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Protease (Subtilisin A from Bacillus licheniformis)

Protease (Subtilisin A from Bacillus licheniformis)
Product code: E-BSPRT-40ML



2 grams - 40ML

Prices exclude VAT

Available for shipping

Content: 0.5 grams - 10 mL or
2 grams - 40 mL or
5 grams - 100 mL or
2.5 grams - 100 mL (ANKOM)
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Formulation: In 50% (v/v) glycerol
Physical Form: Solution
Stability: > 4 years at 4oC
Enzyme Activity: Protease
EC Number:
CAS Number: 9014-01-1
Synonyms: subtilisin; subtilisin A
Source: Bacillus licheniformis
Molecular Weight: 30,250
Expression: Purified from Bacillus licheniformis
Specificity: Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyses peptide amides.
Specific Activity: ~ 6 U/mg of protein (40oC, pH 8.0 on casein)
Unit Definition: One Unit will hydrolyse casein to produce colour equivalent to one μmole (181 µg) of tyrosine per minute at pH 7.0 at 40oC (colour by Folin-Ciocalteu reagent). 
Temperature Optima: 60oC
pH Optima: 7
Application examples: This enzyme is recommended for use in the Megazyme Total Dietary Fiber test method and AOAC INTERNATIONAL Total Dietary Fiber analytical procedures.

High purity Protease (Subtilisin A from Bacillus licheniformis) (liquid) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

For use in Megazyme Total Dietary Fiber test method. 

E-BSPRT-A-100ML specifically to be used with ANKOMTDF Dietary Fiber Analyzer.

Show all analytical enzymes products.

Data booklets for each pack size are located in the Documents tab.

View Megazyme’s latest Guide for Dietary Fiber Analysis.

Megazyme publication

Measurement of available carbohydrates, digestible, and resistant starch in food ingredients and products.

McCleary, B. V., McLoughlin, C., Charmier, L. M. J. & McGeough, P. (2019). Cereal Chemistry, 97(1), 114-137.

Background and objectives: The importance of selectively measuring available and unavailable carbohydrates in the human diet has been recognized for over 100 years. The levels of available carbohydrates in diets can be directly linked to major diseases of the Western world, namely Type II diabetes and obesity. Methodology for measurement of total carbohydrates by difference was introduced in the 1880s, and this forms the basis of carbohydrate determination in the United States. In the United Kingdom, a method to directly measure available carbohydrates was introduced in the 1920s to assist diabetic patients with food selection. The aim of the current work was to develop simple, specific, and reliable methods for available carbohydrates and digestible starch (and resistant starch). The major component of available carbohydrates in most foods is digestible starch. Findings: Simple methods for the measurement of rapidly digested starch, slowly digested starch, total digestible starch, resistant starch, and available carbohydrates have been developed, and the digestibility of phosphate cross‐linked starch has been studied in detail. The resistant starch procedure developed is an update of current procedures and incorporates incubation conditions with pancreatic α‐amylase (PAA) and amyloglucosidase (AMG) that parallel those used AOAC Method 2017.16 for total dietary fiber. Available carbohydrates are measured as glucose, fructose, and galactose, following complete and selective hydrolysis of digestible starch, maltodextrins, maltose, sucrose, and lactose to glucose, fructose, and galactose. Sucrose is hydrolyzed with a specific sucrase enzyme that has no action on fructo‐oligosaccharides (FOS). Conclusions: The currently described “available carbohydrates” method together with the total dietary fiber method (AOAC Method 2017.16) allows the measurement of all carbohydrates in food products, including digestible starch. Significance and novelty: This paper describes a simple and specific method for measurement of available carbohydrates in cereal, food, and feed products. This is the first method that provides the correct measurement of digestible starch and sucrose in the presence of FOS. Such methodology is essential for accurate labeling of food products, allowing consumers to make informed decisions in food selection.

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Megazyme publication

Modification to AOAC Official Methods 2009.01 and 2011.25 to allow for minor overestimation of low molecular weight soluble dietary fiber in samples containing starch.

McCleary, B. V. (2014). Journal of AOAC International, 97(3), 896-901.

