Binding of cellulose sulphates to the 12 S globulin and the low molecular mass basic protein fraction from rape seed in insoluble complexes

The binding of two differently substituted cellulose sulphates (CS) with DS 0.50 and 0.33 to two main rapeseed proteins, the high molecular mass neutral 12 S globulin and the low molecular mass basic protein fraction ("albumin") in insoluble complexes at pH less than Pi (protein) has been studied using turbidimetric titration and chemical analysis of the supernatant after coprecipitation. The binding of both types of CS to the globulin at pH 3.0-2.5 at the precipitation occurs at a substoichiometric CS-protein mass ratio. This result has been obtained both by turbidimetric titration and chemical analysis. Contrary to that, the CS binding to the albumin is substoichiometric according to the turbidimetric titration and stoichiometric according to the chemical analysis. The CS-protein mass ratio in the coprecipitates obtained of pH 2.5 and 3.0 is nearly independent on the CS concentration applied for the precipitation of the albumin. There is a typical dependence on the CS concentration, however, for the globulin at pH 3.0, which becomes less pronounced at pH 2.5. The CS-globulin complexes form sharp turbidimetric titration curves at pH less than Pi (pH 2.5-5.5), the maximum position of which shifts to lower pH with increasing percentage of CS. The analogous titration curves for the CS-albumin complexes are broader, owing to the heterogeneity of the albumin fraction. Both polyanions exert a solubilizing effect at a molar excess on both proteins. Regarding the weight part necessary for precipitation (W insol.), forming stoichiometric complexes (W stoich.) and solubilization (W crit.) of the proteins, the following range can be written: W insol. less than W stoich. less than W crit.     

PROTEIN-PHYTATE INTERACTIONS IN SOYBEANS. II. MECHANISM OF PROTEIN-PHYTATE BINDING AS AFFECTED BY CALCIUM

Phytate and bovine serum albumin were used in a model system to investigate the mechanism of their binding. Ultrafiltration studies using response surface design showed the association of protein and phytic acid to be highly pH dependent. Under acid conditions, the protein formed an insoluble complex with phytic acid. At pH 3.0, a binding constant of 2.3 times 10⁵ was obtained and it was calculated that there are 78 binding sites of the total 93 basic amino acid residues potentially available. This low pH complex was not disrupted at high temperatures but the presence of calcium ions caused dissolution of the precipitate. Calcium produced different effects at higher pH (> 6). Soluble protein-calcium-phytic acid complexes were formed which were less stable to heat and dissociated above pH 10 at high ionic strength. Since this interaction occurred only in the presence of calcium, a salt linkage is implicated in which divalent cations simultaneously bind to the protein and phytic acid in the form of a soluble complex. It is proposed that either the addition of divalent cations at low pH or sequestering agents at high pH would best effect the removal of phytate from soy products by ultrafiltration.     

In vitro analysis of binding capacities of calcium to phytic acid in different food samples

The present work was performed to study the role which plays phytic acid in calcium binding, and to determine the calcium binding capacities in different foods, using in vitro extractions. Different food samples (soybeans, oats, chickpea, rice flour, and corn semolina) were extracted for 4 h at 37 °C using artificial simulated gastrointestinal juice (pepsin) at pH=2. The total calcium and phytic acid concentrations were determined by AAS and capillary electrophoresis, respectively, at pH=2 and pH=8 after neutralisation with a sodium hydroxide solution (3 M). Having determined the binding capacities of calcium in each food, we then use these results to estimate the fraction of calcium available for resorption during the process of digestion, when food moves from the acid pH of the stomach to the alkaline milieu of the intestines. The results obtained for the foods analysed show that the capacity of calcium to bind to phytic acid exhibits a clear pH dependence. The calculated calcium binding capacities, or the molar ratio of calcium to phytic acid in the in vitro extracted foods, varies from 3 mol calcium per mol phytic acid for soybean, chickpea and oats, to 2 mol calcium per mol phytic acid for rice, to1 mol calcium per mol phytic acid in corn semolina. Calcium may bind to one or more of the phosphate groups of phytic acid. Previous studies have demonstrated that phytic acid has the ability to bind minerals, proteins, and starch, and have then considered it as an inhibitor to the bioavailability of minerals and trace elements.     

