Predicting the Buckwheat Flour Ratio for Commercial Dried Buckwheat Noodles Based on the Fluorescence Fingerprint

A rapid method for predicting the buckwheat flour ratio of dried buckwheat noodles was developed by using the fluorescence fingerprint and partial least squares regression. Fitting the calibration model to validation datasets showed R ²=0.78 and SEP=12.4%. The model was refined for a better fit by deleting several samples containing additional ingredients. The best fit was finally obtained (R ²=0.84 and SEP=10.4%) by deleting the samples containing vinegar, green tea, seaweed, polysaccharide thickener, and yam. This result demonstrates that a calibration model with high accuracy could be constructed based on samples similar in material composition. The developed methodology requires no complex preprocessing, enables rapid measurement with a small sample amount, and would thus be suitable for practical application to the food industry.     

基于NIRS法快速检测荞麦蛋白质与淀粉含量的研究

为建立近红外光谱技术测定荞麦蛋白质与淀粉含量的方法,本研究以217份荞麦样品为试验材料,采用最小二乘回归预测和交叉验证构建近红外预测模型。分析表明:前处理采用多元散射校正法(MSC),维数(Rank)分别为5和5,光谱区间6803.9~6094.2/cm所建立的荞麦蛋白质与淀粉含量模型的预测效果较好,其决定系数(R~2)分别为0.9481和0.9167,交叉验证均方根(RMSECV)分别为0.68和2.08,相对分析误差(RPD)分别为4.39和3.46,均大于3.0,外部验证相关系数均大于0.96。本试验所建立的蛋白质与淀粉含量近红外预测模型具有较高的准确度和稳健性,可用于荞麦品质的快速测定。     

染料结合法测定荞麦种子蛋白质含量的研究

以考马斯亮蓝G250为染料,结合经典的凯氏定氮法测定结果,对影响染料结合法测定蛋白质含量的振荡时间、温度、考马斯亮蓝溶液浓度等因素进行了研究,并分析了由考马斯亮蓝染料和蛋白质结合后染料结合量(OD值差)与凯氏法测得的蛋白质百分含量之间的相关性。结果表明:测定的适宜条件是:温度15℃,处理时间50min,考马斯亮蓝溶液的浓度是0.06mg/mL。此条件形成的络合物较稳定,重复性较好,并且所测的染料结合量与凯氏定氮法测得的蛋白质含量间呈极显著的一元线性回归和相关关系。栽培甜荞和栽培苦荞的回归方程分别为:y=15.364x+3.865和y=10.769x+6.287,这两个回归方程差异显著,不能合并,分别适合于快速估计甜荞和苦荞种子的蛋白质含量。     

Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis

Proteins from buckwheat (Fagopyrum esculentum) are strong allergens that can cause serious symptoms, including anaphylaxis, in patients with hypersensitivity. In this study, we successfully developed a modified lactic acid bacterial vector (pNSH) and a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced a major allergenic storage protein of buckwheat, Fagag1 (61.2 kDa, GenBank accession number AF152003), with or without a green fluorescent protein (GFP) tag. GFP fluorescence allows for rapid, simple, and accurate measurement of target protein expression by microscopy or fluorimetry. We describe a convenient method for production of rGFP-Fagag1 fusion and rFagag1 proteins with a good yield in an advantageous probiotic host. We found that in vitro treatment of splenocytes isolated from buckwheat crude protein-immunized mice with rFagag1 increased the expression of allergic inflammation cytokines such as IL-4, IL-13, and IL-17 F. Because it was less antigenic, rGFP-Fagag1 protein from NZ9000 might be of limited use; however, rFagag1 from NZ9000 evoked a robust response as measured by induction of IL-4 and IL-17 F expression levels. The observed allergic activity is indicative of a Th2 cell-mediated immune response and is similar to the effects induced by exposure to buckwheat crude protein. Our results suggest that expression of rFagag1 in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to buckwheat allergens.     

