Transglutaminase polymerisation of buckwheat (Fagopyrum esculentum Moench) proteins

Recently, screening of transglutaminase (TGase) treatment on several gluten-free cereals revealed significant improvements on the baking performances of buckwheat flour by promoting protein networks. In this study, the impact of TGase on the protein fractions of buckwheat flour was investigated in order to better understand the activity and specificity of the enzyme. Albumin, globulin, prolamin and glutelin fractions were extracted from the flour and incubated with TGase. Capillary electrophoresis, two dimensional (2D) gel electrophoresis and size exclusion chromatography (SEC) were performed on each fraction. Capillary electrophoresis and 2D gel electrophoresis revealed that buckwheat main storage proteins, i.e. 2S albumin, 13S and 8S globulin, were cross-linked after TGase treatment. SEC showed the presence of high molecular weight (HMW) protein polymers in the TGase-treated albumin and globulin fraction. Analysis of the amino acid composition of the fractions revealed high amounts of glutamine and lysine residues in all fractions. In conclusion, the increase in the average molecular weight of buckwheat proteins and the formation of HMW protein polymers after TGase treatment are responsible for the improved functionality of buckwheat flour in terms of breadmaking potential. The enzyme was revealed to be not fraction specific as all fractions were TGase-reactive.     

Transglutaminase polymerisation of buckwheat (Fagopyrum esculentum Moench) proteins

Recently, screening of transglutaminase (TGase) treatment on several gluten-free cereals revealed significant improvements on the baking performances of buckwheat flour by promoting protein networks. In this study, the impact of TGase on the protein fractions of buckwheat flour was investigated in order to better understand the activity and specificity of the enzyme. Albumin, globulin, prolamin and glutelin fractions were extracted from the flour and incubated with TGase. Capillary electrophoresis, two dimensional (2D) gel electrophoresis and size exclusion chromatography (SEC) were performed on each fraction. Capillary electrophoresis and 2D gel electrophoresis revealed that buckwheat main storage proteins, i.e. 2S albumin, 13S and 8S globulin, were cross-linked after TGase treatment. SEC showed the presence of high molecular weight (HMW) protein polymers in the TGase-treated albumin and globulin fraction. Analysis of the amino acid composition of the fractions revealed high amounts of glutamine and lysine residues in all fractions. In conclusion, the increase in the average molecular weight of buckwheat proteins and the formation of HMW protein polymers after TGase treatment are responsible for the improved functionality of buckwheat flour in terms of breadmaking potential. The enzyme was revealed to be not fraction specific as all fractions were TGase-reactive.     

江西省大豆种质资源7S和11S球蛋白及其亚基相对含量分析

为明确江西省大豆球蛋白各亚基相对含量及其比例,促进营养或加工品质优良大豆资源的挖掘与应用。本研究采用SDS-PAGE方法结合Gel-Pro Analyzer 4.5软件对供试的131份江西省大豆种质资源球蛋白及其亚基相对含量进行分析。结果表明:江西省大豆资源的7S、11S球蛋白及其亚基相对含量具有丰富的遗传变异,供试材料7S和11S球蛋白相对含量的变幅、平均值及变异系数分别为18.01%~52.21%、32.64%、19.46%和47.79%~81.99%、67.35%、9.43%,11S/7S比值范围为0.92~4.55,平均值为2.19,变异系数为30.64%,且筛选鉴定出3个11S/7S比值大于4.0和4个α′亚基、α亚基或β亚基含量较低的优异大豆种质资源,这些资源的挖掘为大豆品质的改良提供了材料和参考。     

706份中国大豆种质贮藏蛋白7S和11S组分及其亚基相对含量的研究

大豆籽粒贮藏蛋白7S和11S组分及其亚基含量、11S/7S比值与大豆蛋白的营养价值和加工特性密切相关.获得具不同7S和11S组分及其亚基含量、不同11S/7S比值的种质材料是对大豆蛋白的营养价值和功能特性进行遗传育种改良的重要材料基础.本研究利用SDS-PAGE技术,对706份中国大豆种质资源7S、11S组分及其亚基相对含量进行了研究.结果表明:706份我国大豆种质资源中7S、11S组分及其亚基相对含量具有丰富的遗传变异;7S和11S组分含量间存在极显著的负相关(r=-1.00,P＜0.01);603份地方品种和103份新育成品种或主栽品种的7S、11S组分相对含量的平均值和变异幅度分别为40.00%,20.58%～56.65%,60.00%,43.35%～79.42%和38.21%,30.33%～52.67%,61.79%,47.33%～69.67%;11S/7S比值的平均值和变异幅度分别为1.54,0.77～3.86和1.65,0.90～2.30;筛选获得了63份7S、11S组分或亚基含量变异种质.     

