Isolation and chemical composition of surface-active material from human lung lavage

Surface-active material (SF) was isolated from human lung lavage fluid collected at autopsy employing differential and sucrose density gradient centrifugation. The isolated material showed well-defined electron microscopic structure, consisting of clearly preserved, closely packed vesicles with limiting membranes and inclusion bodies. It showed a very high degree of alkaline phosphatase specific activity and was devoid of other subcellular contaminants. The isolated material also showed a high phospholipid/protein ratio and increasing surface activity when monitored at different stages of purification. It contained 68.5% phosphatidylcholine, 11.5% phosphatidylglycerol and relatively smaller amounts of phosphatidylethanolamine and other individual phospholipid (PL) classes. In addition, cholesterol, unesterified fatty acids, triacylglycerols and other neutral lipids were found. Saturated fatty acids, particularly palmitic acid (16∶0), predominated in the major PL fractions. However, various fatty acids of which oleic acid (18∶1) constituted a large proportion also are present. Chemical analysis of the material showed that besides lipids and proteins, nucleic acids, sialic acid, hexose, amino sugars, nitrogen and phosphorus were present. The delipidated material showed the presence of three to four proteins as characterized by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, and gel permeation chromatography on Sephadex G-200 resolved two well-separated peaks. The first fraction contained serum-associated 68 kDa protein, while the second fraction had two apoproteins with molecular weights of 34 kDa and 10 kDa. These two proteins were associated with the SF and they, as well as the whole surface-active material, strongly reacted with the antibody directed against the whole SF in a double-diffusion immunoprecipitation assay. The first Sephadex fraction containing a 68 kDa protein did not produce any precipitation line when reacted against antisera.     

Fractionation and Some Chemical Studies on Ailanthus excelsa Roxb. Seed Protein

The unavailability of protein foods, particularly in the context of population growth, has been an important factor in the protein malnutrition encountered in developing countries. The fractionation, gel filtration and polyacrylamide gel electrophoresis (PAGE) of Ailanthus excelsa seed (a nontraditional source containing 15.81% protein) proteins were carried out in the present study, and their solubility profiles, surface topographies and amino acid compositions were evaluated. The globulin fraction dominated the seed protein composition, accounting for 51.31% (w/w) of the total soluble proteins in the seeds. Protein isolate and protein fractions of A. excelsa seeds showed similar topographical structures to those of other plant seed proteins. Analysis of the isolated proteins identified 17 amino acids, of which nine were essential. Gel filtration on Sephadex G-200 revealed the presence of seven components. PAGE detected different polypeptide bands in the range of 28.8−154.9 kDa in the protein isolate as well as in protein fractions for A. excelsa. The amino acid compositions, the solubility patterns and the high abundances of low molecular weight proteins indicate that the isolated seed protein of A. excelsa may be a potential food protein.     

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.     

Fractionation of Condensed Distillers Solubles and Compositional Characterization of Its Co-Products

Condensed distillers solubles (CDS) was fractionated into a protein-mineral fraction and a glycerol fraction by a chemical method; protein and glycerol-mineral fractions by a physical method; and protein, mineral, and glycerol fractions by a physicochemical method. The co-products from each method, along with CDS, were characterized for concentrations of key constituents (protein, oil, ash, glycerol and other carbohydrates), mineral profile, and amino acid composition. Recovery of mass and main constituents was also investigated. With the chemical method, about two-third of the mass went to the protein-mineral fraction, while by the physical method the equal amounts of mass went to the protein and glycerol-mineral fractions. Protein, minerals, and glycerol were mostly recovered into their respective fractions. CDS and its fractions contained six major minerals (Ca, Mg, P, K, Na, and S) and four trace ones (Cu, Fe, Mn, and Zn). Both chemical and physical treatments caused significant reduction of mineral contents (dry matter basis) in the protein or glycerol fraction. Several amino acids differed significantly in percent relative to total amino acids among fractions but the extent was not substantial for protein-rich fractions. These changes resulting from CDS fractionation are favorable for value added utilization of the new co-products.     

Amino Acid Composition,Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L.) Proteins and Protein Fractionations

As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.     

