Amino Acid Composition Analysis Research Articles

Analysis of Amino and Fatty Acids Composition of Senna alata Seed

Aim: To investigate the amino acids and fatty acids contents of Senna alata seed. Study Design: To Collect and analyze the seed sample of Senna alata from Osun State in South Western Nigeria. Place and Duration of Study: Seed samples of Senna alata were collected from Irepodun Local Government, Osun state, Nigeria at the start of the raining season in March, 2013. Methodology: The collected samples were identified at Taxonomy unit, Department of Biological Sciences, Usmanu Danfodiyo University, Sokoto, as Senna alata. The determination of the amino acid profile in the sample was determined using methods described in literature while the determination of the protein content was done using micro Kjeldahl method. The amino acid was analyzed using a Technicon Sequential Multi-Sample Amino Acid Analyzer (TSM). The fatty acids content was determined by converting the fatty acids to fatty acid methyl esters (FAMEs), after which it was loaded to a gas chromatography coupled with mass spectrometer for analysis. Results: The protein content of the seed was 12.75±0.05% while the amino acids content revealed glutamic acid and aspartic acid as predominant non essential amino acids while leucine as the Original Research Article Adigun et al.; ACSj, 7(1): 1-6, 2015; Article no.ACSj.2015.056 2 most predominant essential amino acid. The sample also contains appreciable amounts of other essential amino acids. The fatty acid content of Senna alata seed oil revealed unsaturated fatty acid; linoleic acid as the major fatty acid (34.26%), followed by saturated fatty acid; palmitic acid (13.73%). The oil was having more unsaturated fatty acid (51.90%) than the saturated fatty acid (43.34%). Conclusion: Senna alata seed is a good source of valine, isoleucine, leucine, phenylalanine, tyrosine and threonine. The high amount of linoleic acid and palmitic acid also suggest the edibility of Senna alata seed oil.

Mapping of QTL Associated with Seed Amino Acids Content in “MD96-5722” by “Spencer” RIL Population of Soybean Using SNP Markers

Limited information is available on the genetic analysis of amino acid composition in soybean seeds. Previously, quantitative trait loci (QTLs) for seed isoflavones, protein, oil, and fatty acids were identified in the MD96-5722 by Spencer and other RIL populations. There were wide variations for these seed constituents among the RIL populations. Therefore, the objective of this study was to identify QTLs controlling different amino acids content in soybean seeds. To achieve this objective, ninety-two F5:7 recombinant inbred lines (RIL), developed from a cross of MD96- 5722 and Spencer, using a total 5376 Single Nucleotide Polymorphism (SNPs) markers, were used. The RILs were genotyped by using 537 polymorphic, reliably segregating SNP markers, developed from the Illumina Infinium SoySNP6K BeadChip array. A total of 13 QTLs were identified with three QTLs for threonine on the linkage group (LG) A1, C2, and B2. Two QTLs were identified for each of the amino acids proline on LG D1a and B2, serine on LG A1 and C2, tryptophan on LG K and G, and cysteine on LG A1 and K. One QTL was identified for arginine on LG N and histidine on LG J. The new QTLs findings for seed amino acid will facilitate the development of soybean cultivars with higher protein and amino acid quality to help meet the industry and consumer needs. * Corresponding author.

Unique Marine Organism : Identification of some Methods for Biomaterial Production

In this work, one dominant species of tunicates in Scandinavian Ocean called Ciona intestinalis has been investigated by extraction and characterization of its cellulose, oil, and amino acids. Potential applications have been evaluated for production of pure crystalline cellulose, biodiesel, and by analysis of amino acid composition of the samples. Pure tunicate cellulose (IC) has been prepared via chemical treatment, using acid, alkali, and the subsequent bleaching. The cellulose percentage, and yield for the chemically pure cellulose obtained were around 96% and 54% respectively. Oil extraction has been done using petroleum ether as the extraction solvent, and then chemical structure and fatty acid composition of the obtained oil have been characterized by NMR (Nuclear Magnetic Resonance Spectroscopy), FTIR (Fourier Transform Infrared Spectroscopy), and GC-MS (Gas Chromatography-Mass Spectrometry). Tunicate's oil has shown high content of free fatty acids together with very low content of glycerol, the latter being the common oil from vegetable origins. Moreover, the composition profile of Tunicate fatty acids seems to be similar to fish oil. However, amino acid composition has shown similarity to egg albumin, implying tunicate to have the capability to be considered as other marine organisms' feed in the near future.

