Influence of sulfur-amino acid content variation in plant vs animal protein on serum and tissue lipids in rats

The objective of the study was to determine the effects of methionine and cysteine supplementation of soy protein isolate and casein on serum and tissue lipid levels in rats. Sixty male, weanling, Wistar-Kyoto rats were fed two sources of protein (casein or soy protein isolate) and three variations of sulfur-amino acid supplementation (none, methionine, or cysteine). At this level of protein intake (10% by weight), rats fed soy-based diets had similar serum lipid concentrations than rats fed casein-based diets. Choline was not added to the diet in order to be able to assess independent influences of methionine and cysteine on lipid metabolism. Overall, serum lipid values were greater in rats fed proteins supplemented with methionine while the addition of cysteine produced lower lipid levels. Liver lipid concentrations were increased tremendously upon cysteine supplementation of soy protein isolate. Protein quality, as determined by protein efficiency ratio, was improved by supplementation of either sulfur-amino acid; however, methionine had the greatest effect. Results indicate that the sulfur-amino acids influence lipid metabolism in the absence of dietary choline. The mechanism by which this occurs is not known.     

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.     

Globulin and albumin proteins in dehulled seeds of three Phaseolus vulgaris cultivars

Albumin and globulin fractions, obtained after modifications of the Osborne's fractionation scheme, were investigated in the seeds of three cultivars of Phaseolus vulgaris, after removal of the seed coats, to prevent interactions of tannins and phenols with the globulins. Also, by using an appropriate buffer to separate the globulins more efficiently, no cross-contaminations were observed in SDS-PAGE between these two fractions. The combination of these two procedures resulted in globulin to albumin ratios 70percnt higher than those published previously with the same cultivars. Amino acid compositions of total globulin, phaseolin and albumin fractions are presented. Whereas the albumins contained much more cysteine than the globulins, the concentrations of methionine were evenly distributed in these two fractions. Also, the bulk of methionine was found in phaseolin; cysteine was found in non-phaseolin proteins. In addition, the globulins contained unexpectedly high amounts of lysine.     

Methionine-containing proteins in twoPhaseolus vulgaris cultivars with different methionine bioavailabilities

The full utilization of legumes as human food is limited by a deficiency of sulfur amino acids, low protein digestibility, low methionine bioavailability and the presence of anti-nutritional factors. A new cultivar ofPhaseolus vulgaris (Carioca 80) has 56.8% available methionine, compared with 29.3% availability in the parent cultivar Carioca. The total methionine content, denaturing gel electrophoretic patterns of methionine-containing proteins, and the percentage of phaseolins (the major storage proteins in Phaseolus) relative to the total protein are similar in the two cultivars. Although the digestibility of the two cultivars is similar, the increased biological value of Carioca 80 may indicate that there are differences in overall bean composition that affect protein hydrolysis and utilization. We suggest the tentative explanation that this is due to differences in the distribution of methionine in the methionine-containing proteins of the two cultivars.     

An improved method for extraction of sorghum polymeric protein complexes

A method for fractionating sorghum proteins using extraction solvents and techniques designed to obtain polymeric protein structures (especially disulfide linked) was developed. Extraction and separation conditions were optimized in terms of completeness of protein extraction, sample stability, and analytical resolution. After pre-extraction of albumins and globulins, a 3-step sequential procedure involving no reducing agents was applied to ground whole sorghum flour. The three fractions obtained represented proportionally different protein polymer contents and molecular weight distribution as evidenced by comparative size exclusion chromatography. Protein composition also varied among the extracts with differences in kafirin composition and non-kafirin proteins detected in the fractions by RP-HPLC and SDS-PAGE analysis. The ability to quantify and further characterize sorghum polymeric protein complexes will be useful for additional studies linking protein structures with functionality and digestibility and variations for these properties within diverse sorghum germplasm.     

