High-performance liquid chromatography of reduced glutenin: Amino acid composition of fractions and components

Summary Reduced glutenin is separated by gel permeation high-performance liquid chromatography into three major and five minor fractions, which significantly differ in their amino acid compositions. By reversed-phase high performance liquid chromatography, about 20 glutenin components are obtained. These can be classified into three groups according to their amino acid compositions: a hydrophilic group with relatively high values of Glx and Phe, a more hydrophobic group with a high content of Gly, and a strongly hydrophobic group with higher values of Val and Leu. Groups 1, 2 and 3 contain middle-, high-and low-molecular-weight (MMW-, HMW-, LMW-) subunits respectively.

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Hochleistungsflüssigchromatographie von reduziertem Glutenin: Aminosäurezusammensetzung von Fraktionen und Komponenten

Zusammenfassung Reduziertes Glutenin wird durch Gelpermeations-Hochleistungsflüssigchromatogra-phie in drei Haupt- und 5 Nebenfraktionen aufgetrennt, die sich in der Aminosaurezusammensetzung deutlich unterscheiden. Durch Umkehrphasen-Hochleistungsflüssigchromatographie werden etwa 20 Gluteninkomponenten erhalten, die anhand der Aminosäurezusammensetzung drei Grundtypen zugeordnet werden können: Eine hydrophile Gruppe mit relativ hohen Werten für Glx und Phe, eine Gruppe mittlerer Hydrophobität mit einem hohen Anteil an Gly sowie eine hydrophobe Gruppe mit einem höheren Gehalt an Val und Leu. Gruppe 1 enthält Subeinheiten mittleren Molekulargewichts (MMW-Subeinheiten), Gruppe 2 hochmolekulare (HMW-) und Gruppe 3 niedermolekulare (LMW-) Subeinheiten.

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Supported by a grant from AIF The authors are grateful to Mrs. Mosler, Mrs. Redler and Mrs. Schützler for excellent assistance     

Isolation and partial characterisation of two low molecular weight durum wheat (Triticum durum) glutenins

Two low molecular weight durum wheat (Triticum durum Desf) glutenins, DSG-1 and DSG-2 (durum-wheat sulphur-rich glutenin fractions) were isolated from two cultivars, Mondur of good technological quality and Kidur of poor technological quality. The glutenin fraction, composed mostly of DSG protein, was extracted using a low concentration of NA-tetradecanoate and then fractionted by by using molecular sieve chromatography (Bio-Gel P 30). The amino acid compositions and the N-terminal sequences of the pure DSG proteins were determined, and their hydrophobic characteristics, calculated on the basis of these data, showed that DSG-2 is more hydrophobic than DSG-1. The amino acid compositions of DSG-1 and DSG-2 were different. The N-terminal amino acids of DSG-1 and DSG-2 were also different but were identical for each of the two cultivars. In the case of DSG-1, in addition to the main chain a minor chain was found in which the first three amino acids of the main chain were missing. The minor chain represented about 30% int he DSG-1 of mondur and almost 50% in Kidur.     

紫米蛋白-多酚复合物的结构组成及分子形貌

为研究紫米中蛋白-多酚复合物的分离纯化及其结构组成与分子形貌,采取Osborne分级提取从紫米中得到紫米蛋白-多酚复合物,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析其分子质量,邻苯二甲醛柱后衍生法分析比较紫米蛋白-多酚复合物以及大米蛋白的氨基酸组成,高效液相色谱-二极管阵列检测器-质谱联用技术分析鉴定紫米蛋白-多酚复合物中花色苷聚合物的基本单体组成,并利用原子力显微镜观察紫米蛋白-多酚复合物的表面形貌特征。结果表明,紫米蛋白-多酚复合物的蛋白质质量分数为（94.3±2.7）%,多酚含量为（10.1±4.0） mg/g;紫米蛋白-多酚复合物与大米蛋白在37 ku以上分子质量处亚基组成存在明显差异;复合物的氨基酸组成更加丰富,有更优的必需氨基酸组成;复合物中花色苷单体组成为矢车菊素-3-O-葡萄糖苷和芍药素-3-O-葡萄糖苷;紫米蛋白-多酚复合物以不规则球形结构为主,其结合单元的粒径大约为4.7 nm左右。本实验对后续探索有色谷物多酚复合物的功能特性、多酚-蛋白质相互作用与生物活性等提供了科学依据。     

