860 DOI 10.1002/star.201500017 Starch/Stärke 2015, 67, 860–883 RESEARCH ARTICLE Determination of total dietary fibre and available carbohydrates: A rapid integrated procedure that simulates in vivo digestion Barry V. McCleary, Naomi Sloane and Anna Draga Megazyme International Ireland, Bray Business Park, Bray, County Wicklow, Ireland The new definition of dietary fibre introduced by Codex Alimentarius in 2008 includes resistant Received: January 23, 2015 starch and the option to include non-digestible oligosaccharides. Implementation of this definition Revised: March 5, 2015 required new methodology. An integrated total dietary fibre method was evaluated and accepted by Accepted: March 5, 2015 AOAC InternationalandAACCInternational(AOACMethods2009.01and2011.25;AACCMethod 32–45.01 and 32–50.01, and recently adopted by Codex Alimentarius as a Type I Method. However, in application of the method to a diverse range of food samples and particularly food ingredients, some limitations have been identified. One of the ongoing criticisms of this method was that the time of incubation with pancreatic a-amylase/amyloglucosidase mixture was 16 h, whereas the time for food to transit through the human small intestine was likely to be approximately 4 h. In the current work, we use an incubation time of 4 h, and have evaluated incubation conditions that yield resistant starch and dietary values in line with ileostomy results within this time frame. Problems associated with production, hydrolysis and chromatography of various oligosaccharides have been addressed resulting in a more rapid procedure that is directly applicable to all foods and food ingredients currently available. Keywords: Available carbohydrates / Codex Alimentarius / Dietary fibre determination / Enzymic / Non-digestible oligosaccharides / Resistant starch : Additional supporting information may be found in the online version of this article at the publisher’s web-site. 1 Introduction of the consumer. Since the definition and analysis of dietary fibre are intimately related, analysis methods should be Interest in dietary fibre is a consequence of the belief that developed to comply with the conceptual definitions. dietary fibre contributes positively to the health/quality of life However, compromises must be accepted due to constraints of cost and time [1]. Available carbohydrates are carbohydrates that are readily Correspondence: Dr. Barry V. McCleary, Megazyme International Ireland, Bray Business Park, Southern Road, Bray, County hydrolysed into D-glucose and D-fructose and absorbed into Wicklow, Ireland the human small intestine. They consist of non-resistant E-mail: [email protected] starch, maltodextrins, sucrose, the glucose component of Fax: þ353-1-286-1264 lactose and free D-glucose and D-fructose. ‘ fi ’ Abbreviations: ACH, available carbohydrates (¼ non-resistant The term dietary bre was coined by Hipsley [2] to cover starch þ maltodextrins þ maltose þ sucrose þ free D-glucose the non-digestible constituents of plants that make up the and D-fructose þ the D-glucose component of lactose); DF, dietary plant cell wall, known to include cellulose, hemicellulose and fibre (¼ HMWDF þ LMWDF) (¼ IDF þ HMWSDF þ LMWSDF); lignin. The aim was to define some property of the HMWDF, high molecular weight dietary fibre (¼ IDF þ HMWSDF); HMWSDF, high molecular weight soluble dietary fibre (¼ SDFP); constituent of the food that could be related to physiological IDF, insoluble dietary fibre; LMWSDF, low molecular weight behaviour in the human small intestine. This definition of soluble dietary fibre (¼ SDFS); NDO, non-digestible dietary fibre was broadened by Trowell et al. [3] to become oligosaccharides (¼ LMWSDF ¼ SDFS); RS, resistant starch; primarily a physiological definition, based on edibility and SDFP, soluble dietary fibre which precipitates in the presence of 76% ethanol; SDFS, soluble dietary fibre that remains soluble in the resistance to digestion in the human small intestine; the presence of 76% ethanol definition included indigestible polysaccharides such as ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com Starch/Stärke 2015, 67, 860–883 861 gums, modified celluloses, mucilages and pectin, and non- Ireland, Woodford Business Park, Santry, Dublin, Ireland) digestible oligosaccharides (NDO). column at the same point as disaccharides such as lactose, Initial efforts to develop a method to satisfy this analytical maltose and sucrose, so it is not included in the analytical requirement focused on the removal of starch and protein. value for SDFS (DP > 3). A solution to this problem is to use Enzymes employed had to meet specific activity require- different chromatographic columns as are described in ments and be devoid of contaminating enzymes active on AOAC Method 2001.03 [7, 34]. Thirdly, on hydrolysis of dietary fibre components. The method that evolved was products high in starch, including certain breads and rice AOAC Official Method 985.29 ‘Total Dietary Fiber in Foods; products, various maltodextrins are produced which are very Enzymatic-Gravimetric Method’ [4, 