The interaction of zinc(II) and phytic acid with soya bean glycinin

Binding of phytic acid to Zn(II) free glycinin was not observed in 0.5 M KCl at pH 6.2. The addition of varying quantities of phytic acid to a Zn(II)- glycinin system at pH 6.2 ([KCl]=0.5 M ) initially resulted in binding of phytate and increased binding of Zn(II) to glycinin probably as a phytate-Zn(II)-glycinin complex. Further addition of phytate resulted in precipitation of zinc and protein. Bovine serum albumin also showed increased affinity for Zn(II) owing to the presence of phytic acid. Phytic acid-Zn(II) precipitates have a capacity of removing glycinin from solution, presumably by surface adsorption. The presence of soluble Zn(II) inhibits this adsorption. Bovine serum albumin is removed from solution by phytic acid-Zn(II) precipitates only when soluble zinc is present.     

Effect of extraction conditions on composition,surface activity and rheological properties of protein isolates from flaxseed (Linum usitativissimum L)

Flaxseed protein isolates were prepared by micellisation (FM) and isoeletric precipitation (FI). The influence of preparation conditions on composition and functional properties was investigated. Contents of 0.6% phytic acid and 2.3% pentosans were found for FI, whereas FM was almost phytic acid‐free and had a low content of pentosans (0.6%). Chromatography and electrophoresis identified the 11S globulin (linin) as the main protein fraction in both isolates. Protein solubility, water‐ and oil‐binding capacities, emulsification and rheological properties of dispersions and gels were measured at pH 8 and 3. For the latter, interactions of protein with phytic acid and pentosans are highly probable. FI possesses a lower solubility (about 40–50%) and an overall higher water‐binding capacity than FM. For FI dispersions a higher storage modulus G′ than loss modulus G″ was measured, clearly pointing to the formation of protein networks. Moreover, FI formed stronger gels than FM (G′ about fivefold). The emulsifying activity, however, was distinctly lower for FI. These results point to enhanced complexation and aggregation of the isoelectric‐precipitated protein isolate. © 2002 Society of Chemical Industry     

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.).     

菜籽预榨-浸出粕中植酸的提取

为实现菜籽粕的综合开发利用,研究了一种从菜籽预榨-浸出粕中两步提取植酸的工艺.考察了酸洗pH、酸洗时间、碱提pH和碱提乙二胺四乙酸二钠(EDTA)浓度等对植酸提取的影响.将经过复合纤维素酶水解的菜籽粕在pH 5.0和50℃下酸洗3h获得酸洗液,然后用EDTA浓度0.1mol/L的碱性溶液在pH 12.0和50℃下碱提酸沉获得乳清液,经以上两步可将菜籽粕中的植酸基本提取完全.将提取液中的粗植酸经氢氧化钙沉淀并酸化,再经离子交换可获得纯度为69.96％、提取率为58.0％的植酸产品.     

Large scale procedure for fractionation of albumins and globulins from pea seeds

The buffer extractable proteins of pea, albumins and globulins, were successively extracted in a large amount at a pilot scale. In order to preserve as much as possible the native structure of proteins, a selective solubilization step procedure was performed. Firstly, albumins were extracted with acetate buffer and secondly globulins with phosphate buffer of high ionic strength. Each extract was desalted by diafiltration without protein precipitation. From 15 kg of flour, 380 g of albumin fraction and 1000 g of globulin fraction were obtained with a protein content of 86.0% and 90.7% of dry matter respectively. The characterisation of albumin and globulin fractions by electrophoresis, ultracentrifugation and anion-exchange chromatography, showed that the cross-contamination of these two fractions was minimal.     

