Assessing Starch Retrogradation from the Perspective of Particle Order.

Starch retrogradation is a complex process involving changes in the multi-scale structure. In particular, the particle order of retrograded starch is unclear. In this study, we measured the radius of gyration (Rg) and radius of particles (R) of retrograded starch using small-angle X-ray scattering. Retrograded starch included various Rg, and the values of Rg depended on the length and state of the starch chains. With time, the standard deviations of R decreased due to the increase in particle uniformity. Based on these results, a new method for assessing the degree of starch retrogradation was established from the perspective of the particle order. The accuracy of the new method was verified through differential scanning calorimetry and scanning electron microscopy. The microstructures of the samples indicated that the retrograded starch granules contained substructures (primary particles) of different sizes. This study provides a new perspective for analyzing the structure of retrograded starch.

Improvement of resistant starch content and thermal-stability of starch-linoleic acid complex: An attempt application in extruded recombinant rice.

The utilization of resistant starch in food industry is restricted due to its susceptibility to thermal degradation. This work aimed to address this issue by preparing a starch-linoleic acid complex (RS5) via extrusion method combined with heat moisture treatment, obtaining VII-type crystal (melting temperature âˆ¼110 °C). The complex obtained through an 8-hour heat moisture treatment exhibited a high RS content of 46.7 %. The glycemic index (pGI) values predicted by two different methods for this complex were 54.5 and 64.2. The complex was further processed into recombinant rice, which exhibited similar textural properties to commercial rice products after cooking. Notably, the recombinant rice maintained an anti-enzyme structure (VII-type complex) as evidenced by its significant resistant starch content of 38.1 %, the lowest pGI values of 59.6 and 72.5. These findings could serve as a useful reference to aid in developing low glycemic index foods based on starch.

Structural changes of thermally treated starch during digestion and the impact on postprandial glucose homeostasis.

Intake of foods upon thermal treatment is typically associated with an elevated postprandial glycemic response, which is one of the risk factors for type 2 diabetes development and progression. In this study, rice starch was thermally treated using aqueous phase (boil), air phase (bake), and lipid phase (fry). Peak blood glucose levels in C57 mice increased by 16.94 %, 12.60 %, and 8.1 % after ingestion of thermally treated starch (20.23, 19.48, and 18.70 mmol/L), compared with raw starch (17.30 mmol/L). The insulin response to the intake of thermally treated starch increased (4.73 %-6.83 % higher than the control), whereas the concentration of GLP-1, a hormone used to promote insulin secretion, decreased (1.54 %-8.56 % lower than the control). Furthermore, thermally treated starch accelerated food absorption by enhancing gastrointestinal digestion, exacerbating postprandial glucose fluctuation at the next meal. Structural characterization showed thermal treatment reduced starch branching density and degree of structure order, which were not conducive to preventing the attack of enzymes. During digestion, they were highly hydrolyzed into low-molecular-weight fragments, and the proportion of ultrashort chains substantially increased. These findings provide a better understanding of the fine structure of starch that promotes hypoglycemia and initially explain how diets high in thermally treated starch impair glucose balance.

Simultaneous improvement of the physical and biological properties of starch films by incorporating steviol glycoside-based solid dispersion.

Bioactive compounds are frequently incorporated into polysaccharides (e.g., starch) to form active biodegradable films for food packaging, but some of them are water insoluble (e.g., curcumin, CUR) that will make the films have undesirable performance. Herein, CUR was successfully solubilized into the aqueous starch film solution by steviol glycoside (STE, a natural sweetener)-based solid dispersion. The mechanisms of solubilization and film formation were explored through molecular dynamic simulation and various characterization methods. The results showed that the amorphous state of CUR combined with micellar encapsulation of STE achieved the solubilization of CUR. STE and starch chains cooperated to form the film via hydrogen bonding, while CUR was uniformly and densely distributed within the film in the form of needle-like microcrystals. The as-prepared film exhibited high flexibility, great moisture barrier, and excellent UV barrier (UV transmittance: ∼0 %). Compared with the film containing CUR alone, the as-prepared film possessed higher release efficiency, antibacterial activity, and pH response sensitivity due to the assistance of STE. Hence, the introduction of STE-based solid dispersion can simultaneously improve the biological and physical properties of starch films, which provides a green, nontoxic, and facile strategy for the perfect integration of hydrophobic bioactive compounds and polysaccharide-based films.

