Karyotype analysis of buckwheat using atomic force microscopy.

Karyotype analysis and classification of buckwheat chromosomes were performed without chemical banding or staining using atomic force microscopy (AFM). Fagopyrum esculentum (common buckwheat) and Fagopyrum tartaricum (Tartarian buckwheat) chromosomes were isolated from root tissues using an enzymatic maceration technique and spread over a glass substrate. Air-dried chromosomes had a surface with ridges, and the height of common and tartary buckwheat were approximately 350 and 150 nm. Volumes of metaphase sets of buckwheat chromosomes were calculated using three-dimensional AFM measurements. Chromosomes were morphologically characterized by the size, volume, arm lengths, and ratios. The calculated volumes of the F. esculentum and F. tartaricum chromosomes were in the ranges of 1.08-2.09 µm3 and 0.49-0.78 µm3, respectively. The parameters such as the relative arm length, centromere position, and the chromosome volumes measured using AFM provide accurate karyomorphological classification by avoiding the subjective inconsistencies in banding patterns of conventional methods. The karyotype evolutionary trend indicates that F. esculentum is an ancient species compared to F. tartaricum. This is the first report of a cytological karyotype of buckwheat using AFM.

Nano-structure of heat-moisture treated waxy and normal starches.

Surface regions of untreated and heat-moisture treated (HMT) normal rice, waxy rice, normal corn, waxy corn, normal potato, and waxy potato starch granules were examined by atomic force microscopy (AFM). AFM images revealed surface roughness of untreated starch granules and protrusions with a diameter of approximately 15-90 nm. After treatment, the smooth surface region on starch granules was observed, especially in normal rice, waxy rice, and normal corn starches. A significant reduction in the size of protrusions on the surface of HMT potato starch granules was also detected. The newly formed structures may act as barriers and retard water penetration into starch granules. The blocklet model of starch granule architecture was also confirmed by the AFM images.

Degradation of microcrystalline cellulose and non-pretreated plant biomass by a cell-free extracellular cellulase/hemicellulase system from the extreme thermophilic bacterium Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii is a cellulolytic/hemicellulolytic anaerobe, which extracellularly secretes various proteins, including multidomain cellulases with two-catalytic domains, for plant biomass degradation. Degradation by C. bescii cells has been well characterized, but degradation by the cell-free extracellular cellulase/hemicellulase system (CEC) of C. bescii has not been as well studied. In the present study, C. bescii CEC was prepared from cell-free culture supernatant, and the degradation properties for defined substrates and non-pretreated plant biomass were characterized. Four multidomain cellulases (Cbes_1857, Cbes_1859, Cbes_1865, and Cbes_1867), composed of the glycoside hydrolase families 5, 9, 10, 44, and 48, were the major enzymes identified in the CEC by mass spectrometry. The CEC degraded xylan, mannose-based substrates, ß-1,4-linked glucans, including microcrystalline cellulose (Avicel), and non-pretreated timothy grass and rice straw. However, degradation of chitin, pectin, dextran, and wheat starch was not observed. The optimum temperatures for degradation activities were 75°C for timothy grass and Avicel, 85°C for carboxylmethyl cellulose, and >85°C for xylan. The optimum pH for these substrates was 5-6. The degradation activities were compared with a CEC derived from the fungus Trichoderma reesei, the most common enzyme used for plant biomass saccharification. The amounts of degraded Avicel, timothy grass, and rice straw by C. bescii CEC were 2.2-2.4-fold larger than those of T. reesei CEC. The high hydrolytic activity of C. bescii CEC might be attributed to the two-catalytic domain architecture of the cellulases.

Effect of sectioning and water on resin-embedded sections of corn starch granules to analyze inner structure.

Resin-embedded sections and paired block surface of corn starch granules were observed using atomic force microscopy (AFM) and scanning electron microscopy to analyze the fine inner structure of starch granules and observe artifacts. Wrinkles were formed on the starch surfaces because of shear stress caused by the knife. Sectioned starches were isotropically expanded by water, and the growth rings and cracks between the growth rings were observed only on the sections. From this result, it was considered that the growth rings clearly showed shrinkage and/or corrosion of both edges of the ring during drying of the sections. Moreover, many small particles (width, ∼30 nm; height, several nanometers) were clearly observed on the growth rings. These particles could be single clusters (∼10 nm) of amylopectin molecules, considering the effect of the AFM tip radius.

Ultrastructural analysis of buckwheat starch components using atomic force microscopy.

UNLABELLED: Morphological and structural features of buckwheat starch granules and nanocrystals were examined using atomic force microscopy and dynamic light scattering. Partially digested starch granules revealed a clear pattern of growth rings with the central core revealing lamellar structure. Atomic force microscopy and dynamic light scattering experiments revealed that the buckwheat starch granules were polygonal in shape and were in the range of 2 to 19 µm in diameter. The optimized acid hydrolysis process produced nanocrystals with the shape of spherical structure with lengths ranging from 120 to 200 nm, and the diameter from 4 to 30 nm from aqueous suspensions of buckwheat starch solution. The sorption isotherms on buckwheat starch nanocrystal/glycerol composite exhibited a 3-stage transition of moisture in the blending. The biocompatible nature of buckwheat starch nanocrystals and their structural properties make them a promising green nanocomposite material. PRACTICAL APPLICATION: Buckwheat starches had never been studied on a nanoscale, but we have achieved new understanding of starch granule morphology and concentric growth rings using nanoscale imaging. Since buckwheat is an underutilized crop, we foresee the potential application of buckwheat starch, starch-based nanocrystals, and nanoparticles, to expand markets and encourage producers to expand their buckwheat acreage. The atomic force image analysis suggests that buckwheat starch could be used as a new biopolymer material in food industries.