Fiber is nutritionally important because it represents the

Transcription

1 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, AGRICULTURAL MATERIALS Gravimetric Determination of Amylase-Treated Neutral Detergent Fiber in Feeds with Refluxing in Beakers or Crucibles: Collaborative Study DAVID R. MERTENS U.S. Department of Agriculture, Agricultural Research Service, U.S. Dairy Forage Research Center, 1925 Linden Dr West, Madison, WI Collaborators: M. Allen; J. Carmany; J. Clegg; A. Davidowicz; M. Drouches; K. Frank; D. Gambin; M. Garkie; B. Gildemeister; D. Jeffress; C-S. Jeon; D. Jones; D. Kaplan; G-N. Kim; S. Kobata; D. Main; X. Moua; B. Paul; J. Robertson; D. Taysom; N. Thiex; J. Williams; M. Wolf As an important constituent of animal feeds, fiber represents the portion of feeds that is bulky and difficult to digest. The neutral detergent fiber (NDF) method, developed over 30 years ago, is the method of choice for measuring total fiber in forages and other feeds. Several modifications that were made to improve its general applicability to all feeds and others developed in individual laboratories often resulted in variability among laboratories in measuring NDF. The amylase-treated NDF (andf) method, therefore, was developed as an accurate and precise method of measuring total insoluble fiber in feeds. A collaborative study was conducted to evaluate the repeatability and reproducibility of the andf method over the full range of animal feed materials. Twelve laboratories representing research, feed company, regulatory, and commercial feed testing laboratories analyzed 11 materials as blind duplicates. The materials represented feed matrixes, including animal products; high-protein, high-fat, and high-pectin feeds; oil seeds; grains; heated by-product feeds; and legume and grass hays and silages. Materials selected varied in chemical composition and contained 0 90% andf, 1 16% ash, 1 20% crude fat, 1 40% crude protein, and 0 50% starch. Correcting results for changes in blanks and reporting results as ash-free andf organic matter (andfom) improved the repeatability and reproducibility of results when andf was <25%. The within-laboratory repeatability standard deviation (s r ) for percentage andfom in feeds varied from 0.21 to 1.82 Submitted for publication July The recommendation was approved by the Methods Committee on Feeds and Fertilizers and Agricultural Related Products as First Action. See Official Methods Program Actions, (2002) Inside Laboratory Management, September/October issue. Corresponding author s davem@dfrc.wisc.edu. and among-laboratory reproducibility standard deviation (s R ) varied from 0.37 to The HORRAT was <2 for all materials except feed materials containing >10% fat. However, standard deviations of repeatability and reproducibility for feeds with >10% fat were similar to those of other materials. It is recommended that the andf method be accepted for Official First Action status. Fiber is nutritionally important because it represents the organic portion of feeds and foods that is the most difficult to digest; nonfiber fractions of feeds are easily and almost completely digestible by most animals. There is a need for a rapid and simple assay to determine the total insoluble fiber content of feeds. Originally fiber was related to the cell wall fraction of plants; however, because fiber occurs in all feeds, fiber methods must be applicable to all feeds and foods. Although neutral detergent (ND) extraction solubilizes some material that is not digested by mammalian enzymes, neutral detergent soluble fiber is often fermented by bacteria in the gut of animals and is digested. Thus, neutral detergent fiber (NDF) is the insoluble fiber in feeds that is either indigestible or slowly digested, and occupies space in the digestive tract of animals. The NDF method was originally developed by Van Soest and Wine (1) for the analysis of total fiber in forages, and numerous modifications have been proposed to extend its application to grains, concentrated feeds, and human foods (2 5). Most of these modifications were based on conceptual improvement in total fiber analysis without ruggedness testing to determine their suitability for all feed matrixes or practical application as a routine method. Unfortunately, this resulted in fiber methods that differed, yet were all called NDF. The difficulty in extracting and washing fibrous residues in some materials and the variety in modifications of the method have led to the perception that NDF is difficult to measure precisely.

2 1218 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 Collaborative Study Optimization and Ruggedness of NDF Methods The critical steps in the NDF and amylase-treated NDF (andf) procedures were investigated by the original developers, who proposed modifications, and the Study Director, who developed the method evaluated in this study. The following factors were evaluated: (1) Time of refluxing. Sixty min of refluxing at boiling temperatures achieves asymptotic extraction for most materials and was selected as an optimum compromise between analysis time and complete extraction (1). It should not vary more than 5 min. (2) Neutral detergent concentration. Sodium lauryl sulfate is used at near saturation concentrations to maximize solubilization of nonfiber with a minimal volume of liquid that must be filtered (1). (3) Neutral detergent ph. Phosphate and borate buffers are used to maintain ph near 7.0 because alkaline or acid conditions can solubilize fiber. The Study Director observed that laboratories were not confirming that ph was near 7.0 and conducted a test to evaluate the effects of ph variation. Results indicated that the ph should be maintained within the range of (6). (4) Triethylene glycol. The original NDF method used ethylene glycol monoethylether to remove nonfiber soluble material from concentrated feeds. This compound is a potential mutagen and was replaced with triethylene glycol. Research confirmed that changing to triethylene glycol did not alter results (J.B. Robertson, Cornell University, Ithaca, NY, personal communication, 1988). (5) Sodium sulfite. Sodium sulfite was included to remove proteinaceous material from fiber residues. When heat-stable amylase was included as a part of the NDF method, sodium sulfite was removed because it has the potential to destroy phenolic compounds (2). However, research by the Study Director indicated that sodium sulfite is critical for removal of proteinaceous matter in heated or cooked feeds, and it was reintroduced in the method that is being evaluated (7). (6) Heat-stable -amylase. Starch was not completely removed by the original NDF method. Numerous modifications of the method used various amylases to remove starch before or during ND extraction. Both the amylase source and the method of using the enzyme alter NDF values, and pre-extraction by incubation has the potential to solubilize fiber. Heating samples and gelatinizing the starch may improve amylase effectiveness in removing starch contamination. Many amylase solutions are crude extracts that contain enzymes that can degrade fiber and must be deactivated by near-boiling temperatures. Alternative methods of using heat-stable amylases have been evaluated by the Study Director and others (4, 8). The approach used in the proposed method uses 2 additions of heat-stable amylase: one in ND after initiation of boiling, and one