Impact of processing conditions on the extractability and molecular weight distribution of proteins in parboiled brown rice

Parboiling, a hydrothermal treatment of paddy or brown rice, impacts the texture and nutritional characteristics of cooked rice. We investigated the impact of parboiling conditions on the extractability and molecular weight (MW) distribution of proteins in brown rice. Brown rice was parboiled using different soaking and steaming conditions. The extractability and MW distribution of proteins extracted with sodium phosphate buffer (50 mmol/L; pH 6.8) containing (i) 2.0% sodium dodecyl sulfate (SDS), (ii) 2.0% SDS/1.0% dithiothreitol (DTT)/6.0 mol/L urea, (iii) 2.0% SDS/1.0% DTT, and (iv) 2.0% SDS/6.0 mol/L urea was examined by size exclusion-high performance liquid chromatography. Depending on the parboiling conditions, protein extractabilities in media (i), (ii), (iii), and (iv) ranged from 14 to 25%, 83 to 100%, 40 to 82%, and 19 to 37%, respectively. Unlike soaking conditions, steaming conditions had pronounced effects on the level of extractable protein. In general, more severe steaming conditions caused greater reductions in protein extractability, indicating a denser protein network. Apparent MW profiles revealed that especially glutelins polymerize upon severe steaming. Albumins, globulins and prolamins either polymerize through disulfide bonds and/or interact with one another through hydrogen bonds or hydrophobic interactions to form a separate protein network or become incorporated in the glutelin network.     

Extractability and chromatographic separation of rye (Secale cereale L.) flour proteins

A size exclusion – high performance liquid chromatography (SE-HPLC) method originally developed for separating wheat, barley or rice proteins was applied to study the extractability and molecular weight (MW) distribution of rye flour proteins. These were extracted with 50 mmol/l sodium phosphate buffer (pH 6.8) containing 2.0% (w/v) sodium dodecyl sulfate (SDS) and, optionally, 1.0% (w/v) dithiothreitol (DTT). About 95% of the proteins were extracted in buffer containing 2.0% SDS. Addition of 1.0% DTT to such buffer increased the protein extractability to 100%, indicating that rye flour contains some proteins cross-linked by disulfide (SS) bonds. The SE-HPLC profiles revealed that rye flour contains SS-linked HMW-secalins and 75 k γ-secalins which elute in specific peaks. Upon reduction, these SS-linked protein aggregates dissociate and some entrapped albumins, globulins and/or ω-secalins are released. Rye flour albumins and globulins elute over the entire SE-HPLC profile. In contrast, the monomeric ω-secalins and 40 k γ-secalins are detected in specific well resolved SE-HPLC peaks. The applied fast and reproducible method can be used to characterise and quantify rye flour proteins and to determine changes as a result of processing.     

Sesame oleosin and prepro-2S albumin expressed as a fusion polypeptide in transgenic rice were split,processed and separately assembled into oil bodies and protein bodies

A recombinant polypeptide containing the precursor protein of a sesame storage protein, 2S albumin, fused to the C-terminus of a sesame oleosin was expressed in transgenic rice seeds under the control of a rice glutelin promoter. The recombinant polypeptide of 32 kDa, equivalent to the resultant molecular mass of sesame oleosin (15 kDa) and prepro-2S albumin (17 kDa), was detected in the endoplasmic reticulum fraction of maturing transgenic rice seeds, but not in the purified oil bodies or the soluble extract of transgenic seeds. However, sesame oleosin presumably fused with a 2 kDa C-terminal appendix originating from the signal sequence of prepro-2S albumin, was found in the purified oil bodies, and mature sesame 2S albumin apparently processed into two subunits (9 and 4 kDa) linked by disulfide bonds was detected in extracts of transgenic seeds. Immunogold labeling revealed that the sesame oleosin and 2S albumin were separately located in oil bodies and protein bodies of embryo cells of transgenic rice seeds. While sesame 2S albumin was also detected in protein bodies of endosperm cells of transgenic seeds, the co-expressed sesame oleosin, probably degraded due to the lack of oil bodies in this tissue, and was not detected. The results provide a new technique for introducing two recombinant polypeptides separately into rice oil bodies and protein bodies from one expression construct.     