丙酮酸肌酸对大鼠胴体组成及机体氨基酸谱的影响

以大鼠为实验对象,灌胃丙酮酸肌酸(CrPyr),通过观测其对机体胴体组成及其血清与组织中氨基酸谱的影响,探讨丙酮酸肌酸对机体中营养物质的代谢流向及其胴体品质的影响。实验选用40 只雄性清洁级SD 大鼠,随机分为对照组(C)、低剂量组(L)、中剂量组(M)和高剂量组(H)4 个处理组,每个处理组按体质量分别灌胃0、750、1500、3000mg/(kg bw·d)的CrPyr。实验为期7 周,第49 天宰杀大鼠,借助反相高效液相色谱(RP-HPLC)方法测定血清、组织中氨基酸含量的变化。结果表明：与对照组相比,在不影响饲料转化率的情况下,灌胃 CrPyr可显著(P ＜ 0.05) 地提高其腿肌的相对质量;灌胃 CrPyr 可显著(P ＜ 0.05)或极显著(P ＜ 0.01)升高大鼠血清和骨骼肌中主要生糖氨基酸的含量,但对肝脏组织的氨基酸谱则没有明显影响。提示,灌胃CrPyr 可能通过升高生糖氨基酸的含量,从而促进大鼠体蛋白的合成。     

离子色谱在中药分析中的应用研究

离子色谱法是用于离子化合物分离的高效液相色谱技术,能够实现中药材中多种强极性物质的定性检测和定量分析,具有操作简便、灵敏度高、选择性好等优点。本文总结了近年来离子色谱在中药亚硫酸盐残留、有机酸、氨基酸和糖类、无机阴离子和金属离子含量分析等多个方面的应用,阐述了离子色谱在中药分析领域的最新进展。     

γ-氨基丁酸检测方法的比较

本文对检测γ-氨基丁酸的纸层析法、高效液相色谱法和氨基酸分析仪法进行了全面的比较研究.结果表明纸层析法检测γ-氨基丁酸,在使用展开剂V（正丁醇）∶V（冰醋酸）∶V（水）=4∶1∶3的条件下,具有简单、快速等优点,但精密度较低.高效液相色谱法以邻苯二甲醛作为衍生剂,用反相C18柱为分离柱,精密度较高,准确度较好,但是操作过程相对繁琐.氨基酸分析仪法采用茚三酮柱后衍生,稳定性好、灵敏度高、操作简便,但要求样品氨基酸浓度在100nmol/mL范围内,使用费用高.     

鳕鱼酶解液苦味肽的纯化鉴定

本文利用DA201-C大孔树脂对鳕鱼肉碱性蛋白酶酶解液进行脱盐后,用SP Sephadex C-25阳离子交换层析和反相高效液相色谱等方法对鳕鱼肉酶解液进行分离纯化,最终得到了浓度较高的两种苦味肽。将得到的苦味肽采用液相色谱-飞行时间串联质谱(LC-TOF-MS/MS)技术对多肽结构进行鉴定,确定其氨基酸序列分别为:HWPWMK(His-Trp-Pro-Trp-Met-Lys)和AVVLII(Ala-ValVal-Leu-Ile-Ile)。同时本文采用感官评定与多肽Q值相结合的方法对苦味进行评价,通过氨基酸分析仪对苦味样品进行氨基酸组成的测定,得出具有苦味的C7和S9组分的Q值分别为6.03 k J/mol和6.79 k J/mol,通过感官评定得出其苦味强度为:9±0.8和8.2±1.6。鉴定得到的苦味肽S9purity和S10purity的Q值分别为:8.85 k J/mol和8.53 k J/mol,苦味强度为:9.4±0.8和8.9±0.9。本文从鳕鱼肉酶解液中分离纯化得到的苦味肽为苦味肽家族增添了新成员,为下一步的研究提供了原料和实验基础。     