in the first residue washing step. Unfortunately enzyme sources changed or were discontinued and new specifications for the type and amount of amylase to use had to be rediscovered. The Study Director, therefore, developed a visual assessment method for determining the amount of any heat-stable amylase source that is active under the conditions used in andf analysis, and this is included as part of the method. (7) Washing method. Rapid rinsing of fibers removes only surface contamination of detergent and soluble matter and results in high fiber values. Research by the Study Director s laboratory indicated that multiple soakings of fibrous residues are required to obtain consistent results and uncontaminated fibrous residues. (8) Reporting fiber as organic matter. Ashing fiber residues and reporting results as andf organic matter (andfom) eliminates some differences in results associated with inadequate washing of fibrous residues. In addition, it allows nutritionists to estimate nonfibrous carbohydrates more accurately by difference (NFC = 100 crude protein crude fat ash andfom), because a portion of ash is not double-subtracted as it would be if andf were used in the calculation. Calculations in the proposed method allow results to be reported to meet the needs of the user. (9) Sample amount and ratio to ND solution. The original NDF method used 100 ml ND with 1 g sample (9). Halving the amount of sample reduced andf by about 1 percentage unit. To reduce filtering problems, the same ratio of sample to ND was maintained, but sample and ND solution amounts were reduced to 0.5 g in 50 ml. (10) Sample grinding. In most near infrared reflectance spectroscopy, samples are ground through a cyclone mill with 1 mm screen. Discrepancies in fiber analysis were observed when these samples were analyzed by chemical methods. Research by the Study Director confirmed that particle size of the sample and andf results were affected by both screen size and grinder type. Although samples that are ground finer are extracted more completely by ND, they are often more difficult to filter. Thus, the proposed method specifies that materials must be ground through a 1 mm screen with a cutter mill or equivalent. (11) Sample drying. Artifact fiber can be created during sample preparation by drying materials at temperatures >60 C (10). Therefore, the method to be evaluated specifies that wet samples be dried at temperatures <60 C. (12) Modifications for problem samples. Samples that are high in starch, pectin, fat, or animal proteins are difficult to filter. Modifications to the basic method have overcome the difficulties inherent in these types of samples (11). As indicated by Horwitz et al. (12), empirical methods of analysis, such as fiber methods, must be followed exactly. This suggests that the various modifications of the original NDF method should have different names because they are different measures of fiber. The proposed fiber method is called andf to distinguish it from the original method and its modifications. The objective of this research was to evaluate the repeatability and reproducibility of the andf method in a collaborative study involving laboratories with different missions and using materials representing the full scope of feed ingredients.

3 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, Table 1. Characteristics of collaborating laboratories Lab No. Type Method apparatus Collaborating Laboratories Amylase source 0 Research (Study Director) Reflux beaker crucible A 1 Research Reflux beaker crucible B 2 Research (dropped out) 3 Research Reflux beaker crucible A 4 Regulatory Reflux in crucible A 5 Regulatory Reflux beaker crucible B 6 Testing Reflux in crucible B 7 Feed company Reflux beaker crucible B 8 Testing Reflux beaker crucible A 9 Testing Reflux beaker crucible A 10 Testing (dropped out) 11 Feed company Reflux beaker crucible B 12 Feed company Reflux beaker crucible B 13 Feed company Reflux in crucible A 14 Regulatory Reflux beaker crucible B The 14 laboratories which agreed to participate in the study represented 3 feed company laboratories, 5 commercial feed testing laboratories, 3 feed regulatory laboratories, and 4 research laboratories. Two feed testing and one research laboratory failed to complete the study; the Study Director s laboratory was substituted for the missing research laboratory and a volunteer feed company laboratory was substituted for one of the feed testing laboratories (Table 1). Participants received no compensation. Each laboratory provided reflux apparatus, filtration vessels, balances, and drying ovens. Collaborators were provided with test samples, heat-stable α-amylase, and chemicals, but were required to mix and standardize the necessary reagents. They were asked to respond to several questionnaires about equipment and routine procedures in their laboratories, provide results of standardizing the amylase working solution, analyze 5 familiarization samples, and provide andf results for 11 collaborative study materials and one blank analyzed as blind duplicates. Collaborators were asked to perform singles analyses of each sample, to report all weights to 4 significant digits, and to submit data on an as-is basis. Materials There are no officially recognized reference materials for fiber, and it is not possible to prepare spiked samples. To determine the general applicability of the andf method, study materials were selected to represent the full range of fiber concentrations and types of animal feeds (Table 2). In addition, materials were included that required the use of each of the modifications described in the method. Materials representing feed matrixes from animal products; high-protein, high-fat, and high-pectin feeds; oil seeds; grains; heated by-product feeds; legume forages; and grass forages (hays and silages) were included in the study. A mixed dairy feed that contained a mixture of animal and plant supplements with added fat was used to represent a variety of feed ingredients. Materials selected for the collaborative study encompassed a wide range of andf concentrations in feeds, varying from 0 to 90%. The materials also varied in concentration of ash (1 16%), fat as measured by ether extract (1 20%), crude protein (1 40%), and starch (0 50%). The diverse chemical composition of the materials used as blind duplicates permitted evaluation of most, if not all, possible interactions of feed composition on andf analysis. Blanks were included to eval- Table 2. Materials used in the study, representing full range of fiber concentrations and types of animal feeds a Material Description DM, % Ash, % EE, % CP, % Starch, % NFC, % ADF, % andfom, % Alfalfa silage Forage, fermented legume Brewer s grains Concentrate, heated by-product Citrus and beet pulp Concentrate, pectic by-product Corn grain with cob Concentrate, containing starch Corn silage Forage, grass with starch Corn stalks Forage, high-fiber grass Dairy mixed feed Concentrate, mixture with fat Grass hay Forage, temperate grass Milk replacer Concentrate, animal fat Roasted soybeans Concentrate, plant fat Sawdust Reference, high cellulose Blank No material a Dry matter (DM), ash, ether extract (EE), crude protein (CP), starch, nonfibrous carbohydrates (NFC = DM ash EE CP andfom), acid detergent fiber (ADF), and amylase-treated neutral detergent fiber organic matter (andfom) of materials used in the collaborative study.