Sulfur Metabolism and Protein Quality of Soybean

Abstract

Soybean is an important source of protein in livestock production, and is of growing importance for human consumption. As a sole dietary protein source, soybean seed protein is deficient in the amino acids methionine, cysteine, and threonine. Increasing the amount of methionine in the amino acid profile of soybean meal would enhance its value for producers and consumers. Methionine contains S, and so its production is necessarily linked to sulfur metabolism within the soybean plant. Sulfur is taken up from the soil in the form of sulfate. During vegetative growth, developing leaves appear to be the predominate site of sulfate reduction and incorporation of reduced S into amino acids. During reproductive growth, developing pods and seeds seem to be the predominate location of sulfate reduction. Sulfate reduction and methionine synthesis are complex and highly regulated processes. Synthesis of storage proteins within the developing seed is sensitive to the amount of methionine present such that provision of extra methionine blocks synthesis of poor quality storage proteins. Sulfur deficiency on the other hand dramatically enhances accrual of poor quality seed storage proteins. It appears that the plant manufacturers higher qualty storage proteins as long as methionine is present in adequate quanitities relative to the non-S-amino acids. Accumulation of poor-quality seed storage proteins thus appears to be a function, at least to some degree, of rate of methionine synthesis within the seed. Efforts to enhance soybean seed protein quality may require enhanced rates of methionine synthesis within the seed to be successful.     

Effect of accelerated aging on maize, sorghum, and sorghum meal

Accelerated aging at 50 °C significantly affected the physical and chemical properties of sorghum and maize. Aging caused associations between starch granules, protein matrix, and cell walls. During aging, floury areas of the endosperm became more corneous; as the endosperm hardened, strong associations between starch and protein developed, causing the endosperm to fracture through endosperm cells instead of along cell walls, which is common for non-aged maize. Aging significantly decreased the pasting viscosity of starch, molecular solubility at 85 °C and the molecular weight of solubilized starch. Solubility of albumins and globulins decreased while solubility of proteins extracted by a reducing agent and/or in alkaline pH increased during aging. Decreased solubility and functionality of starch and protein in aged grain appear to be due to protein oxidation.     

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.     

Protein Characterization of Protein Bodies from Cotyledons of Mucuna pruriens (L.) DC

Seeds of Mucuna pruriens (L.) DC. (Fabaceae) were analyzed for protein composition of protein bodies isolated from cotyledons. Protein bodies were successfully separated by Lympholyte and those of dry seeds, observed by scanning electron microscope, were elliptical or spherical in shape with a diameter of 5–12 μm. Protein content in dry seed protein bodies was 10.6 mg/g dry weight. Globulin was the largest protein fraction isolated (62.5 %), followed by albumin (18.3 %), glutelin (15.8 %) and prolamin (3.4 %). The prolamin fraction and high glutelin content are uncommon in legumes. SDS-PAGE of albumins, globulins, prolamins and glutelins provided different band numbers and molecular weights under reducing and non reducing conditions and suggested that the albumin fraction is rich in disulphide bonds.     

Biochemical Characterisation of a Novel Polymeric Protein Subunit from Bread Wheat (Triticum aestivum L.)

A new wheat endosperm protein subunit that was found in accessions belonging to different collections was identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Insoluble in 0·5 M NaCl, 70% ethanol, dimethyl sulphoxide (DMSO) and 50% propan-1-ol, it appeared in the pellet corresponding to the polymeric proteins along with high (HMW) and low molecular weight (LMW) glutenin subunits (GS). In the reduced form, it had an electrophoretic mobility between those two types of glutenin subunits. The apparent M_r of this novel protein was estimated by SDS-PAGE to be 71 000. N-terminal sequence and amino acid analyses indicated a composition similar to the ω-gliadins encoded by genes located on chromosome 1B. This protein can be ascribed to the D-subunits of LMW-GS with at least one cysteine residue that allows it to form part of the polymeric structure of glutenin, as shown by reaction with a fluorogenic reagent specific for sulphydryl groups. Fractions collected after size exclusion high-performance liquid chromatography (SE-HPLC) fractionation and further characterised by SDS-PAGE, confirm that the protein participates in the glutenin polymeric structure. An increase in its concentration was observed in fractions collected within the polymeric peak as elution time increased, implying that a larger amount of this protein is present in small size polymers. The role of this protein in the complex relationship between endosperm proteins and quality parameters is discussed in relation to its likely role as a chain terminator.     

