Sweet lupine (Lupinus luteus, var. Aurea/Weico and Lupinus albus, var. Multolupa) proteins. II. Separation by electrophoresis

The albumins and globulins extracted from Lupinus luteus var. Aurea/Weico and Lupinus albus var. Multolupa defatted flour samples were analyzed by polyacrilamide gel electrophoresis. The purpose was to determine the percentage distribution of pattern in the electrophoretograms and the relative mobility values of protein. The L. luteus albumins densitogram showed six fractions, two of them, No. 2 and No. 4, represented 58.1% of the total. On the other hand, the globulins densitogram revealed five fractions, numbers 1, 2 and 3 representing 90.7%. The densitogram of peak I from Sephadex G-100 globulins filtration indicated two fractions similar to those number 1 and 2 of the total globulins, showing low relative mobility values (from 0.26 to 0.39). The L. albus albumins resolved in four fractions, with No. 2 being the most prevalent (54.3%). In regard to the L. albus globulins, these showed five fractions identical in distribution with those of peak I, fraction 2 appearing as the most important (48.2%). It was found that Sephadex G-100 did not perform a good separation. As to the relative mobility values of globulins, fractions 2 and 3 were the most prominent (relative mobility of 0.35 and 0.48, respectively). It may be concluded that L. luteus albumins presented more components but with lower relative mobility values than those of L. albus. As for the globulins from both lupine flour samples, the gel separated fractions were classified in two groups of components, those of high and of low relative mobility.     

Possibilities of Lupinus mutabolis and Lupinus albus in the Andean countries

Lupinus albus and Lupinus mutabilis may achieve importance among the andean countries in which soy bean can not grow due to ecological reasons. Both lupin varieties are outstanding because of their high protein and oil content. Its alkaloid content limits the lupins usage; however the bitter substances can be eliminated by means of genetic selection or technological processing. Beside the intoxication caused by alkaloids exists the lupinosis, which is caused by a micotoxin. This disease can be observed when animals pasture forages which suffered under a secundary attack of fungus. According to the results obtained up to date other antimetabolic substances present in the legums have no significant importance. The lupin seed flour is adequate for animal consumption, being used for this effect in different countries. Starting next year there exist the prospects of employing Lupinus mutabilis as an oil source in Peru and Lupinus albus as proteic flour in Chile.     

Electrophoretic study of albumins and globulins of 4 genotypes of Canavalia ensiformis

Polyacrylamide gel electrophoresis (PAGE) and PAGE-SDS were used to study seed albumins and globulins of Canavalia ensiformis. In PAGE the concentration of acrylamide used in the upper gel was 4.0% with a pH of 6.7 and in the lower gel 7.5% and 10% of acrylamide were used with a pH of 8.9. In PAGE-SDS the concentration of acrylamide was 4.4% with a pH of 6.8 in the upper gel and 7.5% and 12.6% with a pH of 8.8 in the lower gel. The material used were the Venezuelan genotypes Tovar, Yaracuy, Original and U-02. The albumins and globulins were extracted with a 0.5 M NaCl solution and then separated by dialysis against water and lyophilized. These protein fractions represented 84.85% of the total amount of protein in seeds. The albumins were separated in PAGE with 7.5% acrylamide into five fractions and globulins into six, with similar electrophoretic patterns between genotypes. In a similar manner, the patterns obtained in PAGE with 10% acrylamide were the same for all genotypes, showing five bands for albumins and three bands for globulins. With PAGE-SDS containing 7.5% of acrylamide, albumins were separated into as many as eight components, and globulins into as many as seven bands with mobilities between 0.2981 and 0.9932, with different patterns for each genotype. Also the patterns PAGE-SDS at 12.6% of acrylamide were different for the genotypes, separating proteins into a greater number of bands. The albumins showed as many as twenty-one bands with mobilities between 0.2603 and 0.7398, and globulins as many as sixteen bands with mobilities between 0.2454 and 0.7390. The PAGE patterns of the genotypes analyzed did not show differences between them. However, with PAGE-SDS different electrophoretic patterns were obtained which varied in the number and intensity of the bands, making it possible to distinguish the genotypes studied. The molecular weight of the albumins varied between 76,000 and 12,000 daltons and of the globulins between 80,000 and 12,000 daltons.     