4 1220 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 Table 3. Homogeneity of andfom (blank-corrected) for 4 sample sets for each material analyzed a Material n Mean, % s r s R RSD r, % RSD R, % 2.8 s r 2.8 s R HORRAT Alfalfa silage Brewer s grains Citrus and beet pulp Corn grain with cob Corn silage Corn stalks Dairy mixed feed Grass hay Milk replacer Roasted soybeans Sawdust Blank, g a s r = Standard deviation of replicated analyses within sample set; s R = standard deviation within and among sample sets; RSD r = repeatability relative standard deviation; RSD R = reproducibility relative standard deviation. uate weighing technique and determine if blank-correction improved precision. Single batches of homogenous materials were obtained from commercial feed suppliers or the U.S. Dairy Forage Research Center (Madison, WI). Materials were dried as needed at 55 C and were ground through a 1 mm screen with a Wiley cutter mill, and thoroughly mixed. Forty sets of samples containing about 2 g of each of the 11 materials were prepared. Samples were measured into glass vials that were sealed in moisture-proof pouches for storage until shipment. All samples were numbered in the order in which they were to be analyzed. Homogeneity of sample sets was verified by selecting 4 sets of samples at random for each material and analyzing them in duplicate in the Study Director s laboratory to provide an estimate of random variation within and among sets of samples. The results of the homogeneity study were analyzed by using the AOAC spreadsheet statistical software recommended for collaborative studies to determine among-sample set instead of among-laboratory variation (Table 3). For most materials, the variation between duplicate analyses within a sample set (s r ) was similar to the variation between samples sets (s R ). This analysis allows variability in homogeneity of sample sets to be compared directly with the variation among collaborating laboratories. Except for milk replacer (MR), which had an andf concentration near zero, the Horwitz ratio (HORRAT; 12) of observed versus expected relative standard deviations (RSDs) among results was below the acceptable threshold of 2.0. For most samples, the HORRAT was between 0.33 and Within the Study Director s laboratory, the andf variation was similar to that of other AOAC Official Methods. The HORRAT values for roasted soybeans (RS), corn grain (CG), dairy mixed feed (DM), and citrus and beet pulp (CB) were between 0.80 and 1.50, suggesting that these materials were less homogeneous or more difficult to analyze for andf. Familiarization samples were sent to each collaborator in a preliminary study to acquaint them with the method and evaluate their ability to handle difficult samples. Familiarization materials included alfalfa silage (AS), barley hay (BH), CG, meat and bone meal (MB), and RS. The latter 3 materials were selected to represent materials that were extremely difficult to analyze for andf. Heat-stable amylase is a critical reagent in the andf method. To verify that alternative sources of amylase can be standardized and used, 3 amylase stock solutions from 2 sources were standardized by each collaborator during the familiarization study, and half of the laboratories were assigned to use one of the 2 sources during the collaborative study (Table 4). Statistical Analyses The experiment was designed as a randomized complete block design using blind duplicates of 11 unidentified materials. Although feeds were not identified, milk replacer, dairy feed, and roasted soybeans were labeled to indicate >10% fat in the first blind duplicate, but not the second. Only one analyst from each laboratory was asked to submit results, and blind duplicates were analyzed on the same day within the Table 4. Sources of stock heat-stable -amylases evaluated and used in the study Amylase type Source Dilution Taka-Therm L-340 Termamyl 120 L ANKOM Technology Corp. (Fairport, NY) Novo Nordisk Biochem (Raleigh, NC) Full strength Full strength; one-half strength

5 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, same run. Block was defined as first replicate, second replicate, and rerun analyses using modifications. In the first replicate, all laboratories were asked to analyze one duplicate of each of the samples in a fixed order of decreasing filtration ease. In the second replicate, one of 12 random orders of analysis for the samples was assigned to each laboratory. Ordering samples in the first replicate was used to determine the effect of a difficult-to-filter sample on the subsequently analyzed samples. The statistical model for analysis of variance (ANOVA) was: Y=µ + Material + Lab + Block + Material*Lab + error (1) where µ = the mean, Material = variation due to materials, Lab = variation among laboratories, Block = variation due to replicate or rerun, Material*Lab = variation due to Material by Laboratory interaction, and error = random variation within laboratory. Block was included in the model to test the effect of order of analysis and was removed from the model when not significant. There are several procedures in the andf method that may not be followed exactly among laboratories. For example, identifying the proper level of amylase to use when determining andf is an integral part of the method because the source and level of enzyme is not specified in the method. A portion of the collaborative study was designed to evaluate the effects of amylase source (A or B) and dose (independently determined by each collaborator). The effect of source and dose of amylase was evaluated on all materials and only on materials containing significant starch [corn silage (CS) and CG] using the ANOVA model: Y=µ + Material + Amylase + Dose + Amylase*Dose + error (2) where Amylase = variation due to source of amylase, Dose and Amylase*Dose = variation explained by linear regression of amylase dose for each source, and all other variables as described previously. Because andf is an empirical method it is important to evaluate the effects of factors that are left to the discretion of laboratories and may differ among them. Each collaborator submitted answers to a questionnaire about factors that might affect results. In addition to amylase source and dose, collaborators were asked to describe for each result the apparatus, filtering aid, and pre-extraction technique used, and to describe the temperature of washing water and the methods for residue washing and weighing. This information was evaluated using appropriate ANOVA models. Initially, outlying results were detected with spreadsheet statistical software provided by AOAC for a blind duplicates design