AOAC Official Methods 2009.01 and 2011.25 have been modified to allow removal of resistant maltodextrins produced on hydrolysis of various starches by the combination of pancreatic α-amylase and amyloglucosidase (AMG) used in these assay procedures. The major resistant maltodextrin, 63,65-di-α-D-glucosyl maltopentaose, is highly resistant to hydrolysis by microbial α-glucosidases, isoamylase, pullulanase, pancreatic, bacterial and fungal α-amylase and AMG. However, this oligosaccharide is hydrolyzed by the mucosal α-glucosidase complex of the pig small intestine (which is similar to the human small intestine), and thus must be removed in the analytical procedure. Hydrolysis of these oligosaccharides has been by incubation with a high concentration of a purified AMG at 60°C. This incubation results in no hydrolysis or loss of other resistant oligosaccharides such as FOS, GOS, XOS, resistant maltodextrins (e.g., Fibersol 2) or polydextrose. The effect of this additional incubation with AMG on the measured level of low molecular weight soluble dietary fiber (SDFS) and of total dietary fiber in a broad range of samples is reported. Results from this study demonstrate that the proposed modification can be used with confidence in the measurement of dietary fiber.

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Megazyme publication

Measurement of total dietary fiber using AOAC method 2009.01 (AACC International approved method 32-45.01): Evaluation and updates.

McCleary, B. V., Sloane, N., Draga, A. & Lazewska, I. (2013). Cereal Chemistry, 90(4), 396-414.

The Codex Committee on Methods of Analysis and Sampling recently recommended 14 methods for measurement of dietary fiber, eight of these being type I methods. Of these type I methods, AACC International Approved Method 32-45.01 (AOAC method 2009.01) is the only procedure that measures all of the dietary fiber components as defined by Codex Alimentarius. Other methods such as the Prosky method (AACCI Approved Method 32-05.01) give similar analytical data for the high-molecular-weight dietary fiber contents of food and vegetable products low in resistant starch. In the current work, AACCI Approved Method 32-45.01 has been modified to allow accurate measurement of samples high in particular fructooligosaccharides: for example, fructotriose, which, in the HPLC system used, chromatographs at the same point as disaccharides, meaning that it is currently not included in the measurement. Incubation of the resistant oligosaccharides fraction with sucrase/β-galactosidase removes disaccharides that interfere with the quantitation of this fraction. The dietary fiber value for resistant starch type 4 (RS4), varies significantly with different analytical methods, with much lower values being obtained with AACCI Approved Method 32-45.01 than with 32-05.01. This difference results from the greater susceptibility of RS4 to hydrolysis by pancreatic α-amylase than by bacterial α-amylase, and also a greater susceptibility to hydrolysis at lower temperatures. On hydrolysis of samples high in starch in the assay format of AACCI Approved Method 32-45.01 (AOAC method 2009.01), resistant maltodextrins are produced. The major component is a heptasaccharide that is highly resistant to hydrolysis by most of the starch-degrading enzymes studied. However, it is hydrolyzed by the maltase/amyloglucosidase/isomaltase enzyme complex present in the brush border lining of the small intestine. As a consequence, AOAC methods 2009.01 and 2011.25 (AACCI Approved Methods 32-45.01 and 32-50.01, respectively) must be updated to include an additional incubation with amyloglucosidase to remove these oligosaccharides.

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Megazyme publication
Determination of insoluble, soluble, and total dietary fiber (codex definition) by enzymatic-gravimetric method and liquid chromatography: Collaborative Study.

McCleary, B. V., DeVries, J. W., Rader, J. I., Cohen, G., Prosky, P., Mugford, D. C., Champ, M. & Okuma, K. (2012). Journal of AOAC International, 95(3), 824-844.

A method for the determination of insoluble (IDF), soluble (SDF), and total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official MethodsSM 985.29, 991.43, 2001.03, and 2002.02, the method quantitates water-insoluble and water-soluble dietary fiber. This method extends the capabilities of the previously adopted AOAC Official Method 2009.01, Total Dietary Fiber in Foods, Enzymatic-Gravimetric-Liquid Chromatographic Method, applicable to plant material, foods, and food ingredients consistent with CODEX Definition 2009, including naturally occurring, isolated, modified, and synthetic polymers meeting that definition. The method was evaluated through an AOAC/AACC collaborative study. Twenty-two laboratories participated, with 19 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 10.45 to 29.90%. Digestion of samples under the conditions of AOAC 2002.02 followed by the isolation, fractionation, and gravimetric procedures of AOAC 985.29 (and its extensions 991.42 and 993.19) and 991.43 results in quantitation of IDF and soluble dietary fiber that precipitates (SDFP). The filtrate from the quantitation of water-alcohol-insoluble dietary fiber is concentrated, deionized, concentrated again, and analyzed by LC to determine the SDF that remains soluble (SDFS), i.e., all dietary fiber polymers of degree of polymerization = 3 and higher, consisting primarily, but not exclusively, of oligosaccharides. SDF is calculated as the sum of SDFP and SDFS. TDF is calculated as the sum of IDF and SDF. The within-laboratory variability, repeatability SD (Sr), for IDF ranged from 0.13 to 0.71, and the between-laboratory variability, reproducibility SD (sR), for IDF ranged from 0.42 to 2.24. The within-laboratory variability sr for SDF ranged from 0.28 to 1.03, and the between-laboratory variability sR for SDF ranged from 0.85 to 1.66. The within-laboratory variability sr for TDF ranged from 0.47 to 1.41, and the between-laboratory variability sR for TDF ranged from 0.95 to 3.14. This is comparable to other official and approved dietary fiber methods, and the method is recommended for adoption as Official First Action.

