A comparative study of physicochemical and conformational properties in three vicilins from Phaseolus legumes: Implications for the structure–function relationship

The polypeptide constituents, physicochemical and conformational properties of three vicilins from kidney, red and mung beans, fractionated using combined acid extraction and ion-exchange chromatographic techniques, were characterized and compared. The tested physicochemical and conformational properties included amino acid composition, surface charge and hydrophobicity, protein solubility (PS; as a function of pH), emulsifying activity index (EAI) and emulsion stability index (ESI), as well as secondary, tertiary and/or quaternary conformations. The amino acid composition (especially relative acidic/basic charged amino acid ratio, and the percentage of polar uncharged amino acids) was an intrinsic physicochemical parameter that largely determined other physicochemical and conformational properties. The PS and EAI (and ESI) of these vicilins at various pH values were associated with each other, and both were closely related to their surface charge and/or hydrophobicity. Far-UV CD spectral analyses indicated that these vicilins exhibited similar secondary structure compositions at pH deviating from the isoelectric point (about pH 4.5). However, their tertiary and/or quaternary conformations, as determined by differential scanning calorimetry, intrinsic fluorescence and near-UV CD spectroscopy, remarkably varied. At pH 7.0 or 9.0, there were close relationships between the EAI and the flexibility in quaternary conformation, and between the ESI and the flexibility in tertiary conformation. This is the first observation to elucidate the importance of tertiary and/or quaternary conformations for the emulsifying properties of legume vicilins. Additionally, an underlying mechanism of the emulsification process for the vicilins (trimeric form) with excellent emulsifying properties was proposed.     

The amino acid composition,solubility and emulsifying properties of sweet potato protein

A protein was purified from the high-protein type sweet potato variety 55-2 available in China. The amino acid composition, solubility and emulsifying properties of the sweet potato protein (SPP) were studied. The SPP was rich in aspartic acid (18.5%) and glutamic acid (9.30%) while essential acid amino acids made up approximately 40.7% of the SPP. The SPP was highly soluble in distilled water over a wide range of pH. However, solubility of the SPP in 1.0 M NaCl and 1.0 M CaCl₂ solutions was low especially at pH below the pI of the SPP. The SPP in CaCl₂ demonstrated emulsifying activity index (EAI) and emulsion stability index (ESI) many folds higher than those in distilled water and NaCl solution (P < 0.05).     

3 种方法提取的澳洲坚果蛋白功能性质与构象的关系

采用3?种蛋白提取方法从澳洲坚果脱脂粉中提取得到3?种分离蛋白,包括碱提酸沉澳洲坚果蛋白（alkaline extraction and acid precipitation extracted macademia protein isolate,MAPI）、三（羟甲基）氨基甲烷提取澳洲坚果蛋白（tris (hydroxymethyl) aminomethane extracted macademia protein isolate,MTPI）以及氯化钠提取澳洲坚果蛋白（sodium chloride extracted macadamia protein isolate,MSPI）,并对其功能和构象进行比较研究。研究发现,在酸性pH值范围内MSPI比MAPI和MTPI有更高的溶解性,而在碱性pH值范围内恰好相反。MSPI比MAPI和MTPI具有更高的乳化活性指数。MAPI、MTPI和MSPI的蛋白质量分数分别为88.9%、80.3%和86.1%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和氨基酸分析表明,3?种分离蛋白具有相似的条带分布和氨基酸组成,但它们的条带强度和氨基酸数量不同。此外,与MAPI和MTPI相比,MSPI具有较高的表面疏水性、荧光强度和较小的粒度。场发射扫描电子显微镜结果表明,MSPI比MAPI和MTPI具有更多的由不同大小球状结构组成的颗粒。     

Role of β-conglycinin and glycinin subunits in the pH-shifting-induced structural and physicochemical changes of soy protein isolate