using a 2.5% significance level. The Cochran test identifies replicate results within a laboratory that are suspect, and the Grubb test determines if the average result of laboratories deviates from those of all laboratories. Cycles of Cochran, single Grubb, and pair Grubb tests were used to identify outliers until no additional removal was necessary or no more than 2/9 of the laboratories were flagged. The ANOVA identified which of the 2 blind replicates was the outlier by using a main effects ANOVA with no interaction terms: Y = Material + Lab + Block + error (3) Predicted results were compared with observed results, and the replicate with the largest residual deviation was identified as the outlier. Laboratory ranking scores (after removal of individual replicate outliers) were used to identify laboratories that were outliers across all materials according to Wernimont (13). All outlying results were checked for calculation and data entry errors, and answers to the questionnaire were scrutinized to determine if the collaborator s procedure differed from the andf method that was being evaluated. Within laboratory repeatability (s r ), among laboratory systematic variation (s L ), and total reproducibility variation (s R ; where s R 2 =s r 2 +s L 2 ) associated with a single analysis were determined by approaches described by Youden and Steiner (14) and Wernimont (13) with spreadsheet statistical software recommended by AOAC. Statistical results were determined for individual materials. In addition, ANOVA using the interaction model (Model 1) was used to determine if results from all materials or classes of materials (forage, concentrates with <10% fat and concentrates with >10% fat) could be pooled. If the interaction term in Model 1 was not significant, it was assumed that the method obtained consistent results among materials, and pooled precision statistics were calculated according to Wernimont (13). True values cannot be determined for fiber because the empirical method defines the fraction. Therefore, there is no direct measure of systematic bias (accuracy) for fiber, and consensus values derived from the collaborative study were used as the point of reference. As suggested by Youden (15), variability in systematic bias was equated with among-laboratory variability (s L ) that does not include within-laboratory variability. Repeatability (s r ) is the within-laboratory precision that was calculated from variation between blind duplicates within laboratories. Reproducibility (s R ) is the total variation associated with a single analysis, which is the sum of systematic bias and repeatability, i.e., s R 2 =s r 2 +s L 2. Repeatability within a laboratory is expected to be 1/2 2/3 of the total reproducibility variation among laboratories. Although specific criteria for the acceptability of a fiber method cannot be imposed, it is expected that the variation among laboratories will be a function of andf concentration. The ratio (HORRAT) of the observed reproducibility relative standard deviation (RSD R ) divided by the expected RSD from the Horwitz equation (16) was used to evaluate the acceptability of the method. A HORRAT <2 was used as an indicator of acceptability (12). AOAC Official Method Amylase-Treated Neutral Detergent Fiber in Feeds Using Refluxing in Beakers or Crucibles First Action 2002 [This method is applicable for the determination of amylase-treated neutral detergent fiber (andf) in forages, grains, grain by-product feeds, animal by-product feeds, oil seeds, oilseed meals and mixed feeds that range in concentration from 1.5 to 100%.]

6 1222 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 Caution: Enzyme (amylase) preparations can cause allergic reactions in hypersensitive individuals; avoid inhaling aerosols or dusts. Cover eyes, nose, mouth, and exposed skin as needed to prevent irritation. Sodium lauryl sulfate is mildly irritating to mucous membranes and may cause allergic reactions in hypersensitive individuals. Do not inhale or allow material to contact skin or eyes. Wear a dust mask and gloves while handling. Triethylene glycol is harmful if inhaled, ingested, or absorbed through the skin. Vapors or mists are irritating to the eyes, mucous membranes, and upper respiratory tract. Contact is irritating to the skin. Wear appropriate clothing and mix in a hood or well-ventilated area. Acetone is a flammable solvent; do not use near open flames. Use in an effective fume hood and do not let vapors accumulate in work area. Avoid inhaling or contact with skin. Evaporate all traces of acetone before placing fiber residues into an oven. Avoid skin contact with hot neutral detergent solution and reagents. Maintain all electrical equipment in suitable working order and ensure that it is properly grounded. See Table for the results of the interlaboratory study supporting acceptance of the method. A. Principle Fiber in feeds is a nutritionally defined entity that represents the indigestible and slowly digesting fraction of feeds. Neutral detergent (ND) solution and heat-stable α-amylase are used to dissolve easily digested proteins, lipids, sugars, starches, and pectins in feeds, leaving a fibrous residue that is primarily cell wall components in plant materials (cellulose, hemicellulose, and lignin) and indigestible nitrogenous matter in animal products. andf is a gravimetric method that best estimates the total insoluble fiber, and is inversely related to digestibility and intake potential of a feed. ND soluble matter is almost completely digested by most animals; however, the digestibility of andf is variable among feeds and is related to lignin and other constituents in fiber. Sodium lauryl sulfate, an anionic detergent, and sodium sulfite are used to solubilize nitrogenous matter; EDTA is used to chelate calcium and enhance removal of pectins at boiling temperatures; triethylene glycol helps remove some nonfibrous matter from concentrated feeds; disodium phosphate and sodium borate are used as buffers to maintain a neutral ph. Hot ND solution has limited ability to solubilize starch; therefore, a heat-stable amylase is used to hydrolyze starch to saccharides that can be easily removed from fiber by filtration. Heat-stable amylases are used in hot solutions to inactivate potential contaminating enzymes in crude amylase extracts that might degrade fibrous constituents. To ensure that the amylase activity is sufficient to remove most starch and to reduce filtering difficulties, the amount of any specific amylase source needed to measure andf is determined under the conditions of the andf method. Although boiling ND solution dissolves most proteins, lipids, and