP 3 (higher maltodextrins). The fraction shown as SDFS denotes the fraction that would be collected as SDFS, however, in this case these are maltodextrins that would be hydrolysed by the PAA/AMG mixture. AOAC Research Institute ERP Use Only

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Figure 2017.xxE. Chromatograms on TSK-Gel G2500PWXL columns of glucose/glycerol mixtures. A mixture of glycerol (100 mg) and glucose (100 mg) was analysed according to the RINTDF procedure. The ethanolic filtrate (for SDFS determination) was concentrated to dryness and re-dissolved in 32 mL of deionised water. A sample of this was analysed by HPLC; a) directly with no desalting and no Bio-Rad ® de-ashing pre-cartridges in place; b) a sample (5 mL) was desalted by mixing with 1.5 g of Amberlite ® FPA53 (OH − ) and 1.5 g of Ambersep ® 200 (H + ) resins over 5 min and the supernatant was analysed by HPLC with no Bio-Rad ® de-ashing pre-cartridges in place; c) Sample b) was analysed with a Bio-Rad ® de-ashing pre-cartridges in place. Desalting 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 ® de-ashing pre-cartridges. This desalting step increases the effectiveness of the de-ashing cartridges and allows up to 10-times more samples to be chromatographed before the need to regenerate or replace the de-ashing cartridges. AOAC Research Institute ERP Use Only

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Figure 2017.xxF. Desalting of samples for HPLC. Five (5) mL of concentrated eluate mixed with 1.5 g of Amberlite ® FPA53 (OH − ) and 1.5 g of Ambersep ® 200 (H + ) resins in a polypropylene tube.

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12 th May, 2017 AOAC International /AACC International / ICC Interlaboratory Evaluation of A Rapid Integrated Total Dietary Fiber Method consistent with the CODEX Definition of Dietary Fiber AOAC Official Method 2017.xx Total Dietary Fiber in Foods Enzymatic-Gravimetric-High Pressure Liquid Chromatography Method General approach: This study was coordinated by Dr. B. McCleary, Megazyme, and was performed in 2016 under the auspices of AACC International and ICC (International Association of Cereal Science and Technology). The study was performed in three stages; In the First Stage , various laboratories were invited to participate. Those who accepted were: 1 Megazyme, Bray, County Wicklow, Ireland. 2 Medallion Laboratories/General Mills, Golden Valley, MN, USA. 3 Agriculture and Agri-Food Canada/Agriculture et Agroalimentaires Canada, University of Manitoba – Winnipeg, Manitoba, Canada. 4 Grain Growers Limited, PO Box 7, North Ryde, NSW, Australia. 5 Sanitarium Development and Innovation Analytical Department, Cooranbong, NSW, Australia. 6 CRDS Tienen, Central Department Research, Development and Services, Tienen, Belgium. 7 Finnish Food Safety Authority Evira, Chemistry and Toxicology Research Unit, Helsinki, Finland. 8 Matsutani Chemical Company, Itami City, Hyogo, Japan. 9 NEOTRON SPA, Stradello Aggazzotti, Modena, Italy. 10 Eurofins Food Testing Netherlands BV, Heerenveen, Netherlands. 11 Kellogg Company, Battle Creek, MI, U.S.A. 12 Japanese Food Research Laboratories, Japan 13 Nestle, Food Science and Technology Carbohydrates, Nestlé Research Centre Lausanne, Switzerland. In the second stage , four samples were sent to each collaborator along with the assay protocol, all enzymes and reagents required to perform the assay. There were no blind duplicates. The collaborators were requested to analyse the samples for HMWDF and SDFS in duplicate and to report back on any particular difficulties experienced so that these could be addressed before the full collaborative study was initiated. Results were received and compared. The only complication was that two laboratories did not have the

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required HPLC columns. These collaborators were requested to concentrate the SDFS fractions and ship them to Megazyme where the HPLC analysis would be performed and results returned to the collaborator. Results from this evaluation were discussed with the particular collaborator, with the aim of identifying problems. All results were shared with all collaborators (specific collaborator results remained confidential). In the third Stage, 16 samples were sent to each collaborator as eight blind duplicates. These were analysed for HMWDF and SDFS. This set of samples contained food materials as well as samples containing resistant starch. Results from this study were received after approximately 3-4 months and were analysed using the AOAC International statistics program.

Barry V. McCleary, PhD, DScAgr. CEO Megazyme. Adjunct Professor, University of Sydney.

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No. labs/analysts 12 13 12 12 13 13 12 13 Mean, % 60.62 23.70 29.37 6.79 16.15 19.28 21.09 10.76 S r 0.74 0.67 0.36 0.29 0.39 0.29 0.43 0.68 S R 4.67 0.99 0.78 0.91 0.85 1.74 0.57 0.86

%RSD r 1.22 2.81 1.22 4.32 2.41 1.51 2.05 6.34 %RSD R 7.70 4.17 2.64 13.38 5.29 9.01 2.72 8.02 Samples: A&D = Fibersym ® ; B&F = kidney beans (canned, washed and lyophilized); C&J = bran cereal; E&H = Defatted cookies containing FOS; G&N = oat bran; I&M = defatted cookies containing polydextrose and RS 2 ; K&O = dark rye crispbread; L&P = whole meal bread. a s r ; Within laboratory variability; RSD r : within laboratory relative variability; s R : between laboratory variability; and RSD R : between laboratory relative

L & P

K & O

I & M

G & N

E & H

C & J

B & F

A & D

Table 2017.xx. Interlaboratory study results for total dietary fiber in foods (RIN-TDF method). Sample/parameter variability.

