Recent advances on bioactive compounds, biosynthesis mechanism, and physiological functions of Nelumbo nucifera.

Nelumbo nucifera Gaertn, commonly known as lotus, is a genus comprising perennial and rhizomatous aquatic plants, found throughout Asia and Australia. This review aimed to cover the biosynthesis of flavonoids, alkaloids, and lipids in plants and their types in different parts of lotus. This review also examined the physiological functions of bioactive compounds in lotus and the extracts from different organs of the lotus plant. The structures and identities of flavonoids, alkaloids, and lipids in different parts of lotus as well as their biosynthesis were illustrated and updated. In the traditional medicine systems and previous scientific studies, bioactive compounds and the extracts of lotus have been applied for treating inflammation, cancer, liver disease, Alzheimer's disease, etc. We suggest future studies to be focused on standardization of the extract of lotus, and their pharmacological mechanisms as drugs or functional foods. This review is important for the lotus-based food processing and application.

Amylose/cellulose nanofiber composites for all-natural, fully biodegradable and flexible bioplastics.

Thermoplastic, polysaccharide-based plastics are environmentally friendly. However, typical shortcomings include lack of water resistance and poor mechanical properties. Nanocomposite manufacturing using pure, highly linear, polysaccharides can overcome such limitations. Cast nanocomposites were fabricated with plant engineered pure amylose (AM), produced in bulk quantity in transgenic barley grain, and cellulose nanofibers (CNF), extracted from agrowaste sugar beet pulp. Morphology, crystallinity, chemical heterogeneity, mechanics, dynamic mechanical, gas and water permeability, and contact angle of the films were investigated. Blending CNF into the AM matrix significantly enhanced the crystallinity, mechanical properties and permeability, whereas glycerol increased elongation at break, mainly by plasticizing the AM. There was significant phase separation between AM and CNF. Dynamic plasticizing and anti-plasticizing effects of both CNF and glycerol were demonstrated by NMR demonstrating high molecular order, but also non-crystalline, and evenly distributed 20 nm-sized glycerol domains. This study demonstrates a new lead in functional polysaccharide-based bioplastic systems.

Generation of short-chained granular corn starch by maltogenic α-amylase and transglucosidase treatment.

We describe a method for permitting efficient modification by transglucosidase (TGA), from glycoside hydrolase family 31 (GH31), sequentially after the pre-treatment by maltogenic α-amylases (MA) from GH13. TGA treatment without MA pre-treatment had negligible effects on native starch, while TGA treatment with MA pre-treatment resulted in porous granules and increased permeability to enzymes. MA→TGA treatments lead to decreased molecular size of amylopectin molecules, increased α-1,6 branching, and increased amounts of amylopectin chains with the degree of polymerization (DP)<10 and decreased amounts of DP 10-28 after debranching. Wide-angle X-ray scattering (WAXS) data showed a general decrease in crystallinity except for a long term (20 h) TGA post-treatment which increased the relative crystallinity back to normal. MA→TGA treatment significantly lowered the starch retrogradation of starch and retarded the increase of storage- and loss moduli during storage. This work demonstrates the potential of sequential addition of starch active enzymes to obtain granular starch with improved functionality.

High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato.

CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato U6 promoters. Highly increased editing efficiencies in the Granular Bound Starch Synthase gene at the protoplast level were obtained by replacement of the Arabidopsis U6 promotor, driving expression of the CRISPR component, with endogenous potato U6 promotors. This translated at the ex-plant level into 35% full allelic gene editing. Indel Detection Amplicon Analysis was established as an efficient tool for fast assessment of gene editing in complex genomes, such as potato. Together, this warrants significant reduction of laborious cell culturing, ex-plant regeneration and screening procedures of plants with high complexity genomes.

Effect of potato fiber on survival of Lactobacillus species at simulated gastric conditions and composition of the gut microbiota in vitro.