nonfibrous carbohydrates, these constituents are nonviscous only in water that is near boiling temperature. Therefore, boiling water is needed for washing fibrous residues and removing nonfibrous matter. Because fiber is particulate matter, mass-action equilibration during soaking is needed to migrate ND and contaminating soluble matter from the interior of fiber particles. Fibrous residues must be soaked, instead of being simply rinsed, in near boiling water to remove nonfibrous matter. Boiling ND solution solubilizes lipids, and acetone soaking of the residue completes the extraction of lipids and pigments in most materials. However, excessive amounts of lipids in materials can complex with the detergent and reduce extraction efficiency. Because extraction of lipids by ND and acetone may be incomplete when feeds contain >10% lipid, these materials are pre-extracted to ensure complete removal of lipid contamination from fiber. Pre-extract materials with 5 10% lipid to minimize filtration difficulties and avoid variable andf results. B. Apparatus (a) Refluxing apparatus. Any conventional apparatus suitable for crude fiber or acid-detergent fiber determinations. Test samples should be extracted in 500 or 600 ml beakers without spouts (Pyrex No. 1040, or equivalent) that are covered with a round cold-water condenser to minimize evaporation. Calibrate heating unit setting so that 50 ml water boils within 4 5 min when cold water condensers are used. Fibertec apparatus 2010 or M6 (Foss North America, Eden Prairie, MN 55344) can be used and should boil 50 ml water within 10 min. (b) Fritted-disk Gooch crucibles. Coarse porosity (pore size µm) crucibles, high-form, ml capacity or Fibertec USP2 (pore size µm, ml capacity). Clean new crucibles and ash at 500 C for 1 h. Clean crucibles after each use by ashing at 500 C for 3 h, removing ash, inverting in detergent solution, and sonicating for 7 10 min. Rinse crucibles in hot water, and soak in room temperature water for at least 30 min. Back-flush crucibles by connecting top of each crucible to a No. 9 ½ stopper or a No. 9 stopper fitted with a No. 4 filter adapter (Cat. No ; VWR, West Chester, PA 19380, that has a port that is connected to a trap and vacuum line. Back-flush each crucible with water by rapidly plunging and removing the bottom of the crucible into water to create a vigorous rinsing action. Occasionally test filtration rate as follows: Fill each crucible with 50 ml distilled water (25 ml for Fibertec USP2 crucibles) and record time required to drain completely without vacuum (should be 180 ± 60 s for Gooch or 75 ± 30 s for USP2). If <100 s (or <35 s for USP2), discard crucible. If <120 s (or <45 s for USP2), check for cracks in fritted disk. If filtration >240 s (or >105 s for USP2), clean crucibles with slow filtration rates using acid or alkaline cleaning solutions. (c) Vacuum filter manifold. Any apparatus similar to that described by Mertens et al. (17) or suitable alternative that allows adequate soaking of fibrous residues (Figure A). Manifold should provide vacuum-tight seal with crucible to reduce foam formation in vacuum lines. Use thick-walled vac-

7 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, Table Interlaboratory study results for amylase-treated neutral detergent fiber in feeds Feed Fiber Mean, % s r s R RSD r, % RSD R, % r a R b Forages andf c Forages andfbc d Forages andfom e Forages andfombc f Concentrates <10% fat andf Concentrates <10% fat andfbc Concentrates <10% fat andfom Concentrates <10% fat andfombc Concentrates >10% fat andf Concentrates >10% fat andfbc Concentrates >10% fat andfom Concentrates >10% fat andfombc All materials andf All materials andfbc All materials andfom All materials andfombc a 2.8 s r. b 2.8 s R. c Amylase-treated neutral detergent fiber (as-received basis). d Amylase-treated neutral detergent fiber, blank-corrected (as-received basis). e Amylase-treated neutral detergent fiber organic matter (as-received basis). f Amylase-treated neutral detergent fiber organic matter, blank-corrected (as-received basis). uum tubing to connect manifold to trap (4 18 L) and vacuum source. Place a vacuum reservoir (18 L) between trap and vacuum source to ensure adequate vacuum capacity to remove foam. Fibertec apparatus may be used for filtration. (d) Boiling water supply. Use continuous boiling water generator as described by Goering and Van Soest (9) or suitable alternative (Figure B). Apparatus must be capable of supplying boiling water (>95 C) in quantity sufficient for all residues to be washed at one time through a nozzle producing a fine stream (flow rate, ml/10 s; a 2.5 ml disposable plastic pipet tip makes an acceptable nozzle). A fine nozzle minimizes water needed to transfer particles to crucible, but provides water pressure needed to remove residues attached to side of beaker. It is critical that water is boiling when added to crucibles, especially for products containing starches, pectic substances, mucilages, or glycoproteins. (Fibertec users: Use syringe with cone-spray nozzle to rinse condensers and 60 ml disposable syringe with 12 gauge needle 10 cm long to dislodge any residues adhering to condensers.) (e) Wash bottle for ND solution. Rinse down particles that are carried up the side of beakers by foaming during refluxing. Use fine nozzle to minimize addition of ND solution during rinsing (flow rate, ml in 3 s with moderate pressure; unclipped nozzle on 500 ml Nalgene Unitary wash bottle or equivalent is acceptable). (Fibertec users: Use extended nozzle with larger opening to adequately rinse particles into ND solution during extraction.) C. Reagents (a) Sodium sulfite. Na 2 SO 3 anhydrous. (b) Dried hominy corn (corn grits, raw). Obtained from food or grocery store. Must be raw or uncooked grits; precooked or instant grits are not acceptable. Grind grits to pass 1 mm screen in a Wiley cutter mill. (c) Burke s iodine solution. 2gKIand1gIin100mL water. Store in amber or opaque bottle. (d) Stock heat-stable -amylase solution. Termamyl 120 by Novozyme Corp. (Cat. No. A3403, Sigma-Aldrich, St. Louis, MO 63178), Spezyme FRED by Genecor International (Cat. No. FAA, Ankom Technology Corp., Fairport, NY 14450) or equivalent liquid heat-stable α-amylases or water extracts of heat-stable α-amylase powders. (e) Working heat-stable -amylase solution. Standardize heat-stable α-amylase stock solution or enzyme powder extract so that 2 additions of 2 ml will remove raw corn starch from 0.5 g of corn hominy grits. Assay as follows: (1) Weigh 0.5 g (±0.005 g) ground, dried hominy corn into each of 6 beakers similar to those used to extract fiber residues described in B(a).