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Determination of Total Dietary Fiber (CODEX Definition) by a Rapid Enzymatic- Gravimetric Method and Liquid Chromatography: Collaborative Study Barry V. McCleary, Jodi Cox, Vincent A. McKie Megazyme, Bray, County Wicklow, Ireland. David Plank Medallion Laboratories/General Mills, Golden Valley, MN, USA. Nancy Ames Agriculture and Agri-Food Canada/Agriculture et Agroalimentaires Canada, University of Manitoba – Winnipeg, Manitoba, Canada. Hayfa Salman Grain Growers Limited, PO Box 7, North Ryde, NSW, Australia. Susan McCarthy Sanitarium Development and Innovation Analytical Department, Cooranbong, NSW, Australia. Saskia Iilians CRDS Tienen, Central Department Research, Development and Services, Tienen, Belgium. Helena Pastell Finnish Food Safety Authority Evira, Chemistry and Toxicology Research Unit, Helsinki, Finland. Toyohide Nishibata Matsutani Chemical Company, Itami City, Hyogo, Japan. Alessandro Santi NEOTRON SPA, Stradello Aggazzotti, Modena, Italy. Peter Sanders Eurofins Food Testing Netherlands BV, Heerenveen, Netherlands. Yulai Jin Kellogg Company, Battle Creek, MI, U.S.A. Mikihiko Yoshida Japanese Food Research Laboratories, Japan. Delphine Curti Nestle, Food Science and Technology Carbohydrates, Nestlé Research Centre Lausanne, Switzerland.

ABSTRACT A rapid, integrated method for measurement of total dietary fiber (TDF) as defined by the Codex Alimentarius Commission was validated for foods. The method is applicable to plant materials, foods, and food ingredients consistent with the 2009 Codex definition (ALINORM 09/32/REP) and it measures insoluble dietary fiber (IDF) and soluble dietary fiber (SDFP plus SDFS). It is an update of AOAC 2009.1, and addresses all of the issues identified by analysts in using that method over the past 8 years. Incubation time with pancreatic α -amylase (PAA) plus amyloglucosidase (AMG) was reduced from 16 h to 4 h, inline with the likely time of residence of food in the small intestine. To ensure complete hydrolysis of non-resistant starch, PAA AOAC Research Institute ERP Use Only

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levels were increased 2-fold and AMG was increased 12-fold and under these conditions, the resistant maltodextrins formed from non-resistant starch under incubation conditions of AOAC Method 2009.01 were no longer produced. Higher molecular weight dietary fiber (HMWDF) [(IDF + SDFP (soluble fiber which precipitates in the presence of 78% aqueous ethanol)] values obtained for samples not containing resistant starch were essentially the same as those obtained with AOAC Method 985.29 and 991.43. The IDF + SDFP values for many samples containing resistant starch (RS), were very similar to those obtained with AOAC Method 2009.01. Samples containing RS 2 (native, high amylose maize starch) and RS 4 (phosphate cross- liked native starches) however, resulted in much higher dietary fiber values. The current method employing TSK-Gel G2500PWXL ® columns for gel permeation chromatography, gives higher dietary fiber values for fructo-oligosaccharides than were obtained in previous methods using the Waters Sugar-Pak ® column because with the Sugar-Pak ® column co-elutes inulinotriose with disaccharides and thus, does not include inulinotriose in the dietary fiber calculations. Glycerol replaces D-sorbitol as the internal standard in-line with AOAC Method 2001.03 as the D-sorbitol is not separated from D-glucose on the TSK columns. An improved method for preparing samples for HPLC involves removing the bulk of the anions and cations (salt) in the sample by adding the sample to cation and anion exchange resins in a polypropylene tube and mixing thoroughly before the gel permeation separation. This step removes ~ 95% of the cations and anions in the sample, allowing more efficient use of expensive desalting pre-columns in simultaneous in-line deionisation and chromatography. With the shorter PAA/AMG incubation time, it is possible to eliminate the use of sodium azide in the incubation buffer solutions. The method described here measures IDF + SDFP gravimetrically and SDFS by HPLC and the values are combined to give TDF. The method was evaluated through an ICC / AACC International collaborative study. A total of 13 laboratories participated, with all laboratories returning valid assay data for most of the 16 test portions (8 blind duplicates) consisting of samples with a range of content of traditional dietary fibers, resistant starch and non-digestible oligosaccharides. In total, only 4 sets of data from the 104 sets submitted were statistically excluded as outliers. The dietary fiber content of the 8 test pairs ranged from 6.79 to 60.6%. TDF was calculated as the sum of HMWDF (IDF + SDFP) and SDFS. For TDF, the within laboratory variability (s r ) ranged from 0.29 to 0.74 and the between laboratory variability (s R ) ranged from 0.57 to 4.67. The within laboratory AOAC Research Institute ERP Use Only

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