Influence of linoleic acid-induced oxidative modifications on physicochemical changes and in vitro digestibility of porcine myofibrillar proteins

Effects of linoleic acid-induced oxidation on the digestibility of myofibrillar proteins (MP) were investigated using an in vitro gastric-intestinal digestion model. The oxidative modifications of MP structure were evaluated by the contents of carbonyl and total sulfhydryls, surface hydrophobicity, SDS-PAGE and Fourier transform infrared spectroscopy (FTIR). Based on the results of digestion rate and digestibility, the MP digestion was strongly affected by the oxidation level, depending on the resultant protein structural changes. With the concentration of oxidized linoleic acid (OLA) below 3 mM, the digestion rate and digestibility of MP were markedly increased mainly due to enhanced protein flexibility and surface hydrophobicity. However, at high OLA concentrations (5–10 mM), the adverse effect in protein digestion was observed, which might be attributed to the accelerated protein aggregation. As for the digestion properties, oxidation lowered its overall nutritional value, as revealed by amino acid composition analysis. Nevertheless, digestion product of MP prior treated with OLA below 1 mM exhibited an enhanced antioxidant activity with a higher oxygen radical absorbance capacity (ORAC) value. This improvement might be mainly attributed to the partial degradation of myosin and troponin-T, which favored their recognition during the intestinal tract, thus increasing the content of Cys and Tyr.

Optimization of production conditions for antioxidant peptides from walnut protein meal using solid-state fermentation

Solid-state fermentation (SSF) of walnut protein meal (WPM) by Bacillus subtilis was optimized for the maximum degree of hydrolysis (DH) and reducing power (RP) using response surface methodology (RSM). Optimum fermentation conditions were fermentation time of 82.01 h, inoculum concentration of 10.40, and water content of 1.50 mL/g. Optimized values were 41.80% and 0.78 for DH and RP, respectively. Walnut peptides (WP) were ultrafiltration membrane fractionated. The WPs-II (molecular weight <5 kDa) fraction showed the highest RP value. WPs-II showed good DPPH free radical scavenging and Fe2+ chelating activities, higher than for GSH (l-glutathione reduced) at the same concentration. Amino acid composition analysis of WPs-II showed that Asp, Glu, and Arg were the major amino acids playing important roles in the antioxidant activity. WPs is an efficient antioxidant suitable for use as a food additive and as a pharmaceutical agent.

Diverse Formulas for Spider Dragline Fibers Demonstrated by Molecular and Mechanical Characterization of Spitting Spider Silk

Spider silks have outstanding mechanical properties. Most research has focused on dragline silk proteins (major ampullate spidroins, MaSps) from orb-weaving spiders. Using silk gland expression libraries from the haplogyne spider Scytodes thoracica, we discovered two novel spidroins (S. thoracica fibroin 1 and 2). The amino acid composition of S. thoracica silk glands and dragline fibers suggest that fibroin 1 is the major component of S. thoracica dragline silk. Fibroin 1 is dominated by glycine-alanine motifs, and lacks sequence motifs associated with orb-weaver MaSps. We hypothesize fibroin 2 is a piriform or aciniform silk protein, based on amino acid composition, spigot morphology, and phylogenetic analyses. S. thoracica's dragline silk is less tough than previously reported, but is still comparable to other dragline silks. Our analyses suggest that dragline silk proteins evolved multiple times. This demonstrates that spider dragline silk is more diverse than previously understood, providing alternative high performance silk designs.