Extractability of protein in physically processed rice bran

Commercially obtained defatted (DF), full-fat stabilized (FFS), and full-fat unstabilized (FFU) rice bran were processed by colloid milling and homogenization to affect bran breakdown and extraction of rice protein. Relative to unprocessed samples, there were moderate to slight increases in the amount of protein extracted from the various fractions of processed bran. Colloid milling and homogenizing slightly influenced the distribution of proteins in the various fractions obtained, with the FFU showing the greatest effect compared to DF and FFS protein fractions. The protein content of the supernatant fraction of FFU bran increased from 21.8 to 33.0% after colloid milling with a further increase to 38.2% after homogenizing, representing an overall increase of 75.2% in protein content. The supernatant fractions of DF bran increased from 13.9 to 14.7% after colloid milling, and to 16.5% after colloid milling and homogenizing, for an overall increase of 18.7%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a molecular weight distribution ranging from 6.0 to 97.4 kDa. Few detectable differences between protein bands of unprocessed and processed DF and FFU bran were observed. However, FFS bran showed breakdown in size distribution of protein after colloid milling and homogenizing, because certain high molecular weight proteins shifted to lower molecular weight units.     

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.     

Cottonseed Meal Protein Isolate as a New Source of Alternative Proteins: A Proteomics Perspective

Cottonseed meal (CSM) is a good source of dietary proteins but is unsuitable for human consumption due to its gossypol content. To unlock its potential, we developed a protein extraction process with a gossypol removal treatment to generate CSM protein isolate (CSMPI) with ultra-low gossypol content. This process successfully reduced the free and total gossypol content to 4.8 ppm and 147.2 ppm, respectively, far below the US FDA limit. In addition, the functional characterisation of CSMPI revealed a better oil absorption capacity and water solubility than pea protein isolate. Proteome profiling showed that the treatment improved protein identification, while SDS-PAGE analysis indicated that the treatment did not induce protein degradation. Amino acid analysis revealed that post-treated CSMPI was rich in branched-chain amino acids (BCAAs). Mass spectrometry analysis of various protein fractions obtained from an in vitro digestibility assay helped to establish the digestibility profile of CSM proteins. Several potential allergens in CSMPI were also found using allergenic prediction software, but further evaluation based on their digestibility profiles and literature reviews suggests that the likelihood of CSMPI allergenicity remains low. Overall, our results help to navigate and direct the application of CSMPIs as alternative proteins toward nutritive human food application.     

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.     

Evaluation of the protein quality of Erythrina edulis (balú)

The protein content (N x 6.25) of the seeds of Erythrina edulis (balú) varies between 18-21%; when the fraction corresponding to non-protein nitrogen is extracted with trichloroacetic acid (10%), this value decreases to 14-15%. Remarkable differences in the distribution of the protein fractions are observed when two schemes of extraction are assayed. The amino acid analysis shows that this legume has similar or higher amounts of most amino acids than those present in other leguminosae; the calculated chemical score and protein score show that methionine is the first limiting amino acid and tryptophan, the second. The protein efficiency ratio (PER) of thermically-treated flours has the highest value at 30 minutes of treatment (1.15).     

Production and characterization of an extensive rapeseed protein hydrolysate

Rapeseed protein isolate has been used as starting material for the generation of an extensive protein hydrolysate. Protein hydrolysis was produced by using sequentially an endopeptidase (Alcalase) and an exopeptidase (Flavourzyme). The final hydrolysate has a 60% degree of hydrolysis and was completely soluble between pH values 2.5 and 7. Molecular weight profile of the protein hydrolysate was characterized by gel filtration chromatography. A reduction in protein size was observed during the hydrolysis process with accumulation of small peptides and free amino acids after Flavourzyme digestion. Amino acid composition of fractions with different molecular weights of the final hydrolysate was analyzed. Some of these fractions, enriched or poor in certain amino acids, could be used for supplementation or treatment of determined clinical syndromes.     

Emulsifying and foaming properties of native and chemically modified peptides from the 2S and 12S proteins of rapeseed (Brassica napus L.)

The 2S and 12S proteins of rapeseed were isolated and subsequently hydrolyzed by pepsin or a combination of pepsin plus trypsin. The resulting hydrolysates had a 15% degree of hydrolysis and were purified by gel filtration chromatography in order to obtain homogeneous peptide fractions. Three major fractions, having an average peptide chain length of 7.5–11 amino acids, were recovered. Purified peptide fractions were acylated with butyric anhydride and sulfamidated with p-toluenesulfonyl chloride. The degree of modification was always higher than 90%. Emulsifying and foaming properties of native and chemically modified peptides were studied and compared to those of sodium dodecyl sulfate (SDS) as standard. A peptide fraction from the 15% hydrolysis of the 12S protein exhibited the best foaming properties. After sulfamidation, this peptide fraction showed a foam formation similar to that of SDS. Whereas the attachment of toluene groups generally improved the surface properties, the incorporation of an aliphatic chain of four atoms of carbon was detrimental in most of the cases. On the other hand, none of the native or hydrophobized peptide fractions was able to form a stable emulsion.     