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Megazyme publication
Determination of total dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid chromatography: collaborative study.

McCleary, B. V., DeVries, J. W., Rader, J. I., Cohen, G., Prosky, L., Mugford, D. C., Champ, M. & Okuma, K. (2010). Journal of AOAC International, 93(1), 221-233.

A method for the determination of total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official MethodsSM 985.29, 991.43, 2001.03, and 2002.02, the method quantitates high- and low-molecular-weight dietary fiber (HMWDF and LMWDF, respectively). In 2007, McCleary described a method of extended enzymatic digestion at 37°C to simulate human intestinal digestion followed by gravimetric isolation and quantitation of HMWDF and the use of LC to quantitate low-molecular-weight soluble dietary fiber (LMWSDF). The method thus quantitates the complete range of dietary fiber components from resistant starch (by utilizing the digestion conditions of AOAC Method 2002.02) to digestion resistant oligosaccharides (by incorporating the deionization and LC procedures of AOAC Method 2001.03). The method was evaluated through an AOAC collaborative study. Eighteen laboratories participated with 16 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 11.57 to 47.83. Digestion of samples under the conditions of AOAC Method 2002.02 followed by the isolation and gravimetric procedures of AOAC Methods 985.29 and 991.43 results in quantitation of HMWDF. The filtrate from the quantitation of HMWDF is concentrated, deionized, concentrated again, and analyzed by LC to determine the LMWSDF, i.e., all nondigestible oligosaccharides of degree of polymerization 3. TDF is calculated as the sum of HMWDF and LMWSDF. Repeatability standard deviations (Sr) ranged from 0.41 to 1.43, and reproducibility standard deviations (SR) ranged from 1.18 to 5.44. These results are comparable to other official dietary fiber methods, and the method is recommended for adoption as Official First Action.

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Megazyme publication
Development and evaluation of an integrated method for the measurement of total dietary fibre.

McCleary, B. V., Mills, C. & Draga, A. (2009). Quality Assurance and Safety of Crops & Foods, 1(4), 213–224.

An integrated total dietary fibre (TDF) method, consistent with the recently accepted CODEX definition of dietary fibre, has been developed. The CODEX Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) has been deliberating for the past 8 years on a definition for dietary fibre that correctly reflects the current consensus thinking on what should be included in this definition. As this definition was evolving, it became evident to us that neither of the currently available methods for TDF (AOAC Official Methods 985.29 and 991.43), nor a combination of these and other methods, could meet these requirements. Consequently, we developed an integrated TDF procedure, based on the principals of AOAC Official Methods 2002.02, 991.43 and 2001.03, that is compliant with the new CODEX definition. This procedure quantitates high- and low-molecular weight dietary fibres as defined, giving an accurate estimate of resistant starch and non-digestible oligosaccharides also referred to as low-molecular weight soluble dietary fibre. In this paper, the method is discussed, modifications to the method to improve simplicity and reproducibility are described, and the results of the first rounds of interlaboratory evaluation are reported.

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Megazyme publication

An integrated procedure for the measurement of total dietary fibre (including resistant starch), non-digestible oligosaccharides and available carbohydrates.

McCleary, B. V. (2007). Analytical and Bioanalytical Chemistry, 389(1), 291-308.

A method is described for the measurement of dietary fibre, including resistant starch (RS), non-digestible oligosaccharides (NDO) and available carbohydrates. Basically, the sample is incubated with pancreatic α-amylase and amyloglucosidase under conditions very similar to those described in AOAC Official Method 2002.02 (RS). Reaction is terminated and high molecular weight resistant polysaccharides are precipitated from solution with alcohol and recovered by filtration. Recovery of RS (for most RS sources) is in line with published data from ileostomy studies. The aqueous ethanol extract is concentrated, desalted and analysed for NDO by high-performance liquid chromatography by a method similar to that described by Okuma (AOAC Method 2001.03), except that for logistical reasons, D-sorbitol is used as the internal standard in place of glycerol. Available carbohydrates, defined as D-glucose, D-fructose, sucrose, the D-glucose component of lactose, maltodextrins and non-resistant starch, are measured as D-glucose plus D-fructose in the sample after hydrolysis of oligosaccharides with a mixture of sucrase/maltase plus β-galactosidase.