Nutritive value of lupine and its potential as human food

The chemical composition and some indices of protein quality were measured in two species of sweet lupine Lupinus albus and Lupinus luteus grown at the Experimental Station in Gorbea, Chile: both samples showed a high protein content (39.5 and 44.6%). The protein efficiency ratio (PER) was measured in the rat and found to be 0.48 and 0.71, respectively, as compared to 2.57 for casein. Supplementation with 0.3% DL-methionine increased significantly those values, thus indicating that lupine protein is deficient in said amino acid. In another experiment the effect of cooking-extrusion on lupine flour (L. albus) was investigated and the chemical composition, protein efficiency ratio, methionine supplementation and digestibility of the protein were measured. The chemical composition was not changed but PER increased from 0.50 for raw lupine to 0.76 for processed lupine (P less than 0.05). Both values increased significantly with the addition of 0.3% DL-methionine. The protein digestibility of the supplemented lupine was not affected by the cooking extrusion process (76.5 and 77.8%, respectively). Supplementation of wheat flour with 5, 10, 15 and 20% lupine flour increased the PER of wheat flour from 0.92, to 1.39 for wheat flour supplemented with 10% lupine flour, and to 1.60 for the 15% level of the supplement. These studies seem to support the conclusion that sweet lupine is an interesting protein resource for human nutrition.     

Ebony (Phitecellobium flexicaule Benth) and proteins fractionation,solubilization, characterization and production of an isolate

Different combinations of pHs (2 to 12) and temperatures (25, 30 and 35 degrees C) were tested to obtain a protein isolate from ebony (Pithecellobium flexicaule, Benth) seeds. Seed proteins contained 54.6% albumins, 32% globulins, 5.7% glutelins and 1.3% prolamins. The isoelectric points for albumins, globulins and glutelins were in the pH range of 2.3-2.7. The average molecular weight of albumins ranged from 92 to 100 kDa and for the four globulin subunits in the range of 28.4 to 57.3 kDa. For isolate production, proteins were sequentially extracted with distilled water and a 5% NaCl solution. The resulting supernatants were mixed. The best extraction was achieved at pH 11 and 25 degrees C. 45.6% of the total seed protein was precipitated at pH 2.6 yielding an isolate with 90% protein (N x 6.25). The isolate contained high quantities of lysine, leucine, threonine and phenylalanine but were low in sulfur containing amino acids methionine and cysteine. The extraction process reduced tannins, phytates and trypsin inhibitor in 53, 70 and 70%, respectively. In vivo protein digestibility of the protein isolate was 85.4% and the corrected digestibility essential amino acid score was of 44% due to the lack of sulfur containing amino acids. In order to upgrade the protein quality of ebony isolate it is recommend to supplement with methionine or sulfur containing rich foods.     

Phenotypic and technological influences of the Lupinus mutabilis (Tarwi) seed on its methionine availability and sulfur content

The present study was carried out to determine the content of available methionine and sulphur in seed cultivars of Lupinus mutabilis from different Andean regions, and to study the influence of processing on methionine and sulphur contents. An additional objective was to evaluate interrelationships among these chemical characteristics and protein quality, as measured by the protein efficiency ratio (PER) method. Results revealed a high variability in the content of available methionine and sulphur between the different ecotypes and varieties of Lupinus mutabilis. Fertilization with CaSO4 (200 kg/ha) did alter the content of available methionine and sulphur in Lupinus albus seeds. Traditional water-debittering of lupines did not affect the methionine content of the seeds, whereas oil-extraction and alcohol-debittering led to a decrease in available methionine (14 and 23% reduction, respectively). Production of a protein isolate further reduced the methionine content (54%). Regression analysis revealed a high correlation between available methionine and sulphur (r = 0.83), between sulphur and PER (r = 0.98) in the processed lupine samples, and lupine mixtures with other protein sources.     