Potato fiber is a side product in starch manufacturing rich in dietary fibers such as pectin, cellulose, hemicellulose and resistant starch. So far, the beneficial properties of potato fiber have been poorly characterized. This study investigated the effect of FiberBind 400, a commercial potato fiber product, on survival of probiotic Lactobacillus strains at simulated gastric conditions and on the composition and metabolic activity of the gut microbiota, using the TIM-2 colon model. Resistant starch and native starch from potato were used as reference substrates. FiberBind 400 had an ability to improve survival of the four tested strains, Lactobacillus fermentum PCC®, L. rhamnosus LGG®, L. reuteri RC-14® and L. paracasei F-19® in a strain-dependent way. The highest effect was observed for L. fermentum PCC® and L. rhamnosus LGG®. The effect of starches on bacterial survival was insignificant. Composition of the fecal microbiota in TIM-2 fermentations was assessed by high-throughput sequencing of 16S rRNA gene amplicon. Fermentation of FiberBind 400 resulted in more diverse microbial communities compared to starches. Changes in microbial abundances specifically mediated by FiberBind 400, included increases in the genera Lachnospira, Butyrivibrio, Mogibacterium, Parabacteroides, Prevotella and Desulfovibrio, and the species B. ovatus, as well as decreases in Ruminococcus torques and unassigned Ruminococcus spp. Shifts in other bacterial populations, such as increased abundances of Oscillospira, Enterococcus, Bacteroidales, Citrobacter, along with reduction of Roseburia, Ruminococcus, and Faecalibacterium prausnitzii were not significantly different between the substrates. Cumulative production of individual short-chain fatty acids was similar between potato fiber and starches. The study demonstrated that FiberBind 400 had a potential to protect probiotic Lactobacillus strains during the passage through the gastrointestinal tract and selectively modulate the gut bacterial populations. This knowledge can support application of potato fiber as a functional food ingredient with added biological benefits.

Short-time microwave treatment affects the multi-scale structure and digestive properties of high-amylose maize starch.

Microwave processing is a suitable technology for starch-based food processing. This work investigated the changes of structures and properties of high-amylose maize starch (HAMS) during short-time microwave irradiation (1-4 min). After 1 min of treatment, short amylopectin chains (DP 6-36) and intermediate amylose chains (DP 150-2000) of HAMS were partially broken down. Compared with native HAMS, treated HAMS (1 min) had the higher relative crystallinity, the intensity of the 9 nm lamellar peak, and fluorescence intensity under CLSM. Moreover, 1-min microwaving caused the lower viscosity and higher resistant starch content of HAMS. In the 2-4 min of treatment, the crystallinity, intensity of the lamellar peak and fluorescence intensity of HAMS granules decreased significantly, but no breakdown of starch molecule chains was observed, suggesting the realignment of the crystalline region during the process. Correspondingly, the viscosity increased and resistant starch content decreased. Our study provides a deeper understanding of the mechanistic effects of short-time microwave irradiation on high-amylose starch, which is of value for the processing of HAMS to produce novel functionality and nutritional values.

The effect of pectins on survival of probiotic Lactobacillus spp. in gastrointestinal juices is related to their structure and physical properties.

Pectins are plant polysaccharides used in food industry as gelling and stabilizing agents. This study investigated the ability of pectins to improve survival of probiotic species Lactobacillus fermentum PCC, L. reuteri RC-14, L. rhamnosus LGG and L. paracasei F-19 in simulated gastric solution in relationship to their structural and physical properties. Electrostatic interactions between pectins and bacteria were evaluated by the Zeta-potential approach. Bacterial survival was assessed by flow cytometry and plate counting. L. fermentum PCC and L. reuteri RC-14 were more resistant to gastric conditions; their survival rate was further improved in the presence of five out of ten tested pectins. Additionally, two of the pectins had a positive effect on viability of the less resistant L. rhamnosus LGG and L. paracasei F-19. The beneficial effect was generally observed for the high-methoxylated pectins, indicating that substituted polygalacturonic acid in the backbone is essential for bacterial protection. Other pectin features associated with improved survival, included less negative Zeta-potential, higher molecular weight, as well as lower values of hydrodynamic sizes, viscosity and degree of branching. The study indicates that pectins have a potential to protect probiotic bacteria through the gastro-intestinal transit and identifies the features linked to their functionality.

Highly phosphorylated functionalized rice starch produced by transgenic rice expressing the potato GWD1 gene.