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Megazyme publication
Measurement of novel dietary fibres.

McCleary, B. V. & Rossiter, P. (2004). Journal of AOAC International, 87(3), 707-717.

With the recognition that resistant starch (RS) and nondigestible oligosaccharides (NDO) act physiologically as dietary fiber (DF), a need has developed for specific and reliable assay procedures for these components. The ability of AOAC DF methods to accurately measure RS is dependent on the nature of the RS being analyzed. In general, NDO are not measured at all by AOAC DF Methods 985.29 or 991.43, the one exception being the high molecular weight fraction of fructo-oligosaccharides. Values obtained for RS, in general, are not in good agreement with values obtained by in vitro procedures that more closely imitate the in vivo situation in the human digestive tract. Consequently, specific methods for the accurate measurement of RS and NDO have been developed and validated through interlaboratory studies. In this paper, modifications to AOAC fructan Method 999.03 to allow accurate measurement of enzymically produced fructo-oligosaccharides are described. Suggested modifications to AOAC DF methods to ensure complete removal of fructan and RS, and to simplify pH adjustment before amyloglucosidase addition, are also described.

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Megazyme publication
Dietary fibre analysis.

McCleary, B. V. (2003). Proceedings of the Nutrition Society, 62, 3-9.

The 'gold standard' method for the measurement of total dietary fibre is that of the Association of Official Analytical Chemists (2000; method 985.29). This procedure has been modified to allow measurement of soluble and insoluble dietary fibre, and buffers employed have been improved. However, the recognition of the fact that non-digestible oligosaccharides and resistant starch also behave physiologically as dietary fibre has necessitated a re-examination of the definition of dietary fibre, and in turn, a re-evaluation of the dietary fibre methods of the Association of Official Analytical Chemists. With this realisation, the American Association of Cereal Chemists appointed a scientific review committee and charged it with the task of reviewing and, if necessary, updating the definition of dietary fibre. It organised various workshops and accepted comments from interested parties worldwide through an interactive website. More recently, the (US) Food and Nutrition Board of the Institute of Health, National Academy of Sciences, under the oversight of the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, assembled a panel to develop a proposed definition(s) of dietary fibre. Various elements of these definitions were in agreement, but not all. What was clear from both reviews is that there is an immediate need to re-evaluate the methods that are used for dietary fibre measurement and to make appropriate changes where required, and to find new methods to fill gaps. In this presentation, the 'state of the art' in measurement of total dietary fibre and dietary fibre components will be described and discussed, together with suggestions for future research.

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Megazyme publication

Two issues in dietary fiber measurement.

McCleary, B. V. (2001). Cereal Foods World, 46, 164-165.

Enzyme activity and purity of these topics, the easiest to deal with is the importance of enzyme purity and activity. As a scientist actively involved in polysaccharide research over the past 25 years, I have come to appreciate the importance of enzyme purity and specificity in polysaccharide modification and measurement (7). These factors translate directly to dietary fiber (DF) methodology, because the major components of DF are carbohydrate polymers and oligomers. The committee report published in the March issue of Cereal FOODS WORLD refers only to the methodology for measuring enzyme purity and activity (8) that led up the AOAC method 985.29 (2). In this work enzyme purity was gauged by the lack of hydrolysis (i.e., complete recovery) of a particular DF component (e.g. β-glucan, larch galactan or citrus pectin). Enzyme activity was measured by the ability to completely hydrolyze representative starch and protein (namely wheat starch and casein). These requirements and restrictions on enzyme purity and activity were adequate at the time the method was initially developed and served as a useful working guide. However, it was recognized that there was a need for more stringent quality definitions and assay procedures for enzymes used in DF measurements.

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Megazyme publication

Measuring dietary fibre.

McCleary, B. V. (1999). The World of Ingredients, 50-53.

Interest in dietary fibre is undergoing a dramatic revival thanks in part to the introduction of new carbohydrates as dietary fibre components. Much emphasis is being placed on determining how much fibre is present in a food. Linking a particular amount of fibre to a specific health benefit is now an important area of research. Total Dietary Fibre. The term “dietary fibre” first appeared in 1953 and referred to hemicelluloses, celluloses and lignin (1). In 1974, Trowell (2) recommended this term as a replacement for the no longer acceptable term “crude fibre” Burkitt (3) has likened the interest in dietary fibre to the growth of a river from its first trickle to a mighty torrent. He observes that dietary fibre “was viewed as merely the less digestible constituent of food which exerts a laxative action by irritating the gut “thus acquiring the designation “roughage” a term which was later replaced by “crude fibre” and ultimately by “dietary fibre” Various definitions of dietary fibre have appeared over the years, partly due the various concepts used in deriving the term (i.e. origin of material, resistance to digestion, fermentation in the colon etc.), and partly to the difficulties associated with its measurement and labelling (4). The principle components of dietary fibre, as traditionally understood, are non-starch polysaccharides, which in plant fibre are principally hemicelluloses and celluloses, and the non-carbohydrate phenolic components, cutin, suberin and waxes with which they are associated in Nature.