Fractionation of sunflower seed proteins

Fractionation of sunflower seed salt-soluble proteins, which amount to nearly 80% of the total seed nitrogen, has been performed by a method we proposed in 1970 and which was confirmed by several others. Three varieties of seeds have been investigated: ‘Armavirec,’ ‘Peredovik’, and a pure strain. The occurrence of three groups of proteic fractions was confirmed. Their proportions, which fluctuate with varieties, are roughly: 20% for “light” (low molecular weight) albumins, 5–10% for “heavy albumins,” and 70–80% for globulins. The first group was isolated by Sephadex G-50 chromatography from the other two, which were separated by dialysis. A second chromatography of these three groups on Sephadex G-200 has been realized (with preliminarily calibrated columns for molecular weight evaluations). “Light” albumins appear as a rather homogeneous constituent with a molecular weight of 14,000 and an aminoacid composition showing high amounts of methionine, cystine, arginine and glutamine. “Heavy” albumins, which are still mixed with globulin fractions after dialysis, have a molecular weight of 48,000 and a very different aminoacid composition with a high level of lysine. Globulins are composed of at least four different fractions, two of which (M=12,000 and M=25,000) are presumably subunits of the other two and have significantly different aminoacid compositions.     

Effect of heat treatment and pH on the thermal,surface, and rheological properties of <Emphasis Type="Italic">Lupinus albus</Emphasis> protein

Endosperm from hand-dissected and- dehulled Lupinus albus seeds was milled into meal, sieved through a 40-mesh screen, and suspended in phosphate buffers (pH 4, 6.8, and 8) at 20% (wt/vol). The suspensions were treated at 75, 90, or 100°C for 1 h. The heat-treated protein was characterized by SDS-PAGE, free zone capillary electrophoresis (FZCE), and DSC; and its surface hydrophobicity, surface tension, and rheological properties were examined. The presence of high M.W. aggregates was apparent from SDS-PAGE and FZCE results. Solubility was lowest at pH 4 and 100°C. DSC analysis was performed on low moisture content samples (3.1%) and 20% (wt/vol) suspensions. DSC analysis at 3.1% moisture content showed a glass transition around 85°C and an exothermic transition at 160°C, whereas the protein suspension showed a more thermally stable protein as indicated by the higher ΔH values. Lupin protein was surface active as demonstrated by its effectiveness in reducing the surface tension of the aqueous phosphate buffer. Surface hydrophobicity of the heat-treated protein decreased as the treatment temperature increased, which supports the SDS-PAGE results. The highest level of aggregation was noted at 90°C and pH 6.8 as indicated by low surface hydrophobicity values. Rheological studies showed direct relationships between the shear storage modulus (G′) of the lupin meal suspension and both pH and temperature treatment, although this effect is minimal at the highest temperature (100°C) and pH 6.8.     

Peanut alkaline lipase

Peanut alkaline lipase, (glycerol ester hydrolase EC 3.1.1.3), pH optimum 8.5, was isolated from acetone powders prepared from developing and germinated peanut seed (arachis hypogaea L. var. NC-2). Enzyme activity/seed increased in successive developmental stages. The course of the hydrolytic reaction was linear with regard to enzyme concentration and all times tested up to periods exceeding 60 min. Km for the reaction was determined to be 2.6 times 10-4M. Molecular weight of peanut lipase, as estimated by Sephadex gel filtration and sodium dodecyl sulfate gel electrophoresis, was ca. 55,000.     

Effect of succinylation of cottonseed flour during protein extraction on the yield and some of the properties of protein isolates

A new method has been developed to improve extraction and recovery of protein isolates, which are highly water-soluble, low in sensitivity to calcium precipitation and light-colored, from defatted cottonseed flour. Optimal conditions include extraction of flour with acidified n-butanol (pH 4.5) to remove chromophores, succinylation of proteins at pH 8.5 using succinic anhydride at a concentration of 30% on protein basis, and precipitation of protein at pH 4.5. The resulting succinylated isolates contain more hydrophobic and neutral amino acids than untreated isolates. Gel filtration chromatography and polyacrylamide gel electrophoresis of protein isolates (water-, salt [4% NaCl]- and alkali [0.2% NaOH]-soluble), sequentially fractionated from succinylated and untreated flours, suggest that succinylation converts much of the salt- and alkali-soluble proteins to water-soluble forms. Succinylation increases emulsion stability of isolates. The 3 succinylated isolates showed similar Chromatographie patterns on Sephadex G-100 columns and clectrophoretic mobilities in polyacrylamide gels, whereas the corresponding 3 isolates from untreated flour had different gel patterns and mobilities. Mobilities of major protein components of the 3 isolates were increased by succinylation.     