Possible nutritional implications of varietal influence on the 7S/11S seed globulin ratios in Amaranth

7S/11S seed globulin ratios were determined for sevencurrently available Amaranthus hybrid linesi.e., Amaranthus K266, K283, K343, K432, K433,K436 and MT-3. Of the seven Amaranthus linesinvestigated, four lines had 7S/11S globulin ratiosranging from 0.47 to 0.81, while three lines werefound to contain exclusively the 11S globulin form. In general, 7S globulins contained lower levels ofessential amino acids such as tryptophan, methionine,lysine, histidine, phenylalanine, valine andisoleucine than the 11S globulins.     

东北大豆品种贮藏蛋白7S和11S组分及其亚基相对含量分析

利用聚丙烯酰胺凝胶电泳分析了黑龙江、吉林、辽宁和内蒙古4省(区)近10 a来通过审定的163个大豆品种的11s和7s球蛋白亚基组成、相对百分含量和11s/7S比值,并对其进行了省份问比较分析和相关性分析.结果表明:品种问大豆蛋白各亚基的相埘含量存在较大差异;163份供试材料的7s、11S球蛋白平均相对含量分别为28.95%和56.30%.变幅分别为18.40%～36.20%和47.90%～71.50%,11S/7S比值的变异幅度在1.34～3.32之间,平均值为1.97;初步筛选出α和A3缺失或稀少的特异大豆品种9份;吉林、黑龙江、辽宁和内蒙古4个地区大豆群体蛋白亚基组成及11S/7S比值存在显著差异.7S和11S组分含量问存在极显著的负相关(r=-0.2862,P<0.01).东北大豆品种贮藏蛋白7S和11S组分及其亚基相对含量存在显著变异,为大豆品质育种和豆制品加工业原料选择提供了丰富的物质基础.     

Physicochemical and functional properties of 11S globulin from chan (Hyptis suaveolens L. poit) seeds

The 11S globulin is one of the most abundant and important storage proteins of the chan seed (Hs11S). Therefore, we investigated its physicochemical and functional properties. Studying parameters related to emulsifying properties, a critical micelle concentration (CMC) at 0.6 mg/mL and a surface activity until 55.5 mN/m were determined using a DuNouy tensiometer. Zeta potential revealed isoelectric points (pI) with a clear NaCl influence (without NaCl pI = 3.5, at 0.05 M NaCl pI = 3, at 0.5 M NaCl pI = 2.5) and electrostatic potential dependent upon the pH. The Hs11S was highly soluble at pH 7.6 (≥94.5%) with salt variations (0–0.4 M). The thermostability of Hs11S by dynamic light scattering showed that it organizes in oligomeric forms: trimer, tetramer, hexamer, octamer, dodecamer and aggregates. Oscillatory rheology studies indicate the onset for the heat-induced gelation of Hs11S which occurred around 74–76 °C, developing strong gels with an elasticity of ∼10³ Pa, while a denaturation temperature of 94.1 °C and an enthalpy of 44.49 kJ/mol were found by differential scanning calorimetry. According to ΔH_expTm/ΔH_vHTm and ΔT_1/2 data, the Hs11S denaturation is not a cooperative process. Our results indicate that Hs11S might be used as potential thermostable ingredient in food systems.     

Bread making quality of durum wheat genotypes with some novel glutenin subunit compositions

Durum wheat genotypes with some novel high M_r (high molecular weight, HMW) and low M_r (low molecular weight, LMW) glutenin subunits were grown in Sicily for two years of testing in order to compare their rheological and baking properties with respect to commercial durum wheat cultivars. Good bread making quality, as measured by Alveograph W and P/L, Farinograph and Mixograph parameters, and loaf volume was observed in genotypes combining high M_r subunits 2⁺, 1 or 11 encoded at the Glu-A1 locus with the so-called LMW-2 subunit group encoded at the Glu-B3 locus. The cultivar Avanzi, which carries high M_r subunit 2⁺ and LMW-2-like subunits, and the cultivars Dritto and Keops, which contain novel high and low M_r subunits, gave higher loaf volumes than control cultivars. The LMW-2 group subunits were found to be the main factor in determining dough strength (Alveograph W). The increase in the amount of high M_r subunits in genotypes with one expressed Glu-A1 gene may account for their improved rheological and baking properties.     