Soy β-conglycinin (7S) and glycinin (11S) were incubated up to 4 h in acidic (pH 1.5 to 3.5) or alkaline (pH 10 to 12) solutions to induce protein structural unfolding followed by refolding 1 h at pH 7.0, a process known as pH-shifting. The pH-shifting markedly increased (P < 0.05) emulsifying activity of 11S and to a lesser extent 7S; the former also produced more uniform oil droplets. The emulsifying activity improvements were accompanied by a significant rise in protein surface hydrophobicity, slight loss of the secondary structure (circular dichroism), and substantial dissociation of disulfide-linked basic and acidic 11S subunits. The findings suggested that 11S globulins of soy protein isolate (SPI) were more responsive to pH-shifting treatments than were 7S globulins, and the resulting emulsifying activity enhancements of 11S, in parallel with that of SPI, were indicative of its determinant role in the overall emulsifying properties of pH-shifting-treated SPI. PRACTICAL APPLICATION: Extreme alkaline (pH 12) and acidic (pH 1.5) medium treatments can significantly modify the structure and enhance the emulsifying properties of both β-conglycinin and glycinin components of SPI. The functionality improvement by the pH processes is more remarkable for the glycinin protein fraction. Therefore, SPI enriched with glycinin seems to be particularly suitable for extreme acidic or alkaline processes to produce surface-active functional ingredients for food applications.     

Structure–physicochemical function relationships of 7S globulins (vicilins) from red bean (Phaseolus angularis) with different polypeptide constituents

Red bean 7S globulins (vicilins) with different polypeptide constituents or heterogeneity were fractionated using acidic extraction (at 0.5 M NaCl) and anion-exchange column chromatography. The physicochemical and conformational properties, including amino acid composition, net charge and/or surface hydrophobicity (H₀), protein solubility (PS), thermal and emulsifying properties, as well as secondary, tertiary and/or quaternary conformations, were evaluated. There were distinct differences in zeta potential, H₀, DSC characteristics, emulsifying activities and tertiary and/or quaternary conformations among the vicilins with different polypeptide constituents, but the PS and secondary conformation were slightly different. The PS as a function of pH and thermal stability were closely related to their surface charge and/or hydrophobicity. The emulsifying ability and the emulsion stability were closely dependent on the PS and H₀, and even the flexibility in tertiary and/or quaternary conformations. These results suggested good relationships between the physicochemical functions and conformational features of red bean vicilins, which could be of great help for further utilization of legume proteins as a potential functional ingredient.     

Soluble protein fractions from pH and heat treated sodium caseinate: physicochemical and functional properties

The physicochemical (solubility and hydrophobicity), and functional (emulsifying activity index and emulsifying capacity) properties of soluble sodium caseinate fractions were studied as a function of pH (3–8) and temperature (50–100°C). Solubility was determined by measuring protein with the Bradford and 280 nm absorbency methods. Hydrophobicity was determined fluorometrically with 1-anilino-8-naphtalenesulfonate (ANS), and cis-parinaric acid (CPA). Sodium caseinate solubility was minimal at pH 3.75–4 but the ANS and CPA-hydrophobicities and the functional properties of the soluble proteins increased in this pH range. Circular dichroic and 280 nm absorptivity measurements detected conformational changes. SDS-PAGE and reversed phase HPLC revealed substantial losses of αs₁ and β caseins following pH and heat treatment (pH 3.75 and 92.5°C) and the concomitant appearance of modified compounds. Under these same conditions, the o-phtaldialdehyde values increased suggesting partial hydrolysis of sodium caseinate. The soluble protein fractions from sodium caseinate heat treated near the pI of the caseins were shown to have enhanced emulsifying activity and capacity.     

杏鲍菇分离蛋白和清蛋白的理化性质及功能分析

以杏鲍菇为原料,分别利用碱溶酸沉法提取杏鲍菇分离蛋白（Pleurotus eryngii protein isolate,PEPI）、Osborne法分离主要蛋白组分,并研究其理化性质和功能分析。结果表明,杏鲍菇的蛋白质量分数为17.57%（以干质量计）,以杏鲍菇清蛋白（Pleurotus eryngii albumin,PEA）为主,占总分离蛋白组分的81.12%。PEPI和PEA中均含18?种氨基酸,且必需氨基酸含量分别占总氨基酸含量的40.80%和40.51%。与PEPI相比,PEA的表面疏水性（265.25）显著高于PEPI（164.27）（P＜0.05）,而总巯基、二硫键含量较低,分别为61.53?μmol/g和10.39?μmol/g;热变性温度（100.98?℃）低于PEPI（108.27?℃）,且PEA持水性（1.64?mL/g）、持油性（5.59?mL/g）显著低于PEPI（3.58、8.36?mL/g）（P＜0.05）。PEPI和PEA的溶解性、起泡性、泡沫稳定性、乳化性及乳化稳定性随pH值的变化趋势均相似,在等电点时均为最低。傅里叶变换红外光谱显示PEPI和PEA的二级结构主要是β-折叠和β-转角,扫描电镜观察PEPI呈蜂巢结构。相比PEA,PEPI具有更好的理化性质和功能特性。     