A Compositional Study of Amaranth Grain

The chemical composition of ten amaranth seed samples was determined. The saccharide content was determined using gas chromatography and high performance liquid chromatography. Sucrose was the major sugar followed by raffinose. Inositol, stachyose, and maltose were found in small amounts in most of the samples. Autolysis for 16 hr at pH 5.0 and 6.5 resulted in decreased sucrose and raffinose concentrations. Maltose was liberated by autolysis at pH 6.5 but not at pH 5.0. Inositol increased after autolysis. It was concluded that invertase, amylase, and phytase occur in the grain. Physicochemical properties of isolated amaranthus starch were measured and compared with analogous values reported for wheat starch. The lipids from representative amaranth grain varieties were analyzed for fatty acid composition. Squalene was present in the oil in large amounts, compared to other grains. The amino acid composition of the grain was used to calculate the chemical score (73) and the nitrogen to protein conversion factor (5.85). Leucine was found to be the limiting amino acid. Tannin and vitamin levels typical of other grains were detected. Mineral and proximate compositions were similar to previously reported values.     

Amino acid analyses of glutenins and gliadins

The amino acid compositions of glutenins and gliadins from two strong and two weak wheats have been compared. Glutenin appears to have an amino acid analysis generally similar to that of gliadin but there are individual differences. Glutenin possesses much higher proportions of lysine, glycine and tryptophan and somewhat higher proportions of arginine, tyrosine, threonine, (aspartic acid + asparagine), serine and alanine. Gliadin is richer in proline, (glutamic acid + glutamine), cystine, phenylalanine, amide nitrogen and isoleucine. No definite differences were observed in contents of methionine, valine, leucine or histidine. The possible significance of these results in explaining the properties of glutenin and gliadin is discussed, and the results are compared on a diagram with some analyses in the literature. No significant differences in amino acid composition from variety, or from strong wheats as against weak, were observed among glutenins or gliadins. The calculated factor for converting weight of nitrogen to glutenin or gliadin is 5.6. The mean percentage of (glutamic + aspartic) acids in the amide form is 82 for glutenin and 92 for gliadin.     

减少后酵固形物对黄酒品质及风味的影响

为改善传统酿造黄酒的风味,采用减少固形物方法酿造黄酒,并用高效液相色谱、固相微萃取气相色谱质谱（SPME-GC-MS）联用法比较了传统黄酒和减少后酵固形物的黄酒之间常规理化成分、氨基酸含量、风味成分等。结果表明,减少后酵固形物方法酿造黄酒,对黄酒常规成分影响不大,能改善黄酒风味。     

蚕蛹蛋白及其水解产物中氨基酸组成分析

采用FMOC-OPA柱前衍生化-高效液相色谱法测定蚕蛹蛋白及水解液氨基酸含量,同时测定血管紧张素转化酶(ACE)抑制活性,研究蚕蛹蛋白水解后氨基酸组成的改变及对ACE抑制活性的影响。结果表明:水解液10kD以下组分(SN2)中疏水性氨基酸含量为49.15%,显著高于10kD以上组分(SN1)中的40.55%及蚕蛹蛋白中的45.21%,其中对ACE抑制活性影响较大的脯氨酸、芳香族氨基酸含量为7.75%和19.20%,高于SN1中的5.62%和13.59%及蚕蛹蛋白中的7.42%和15.81%,并且SN2中ACE抑制率为47.6%显著高于SN1和蚕蛹蛋白中的抑制率,表明水解后疏水性氨基酸组成的改变与ACE抑制活性的变化趋势相同,且ACE抑制活性主要集中于SN2。     

Pilot scale preparation of wheat gluten protein fractions I-Influence of process parameters on their protein composition

Commercial wheat gluten was fractionated on a pilot plant scale to produce gliadin-and glutenin-rich fractions. Acetic acid solutions (0.01–0.05 M) were blended with dry gluten (16, 10 or 7 volumes unit^-1 of dry gluten ratio) and the slurry was separated by continuous centrifugation to yield a gliadin-rich supernatant, which was then concentrated by ultrafiltration and spray-dried. the pellet obtained was optionally rinsed by water or acetic acid and separated in a second stage. the second supernatant was treated as the first one, and the final residue, insoluble and glutenin-rich, was then dispersed in ammonia and spray-dried. the protein distributions in supernatants and residues depended on pH and ranged from 20/80 to 40/60 for the first stage, and from 40/60 to 80/20 for the sum of the two stages. the compositions of the fractions were measured by solubility tests and size exclusion high performance liquid chromatography (SE-HPLC) profiles. A [-1, +1] selectivity scale was created: each fraction was compared to pure gliadins (-1), gluten (0) and pure glutenins (+1). the best combination between yields of soluble and insoluble fractions (40/60 distribution) and selectivities for gliadins and glutenins (-0.32 and +0.26 respectively) was obtained at ratio 16 and a 0.01 M molarity. Though the selectivities were limited because gliadins and medium-size glutenin polymers have similar solubilities in acid solutions, this process permits preparation of fractions which differ widely from gluten in contents of high molecular weight glutenin polymers.     