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Determination of vitamin A and vitamin E esters in infant formulae and fortified milk powders by HPLC: Use of internal standardisation.

Woollard, D. C., Bensch, A., Indyk, H. & McMahon, A. (2016). Food chemistry, 197, 457-465.

An HPLC method is described using normal phase conditions with an unbonded silica column to determine concentrations of supplementary vitamin A and vitamin E esters and β-carotene in infant formulae. The method utilises selective dual-channel fluoresence for vitamins A and E and visible absorbance for β-carotene. An attribute of the method is the use of retinol propionate, α-tocopheryl propionate and all-E-β-apo-8′-carotenoic acid ethyl ester internal standards to compensate for analytical variations associated with these labile vitamins. Extraction is performed without saponification, with the aid of protease to remove vitamin encaspsulation and facilitate vitamin partition into hydrocarbon solvent. Figures of merit indicate the method is suitable for its intended purpose in the highly regulated infant formula environment, including liquid formulations. The method is extendable to whole milk powders where total vitamin A content data can be calculated by summing the innate long-chain vitamin A esters with the added esters.

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The Potential Use of Fermented Chickpea and Faba Bean Flour as Food Ingredients.

Chandra-Hioe, M. V., Wong, C. H. & Arcot, J. (2016). Plant Foods for Human Nutrition, 71(1), 90-95.

Apart from being a rich and inexpensive protein source, legumes provide essential vitamins, minerals and dietary fibre. Considering the nutritional benefits, legumes flour can potentially be incorporated in the development of new products. The aim of this study was to investigate whether fermentation affects the protein content, in vitro protein digestibility, trypsin inhibitor activity and the functionality of proteins in faba bean, desi and kabuli chickpea. Australian grown chickpea and faba bean were selected and initially soaked, de-hulled, dried and milled into flour. This was fermented with lyophilised yoghurt cultures in a 30°C orbital shaker for 16 h. While protein contents in fermented desi and kabuli flour were lower than their raw counterparts (p > 0.05), it was significantly higher in fermented faba bean. A significant increase (9.5 %) in in vitro protein digestibility was found in fermented desi. Trypsin inhibitor activity in fermented desi, kabuli and faba bean reduced by 2.7, 1.1 and 4.7 %, respectively (p > 0.05). Overall, the in vitro protein digestibility in flour samples increased, while simultaneously reducing the trypsin inhibitor activity. The water absorption capacity of the fermented kabuli flour significantly increased by 11.3 %. All fermented flour samples had significantly higher oil absorption capacity than their corresponding raw flour that was likely due to increased insoluble hydrophobic protein. Although, the foaming capacity in all fermented flour samples was significantly lower than their respective raw samples, only fermented desi and faba bean flour showed lower foaming stability (p > 0.05). The present study suggests that fermented legume flour could fulfill the demand for innovative products of higher nutritional value.

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Improvement of the AOAC 2009.01 total dietary fibre method for bread and other high starch containing matrices.

Brunt, K. & Sanders, P. (2013). Food Chemistry, 140(3), 574-580.

The dietary fibre (DF) content in wheat grain based food products have been established with both the classical AOAC 985.29 dietary fibre and the new AOAC 2009.01 total dietary fibre protocol. There is a good agreement between the high molecular weight dietary fibre (HMWDF) contents measured with the AOAC 2009.01 method and (DF) content measured with the classical AOAC 985.29 method in wheat grain based food products. With the AOAC 2009.01 method also a significant amount of low molar weight dietary fibre (LMWDF), ranging from 1% to 3% w/w, was measured which is not quantified with the AOAC 985.29 method. With semi-preparative GPC the LMWDF (DP ≥ 3) fractions in the wheat grain based food products were isolated. The monosaccharide composition of the dissolved LMWDF constituents was determined. Glucose was by far the most abundant monosaccharide present with arabinose, galactose, xylose and mannose as minor constituents. It appeared that the LMWDF contains still not fully converted digestible starch/malto-oligosaccharide fragments with DP ≥ 3, which are erroneously quantified as LMWDF. By introducing an extra AMG hydrolysis step in the AOAC 2009.01 protocol after evaporation of the ethanol and dissolving the residue in deionised water, these malto-oligosaccharides are fully hydrolysed resulting in that way in a correct and lower LMWDF content.