Qualitätsbeurteilung von Proteinkonzentraten und -isolaten aus dem Ölkuchen von Lupinus mutabilis

Evaluation of the Quality of Protein Concentrates and Protein Isolates from the Oil Cake of Lupinus mutabilis The seeds of Lupinus mutabilis contain rather high concentrations of protein amounting to 40%. Their relative high fat content of about 18% made their use as oil seeds possible. The lupine oil was extracted using a process of extraction and manufacturing of the soya oil. The oil cake was debittered with aqueous ethanol (80% ethanol) to a protein concentrate (> 60% protein) from which a protein isolate (> 90% protein) was prepared using an acidic sedimentation. The preparation of both products led to an increase of the protein concentrations (? N × 6.25). In both preparations the S-containing amino acids remained the limiting factor of the protein quality. The alkaloid content decreased from 3.3% in the seeds to 0.06% in concentrates and to 0.02% in the isolate. The protein isolate was free from HCN, trypsin inhibiting factors and haemagglutinins. The evaluation of the protein quality with feeding experiments with rats (PER test) showed that the quality of protein concentrate was about 39% of that of casein (0.9/2.5). While acidifying the debitterning medium to pH 5 did not affect the protein quality, changing the medium to pH 9 led to a concentrate, the protein of which was not available to the rats. Adding 0.2 g DL-Methionine to 100 g diet improved its protein quality to about 84%. The preparation of the protein isolate affected the bioavailability of the S-amino acids, which could not be recognized with the chemical analysis. The quality of the protein isolate amounted to only 19% of that of casein. The supplementation with 0.2% DL-Methionine in the diet improved it to 89%. The digestibility of lupine protein amounted to 83%. In mixtures of wheat protein with lupine protein concentrates (90/10) the limiting amino acids of both proteins could mostly be compensated; the calculated PER-value was 2.3 (78% of that of casein). This result shows that protein concentrates from lupine oil cake could be successfully added to bread and bakery products.     

Gewinnung und Entbitterung von Speiseöl und Proteinkonzentrat aus Samen von Lupinus mutabilis

Production and Debittering of Edible Oil and a Protein Concentrate from Seeds of Lupinus mutabilis Lupine oil was produced from seeds of L. mutabilis using an extraction and refining process of soya oil. The refining included a step of debittering by washing with diluted acids. This decreased the alkaloid content from 0.14 % in the raw oil to 5 ppm in the endproduct, a content of rest alkaloids, which can be considered as unobjectionable. The oil alkaloids are not identical with those of the seeds. While in seeds Lupanin is the main alkaloid fraction, in the oil 13-Hydroxylupanin and N-Methyl angustofolin are dominant. The raw oil contained 800 ppm γ-Tocopherol 39 ppm α-Tocopherol. During the refining process the Tocopherol content decreased from about 840 ppm to 530 ppm total Tocopherol in the endproduct. The oil-cake contained about 4 % alkaloids. With aqueous alcohol (70 %?90 % ethanol) was debittered to a protein concentrate, which contained 73% protein and 0.06% rest alkaloids. By changing the pH value of the debittering medium both in the acid (pH 5) and alkaline (pH 9) range the alkaloid extraction could be improved and the loses of protein could be diminished. Qualitatively the alkaloid pattern of the protein concentrate was similar to that of seeds, although the hydroxylupanin fraction increased from 32.7% of total alkaloids before the debittering to 42.3% in the debittered concentrate. This is advantageous because the toxicity of hydroxylupanin is only about 1/10 of that of Lupanin.     