Modeling the dynamic composition of engineered cartilage

Mathematical models to describe extracellular matrix (ECM) deposition and scaffold degradation in cell-polymer constructs for the design of engineered cartilage were developed and validated. The ECM deposition model characterized a product-inhibition mechanism in the concentration of cartilage molecules, collagen and glycosaminoglycans (GAG). The scaffold degradation model used first-order kinetics to describe hydrolysis (not limited by diffusion) of biodegradable polyesters, polyglycolic acid and polylactic acid. Each model was fit to published accumulation and degradation data. As experimental validation, cell-polymer constructs (n=24) and unseeded scaffolds (n=24) were cultured in vitro. Biochemical assays for ECM content and measurements of scaffold mass were performed at 1, 2, 4, 6, 8, or 10 weeks (n=8 per time point). The models demonstrated a strong fit with published data and experimental results (R(2)=0.75 to 0.99) and predicted the temporal total construct mass with reasonable accuracy (30% RMS error). This approach can elucidate mechanisms governing accumulation/degradation and may be coupled with structure-function relationships to describe time-dependent changes in construct elastic properties.     

自交可育甜荞、金苦荞、米苦荞不同品系总黄酮、粗蛋白及其蛋白组分含量研究北大核心CSCD

为探究荞麦新品种的营养保健价值,该文对自交可育甜荞、金苦荞、米苦荞共56个不同品系荞麦种子的粗蛋白、总黄酮、蛋白组分含量及其果实性状的变异进行了研究。结果表明:(1)甜荞、金苦荞、米苦荞种子中粗蛋白含量平均值分别为13.19%、15.44%、11.75%,总黄酮含量平均值分别为0.14%、2.50%、2.09%,清蛋白含量的平均值分别为5.22%、6.13%、4.56%,球蛋白含量的平均值分别为1.29%、1.15%、0.91%,醇溶蛋白含量的平均值分别为0.42%、0.58%、0.55%,谷蛋白含量的平均值分别为2.66%、3.36%、2.80%,三种荞麦的蛋白组分均符合清蛋白>谷蛋白>球蛋白>醇溶蛋白。(2)果实性状中,甜荞果实千粒重、果实面积、果实直径的变异系数最大,米苦荞果实周长、果实长宽比、果实长、果实宽和50 mL容重的变异系数最大。(3)相关分析表明甜荞种子粗蛋白含量与果实长宽比、果实长,金苦荞种子粗蛋白含量与果实周长、果实长,米苦荞种子粗蛋白含量与果实宽、总黄酮含量与果实面积、果实宽、果实直径、50 mL容重的相关性均达到了显著或极显著水平。(4)该研究筛选出甜荞(1808-166贵甜2号优系)、金苦荞(多苦74、多苦78)、米苦荞(1906-136黑米荞麦、43-2)等高蛋白含量或高黄酮含量的荞麦品系。该研究结果对荞麦优良品种的选育和荞麦新产品的开发具有一定指导意义。     

Silages containing buckwheat and chicory: quality, digestibility and nitrogen utilisation by lactating cows

The suitability of silages containing buckwheat (Fagopyrum esculentum) and chicory (Cichorium intybus) for the nutrition of dairy cows was determined. Buckwheat and chicory were sown in mixture with ryegrass (Lolium multilorum), and a pure ryegrass culture served as a control forage. Swards were harvested 55 d after sowing and were ensiled after wilting, without additives in small round bales. Finally, buckwheat and chicory made up the dietary dry matter (DM) proportions of 0.46 and 0.34, respectively. Concentrates were restricted to 2 kg/d. Diets were fed to 3 x 6 late-lactating cows for 15 d at ad libitum access. During the collection period (days 10-15) amounts of feed intake and faeces, urine and milk were recorded and samples were taken. Ensilability was good for buckwheat and ryegrass swards, but was so less for the chicory sward, which was rich in total ash. The buckwheat silage was rich in acid detergent fibre (445 g/kg DM) and lignin (75.7 g/kg DM) and contained less crude protein (135 g/kg DM) and ether extract (15.8 g/kg DM) than the other silages. Consistent with that, the apparent digestibility of the organic matter and fibre were lowest when feeding this silage. The potassium concentrations in the chicory and ryegrass silages were high (61 g/ kg) and lower in buckwheat (47 g/kg). No significant treatment effects on intake, body weight, milk yield or milk composition as well as plasma beta-hydroxybutyrate and non-esterified fatty acids occurred. Being lowest in nitrogen (N) content, the buckwheat silage resulted in the lowest urine N losses and the most efficient N utilisation for milk protein synthesis, but this at cost of body N retention. The results show that silages containing buckwheat and chicory may be used as components of the forage part of dairy cows' diets even though they were found to have a lower feeding value than ryegrass silage.     