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Flaxseed gum from flaxseed hulls: Extraction, fractionation, and characterization.

Qian, K. Y., Cui, S. W., Wu, Y. & Goff, H. D. (2012). Food Hydrocolloids, 28(2), 275-283.

Soluble dietary fibre with low viscosity has the potential to deliver acceptable mouthfeel and texture when included in the diet in a significant amount to show health benefits. Soluble flaxseed gum (SFG) was reported to have low viscosity, thus has potential in applications as a fibre fortifier. In the present work, SFG extracted from flaxseed hulls was fractionated into a neutral fraction gum (NFG) and an acidic fraction gum (AFG) using ion exchange chromatography. The protein content in SFG (11.8%) and AFG (8.1%) were completely removed by protease to obtain two more protein-free fractions, SFGnP and AFGnP; NFG contained no protein. The uronic acid content in NFG (1.8%) was mostly eliminated, whereas in AFC increased to 38.7%. NFG consisted of high molecular weight (MW) (1470 kDa) arabinoxylans and exhibited pseudoplastic flow behaviour; whereas AFG was mainly composed of rhamnogalacturonans with a higher MW fraction (1510 kDa) and a lower MW fraction (341 kDa) and showed Newtonian flow behaviour. The ranking of intrinsic viscosities (mL g-1) in decreasing order was: SFG (446.0) > NFG (377.5) > AFG (332.5). AFG was expected to have higher chain flexibility for its lower value of Huggins constant (0.16) compared to that of NFG (0.54) and SFG (0.48). In comparison with SFGnP, AFGnP and NFG, SFG and AFG showed better surface activities and emulsifying stabilities due to the presence of protein in both fractions.

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Starch transformation in bran-enriched extruded wheat flour.

Robin, F., Théoduloz, C., Gianfrancesco, A., Pineau, N., Schuchmann, H. P. & Palzer, S. (2011). Carbohydrate Polymers, 85(1), 65-74.

Wheat flour was extruded at different conditions of barrel temperature (120°C and 180°C), water content (18% and 22%) and screw speed (400 rpm and 800 rpm) with an increasing concentration of wheat bran fibers (2.8%, 12.6% and 24.4%). In the tested extrusion conditions, starch crystallites were fully dissociated. The estimated starch solubility was influenced by the process conditions and ranged from 24.1% to 63.1%. At same process conditions, the starch solubility was increased only at the highest bran level. The bran concentration influenced the glass transition temperature, melting temperature and sorption isotherm of the unprocessed wheat flour. At the extrusion conditions, it showed that higher bran levels led to a higher amount of free water and a decrease in starch glass transition temperature of up to 13 K. The differences in starch transformation, induced by the concentration of bran, might contribute to the modulation of the expansion properties of bran-containing starchy foams.

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Water extract of Triticum aestivum L. and its components demonstrate protective effect in a model of vascular dementia.

Han, H. S., Jang, J. H., Jang, J. H., Choi, J. S., Kim, Y. J., Lee, C., Lim, S. H., Lee, H. K. & Lee, J. (2010). Journal of Medicinal Food, 13(3), 572-578.

Although vascular dementia is the second leading cause of dementia and often underdiagnosed, there are no drugs yet approved for the treatment of vascular dementia. In this study, it is demonstrated that water extract of Triticum aestivum L. (TALE) and some of its components have protective effects against vascular dementia-induced damage by preserving the myelin sheath and inhibiting astrocytic activation. The memory test used a vascular dementia model utilizing bilateral ligation of the carotid arteries of rats. TALE, some of its components, such as starch, total dietary fiber (TDF), arabinoxylan, β-glucan, and degraded products of arabinoxylan, such as arabinose and xylose, were administered to the animals from day 8 to day 14, following the surgery. Twenty-one days after the surgery, the water maze test was performed for 5 days, and the time taken to find the platform during training trials (mean escape latency) was measured. The mean escape latency was decreased consistently in the TALE-, starch-, TDF-, arabinoxylan-, and arabinose-treated groups, compared with that in the vascular dementia group. To measure brain damage, Luxol fast blue staining and immunohistochemistry of myelin basic protein (MBP) were performed to observe myelin sheath in the white matter, and immunohistochemistry of glial fibrillary acidic protein (GFAP) was performed to observe the astrocytic reaction. Vascular dementia reduced the MBP level and increased the GFAP level. Arabinose effectively inhibited the MBP and GFAP change, whereas arabinoxylan inhibited the GFAP change only. These results suggest that TALE and some of its components can be used as a medicinal material for the development of neuroprotective agents against vascular dementia.