Characteristics of Canavalia proteins

The purpose of this work was the isolation and characterization of grain protein from five Venezuelan Genotypes (U-02, Yaracuy, Valle De La Pascua, Originally Tovar) of Jack Bean (Canavalia ensiformis). The average protein content from these genotypes was 31.37%, it ranged between 28.44% and 33.05%. The protein isolation was performed by solubility extraction procedures, showed: 84.57% of albumins, globulins and non proteic nitrogen and 15.43% of alcohol insoluble reduced glutelin (AIG). The content of anti-nutritional factors (canavanine and hemagglutination title) found in protein fractions were respectively: Albumins 1.96%, +4; globulins 0.17%, +5 and AIG 0.22%, +1. It was observed that protein fractions of genotype Tovar had the lowest canavanine values (0.79%, 0.02% and 0.00% respectively). The globulins gave the highest in vitro protein digestibility (65.20%) followed by Albumins (58.90%) and AIG (37.28%).     

Isolation and chemical investigation of teak (Tectona grandis linn) seed proteins

Proteins were extracted from the deoiled seeds ofTectona grandis Linn., Fam. Verbenaceae, a quality lumber source, in aqueous solutions of various pH’s or by different concentrations of NaCl at pH 8.0. Chemical analysis of isolated protein identified 15 amino acids, of which eight were essential. Gel filtration on Sephadex G-200 revealed the presence of six components, whose molecular weights were determined by two comparable standard methods. Seven components were resolved electrophoretically (SDS-PAG electrophoresis) and their molecular weights were found to be 118,900, 92,300, 72,400, 62,400, 43,600, 39,800 and 32,400.     

Characterization of proteins in Cuphea (PSR23) seeds

This study characterized the proteins in Cuphea (PSR23) seed to provide fundamental information on their size, amino acid profile, solubility classes, and solubility behavior. The seed contained 32% (dry basis, db) oil and 21% (db) crude protein. Over 70% of the protein was extracted at pH 11.6. Nonprotein nitrogen accounted for 9% of the total N content. Compared with the Food and Agriculture Organization/World Health Organization/United Nations University suggested pattern of requirements, Cuphea PSR23 seed protein had sufficient amounts of methionine+cystine-cysteine, considerable amounts (90%) of valine, phenylalanine+tyrosine, but was practically devoid of tryptophan. Lysine was the second-most limiting essential amino acid at 68%. Glutelins and albumins accounted for 83.5 and 15.4%, respectively, of the total protein extracted. SDS-PAGE showed that Cuphea protein subunits had M.W. ranging from <6.5 to 110 kDa. Dominant protein subunits in albumins had M.W. of 30, 40, 50, and 86 kDa. Glutelins had two major protein subunits with M.W. of 15 and 30 kDa. The distribution of essential amino acids was better in the albumin and glutelin fractions than in the defatted meal.     

Q Sepharose^TM XL强阴离子交换色谱快速纯化猪心中肌红蛋白和细胞色素C

以Q SepharoseTM XL强阴离子交换色谱结合Sephadex G-75分子排阻色谱快速提取纯化猪心中肌红蛋白（Myoglobin,Mb）和细胞色素C（Cyt C）。在pH值为7.6-8.5范围内,Cyt C吸附在Q SepharoseTMXL强阴离子交换柱上,而Mb会缓慢通过。所以选择QSepharoseTMXL强阴离子交换柱的上样pH值为7.9,洗脱pH值为7.2。电泳检测证明,经过QSepharoseTMXL柱初步纯化所得Mb和Cyt C的产率分别为89.7%和93.2%;再经过Sephadex G-75分子排阻色谱可得到纯的Mb和Cyt C,其产量分别为77.43mg.（kg猪心）-1和86.42mg.（kg猪心）-1。使用QSepha-roseTMXL强阴离子交换色谱能从猪心中快速制备纯的Mb和Cyt C。     