Isolation of the 12 S globulin from Rapeseed (Brassica napus L.) and characterization as a “neutral” protein On seed proteins. Part 13

An isolation procedure for the 12 S rapeseed globulin is described which includes precipitation by dialysis, purification using gel chromatography on Sephadex G-200, and ion-exchange chromatography on DEAE-Sephadex A-50. The isolated globulin represents a neutral protein with an isoelectric point at pH 7.25 — determined by isoelectric focusing — and a relation of the acidic to basic amino acid residues (I Glu, Asp — Amide ammonia: ∑ Arg, Lys, His) of 1.0. As in other storage globulins high contents of glutamic (19%) and aspartic (10%) acid and a low content of sulphur containing amino acids are characteristic for the amino acid composition. Amongst the basic amino acids arginine has the highest percentage (7%). Opposite to results of other authors the sugar content of the globulin is low (0.5%). From the amino acid composition an average hydrophobicity according to Bigelow was calculated which amounts to be 1041 cal/res. (4.36 kJ/res.).     

冻融次数对镜鲤鱼肌原纤维蛋白功能和结构特性变化的影响

本实验通过测定肌原纤维蛋白溶解性、乳化性、δ-电势、巯基含量、自由氨基酸含量、二聚酪氨酸含量、表面疏水性和α-螺旋含量的变化,探究反复冻融过程引起镜鲤鱼肌原纤维蛋白功能特性和结构特性的变化。结果表明：当冻融次数增加到5 次时,肌原纤维蛋白的溶解度、乳化活性、乳化稳定性分别显著下降至62.85%、20.67 m2/g、34.83%（P＜0.05）;在冻融过程中随着冻融次数的增加,蛋白质的δ-电势、表面疏水性和二聚酪氨酸含量不断增加,而巯基、自由氨基酸和α-螺旋含量不断下降。蛋白质的这些变化表明,冷冻-解冻循环破坏了镜鲤鱼肌原纤维蛋白的完整结构,降低了蛋白质的功能特性。     

Characterization and formation mechanism of proteins in the secondary precipitate of soy sauce

To reveal the characteristics and formation mechanism of proteins in the secondary precipitate of soy sauce (SPSS), proteins in the supernatant of soy sauce were used as control, and SPSS was prepared by centrifugation in combination with lyophilization. The proteins in SPSS were isolated and identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. Acidic polypeptide A_1a of soy glycinin G1 (32–35 kDa) and basic polypeptide B₃ of soy glycinin G4 (23 kDa) were verified as the predominant proteins (95.2 %) in SPSS. The amino acid composition, average hydrophobicity (HΦ _avg), and the secondary structure of the proteins in SPSS were investigated by high-performance liquid chromatography and Fourier transform infrared spectroscopy. Results revealed that SPSS contained not only proteins but also free amino acids; the significantly higher average hydrophobicity and much lower contents of β-sheet and random coil were the main characteristics and reasons for the formation of proteins in SPSS.     

Protein quality and microbiological changes in aerobically- or vacuum-packaged, irradiated fresh pork loins

The effect of gamma-irradiation on the physicochemical, organoleptic and microbiological properties of pork was studied, during 43 days of storage at 4±1°C. Irradiation treatments were carried out under air or vacuum packaging on fresh pork loins at a dose of 6 kGy, at two different dose rates: 2 kGy/h and 20 kGy/h. The loins were evaluated for protein sulphydryl content and emulsifying capacity, surface hydrophobicity of proteins and sensorial evaluation. Regardless of the type of packaging and dose rate of irradiation, all irradiated pork samples were effectively prevented from bacterial spoilage for at least 43 days. Meat redness and texture of irradiated loins were relatively well preserved during the storage period, especially when samples were stored under vacuum. Overall, the physicochemical and organoleptic changes in pork loins appeared to be relatively little affected by the 6 kGy dose. No marked changes in emulsifying capacity and protein sulphydryl content of proteins were noted throughout the storage period. However, the hydrophobicity was reduced (P?0.05) by the faster dose rate of irradiation and by longer storage.     

