Measurement of Dietary Fiber: Which AOAC Official Method of AnalysisSM to Use.

A broad range of AOAC Official Methods of AnalysisSM (OMA) have been developed and approved for the measurement of dietary fiber (DF) and DF components since the adoption of the Prosky method (OMA 985.29). OMA 985.29 and other OMA were developed to support the Trowell definition of DF. However, these methods do not measure DF as defined by the "new," physiologically relevant, Codex Alimentarius definition. Methodology to support the Codex definition has been developed and updated in recent years. In this article, the relevance of each OMA in supporting the Codex definition of DF is described and suggestions are presented on the most appropriate method, together with proposals for changes in title and application statements for the "historic" OMA methods.

Validation of the Test Method-Determination of Available Carbohydrates in Cereal and Cereal Products, Dairy Products, Vegetables, Fruit, and Related Food Products and Animal Feeds: Collaborative Study, Final Action 2020.07.

BACKGROUND: A simple, accurate, and reliable method to measure available carbohydrate components of food products, including cereal and dairy products, fruits, vegetables, processed food, food ingredients, and animal foods, was developed by Megazyme (product K-AVCHO, Bray, Ireland). A single-laboratory validation of the enzymatic method resulted in First Action status as Official Method of AnalysisSM2020.07. OBJECTIVE: A collaborative study was conducted to evaluate the repeatability and reproducibility of Official Method 2020.07 for the measurement of available carbohydrates, including digestible starch, lactose, sucrose, isomaltose, maltose, glucose, fructose, and galactose in a broad range of food and feed products. METHOD: Samples are defatted if containing >10% fat content, and incubated with pancreatic α-amylase and amyloglucosidase under conditions that simulate those in the small intestine (pH 6, 37°C, 4 h). The reaction solution is clarified and diluted, and an aliquot is incubated with sucrase, maltase, oligo-1,6-α-glucosidase, and ß-galactosidase to hydrolyze sucrose, maltose, isomaltose, and lactose to glucose, fructose, and galactose, which are then measured enzymatically. The multi-laboratory validation (MLV) matrixes included cereal, animal feeds, fruit, vegetables, infant formula, powdered milk drink, a dessert product, and mushrooms. Additional materials were analyzed by collaborators as "practice samples." RESULTS: All MLV matrixes resulted in repeatability relative standard deviations (RSDr) <3.91% and reproducibility relative standard deviations (RSDR) ranging from 3.51 to 11.58% with 9 of the 10 matrixes having RSDR of <6.19%. For the practice samples, the RSDR ranged from 2.7 to 11.4% with 7 of the 8 samples having RSDR of <4.4%. CONCLUSIONS: Official Method 2020.07 meets the AOAC requirements for repeatability and reproducibility, and the data support Final Action status. HIGHLIGHTS: Official Method 2020.07 is a robust, simple to use, and reproducible method for the analysis of available carbohydrates in a wide range of matrixes.

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

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

Lactose Concentration in Low-Lactose and Lactose-Free Milk, Milk Products, and Products Containing Dairy Ingredients by High Sensitivity Enzymatic Method (K-LOLAC), Collaborative Study: Final Action 2020.08.

BACKGROUND: The AOAC Stakeholder Panel on Strategic Food Analytical Methods issued a call for methods in 2018 for the measurement of lactose in low-lactose and lactose-free products under Standard Method Performance Requirement (SMPR®) 2018.009. Megazyme's Lactose Assay Kit (K-LOLAC) was reviewed and accepted as a First Action Official MethodSM in 2020 (2020.08). OBJECTIVE: A collaborative study was conducted to evaluate the to evaluate the reproducibility of AOAC Official MethodSM2020.08 for the measurement of lactose concentration in low-lactose and lactose-free milk, milk products, and products containing dairy ingredients. METHOD: Samples are deproteinated and clarified by treatment with Carrez reagents, and then free glucose is removed using a glucose oxidase and catalase treatment system. Quantification of lactose is based on the hydrolytic activity of ß-galactosidase, which hydrolyses lactose to glucose and galactose. Any remaining free D-glucose is first measured using a hexokinase (HK)/glucose 6-phosphate dehydrogenase (G-6PDH)/6-phosphogluconate dehydrogenase (6-PGDH) based assay procedure, and then ß-galactosidase is added to hydrolyze the lactose in the same reaction vessel with concurrent measurement of the released D-glucose. The samples analyzed included a number of lactose-free and low-lactose milk samples, lactose-free infant formula, lactose-free milkshake, lactose-free adult nutritional drink, lactose-free cream, and lactose-free cheese. RESULTS: All materials had repeatability relative standard deviations (RSDr) <7%. The reproducibility relative standard deviation (RSDR) varied from 3.8 to 14.9% with seven of the 10 test samples having an RSDR of <10%. CONCLUSIONS: The Lactose Assay Kit (K-LOLAC) meets the requirements for reproducibility set out under SMPR 2018.009. HIGHLIGHTS: The Lactose Assay (K-LOLAC) is a robust, simple, and reproducible method for analysis of lactose in foodstuffs and beverages.