Proteins from Crambe abyssinica oilseed. I. Isolation procedure

Crambe abyssinica is a promising new oil crop because of the specific properties of its oil. However, little information is available concerning the properties of the proteins, which constitute a major component of the seed. Therefore, a method for the isolation of proteins from Crambe seeds was developed. Protein extractability for whole and dehulled Crambe meal was studied as a function of pH. Highest extractability was obtained with dehulled meal at pH 11. Double extraction at this pH increased the extractability to about 66%. Protein precipitation from the above-mentioned extract was studied as a function of pH with and without addition of a precipitating agent, i.e., carboxymethylcellulose (CMC). Addition of CMC resulted in a protein recovery of about 75% at pH 4.4. Without CMC, about half of the protein was recovered by isoelectric precipitation. The remaining soluble protein could be concentrated by ultrafiltration with a recovery of about 65%. The developed process, not including CMC addition, results in two protein fractions, i.e., an isoelectric precipitate (protein content 75%) and a retentate (protein content 87%), which together account for about 50% of the protein present in Crambe meal. Application of heat decreased protein extractability, but the protein contents of the resulting fractions were comparable to those from non-heat-treated meal.     

酸模叶蓼和绵毛酸模叶蓼中脂肪酸的研究

用索氏提取法提取脂溶性成分,GC-MS联用法结合计算机检索,首次对酸模叶蓼及其变种绵毛酸模叶蓼的脂肪酸成分进行了分离和鉴定。从酸模叶蓼和绵毛酸模叶蓼中各分离鉴定出21个和13个化合物,分别占峰面积的63.26%和74.73%,其中,脂肪酸成分占53.53%和35.58%,甾类成分占9.73%和39.15%。用峰面积归一化法测定了各成分的相对质量分数,酸模叶蓼中主要成分为:花生酸(11.82%),亚麻酸(10.36%),棕榈酸(9.79%),亚油酸(8.51%),22,23-二氢豆甾醇(5.88%),γ-谷甾醇(3.85%),山嵛酸(3.15%),木蜡酸(2.6%)。绵毛酸模叶蓼主要成分为:γ-谷甾醇(20.61%),22,23-二氢豆甾醇(18.54%),亚油酸(10.41%),棕榈酸(7.42%),油酸(7.50%),花生酸(3.21%),硬脂酸(2.38%)。该文报道工作的新颖性,已为河南大学图书馆2008年9月9日出具的第CX2008008号《科技查新报告》所证实。     

Fractionation and characterization of proteins from <Emphasis Type="Italic">Gevuina avellana</Emphasis> and <Emphasis Type="Italic">Rosa rubiginosa</Emphasis> seeds

Gevuina avellana and Rosa rubiginosa proteins were evaluated for their potential food use. The proteins were sequentially separated into five fractions according to their solubilities in deionized water, 0.5 M NaCl, 70% (vol/vol) isopropyl alcohol, 50% (vol/vol) glacial acetic acid, and 0.1 M NaOH. The five fractionated protein groups were then characterized by SDS-PAGE and gel filtration chromatography to determine their M.W. profiles. Ninety-six percent of G. avellana total protein was solubilized in three extraction stages, and 88% of R. rubiginosa total protein was solubilized in one extraction stage. Albumins were the major protein fraction in G. avellana and glutelins-1 the most abundant in R. rubiginosa. The protein solubility profile determined over the pH range 1–12 showed minimal solubilities at pH 3–5 and pH 3–7 for G. avellana and R. rubiginosa, respectively. Electrophoretic studies revealed the existence of proteins composed of two major kinds of polypeptides linked together via disulfide bonds and with molecular masses ranging from 13 to 119 kDa. Gel filtration chromatography profiles of globulins and albumins were studied for both seeds. Isoelectric focusing showed an isoelectric point in the ranges of 4.5–6 and 3–6.5 for G. avellana and R. rubiginosa proteins, respectively.     

一种竹腐朽菌胞外木聚糖酶的分离和特性研究

从腐朽的竹子上分离得到一株产木聚糖酶侧耳真菌Pleurotus sp.GH196,研究了其液体振荡培养的产酶条件。适宜碳源是1%稻草粉与1%麸皮组成的复合碳源,氮源是0.2%NH4NO3。经过硫酸铵分级沉淀、DEAE-Sephadex A-25离子交换层析、Sephadex G-100凝胶过滤三步纯化得到木聚糖酶的一个主要酶活组分A。该木聚糖酶在40～50℃、pH 4.0～5.5可以保持较好的稳定性,酶活最适反应条件是温度55℃、pH 4.5。