Combining maxRatio analysis with real-time PCR and its potential application for the prediction of Meloidogyne incognita in field samples

Diagnosing and quantifying plant-parasitic nematodes is critical for efficient nematode management. Several studies have been performed intending to demonstrate nematode quantification via real-time quantitative PCR. However, most of the studies used dilution of DNA templates to make standard curves, while few studies used samples with different nematode numbers to make the standard curve, resulting in a high standard error. The objective of the present study was to develop a high quality standard curve using samples containing different numbers of the root-knot nematode Meloidogyne incognita and evaluate the results of real time qPCR with maxRatio analysis. The results showed that a high quality standard curve was obtained with different nematode numbers using specific primers and cycle threshold (Ct)-PCR (R(2)=0.9962, P<0.001, n=9). With the maxRatio analysis, the fractional cycle number (FCN)-PCR cycle curve and adjusted FCN (FCNadj)-PCR cycle curve had similar patterns as those of the Ct-PCR cycle curve. For quantification of nematodes in field soil samples, qPCR estimations with a FCNadj-PCR cycle standard curve was very close to microscope counting of second-stage juveniles (R(2)=0.9064, P<0.001, n=10), qPCR estimations with a FCN-PCR cycle standard curve was comparably good (R(2)=0.8509, P<0.001, n=10), and the biases with a Ct-PCR cycle standard curve were large (R(2)=0.7154, P<0.001, n=10). Moreover, we found that the concentration of Triton X-100 had less of an effect on FCN as compared to Ct, with delta FCN 0.52, and delta Ct 3.94 at 0.8% Triton. The present study suggests, that combined with maxRatio methods, real time qPCR could be a practical approach for quantifying M. incognita in field samples.     

Epitope mapping and identification on a 3D model built for the tartary buckwheat allergic protein TBb

Allergic protein TBb, a major allergen in tartary buckwheat, was divided into four epitope-containing fragments and was named F1, F2, F3, and F4, respectively. Results of immunological assays revealed that F2 had the strongest IgE-binding activity to patient's sera, which indicated that it might contain the linear IgE-binding epitope of TBb. According to the results of sequence analysis and molecular modeling of tartary buckwheat allergen, three mutants of F2 gene (R139A, R141A, and D144A) were reconstructed using site-directed mutagenesis, and each mutant was expressed in Escherichia coli BL21 (DE3). Following purification by Ni(2+) affinity chromatography, enzyme-linked immunosorbent assay and dot blot were performed for wild-type F2 and its mutants using sera from buckwheat-allergic patients and a negative control (non-allergic patient). Results showed that mutants R139A and D144A had weaker IgE-binding activity to patient's sera than wild-type F2, implying that Arg(139) and Asp(144) might be involved in the allergic activity of TBb. However, R141A had the weakest IgE-binding activity, suggesting that Arg(141) may be the critical amino acid of TBb. This is the first report on the epitope mapping and identification of TBb. Our findings will contribute to the production of TBb hypoallergens and to allergen-specific immunotherapy for tartary buckwheat allergy.     

