Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

Oligochitosan inhibits Phytophthora capsici by penetrating the cell membrane and putative binding to intracellular targets

The mechanism of action of oligochitosan, which has shown great antifungal activity against Phytophthora capsiciin vitro, was studied using 2-aminoacridone-labeled oligochitosan (2-AMAC-oligochitosan) and a gel-retardation experiment. Internalization of 2-AMAC-oligochitosan in cysts, germtubes and sporangia of P. capsici was confirmed by confocal laser scan microscopy (CLSM), and the degree of uptake depended on the incubation concentration. 2-AMAC-oligochitosan localized mainly in the cytoplasm and showed no binding to both cell wall and cell membrane. Mannose, an inhibitor for oligochitosan uptake by macrophages, could not inhibit the internalization of oligochitosan in P. capsici. The gel-retardation experiment showed that oligochitosan bound strongly to DNA and RNA of P. capsici. These results indicate that oligochitosan exerts its antifungal activity by penetrating the cell membrane and putative binding to intracellular targets such as DNA and RNA.     

Histological investigation of stripe rust (Puccinia striiformis f.sp. tritici) development in resistant and susceptible wheat cultivars

The wheat cultivar Kariega expresses complete adult plant resistance against stripe rust, whereas cv. Avocet S is susceptible. Using confocal laser scanning microscopy, initial fungal penetration into flag leaves was identical in both cultivars, with directional germ-tube growth towards stomata that were penetrated without the formation of an appressorium, followed by differentiation of a substomatal vesicle, infection hyphae, haustorial mother cells and haustoria. During the following 4 days, further fungal development occurred more quickly in the resistant than in the susceptible cultivar. However, by 7 days postinoculation (dpi) the situation changed, with exponential growth of the pathogen occurring only in the susceptible line. Induced cellular lignification, a typical defence reaction of cereals, was observed at 4 dpi in the resistant cultivar, and 2 days later lignified tissue completely surrounded the fungal colonies. In the susceptible cultivar, isolated lignified host cells occurred at 6 dpi, and long, unbranched fungal hyphae outgrowing the resistance reaction were observed.     

抗菌肽CRAMP的安全性、稳定性及其在清除铜绿假单胞菌生物被膜中的作用

旨在考察鼠源抗菌肽CRAMP的安全性、稳定性及其在清除铜绿假单胞菌生物被膜中的作用。采用兔血红细胞悬液的溶血性和小鼠腹腔巨噬细胞(RAW264.7)的细胞毒性考察CRAMP的安全性;采用不同温度、蛋白酶、金属离子和酸碱梯度对CRAMP抗菌活性的影响,考察其稳定性;通过在6孔细胞培养板中构建铜绿假单胞菌(PAO1株)成熟生物被膜,以人源抗菌肽LL-37和3种抗菌药为对照,采用结晶紫染色法检测生物被膜量,平板菌落计数法对生物被膜活菌数进行计数,苯酚-硫酸法结合Folin-酚试剂法检测生物被膜胞外基质含量,激光扫描共聚焦显微镜(CLSM)观察CRAMP干预PAO1生物被膜形态学的变化。结果显示,CRAMP在所测试浓度下对兔血红细胞溶血率均<2%;在62.5~125 mg·L^-1时对 RAW264.7没有细胞毒性。温度(25~100 ℃)、两种盐离子(Na⁺、K⁺)以及pH为5~10时对CRAMP的抗菌活性没有影响;胃蛋白酶、木瓜蛋白酶、胰蛋白酶均不同程度影响了CRAMP的抗菌活性。6孔细胞培养板中,4倍MIC 的CRAMP极显著降低了PAO1生物被膜量(减少率为76.74%,P<0.01),极显著减少了生物被膜中的活菌数量(减少了1.2个CFU·mL^-1,P<0.01),效果优于LL-37和3种抗菌药。CRAMP组的胞外基质显著低于空白对照组和LL-37组(P<0.05)。CLSM结果显示：与对照组相比较,4MIC浓度下,CRAMP组的细菌总荧光强度(PI+SYTO)显著低于空白对照组和LL-37组(P<0.05);与空白对照组相比,CRAMP组和LL-37组的死菌荧光强度/活菌荧光强度比值 (PI/SYTO)均极显著增大(P<0.01)。综上表明,CRAMP具有低溶血性、低细胞毒性,除蛋白酶作用不稳定外,具有较好的稳定性,对PAO1成熟生物被膜具有明显的清除作用,且效果优于LL-37,具有良好的药物开发前景。     

小麦矮腥黑粉菌侵染小麦子房的激光共聚焦显微观察方法

本试验利用碘化丙啶(propidium iodide)和Alexa Flour 488(AF 488)标记的麦胚凝集素(WGA)分别对小麦子房细胞和小麦矮腥黑粉菌进行染色,结合激光共聚焦显微镜成像系统获取小麦子房的三维立体图像。该技术可获得清晰的小麦子房细胞图像,并观察小麦矮腥黑粉菌在小麦子房中的侵染状况。该方法将为研究病原菌在寄主体内的分布提供可参考依据。     