Biochemical and chemical partial characterization of Bauhinia forficata Link seeds

Seeds of Bauhinia forficata species were submited to biochemical characterization concerning fatty acids analysis, protein fractionization, and hemaglutinanting activity. The seed elementary analysis showed a high protein and lipids contents with 21.24% and 19.45% respectively. The more abundant fatty acid was linoleic acid with 46.47% of the lipidic fraction. With the exception of prolamins, the different proteic fractions (albumin, globulins, acid and basic glutelins) showed hemaglutinanting activity against rabbit red cells no treated and treated with proteolitic enzymes. The fraction acid glutelin showed the higher specific hemaglutinanting activity (1072.25 UH/mg P) against rabbit blood pre-treated with trypsin. Glutamin (16.20%) and Valin (11.07%) were the more abundant amino acids in the seeds. Therefore, B. forficata represent a possible optional source of food because exhibit a high energetic values.     

<Emphasis Type="Italic">Lesquerella fendleri</Emphasis> protein fractionation and characterization

Lesquerella fendleri is a promising new crop whose seed contains hydroxy FATG with potential industrial uses as well as substantial amounts of valuable gums. The defatted L. fendleri seeds also contain more than 30% protein. The objective of this study is to process and characterize this protein component for possible future uses in food. Hexane-defatted seed has more than 30% protein content. Defatted lesquerella meal was extracted sequentially with 0.5 M sodium chloride (2×), water, 70% ethanol, and 0.1 N sodium hydroxide (2×). Each sodium chloride extract was dialyzed against deionized water and centrifuged to separate the water-soluble fraction (albumin) from the salt-soluble fraction (globulin) before freeze-drying. The ethanol extract and the neutralized sodium hydroxide extracts (glutelin) were dialyzed against water and freeze-dried. Albumin had the highest proportion of lysine and sulfur amino acids per 16 g nitrogen among all the fractions analyzed. Glutelin and globulin accounted for the highest amount of protein nitrogen. SDS-PAGE of the reduced albumin, globulin, and glutelin showed the presence of several protein bands with M.W. ranging from 7 to 98 kDa. Nitrogen solubility of defatted lesquerella meal from pH 2 to 12 indicated a solubility minimum of 15% around pH 4.2 and a solubility of 75% at pH 11.5. Nonprotein nitrogen of defatted meal was 12% of total nitrogen. Defatted lesquerella meal has the potential for food use based on good nitrogen solubility and good amino acid composition.     

Fate of minor free amino acids and phospholipids in crude tallow during steam splitting

Minor free amino acids and phospholipids contained in crude tallow were monitored during steam splitting of crude tallow. The bulk of the phospholipids was found in the glycerol sidestream after splitting. Phosphatidic acid and phosphatidylcholine were present in both crude tallow and the glycerol fraction. Phosphatidylserine and phosphatidylethanolamine present in crude tallow were hydrolized with the glycerides. Because of this hydrolysis, high amounts of serine and ethanolamine are found in the fatty acid and glycerol fractions. In addition to constituent amino acids of proteins present in crude tallow, other biological amino acids such as taurine and ornithine were also present.     

Effect of Peptide Size on Antioxidant Properties of African Yam Bean Seed (Sphenostylis stenocarpa) Protein Hydrolysate Fractions

Enzymatic hydrolysate of African yam bean seed protein isolate was prepared by treatment with alcalase. The hydrolysate was further fractionated into peptide sizes of <1, 1-3, 3-5 and 5-10 kDa using membrane ultrafiltration. The protein hydrolysate (APH) and its membrane ultrafiltration fractions were assayed for in vitro antioxidant activities. The <1 kDa peptides exhibited significantly better (p < 0.05) ferric reducing power, diphenyl-1-picryhydradzyl (DPPH) and hydroxyl radical scavenging activities when compared to peptide fractions of higher molecular weights. The high activity of <1 kDa peptides in these antioxidant assay systems may be related to the high levels of total hydrophobic and aromatic amino acids. In comparison to glutathione (GSH), the APH and its membrane fractions had significantly higher (p < 0.05) ability to chelate metal ions. In contrast, GSH had significantly greater (p < 0.05) ferric reducing power and free radical scavenging activities than APH and its membrane fractions. The APH and its membrane fractions effectively inhibited lipid peroxidation, results that were concentration dependent. The activity of APH and its membrane fractions against linoleic acid oxidation was higher when compared to that of GSH but lower than that of butylated hydroxyl toluene (BHT). The results show potential use of APH and its membrane fractions as antioxidants in the management of oxidative stress-related metabolic disorders and in the prevention of lipid oxidation in food products.     