8 1224 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 Figure A. Schematic drawing of vacuum filter manifold: (A) side view; (B) top view; and (C) end view (ref. 17).

9 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, Figure B. Water heating unit. Heating unit on right consists of a3lround flask modified to contain 2 standard taper female joints for cold water condensers at the top and a glass column (6 cm diameter, 17 cm length) at the bottom to accommodate a 1000 watt quartz immersion heater, 455 mm long and 19 mm diameter (Cat. No L, Vycor sheathed heating element, Corning Glass, Corning, NY, vycor@corning.com, or equivalent), inserted through the original joint at top. Cold water enters through a port at the bottom of the column and leaves through a port in the upper 2/3 of the round flask. The tank at the left contains a float valve adjusted to maintain a water level in the heating unit about 3/4 full. (2) Preheat calibrated reflux units, prepare ice bath for cooling beakers (must contain enough ice to maintain temperature below 1 C), and prepare tempering bath [shallow pan containing enough water at exactly 20 C to exceed depth of solutions in beakers and maintain a final temperature between 19.5 and 20.5 C at the end of step (8)]. (3) Add 50 ml ND solution, (f), (do not add sodium sulfite), swirl beaker, and place on preheated refluxing apparatus at 1 min intervals. (4) After ND begins to boil (ca 5 min), add one of 6 doses stock or powder extract solution (geometric progression, e.g., 0, 0.025, 0.05, 0.10, 0.20, and 0.40 ml; exact doses will depend on source of amylase) to beakers in ascending order. (5) Reflux for 10 min, remove at 1 min intervals; add second dose of amylase (matching the first), swirl, and rinse sides of beaker using minimum of room temperature ND solution. (6) React 60 s and filter through glass wool or 2 layers of cheesecloth into 100 ml glass beaker. Prepare blank by adding 2 intermediate doses, e.g., 0.10 and 0.20 ml stock solution, to 40 ml room temperature ND in 100 ml glass beaker. (7) Place beakers, except blank, in ice bath. Remove from ice bath after 5 min (temperature of solutions should be ca 21 C) and place all beakers in tempering bath (20 C). (8) After solutions are 20 ± 0.5 C (may take 5 min or more), remove beakers from tempering bath and arrange in order of increasing enzyme doses on white background. (9) Quickly add 0.5 ml Burke s iodine solution to beakers, and mix. (10) Do not look at beakers. After 90 s, view through solutions from above and make a quick decision (within 30 s) about color of each solution, using the following scale: Purple = not adequate enzyme; pink-amber or amber = not adequate enzyme; pale yellow = adequate enzyme. Compare to blank. Brown tint of enzyme solution should not be confused with pink-amber or amber. If color differences are unclear, place beakers in tempering bath for 5 min and repeat steps (8) (10). (11) After lowest dose that is pale yellow (V 2 ) and next lowest dose (V 1 ) that is pink-amber or amber are identified using a geometric progression of doses, do final standardization using the dose below the pink-amber one (V 1 ) and a linear progression of doses (0.25*V 1 ) between V 1 and V 2 (e.g., if 0.05 ml treatment is amber [V 1 ] and 0.10 ml treatment is pale yellow [V 2 ], use doses of 0.025, 0.05, , 0.075, , and 0.10 ml) stock solution in the final standardization. (12) The lowest dose that is pale yellow (and exceeds next highest inadequate dose with pink-amber or amber solution) represents the volume of amylase stock solution or extract (Vs) used to make amylase working solution. (13) Record date, batch or lot of amylase, doses tested, amount of iodine solution used, and color and final temperature (before iodine addition) of each dose in reagent log book. (14) Determine number (n) of test samples to be analyzed in the following 5 days or less. To add 2 ml amylase working solution twice for each test requires a total volume of amylase working solution of (n) 4 ml. Mix (n) 2 (Vs) ml amylase stock solution with (n) [4 2 (Vs)] ml water. Store working amylase solution in refrigerator using stoppered container no longer than 5 days. (15) Confirm adequacy of amylase working solution by repeating standardization procedure using 0.5 g corn grits with 0, 2, and 4 ml working solution (each added at boiling and after removal from refluxing apparatus). If there is no appreciable difference in color between 2 and 4 ml working solution, then 2 additions of 2 ml are adequate for the andf method. Notes: Time and temperature are critical for proper assessment of adequate amylase dose. The solutions must be 20 C and the decision about color must be made within s after addition of iodine solution. Pink-amber or amber colors fade quickly, and waiting longer than 120 s before making a decision will result in a dosage that is too low. Initial doses of stock solution or extract may have to be adjusted, and the standardization rerun. If maximum dose (0.4 ml) results in purple or pink-amber color, extend the geometric progression of doses starting at 0.4 ml, and rerun initial standardization. If minimum dose (0.025 ml) is yellow, decrease the geometric progression to between 0 and ml, and rerun initial standardization. Each new source or lot of enzyme should be standardized; a single lot that is being used over a period of time should be