Starch phosphorylation occurs naturally during starch metabolism in the plant and is catalysed by glucan water dikinases (GWD1) and phosphoglucan water dikinase/glucan water dikinase 3 (PWD/GWD3). We generated six stable individual transgenic lines by over-expressing the potato GWD1 in rice. Transgenic rice grain starch had 9-fold higher 6-phospho (6-P) monoesters and double amounts of 3-phospho (3-P) monoesters, respectively, compared to control grain. The shape and topography of the transgenic starch granules were moderately altered including surface pores and less well defined edges. The gelatinization temperatures of both rice flour and extracted starch were significantly lower than those of the control and hence negatively correlated with the starch phosphate content. The 6-P content was positively correlated with amylose content and relatively long amylopectin chains with DP25-36, and the 3-P content was positively correlated with short chains of DP6-12. The starch pasting temperature, peak viscosity and the breakdown were lower but the setback was higher for transgenic rice flour. The 6-P content was negatively correlated with texture adhesiveness but positively correlated with the cohesiveness of rice flour gels. Our data demonstrate a way forward to employ a starch bioengineering approach for clean modification of starch, opening up completely new applications for rice starch.

Influence of amylopectin structure and degree of phosphorylation on the molecular composition of potato starch lintners.

Morphology, molecular structure, and thermal properties of potato starch granules with low to high phosphate content were studied as an effect of mild acid hydrolysis (lintnerization) to 80% solubilization at two temperatures (25 and 45°C). Light microscopy showed that the lintners contained apparently intact granules, which disintegrated into fragments upon dehydration. Transmission electron microscopy of rehydrated lintners revealed lacy networks of smaller subunits. The molecular composition of the lintners suggested that they largely consisted of remnants of crystalline lamellae. When lintnerization was performed at 45°C, the lintners contained more of branched dextrins compared to 25°C in both low and intermediate phosphate-containing samples. High-phosphate-containing starch was, however, unaffected by temperature and this was probably due to an altered amylopectin structure rather than the phosphate content. After lintnerization, the melting endotherms were broad with decreased onset and increased peak melting temperatures. The relative crystallinity was lower in lintners prepared at 45°C. A hypothesis that combines the kinetics of lintnerization with the molecular and thermal characteristics of the lintners is presented.

Structural and physical properties of granule stabilized starch obtained by branching enzyme treatment.

Chemical cross-linking of starch is an important modification used in the industry for granule stabilization. It has been demonstrated that treatment with branching enzyme (BE) can stabilize the granular structure of starch and such treatment thereby provides a potential clean alternative for chemical modification. This study demonstrates that such BE-assisted stabilization of starch granules led to partial protection from BE catalysis of both amylose (AM) and amylopectin (AP) in their native state as assessed by triiodide complexation, X-ray diffractometry (XRD) and differential scanning calorimetry (DSC). The granule stabilizing effects were inversely linked to hydration of the starch granules, which was increased by the presence of starch-phosphate esters and suppressed by extreme substrate concentration. The data support that the granule stabilization is due to the intermolecular transglycosylation occurring in the initial stages of the reaction prior to AM-AP phase separation. The enzyme activity needed to obtain granule stabilization was therefore dependent on the hydration capability of the starch used.

Hydration properties and phosphorous speciation in native, gelatinized and enzymatically modified potato starch analyzed by solid-state MAS NMR.

Hydration of granular, gelatinized and molecularly modified states of potato starch in terms of molecular mobility were analyzed by (13)C and (31)P solid-state MAS NMR. Gelatinization (GEL) tremendously reduced the immobile fraction compared to native (NA) starch granules. This effect was enhanced by enzyme-assisted catalytic branching with branching enzyme (BE) or combined BE and ß-amylase (BB) catalyzed exo-hydrolysis. Carbons of the glycosidic α-1,6 linkages required high hydration rates before adopting uniform chemical shifts indicating solid-state disorder and poor water accessibility. Comparative analysis of wheat and waxy maize starches demonstrated that starches were similar upon gelatinization independent of botanical origin and that the torsion angles of the glycosidic linkages were averages of the crystalline A and B type structures. In starch suspension phosphorous in immobile regions was only observed in NA starch. Moreover phosphorous was observed in a minor pH-insensitive form and as major phosphate in hydrated GEL and BE starches.

Effect of amylose deposition on potato tuber starch granule architecture and dynamics as studied by lintnerization.

