Maltotetraose

A simple method for measurement of the amylolytic activity of malt has been developed and fully evaluated. The method, termed the Maltose Value (MV) is an extension of previously reported work. Here, the MV method has been studied in detail and all aspects of the assay (sample grinding and extraction, starch hydrolysis, maltose hydrolysis and determination as glucose) have been optimised. The method is highly correlated with other dextrinising power methods. The MV method involves extraction of malt in 0.5% sodium chloride at 30°C for 20 minutes followed by filtration; incubation of an aliquot of the undiluted filtrate with starch solution (pH 4.6) at 30°C for 15 min; termination of reaction with sodium hydroxide solution; dilution of sample in an appropriate buffer; hydrolysis of maltose with a specific α-glucosidase; glucose determination and activity calculation. Unlike all subsequent reducing sugar methods, the maltose value method measures a defined reaction product, maltose, with no requirement to use equations to relate analytical values back to Lintner units. The maltose value method is the first viable method in 130 years that could effectively replace the 1886 Lintner method.

The pseudo-tetrasaccharide acarbose, produced by Actinoplanes sp. SE50/110, is a α-glucosidase inhibitor used for treatment of type 2 diabetes patients. In industrial production of acarbose, by-products play a relevant role that complicates the purification of the product and reduce yields. Here, we report that the acarbose 4-α-glucanotransferase AcbQ modifies acarbose and the phosphorylated version acarbose 7-phosphate. Elongated acarviosyl metabolites (α-acarviosyl-(1,4)-maltooligosaccharides) with one to four additional glucose molecules were identified performing in vitro assays with acarbose or acarbose 7-phosphate and short α-1,4-glucans (maltose, maltotriose and maltotetraose). High functional similarities to the 4-α-glucanotransferase MalQ, which is essential in the maltodextrin pathway, are revealed. However, maltotriose is a preferred donor and acarbose and acarbose 7-phosphate, respectively, serve as specific acceptors for AcbQ. This study displays the specific intracellular assembly of longer acarviosyl metabolites catalyzed by AcbQ, indicating that AcbQ is directly involved in the formation of acarbose by-products of Actinoplanes sp. SE50/110.

Four different soluble fibres were evaluated as sugar replacers in short dough biscuits: two resistant dextrins (Nutriose® FM06 and Promitor® SGF 70R) and two inulin-derived fibres (Orafti® HSI and Fibruline™ Instant). The degree of polymerisation of the fibres was analysed, and dough viscoelastic properties were assessed. Weight loss during baking, dimensions, textural properties, surface colour and sensory profile were evaluated. Higher degree of polymerisation fibres (e.g. Fibruline) limited water availability for syrup formation, restricting dough expansion and resulting in smaller, more compact, and harder biscuits than control. Biscuits with inulin derived fibres with a lower degree of polymerisation (e.g. Orafti) showed similar dimensions to control biscuits. In general, sucrose reduction gave place to biscuits with lower resistance to penetration and fracture strength due to less sugar recrystallisation in the final biscuit. In contrast, when dextrin-type fibres were used the rheological behaviour of the dough, spreading during baking, and resistance to penetration were similar to the control as the fibres showed an anti-plasticising effect similar to sucrose. However, all reduced sugar biscuits were significantly firmer and crunchier in sensory profile suggesting further optimisation is needed, potentially by modification of the fibre structure or baking method.

Exploring new raw starch-hydrolyzing α-amylases and understanding their biochemical characteristics are important for the utilization of starch-rich materials in bio-industry. In this work, the biochemical characteristics of a novel raw starch-degrading α-amylase (HL11 Amy) from Roseateles terrae HL11 was firstly reported. Evolutionary analysis revealed that HL11Amy was classified into glycoside hydrolase family 13 subfamily 32 (GH13_32). It contains four protein domains consisting of domain A, domain B, domain C and carbohydrate-binding module 20 (CMB20). The enzyme optimally worked at 50°C, pH 4.0 with a specific activity of 6270 U/mg protein and 1030 raw starch-degrading (RSD) U/mg protein against soluble starch. Remarkably, HL11Amy exhibited activity toward both raw and gelatinized forms of various substrates, with the highest catalytic efficiency (k_cat/K_m) on starch from rice, followed by potato and cassava, respectively. HL11Amy effectively hydrolyzed cassava pulp (CP) hydrolysis, with a reducing sugar yield of 736 and 183 mg/g starch from gelatinized and raw CP, equivalent to 72% and 18% conversion based on starch content in the substrate, respectively. These demonstrated that HL11Amy represents a promising raw starch-degrading enzyme with potential applications in starch modification and cassava pulp saccharification.