10 1226 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, 2002 checked every 6 months for activity. Excess enzyme is not beneficial and can be detrimental. Concentrated enzyme solutions are not recommended as working solutions because a 1 drop error when dispensing enzyme contains significant activity that can affect results. Many amylase extracts are crude mixtures that may contain fibrolytic and proteolytic activities. Heat-stable amylase solution must be used in hot liquids (>80 C) to inactivate contaminating enzymes and minimize fiber loss. (f) Neutral-detergent (ND) solution. Measure 990 ml water. Pour ca half of water into a flask, add 4.0 g NaOH, 14.6 g EDTA, 4.56 g dibasic sodium phosphate (Na 2 HPO 4 ), 6.81 g sodium borate decahydrate (Na 2 B 4 O 7 10H 2 O), and mix until dissolved (heat if necessary). Under a hood, add 30 g sodium lauryl sulfate and ½ of remaining measured water. Mix until detergent is dissolved and add 10 ml triethylene glycol to suppress foam. Add remaining water and thoroughly mix. Verify that ph is between 6.95 and 7.05, and adjust with concentrated HCl or NaOH, as required. If ph is off by >0.5, discard. Store ND solution at room temperature or, if cool storage causes precipitation, warm to 25 C, and mix before use. Record date ND solution was prepared, ph measurements, and adjustments in reagent log book. Note: NaOH and EDTA can be replaced with 18.6 g disodium EDTA. (g) Filter aid. (1) Silica sand. Sand, cristobalite, acid purified, mesh (Fluka, Buchs, Switzerland, Cat. No or equivalent). (2) Glass microfiber mats cm Whatman GF/D or equivalent. (h) Crucible cleaning solutions. (1) Acid. Chromic acid [see Definition of Terms (13)] or prepare from 2.5 L H 2 SO 4 and one package Nochromix (Godax Laboratories, Inc., Takoma Park, MD 20912, or equivalent inorganic oxidizer. (2) Alkaline. Dissolve 5 g Na 2 EDTA 2H 2 O50gNa 3 PO 4, and 200 g KOH in 1 L water. Cleaning procedure. After testing filtration rate of crucibles, place crucibles with slow filtration rates in a shallow glass or enamel pan and add ca 40 ml acid cleaning solution to each crucible. Let acid cleaning solution filter through crucible and soak for 1 h. Rinse with water by back flushing and retest filtration rate. Clean crucibles with slow rates with the alkaline cleaning solution. Place crucibles in a shallow pan and add 50 ml alkaline cleaning solution and heat to C. Let alkaline cleaning solution filter through the crucible and then back flush each crucible until it is one-half full of solution. Let solution filter through crucible and back flush crucible until it is one-half full. Let solution filter through crucible. Remove from alkaline solution and back flush each crucible with water. After it has cooled, alkaline cleaning solution can be saved and reused. Use alkaline cleaning solution sparingly because it dissolves glass and weakens the fritted disks in crucibles. Retest crucible filtration rate; do not use those with slow rates for andf analysis. D. Test Sample Preparation Dry wet laboratory samples at <60 C to prevent creation of artifact fiber. Fiber extraction is affected by particle size of test sample. Grind representative samples to obtain geometric mean particle size of mm.a 1 mmscreen in a cutter mill (Wiley) or 2 mm screen in cyclone or centrifugal mill is generally acceptable. Cyclone or centrifugal mills throw particles through the screen at an angle, which effectively reduces the size of the aperture that particles pass through. On material ground to finer particle size, fiber must be determined by microfiber filter mats to minimize particle loss; however, results may have negative bias. Grinding segregates the material, with highest fiber material passing out of grinder last. Do not discard material in grinder; combine it with material in grinder receptacle. Mix ground material by placing on creased sheet of paper (ca cm). Lift corners of paper to roll material to diagonal corner, turn paper 90, and roll; repeat 10 times. Transfer material to suitable container. Ground test samples can be pre-extracted with acetone or ether to remove fat, but do not dry at >60 C. Products containing >5% fat should be pre-extracted; those with >10% fat must be pre-extracted to remove fat. To pre-extract with acetone, weigh test portion into crucible, place on filtering manifold, extract with ml acetone 4 times (allow material to soak at least 5 min, and stir 3 times during each soaking), apply vacuum to remove traces of acetone, air-dry for min, ensure that all traces of acetone are removed, and transfer to reflux beaker. Use same crucible to collect fiber residue for test portion after ND extraction. If filtering aid is used, dry and weigh it with crucible, and then transfer it to a small beaker before test sample is weighed into crucible. After pre-extracted residues have been transferred from the crucible into the refluxing beaker, replace filtering aid in crucible before filtering ND-extracted residues. [Fibertec users: Add silica sand to USP2 crucible, dry and weigh it before adding test sample; then pre-extract with ml acetone 4 times (allow material to soak at least 5 min and stir with back pressure 3 times) using cold filtration device. After removing all traces of acetone, transfer crucible containing silica sand and pre-extracted test portion to the hot extraction device for ND extraction.] E. Determination Dry at 105 C for >4 h and weigh empty crucibles (hot directly from oven or room temperature after desiccation). Record empty crucible weight for test portions (We) or blanks (Be) to nearest g. Mix material thoroughly and weigh 0.5 (±0.0500) g air-dry feed, or equivalent amount of wet material into refluxing beaker. (Fibertec users: Weigh test portion into dried and preweighed USP2 crucible.) Record final weight of test portion to nearest g (S). If results are to be reported on dry matter basis, weigh a second test portion at the same time for dry matter determination. Include in-house reference sample and 2 blanks for first test samples in a run and add one reference and one blank for each additional test samples. Preheat calibrated reflux units. Add 0.5 (±0.1) g sodium sulfite using calibrated scoop (EKCO Housewares Pinch measuring spoon, World Kitchen, Inc., Elmira, NY, or equivalent) and 50 (±5) ml ND to each refluxing beaker and swirl (critical for starchy feeds that stick to bottom during refluxing). (Fibertec users: Use back