The effects of malting and mashing on barley protein extractability

Proteins in unmalted and malted barley and in brewers’ spent grain (BSG) obtained after mashing were fractionated on the basis of their differential extractability in different media and characterised by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Albumins and globulins were first extracted with 5.0% NaCl and hordeins (barley prolamins) were extracted with 55.0% 1-propanol in the presence, or absence, of 1.0% DTT. Glutelins were then extracted with 2.0% SDS/6.0 M urea/1.0% DTT or with 55.0% 1-propanol/6.0 M urea/1.0% DTT/0.036 M Tris-HCl (pH 8.4). Under non-reducing conditions, monomeric C hordeins and some B hordeins were extracted from unmalted barley, whereas most if not all B, C and D hordeins were extracted under reducing conditions. During malting, disulfide bonds are reduced and B and D hordeins are broken down by proteolysis. No D hordeins were extracted from malt and nearly the same levels of malt B hordeins were extracted both under non-reducing and reducing conditions. B hordeins present in BSG proteins were only extractable under reducing conditions. Whereas most of the C hordeins were extracted from BSG under non-reducing conditions, more C hordeins were extracted under reducing conditions. Mashing probably induced disulfide bond formation resulting in aggregation. Although earlier literature suggested the formation of an aggregate composed of B and D hordein (and glutelin) during mashing, the present work suggests the formation of an aggregate composed of B hordeins in which C hordeins are entrapped.     

Effects of glutelin and globulin on the physicochemical properties of rice starch and flour

The effect of two rice endosperm proteins, glutelin and globulin, on the physicochemical properties of rice starch and flour was investigated. Albumin, globulin, prolamin and glutelin were sequentially extracted from defatted rice flour with de-ionised water, 1.5 M NaCl, propan-2-ol and 0.1 M NaOH, respectively, followed by dialysis and lyophilisation. Globulin and glutelin were then added to pure rice starch at various concentrations, separately and together, and the pasting and textural properties of mixtures were analysed by the Rapid Visco Analyser (RVA) and TA-XT2 textural analyser, respectively. The presence of glutelin in rice starch caused an increase in pasting temperature but a decrease in the viscosity parameters of the starch paste. The concentration of glutelin was also positively correlated with the hardness and adhesive properties of the starch gel. The presence of globulin, on the other hand, resulted in a decrease in all the pasting and textural parameters except gel hardness and the changes were linearly correlated with the concentration of the protein for most of the physical parameters. When the two proteins were added to rice starch together, the outcomes in pasting and textural properties were generally dependent upon the relative concentrations of the two proteins, but were also influenced by the presence of the other two protein fractions, albumin and prolamin. The presence of globulin initially accelerated the rate of water absorption by starch during cooking while the presence of glutelin slowed it down, but in both cases, the ultimate amount of water absorbed was significantly lower than that by pure starch. The contrasting effects of the different protein fractions mean that it might be possible to manipulate the textural properties of rice starch and flour to achieve desirable sensory outcomes by varying the proportions of the protein fractions in product formulations.     

Optimization of High-Protein Glutinous Rice Flour Production Using Response Surface Method

A response surface method was employed to study the effect of α-amylase concentration, hydrolysis temperature and time on the production of high protein glutinous rice flour (HPGRF). The suspension of glutinous rice flour (15%) that contained 6.52% protein was gelatinized and subsequently hydrolyzed by thermostable α-amylase. The hydrolysis yielded 0.144–0.222 g/g HPGRF with 29.4%–45.4% protein content. Hydrolysis time exerted a significant effect, while enzyme concentration and hydrolysis temperature showed insignificant effect on the protein content and production yield of HPGRF. The result of response surface method showed that the optimum condition for the production of HPGRF that contained at least 36% protein was treating gelatinized 15% glutinous rice flour suspension with 0.90 Kilo Novo α-amylase Unit (KNU)/g α-amylase at 80 °C for 99 min. By carrying out the predicted hydrolysis condition, HPGRF with 35.9% protein and 61.8% carbohydrates was resulted. The process yielded 0.172 g/g HPGRF. HPGRF contained higher amount of essential amino acids compared to glutinous rice flour. HPGRF had higher solubility and lower swelling power, and also showed no pasting peak compared with glutinous rice flour.     