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Precooked Bran‐Enriched Wheat Flour Using Extrusion: Dietary Fiber Profile and Sensory Characteristics.

Gajula, H., Alavi, S., Adhikari, K. & Herald, T. (2008). Journal of Food Science, 73(4), S173-S179.

The effect of precooking by extrusion processing on the dietary fiber profile of wheat flour substituted with 0%, 10%, 20%, and 30% wheat bran was evaluated. Depending on the level of bran, total dietary fiber (TDF) and soluble dietary fiber (SDF) in uncooked flours ranged from 4.2% to 17.2% and 1.5% to 2.4%, respectively. Precooking by extrusion significantly increased SDF in flours (by 22% to 73%); although in most cases it also led to a significant decrease in TDF. Cookies and tortillas produced from uncooked and precooked flours with 0% and 20% substituted bran were evaluated for consumer acceptability using a 9-point hedonic scale. With a few exceptions, all cookies had scores ranging from 6 to 7 (“like slightly” to “like moderately”) for each attribute, including overall acceptability, appearance, texture, crumbliness, and flavor. Tortillas were rated for the same attributes except for crumbliness, which was replaced with chewiness. In most cases, tortilla scores ranged from 5 to 7 (“neither like nor dislike” to “like moderately”). Consumer acceptability scores of cookies from uncooked flour did not change significantly with increase in bran substitution from 0% to 20%. However, consumer scores for tortillas did decrease significantly with increase in bran level. Extrusion precooking of the flours did not improve the consumer acceptability of cookies and tortillas; however, it did improve their dietary fiber profile by increasing the SDF significantly.

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Natural occurrence of ochratoxin A and antioxidant activities of green and roasted coffees and corresponding byproducts.

Napolitano, A., Fogliano, V., Tafuri, A. & Ritieni, A. (2007). Journal of Agricultural and Food Chemistry, 55(25), 10499-10504.

Ochratoxin A is an important mycotoxin that can enter the human food chain in cereals, wine, coffee, spices, beer, cocoa, dried fruits, and pork meats. Coffee is one of the most common beverages and, consequently, it has a potential risk factor for human health related to ochratoxin A exposure. In this study, coffee and corresponding byproducts from seven different geographic regions were investigated for ochratoxin A natural occurrence by HPLC-FLD, nutritional characterization, and antioxidant activities by spectrophotometric assay. The research focused on composition changes in coffee during the processing step “from field to cup”. Costa Rica and Indian green coffees were the most contaminated samples, with 13 and 11 µg/kg, respectively, while the Ethiopian coffee was the least contaminated, with 3.8 µg/kg of ochratoxin A. The reduction of ochratoxin A contamination during the roasting step was comparable for any samples that were considered under the recommended level of 4 µg/kg. Total dietary fibers ranged from 58.7% for Vietnam and 48.6% for Ivory Coast in green coffees and ranged from 58.6% for Costa Rica to 61.2% for India in roasted coffee. Coffee silverskin byproduct obtained from Ivory Coast was the highest, with 69.2 and 64.2% of insoluble dietary fibers, respectively.

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A simplified modification of the AOAC official method for determination of total dietary fiber using newly developed enzymes: preliminary interlaboratory study.

Kanaya, K., Tada, S., Mori, B., Takahashi, R., Ikegami, S., Kurasawa, S., Okuzaki, M., Mori, Y., Innami, S. & Negishi, Y. (2007). Journal of AOAC International, 90(1), 225-237.

A preliminary interlaboratory study was conducted to evaluate the validity of the modified AOAC method for determination of total dietary fiber by Tada and Innami, in which the 3-step enzymatic digestion process in AOAC Method 991.43 is modified to a 2-step process without pH adjustment. Total dietary fiber contents in 8 representative foodstuffs were measured using both the original AOAC Method 991.43 and the modified method in 6 research facilities in Japan. Repeatability relative standard deviations, reproducibility relative standard deviations, and Horwitz ratio values from the modified method were equivalent to those from AOAC Method 991.43, except in the rice sample. However, this exceptional case shown in the modified method was entirely dissolved by the addition of -amylase stabilizing agents. The modified method, which shortens the process of enzymatic digestion from 3 to 2 steps and in which only reaction temperature is adjusted under the same pH, was found not only to give accurate values comparable to the original method, but also to substantially reduce the labor required by the laboratory staff in the process of routine analysis. This study revealed that the validity of the modified method was further ensured by adding -amylase stabilizing agents to the reaction system.

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Treatment of cereal products with a tailored preparation of Trichoderma enzymes increases the amount of soluble dietary fiber.