双液相萃取菜籽粕蛋白的萃取沉淀特性

本文以双液相萃取菜粕为研究对象，测定了不同ｐＨ下菜粕中蛋白质、植酸和总磷的溶解曲线以及室温下蛋白质的沉淀曲线。试验表明，在４０℃，ｐＨ＝１２时，双液相萃取粕蛋白质萃取率为７．１５％，植酸萃取率为３３．６％。双液相萃取粕蛋白质的等电点在ｐＨ＝４～６，ｐＨ＝５时蛋白质沉淀率为７２．１％。     

The effects of phytic acid on the Maillard reaction and the formation of acrylamide

Phytic acid, myo-inositol hexaphosphoric acid, exists in substantial (1–5%) amounts in edible plant seeds. In this study the effects of phytic acid on the Maillard reaction and the formation of acrylamide were investigated. Both phytic acid and phosphate enhanced browning in glucose/β-alanine system, but phytic acid was less effective than phosphate. Higher pH favoured the catalytic activities for both of them. The influence of the types of sugar and amino acid on the reaction was also examined. Browning was suppressed by the addition of calcium and magnesium ions, but an additive effect was observed for ferrous ions and phytic acid in glucose/β-alanine solution at pH 8.0. Both phytic acid and phosphate promoted the polymerisation of the reaction intermediates. The kinetics of Maillard reaction was first-ordered reaction in the presence of phytic acid. Phytic acid was less effective than phosphate in the formation of acrylamide. When potato slices were treated with sodium phytate and calcium chloride successively, the formation of acrylamide was greatly suppressed.     

Measurement of protein in tannin-protein precipitates using ninhydrin

A method was tested for quantifying protein in precipitated tannin-protein complexes in which protein was hydrolysed with acid and the amino acids released were measured with ninhydrin. Unlike previously published methods, this technique requires no prior separation of tannin and protein and can be used to compare the binding of different soluble proteins to tannins under identical conditions. The method was used to compare the precipitation of bovine serum albumin, porcine pancreatic protease, β-glucosidase and γ-globulin by tannic acid. The amount of tannic acid required to precipitate maximal amounts of protease and β-glucosidase was approximately 7–8 times that required to cause maximal precipitation of albumin and γ-globulin when tested with 2 mg ml^?1 protein. All protein preparations contained a fraction (10–40% of total protein) which was not precipitated by tannic acid. Protein in tannin-protein precipitates produced in a standard haemanalysis assay were also measured with ninhydrin. Per cent precipitation at each tannic acid concentration as measured with ninhydrin was identical to that determined by haemanalysis within the linear portions of the binding curves produced by the two methods. These results confirm that the ninhydrin method accurately measures precipitation of protein by tannin.     

Rapeseed protein — polyanion interactions. Soluble complexes between the 2 S protein fraction (napin) and phytic acid

The formation of electrostatic complexes between the low molecular mass basic rapeseed protein fraction (napin) and phytic acid was studied using turbidimetry, potentiometry, gel chromatography, gel electrophoresis, dynamic light scattering and circular dichroism spectroscopy. In the first step insoluble “substoichiometric” complexes are formed in which the positive charge of the protein are not completely neutralized. Soluble negatively charged oligomeric complexes are formed with an excess of phytic acid. Protein dimers dominate over the monomer and small amounts of higher oligomers. Dimers and larger soluble aggregates are the main components after heating. The critical phytic acid-protein ratio for solubilizing the system represents the threshold value for the heat-stabilizing effect of phytic acid. Complexing with phytic acid does not influence significantly the secondary structure of the protein. Buffers and neutral salts weaken the phytic acid binding to the protein and decrease the molecular mass of the complexes.     