3 种有机酸在不同pH值条件下酸沉处理对米糠蛋白结构的影响

以传统碱溶酸沉制备米糠蛋白所用的盐酸为对照,采用酒石酸、苹果酸、柠檬酸3?种有机酸分别在pH?4.0、3.5、3.0、2.0条件下酸沉制备米糠蛋白,研究有机酸酸沉对米糠蛋白结构的影响。结果表明,同种有机酸在不同pH值条件下酸沉,当酸沉pH值下降时,米糠蛋白总巯基含量和α-螺旋相对含量无显著变化,游离巯基含量和无规卷曲相对含量上升,β-折叠和β-转角相对含量下降,内源荧光强度和表面疏水性先增加后下降。相比盐酸酸沉,有机酸在相同pH值条件下酸沉制备米糠蛋白的总巯基、二级结构组成和表面疏水性无显著差异,但游离巯基含量下降、内源荧光强度升高。分子质量分布、粒径分布及电泳结果表明,较低pH值条件下的酸沉可导致米糠蛋白结构发生部分解离和聚集,但没有形成新的亚基和高分子质量聚集体。     

椰子分级蛋白组分制备及理化性质研究

目的 制备椰子分级蛋白组分并研究其理化性质。方法 对椰子蛋白进行分级并制备相应组分,测定椰子蛋白分级组分的氨基酸组成、疏水性、游离巯基含量、溶解度、乳化性和起泡性,以评估其功能特性。结果 椰子蛋白含有18种氨基酸, 富含精氨酸和谷氨酸,必需氨基酸含量丰富、比例合理,是营养良好的蛋白质来源。所有样品在蛋白质的等电点处溶解度最差、起泡性最差而泡沫稳定性却最好。随着pH增加,溶解度整体趋势先降低后增加,乳化能力总体呈现增加的趋势,而较高的pH下表现出更好的乳化稳定性。结论 椰子蛋白具有良好的营养价值及优良的溶解度、乳化性及起泡性,可为椰子蛋白在食品加工中的应用提供理论参考。     

pH值对大豆11S球蛋白结构和表面疏水性的影响

采用Lowry法、8-苯氨基萘-1-磺酸铵盐（8-anilinonaphthalene-1-sulfonic acid ammonium salt,ANS）荧光探针法研究pH值对大豆11S球蛋白的溶解性和表面疏水性的影响,并利用圆二色光谱和荧光光谱对不同pH值条件下11S球蛋白二级结构和三级结构进行分析,为研究大豆蛋白结构与表面疏水性之间的构效关系提供理论基础。结果表明：除等电点外,大豆11S球蛋白溶解性和表面疏水性呈负相关,并且随着pH值的升高,大豆球蛋白二级结构中发生β-折叠和无规卷曲向α-螺旋的转变,三级结构中色氨酸（Trp）残基微环境极性降低。大豆球蛋白的表面疏水性与α-螺旋结构含量呈负相关。     

Physicochemical and functional properties of the protein isolate and major fractions prepared from Akebia trifoliata var. australis seed

The physicochemical and functional properties of protein isolate (API) and major protein fractions prepared from Akebia trifoliata var. australis seed were investigated. The seed contained 38.83% of oil and 17.23% of protein. Albumin (51.65%) and glutelin (46.40%) were the predominant fractions in the protein component of the seed. The major amino acids were found to be glutamic acid and aspartic acid, while the contents of sulphur-containing amino acids and threonine were very low. One to eight distinct bands with molecular weight (MW) ranging from 12.0 to 50.0 kDa were displayed by SDS–PAGE. The solubilities of API, albumin and glutelin from seeds of the A. trifoliata var. australis were the lowest at pH 4.0–5.0. The high surface hydrophobicity indices of these three proteins were observed at pH 7.0, while the excellent emulsifying properties were displayed at pH 9.0. Circular dichroism measurements indicated that API, albumin and glutelin were rich in β-strand and random coil structures.     

Physicochemical characteristics and functional properties of grape (Vitis vinifera L.) seeds protein

The amino acid composition, physicochemical and functional properties of grape (Vitis vinifera L.) seeds protein (GSP) were evaluated and compared with those of soybean protein isolate (SPI). Amino acid analyses of GSP revealed high levels of glutamic/glutamine, glycine and aspartic/asparagines. SDS‐PAGE analysis demonstrated that globulin was the major protein component in GSP, whose subunit molecular weights were mainly varied from 25.5 to 40.0 kDa. The isoelectric pH of GSP was found to be at the acidic pH of around 3.8. At all the pHs tested except pH 2.0, no significant changes of GSP secondary structure were observed. GSP exhibited beneficial functional properties such as preferable solubility and emulsifying activity, while the foaming properties and water holding capacity were relatively poor compared to SPI. It could then be employed to soup, sauce, beverage or meat product for improving nutritional and sensory quality of these foods at appropriate pHs.     