Construction of TiO2/starch nanocomposite cryogel for ethylene removal and banana preservation.

Perishability caused by natural plant hormone ethylene has attracted great attention in the field of fruit and vegetable (F&V) preservation. Various physical and chemical methods have been applied to remove ethylene, but the eco-unfriendliness and toxicity of these methods limit their application. Herein, a novel starch-based ethylene scavenger was developed by introducing TiO2 nanoparticles into starch cryogel and applying ultrasonic treatment to further improve ethylene removal efficiency. As a porous carrier, the pore wall of cryogel provided dispersion space, which increased the area of TiO2 exposed to UV light, thereby endowing starch cryogel with ethylene removal capacity. The photocatalytic performance of scavenger reached the maximum ethylene degradation efficiency of 89.60 % when the TiO2 loading was 3 %. Ultrasonic treatment interrupted starch molecular chains and then promoted their rearrangement, increasing the material specific surface area from 54.6 m2/g to 225.15 m2/g and improving the ethylene degradation efficiency by 63.23 % compared with the non-sonicated cryogel. Furthermore, the scavenger exhibits good practicability for removing ethylene as a banana package. This work provides a new carbohydrate-based ethylene scavenger, utilizing as a non-food contact inner filler of F&V packaging in practical applications, which exhibits great potential in F&V preservation and broadens the application fields of starch.

Structure, thermal stability, and in vitro digestibility of rice starch-protein hydrolysate complexes prepared using different hydrothermal treatments.

In this study, rice starch-protein hydrolysate (WPH-S) complexes with high resistant starch (RS) content were prepared by heat-moisture treatment (HMT) and annealing (ANN). The effects of different hydrothermal treatments on the structure and thermal stability of the WPH-S complexes and their relationship with starch digestibility were further discussed. The results showed that RS contents of ANN-WPH-S complexes (35.09-40.26 g/100 g) were higher than that of HMT-WPH-S complexes (24.15-38.74 g/100 g). Under hydrothermal treatments, WPH decreased the hydrolysis kinetic constant (k) of starch form 4.07 × 10-2-4.63 × 10-2 min-1 to 3.29 × 10-2-3.67 × 10-2 min-1. HMT and ANN promoted hydrogen bonding between WPH and starch molecules, thus increasing the molecular size of starch. In addition, the shear stability of WPH-S mixture was improved with the hysteresis loop area decreased after HMT/ANN treatments, resulting in a more stable structure. Most importantly, the hydrothermal treatment made the scatterers of WPH-S complexes denser and the surface smoother. Especially after ANN treatment, the WPH60-S complex formed a denser aggregate structure, which hindered the in vitro digestion of starch to a certain extent. These results enrich our understanding of the regulation of starch digestion by protein hydrolysates under different hydrothermal treatments and have guiding significance for the development of foods with a low glycemic index.

Starch digestion retarded by wheat protein hydrolysates with different degrees of hydrolysis.

Wheat protein hydrolysates (WPH) were prepared by pepsin hydrolysis for 30, 60, and 120 min (WPH30, WPH60, and WPH120). The mixed system of rice starch and WPH was hydrothermally treated to explore the effect of WPH with different degrees of hydrolysis on starch digestion. WPH reduced the first-order rate coefficient (k) of starch digestion. Especially, WPH30 reduced the k value the most and formed the highest slowly digestible starch content due to the entanglement of starch chains and long-chain peptides. WPH60 and WPH120 with more hydrophobic peptides and polar amino acids than WPH30 tended to form hydrogen bonds with starch molecules due to less steric hindrance. Particularly, the complexation of WPH60 promoted the formation of dense aggregate structure and hindered the enzymatic hydrolysis of starch to a certain extent, thereby increasing the resistant starch content. These findings provide significant guidance for the preparation of hypoglycemic reformed food.

Mechanism of peptides from rice hydrolyzed proteins hindering starch digestion subjected to hydrothermal treatment.