In vitro amino acid digestibility of food proteins as measured by the digestion cell technique

The digestibility of proteins and individual amino acids of nineteen selected foods was determined by anin vitro assay. Samples were hydrolysed with pepsin for 30 minutes in an acidic medium; the pH was then raised to 7.5 and the mixture poured into the dialysis bag (molecular weight cut-off 1000) of a digestion cell with pancreatin. Digestion products, mixtures of free amino acids and low molecular weight peptides which pass through the dialysis membrane, were collected for 6 hours by sodium phosphate buffer circulation. All proteins from animal sources displayed a digestibility similar to casein, except for breakfast sausage. Vegetable proteins showed intermediate digestibility, except for cereals (lower) or peanut butter (higher). Target amino acids of enzymes were generally more readily hydrolysed. However, compared to other animal proteins, glycine in milk products, valine, isoleucine, methionine and lysine in breakfast sausage and hot dog, and histidine in tuna were more easily released. Overheating of non-fat dried milk not only reduced the lysine digestibility, but also that of methionine, phenylalanine, histidine and cystine. Among vegetable proteins, wheat products were characterized by a relatively greater release of threonine, isoleucine and histidine, and peas by a lower digestibility of methionine and lysine. Proline of soy isolate and isoleucine of pinto bean were resistant to hydrolysis while arginine of pinto beans and of rice-wheat-gluten was easily released.     

Nutritional assessment of seed proteins in rice bean [Vigna Umbellata (Thumb.) Ohwi and Ohashi

Seeds of three rice bean accessions had 17.26 to 21.42% protein, 3.46 to 4.03% fat, 61.09 to 64.73% carbohydrates 3.99 to 4.58% ash and 5.22 to 7.43% fiber (dry weight basis).The most limiting amino acids in the seed meal, albumin and globulin fractions, were methionine and cysteine with chemical scores of these fractions being 38% to 59%. The amino acid pattern of globulin and seed meal were similar.Thein vitro protein digestibility (IVPD) ranged from 82 to 86% for the seed meal, 86 to 88.5% for the albumin and 75.9 to 83.3% for the globulin. Relative nutritive values (RNV) of raw mature seed of two accessions were 22.6% and 42.4% and increased to 55.6% to 79.4% after boiling and roasting.Part of MS thesis of the senior author.     

Functional and Rheological Properties of Amaranth Albumins Extracted From Two Mexican Varieties

The functional and rheological properties of amaranth albumins isolates extracted from two new Mexican varieties were determined. Functional properties tested were protein solubility, foaming, water and oil absorption capacities, emulsifying activity, and emulsion stability. The maximum solubility values for both amaranth albumins were found above pH 6 and values were compared to the solubility of egg albumins. Albumins from amaranth showed excellent foaming capacity and foaming stability at pH 5, suggesting that this protein could be used as whipping agents as egg albumins, also the water and oil absorption capacities reached their maximum values at acidic pH, suggesting that amaranth albumins could be appropriate in preparation of acidic foods. The rheological test based on farinograms and alveograms showed that wheat flour supplemented with 1% amaranth albumins improves the dough properties due to higher mixing stability and the bread had better crumb characteristics. In addition of the known high nutritional values of amaranth albumins, our results indicate the high potential for use of these proteins as an ingredient in food preparations.     

Amaranth seed proteins: Effect of defatting on extraction yield and on electrophoretic patterns

Acetone and hexane were used to know the effect of defatting amaranth flour on the extraction yield of protein fractions and on the electrophoretic patterns. It was found that albumins (33%) and globulins (20%) did not present yield changes when using these two solvents, but it was noted that with hexane compared to acetone, prolamins extraction was reduced by half (3.0 to 1.6%) whereas glutelins increased from 26.5 to 30%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of prolamins extracted with acetone and hexane showed one band of low molecular weight (22 KDa) and five bands between 52 to 22 KDa, respectively. No electrophoretic changes were observed in the remaining fractions.     