Fractionation and Characterization of the Macromolecular Meaty Flavor Enhancer from Beef Meat Extract

ABSTRACT: Macromolecular meaty flavor enhancer was fractionated from a commercial beef extract. The macromolecular fraction was obtained by dialysis and separated by anion-exchange chromatography, Cu²⁺-chelate chromatography, and gel filtration chromatography. Two fractions were isolated as active meaty flavor enhancers. To elucidate the partial structures, the active fractions were treated with endoprotease Glu-C followed by high-performance liquid chromatography (HPLC) separation of the peptide fragments. Determinations of the amino acid compositions and amino acid sequences of the isolated fragments showed that the 2 active fractions consisted of collagen and tropomyosin. The macromolecular material obtained from heated collagen and tro-pomyosin in the low-molecular-weight fraction of beef soup stock enhanced the meaty flavor. These results suggested that collagen and tropomyosin were precursors of the macromolecular meaty flavor enhancer.     

LTQ-Orbitrap 液- 质联用技术对水牛奶酪蛋白的鉴定

建立一种快速、高效的分析酪蛋白组分氨基酸序列的LTQ-Orbitrap质谱方法。为了研究水牛奶酪蛋白、乳牛奶酪蛋白和山羊奶酪蛋白的氨基酸序列组成及氨基酸的替换现象,采用LTQ-Orbitrap液-质联用技术分别对乳源酪蛋白的4种主要组分进行分析,搜索数据库获得4种组分的氨基酸全序列。与水牛奶4种酪蛋白组分进行比对。结果表明,乳牛奶的4种酪蛋白发生氨基酸替换的部位和比率明显小于山羊奶。这意味着与水牛奶酪蛋白相比,乳牛奶酪蛋白氨基酸的稳定性优于山羊奶酪蛋白。     

质谱技术在氨基酸分析方法中的应用

氨基酸分析方法是测定含游离氨基酸,多肽或蛋白质样品中氨基酸含量的方法。在过去几十年中,氨基酸分析主要是由经典的柱后衍生-离子交换色谱和柱前衍生-高效液相色谱完成的。随着现代氨基酸分析技术的发展,高通量和高灵敏性的质谱被广泛应用于氨基酸分析中,并发挥了重要的作用。作者就近年来国内外气相色谱、液相色谱和毛细管电泳与质谱联用技术在氨基酸分析中的应用进行了综述并比较了这几种氨基酸分析技术。     

Extractability and Chromatographic Characterization of Wheat (Triticum aestivum L.) Bran Protein

About 70% of the protein for human consumption is derived from plants, with cereals as the most important source. Wheat bran protein has a more balanced amino acid profile than that of flour. We here for the first time report the amino acid, size exclusion, and SDS‐PAGE profiles of bran Osborne protein fractions (OPFs). Moreover, we also investigated how OPFs are affected when physical barriers which entrap proteins in bran tissues are removed. Albumin/globulin is the most abundant OPF. It is richer in lysine and asparagine/aspartic acid than other OPF. Most bran albumin/globulin proteins have a molecular weight (MW) lower than 30 k and their chromatographic profiles differ from those of flour. The prolamin has high levels of proline and glutamine/glutamic acid. It is rich in proteins with a MW of 30 to 45 k and about 66 k reflecting contamination with gliadin from endosperm. The glutelin has high levels of glycine, proline, and glutamine/glutamic acid. Its protein is of intermediate and high MW with little protein with MW lower than 30 k. The high (MWs from 80 to 120 k) and low (MW around 45 k) MW glutenin subunits of flour are also present in bran. The glutelin of wheat endosperm is named glutenin. Ball milling releases albumin/globulin and glutelin but not prolamin. Not all glutelin was endosperm glutenin as a substantial part was entrapped in the aleurone cells.     