Surplus bread is a major bakery side stream that should be strictly kept within the human food chain to reduce waste and ensure resource efficiency in baking processes. Optimally, surplus bread should be recycled as a dough ingredient, however, this is known to be detrimental to the volume and texture of bread. The purpose of this study was to investigate how gelatinized starch in surplus bread, untreated or enzymatically hydrolyzed, affects dough development, bread volume and textural attributes. Starch was hydrolyzed to various degrees using commercial α-amylase and amyloglucosidase. Bread hydrolysates containing different carbohydrate profiles (untreated, 75%, 57%, and 26% starch remaining) were evaluated as dough ingredients. More complete starch hydrolysis resulted in better dough visco-elastic properties and higher dough level, and reduced dough water absorption by 13%. Nonetheless, breads containing hydrolysate with high-malto-oligosaccharides had the lowest intrinsic hardness and similar volume yield when compared to control bread. Furthermore, compared to untreated slurry, the hydrolysate with high-malto-oligosaccharides, reduced crumb hardness by 28% and staling rate by 42%, and increased specific volume by 8%. The present findings show that enzymatic hydrolysis dramatically transforms the impact of gelatinized starch. Thus, by selecting correct bioprocessing approaches, bread recycling performance may be significantly improved.

Resistant starch type 3 (RS-3) holds great potential as a prebiotic by supporting gut microbiota following intestinal digestion. However the factors influencing the digestibility of RS-3 are largely unknown. This research aims to reveal how crystal type and molecular weight (distribution) of RS-3 influence its resistance. Narrow and polydisperse α-glucans of degree of polymerization (DP) 14-76, either obtained by enzymatic synthesis or debranching amylopectins from different sources, were crystallized in 12 different A- or B-type crystals and in vitro digested. Crystal type had the largest influence on resistance to digestion (A >>> B), followed by molecular weight (Mw) (high DP >> low DP) and Mw distribution (narrow disperse > polydisperse). B-type crystals escaping digestion changed in Mw and Mw distribution compared to that in the original B-type crystals, whereas A-type crystals were unchanged. This indicates that pancreatic α-amylase binds and acts differently to A- or B-type RS-3 crystals.

An accurate high-performance anion-exchange chromatography (HPAEC) method is presented to measure the inhibition property of flavonoids against mammalian starch digestive enzymes, because flavonoids interfere with commonly used 3,5-dinitrosalicylic acid (DNS) and glucose oxidase/peroxidase (GOPOD) methods. Eriodictyol, luteolin, and quercetin increased absorbance values (without substrate) in the DNS assay and, with substrate, either overestimated or underestimated values in the DNS and GOPOD assays. Using a direct HPAEC measurement method, flavonoids showed different inhibition properties against α-amylase and α-glucosidases, showing different inhibition constants (K_i) and mechanisms. The double bond between C2 and C3 on the C-ring of flavonoids appeared particularly important to inhibit α-amylase, while the hydroxyl group (OH) at C3 of the C-ring was related to inhibition of α-glucosidases. This study shows that direct measurement of starch digestion products by HPAEC should be used in inhibition studies, and provides insights into structure-function aspects of polyphenols in controlling starch digestion rate.

Measuring reducing sugar is a common practice in carbohydrate research, and the colorimetric assay developed by Somogyi and Nelson has a high sensitivity in a broad concentration range. However, the method is time-consuming when analyzing a large number of samples. In this study, a modified Somogyi-Nelson assay with excellent accuracy and sensitivity was developed using a 96-well microplate. This microassay greatly improves the analytic capacity and efficacy of the method.