Effect of pH and Heat on the Binding of Iron, Calcium, Magnesium, and Zinc and the Loss of Phytic Acid in Soy Flour

The effect of pH and heat treatments on the binding of iron, calcium, magnesium and zinc and the loss of phytic acid in defatted soy flour was investigated. The soy flour was found to bind more iron, calcium, and magnesium at pH 6.8 than at pH 5.0, but the reverse situation occurred with zinc. Boiling caused a significant increase in binding of zinc and magnesium at both pH values, but was pH dependent for iron and calcium. Toasting caused a significant increase in binding of zinc and calcium at both pH values and a pH variable effect on iron and magnesium. Phytic acid analysis under the same conditions suggested that the degree of binding of these minerals did not correlate with the presence of phytic acid.     

Gelling properties of protein fractions and protein isolate extracted from Australian canola meal

Gelling properties of canola albumin and globulin fractions, and canola protein isolate (CPI) were examined in this study. The effects of pH and salt concentration on canola protein gelling properties were studied primarily by means of dynamic oscillatory rheology and gel texture analysis. The findings were supported by confocal laser scanning microscopy (CLSM) images of the gels, isoelectric point, and solubility measurement data. All canola proteins showed typical heat-set gel protein profiles. Gels formed at higher pH had better gelling properties including higher overall resistance to deformation (G*), higher gel elasticity (low tan δ ), higher fracture stress and firmness, and denser gel microstructure. Isoelectric points of canola proteins used in this study were in the range of pH 3.0–4.7 where low protein solubility was observed. The albumin fraction was able to form a very weak gel at pH 4, whereas the globulin fraction and CPI precipitated due to loss of protein surface charge. The effects of NaCl on gelling were protein sample dependent. The presence of NaCl negatively affected gelling properties of albumin and globulin fractions, with decreases in overall resistance to deformation (G*), and fracture stress and firmness, but positively affected CPI gels in the same aspects. The elasticity (tan δ) of all canola protein gels remained constant in the presence of NaCl. Frequency sweep analysis revealed that the albumin fraction and CPI formed weak gels, whereas the globulin fraction formed a strong gel. Strain sweep analysis further confirmed that the globulin fraction formed a stronger gel with a critical strain of at least 10%. This study demonstrates the high potential of canola proteins, particularly the globulin fraction, as a prospective gelling agent.     

Constitution of leguminous seeds. A note on protein-phytic acid interactions during isolation of acid-soluble protein from phaseolus beans

Acid-soluble proteins isolated from three types of Phaseolus beans (white kidney beans, navy beans and lima beans) were found to contain phytic acid. The amount of phytic acid complexed by the proteins was unaffected by the phytic acid content of the bean extracts from which the proteins were isolated but depended on the number of positively charged basic groups which were available for reaction with the phytate anion. It was found that the Neuberg formula for phytic acid (C₆H₂₄O₂₇P₆) represents more accurately the molecular formula of phytic acid associated with the isolated proteins, than does the Anderson formula (C₆H₁₈O₂₄P₆).     

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。     

Phytic acid interactions in food systems

Phytic acid is present in many plant systems, constituting about 1 to 5% by weight of many cereals and legumes. Concern about its presence in food arises from evidence that it decreases the bioavailability of many essential minerals by interacting with multivalent cations and/or proteins to form complexes that may be insoluble or otherwise unavailable under physiologic conditions. The precise structure of phytic acid and its salts is still a matter of controversy and lack of a good method of analysis is also a problem. It forms fairly stable chelates with almost all multivalent cations which are insoluble above pH 6 to 7, although pH, type, and concentration of cation have a tremendous influence on their solubility characteristics. In addition, at low pH and low cation concentration, phytate‐protein complexes are formed due to direct electrostatic interaction, while at pH >6 to 7, a ternary phytic acid‐mineral‐protein complex is formed which dissociates at high Na concentrations. These complexes appear to be responsible for the decreased bioavailability of the complexed minerals and are also more resistant to proteolytic digestion at low pH. Development of methods for producing low‐phytate food products must take into account the nature and extent of the interactions between phytic acid and other food components. Simple mechanical treatment, such as milling, is useful for those seeds in which phytic acid tends to be localized in specific regions. Enzyme treatment, either directly with phytase or indirectly through the action of microorganisms, such as yeast during bread‐making, is quite effective, provided pH and other environmental conditions are favorable. It is also possible to produce low‐phytate products by taking advantage of some specific interactions. For example, adjustment of pH and/or ionic strength so as to dissociate phytate‐protein complexes and then using centrifugation or ultrafiltration (UF) has been shown to be useful. Phytic acid can also influence certain functional properties, such as pH‐solubility profiles of the proteins and the cookability of the seeds.     

