Pullulanase M2 (Bacillus licheniformis)

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.

A new super-branched amylopectin with longer exterior chains was produced from normal maize starch by modification with branching enzyme followed by 4-α-glucanotransferase, and applied for co-entrapment of a curcumin-loaded emulsion in alginate beads. The network structure of the gel beads was obtained with Ca²⁺-cross-linked alginate and a modest load of retrograded starch. The dual enzyme modified starch contained more and longer α-1,6-linked branch chains than single enzyme modified and unmodified starches and showed superior resistance to digestive enzymes. Alginate beads with or without starch were of similar size (1.69–1.74 mm), but curcumin retention was improved 1.4–2.8-fold in the presence of different starches. Thus, subjecting the curcumin-loaded beads to in vitro simulated gastrointestinal digestion resulted in retention of 70, 43 and 22 % of the curcumin entrapped in the presence of modified, unmodified, or no starch, respectively. Molecular docking provided support for curcumin interacting with starch via hydrogen bonding, hydrophobic contacts and π-π stacking. The study highlights the potential of utilizing low concentration of dual-enzyme modified starch with alginate to create a versatile vehicle for controlled release and targeted delivery of bioactive compounds.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBD_St1 and SBD_St2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBD_GA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBD_GA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3–9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBD_GA-TuαGT and-SBD_St2-TuαGT showed K_d of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBD_St1-TuαGT had 3-5-fold lower affinity. SBD_St2 contributed more than SBD_St1 to activity, substrate binding, and the stability of TuαGT fusions.

The structural, physicochemical and digestive properties of rice starch modified by the combination of different temperature (60, 70, 80, 90 and 100°C) preheating and pullulanase (PUL60, PUL70, PUL80, PUL90 and PUL100) treatments were investigated. The PUL60 treatment mainly modified the surface layer of starch granules, which increased the amylose content and damaged some ordered structures, resulting in slight decreases of gel strength and estimated glycemic index (eGI). With the increase of preheating temperature, PUL could act on more enzymatic sites to release a large amount of linear chains, reduce the ordered degree, and transform the A-type crystalline structure into B-type. The low molecule interaction strength between linear chains weakened the gel network structure, and some stable crystal structures formed by longer chains resisted the enzyme digestion. The gel strength and eGI value of PUL70 starch decreased significantly, and the properties of PUL80–100 starches tended to be stable, showing a further significant decrease of gel strength and a slight reduction of eGI value. Therefore, the preheating treatments at 60, 70 and 80°C were suitable for the PUL modification of rice starch to obtain strong, medium and weak gel strength respectively, and the digestibility decreased with increasing preheating temperature.

Due to the semi-crystalline structure of native starch granules, enzymatic modification of these solid, raw, entities by branching enzyme (BE) is limited. Here, we describe a method to efficiently modify starch by BE after maltogenic α-amylases pre-treatment. This pre-treatment produced pores at the starch granule surface, which decreased the granular yield, but increased the branching degree in starch molecules. BE post-treatments recovered the yield, increased the content of long amylose chains, and the starch crystallinity. WAXS analysis showed that BE transformed the unresolved doublet peak at 2θ 17° and 18° to a strong peak at 2θ 17°, i.e. transformed the granules from the A-type to a mixed A-, B-type allomorph. Syneresis of starch gels increased with increasing BE concentrations and increased the content of slowly digested starch in retrograded starch preparations. Rheology data demonstrated that low and medium BE concentrations produced starch gels with higher G′ and G″ after storage for 1day, whereas high BE concentrations reduced both G′ and G’’. Our data demonstrate the potential of clean, enzyme-based protocols using sequential addition of starch active enzymes for post-harvest modification of raw starch granules to obtain clean and functional starch.

Sorghum bicolor is a C₄ grass widely cultivated for grain, forage, sugar, and biomass. The sorghum Dry Stalk (D) locus controls a qualitative difference between juicy green (dd) and dry white (D‐) stalks and midribs, and co‐localizes with a quantitative trait locus for sugar yield. Here, we apply fine‐mapping and genome‐wide association study (GWAS) to identify a candidate gene underlying D, and use nearly isogenic lines (NILs) to characterize the transcriptional, compositional, and agronomic effects of variation at the D locus. The D locus was fine‐mapped to a 36 kb interval containing four genes. One of these genes is a NAC transcription factor that contains a stop codon in the NAC domain in the recessive (dd) parent. Allelic variation at D affects grain yield, sugar yield, and biomass composition in NILs. Green midrib (dd) NILs show reductions in lignin in stalk tissue and produce higher sugar and grain yields under well‐watered field conditions. Increased yield potential in dd NILs is associated with increased stalk mass and moisture, higher biomass digestibility, and an extended period of grain filling. Transcriptome profiling of midrib tissue at the 4-6 leaf stages, when NILs first become phenotypically distinct, reveals that dd NILs have increased expression of a miniature zinc finger (MIF) gene. MIF genes dimerize with and suppress zinc finger homeodomain (ZF‐HD) transcription factors, and a ZF‐HD gene is associated with midrib color variation in a GWAS analysis across 1,694 diverse sorghum inbreds. A premature stop codon in a NAC gene is the most likely candidate polymorphism underlying the sorghum D locus. More detailed understanding of the sorghum D locus could help improve agronomic potential in cereals.