Clarifying the interactions between food components is critical in designing carbohydrate-based foods with low digestibility. To date, the hindering effect of starch-protein interactions on starch digestion has attracted extensive attention. In this study, rice proteins were further hydrolyzed, and rice peptides (RP) with different molecular weights were obtained by ultrafiltration. The effects and possible mechanisms of RP with different molecular weights on the structure, thermal properties, and in vitro digestibility of cooked rice starch were investigated. All peptides slowed the digestion of rice starch in a concentration-dependent manner. A concentration of 10% RP>10 decreased the rapidly digestible starch content from 68.02 to 45.90 g/100 g, and increased the resistant starch content from 17.54 to 36.54 g/100 g. The addition of RP improved the thermal stability of the starch and reduced the amount of leached amylose. Infrared analysis shows that strong hydrogen bonds formed between RP (especially RP>10) and starch during co-gelatinization. In addition, RP improved the compactness of aggregated structure and played an important role in hindering the enzymatic hydrolysis of starch. These results enrich the theory of starch-protein interactions and have important implications for the development of carbohydrate-based foods with low digestibility and protein functional foods.

Immobilization of D-allulose 3-epimerase into magnetic metal-organic framework nanoparticles for efficient biocatalysis.

D-allulose is a rare low-calorie sugar that has many fundamental biological functions. D-allulose 3-epimerase from Agrobacterium tumefaciens (AT-DAEase) catalyzes the conversion of D-fructose to D-allulose. The enzyme has attracted considerable attention because of its mild catalytic properties. However, the bioconversion efficiency and reusability of AT-DAEase limit its industrial application. Magnetic metal-organic frameworks (MOFs) have uniform pore sizes and large surface areas and can facilitate mass transport and enhance the capacity for enzyme immobilization. Here, we successfully encapsulated cobalt-type AT-DAEase into the cobalt-based magnetic MOF ZIF-67@Fe3O4 using a self-assembly strategy. We confirmed the immobilization of enzyme AT-DAEase and characterized the enzymatic properties of the MOF-immobilized AT-DAEase@ZIF-67@Fe3O4. The AT-DAEase@ZIF-67@Fe3O4 nanoparticles had higher catalytic activity (65.1 U mg-1) and bioconversion ratio (38.1%) than the free AT-DAEase. The optimal conditions for maximum enzyme activity of the AT-DAEase@ZIF-67@Fe3O4 nanoparticles were 55 °C and pH 8.0, which were significantly higher than those of the free AT-DAEase (50 °C and pH 7.5). The AT-DAEase@ZIF-67@Fe3O4 nanoparticles displayed significantly improved thermal stability and excellent recycling performance, with 80% retention of enzyme activity at a temperature range of 45-70 °C and > 45% of its initial activity after eight cycles of enzyme use. The AT-DAEase@ZIF-67@Fe3O4 nanoparticles have great potential for large-scale industrial preparation of D-allulose by immobilizing cobalt-type AT-DAEase into magnetic MOF ZIF-67@Fe3O4.

Superhydrophobic modular cryogel with variable magnetic-actuated motion direction for discrete small-scale oil spill cleanup.

Smart superhydrophobic sorbents are in high demand for cleaning oil spills that could endanger the aquatic ecosystem. Herein, we demonstrated the fabrication of a superhydrophobic and magnetic modular cryogel (SNS@Fe-PSC) containing three starch-based modules, namely, a superhydrophobic nano-coating, a magnetic nanocomposite insertion, and a high-strength starch/polyvinyl alcohol composite substrate. The surface chemical composition and hierarchical micro/nanostructures of this material were investigated in detail. The modular cryogel had a high water contact angle (>151°) and low sliding angle (<9°), as well as excellent water-repellent, self-cleaning, and anti-fouling properties. This material also exhibited good durability owing to its stable chemical bonding and structural support. SNS@Fe-PSC could be applied to remove oil from water effectively. Moreover, the magnetic module (saturation magnetization, 5.04 emu/g) allowed the as-obtained material to be propelled and controlled by a magnet on the surface of water. Variable magnetic-actuated motion direction could be realized by adjusting the position and amount of magnetic modules inserted to the cryogel.

Preparation and characterization of non-crystalline granular starch with low processing viscosity.