Functionality of Succinylated Brazil Nut (<Emphasis Type="Italic">Bertholletia excelsa HBK</Emphasis>) Kernel Globulin

One of the possible ways to improve the utilisation of defatted Brazil nut kernel flour, a by-product of oil extraction industries, is to improve its functional properties by chemical modification as it possesses very modest functional characteristics. Succinylated Brazil nut kernel globulin at 55.8%, 62.4% and 72.0% level showed a positive effect on functionality. The solubility of acylated globulin was improved above pH 4.0 butwas reduced in the pH range of 3.0-4.0. Water absorption (1.96-4.00, 4.12, and 4.21 ml/g protein), oil absorption capacity (1.44-2.72, 2.80 and 2.94 ml/g protein) and apparent viscosity of the succinylated globulin increased with increase in the level of succinylation. The extent of modi-fication also influenced emulsifying capacity, which showed a decrease at pH 3.0, but was increased at pH 5.0, 7.0 and 9.0. Highest emulsion activity (approximately 63.0%) was observed at pH 3.0, followed by pH 9.0 and pH 7.0 and, least (about 11.8%) at pH 5.0. Emulsion stability also followed similar behaviour as that of emulsion activity. The improved functional properties of succinylated Brazil nut kernel globulin could be explored in a variety of food formulations such as high protein drinks, soups, bakery and meat products as well as in salad dressings and mayonnaise as an emulsifier.     

Purification and Characterization of a Fucoidanase (FNase S) from a Marine Bacterium Sphingomonas paucimobilis PF-1

The Search for enzyme activities that efficiently degrade marine polysaccharides is becoming an increasingly important area for both structural analysis and production of lower-molecular weight oligosaccharides. In this study, an endo-acting fucoidanase that degrades Miyeokgui fucoidan (MF), a sulfated galactofucan isolated from the sporophyll (called Miyeokgui in Korean) of Undaria pinnatifida, into smaller-sized galactofuco-oligosaccharides (1000–4000 Da) was purified from a marine bacterium, Sphingomonas paucimobilis PF-1, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose column chromatography, and chromatofocusing. The specific activity of this enzyme was approximately 112-fold higher than that of the crude enzyme, and its molecular weight was approximately 130 kDa (FNase S), as determined by native gel electrophoresis and 130 (S1), 70 (S2) and 60 (S3) kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of FNase S were pH 6.0–7.0 and 40–45 °C, respectively. FNase S activity was enhanced by Mn²⁺ and Na⁺ (115.7% and 131.2%), but it was inhibited by Ca²⁺, K⁺, Ba²⁺, Cu²⁺ (96%, 83.7%, 84.3%, and 89.3%, respectively), each at 1 mM. The K_m, V_max and K_cat values of FNase S on MF were 1.7 mM, 0.62 mg·min⁻¹, and 0.38·S⁻¹, respectively. This enzyme could be a valuable tool for the structural analysis of fucoidans and production of bioactive fuco-oligosaccharides.     

东北地区5个大豆品种球蛋白含量的分析及利用

利用线性梯度SDS—PAGE方法分析东北地区(黑龙江省、吉林省和辽宁省)11个地点的5个大豆品种的7S和11S球蛋白含量。结果分析表明：7S和11S含量呈显著负相关关系。同一品种7S球蛋白和11S球蛋白含量在不同地区差异较大；7S和11S球蛋白含量在品种间和地点间差异显著或板显著，地点和品种间互作则不显著；利用11S／7S值可以评价大豆蛋白质营养品质的优劣。5个大豆品种中九农7714和东农42品质较佳。     

Characterization of HMW glutenin subunits in common wheat and related species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

The sample preparation method of high molecular weight glutenin subunits (HMW-GS) for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, without a separation step, by high-performance liquid chromatography (HPLC) was established in this study. Three major factors influencing mass spectra—the ratio of components of the solvent, the resolving time, and the sample volume—were optimized using HMW-GS mixtures extracted from Chinese cultivar Jing 411. The results showed that the optimized method for sample preparation was to resolve HMW-GS from 20 mg in an hour with 50 μl solvent of 0.4% TFA, 30.0% ACN and 69.6% H₂O. The stable mass spectra and accurate molecular weights of 16 major HMW glutenin subunits from common wheat and related species were obtained using the optimized MALDI-TOF-MS method. Seven subunits, where each was from 2–5 cultivars, showed very similar molecular weights. The determined molecular weights of 11 subunits were close to those calculated from their coding sequences. In addition, no positive reaction between HMW-GS and GelCode^® Glycoprotein Staining Reagent was observed. These results suggested that HMW-GS lack extensive post-translational modifications (PTMs), but low levels of glycosylation or phosphorylation present in some subunits cannot be ruled out. Because of its ability to obtain a rapid, complete and precise profile of HMW glutenin subunits without purifying procedures, MALDI-TOF-MS is expected to be a powerful technique for structural and functional studies of HMW glutenin subunits as well as other cereal proteins.     

