New developments in the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase.
McCleary, B. V. & Monaghan, D. (2000). “Proceedings of the Second European Symposium on Enzymes in Grain Processing”, (M. Tenkanen, Ed.), VTT Information Service, pp. 31-38.
Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.
Measurement of cereal α-Amylase: A new assay procedure.
McCleary, B. V. & Sheehan, H. (1987). Journal of Cereal Science, 6(3), 237-251.
A new procedure for the assay of cereal α-amylase has been developed. The substrate is a defined maltosaccharide with an α-linked nitrophenyl group at the reducing end of the chain, and a chemical blocking group at the non-reducing end. The substrate is completely resistant to attack by β-amylase, glucoamylase and α-glucosidase and thus forms the basis of a highly specific assay for α-amylase. The reaction mixture is composed of the substrate plus excess quantities of α-glucosidase and glucoamylase. Nitrophenyl-maltosaccharides released on action of α-amylase are instantaneously cleaved to glucose plus free p-nitrophenol by the glucoamylase and α-glucosidase, such that the rate of release of p-nitrophenol directly correlates with α-amylase activity. The assay procedure shows an excellent correlation with the Farrand, the Falling Number and the Phadebas α-amylase assay procedures.
A new procedure for the measurement of fungal and bacterial α-amylase.
Sheehan, H. & McCleary, B. V. (1988). Biotechnology Techniques, 2(4), 289-292.
A procedure for the measurement of fungal and bacterial α-amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end) p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and α-glucosidase, and is absolutely specific for α-amylase. The assay procedure is simple, reliable and accurate.
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.
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.
Measurement of resistant starch.
McCleary, B. V. & Monaghan, D. A. (2002). Journal of AOAC International, 85(3), 665-675.
A robust and reliable method was developed to measure resistant starch (RS), i.e., starch that enters the large intestine. In vivo conditions were reflected as much as possible while a user-friendly format was maintained. Parameters investigated included α-amylase concentration, pH of incubation, maltose inhibition of α-amylase, the need for amyloglucosidase inclusion, the effect of shaking and stirring on determined values, and problems in recovering and analyzing the RS-containing pellet. The RS values obtained were in good agreement with published in vivo data. An interlaboratory evaluation of the method has been completed (First Action Method 2002.02).
Measurement of resistant starch by enzymatic digestion in starch and selected plant materials: Collaborative study.
McCleary, B. V., McNally, M. & Rossiter, P. (2002). Journal of AOAC International, 85(5), 1103-1111.
Interlaboratory performance statistics was determined for a method developed to measure the resistant starch (RS) content of selected plant food products and a range of commercial starch samples. Food materials examined contained RS (cooked kidney beans, green banana, and corn flakes) and commercial starches, most of which naturally contain, or were processed to yield, elevated RS levels. The method evaluated was optimized to yield RS values in agreement with those reported for in vivo studies. Thirty-seven laboratories tested 8 pairs of blind duplicate starch or plant material samples with RS values between 0.6 (regular maize starch) and 64% (fresh weight basis). For matrixes excluding regular maize starch, repeatability relative standard deviation (RSDr) values ranged from 1.97 to 4.2%, and reproducibility relative standard deviation (RSDR) values ranged from 4.58 to 10.9%. The range of applicability of the test is 2-64% RS. The method is not suitable for products with <1% RS (e.g., regular maize starch; 0.6% RS). For such products, RSDr and RSDR values are unacceptably high.
Measurement of α-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the ceralpha assay: Collaborative study.
McCleary, B. V., McNally, M., Monaghan, D. & Mugford, D. C. (2002). Journal of AOAC International, 85(5), 1096-1102.
This study was conducted to evaluate the method performance of a rapid procedure for the measurement of α-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing α-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable α-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, α-glucosidase, and β-amylase. When the substrate is cleaved by endo-acting α-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of α-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 µmol p-nitrophenyl (in the presence of excess quantities of α-glucosidase) in 1 min at 40°C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal α-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%.
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.
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.
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.
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.
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.
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 Method2009.01, 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.
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.
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.
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.
The effect of granule size on the digestibility of wheat starch using an in vitro model.
Teo, M. L. L. C. & Small, D. M. (2012). World Academy of Science, Engineering and Technology, 6(9), 701-705.
Wheat has a bimodal starch granule population and the dependency of the rate of enzymatic hydrolysis on particle size has been investigated. Ungelatinised wheaten starch granules were separated into two populations by sedimentation and decantation. Particle size was analysed by laser diffraction and morphological characteristics were viewed using SEM. The sedimentation technique though lengthy, gave satisfactory separation of the granules. Samples (<10µm, >10µm and original) were digested with α-amylase using a dialysis model. Granules of <10µm showed significantly higher rate of reducing sugar release than those >10µm (p<0.05). In contrast, the rate was not significantly different between the original sample and granules >10µm. Moreover, the digestion rate was dependent on particle size whereby smaller granules produced higher rate of release. The methodology and results reported here can be used as a basis for further evaluations designed to delay the release of glucose during the digestion of native starches.
Assessing the susceptibility of amylose–lysophosphatidylcholine complexes to amylase by the use of iodine.
Ahmadi‐Abhari, S., Woortman, A. J. J., Hamer, R. J. & Loos, K. (2014). Starch‐Stärke, 66(5-6), 576-581.
The formation of amylose–lysophosphatidylcholine (LPC) inclusion complexes renders amylose less susceptible to amylase digestion. In order to better understand this phenomenon on a structural level, the complexation of 9% wheat starch suspensions with 0, 2, 3, and 5% exogenous LPC was developed in RVA. Amylose–LPC inclusion complexes were isolated after 15, 30, 60, 120, and 240 min in vitro digestion of the wheat starch suspensions to quantify the amount of non-complexed amylose by spectrophotometry. The samples were dissolved in DMSO containing 0.5% LiBr and exposed to iodine. In addition, parts of the digesta were defatted and subjected to the same procedure to elucidate the total amount of amylose that remained undigested. In this way, more insight was obtained into the protective effect of amylose–LPC complex formation on digestion of starch. This study confirms that the amylose susceptibility to amylolysis decreases in the presence of LPC. Higher LPC concentrations not only induced the formation of more amylose inclusion complexes but also resulted in more stable complexes which remained undigested as well as longer amylose chains after enzyme hydrolysis, due to the presence of LPC inside the amylose helix. In addition, a higher melting enthalpy of the amylose–LPC complexes in the digesta demonstrates the protective effect of LPC during enzyme hydrolysis.
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.