Nutritional Evaluation, Characterization and Antioxidant Activity of Radix Isatidis Protein Hydrolysates under Simulated Gastrointestinal Digestion

In this study, the antioxidant activities of alcalase-treated Radix Isatidis protein hydrolysates (RIPH) were compared during a two-stage gastrointestinal (GI) digestion in vitro. Compared with the blank, the enzymatic breakdown of the GI digests increased their 1,1-diphenyl-2-picrylhydrazyl (DPPH) (1.2-fold increase), hydroxyl (1.9-fold increase), (5.6-fold increase) in the radical scavenging activities and the reducing power (4.0-fold increase). The investigated items of RIPH consisted of the degrees of hydrolysis (DH), the compositions of amino acid, the nutritional parameters and the molecular weight distributions (MWD). The analysis of amino acid compositions of RIPH showed that essential amino acids such as isoleucine, threonine, tryptophan, valine and histidine took a high portion. The final Radix Isatidis protein hydrolysate digests (FRIPHD) contained the free amino acids of about 29.4%. Results from this study showed that RIPH is a good source of natural antioxidants that the formed peptides can resist physiological digestion in GI digestive system.

Účinek vybraných obilnin obsažených v krmné dávce na obsah aminokyselin v mléce

The aim of this study was to evaluate the possible effects of maize replacement in feeding rations on the amino acid content in cows´ milk. Cows were fed total mix ration based on the maize, clover silage and hay. There was a difference in the concentrate of the feeding ration. The first group (fed maize) was the control group, another two groups were experimental, one fed wheat and second fed triticale. During six weeks, totally 26 milk samples were taken from dairy cows of Czech Pied breed. Feed groups were preferably balanced in terms of milk yield, stage and number of lactations. The samples of feedstuffs as well as milk were modified for the analysis using acidic and oxidative hydrolyses. The analysis of amino acids content and composition of the sample hydrolysates was performed chromatographically by an AAA 400 analyzer, using Na-citrate buffers and ninhydrin detection. Total nitrogen content was determined according to Kjehldahl and the crude protein of the samples was determined by conversion from the nitrogen content multiplied by appropriate factor. The high content of crude protein in wheat did not influenced composition of milk from dairy cows fed this type of feedstuff. With respect to resulting amino acid content and composition of milk samples, none of the tested grains can be re­com­men­ded as a full-value maize replacement. Each feedstuff is an abundant source of several and ty­pi­cal amino acids in milk. However, triticale (cultivar Kitaro) seems to be acceptable replacement of maize owing to better crude protein efficiency, composition and health indicators of milk quality.

Optimization of Enzyme Digestion Conditions for Quantification of Glycated Hemoglobin Using Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

Glycated hemoglobin (HbA1c) is used as an index of mean glycemia over prolonged periods. This study describes anoptimization of enzyme digestion conditions for quantification of non-glycated hemoglobin (HbA0) and HbA1c as diagnosticmarkers of diabetes mellitus. Both HbA0 and HbA1c were quantitatively determined followed by enzyme digestion using isotopedilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with synthesized N-terminal hexapeptides asstandards and synthesized isotope labeled hexapeptides as internal standards. Prior to quantification, each peptide was additionallyquantified by amino acid composition analysis using ID-LC-MS/MS via acid hydrolysis. Each parameter was consideredstrictly as a means to improve digestion efficiency and repeatability. Digestion of hemoglobin was optimized when using100 mM ammonium acetate (pH 4.2) and a Glu-C-to-HbA1c ratio of 1:50 at 37oC for 20 h. Quantification was satisfactorilyreproducible with a 2.6% relative standard deviation. These conditions were recommended for a primary reference method ofHbA1c quantification and for the certification of HbA1c reference material.

Comparative Analysis of Amino Acid Composition in Antarctic Krill and White Shrimp

The purpose of this experiment is to evaluate the nutritional value of amino acids and its commercial value in the fish processing sector in Antarctic krill with cross-referenced in the white shrimp. For determination of the composition of 17 amino acids in the two shrimp muscle, free amino acids were measured using sulfosalicylic acid method, hydrolysed amino acid using hydrochloric acid hydrolysis.17 kinds of hydrolysed amino acids are measured in the muscle of Antarctic krill and White shrimp, which total content is 513.59±18.56mg/g and 537.61±16.8mg/g respectively.16 kinds of free amino acids are measured, which total content is 616.88±44.61mg/100g and 1276±75.67mg/100g respectively, while cysteine is not found. According to amino acid score (AAS), chemical score (CS), the limiting amino acid of Antarctic krill and White shrimp is valine and methionine + cysteine. The umani amino acids accounted for 27.76% of free amino acids and 35.69% of hydrolysed amino acids in Antarctic krill, while White shrimp is 41.08% and 31.52% respectively. The amino acids in Antarctic krill, TAV of which is greater than 1, include glutamic acid, alanine, lysine and arginine.