Mineral element distributions in milling fractions of Chinese wheats

Malnutrition related to micronutrient deficiency can create immense economic and societal problems. The objective of this study was to quantify the mineral element concentration distribution in milled fractions, using 43 common wheat (Triticum aestivum L.) cultivars sown in Jinan, China during the 2005–2006 crop season. All 43 cultivars had low Fe (average 28.2 mg Kg⁻¹) and Zn (28.6 mg Kg⁻¹) concentrations, and wide ranges of variation for mineral element concentrations. Highly significant effects among milling fractions and cultivars on all traits were observed, with fraction effect being the larger. There was an uneven distribution of mineral element concentrations in wheat grain. Shorts and bran fractions had the highest mineral element concentrations, whereas flours from break and reduction had low concentrations. Compared with those in the central endosperm, the concentration of inorganic phosphorus (Pi) decreased the most with decreasing flour yield, whereas the concentration of phytic acid P (PAP), phytase activity, and Ca decreased the least. Pi was the most concentrated element in the aleurone, whereas PAP, phytase activity, and Ca were the least, compared to those in the central endosperm. Milling technique through adjusting flour yield can be used to improve the element composition of flour.     

Characterization of rice storage proteins by SE-HPLC and micro z-arm mixer

While the effect of protein content and composition on the functional properties of wheat flour is well studied, our knowledge on the same properties of rice flour is limited. This work was conducted to study the relationship between the dough mixing properties of flour from different rice cultivars and protein content and composition. An efficient sonication-based two-step extraction procedure was applied to isolate rice flour proteins. The size-exclusion HPLC (SE-HPLC) method, originally developed for separating wheat proteins, was applied with some minor modifications in order to study the size distribution of rice flour proteins. Four fractions were distinguished on the SE-HPLC profile and were further characterized by SDS-PAGE. Fractions I–III consisted of glutelins, while fraction IV contained albumin, globulin and prolamin proteins. When rice dough was characterized on the basis of mixing parameters in a micro z-arm mixer, significant differences were observed depending on the protein composition of the flour. Statistical analysis results indicated that the functional properties of the flour from different rice cultivars were associated with the amount of polymeric proteins and their size distribution.     

Stability of wheat proteins in solution

Gluten from defatted wheat flour was used to study the stability of protein extracts during various steps that are routinely made in protein analyses. These included aqueous extraction and short-term storage, handling of chromatographic fractions, freeze-drying and reconstitution. Protein composition was monitored by size-exclusion high performance liquid chromatography (SE-HPLC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Distilled, deionized and sterilized water were used to prepare the gluten solutions (TFA or HCl solutions at pH 3.0) and to prepare elution solvents. In distilled or deionized water there was a decrease of polymeric protein and a corresponding increase in the monomeric components with time unless initial heating was applied. Protein extracts obtained from gluten and solvents prepared with freshly sterilized water showed no change during storage for 4 days at a temperature of 25 °C. Microbiological assays of the water samples supported the view that microbial contaminants were the source of proteases. After elution from SE-HPLC column with acetonitrile/water followed by freeze-drying and re-dissolution, the SE-HPLC profile of the monomeric proteins was unchanged. However, polymeric proteins aggregated, lost solubility and showed an altered SE-HPLC profile. The effect appeared to be due to the presence of acetonitrile and could be avoided by membrane concentration instead of freeze-drying.     

Nutritional potential ofVigna minima (Roxb.) Ohwi and Ohashi:

Quantitative variation in different fractions of seed proteins and their amino acid levels in populations ofVigna minima (Roxb.) Ohwi and Ohashi and inV. umbellata cv. IC 1568 — the rice bean — were investigated. Globulin I fraction, together with globulin II, constitutes 38 to 54 per cent of the total seed protein. The alkali soluble (glutelin) fraction is the second largest fraction. Both these fractions show broad range of variation, suggesting a broad genetic base. The profiles are population specific; the coastal population, which contains higher seed protein also possesses maximum levels of globulin I and glutelin fraction suggesting its potentiality for breeding lines with high protein content, high nutritive value, and salt tolerance. Protein content is positively correlated with globulin I and glutelin fractions, which are in turn positively correlated with each other. The amino acid profiles are specific not only to the fractions but also to the populations. The range of variation in the levels of all amino acids in different fractions is broad suggesting substantial genetic diversity. The average levels of lysine and sulphur amino acids are high in globulin I and glutelin fractions.     