The Challenge of Measuring Sweet Taste in Food Ingredients and Products for Regulatory Compliance: A Scientific Opinion.

The Codex Alimentarius Commission, a central part of the joint Food and Agricultural Organization/World Health Organizations Food Standards Program, adopts internationally recognized standards, guidelines, and code of practices that help ensure safety, quality, and fairness of food trade globally. Although Codex standards are not regulations per se, regulatory authorities around the world may benchmark against these standards or introduce them into regulations within their countries. Recently, the Codex Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) initiated a draft revision to the Codex standard for follow-up formula (FUF), a drink/product (with added nutrients) for young children, to include requirements for limiting or measuring the amount of sweet taste contributed by carbohydrates in a product. Stakeholders from multiple food and beverage manufacturers expressed concern about the subjectivity of sweetness and challenges with objective measurement for verifying regulatory compliance. It is a requirement that Codex standards include a reference to a suitable method of analysis for verifying compliance with the standard. In response, AOAC INTERNATIONAL formed the Ad Hoc Expert Panel on Sweetness in November 2020 to review human perception of sweet taste, assess the landscape of internationally recognized analytical and sensory methods for measuring sweet taste in food ingredients and products, deliver recommendations to Codex regarding verification of sweet taste requirements for FUF, and develop a scientific opinion on measuring sweet taste in food and beverage products beyond FUF. Findings showed an abundance of official analytical methods for determining quantities of carbohydrates and other sweet-tasting molecules in food products and beverages, but no analytical methods capable of determining sweet taste. Furthermore, sweet taste can be determined by standard sensory analysis methods. However, it is impossible to define a sensory intensity reference value for sweetness, making them unfit to verify regulatory compliance for the purpose of international food trade. Based on these findings and recommendations, the Codex Committee on Methods of Analysis and Sampling agreed during its 41st session in May 2021 to inform CCNFSDU that there are no known validated methods to measure sweetness of carbohydrate sources; therefore, no way to determine compliance for such a requirement for FUF.

Determination of Lactose Concentration in Low-Lactose and Lactose-Free Milk, Milk Products, and Products Containing Dairy Ingredients, Enzymatic Method: Single-Laboratory Validation First Action Method 2020.08.

BACKGROUND: The AOAC Stakeholder Panel on Strategic Food Analytical Methods issued a call for methods for the measurement of lactose in low-lactose and lactose-free products under Standard Method Performance Requirement (SMPR®) 2018.009. Megazyme's Lactose Assay Kit (K-LOLAC) was developed specifically to address the need for accurate enzymatic testing in lactose-free samples. OBJECTIVE: K-LOLAC was validated for measurement of lactose in low-lactose and lactose-free milk, milk products, and products containing dairy ingredients. A single-laboratory validation (SLV) of the method is reported. METHOD: K-LOLAC is an accurate and sensitive enzymatic method for the rapid measurement of lactose in low-lactose or lactose-free products. Validation analysis was performed on a sample set of 36 commercial food and beverage products and a set of 10 certified reference materials. Parameters examined during the validation included working range and linear range, selectivity, LOD, LOQ, trueness (bias), precision (repeatability and intermediate precision), robustness, and stability. RESULTS: For all samples tested within the lower range (10-100 mg/100 g or mL), recoveries varied from 93.21-114.10%. Recoveries obtained for samples in the higher range (>100 mg/100 g or mL) varied from 94.44-108.28%. All materials had repeatability relative standard deviations (RSDr and RSDir) of <9%. CONCLUSIONS: The commercial K-LOLAC assay kit, as developed by Megazyme, meets the requirements set out under SMPR 2018.009. HIGHLIGHTS: K-LOLAC is a robust, quick, and easy method for analysis of lactose in foodstuffs and beverages.