Extraction of amino acids from protein hydrolysates by electrodialysis

Protein hydrolysates were obtained by acid hydrolysis from animal or human residues, such as poultry feathers, ox blood and human hair. After neutralization and discolouration with active charcoal, the hydrolysates were treated by successive electrodialysis (ED) in order to extract amino acids into several fractions. The current density and pH were optimized for each ED operation performed with preindustrial pilot scale equipment. The first step was the demineralization of amino acid mixtures using an ED stack with two compartments. The salt removal was achieved with extraction degrees higher than 90% and current efficiencies of about 80%. In the most favourable case, the amino acid losses did not exceed 10%. The second step was the extraction of the charged amino acids using an ED stack with four compartments. Three fractions were obtained, corresponding to the acidic, basic and neutral amino acids. The extraction degrees varied from 80% to 100%. In the third step, the fractionation of basic amino acids on the one hand, and neutral amino acids on the other hand, was carried out with enrichment degrees varying from 50% to 80%. © 1998 SCI     

Fatty acid composition of polar lipids in goats' milk

Silicic acid column chromatography was used to separate the polar lipids of goats' milk into glycolipid, phosphatidylethanolamine, phosphatidylserine plus phosphatidylinositol, phosphatidylcholine, and sphingomyelin fractions. Each fraction was purified by column chromatography and its fatty acid profile determined by gas liquid chromatography and mass spectrometry. The glycerophospholipids each contained 18∶1 as the predominant fatty acid (∼45%). The sphingolipids contained a high percentage of long-chain saturated fatty acids (C₂₂ to C₂₄>45%); the glycolipid fraction also contained ca. 2% 2-hydroxy fatty acids. The data represent a comprehensive cross-sectional study of the major polar lipids found in goats' milks.     

Host egg kairomones essential for egg-larval parasitoid,Ascogaster reticulatus Watanabe (Hymenoptera: Braconidae)

Several components of an internal kairomone were identified inside eggs of the host,Adoxophyes sp. (Lepidoptere: Tortricidae), that releases egg deposition of the egg-larval parasitoid,Ascogaster reticulatus Watanabe (Hymenoptera: Braconidae). Pupal hemolymph with the same activity as an internal host egg kairomone was used as a convenient test sample. Heat-treated pupal hemolymph was chromatographed on a Sephadex G-25 column. Each fraction was bioassayed and reacted with ninhydrin. The active fractions were ninhydrin-positive. Each fraction was placed onto an araino acid analyzer, which showed that the amino acids were most abundant in active fractions. Among 22 amino acids, alanine, arginine, glycine, histidine, isoleucine, leucine, methionine, proline, serine, tryptophan, and valine were active. The mixture of these active amino acids was as active as the egg-mass homogenate at the same ratio and concentration, suggesting that the most important component as the kairomone in a host egg is the mixture of several amino acids.     

Protein fraction distribution in milling and screened physical fractions of grain amaranth

The purpose of the study was to establish the protein distribution based on solubility in physical fractions of amaranth flour, in particular between the flour from the germ and that from the perisperm. The protein distribution was obtained applying a series of solvents sequentially utilized in the classical methodology of Osborne & Mendel. The sample of A. cruentus weighing 2000 g was divided into 4 subsamples of 500 g each. One was left as the control while the other 3 were ground individually with a mill. Each flour was screened through 18, 20, 30 and 40 mesh screens, so that 5 fractions were obtained from each of the whole grain flours. Samples of each screened fractions were observed by stereoscopy and analyzed for moisture, fat and protein. This characterization suggested that the fraction above the 30 mesh screen and the flour which passed the 40 mesh screen probably were the perisperm and germ respectively. The 30 mesh sample contained 2.34 fat and 9.05% protein while the 40 mesh contained 16.18% fat and 26.46% protein. The extraction and partitioning of the proteins indicated that the most important fractions in germ and perisperm were the water soluble and glutelins measured by Kjeldahl. The relationship of the water soluble + globulin to glutelins ratio was 2.1 to 1 in the whole grain, 1.9 to 1 in the perisperm and 1.7 to 1 in the germ. The distribution of proteins was very much alike between germ and perisperm. The levels of prolamines were quite low. The protein extraction of the perisperm proteins retained on the 30 mesh screen was low (71.1%) measured by Kjeldahl and 47.4% with the Bradford method to measure protein.