Impact of redox agents on the physico-chemistry of wheat gluten proteins during hydrothermal treatment

The impact of the oxidants potassium bromate and potassium iodate and the reducing agent dithiothreitol (DTT) on the rheological behaviour of 20% (w/v) gluten-in-water suspensions during thermal treatment was monitored with the rapid visco analyser (RVA). The suspensions were subjected to a linear temperature increase from 40 to 95 °C in 14 min, a holding step of 40 min at 95 °C, a cooling step (7 min) with a linear temperature decrease to 50 °C, and a final holding step at 50 °C (13 min). Potassium iodate (1.18 and 1.77 μmol/g protein) and potassium bromate (1.52 and 15.2 μmol/g protein) decreased RVA viscosities in the holding step and increased sodium dodecyl sulphate (SDS) protein extractabilities suggesting a greater heat resistance and decreased gliadin–glutenin cross-linking. In contrast, in the presence of DTT (1.65 and 3.30 μmol/g protein) RVA viscosity increased at lower temperatures and lowered SDS extractabilities. It is postulated that low concentrations of reducing agent facilitate gliadin–glutenin cross-linking during heating while oxidants hinder gluten polymerization due to decreased levels of free sulphydryl groups and less flexibility of the glutenin chains.     

Characterisation and functional properties of Australian rice protein isolates

Albumin, globulin, glutelin and prolamin fractions were isolated from an Australian rice variety (cv. Langi) and characterised by yield, protein content and molecular weight profile using both capillary electrophoresis (SDS-CE) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The influence of pre-extraction enzymatic hydrolysis of starch and heating to 70 °C was also investigated, as was the extraction of the glutelin fraction without prior removal of the albumin and globulin fractions. Pre-extraction treatment affected mainly the albumin fraction, increasing dry matter yield but reducing protein content. SDS-CE was able to separate the protein fractions over a wider molecular weight range than SDS-PAGE, and the peaks from SDS-CE showed slightly higher molecular weight compared to equivalent bands from SDS-PAGE. The glutelin fraction extracted without prior removal of albumin and globulin fractions had different characteristics compared to those obtained by conventional extraction methods. Pre-extraction hydrolysis of starch did not significantly affect the emulsifying, foaming and gelling properties of extracted protein. Although rice glutelin had poor solubility, emulsifying and foaming properties in aqueous systems, it had good gelling properties which could be important for food applications.     

Evaluation of rice flour modified by extrusion cooking

Flour from long-grain, high-amylose, milled rice was extruded in a double screw extruder. Response surface methodology (RSM) using a face-centered cube design was used to evaluate the effects of operating variables, namely the screw speed (200–300 rpm), barrel temperature (100–160 °C), and feed moisture content (16–22%) on some functional, physical, pasting, and digestibility characteristics of the extrudates. Regression analyses showed that water absorption index (WAI) was significantly (P<0.05) affected by all linear, quadratic, and interaction terms. Viscosity values of extruded rice flours were far less than those of their corresponding unprocessed rice flour dispersed in the Micro Visco Amylo Graph (MVAG) indicating that the starches had been partially pregelatinized by extrusion process. Peak viscosity indicated a high positive correlation with hot paste viscosity (HPV) and cold paste viscosity (CPV) with r>0.700 (P<0.01). The effects of processing on the in vitro digestibility of starch fractions in rice extrudates was tested using controlled enzymatic hydrolysis with alpha-amylase and glucoamylase. The starch-digestion rate depended mainly on processing conditions. Rapidly digestible starch (RDS) was found to correlate negatively with slowly digestible starch (SDS) (r=−0.964, P<0.01) and with resistant starch (RS) (r=0.793, P<0.01), respectively. Whereas SDS correlated positively with RS (r=0.712, P<0.01).     