A comparative in silico characterization of functional and physicochemical properties of 3FTx (three finger toxin) proteins from four venomous snakes.

Snake venom is an abundant resource of diverse pharmacologically bioactive proteins and peptides and a good natural source of drug lead compounds and used as important research tools in the field of toxicology, pharmacology and neuroscience. Three finger toxins (3FTx) is an important super-family of snake venom proteins which has a conserved three finger like appearance in three dimensional structures. Members of 3FTx family show a wide array of pharmacological effects by targeting different receptors and ion channels with high specificity and many of them are being investigated as potential drug target. Therefore, with a vision to verdict a new edge and attempt we determined the amino acid compositional (%) profile, physiochemical properties, secondary structural and functional analysis and phylogenetic relationship of three finger toxins present in four different elapid snake species namely, Naja naja, Astrotia stokesii, Hydrophis cyanocintus and Pelamis platura using different bioinformatics tools. From the outcome of the current studies, it will be possible to know about a range of biological functions which are responsible mainly for the glowing amino acid composition profile of these proteins. Amino acid composition (%) profile although represents differential amount of different amino acid residues which encompasses a family precise model but all the protein sequence have a conserved amount of cysteine. The analysis of physicochemical properties can be used as a basic approach to contribute in developing rational drug through protein engineering and understanding different physiological function which will be beneficial for the welfare of human being.

Application of quantitative fingerprint to amino acids composition analysis of Xingnao Tongluo injection

To control the quality of the product, quantitative fingerprint was used to evaluate the composition of the amino acids in the Xingnao Tongluo injection. The method of the quantitative fingerprint to the amino acids composition was established through AccQ Tag precolumn derivatization. The quality was evaluated by the quantitative test of the amino acids and the similarity in ten batches. The Xingnao Tongluo injection contained 12 amino acids and the contents of these amino acids were stable. All the ten batches of the samples had similarity of more than 0.90. The method was accurate, feasible and could be a simple and effective way to evaluate the quality of the traditional Chinese medicine.

Validation of a reverse-phase high-performance liquid chromatography method for the determination of amino acids in gelatins by application of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent

In-house method validation was conducted to determine amino acid composition in gelatin by a pre-column derivatization procedure with the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation for 18 amino acids in less than 40min; the overall detection and quantitation limit for amino acids fell into ranges of 5.68–12.48 and 36.0–39.0pmol/μl, respectively; the matrix effect was not observed, and the linearity range was 37.5–1000pmol/μl. The accuracy (precision and recovery) analyses of the method were conducted under repeatable conditions on different days in random order. Method precision revealed by HorRat values was significantly less than 2, except for histidine with a precision of 2.19, and the method recoveries had a range of 80–115% except for alanine which was recovered at 79.4%. The findings were reproducible and accurately defined, and the method was found to be suited to routine analysis of amino acid composition in gelatin-based ingredients.

Characterization, mechanism of anticoagulant action, and assessment of therapeutic potential of a fibrinolytic serine protease (Brevithrombolase) purified from Brevibacillus brevis strain FF02B.