Non-crystalline granular starch (NCGS) has advantages in the deep processing of starch owing to its unique structure and function. In this study, NCGS was successfully prepared at a baking temperature of 210 °C, and the morphology, structure, pasting properties, and rheological properties of the NCGS were systematically studied. Compared with native starch, NCGS showed a lower processing viscosity and rapid reduction in the peak viscosity from 3795 to 147 cP. Furthermore, NCGS exhibited impaired short- and long-range ordered structures, as indicated by the lower ratio of absorbance at 1047/1015 cm-1 and decreased crystallinity compared to native starch. Additionally, amylose and amylopectin with long and medium chains in NCGS were degraded into short chains, resulting in an increase in amylose content and branch density. The analysis of the physicochemical properties of NCGS, especially the low processing viscosity, is of great importance for the industrial application of starch, particularly in terms of improving the yield, saving energy, and reducing environmental pollution.

PlantDeepSEA, a deep learning-based web service to predict the regulatory effects of genomic variants in plants.

Characterizing regulatory effects of genomic variants in plants remains a challenge. Although several tools based on deep-learning models and large-scale chromatin-profiling data have been available to predict regulatory elements and variant effects, no dedicated tools or web services have been reported in plants. Here, we present PlantDeepSEA as a deep learning-based web service to predict regulatory effects of genomic variants in multiple tissues of six plant species (including four crops). PlantDeepSEA provides two main functions. One is called Variant Effector, which aims to predict the effects of sequence variants on chromatin accessibility. Another is Sequence Profiler, a utility that performs 'in silico saturated mutagenesis' analysis to discover high-impact sites (e.g., cis-regulatory elements) within a sequence. When validated on independent test sets, the area under receiver operating characteristic curve of deep learning models in PlantDeepSEA ranges from 0.93 to 0.99. We demonstrate the usability of the web service with two examples. PlantDeepSEA could help to prioritize regulatory causal variants and might improve our understanding of their mechanisms of action in different tissues in plants. PlantDeepSEA is available at http://plantdeepsea.ncpgr.cn/.

Effect of cooling rate on long-term recrystallized crystal of rice starch in the presence of flavor compounds.

This study evaluated the effect of cooling rate on starch recrystallization in the presence of 2,3-butanedione and 2-acetyl-1-pyrroline, which could form B-type and V-type complexes with starch, respectively. Rapid cooling resulted in poor perfection and high heterogeneity of both B-type and V-type recrystallized crystal. For B-type crystal, rapid cooling changed nucleation mode from instantaneous (Avrami index n < 1) to continuous mechanism (1 ≤ n ≤ 2), and decreased recrystallization rate from 0.0502 to 0.0160 d-n, indicating the increased retention of starch on 2,3-butanedione. V-type crystal was formed at initial stages of recrystallization, and inhibited the growth of B-type crystal. The loose crystalline obtained by rapid cooling is conducive to the retention of flavor compounds for B-type complexes (especially ≤14 days) and V-type complexes (especially ≤1 day). These results could provide guidance for maintaining fragrance of instant rice during long-term storage.

Characterization and application of a putative transcription factor (SUT2) in Pichia pastoris.

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the - 973 base pair (bp) to - 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, "A" at the 22nd bp upstream of ATG, was determined with 5'-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system.

Effects of starchy seed crystals on the retrogradation of rice starch.

This study aimed at exploring the effects of exogenous seed crystals on the starch recrystallization. A-type and B-type seed crystals from rice starch were added to rice starch paste during cooling to analyze the changes in retrogradation process. DSC results indicated that B-type seed improved the uniformity of crystal size and the degree of crystal perfection; it also affected the nucleation mode of retrograded starches at 4 °C, while A-type seed did not affect these properties. The XRD showed that the unit cell was compact, and the crystal grain became larger upon the introduction of B-type seed, while A-type seemed to loosen the cell. The SAXS reflected that B-type seed increased the thickness of the imperfect lamellar structure formed during recrystallization, while A-type seed decreased it. These findings might be due to the molecular segment arrangement on the nuclei growth surface affecting the arrangement of starch molecular segments approaching the nuclei.

Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology.