Measurement of Available Carbohydrates in Cereal and Cereal Products, Dairy Products, Vegetables, Fruit, and Related Food Products and Animal Feeds: First Action 2020.07.

BACKGROUND: The level of available carbohydrates in our diet is directly linked to two major diseases: obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. OBJECTIVE: The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose, and galactose. METHOD: Non-resistant (digestible) starch is hydrolyzed to glucose and maltose by pancreatic α-amylase (PAA) and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose, and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. RESULTS: A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. CONCLUSIONS: A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (RSDr, %) was <3.58% (w/w) across a range of samples containing 44.1-88.9% available carbohydrates. The LOD and LOQ obtained were 0.054% (w/w) and 0.179% (w/w), respectively. The method is all inclusive, specific, robust, and simple to use. HIGHLIGHTS: A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose, and galactose, information of value in determining the glycemic index of foods.

Determination of Ethanol Concentration in Kombucha Beverages: Single-Laboratory Validation of an Enzymatic Method, First Action Method 2019.08.

Kombucha is a fermented, lightly effervescent sweetened black or green tea drink. It is marketed as a functional beverage based on its proposed health benefits. Kombucha is produced by fermenting tea using a "symbiotic colony of bacteria and yeast" (SCOBY). Kombucha is marketed as a non-alcoholic beverage, however due to the production process employed, there is a high possibility that the Kombucha products will contain low levels of ethanol. Kombucha is sold in a raw and unpasteurized form and, if kept at temperatures above 4 °C, the possibility exists that it will continue to ferment, producing ethanol. This possibility of continued fermentation may lead to an increase in ethanol content from levels below 0.5%ABV at time of production to higher levels at time of consumption. Thus, there is a potential for levels rising to greater than 0.5%ABV, the threshold for certification as a non-alcoholic beverage. It is essential that Kombucha manufacturers have the capacity to accurately and quickly test for ethanol in their products. The Ethanol Assay Kit is an enzymatic test kit developed by Megazyme for the determination of ethanol in a variety of samples. The kit has been validated in a single laboratory for use with Kombucha fermented drinks, fruit juices, and low-alcohol beer samples. The commercially available Ethanol Assay Kit (Megazyme catalogue no. K-ETOH) contains all components required for the analysis. Quantification is based on the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase and further oxidation of acetaldehyde by acetaldehyde dehydrogenase with conversion of NAD+ to NADH. The single laboratory validation (SLV) outlined in this document was performed on a sample set of eight different commercial Kombucha products purchased in Ireland, a set of five Cerilliant aqueous ethanol solutions, two BCR low-alcohol beer reference materials, two alcohol-free beer samples, and two fruit juice samples against SMPR 2016.001 (1). Parameters examined during the validation included Working range, Selectivity, Limit of Detection (LOD), Limit of Quantification (LOQ), Trueness (bias), Precision (reproducibility and repeatability), Robustness, and Stability. The Ethanol Assay is a robust, quick and easy method for the measurement of ethanol in Kombucha. Our data suggests this method is also reliable for similar matrices, such as low-alcohol beer and fruit juice. The assay meets all requirements set out in in AOAC SMPR 2016.001.

Determination of Fructan (Inulin, FOS, Levan, and Branched Fructan) in Animal Food (Animal Feed, Pet Food, and Ingredients): Single-Laboratory Validation, First Action 2018.07.