In this study, biochemical and pharmacological characterization of Brevithrombolase, a fibrinolytic serine protease purified from Brevibacillus brevis strain FF02B has been reported. An assessment of its thrombolytic potency has also been made. The molecular mass of this monomeric protease was determined as 55 kDa, and 56043 Da, respectively, by SDS-PAGE and MALDI-TOF-MS. In the analytical studies, the N-terminal sequence of Brevithrombolase was found to be blocked; however, the peptide mass fingerprinting and amino acid composition analyses demonstrated the similarity of Brevithrombolase with endopeptidases in possessing serine in their catalytic triad. This finding was confirmed by the observation that the serine protease inhibitors decrease the catalytic (fibrinolytic) activity of Brevithrombolase. The secondary structure of Brevithrombolase was composed of 30.6% alpha helix and 69.4% random coil. Brevithrombolase showed the Km and Vmax values towards the chromogenic substrate for plasmin at 0.39 mM and 14.3 μmol/min, respectively. Brevithrombolase demonstrated optimum fibrinolytic activity at pH 7.4 and 37 °C, and showed marginal hydrolytic activity towards globulin, casein and fibrinogen. The anticoagulant potency of Brevithrombolase was comparable to the low molecular mass heparin/antithrombin-III and warfarin. Among the three enzymes-Brevithrombolase, plasmin and streptokinase-the fibrinolytic activity and in vitro thrombolytic potency of Brevithrombolase was found to be superior. The RP-HPLC and SDS-PAGE analyses suggested a similar pattern of fibrin degradation by Brevithrombolase and plasmin, indicating that former enzyme is a plasmin-like fibrinolytic serine protease. Brevithrombolase did not show in vitro cytotoxicity on HT29 and HeLa cells or hemolytic activity. At a dose of 10 mg/kg, Brevithrombolase did not exhibit lethality or toxicity on Wistar strain albino rats. Brevithrombolase did not inhibit factor Xa, and its mechanism of anticoagulant action was associated with the enzymatic cleavage of thrombin. The combined properties of Brevithrombolase indicate its therapeutic potential in peptide-based cardiovascular drug development.

Chemical properties and antioxidant activity of beverage with ethanol extracts from the roasted Gugija (Lucii fructus) (647.41)

The antioxidant activity of beverage with ethanol‐extract from the roasted Gugija (Lucii fructus) were studied along with free amino acid and mineral composition analysis. Antioxidant activity determined by DPPH and ABTS radical scavenging activity. Five major free amino acids of the Gugija beverage were methionine (20.91 mg%), aspartic acid (16.91), alanine (12.52 mg%), %), proline (11.39 mg%), and arginine (7.53 mg%). The three major mineral in beverage with the ethanol‐extracts of the roasted Gugija were potassium, magnesium and calcium at 472.9, 16.47 and 9.66 mg% , respectively. Electron donating ability and ABTS radical scavenging activity of the Gugija beverage were 94.8 and 94.9%, respectively, of control L‐ascorbic acid, whereas those of ethanol‐extract were 85.4%, and 80.4%, respectively. Contents of total polyphenols and flavonoid of Gugija beverage were 325.5 mg tannic acid/g and 97.9 mg quercetin /g, respectively. The total polyphenol content and flavonoid was higher in Gugija beverage, which correspond to 3.77 and 4.49 times, respectively of that of ethanol extract. High DPPH and ABTS radical scavenging activity of the Gugija beverage were possibly related with amino acid composition as well as high contents of polyphenols and flavonoids in beverage

Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection.

Studies of protein nutrition and biochemistry require reliable methods for analysis of amino acid (AA) composition in polypeptides of animal tissues and foods. Proteins are hydrolyzed by 6M HCl (110°C for 24h), 4.2M NaOH (105°C for 20 h), or proteases. Analytical techniques that require high-performance liquid chromatography (HPLC) include pre-column derivatization with 4-chloro-7-nitrobenzofurazan, 9-fluorenyl methylchloroformate, phenylisothiocyanate, naphthalene-2,3-dicarboxaldehyde, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and o-phthaldialdehyde (OPA). OPA reacts with primary AA (except cysteine or cystine) in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct. OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline, respectively, in the presence of chloramine-T plus sodium borohydride at 60°C, or with S-carboxymethyl-cysteine formed from cysteine and iodoacetic acid at 25°C. Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 50 fmol for AA. This technique offers the following advantages: simple procedures for preparation of samples, reagents, and mobile-phase solutions; rapid pre-column formation of OPA-AA derivatives and their efficient separation at room temperature (e.g., 20-25°C); high sensitivity of detection; easy automation on the HPLC apparatus; few interfering side reactions; a stable chromatography baseline for accurate integration of peak areas; and rapid regeneration of guard and analytical columns. Thus, the OPA method provides a useful tool to determine AA composition in proteins of animal tissues (e.g., skeletal muscle, liver, intestine, placenta, brain, and body homogenates) and foods (e.g., milk, corn grain, meat, and soybean meal).