Pinhão seed is an unconventional source of starch and the pines grow up in native forests of southern Latin America. In this study, pinhão starch was adjusted at 15, 20 and 25% moisture content and heated to 100, 110 and 120°C for 1 h. A decrease in λ max (starch/iodine complex) was observed as a result of increase in temperature and moisture content of HMT. The ratio of crystalline to amorphous phase in pinhão starch was determined via Fourier transform infra red by taking 1045/1022 band ratio. A decrease in crystallinity occurred as a result of HMT. Polarised light microscopy indicated a loss of birefringence of starch granules under 120°C at 25% moisture content. Granule size distribution was further confirmed via scanning electron microscopy which showed the HMT effects. These results increased the understanding on molecular and structural properties of HMT pinhão starch and broadened its food and nonfood industrial applications.

Research on prebiotics and other novel health-promoting food components has been active for over a decade. Arabinoxylan (AX) derived arabinoxylooligosaccharides (AXOS), which may have various chemical structures, depending on the xylan source and the degradation method used, stand increasingly in the spotlight as potential prebiotics. During the past decade, the studies of the possibilities to produce the AXOS by using biocatalytic conversion have received more attention. In addition, there is an interest in the use of novel cereal biomass for the production of AXOS. The aim of this study was to investigate the influence of various commercial enzyme preparations on the degradability of insoluble arabinoxylans in wheat and rye wholemeal treated by extrusion, identify and quantify xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) in treated media. The enzymatic degradation of rye and wheat cell wall materials was monitored by HPSEC, HPAEC and MALDI-TOF-MS techniques. It was noticed that there is no significant difference between extruded and natural cereals, and type of cereals had not significant influence on XOS and AXOS production. The most effective biocatalysts were hemicellulases expressed in the enzyme preparations from Trichoderma and Aspergillus spp. (Depol 692), Humicola and Bacillus spp. (Ceremix Plus). Degradability of rye and wheat cell wall materials by these enzyme preparations obtained break down percentages of 70–87% and 67–77%, respectively. After enzymatic treatment, only small amounts of xylose, xylobiose, and xylotriose was eluted compare to the amount of more complex oligosaccharides with higher degree of polymerization (DP). The mass spectra of oligosaccharides indicated the presence of a homologous series of pentoses ranging from DP 4 to 15. This indicates that chosen enzyme preparations acted well on wheat and rye biomass, and released quite high amounts of XOS and AXOS.

New reactive unsaturated starch derivatives, 1-allyloxy-2-hydroxy-propyl-starches (AHP-starches), were synthesized by the reaction of waxy maize starch (WMS) and amylose-enriched maize starch (AEMS) with allyl glycidyl ether in a heterogeneous alkaline suspension containing NaOH and Na₂SO₄. The degree of substitution (DS) was determined by ¹H NMR spectroscopy, and a DS of 0.20 ± 0.01 was found for both AHP-WMS and AHP-AEMS, respectively. The AHP derivatives of WMS and AEMS were further characterized with ¹H and ¹³C NMR. It was shown that the AHP substitution was located on the C-6 hydroxyl group of the glucose residues in the starch. The substitution pattern of the AHP groups along the polymer chain was randomly clustered, as determined by enzymatic digestion using pullulanase, α-amylase, and amyloglucosidase, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the digestion products. With X-ray diffraction and scanning electron microscopy, no changes in the granular morphology and crystallinity between the unmodified starches and AHP-starches were detected.

Starches from cowpea and chickpea seeds were isolated and their properties were compared with those of commercial yellow pea starch. Amylose contents were 25.8%, 27.2%, and 31.2%, and the volume mean diameter of granules, determined in the dry state, were 15.5, 17.9, and 33.8 µm for cowpea, chickpea and yellow pea starches, respectively. All three legume starches showed a C-type X-ray diffraction pattern and two-stage swelling pattern. Amylopectin populations were isolated and the unit chain profiles were analyzed by HPLC after debranching with pullulanase. The degree of polymerization (DP) of short chain populations was about 6–50 and the populations of long chain had a DP of 50–80. Cowpea showed a lower weight ratio of short:long chains than chickpea and yellow pea starches. The larger portion of long side chains in cowpea amylopectin can be correlated with a higher gelatinization temperature, greater pasting peak and a slight difference in crystalline structure found for cowpea starch. Chickpea and yellow pea starches exhibited similarity in unit chain profile of amylopectin as well as in gelatinization temperature and pasting profile, while they differed in amylose content, particle size and syneresis. It is assumed that the chain length distribution of amylopectin has a large influence on starch properties.