Bacillolysin,papain, and subtilisin improve the quality of gluten-free rice bread

Protease has been shown to be an effective food additive for improving the quality of gluten-free rice bread. In this study, we found that bacillolysin (Protin SD-NY10, metallo protease), papain (cysteine protease), and subtilisin (Protin SD-AY10, serine protease) increased the specific volume of gluten-free rice breads by 30–60% compared with untreated breads. These proteases also decreased crumb hardness by 10–30% compared with untreated breads. Many fine bubble cells were observed in the crumb of the protease-treated rice breads using scanning electron microscopy. Optical microscopic observation revealed fine networks of small protein aggregates stained by Coomassie Brilliant Blue (CBB) in the rice batter of the improved gluten-free rice breads. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein in the rice batter suggested that the amount of low molecular weight proteins (less than 10 kDa) increased with the use of Protin SD-NY10, Protin SD-AY10 and papain treatments compared with untreated rice batter. Thus, we considered that the small proteins aggregates were formed through disulfide bonds. This fine network was effective for retaining CO₂ gas during the fermentation process, resulting in an increase in the specific volume and a decrease in the crumb hardness of gluten-free rice bread.     

The use ofEscherichia coli mutants to measure the bioavailability of essential amino acids in foods

The -galactosidase-based assay for lysine developed by Tuffnell & Payne was used to measure the bioavailabilities of cyst(e)ine, methionine, threonine and tryptophan in pronase digests of 17 foods. The digests were assayed by estimating the -galactosidase synthesis responses of fiveEscherichia coli mutants, each requiring one of the respective amino acids for protein synthesis. Deletion mutants were used whenever possible in order to ensure strain stability. Single digests of each food were assayed with 3 or 4 separate cultures of each mutant and the results were compared with those from the corresponding chemical assay. Omitting the anomalously low values for one food, the rank correlation coefficients of the bio- and chemo-assay values were 0.61 (cysteine), 0.91 (lysine), 0.95 (methionine), 0.64 (threonine) and 0.85 (tryptophan). Mean (± S.D.) relative amino acid bioavailabilities (casein = 100%) for the 17 foods were: cysteine, 53±23; lysine, 90±10; methionine, 95±18; threonine, 89±13; and tryptophan, 89±25. The cysteine mutant appeared to give unusually low bioavailability values except for the milk products. These amino acid mutants afford a rapid method for assaying the bioavailabilities of at least four of the five amino acids studied.     

Glutathione and related thiol compounds II. The importance of protein bound glutathione and related protein-bound compounds in gluten proteins

Protein-bound glutathione (PSSG) and protein-bound related thiol compounds, i.e. cysteine (PSSCys), glutamyl-cysteine (PSSGlu-Cys) and cysteinyl-glycine (PSSCys-Gly), were analysed in proteins of Osborne fractions, i.e. gliadin, glutenin and gliadin-, glutenin-subfractions separated by gel filtration chromatography, gel protein and the total gluten proteins separated from wheat varieties with varying breadmaking performances. The results showed that PSSG and some protein-bound related thiol compounds were found in monomeric gliadins, indicating that glutathione and some related thiol compounds are able to form disulphide bonds (SS) with sulphydryl group (SH) of those proteins and the formation of those disulphide bonds may prevent those monomeric proteins from binding to other proteins. It was also observed that a larger amount of PSSG in glutenin proteins was negatively correlated with the molecular weight (M_w) distribution of glutenin polymers, suggesting that PSSG and protein-bound related thiol compounds may play an important role in controlling polymerisation of glutenin. Furthermore, it was found that the level of PSSG in gel protein from flours with poor breadmaking performances was constantly higher and significantly different (p<0.05) from that of flours with good breadmaking performance. The same trend was observed with gluten samples from breadmaking and biscuitmaking flours.     