Purification and Characterization of Pepsin-Solubilized Collagen from Skin of Sea Cucumber Holothuria parva

Pepsin-solubilized collagen (PSC) was extracted from the skin of sea cucumber Holothuria parva and was fractionally characterized. The PSC from H. parva skin consisted of three α1 chains (α1)3, in contrast to calf skin collagen type I with two α1 and one α2 chains (α1)2α2 with approximately 130kDa each. The maximum transition (Tm) and denaturation temperature (Td) of PSC were determined to be 46.94 and 32.5°C, respectively. The amino acid composition analysis revealed that glycine, proline, alanine, and hydroxyproline were the abundant amino acids available in extracted PSC. The results showed that the isolated collagen from H. parva has some similar characteristics to previously reported collagens used in food and pharmaceutical industries.

Biochemical characterisation and assessment of fibril-forming ability of collagens extracted from Bester sturgeon Huso huso × Acipenser ruthenus

Collagens purified from Bester sturgeon organs were characterised biochemically, and their fibril-forming abilities and fibril morphologies formed in vitro clarified. Yields of collagens were 2.1%, 11.9%, 0.4%, 18.1%, 0.4%, 0.8% and 0.03% (collagen dry weight/tissue wet weight) from scales, skin, muscle, swim bladder, digestive tract, notochord and snout cartilage, respectively. Using SDS–PAGE and amino acid composition analyses, collagens from scales, skin, muscle, the swim bladder and digestive tract were characterised as type I, and collagens from the notochord and snout cartilage as type II. Denaturation temperatures of the collagens, measured using circular dichroism, were 29.6, 26.8, 29.0, 32.9, 31.6 and 36.3°C in scales, skin, muscle, swim bladder, digestive tract, and notochord, respectively. For fibril formation, swim bladder and skin collagen showed a more rapid rate of increase in turbidity, a shorter time to attain the maximum turbidity, and formed thicker fibrils compared with porcine tendon type I collagen.

Biochemical characterization of psychrophilic Mn-superoxide dismutase from newly isolated Exiguobacterium sp. OS-77

Many types of superoxide dismutases have been purified and characterized from various bacteria, however, a psychrophilic Mn-superoxide dismutase (MnSOD) has not yet been reported. Here, we describe the purification and the biochemical characterization of the psychrophilic MnSOD from Exiguobacterium sp. strain OS-77 (EgMnSOD). According to 16S rRNA sequence analysis, a newly isolated bacterium strain OS-77 belongs to the genus Exiguobacterium. The optimum growth temperature of the strain OS-77 is 20 °C. The EgMnSOD is a homodimer of 23.5 kDa polypeptides determined by SDS-PAGE and gel filtration analysis. UV-Vis spectrum and ICP-MS analysis clearly indicated that the homogeneously purified enzyme contains only a Mn ion as a metal cofactor. The optimal reaction pH and temperature of the enzyme were pH 9.0 and 5 °C, respectively. Notably, the purified EgMnSOD was thermostable up to 45 °C and retained 50% activity after 21.2 min at 60 °C. The differential scanning calorimetry also indicated that the EgMnSOD is thermostable, exhibiting two protein denaturation peaks at 65 and 84 °C. The statistical analysis of amino acid sequence and composition of the EgMnSOD suggests that the enzyme retains psychrophilic characteristics.

The effect of water stress on nitrogen metabolism of horsegram Dolichos biflorus L.

Horsegram plants were raised in terule soil in earthen pots and subjected to various durations of water stress (7, 14 and 21 days). Total nitrogen contents were reduced in water-stressed plants but differently in different plant parts. Nitrate content and nitrate reductase activity decreased with stress. This species possesses a good capacity for accumulation of free proline under water stress. Analysis of amino acid composition also revealed marked changes in the levels of various amino acids in water-stressed plants. Accumulation of γ-aminobutyric acid in water-stressed plants was observed.