11 MERTENS: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 6, pressure to mix reagents.) Do not add ND and sodium sulfite to test portions more than 60 min before refluxing. Heat to boiling within 4 5 min, add 2 ml working amylase solution, resuspend particles stuck to bottom or sides, and swirl. (Fibertec users: Use back pressure to mix amylase with ND solution and test portion.) Reflux for 60 min at boiling temperature that creates vigorous particle movement, but not excessive foaming that carries particles up the side of the beaker. Mixtures may foam vigorously for 1 2 min (do not reduce temperature of heating unit). Rinse sides of beaker with minimum amount of ND using bottle with fine nozzle 5 10 min after amylase is added, and rinse as needed to resuspend particles on side of beaker (twice maximum). Remove extracted mixture from heating unit and let particles settle for s. Before transfer (Fibertec users: Before initial filtration), observe mixture to determine if lipid globules are present on surface or if solution is milky, which indicates that test sample should be rerun after acetone pre-extraction. Place Teflon stirring rod in crucible and preheat by adding 40 ml boiling water for s. (Fibertec users: Ignore.) Remove water with vacuum, and immediately decant top ml of solution, keeping beaker inverted over crucible. (Fibertec users: Ignore.) Use minimum vacuum to evacuate excess liquid, but close vacuum before residue becomes dry. Note: Excessive vacuum and evacuating to dryness cause some residues to clog crucible and not wash properly. Rinse all unattached particles into crucible using fine stream of boiling water. (Fibertec users: Ignore.) Fill crucible half full with hot water. Add 2 ml working amylase solution and stir. (Fibertec users: Use back pressure to mix amylase in initial water soak.) React with amylase for minimum of s while scraping remaining particles from bottom and sides of reflux beaker with rubber policeman. Evacuate amylase solution and transfer any remaining residue from reflux beaker into crucible with ml boiling water. Two rinses are usually sufficient. After transferring residues from beaker, fill crucible 3/4 full with boiling water and soak for 1 3 min. (Fibertec users: Remove amylase-water soak after minimum reaction of 60 s. Crucibles can be removed from hot to cold filtration unit for remaining hot water soaks for residues that are easy to filter. This allows next set of test samples to begin ND extraction on hot filtration unit. Residues that are difficult to filter can be washed on Fibertec heating unit with heat reduced to minimize particle agitation.) Evacuate water, add ml boiling water, soak 3 5 min, and repeat. If residues are difficult to filter after first soak, add additional 2 ml working amylase solution. If residues appear translucent and become more difficult to filter with each additional soaking, eliminate third water soak. If plugged, crucible can be back-flushed by removing it from filter manifold and reinserting it. (Fibertec users: Use minimal back pressure to open crucibles and improve filtration.) Evacuate water, refill crucible with ml acetone, stir to disperse particles, soak 3 5 min, and repeat, rinsing stir rod to remove attached fiber particles. (Fibertec users: Move crucibles to cold extraction unit for acetone soaks.) Note: After the last water soak, do not evacuate fiber residues to dryness, but remove water to leave a damp or moist residue, before adding acetone. Excessive evacuation clumps the residues and makes acetone extraction difficult. Vacuum residue dry, remove crucible from manifold, and air dry for min to remove acetone. Dry crucibles at 105 C for minimum of 8 h and weigh (Wf and Bf). Ignite crucible and fiber in 500 C furnace for 5 h or until C-free. Temper in 105 C oven for at least 1 h and weigh (Wa and Ba). Weigh crucibles containing fiber or ash residues (hot or desiccated to room temperature) in same order as empty crucibles. Modifications for specific types of test samples. (a) If extracted ND solution appears milky and opaque and filtration is slow during transfer of residues or after first water soaking, high starch is suspected. Add additional treatment with 2 ml amylase during second water soaking. Shorten soaking times to minimum to keep soaking solutions as hot as possible (>85 C). (b) If residue clogs crucible during transfer and additional amylase does not improve filtration, feed material may contain proteinaceous, gum, or mucilage residues (meat products and some oil seed meals). Preheating crucible with boiling water is crucial for filtering these materials. The best filter aid for these materials is g (6 8 g for Fibertec USP2) of silica sand, C(g)(1). The gummy substances in these feed materials will stick to sand particles which prevents them from clogging the fritted disk and allows residues to be washed. All filter aids must be added to crucibles (including blanks) before initial weights are recorded. (c) If fiber residue has glossy, translucent sheen and filtration becomes more difficult with each water soaking, pectic substances are suspected. Preheat crucible with boiling water and transfer residues as quickly as possible without settling when removed from reflux unit. Reduce all soaking times to minimum to maintain temperature >85 C to prevent cooling and jelling in crucible. Filtering aids may improve filtration (in order of preference): g (6 8 g for Fibertec USP2) silica sand, 0.25 g (0.15 g for Fibertec USP2) glass wool, and glass microfiber mats, C(g)(2). (d) If fat globules are observed floating on surface of ND or wash water and residue is difficult to filter, or if material is known to contain >10% fat, pre-extract it with acetone or ether (see D). (e) If material contains fine particles, flocculant precipitates, dirt (fine clay), or fecal matter, but not pectic substances or starch, increase settling time to maximum of 2 min after removal from refluxing unit and use filter aid in crucible. Filter aids (in order of preference) include glass microfiber mats, ceramic fiber, g silica sand, and 0.25 g glass wool. Microfiber mats can be gently scraped to renew surface during filtration. (f) If all other modifications fail, reduce test portion amount to 0.3 g and repeat analysis with filter aid in crucible. Reducing test portion will magnify effects of weighing errors and increase variation in results. Sometimes reducing test portion amount and increasing ND to ml is beneficial. If fiber is <1.5%, do not reduce test portion amount, and if filtration is not possible, report results as difficult to analyze, fiber <1.5%. (g) Do not add acetone before all rinse water has been removed. Although this occasionally improves filtering, it does not remove detergent or detergent solubles from residues.