Protein quality and identification of the storage protein subunits of tofu and null soybean genotypes,using amino acid analysis,one- and two-dimensional gel electrophoresis,and tandem mass spectrometry

The protein quality of 11 null and 2 tofu soybean genotypes were determined from their total protein content, their amino acid composition, and their glycinin and β-conglycinin contents. There were highly significant differences (P < 0.001) in their total storage proteins, and amino acid contents. Total protein among these genotypes ranged from 33 to 37%, with arginine being the third highest amino acid (7.4–10.9 g/100 g protein) followed by glutamic and aspartic acids. Methionine accounted for only 1.6–2.4 g/ 100 g of protein. All genotypes contained a good balance of essential amino acids (EAA₉), ranging from 43.5 to 47.3% of the total protein, limited only in methionine and possibly threonine and valine. Two-dimensional gel electrophoretic (2-DE) reference maps, using narrow range immobilized pH gradient (IPG) strips, revealed unique differences in the proteome, and subunit expression of glycinin and β-conglycinin, among these null genotypes, which can then be correlated with their protein quality. Out of a total of 111 basic (pH 6–11), and 223 acidic (pH 4–7) protein spots separated by 2-DE, 41 soybean storage protein spots were excised, and identified by liquid chromatography on-line with electro spray LCQ DecaXP tandem quadrupole time-of-flight mass spectrometry (LC/MS/MS). These methods will enable accurate evaluation of protein quality in soybeans, based on their protein digestibility-corrected amino acid score, assessment of the genetic variability of soybean genotypes, and serve as very effective tools for assisting plant breeders in their selection of high quality soybean varieties.     

紫苏籽中不同蛋白组分的功能性质研究

以紫苏籽为原料,经粉碎过60目筛后石油醚脱脂得到紫苏籽脱脂粉,然后采用不同方法提取得到紫苏籽分离蛋白、清蛋白和球蛋白,研究了3种蛋白的氨基酸组成及持水性、溶解性、乳化性等功能特性。结果表明:紫苏籽脱脂粉中蛋白质含量丰富,不同紫苏籽蛋白的氨基酸组成相近,其中谷氨酸含量最高,且均含有8种必需氨基酸;分离蛋白的热变性温度稍高于其他两种蛋白;清蛋白的持水性、持油性较好;在pH 1～10范围内,3种蛋白的溶解性均呈现出U型变化趋势,其中球蛋白的溶解性最好;在不同pH下,球蛋白的乳化活性和乳化稳定性高于其他两种蛋白。     

热诱导大豆蛋白纤维聚集体的分离及性质研究

酸性热处理条件下,随着大豆分离蛋白(SPI)自发形成纤维聚集体,仍有部分未自组装聚集的多肽分子存在其中,二者比例对蛋白功能性质产生较大影响。本文以超滤手段对热诱导大豆蛋白纤维聚集体和多肽进行大量分离,确定分离方法并研究两者的理化性质。结果表明:100 ku超滤膜反复分离两次即能获得较好的分离效果;SPI经热处理后,其等电点溶解度提高,但中性p H下溶解度变小,且蛋白水解致使其表面电位绝对值明显提高;分离所得纤维聚集体的氨基酸组成和表面电位与热处理蛋白相似,在等电点和中性p H溶解度更低;多肽则含有较少疏水氨基酸和较多负电氨基酸,在p H 2.0~9.0溶解度较好,其在酸性p H下电荷量和静电排斥力较低,导致其以无定形聚集体的形式存在,而中性p H其电荷量较高导致多肽分子间相互作用减弱,聚集体解离。     

Isolation and characterization of proteins from soymilk residue (okara)

Protein was extracted from okara at pH 9.0 and 80 °C for 30 min, giving a recovery of 53% protein. The extracted protein was isolated by isoelectric precipitation at pH 4.5, and the dried, defatted protein isolates (prepared at 25 and 80 °C) had over 80% protein.

The okara protein isolates have essential amino acid profiles similar to the FAO scoring pattern, and high in vitro protein digestibility, with methionine and cysteine as the limiting amino acids. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that okara protein isolates had a large quantity of high molecular weight components suggesting protein aggregation. Differential scanning calorimetry and hydrophobicity data suggested extensive protein unfolding in the okara products.

Okara protein isolates had lower solubility than a commercial soy protein isolate at both acidic and alkaline pH, probably due to protein aggregation. Other functional properties, including emulsifying, water and fat binding, and foaming properties, were found to be comparable to the commercial soy isolate.