Raman spectroscopic study of rice globulin

The conformation of rice globulin was studied by Fourier-transform Raman spectroscopy as influenced by different buffer environments and heat treatments. The Raman spectrum of the native protein showed a predominance of α-helical structures as indicated by major amide I and III bands at 1657 and 1270 cm⁻¹, respectively. Highly acidic and alkaline pH conditions induced band shifts and intensity changes in amide I, amide III, and C–H (bending and stretching) vibrations, indicating protein denaturation. Addition of dithiothreitol and β-mercaptoethanol led to changes in S–S stretching vibration, whereas ethylene glycol and urea caused marked changes in tryptophan, tyrosine Fermi doublet and C–H band intensities. Heating at 100 °C resulted in progressive denaturation as indicated by band shifts and intensity changes of major spectral regions. Our results revealed that hydrophobic interactions and disulfide bonds play a major role in stabilizing the conformation and in thermal aggregation of rice globulin.     

椰麸组分蛋白的氨基酸组成、结构与乳化性分析

为提高椰子加工副产物的利用,本研究从椰麸中依次提取4种组分蛋白,包括清蛋白、球蛋白、谷蛋白-1和谷蛋白-2.分析这4种组分蛋白的氨基酸组成,并利用SDS-PAGE技术分析其亚基组成和分子量.同时分析其表面疏水性,并研究不同离子强度对椰麸各组分蛋白的乳化性和乳化稳定性的影响.结果表明,椰麸蛋白质中清单白、球蛋白、谷蛋白-...     

气象因子对大豆主要贮藏蛋白组分及亚基含量的影响

以20个大豆品种为材料，同年同地点分六个播期种植，考察了整个生长期以及营养生长和生殖生长两个时期的气象因子(总积温、总日照时数、总降水量、日均温差和日均相对湿度)，对春大豆和夏大豆籽粒总蛋白以及主要贮藏蛋白组分、亚基相对百分含量的影响。结果表明：(1) 大豆籽粒总蛋白含量以及组分、亚基相对百分含量会随播期的改变而变化。(2)整个生育期间，5个气象因子极显著影响20份大豆总蛋白含量，对春大豆总蛋白含量的影响也达极显著水平，仅总积温、总日照时数和总降水量对夏大豆总蛋白含量影响达显著水平。(3)整个生育期的总积温和总日照时数与20份大豆蛋白11S相对百分含量和11S/7S比值显著负相关，总积温与20份大豆蛋白7S含量显著正相关；但气象因子对春、夏大豆的7S、11S相对百分含量和11S/7S比值的影响不显著。(4)整个生育期间气象因子对各组分亚基的影响程度存在差异；夏大豆各蛋白组分亚基的相对百分含量受两个生育时期的气象因子的影响皆不显著，而春大豆许多蛋白亚基相对百分含量受两个生育时期的气象因子的影响达到显著或极显著水平。     

Extractability and chromatographic separation of rice endosperm proteins

The molecular weight (MW) distribution of proteins extracted with different solvents from defatted rice endosperm was examined by size exclusion-high performance liquid chromatography (SE-HPLC) with 2.0% sodium dodecyl sulfate (SDS) (w/v) as mobile phase. The resulting protein peaks were further characterized by SDS-PAGE. Under the experimental conditions, 2.0% SDS extracted 64% of the proteins. Adding 6.0 M urea resulted in a 15% increase in extractability (up to 79%). With using 20–100 mM NaOH, 70–81% of the proteins were extractable. Maximum extractability was reached with 2.0% SDS, 6.0 M urea and 0.5–1.5% dithiothreitol (DTT). Apparent MW profiles of rice endosperm proteins allowed classification into six fractions of decreasing apparent MW. Fraction VI contained the low MW albumin, globulin, and prolamin protein material. Fractions IV and V originated from α and β glutelin subunits, respectively. The polypeptides of fraction III consisted of an α and a β subunit linked by an intermolecular disulfide bond. The polypeptides of fractions I and II were dimers, trimers or more highly polymerized forms of the (α–β) glutelin subunit dimer in fraction III. While the work confirmed that rice glutelin is composed of polymers of α and β subunits, remarkably, higher MW glutelin aggregates (fractions I–III) only partly dissociated on reduction. Low MW protein material (fraction VI) was entrapped in the aggregated protein network and was released on reduction. The rapid and reproducible SE-HPLC method developed for rice protein separation allows a more quantitative approach than SDS-PAGE.     

