超声提取-离子色谱法测定芹菜中氯量

研究了超声提取-离子色谱法测定芹菜中氯含量的方法。芹菜经超声提取20 min后,以浓度4.5 mmol/L Na₂CO₃和0.8 mmol/L NaHCO₃为淋洗液,经IonPacAG23分离测定。本方法具有快速、灵敏、准确度高,适合于芹菜中氯量的测定。     

不同温度下TMV侵染枯斑三生烟的LncRNA差异表达

【目的】 筛选不同温度下烟草花叶病毒（Tobacco mosaic virus,TMV）侵染后枯斑三生烟（Nicotiana tabacum var. Samsun NN）差异表达的长链非编码RNA（long non-coding RNA,lncRNA）,研究lncRNA在枯斑三生烟抗性反应中的作用。【方法】 N基因的温度敏感性使枯斑三生烟在25℃时具备对TMV的抗性、在31℃抗性丧失,在这两个温度条件下对枯斑三生烟接种TMV和磷酸盐缓冲盐水（phosphate buffered saline,PBS）,48 h后提取系统叶总RNA,构建链特异性文库后进行深度测序。对测序结果进行过滤后利用HTSeq将有效数据与近缘品种TN90（N. tabacum var. TN90）基因组比对,筛选得到lncRNA后利用FPKM法估计lncRNA的表达水平。通过edgeR筛选差异表达lncRNA（differentially expressed lncRNA,DElncRNA）,并利用qRT-PCR技术对这一结果进行验证。通过共定位及共表达分析预测DElncRNA的靶基因,通过参考基因组注释、GO和KEGG富集分析研究靶基因的功能。【结果】 4个处理共12个样本经lncRNA-seq各测得约8 000万条clean reads,共获得4 737条已知lncRNA、40 169条新lncRNA。其中64个lncRNA在不同温度条件下TMV侵染后存在差异表达,qRT-PCR测定结果显示这些lncRNA的测序正确率在80%左右,表明本研究所得测序数据具备较高的可信度。对DElncRNA进行靶基因预测,发现一些基因同时被25℃下调和31℃上调的DElncRNA靶向。靶基因注释功能丰富,主要参与植物抗病、激素和代谢等生理过程。部分可能与激素通路相关的lncRNA,在25℃下TMV侵染时呈现下调趋势,而在31℃下TMV侵染则呈现上调趋势。GO富集分析显示靶基因主要参与构成膜、囊泡等组分,具备钙、钾离子通道抑制剂活性等分子功能,使相应离子得以转运引发随后的反应,同时也参与发病、抗原加工和呈现、细胞分裂素代谢等生理过程。KEGG分析发现靶基因显著富集在植物激素信号转导通路,25℃下调和31℃上调的DElncRNA靶基因同时富集在激素信号传导、ABC运输蛋白、苯丙烷类生物合成等通路。【结论】 不同温度（25℃和31℃）条件下TMV侵染枯斑三生烟后,长链非编码RNA差异表达,DElncRNA通过作用于激素信号传导、物质转运等过程参与寄主系统获得性抗性反应。研究结果可为揭示植物系统获得性抗性中lncRNA的调控功能以及新型抗病毒技术开发提供依据。     

全麦粉和小麦粉中烷基间苯二酚同系物组成的对比分析

【目的】 建立全麦粉标记物烷基间苯二酚（ARs）的高效液相检测方法,对全麦粉和小麦粉中ARs同系物组成进行分析,实现全麦粉真伪品质评价。【方法】 探究高效液相条件,建立ARs同系物5-十七烷基间苯二酚（C17:0）、5-十九烷基间苯二酚（C19:0）、5-二十一烷基间苯二酚（C21:0）、5-二十三烷基间苯二酚（C23:0）和5-二十五烷基间苯二酚（C25:0）的高效液相检测方法,并进行方法学评价。以国内外全麦粉、小麦粉及小麦麸皮等共47份样品为研究对象,测定ARs同系物的含量,分析其ARs同系物组成,并进行热图分析和主成分分析。【结果】 选用ZORBAX SB-C18（150 mm×4.6 mm,5 μm,Agilent）色谱柱,流动相选用0.1%甲酸水溶液和0.1%甲酸甲醇溶液进行梯度洗脱,在紫外检测波长280 nm、柱温35℃、流速0.5 mL·min^-1、进样量20 μL条件下,可在35 min内有效分离5种ARs同系物,各个同系物的色谱峰峰形对称,分离度好。经方法学评价,该方法具有良好的精密度和灵敏度,标准曲线线性相关系数均大于0.999,检出限为0.06—0.19 μg·mL ^-1,回收率为81.16%—112.92%,精密度标准偏差为0.02%—4.02%,可作为ARs同系物的测定方法。通过热图聚类分析可知不同检测样品中ARs同系物组成及总量存在显著差异（P<0.05）。麦粉样品ARs同系物组成中C21:0最多,占33%—61%;C19:0次之,占12%—44%;C17:0、C23:0和C25:0的含量占比较小。美国、加拿大全麦粉的ARs同系物组成丰富度和含量相近,国外全麦粉中ARs同系物组成含量高于国内全麦粉。国内全麦粉中的ARs同系物组成比小麦粉中的ARs同系物组成丰富度高,但个别小麦粉除外。小麦麸皮F₁中ARs同系物组成和总含量显著高于全麦粉和小麦粉,其ARs总含量高达1 795.78 μg·g^-1,与其他样品相比差异显著（P<0.05）。经主成分分析得出,麦粉样品间的主要差异指标为ARs同系物组成含量,作为第1主成分,其累积贡献率为98.40%。小麦麸皮与其他麦粉样品的相对分散最远,中国小麦粉的分散度最大。美国全麦粉和加拿大全麦粉几乎重叠,而中国全麦粉和部分小麦粉与美国、加拿大全麦粉部分交叉。【结论】 本研究建立的液相方法能够有效、快速的对全麦粉中ARs同系物进行定量,适用于麦类全谷物产品中ARs同系物含量的测定。同时,通过全麦粉和小麦粉中ARs同系物组成对比分析可知,全麦粉中的ARs同系物组成丰富度优于小麦粉中的ARs同系物组成丰富度。     