Traditional enzyme-based methods for measurement of fructan were designed to measure just inulin and branched-type (agave) fructans. The enzymes employed, namely exo-inulinase and endo-inulinase, give incompletely hydrolysis of levan. Levan hydrolysis requires a third enzyme, endo-levanase. This paper describes a method and commercial test kit (Megazyme Fructan Assay Kit) for the determination of all types of fructan (inulin, levan, and branched) in a variety of animal feeds and pet foods. The method has been validated in a single laboratory for analysis of pure inulin, agave fructan, levan, and a range of fructan containing samples. Quantification is based on complete hydrolysis of fructan to fructose and glucose by a mixture of exo-inulinase, endo-inulinase, and endo-levanase, followed by measurement of these sugars using the PAHBAH reducing sugar method which gives the same color response with fructose and glucose. Before hydrolysis of fructan, interfering sucrose and starch in the sample are specifically hydrolyzed and removed by borohydride reduction. The single-laboratory validation (SLV) outlined in this document was performed on commercially available inulin (Raftiline) and agave fructan (Frutafit®), levan purified from Timothy grass, two grass samples, a sample of legume hay, two animal feeds and two barley flours, one of which (Barley MAX®) was genetically enriched in fructan through plant breeding. Parameters examined during the validation included working range, target selectivity, recovery, LOD, LOQ, trueness (bias), precision (repeatability and intermediate precision), robustness, and stability. The method is robust, quick, and simple.

A novel enzymatic method for the measurement of lactose in lactose-free products.

BACKGROUND: In recent years there has been a surge in the number of commercially available lactose-free variants of a wide variety of products. This presents an analytical challenge for the measurement of the residual lactose content in the presence of high levels of mono-, di-, and oligosaccharides. RESULTS: In the current work, we describe the development of a novel enzymatic low-lactose determination method termed LOLAC (low lactose), which is based on an optimized glucose removal pre-treatment step followed by a sequential enzymatic assay that measures residual glucose and lactose in a single cuvette. Sensitivity was improved over existing enzymatic lactose assays through the extension of the typical glucose detection biochemical pathway to amplify the signal response. Selectivity for lactose in the presence of structurally similar oligosaccharides was provided by using a ß-galactosidase with much improved selectivity over the analytical industry standards from Aspergillus oryzae and Escherichia coli (EcLacZ), coupled with a 'creep' calculation adjustment to account for any overestimation. The resulting enzymatic method was fully characterized in terms of its linear range (2.3-113 mg per 100 g), limit of detection (LOD) (0.13 mg per 100 g), limit of quantification (LOQ) (0.44 mg per 100 g) and reproducibility (≤ 3.2% coefficient of variation (CV)). A range of commercially available lactose-free samples were analyzed with spiking experiments and excellent recoveries were obtained. Lactose quantitation in lactose-free infant formula, a particularly challenging matrix, was carried out using the LOLAC method and the results compared favorably with those obtained from a United Kingdom Accreditation Service (UKAS) accredited laboratory employing quantitative high performance anion exchange chromatography - pulsed amperometric detection (HPAEC-PAD) analysis. CONCLUSION: The LOLAC assay is the first reported enzymatic method that accurately quantitates lactose in lactose-free samples. © 2018 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Total Dietary Fiber (CODEX Definition) in Foods and Food Ingredients by a Rapid Enzymatic-Gravimetric Method and Liquid Chromatography: Collaborative Study, First Action 2017.16.

A method for measurement of total dietary fiber (TDF) has been validated. This method is applicable to plant materials, foods, and food ingredients as consumed, consistent with the 2009 CODEX definition (ALINORM 09/32/REP), and measures insoluble dietary fiber (IDF) and soluble dietary fiber (SDF), comprising SDF that precipitates in the presence of 78% ethanol (SDFP) and SDF that remains soluble in the presence of 78% ethanol (SDFS). The method is an update of AOAC Method 2009.01 and addresses each of the issues identified by analysts in using that method over the past 8 years. A total of 13 laboratories participated in the study, 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 nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 6.90 to 60.37 g/100 g. TDF was calculated as the sum of IDF plus SDFP measured gravimetrically and SDFS measured by HPLC. The repeatability SD ranged from 0.27 to 0.76 g/100 g, and the reproducibility SD ranged from 0.54 to 3.99 g/100 g. The RSDr ranged from 1.22 to 6.52%, and the RSDR ranged from 2.14 to 10.62%.

Undefined cellulase formulations hinder scientific reproducibility.

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.

Novel substrates for the automated and manual assay of endo-1,4-ß-xylanase.

endo-1,4-ß-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-ß-45-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

A novel automatable enzyme-coupled colorimetric assay for endo-1,4-ß-glucanase (cellulase).

endo-1,4-ß-Glucanase (endo-cellulase, EC 3.2.1.4) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In 2014, a novel enzyme-coupled assay that addressed many of the limitations of the existing assay methodology was reported. This involved the use of a bifunctional substrate chemically derived from cellotriose. Reported herein is a much improved version of this assay employing a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-ß-D-cellopentaoside. Graphical Abstract Principle of the CELLG5 assay.