Napolitano, A., Lanzuise, S., Ruocco, M., Arlotti, G., Ranieri, R., Knutsen, S. H., Lorito, M. & Fogliano, V. (2006). Journal of Agricultural and Food Chemistry, 54(20), 7863-7869.

Nutritionists recommend increasing the intake of soluble dietary fiber (SDF), which is very low in most cereal-based products. Conversion of insoluble DF (IDF) into SDF can be achieved by chemical treatments, but this affects the sensorial properties of the products. In this study, the possibility of getting a substantial increase of SDF from cereal products using a tailored preparation of Trichoderma enzymes is reported. Enzymes were produced cultivating Trichoderma using durum wheat fiber (DWF) and barley spent grain (BSG) as unique carbon sources. Many Trichoderma strains were screened, and the hydrolysis conditions able to increase by enzymatic treatment the amount of SDF in DWF and BSG were determined. Results demonstrate in both products that it is possible to triple the amount of SDF without a marked decrease of total DF. The enzymatic treatment also causes the release of hydroxycinnamic acids, mainly ferulic acid, that are linked to the polysaccharides chains. This increases the free phenolic concentration, the water-soluble antioxidant activity, and, in turn, the phenol compounds bioavailability.

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Characterization of a new potential functional ingredient: coffee silverskin.

Borrelli, R. C., Esposito, F., Napolitano, A., Ritieni, A. & Fogliano, V. (2004). Journal of Agricultural and Food Chemistry, 52(5), 1338-1343.

Dietary fiber (DF) is one of the main dietary factors contributing to consumers' well-being. In this work the possibility of using the roasted coffee silverskin (CS), a byproduct of roasted coffee beans, as a DF-rich ingredient has been evaluated. The results of our investigation showed that this material has 60% total DF, with a relevant component (14%) of soluble DF. Although a small amount of free phenol compounds is present in CS, it has a marked antioxidative activity, which can be attributed to the huge amount of Maillard reaction products, the melanoidins. Static batch culture fermentation experiments showed that CS induces preferential growth of bifidobacteria rather than clostridia and Bacteroides spp. CS can be proposed as a new potential functional ingredient in consideration of the high content of soluble DF, the marked antioxidant activity, and the potential prebiotic activity.

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Enzyme-aided investigation of the substituent distribution in cationic potato amylopectin starch.

Richardson, S., Nilsson, G., Cohen, A., Momcilovic, D., Brinkmalm, G. & Gorton, L. (2003). Analytical Chemistry, 75(23), 6499-6508.

The distribution of substituents along the polymer chain in cationic potato amylopectin starch, modified in solution, granular slurry, or dry state, was investigated. The starch derivatives were successively hydrolyzed by different enzymes, followed by characterization of the hydrolysis products obtained by means of electrospray mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). ESI-MS and MALDI-MS were proved to be appropriate techniques for identification of the substituted hydrolysis products, for which there are no standard compounds available. No highly substituted oligomers were found in the hydrolysates, which was taken as an indication of a more or less homogeneous distribution of cationic groups in the amylopectin molecules. Furthermore, from the results obtained it was suggested that the enzymes cleave glucosidic linkages only between unsubstituted glucose units and, preferentially, linkages in sequences containing more than two adjacent unsubstituted units. The determination of the amount of unsubstituted glucose produced from every successive hydrolysis step revealed slight differences between the different starch samples with respect to the homogeneity of the substitution pattern. Among the three samples under investigation, starch cationized in solution was found to have the most and dry-cationized starch the least homogeneous distribution of substituents.

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Starch from hull-less barley: Ultrastructure and distribution of granule-bound proteins.

Li, J. H., Vasanthan, T., Hoover, R. & Rossnagel, B. G. (2003). Cereal Chemistry, 80(5), 524-532.

Starch granules isolated from waxy, normal, and high-amylose hullless barley grains were examined by transmission electron microscopy with cytochemical techniques. The micrographs showed two distinct regions of different sizes: 1) densely packed granule growth rings (which varied in size and number depending on the genotype), and 2) a loose filamentous network located in the central region of the granule. The granule ring width decreased with increasing amylose content. In all three genotypes, the growth rings closer to the granule surface were narrower in width than those within the granule interior. The waxy starch had wider intercrystalline amorphous growth rings, semicrystalline growth rings, and more open crystalline lamellae than normal and high-amylose starches. Granule bound proteins (mainly integral proteins) were located in the central and peripheral (growth ring) regions of the granule.

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Safety Information
Symbol : GHS05, GHS08
Signal Word : Danger
Hazard Statements : H318, H334
Precautionary Statements : P261, P280, P284, P304+P340, P305+P351+P338, P310
Safety Data Sheet
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