An improved method for extraction of sorghum polymeric protein complexes

A method for fractionating sorghum proteins using extraction solvents and techniques designed to obtain polymeric protein structures (especially disulfide linked) was developed. Extraction and separation conditions were optimized in terms of completeness of protein extraction, sample stability, and analytical resolution. After pre-extraction of albumins and globulins, a 3-step sequential procedure involving no reducing agents was applied to ground whole sorghum flour. The three fractions obtained represented proportionally different protein polymer contents and molecular weight distribution as evidenced by comparative size exclusion chromatography. Protein composition also varied among the extracts with differences in kafirin composition and non-kafirin proteins detected in the fractions by RP-HPLC and SDS-PAGE analysis. The ability to quantify and further characterize sorghum polymeric protein complexes will be useful for additional studies linking protein structures with functionality and digestibility and variations for these properties within diverse sorghum germplasm.     

Effects of germination and kilning on the phenolic compounds and nutritional properties of quinoa (Chenopodium quinoa) and kiwicha (Amaranthus caudatus)

Quinoa (Chenopodium quinoa) and kiwicha (Amaranthus caudatus) are nutritious pseudocereals that originate from the Andean region. The aim of this research was to study the effect of germination and the subsequent kilning on the phenolic compounds and proximate composition in selected Peruvian varieties of quinoa (“Chullpi”) and kiwicha (“Oscar Blanco”). The germination process was carried out for 24, 48 and 72 h at 22 °C, and the kilning was performed with samples germinated for 72 h by drying the seeds at 90 °C for 5 min. Both processes increased the protein content of the samples. However, lipid content was reduced during germination. On the other hand, germination and kilning clearly increased the concentration of total phenolic compounds in both quinoa and kiwicha. Germination for 72 h either with or without kilning process resulted in a significant (p < 0.05) increase in the total content of phenolics compared to untreated materials, which was especially due to coumaric acid and a kaempferol tri-glycoside in quinoa and caffeoylquinic acid in kiwicha. Based on the results, germination and kilning may improve the nutritional quality of the Andean grains, encouraging the usage of the processed grains as ingredients in functional products for people with special gluten-free or vegetarian diets.     

Recent progress in analytical method development to ensure the safety of gluten-free foods for celiac disease patients

As laid down by the Codex Alimentarius, products bearing a gluten-free label must not contain gluten levels above 20 mg/kg to be safe for consumption by celiac disease patients. Analytical methods to detect gluten from wheat, rye and barley need to be sufficiently sensitive, specific, suitable for routine analyses and validated by collaborative studies. With continuous progress in the field of gluten analysis, the aim of this paper is to provide an up-to-date overview of legislation regarding gluten-free products worldwide, as well as immunochemical, proteomics-based, genomics-based and other methods designed to analyse gluten traces. While ELISA test kits and PCR are still most widely used in quality control, liquid chromatography tandem mass spectrometry (LC-MS/MS) is gaining more and more importance by providing unprecedented insights into gluten. Several other methods such as immunosensors, other sensors and microarrays are being developed. The pro's and con's of the different methods are discussed as well as the remaining challenges, including the need for improved extraction procedures, comprehensive reference materials and independent reference methods.     