刺参水煮液糖蛋白组成成分分析

目的：刺参水煮液是刺参加工过程中的副产物,通过对刺参水煮液营养成分的分析有助于将这种海产珍品加工废弃物转化为高附加值产品。方法：刺参水煮液经醇沉、透析、冻干,得到刺参水煮液糖蛋白（glycoproteinin sea cucumber boiling water,GSCB）粗制品;采用液相色谱和SepharoseCL-6B凝胶层析柱确定其糖蛋白组成成分及分子质量;采用液相色谱-质谱对1-苯基-3-甲基-5-吡唑啉酮（1-phenyl-3-methyl-5-pyrazolone,PMP）衍生化的GSCB单糖组成进行分析;采用液相色谱法对其氨基酸组成进行分析。结果：GSCB主要含有2 种糖蛋白,分子质量分别为964.3 kD和2.5 kD。GSCB含有氨基葡萄糖、甘露糖、氨基半乳糖、葡萄糖、半乳糖、岩藻糖6 种单糖;含有18 种氨基酸,其中含有8 种人体必需氨基酸,占氨基酸总量的38.38%。此外,半必需氨基酸组氨酸的含量在18 种氨基酸中占比最高,占比达到13.21%。结论：GSCB中含有酸性黏多糖物质,并含有多种必需氨基酸和半必需氨基酸,是一种良好的营养糖蛋白物质。对其深入开发将有利于变废为宝,开发出活性良好的功能保健食品。     

游离氨基酸检测方法及其应用

游离氨基酸是动植物体中重要的活性成分和风味物质,随着现代科学技术的进步,游离氨基酸的检测方法多样且应用研究进展迅速,汇总整理各类检测方法及其相关应用不可或缺。综述了常用于检测分析游离氨基酸的分光光度法、离子色谱-积分脉冲安培法、氨基酸分析仪法、高效液相色谱法和液相色谱-串联质谱法5种方法,以及2种新技术,分析了各检测方法的原理及优缺点,并对各检测方法列举了相关应用研究。现代新型技术的发展和推广,多元化离子化方式、高分辨质谱、多级串联质谱等技术将会使游离氨基酸分析的灵敏度、准确度、分析速度及自动化程度均提高到一个崭新的水平,选择性好、准确度高、前处理简便的检测手段将是游离氨基酸检测分析技术的未来发展趋势。     

Free amino acids and dipeptides in porcine muscles: differences between `red' and `white' muscles

Three porcine muscles (m. longissimus dorsi, masseter and trapezius), chosen to represent the three main metabolic types, from 18 carcasses had their free amino acids and dipeptides quantified by reverse-phase high performance liquid chromatography (HPLC) in aqueous extracts derivatized with phenyl isothiocyanate. Of the 25 measured compounds, four amino acids and the dipeptide carnosine were closely related to the metabolic type of the three muscles. Masseter, a red oxidative muscle, had the highest contents of aspartic acid, glutamine and taurine. Longissimus dorsi, a white glycolytic muscle was characterised by the highest contents of β-alanine and carnosine. Trapezius, an intermediate muscle, had intermediate contents. These results show that free amino acid and dipeptide contents could partly explain differences in taste of muscles from the same species.     

Purification and characterisation of angiotensin I converting enzyme (ACE) inhibitory peptides derived from enzymatic hydrolysate of ovotransferrin

Three novel peptides, IQW, IRW and LKP, were predicted in our previous study in the thermolysin–pepsin ovotransferrin hydrolysate. The aims of the present study were to purify the peptides, and determine if the predicted peptides purified from the hydrolysate would have the same activity as the synthetic ones. We also determined the stability of the peptides under simulated gastrointestinal condition. IQW, IRW and LKP were then successfully purified from crude ovotransferrin hydrolysate through multi-step chromatographic purification comprising of cation exchange chromatography followed by three-step reverse-phase high performance liquid chromatography (RP-HPLC), and their sequences were analysed by UPLC-MS/MS. Our results showed that their activities were comparable to the synthetic ones. Simulated gastrointestinal incubation showed that IRW was degraded into a dipeptide of IR and a free amino acid of W by pancreatin, LKP was degraded into a dipeptide of KP and a free amino acid of L by mucosal peptidase, while IQW was stable against the digestive enzymes.