The effect of amylose deposition on the amylopectin crystalline lamellar organization in potato starch granules was studied by mild acid, so-called lintnerization, of potato tuber starch transgenically engineered to deposit different levels of amylose. The starch granules were subjected to lintnerization at different temperatures (25, 35, and 45°C) and to two levels of solubilization, ∼ 45 and 80%. The rate of the lintnerization increased with temperature but was suppressed by amylose. The molecular size of the lintner dextrins increased with temperature, but this effect was suppressed by the presence of amylose. At high temperatures and low-amylose content, the degree of branches was high with the concomitant increase in size in the dextrins. A portion of the branches was resistant to debranching enzymes possibly due to specific structural formations. The effects of temperature suggested a unique granular architecture of potato starch, and a model showing the dependence of temperature on the dynamic arrangement of amylopectin and amylose in the crystalline and amorphous lamellae for the potato starch is suggested.

Characterisation of the arabinose-rich carbohydrate composition of immature and mature marama beans (Tylosema esculentum).

Marama bean (Tylosema esculentum) is an important component of the diet around the Kalahari Desert in Southern Africa where this drought resistant plant can grow. The marama bean contains roughly 1/3 proteins, 1/3 lipids and 1/3 carbohydrates, but despite its potential as dietary supplement little is known about the carbohydrate fraction. In this study the carbohydrate fraction of "immature" and "mature" marama seeds are characterised. The study shows that the marama bean contains negligible amounts of starch and soluble sugars, both far less than 1%. The cell wall is characterised by a high arabinose content and a high resistance to extraction as even a 6M NaOH extraction was insufficient to extract considerable amounts of the arabinose. The arabinose fraction was characterised by arabinan-like linkages and recognised by the arabinan antibody LM6 and LM13 indicating that it is pectic arabinan. Two pools of pectin could be detected; a regular CDTA (1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid) or enzymatically extractable pectin fraction and a recalcitrant pectin fraction containing the majority of the arabinans, of which about 40% was unextractable using 6M NaOH. Additionally, a high content of mannose was observed, possibly from mannosylated storage proteins.

Phosphate esters in amylopectin clusters of potato tuber starch.

Starch phosphate is important in starch metabolism and in order to deduce its location and structural effects in clusters and building blocks of amylopectin, these were isolated from a normal potato (WT) and two starches with antisense suppressed glucan water dikinase (asGWD) activity and starch branching enzyme (asSBE) activity possessing suppressed and increased phosphate contents, respectively. Neutral N-chains and phosphorylated P-chains of the amylopectin macromolecules were similar in WT and asGWD, whereas asSBE possessed considerably longer P-chains. Cluster ß-limit dextrins were isolated by α-amylase treatment and successive ß-amylolysis. Cluster sizes were generally smaller in asSBE. The building block composition of neutral N-clusters were very similar in WT and asGWD, while asSBE was different, containing less blocks with degree of polymerization (DP)>14. Phosphate content of the P-clusters of WT and asGWD was rather similar, while asSBE contained highly phosphorylated P-clusters with proportionally more P-chains and a low degree of branching. The average chain lengths of the P-clusters were, however, similar in all samples. Our data demonstrate only minor effect on the cluster structure in relation to phosphate deposition suggesting conserved reaction patterns of starch phosphorylation. Models are suggested to account for the principle structural and functional effects of starch phosphate esters.

Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism.

Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.

Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato.

Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the first detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking StDPE2. In addition, they contained a range of malto-oligosaccharides from maltose to maltoheptaose. Plants repressed in both isoforms had chlorotic leaves and did not grow as well as either the controls or lines where only one of the isoforms was repressed. Examination of photosynthetic parameters suggested that this was most likely due to a decrease in carbon assimilation. The subcellular localisation of StDPE2 was re-addressed in parallel with DPE2 from Arabidopsis thaliana by transient expression of yellow fluorescent protein fusions in tobacco. No translocation to the chloroplasts was observed for any of the fusion proteins, supporting a cytosolic role of the StDPE2 enzyme in leaf starch metabolism, as has been observed for Arabidopsis DPE2. It is concluded that StDPE1 and StDPE2 have individual essential roles in starch metabolism in potato and consequently repression of these disables regulation of leaf malto-oligosaccharides, starch content and photosynthetic activity and thereby plant growth possibly by a negative feedback mechanism.