Simultaneous quantitation of 7-methyl- and O6-methylguanine adducts in DNA by liquid chromatography-positive electrospray tandem mass spectrometry

A methodology has been developed and validated for the simultaneous quantitation of O6-methyl- and 7-methylguanine in DNA isolated from in vitro exposure to the model alkylating agents: N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS). After exposure, DNA was isolated and directly hydrolyzed under acid conditions to hydrolytes containing DNA bases (modified and unmodified). The hydrolytes were used for direct O6- and 7-methylguanine quantitation using a rapid and selective liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS). The lower limits of quantitation for O6-methyl- and 7-methylguanine were 75.8 and 151.5 fmol, respectively. Linearity of the calibration curve was greater than 0.999 from 75.8 to 151,600.0 fmol for O6-methylguanine and 0.999 from 151.5 to 303,200.0 fmol for 7-methylguanine. The intra-day assay precision relative standard deviation (R.S.D.) values for O6-methylguanine for quality control (QC) samples were < or =9.2% with accuracy values ranging from 90.8 to 118%, and for 7-methylguanine the R.S.D. values for QC samples were < or =11%, with accuracy values ranging from 92.9 to 119%. The inter-day assay precision (R.S.D.) values for O6-methylguanine QC samples were < or =7.9% with accuracy values ranging from 94.5 to 116%, and for 7-methylguanine QC samples were < or =7.1% with accuracy values ranging from 95.2 to 110.2%. This method was used for simultaneous determination of the levels of 7-methyl- and O6-methylguanine in DNA acidic hydrolytes present in a series of incubations from salmon testis DNA treated with either MNU or MMS.     

An anthocyanin compound in buckwheat sprouts and its contribution to antioxidant capacity

The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.     

荞麦及其野生种遗传多样性分析

荞麦起源于我国西南地区,该地区分布有大量荞麦地方品种和野生近缘种。本研究利用SSR分子标记,对从我国西南地区收集的81份荞麦及其野生资源进行遗传多样性分析。结果显示,利用19对SSR引物共检测出84个等位基因,每对SSR引物平均扩增出4.421个等位基因,19对SSR引物的平均Shannon's信息指数为0.985,平均PIC为0.478。81份荞麦及其野生资源的相似系数范围为0.500~1.000,分析发现大粒组荞麦种(甜荞及其近缘种、苦荞及小米荞、金荞)与小粒组硬枝万年荞亲缘关系比较近。通过聚类分析在遗传相似系数为0.732时,可将81份荞麦种质分为4个类群,第Ⅰ类由小粒组齿翅野荞、细柄野荞、小野荞麦、疏穗小野荞麦和硬枝万年荞组成;第Ⅱ类由甜荞和甜荞近缘种组成;第Ⅲ类都是由金荞组成;第Ⅳ类是由苦荞和小米荞组成。在遗传系数为0.920时,小粒组荞麦种可以明显区分出疏穗小野荞麦、硬枝万年荞、齿翅野荞及其细柄野荞。研究表明,19对SSR引物多态性较高,能较好反应荞麦及其野生种质的遗传多样性,本研究的结果为荞麦属种之间的亲缘关系分析和荞麦起源进化研究提供科学依据。     