Properties of protein concentrates and hydrolysates from Amaranthus and Buckwheat

Protein concentrates and pepsin hydrolysates were made after isoelectric precipitation of the proteinaceous liquor from wet-milling of grain of five Amaranthus and one buckwheat genotype. The Amaranthus protein concentrates exhibited better solubility, foaming, and emulsification than two commercial soy protein controls. Many protein properties depend on solubility, and Amaranthus protein concentrates were more soluble than soy protein isolate. The buckwheat protein concentrate was highly soluble with excellent emulsification, but poor foaming ability. Partial pepsin hydrolysis further improved solubility of the protein concentrates and also altered their foaming property. Fractionation and subsequent characterization of protein concentrates revealed that glutelins, albumins, and globulins predominated, with prolamins present in minor quantity. SDS-PAGE showed that globulins and glutelins were comprised of several subunits with varying molecular weights from relatively high to low while albumins were mostly of low molecular weight. The prolamin fraction of the buckwheat concentrate was comprised of intermediate to low molecular weight subunits while those of Amaranthus concentrates were only of low molecular weight. This study demonstrated the feasibility of producing potentially useful functional protein concentrates as by-product of Amaranthus and buckwheat starch extraction.     

High Carbon Requirements for Seed Production in Soybeans [Glycine max (L.) Merr.]

Abstract

In soybeans, increase in the dry weight of seed (IWS) during the seed-filling period (SFP) is nearly half that of the whole plant (IWP). Hence, the contributions of assimilate during SFP to seed growth in soybeans is apparently lower than that in other cereal crops. However, analysis of the assimilate budget for seed production based on dry matter could be misleading in soybeans because the carbon (C) contents of seed and that of other organs are different due to the differences in lipid and protein content and respiration loss in each organ results in underestimation of the C contribution to seed production. In field soybeans, irrespective of shading and thinning, IWS/IWP and harvest index (HIW) for dry weight were slightly lower than ICS/ICP and HIC for C, respectively. Of the ¹³C fed at the vegetative stage and early SFP, 3.4% and 16.4%, respectively were accumulated in the seed, 0.9% and 28.2%, respectively, in the pod shell and 26.8% and 37.8%, respectively were respired before maturity. Ninety-six percent of seed C was assimilated during the SFP. Respiratory loss of ¹³C fed at the early SFP was larger than that from ¹³C at the vegetative stage, showing that seed production requires much more C than leaf or stem growth. These results suggest that the difference in C contents between plant organs has little effect on the estimation of assimilate partition into soybean seeds based on dry matter, and high respiration loss for seed growth reduces the partition of dry matter into seeds.     

Chemical and biological characteristics of a West African weaning food supplemented with compea (Vigna unguiculata)

The effects of cowpea and amino acid supplementation on the protein quality and chemical characteristics of a maize-based West African traditional weaning food were studied. Process optimization for improved nutritive value was also determined. Supplementation of the traditional weaning food with cowpea increased the lysine, tryptophan and threonine content while the sulphur-amino acids decreased with increasing levels of cowpea. Further supplementation of 70:30 maize/cowpea blends with lysine, threonine or methionine did not significantly improve (p>0.05) protein quality in terms of the biological value (BV) and net protein utilization (NPU), although significant increases in the protein scores were noted. However, considerable improvements in the BV and NPU were recorded in blends fortified with either tryptophan alone or a combination of lysine, tryptophan, methionine and threonine. Cooking whole cowpea seeds for 45 min before incorporating in the blend formulation also significantly improved (p<0.05) the protein quality of maize/cowpea blends. The BV and NPU of blends containing 30% pre-cooked cowpea increased from 52 to 76% and 50 to 71% respectively compared to pure maize porridge. The protein content increased from 10 to 14% and the utilizable proteins more than doubled. The weight increase of experimental rats fed with these blends was comparable to that of rats on casein diet. A 30% supplementation of the maize-based weaning food with cowpea therefore greatly enhances the nutritive value especially when the cowpea is pre-cooked for 45 min. Use of chemical scores alone for such blends cannot be a reliable index of blend quality.