A highly efficient and targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system was constructed for Pichia pastoris (syn Komagataella phaffii). Plasmids containing single guide RNA and the methanol expression regulator 1 (MXR1) homology arms were used to precisely edit the transcriptional activator Mxr1 on the P. pastoris genome. At the S215 amino acid position of Mxr1, one, two, and three nucleotides were precisely deleted or inserted, and S215 was also mutated to S215A via a single-base substitution. Sequencing of polymerase chain reaction (PCR) amplicons in the region spanning MXR1 showed that CRISPR/Cas9 technology enabled efficient and precise gene editing of P. pastoris. The expression levels of several of the Mxr1-targeted genes, AOX1, AOX2, DAS1, and DAS2, in strains containing the various mutated variants of MXR1, were then detected through reverse transcription PCR following induction in methanol-containing culture medium. The frameshift mutations of Mxr1 led to almost zero transcription of AOX1, DAS1, and DAS2, while that of AOX2 was reduced to 60%. For the Mxr1 S215A mutant, the transcription of AOX1, AOX2, DAS1, and DAS2 was also reduced by nearly 60%. Based on these results, it is apparent that the transcription of AOX1, DAS1, and DAS2 is exclusively regulated by Mxr1 and serine phosphorylation at Mxr1 residue 215 is not critical for this function. In contrast, the transcription of AOX2 is mainly dependent on the phosphorylation of this residue. CRISPR/Cas9 technology was, therefore, successfully applied to the targeted editing of MXR1 on the P. pastoris genome, and it provided an effective method for the study of this transcription factor and its targets.

Pasting, rheology, and fine structure of starch for waxy rice powder with high-temperature baking.

The pasting and rheological properties of waxy rice powder (WRP) with high-temperature baking (140-180 °C) and the fine structure of its starch were investigated. Rapid visco-analysis showed that the viscosity and breakdown value of baked WRP decreased obviously compared to its native counterpart, and this trend was greatly increased under higher temperature. Rheological analysis indicated that the baked WRP had relatively more elastic component compared to viscosity component but was less thixotropic than native WRP, suggesting the swelling of starch granules and the leaching of starch molecules were retarded when the WRP was baked at high temperature. Additionally, WRP was severely degraded and further re-associated and interacted with other granules during high-temperature baking. Changes in starch fine structure contributed to the alteration of pasting and rheological properties of WRP. These findings suggest that high-temperature baking induced structural changes of starch and thus reduced the viscosity of WRP.

[Effect of N-terminal truncation of Bacillus acidopullulyticus pullulanase on enzyme properties and functions].

We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT.

Highly branched dextrin prepared from high-amylose maize starch using waxy rice branching enzyme (WRBE).

Branching enzyme (BE, EC 2.4.1.18) was isolated from the developing waxy rice endosperm and used to prepare a highly branched dextrin based on high-amylose maize starch (HAMS) as a substrate. The molecular mass of the starch initially degraded quickly from 2.5 × 10(7) to 4.1 × 10(5)Da, and then stabilized, with a minimal increase during the BE treatment. The resultant branched dextrin had a narrow size distribution, with a mean molecular weight of 5.1 × 10(5)Da and a polydispersity index (PI) of 1.567. The results of high-performance anion exchange chromatography indicated that the degree of polymerization (DP) of the branched chains ranged from 3 to 27; approximately 75.26% of these chains were short (DP<10). These findings suggest that the isolated BE can cleave long chains into oligosaccharides, subsequently transferring oligosaccharides into highly branched dextrins with a narrow size distribution and short side chains.

Breast cancer intrinsic subtype classification, clinical use and future trends.

Breast cancer is composed of multiple subtypes with distinct morphologies and clinical implications. The advent of microarrays has led to a new paradigm in deciphering breast cancer heterogeneity, based on which the intrinsic subtyping system using prognostic multigene classifiers was developed. Subtypes identified using different gene panels, though overlap to a great extent, do not completely converge, and the avail of new information and perspectives has led to the emergence of novel subtypes, which complicate our understanding towards breast tumor heterogeneity. This review explores and summarizes the existing intrinsic subtypes, patient clinical features and management, commercial signature panels, as well as various information used for tumor classification. Two trends are pointed out in the end on breast cancer subtyping, i.e., either diverging to more refined groups or converging to the major subtypes. This review improves our understandings towards breast cancer intrinsic classification, current status on clinical application, and future trends.