Identification and Biological Characterization of Angiogenic and Tumor Growth Inhibitors derived from Sinica cetorhinus maximum Cartilage

Shark (Sinica cetorhinus maximum) cartilage was extracted in 1 mol/L Gu- HCl guanidine. Two purified active proteins with apparent molecular weights of 15.2×10³ Da and 8.0×10³ Da (designated as Sp15 and Sp8, respectively) were obtained through ultrafiltration and Superdex 75 chromatography. The activities of the samples were studied in terms of their potential inhibition of vascular endothelial cell growth in vitro, of angiogenesis both in rabbit cornea and chick embryo chorioallantoic membrane (CAM) assay models in vivo, and of growth of transplanted S180 sarcoma in mice in vivo. The results showed that Sp15 expressed a typical lysozymatic activity up to 223,000 U/mg and its N-terminus was highly homologous to lysozymes of various mammalian origins. Sp15 exhibited a strong anti-angiogenic activity only in vitro, whereas Sp8 shared this effect both in vitro and in vivo. Both Sp15 and Sp8 provided an effective anti-tumor activity in mice bearing transplanted S180 sarcoma. These results suggest that Sp15 is a shark cartilage-derived lysozyme that participates in the defense to bacterial invasion to the body, while Sp8 is an angiogenic inhibitor that mediates at least part of the anti-tumor activity associated with shark cartilage probably through the inhibition of tumor-induced angiogenesis.     

小麦面粉蛋白质量评价模型构建及蛋白质量再评价

小麦面粉蛋白的含量和类型决定着小麦面粉的加工品质。为量化比较小麦面粉蛋白对品质影响的差异,以11个不同品质类型的品种为材料,分析了面粉蛋白巯基集团与面粉质量的相关性,发现自由巯基含量与面团稳定时间有极显著正相关性,与面筋指数有显著正相关性;基于面粉蛋白的自由巯基和分子内二硫键含量差异,建立了一个简单的品质贡献量化评价模型;依托蛋白质巯基预测结果,对90个不同类型的面粉蛋白的品质贡献进行了量化比较。结果表明,高分子量麦谷蛋白亚基中得分较高的是1Dy10、DX5和1Dy3;低分子量麦谷蛋白亚基中,位于 Glu-B3、 Glu-D3位点的蛋白得分达到7.2分,高于高分子量麦谷蛋白最高分的1Dy10(6.3分)。因为低分子量麦谷蛋白在面粉中的含量远超高分子量麦谷蛋白,推测面团强度的主要决定因素是低分子量麦谷蛋白,而不是传统观点认为的高分子量麦谷蛋白亚基。另外,一些燕麦类似蛋白和部分醇溶蛋白也对面团强度有一定贡献。     

Characterization of seed proteome in Brachypodium distachyon

Brachypodium distachyon, an emerging model plant system for some economically important temperate grasses such as wheat, barley and switchgrass, has recently caught wide attention in modern biological research. In the current study, the glutenin, albumin and globulin components of 13 B. distachyon accessions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) followed by peptide mass finger printing (PMF) and MS/MS protein identification. Abundant wheat low molecular weight glutenin subunit (LMW-GS) like proteins and a few high molecular weight glutenin subunits (HMW-GS) with low expression level were detected in B. distachyon. A total of 18 storage proteins and 15 albumin proteins were identified through PMF and MS/MS. The results demonstrated that the major seed storage proteins in B. distachyon are wheat LMW-GS like proteins and globulins. The identified albumins and globulins were mostly various enzymes that were classified into five groups according to their functions. The 2-DE spot distribution and MS results suggested that post-translational modifications (PTMs) such as phosphorylations and glycosylations are common phenomena in B. distachyon seed proteome.     

提取条件对大豆7S和11S球蛋白凝胶性的影响

以低温脱脂大豆粕为原料,采用碱溶酸沉法浸提7S和11S球蛋白,用凝胶特性来评价提取条件对7S和11S球蛋白的影响。研究表明,提取7S和11S球蛋白时,浸提时间、浸提温度、pH值以及静置条件对蛋白质的结构均有一定的影响。通过质构仪进行物性测定进而分析蛋白质凝胶特性,得到了在浸提温度为45℃、浸提时间为40 min、11S球蛋白的酸沉pH值为6.4、7S球蛋白的酸沉pH值为4.7、4℃静置的条件下,7S和11S球蛋白产生的凝胶其硬度和弹性均处于较佳的状态,扩大了其在食品中的可应用性。