An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley ß-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.

Measurement of ß-Glucan in Mushrooms and Mycelial Products.

A robust and reliable method has been developed for the measurement of ß-glucan in mushroom and mycelial products. Total glucan (plus free glucose and glucose from sucrose) was measured using controlled acid hydrolysis with H2SO4 and the glucose released specifically was measured using glucose oxidase/peroxidase reagent. α-Glucan (starch/glycogen) plus free glucose and glucose from sucrose were specifically measured after hydrolysis of starch/glycogen to glucose with glucoamylase and sucrose to glucose plus fructose with invertase and the glucose specifically measured with GOPOD reagent. ß-Glucan was determined by the difference. Several acid and enzyme-based methods for the hydrolysis of the ß-glucan were compared, and the best option was the method using H2SO4. For most samples, similar ß-glucan values were obtained with both the optimized HCl and H2SO4 PROCEDURES: However, in the case of certain samples, specifically Ganoderma lucidum and Poria cocus, the H2SO4 procedure resulted in significantly higher values. Hydrolysis with 2 N trifluoroacetic acid at 120°C was found to be much less effective than either of the other two acids evaluated. Assays based totally on enzymatic hydrolysis, in general, yielded much lower values than those obtained with the H2SO4 procedure.

Novel assay procedures for the measurement of α-amylase in weather-damaged wheat.

BACKGROUND: The measurement of α-amylase (EC 3.2.1.1) in sprout-damaged grains is a crucial analysis yet a problematic one owing to the typically low α-amylase levels in ground wheat samples. A number of standardised methods such as the Falling Number method and the Ceralpha method exist which are routinely used for the assay of α-amylase. These methods, however, are either highly substrate-dependent or lack the required sensitivity to assess sprout damage. RESULTS: Novel colorimetric and fluorometric reagents have been prepared (Amylase HR, Amylase SD, BzCNPG7 reagent and BzMUG7 reagent) for the direct and specific assay of α-amylase activity in sprout-damaged wheat. Assays employing these reagents have been developed and optimised to include a decolourisation step using activated charcoal. When used in a convenient assay format, Amylase SD--containing EtNPG7 (II) as the colorimetric substrate and α-glucosidase as the ancillary enzyme--was found to be an excellent reagent for the assessment of sprout damage in wheat with incubation times as short as 5 min. CONCLUSION: The assay using Amylase SD is completely specific for α-amylase. The use of the Amylase SD assay represents a sensitive and valid alternative to the traditionally used Falling Number values for the assessment of sprout damage in wheat samples.

A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-ß-Xylanase.

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass.

BACKGROUND: Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails. RESULTS: It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition. CONCLUSIONS: These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.

Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by ß-xylanase, α-L-arabinofuranosidase and ß-xylosidase.

A range of α-L-arabinofuranosyl-(1-4)-ß-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 ß-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and ß-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum ß-xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 3(2)-α-L-Araf-(1-4)-ß-D-xylobiose (A(3)X), 2(3)-α-L-Araf-(1-4)-ß-D-xylotriose (A(2)XX), 3(3)-α-L-Araf-(1-4)-ß-D-xylotriose (A(3)XX), 2(2)-α-L-Araf-(1-4)-ß-D-xylotriose (XA(2)X), 3(2)-α-L-Araf (1-4)-ß-D-xylotriose (XA(3)X), 2(3)-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(2)XX), 3(3)-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(3)XX), 2(3),3(3)-di-α-L-Araf-(1-4)-ß-D-xylotriose (A(2+3)XX), 2(3),3(3)-di-α-L-Araf-(1-4)-ß-D-xylotetraose (XA(2+3)XX), 2(4),3(4)-di-α-L-Araf-(1-4)-ß-D-xylopentaose (XA(2+3)XXX) and 3(3),3(4)-di-α-L-Araf-(1-4)-ß-D-xylopentaose (XA(3)A(3)XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A(2,3)XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with ß-xylosidase and ß-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.