The amyloplast proteome of potato tuber.

Potato (Solanum tuberosum) is the fourth largest crop worldwide in yield, and cv. Kuras is the major starch potato of northern Europe. Storage starch is packed densely in tuber amyloplasts, which become starch granules. Amyloplasts of soil-grown mini-tubers and agar-grown micro-tubers of cv. Kuras were purified. The mini-tuber amyloplast preparation was enriched 10-20-fold and the micro-tuber amyloplast approximately fivefold over comparative total protein extracts. Proteins separated by SDS-PAGE were digested with trypsin, analysed by mass spectrometry and identified by mascot software searches against an in-house potato protein database and the NCBI non-redundant plant database. The differential growth conditions for mini- and micro-tubers gave rise to rather different protein profiles, but the major starch granule-bound proteins were identical for both and dominated by granule-bound starch synthase I, starch synthase II and alpha-glucan water dikinase. Soluble proteins were dominated by starch phosphorylase L-1, other large proteins of the classes 'starch and sucrose metabolism', 'pentose phosphate pathway', 'glycolysis', 'amino acid metabolism', and other proteins such as plastid chaperonins. The majority of the identified proteins had a predicted plastid transit peptide, supporting their presence in the amyloplast. However, several highly expressed proteins had no transit peptide, such as starch phosphorylase H, or had a predicted mitochondrial location. Intriguingly, all polyphenol oxidases, a family of enolases, one transketolase, sulfite reductase, deoxynucleoside kinase-like and dihydroxy-acid dehydrase had twin-arginine translocation motifs, and a homologue to dihydrolipoamide dehydrogenase had a Sec (secretory) motif; these motifs usually target thylakoid-like structures.

Structural and thermodynamic properties of starches extracted from GBSS and GWD suppressed potato lines.

A combined DSC-SAXS approach was employed to study the effects of amylose and phosphate esters on the assembly structures of amylopectin in B-type polymorphic potato tuber starches. Amylose and phosphate levels in the starches were specifically engineered by antisense suppression of the granule bound starch synthase (GBSS) and the glucan water dikinase (GWD), respectively. Joint analysis of the SAXS and DSC data for the engineered starches revealed that the sizes of amylopectin clusters, thickness of crystalline lamellae and the polymorphous structure type remained unchanged. However, differences were found in the structural organization of amylopectin clusters reflected in localization of amylose within these supramolecular structures. Additionally, data for annealed starches shows that investigated potato starches possess different types of amylopectin defects. The relationship between structure of investigated potato starches and their thermodynamic properties was recognized.

A novel type carbohydrate-binding module identified in alpha-glucan, water dikinases is specific for regulated plastidial starch metabolism.

The phosphorylation of the amylopectin fraction of starch catalyzed by the alpha-glucan, water dikinase (GWD, EC 2.7.9.4) plays a pivotal role in starch metabolism. Limited proteolysis of the potato tuber (Solanum tuberosum) GWD (StGWD, 155 kDa) by trypsin primarily produced stable fragments of 33 and 122 kDa, termed the SBD fragment and N11, respectively, as generated by trypsin cleavage at Arg-286. SBD and N11 were generated using recombinant DNA technology and purified to near homogeneity. Tandem repeat sequences, SBD-1 and SBD-2, of a region that is significantly similar in sequence to N-terminal regions of plastidial alpha-amylases are located in the N-terminus of StGWD. The SBD-1 motif is located within the sequence of the SBD fragment, and our results demonstrate that the fragment composes a new and novel carbohydrate-binding module (CBM), apparently specific for plastidial alpha-glucan degradation. By mutational analyses of conserved Trp residues located within the SBD-1 motif, W62 and W117, we show that these aromatic residues are vital for carbohydrate binding. N11 still possessed starch phosphorylating activity, but with a 2-fold higher specific activity compared to that of wild type (WT) StGWD using potato starch as the glucan substrate, whereas it had double the K(m) value for the same substrate. Furthermore, investigation of the chains phosphorylated by WT StGWD and N11 shows that N11 exhibits a higher preference for phosphorylating shorter chains of the amylopectin molecule as compared to WT. From analyses of the glucan substrate specificity, we found up to 5-fold higher specific activity for N11 using amylose as the substrate.