甜荞花青素合成相关基因FeR2R3-MYB的克隆与表达分析

MYB 是一类常见的转录因子,广泛参与植物花青素生物合成的调控。为探究 MYB转录因子在甜荞花青素生物合成中的调控作用,该研究从红花甜荞和白花甜荞转录组学数据中筛选并克隆出一个和花青素生物合成相关的MYB基因,将其命名为 FeR2R3-MYB,GenBank 登录号为 MT151381.1,并对该序列进行生物信息学分析,以及利用 qRT-PCR 分析FeR2R3-MYB基因在白花甜荞和红花甜荞中的表达特征。结果表明:(1)FeR2R3-MYB基因全长 831 bp,编码 276 个氨基酸,蛋白的相对分子质量为 30.95 kD,理论等电点(pI)为 8.73,蛋白的不稳定指数为 69.64,属于不稳定蛋白,总疏水值为-0.679,整条肽链呈现亲水特性。(2)FeR2R3-MYB 具有典型的 R2R3-MYB 结构域,属于 R2R3-MYB 亚家族。(3)FeR2R3-MYB 与同属蓼科的苦荞和虎杖亲缘关系比较近。(4)FeR2R3-MYB 的启动子序列共含有 9 个光照响应元件、17 个转录因子结合位点、4 个非生物响应元件和 2 个激素响应元件。(5)亚细胞定位发现 FeR2R3-MYB 只在细胞核中表达。(6)FeR2R3-MYB 基因的表达量在叶片和花序中红花甜荞均高于白花甜荞,推测 FeR2R3-MYB 基因可以正向调节甜荞花青素生物合成。综上所述,该研究结果为进一步深化 FeR2R3-MYB 基因在甜荞花青素生物合成途径中的功能及表达调控方面的研究提供了基础。     

Epitope mapping and immunological characterization of a major allergen TBa in tartary buckwheat

Predicted by an antigenic program, full-length tartary buckwheat allergen (TBa) is divided into six fragments: E1, E2, E12, E3, E4 and E34. Immunological assays revealed that E1 has the greatest binding activity to patients’ serum IgE. Five mutants of E1 gene (L39R, L42R, L47R, V52R and L54R) were constructed using site-directed mutagenesis and each protein was expressed in Escherichia coli BL21. Following purification by Ni²⁺ affinity chromatography, ELISA and dot-blot were performed for wild type E1 and its mutants using sera from buckwheat allergic patients and healthy controls. Mutants L42R, L47R, and L54R had weaker IgE binding activity to patient’s sera than wild-type E1 implying that Leu42, Leu47, and Leu54 might be involved in the allergic activity of TBa.     

Muscle hypertrophy in rats fed on a buckwheat protein extract.

Growing rats were examined for the influence of a buckwheat protein diet on muscle weight and protein. In experiment 1, the rats were fed on a diet containing either casein or a buckwheat protein extract (BWPE) as the protein source (10%, 20% or 30%) for 5 wk. The relative weights (g per kg of body wt) of the gastrocnemius, plantaris and soleus muscles were higher in the BWPE-fed animals than in the casein-fed ones, but were unaffected by the dietary level of protein. These differences were not associated with growth. In experiment 2, the rats were fed on either a casein or BWPE diet at the 20% protein level for 5 wk. BWPE intake significantly elevated the gastrocnemius muscle weight, carcass protein and water, and reduced carcass fat. These results demonstrate that BWPE consumption causes muscle hypertrophy, elevates carcass protein and water, and reduces body fat.     