5]. Subsequently, the resistant to further hydrolysis by AMG or PAA [35–37]. method was extended to allow measurement of total, soluble However, these oligosaccharides are readily hydrolysed [8] by and insoluble dietary fibre in foods (AOAC Official Method a mucosal a-glucosidase preparation from the small 991.43) [6], and various other modified methods for fibre intestine of pig [38], indicating that they should not be determination have been approved by AOAC International included in the SDFS fraction of dietary fibre (DF). A fourth [7] and accepted by Codex Alimentarius [8, 9]. challenge experienced in the use of AOAC Method 2009.01 is In parallel research in the UK, Englyst et al. [10] developed the considered underestimation of phosphate cross-linked W W methods for the measurement of available starch and non- starch (Resistant Starch 4, e.g. FiberRite and Fibersym , starch polysaccharides (NSP) [11–14], based on original work MGP Ingredients, Cray Business Park, Atchison, Kansas, W W of Southgate [15, 16]. This NSP procedure measures only USA). Much higher DF values for FiberRite and Fibersym non-starch polysaccharides; resistant starch (RS) is deliber- are obtained [39] using the Prosky TDF method (AOAC ately removed and NDOs are not measured. Method 985.29) [5]. A fifth concern in the use of AOAC Based on the recommendation for endorsement of the Method 2009.01, relates to the use of sodium azide as a Codex Committee on Nutrition and Foods for Special Dietary preservative in the buffer. While the concentration employed Uses (CCNFSDU) in November 2008, a definition for dietary is low (0.02% w/v), it is still of concern to some analysts. With fibre was adopted in June 2009 by the Codex Alimentarius the extended incubation conditions described in AOAC Commission (CAC) [17]. The definition includes carbohy- Method 2009.01 (16 h, 37°C, pH 6), inclusion of an drate polymers that are not hydrolysed by the endogenous antimicrobial agent is essential. However, with a shorter enzymes in the small intestine of humans and thus includes incubation time of just 4 h, the sodium azide could RS. However, decisions concerning the inclusion, or not, of potentially be removed altogether or alternatively replaced oligosaccharides of degrees of polymerisation (DP) of 3–9 with another preservative such as a few drops of toluene. were left to the discretion of national authorities [18]. The aim of this research was to develop a method for the A method that appeared to satisfy this definition was measurement of dietary fibre that resolves each of the published in 2007 [19] and this method was successfully challenges detailed above. Part of this process involved a evaluated in interlaboratory evaluations [20, 21]. In this comparison of AOAC Method 2002.02 [22] and the Englyst method, samples are incubated with pancreatic a-amylase et al. [10] methods for measurement of resistant starch, in an (PAA) and amyloglucosidase (AMG) under near physiolog- attempt to understand why the methods give similar RS ical conditions (37°C, pH 6) as previously described for values across a range of samples, even though the incubation measurement of resistant starch (AOAC Method 2002.01) [7, times with PAA/AMG are very different. The ultimate goal 22, 23]. This method allows the measurement of high being to develop a method that more closely simulates molecular weight dietary fibre (HMWDF) which included physiological conditions. Problems associated with measure- insoluble dietary fibre (IDF) and high molecular weight ment of FOS and production of resistant maltodextrins from soluble dietary fibre (HMWSDF) which precipitates in the starch have also been addressed. A second aim was to presence of 78% aqueous ethanol (SDFP) and also low incorporate quantitative measurement of available carbohy- molecular weight soluble dietary fibre (LMWSDF) that drates into the overall analysis. remains soluble in the presence of 78% aqueous ethanol (SDFS). However, in the application of this method to a range of food products and ingredients, several challenges/ 2 Materials and methods concerns were identified. Firstly, an incubation time with PAA plus AMG of 16 h was considered not to simulate 2.1 Materials physiological conditions. Based on numerous studies in literature, a more likely residence time for food in the small D/L-Maleic acid (cat. no. M-0375), bovine serum albumin (cat. intestine is 4 Æ 1 h [24–33]. Secondly, most commercially no. A-2153), dimethyl sulphoxide (cat. no. D-8779) and available fructo-oligosaccharides (FOS) contain the trisac- sodium azide (cat. no. S-8032) were from Sigma–Aldrich charide, fructosyl-b-(2-1)-fructosyl-b-(2-1)-fructose (F3). This Ireland Ltd. (Dublin, Ireland). Acetic acid (glacial) GR, 1 oligosaccharide elutes from the Waters Sugar-Pak (Waters sodium hydroxide and calcium chloride (CaCl2.2H2O) were ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com 862 B. V. McCleary et al. Starch/Stärke 2015, 67, 860–883 from Merck (Darmstradt, Germany). Partially degraded 2.2 Methods W chicory inulin (Raftilose P-95 ) was a kind gift from Raffinerie Tirlemontoise S.A.