The impact of heating and cooling on the physico-chemical properties of wheat gluten–water suspensions

The rapid visco analysis (RVA) system was used to measure rheological behaviour in 20% (w/v) gluten-in-water suspensions upon applying temperature profiles. The temperature profiles included a linear temperature increase, a holding step, a cooling step with a linear temperature decrease to 50 °C, and a final holding step at 50 °C. Temperature and duration of the holding phase both affected RVA viscosity and protein extractability. Size-exclusion and reversed-phase HPLC showed that increasing the temperature (up to 95 °C) mainly decreased glutenin extractability. Holding at 95 °C resulted in polymerisation of both gliadin and glutenin. Above 80 °C, the RVA viscosity steadily increased with longer holding times while the gliadin and glutenin extractabilities decreased. Their reduced extractability in 60% ethanol showed that γ-gliadins were more affected after heating than α-gliadins and ω-gliadins. Enrichment of wheat gluten in either gliadin or glutenin showed that both gliadin and glutenin are necessary for the initial viscosity in the RVA profile. The formation of polymers through disulphide bonding caused a viscosity rise in the RVA profile. The amounts of free sulphydryl groups markedly decreased between 70 and 80 °C and when holding the temperature at 95 °C.     

Improved functional properties for soy–wheat doughs due to modification of the size distribution of polymeric proteins

Physical modification of soy flour was shown to greatly improve the dough and baking qualities of soy–wheat (1:1) composite doughs, compared to raw soy flour, giving better stability and R_max, although extensibility was still below that of the wheat dough.Reasons for improvements caused by the physical-modification process were sought by determining the relative size distribution of proteins in the soy–wheat composite doughs by size-exclusion high-performance liquid chromatography (SE-HPLC). Results were expressed as the proportion of ‘unextractable polymeric protein’ (%UPP)—the proportion of the protein that is over 100,000 Da and only extractable after sonication. Protein extracts from the soy–wheat dough were sampled at different stages of dough mixing and fermentation, and their molecular-size distributions evaluated.Unextractable soy proteins were lower in raw soy flour (only 8% UPP) than in two physically-modified soy flours (19 and 34% UPP, respectively). Unextractable polymeric protein was much greater for wheat flour (57% UPP). After mixing a 1:1 soy–wheat composite dough, the %UPP was 36 and 22 (for the two types) when made from physically modified soy flours, compared to 8 for a composite dough using raw soy flour, and 43 for a wheat-only dough. The higher proportion of UPP for the wheat-modified soy doughs was taken as a reason for this composite dough providing better dough and baking qualities. Prolonged fermentation time caused a decrease in UPP percentages for all composite doughs and for the wheat-only dough.     

Properties of poorly digestible fraction of protein bodies of cooked milled rice

Pepsin-treated protein bodies and human fecal protein particles from IR480-5-9 cooked milled rice (Oryza sativa L.) consisted mainly of giutelin-type polypeptides with a mean molecular weight (MW) of 16 000. They had similar MW, amino acid pattern, and isoelectric focusing characteristics as the 70% 2-propanol—0.6% -mercaptoethanol soluble fraction of rice glutelin. The lipids of the pepsin-treated protein bodies had an identical lipid fraction ratio and fatty acid composition as the lipids of whole protein bodies.     

Submergence tolerance and yield performance of lowland rice as affected by agronomic management practices in eastern India