Preparation and characterization of high‐quality rice bran proteins

Rice bran contains 120–200 g kg^?1 protein in addition to a large amount of fat, carbohydrate, and phytic acid. Rice bran protein (RBP) fractions were refined by a two‐step preparation to eliminate residual carbohydrate. The first step involved the sequential extraction of defatted rice bran into RBP fractions using their distinct solubility to give 37 g kg^?1 of albumin, 31 g kg^?1 of globulin, 27 g kg^?1 of glutelin, and 2 g kg^?1 of prolamin. In the second step, carried out by dissolving in respective solvent and isoelectric precipitation, the protein content of each fraction increased from 69% to 97% for albumin, from 71% to 90% for globulin, from 74% to 83% for glutelin, and from 18% to 20% for prolamin. The low protein content in the prolamin fraction might be due to its low solubility in the protein assay. Emulsifying stability index and surface hydrophobicity increased in the second‐step preparation of albumin and globulin, but not of glutelin. Emulsifying properties of RBPs were lower than that of a soybean protein isolate. Denaturation temperatures and enthalpy values of denaturation for albumin, globulin, glutelin, and prolamin were 50.1 °C/1.2 J g^?1, 79.0 °C/1.8 J g^?1, 74.5 °C/3.0 J g^?1, and 78.5 °C/8.1 J g^?1, respectively. No significant differences in the denaturation temperatures and enthalpy values of denaturation of RBP fractions were obtained with these two‐step preparations (P < 0.05). Copyright © 2007 Society of Chemical Industry     

Determination of uric acid precursors in dried yeast and other forms of single-cell protein

Total purine content, expressed as μmol/g, or as percentage RNA of a conventionally defined composition, is suggested as a convenient index of the amount of uric acid precursors in SCP. A simple but reproducible analytical procedure was based on the extraction of RNA and nucleotides from dried microbial cells with 1 N-perchloric acid at room temperature, followed by hydrolysis to free bases by heating the cell-free extract for 1 h at 100°C. After precipitation as the silver complexes, washing, and regeneration with HCI, purines (mainly adenine + guanine) were determined by spectrophotometry at 252 nm. If absorbance at 264.5 nm was measured as well, adenine and guanine could be determined separately. Results with dried yeast and with fungal mycelium were confirmed by cation-exchange chromatography, when purines were eluted from the column by citrate buffers (pH 3.0–5.0).     

Isoelectric protein precipitation from mild-acidic extracts of de-oiled sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) press cake

Sunflower protein extraction using sodium chloride solutions allows high extraction yields at pH ~6 while preventing oxidation and covalent binding of phenolic compounds to the proteins, which usually occurs under alkaline conditions. Since protein solubility is enhanced at high NaCl concentrations, isoelectric precipitation had to be adapted in the present study besides developing subsequent desalting steps. Precipitation losses decreased from 62 to 31% with increasing NaCl (0.6–2.0 mol/L) and protein concentrations of the solution (6–14 mg/mL) and decreasing pH of precipitation (pH 4.5–3.0). Maximum yields were achieved at low temperature (8 °C) and upon instant acid addition. After adsorptive removal of co-extracted phenolics from the protein extracts, overall protein yields were considerably higher after precipitation at pH 3.5 compared with 4.5, but only slightly higher after washing of the precipitates. The physico-chemical properties of the protein isolates did not differ significantly except for the marked protein denaturation upon precipitation at pH 3.5. From the proposed process, light-coloured protein isolates of high purity (>98%) are obtained, which are suitable for food use.