荧光指示剂孵育法测定小麦叶肉细胞原生质体胞质游离Ca2+的变化

研究以孵育法将Ca^2 荧光探针Fluo-3AM载人小麦叶肉细胞原生质体胞质中,并结合激光共聚焦扫描显微技术,建立了测定原生质体胞质游离Ca^2 动态变化的试验方法。     

Effects of drought on the morphological and physicochemical characteristics of starch granules in different elite wheat varieties

In this study, scanning electron microscopy (SEM) revealed the formation of pits and pores on the surfaces of starch granules in response to drought stress, with substantially more pronounced effects in the ordinary yield potential wheat cv. Xindong 23 than the excellent yield potential wheat cv. Xindong 20. Drought induced a significant reduction in starch granule sizes in both wheat varieties, though the reduction observed in Xindong 23 was six times more pronounced than that observed for Xindong 20. Amyloglucosidase and α-amylase treatment of starch from wheat grown in drought conditions released significantly more reducing sugars compared with samples from irrigated controls. SEM and confocal laser scanning microscopy (CLSM) revealed that starch granules from the two wheat varieties grown under drought conditions had substantially increased fluorescence after treatment with proteolytic enzymes and staining with methanolic merbromin and 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde dyes. Analysis of pasting properties showed significant increases of peak viscosity, trough viscosity, break down, and setback following drought stresses. Furthermore, drought induced a significant reduction in the water binding capacity and increased damage to starch only in Xindong 23. These results provide insight into the potential mechanisms through which drought influences the ultrastructures and physicochemical properties of starch in wheat.     

Effect of curdlan on the quality of frozen-cooked noodles during frozen storage

Frozen-cooked noodles (FCN) and its components undergo quality changes during frozen storage, such as reduced textural and cooking qualities, weakened gluten network, and damaged starch properties; thus, storage condition is a critical factor affecting the final quality of FCN. In this study, in view of the thermoirreversible high-strength gel property (at ≥ 80 °C) of curdlan, strong hydrophilicity, and freeze-thawed stability of high-strength gel prepared from curdlan powder, the effect of curdlan on the quality of FCN during frozen storage was first evaluated. The results showed that curdlan was effective in reducing cooking loss, enhancing water absorption, and improving textural properties of FCN; the improving effect presented a trend of first increasing and then decreasing with the amount of increasing curdlan (0.1%–0.9%); and the addition of 0.5% curdlan was most effective in improving the quality of FCN. Thermal gravimetric analysis indicated that curdlan enhanced thermal stability of FCN, implying curdlan could strengthen the gluten network. Meanwhile, structural observations revealed that, during frozen storage, FCN with added curdlan exhibited a more continuous and compact gluten network accompanied with more uniform and smaller ice crystals. Thus, curdlan is desirable to be used as a novel gum in FCN to provide specific functionality and minimize the negative effect of frozen storage. This study provides new insights into the quality improvement of FCN and further expands the application potential of curdlan in food industry.     

Modification of protein structure and dough rheological properties of wheat flour through superheated steam treatment

Native (NF, 13.5% w.b) and moistened (MF, 27% w.b) wheat flours were treated with superheated steam (SS) at 170 °C for 1, 2 and 4 min, and their protein structure as well as dough rheological properties were analyzed. Confocal laser scanning microscopy (CLSM) and SDS-PAGE patterns indicated the formation of protein aggregates with reduced SDS extractability after treatment. Farinograph and dynamic rheometry measurements showed that the strength as well as elastic and viscous moduli of the dough made from SS-treated flours progressively increased with SS treatment time. And both the improvements were more pronounced for superheated steam-treated moistened flours (SS-MF) than for superheated steam-treated native flours (SS-NF). Size-exclusion high performance liquid chromatography (SE-HPLC) analysis demonstrated that dough rheological parameters have positive correlations with SDS unextractable polymeric proteins (UPP) contents. SS treatment on flours led to a transition of protein secondary structures to more ordered form (α-helix and β-sheet). Additionally, free sulfhydryl (SH) contents decreased after treatment, which implied that disulfide bonds accounted for protein extractability loss and dough rheological properties improvement. Elevated moisture level promoted the modification of both protein structure and dough behaviors of flours during SS treatment.     

Rheological properties and baking performance of rye dough as affected by transglutaminase

Since protein aggregation and formation of a continuous protein matrix in rye dough is very limited, an enzyme-induced protein aggregation method to improve the baking properties was investigated. The effects of microbial transglutaminase (TG) on the properties of rye dough were studied by rheological tests, confocal laser scanning microscopy (CSLM), standard-scale baking tests and crumb texture profile analysis. Addition of TG in the range of 0-4000 Ukg⁻¹ rye flour modified the rheological properties of rye flour dough, resulting in a progressive increase of the complex shear modulus (|G∗|) and in a decrease of the loss factor (tan δ) due to protein cross-linking or aggregation. CLSM image analysis illustrated a TG-induced increase of the size of rye protein complexes. Standard baking tests showed positive effects on loaf volume and crumb texture of rye bread with TG applied up to 500 Ukg⁻¹ rye flour. Higher levels of TG (500 U ≤ TG ≤ 4000 U) had detrimental effects on loaf volume. Increasing TG concentration resulted in an increase of crumb springiness and hardness. In conclusion, the results of this work demonstrated that TG can be used to improve the bread making performance of rye dough by creating a continuous protein network.