Rice is subjected to excessive waterlogging and flash-flooding on large areas in south and south-east Asia. Besides cultivars, submergence tolerance of plants is influenced by various agronomic practices. A field experiment was conducted at Cuttack, India during 1994–1995 to study the effect of method of stand establishment (direct seeding and transplanting), vigour of seed (low and high-density) or seedlings (N-fertilized and unfertilized), plant population (normal and 50% more) and N fertilizer (single basal and split application) on yield performance of lowland rice under conditions of natural submergence and simulated flash-flooding (impounding up to 90 ± 3 cm depth for 10 days at vegetative stage). Flooding reached a maximum depth of 80 cm in 1994 and 52 cm in 1995 under natural submergence. The crop performance was better in 1994 due to timely sowing in dry soil and delayed accumulation of water (43 days after sowing) than in 1995 when sowing was done late in saturated soil followed by early water accumulation (28 days after sowing). Grain yield of rice decreased by 30.0–33.6% due to simulated flash-flooding compared with natural submergence, and by 21.4–33.1% due to transplanting in July compared with direct seeding in May-end/early June. The yield of direct-sown crop increased by using high-density seed of 22.9–23.0 mg weight (5.2–9.0%), higher seed rate of 600 m⁻² (2.2–2.3%) and basal fertilization at 40 kg N ha⁻¹ (19.4–25.7%) compared with low-density seed (19.4–20.1 mg), 400 seed m⁻² and no N, respectively. The yield of transplanted crop increased by using N-fertilized seedlings of 0.49–1.65 g weight (29.5–38.5%), higher number of seedlings at 155 m⁻² (3.5–16.7%) and basal fertilization at 40 kg N ha⁻¹ (31.9–32.5%) compared with unfertilized seedlings (0.19–0.79 g), 115 seedlings m⁻² and no N. Split application of 40 kg N ha⁻¹ — 50% each at basal and top dressing (105–115 days of growth after flash-flooding) — improved yield significantly (10.1–13.1%) over single basal application under simulated flash-flooding, but not under natural submergence conditions. Regression analysis indicated that relative contribution of various factors in increasing grain yield was in order: N fertilizer > seed density > seed m⁻² in direct-sown rice, and N fertilizer > seedlings m⁻² > seedling dry weight in transplanted rice. It was concluded that grain yield of flood-prone lowland rice can be increased by establishing the crop early through direct seeding using high-density seed and basal N fertilization.     

稻米蛋白质组分及总蛋白质含量与淀粉RVA谱特征值的关系

 利用低谷蛋白含量（low glutelin content, LGC）粳稻品种W1721与大面积应用的杂交粳稻恢复系轮回422构建了重组自交系群体。根据种子总蛋白质的SDS PAGE带型,推断各单株LGC位点的基因型。在排除脂肪的影响下,比较了基因型LGCLGC（谷蛋白含量较醇溶蛋白低）与基因型lgclgc（谷蛋白含量较醇溶蛋白高）在RVA谱特征值上的差异,以及同一基因型内总蛋白质含量与淀粉RVA谱特征值的关系。结果表明基因型LGCLGC的崩解值和回复值的平均值分别显著和极显著大于基因型lgclgc;LGC对崩解值和回复值的相对加性效应分别为3.91%和4.45%,贡献率分别为4.81%和12.81%。LGCLGC型的稻米,总蛋白质含量与崩解值和消减值分别存在显著的负线性回归和正线性回归关系,贡献率分别为9.86%和11.48%;lgclgc型的稻米,总蛋白质含量与消减值和回复值都存在极显著的负线性回归关系,贡献率分别为1341%和27.88%。结合前人研究认为,稻米食味品质受谷蛋白相对于醇溶蛋白的含量以及总蛋白质含量的影响;而总蛋白质含量对食味品质的影响因谷蛋白相对于醇溶蛋白的含量的不同而异。因此,育种工作中应当把谷蛋白相对于醇溶蛋白的含量以及总蛋白质含量同时作为选择指标。     

Differential accumulation of sulfur-rich and sulfur-poor wheat flour proteins is affected by temperature and mineral nutrition during grain development

Hard red spring wheat (Triticum aestivum cv Butte86) was grown under controlled environmental conditions and grain produced under 24/17 °C, 37/17 °C or 37/28 °C day/night regimens with or without post-anthesis N supplied as NPK. Flour proteins were analyzed and quantified by differential fractionation and RP-HPLC, and endosperm proteins were assessed by two-dimensional gel electrophoresis (2-DE). High temperature or NPK during grain fill increased protein percentage and altered the proportions of S-rich and S-poor proteins. Addition of NPK increased protein accumulation per grain under the 24/17 °C but not the 37/28 °C regimen. However, flour protein composition was similar for grain produced with NPK at 24/17 °C or 37/28 °C. 2-DE of gluten proteins during grain development revealed that NPK or high temperature increased the accumulation rate for S-poor proteins more than for S-rich proteins. Flour S content did not indicate S-deficiency, however, and addition of post-anthesis S had no effect on protein composition. Although, high-protein flour from grain produced under the 37/28 °C regimen with or without NPK had loaf volumes comparable to flour produced at 24/17 °C with NPK, mixing tolerance was decreased by the high temperature regimen.