黑苦荞茎叶对2型糖尿病小鼠的治疗作用及其对其胰腺、脾脏的影响

目的:观察黑苦荞茎叶(BBL)对2型糖尿病小鼠治疗作用及其对胰腺、脾脏的影响。方法:将40只SPF级雄性C57/B16小鼠随机分为正常对照组(n=10)和实验组(n=30),实验组给予高糖高脂饲料喂养1个月后,每只小鼠在禁食12h后腹腔注射链脲佐菌素35mg/kg,注射后72h取尾血测血糖≥13.8mmol/L为糖尿病小鼠。成功建立2型糖尿病(T2DM)模型的30只小鼠随机分为3组(n=10):模型对照组(DM组)、低剂量黑苦荞茎叶治疗组(DM+L)组、高剂量黑苦荞茎叶治疗(DM+H)组。对照组和模型组小鼠每日给予生理盐水灌胃,两个黑苦荞茎叶组分别给予0.21g/kg·d-1和0.42g/kg·d-1的黑苦荞茎叶灌胃,14d后杀死小鼠,取血和胰腺、脾脏,检测血清中葡萄糖(FBG)、甘油三酯(TG)、总胆固醇(TCH)、胰岛素和脾脏组织中TNF-α蛋白的含量,观察胰腺组织形态学,称量小鼠体重和脾脏重量、计算脾脏系数,检测胰腺组织中IRS-1mRNA的表达和胰腺组织内胰岛素受体底物IRS-1蛋白表达。结果:与对照组相比,模型组小鼠血清FBG、TC、TCH水平明显升高,使血胰岛素水平明显下降,脾脏组织TNF-α蛋白表达水平显著升高,胰腺组织中IRS-1mRNA表达和IRS-1的蛋白表达水平显著降低(P<0.05);与模型组相比,治疗组小鼠的血清FBG、TC、TCH水平明显降低,血胰岛素水平显著升高,小鼠脾脏系数、脾脏组织TNF-α蛋白表达水平、胰腺组织IRS-1mRNA表达和IRS-1的蛋白表达水平显著升高(P<0.05)。高剂量黑苦荞茎叶(0.42g/kg·d-1)治疗作用更加明显。结论:黑苦荞茎叶对2型糖尿病小鼠具有显著的降血糖、降血脂的治疗效果,对糖尿病小鼠胰腺、脾脏具有一定的保护作用。     

Rapid analysis of sugars in fruit juices by FT-NIR spectroscopy.

A simple analytical procedure using FT-NIR and multivariate techniques for the rapid determination of individual sugars in fruit juices was evaluated. Different NIR detection devices and sample preparation methods were tested by using model solutions to determine their analytical performance. Aqueous solutions of sugar mixtures (glucose, fructose, and sucrose; 0-8% w/v) were used to develop a calibration model. Direct measurements were made by transflection using a reflectance accessory, by transmittance using a 0.5-mm cell, and by reflectance using a fiberglass paper filter. FT-NIR spectral data were transformed to the second derivative. Partial least-squares regression (PLSR) was used to create calibration models that were cross-validated (leave-one-out approach). The prediction ability of the models was evaluated on fruit juices and compared with HPLC and standard enzymatic techniques. The PLSR loading spectra showed characteristic absorption bands for the different sugars. Models generated from transmittance spectra gave the best performance with standard error of prediction (SEP) <0.10% and R(2) of 99.9% that accurately and precisely predicted the sugar levels in juices, whereas lower precision was obtained with models generated from reflectance spectra. FT-NIR spectroscopy allowed for the rapid ( approximately 3 min analysis time), accurate and non-destructive analysis of sugars in juices and could be applied in quality control of beverages or to monitor for adulteration or contamination.     

PCR method for detecting trace amounts of buckwheat (Fagopyrum spp.) in food

Buckwheat often causes severe allergic reactions, even when its ingestion level is extremely low. Therefore, buckwheat is listed in several countries as a common food allergen. In addition to common buckwheat and Tartarian buckwheat that are cultivated and consumed widely, wild buckwheat may be potentially allergenic. Food containing undeclared buckwheat poses a risk to patients with the buckwheat allergy. We describe in this report a PCR method to detect buckwheat DNA by using primers corresponding to the internal transcribed spacer region and the 5.8S rRNA gene. The method is buckwheat-specific and compatible with both cultivated and wild buckwheat of the Fagopyrum spp. Its sensitivity was sufficient to detect 1 ppm (w/w) of buckwheat DNA spiked in wheat DNA. This method should benefit food manufacturers, clinical doctors, and allergic patients by providing information on the presence of buckwheat contamination in food.     

A neural network for predicting the stability of DNA/DNA duplexes

A back-propagation neural network method has been developed to predict the stability of DNA/DNA duplexes. Calculated Tm with the present parameters fits the experimental values within reasonable errors (AD = 1.86 K, SEP = 1.99151, R2 = 0.9894 for NN1; AD = 1.59667 K, SEP = 2.03824, R2 = 0.99371 for NN2) and it has the advantage that the determinations of thermodynamic parameters are not needed.