茶红素分离及稳定性研究

采用茶汤有机试剂萃取分离,并分别经过反相C_(18)柱层析纯化,制备出乙酸乙酯层、正丁醇层和酸性正丁醇层三部分茶红素。根据紫外可见光波扫描计算出色价,并研究了茶红素在不同条件下的稳定性。结果表明,30%、50%和70%甲醇梯度洗脱,纯化后3类茶红素液相检测发现无咖啡碱和茶黄素,仅有少量儿茶素杂质。纯化后色素色价分别为乙酸乙酯层128.04,正丁醇层91.04,酸性正丁醇层76.16。遮光处理,p H=3酸性条件,45℃以下贮藏,有利于茶红素的稳定性。     

水稻苗期根系铵钾离子吸收的交互作用研究

【目的】为探讨水稻幼苗根系NH_4~+、K~+吸收的交互作用,深化水稻养分吸收理论,【方法】采用溶液培养的方法,对低钾及高钾浓度下水稻在有铵和无铵时的K~+吸收动力学特征进行了研究,对不同钾浓度下水稻根系NH_4~+的吸收速率进行了比较。【结果】1)当K~+0.2 mmol/L时,水稻根系通过高亲和转运系统吸收K~+服从Michaelich-Menten动力学方程;NH_4~+的存在显著降低K~+的最大吸收速率(Vmax),且降幅随着NH_4~+浓度的增加而增大;NH_4~+对水稻根表载体与K~+的亲和力(Km)影响较小,在1.62 mmol/L NH_4~+浓度下,水稻品种齐粒丝苗和沪科3号的Km分别下降了12.33%和16.46%,远低于Vmax 47.30%和39.21%的降幅。2)当K~+0.5 mmol/L时,水稻根系K~+低亲和转运系统发挥作用,K~+吸收速率随浓度的增加而不断增加,呈不饱和特征;但在相同K~+浓度下,水稻根系的K~+吸收速率随NH_4~+浓度的增加而下降。3)水稻根系对NH_4~+的吸收速率随着NH_4~+浓度的增加而增加;在相同NH_4~+浓度下,水稻根系对NH_4~+的吸收速率受K~+浓度的影响很小。【结论】NH_4~+抑制水稻苗期根系K~+的高亲和转运和低亲和转运,NH_4~+对K~+高亲和吸收的影响主要是由于铵竞争细胞膜上的钾载体所致;外界K~+浓度的变化对水稻幼苗的NH_4~+吸收速率影响很小。水稻铵钾的交互作用主要表现在NH_4~+对K~+吸收的抑制作用。     

Polymerisation of gluten proteins in developing wheat grain as affected by desiccation

The breadmaking quality of wheat is affected by the composition of gluten proteins and the polymerisation of subunits that are synthesised and accumulated in developing wheat grain. The biological mechanisms and time course of these events during grain development are documented, but not widely confirmed. Therefore, the aim of this study was to monitor the accumulation of gluten protein subunits and the size distribution of protein aggregates during grain development. The effect of desiccation on the polymerisation of gluten proteins and the functional properties of gluten were also studied. The results showed that the size of glutenin polymers remained consistently low until yellow ripeness (YR), while it increased during grain desiccation after YR. Hence, this polymerisation process was presumed to be initiated by desiccation. A similar polymerisation event was also observed when premature grains were dried artificially. The composition of gluten proteins, the ratios of glutenin to gliadin and high molecular weight-glutenin subunits to low molecular weight-glutenin subunits, in premature grain after artificial desiccation showed close association with the size of glutenin polymers in artificially dried grain. Functional properties of gluten in these samples were also associated with polymer size after artificial desiccation.     

Disulphide structure of high-molecular-weight (HMW-) gliadins as affected by terminators

This study aimed at elucidating SS-bonds of HMW-gliadins (HGL) from wheat with the focus on terminators of glutenin polymerisation. HGL from wheat flour extracts non-treated or treated with the S-alkylation reagent N-ethylmaleinimide (NEMI) were compared. HGL from wheat flour Akteur were isolated, hydrolysed with thermolysin and the resulting peptides pre-separated by gel permeation chromatography and analysed by liquid chromatography/mass-spectrometry using alternating electron transfer dissociation/collision-induced dissociation. Altogether, 22 and 28 SS-peptides from samples without and with NEMI treatment, respectively, were identified. Twenty-six peptides included standard SS-bonds of α- and γ-gliadins, high-molecular-weight and low-molecular-weight glutenin subunits. Eleven SS-bonds were identified for the first time. Fifteen peptides unique to HGL contained cysteine residues from gliadins with an odd number of cysteines (ω5-, α- and γ-gliadins). Thus, gliadins with an odd number of cysteines, glutathione and cysteine had acted as terminators of glutenin polymerisation. Decisive differences between samples without and with NEMI treatment were not obvious showing that the termination of polymerisation was already completed in the flour. The two HGL samples, however, were different in the majority of ten peptides that included disulphide-linked low-molecular-weight (LMW) thiols such as glutathione and cysteine with the former being enriched in the non-treated HGL-sample.