Amino Acids In Protein Hydrolysates Research Articles

ORIGIN OF TWENTY PROTEINOGENIC AMINO ACIDS

“Protein structure alphabet” – that is a name of L-configuration amino acids, which always or frequently occur in protein hydrolysates. Discovery of proteinogenic amino acids was a quite long process and took many years from 1806 to1935.   According to Wicker and Schmidt a presence of amino acids in protein hydrolysate is considered to be established if at least two researchers derived them independently of each other and if its structure is confirmed by synthetic pathway [1]. Even today these requirements are valid in relation to newly discovered amino acids. Each amino acid, except for a name foreseen by the international nomenclature has its trivial (conventional) name, which points either at the source, from which this amino acid was derived first, or notes some of its special characteristics. The presented work provides chronological review of the history of discovery of twenty proteinogenic amino acids.&nbsp

RELATIONSHIP BETWEEN BITTERNESS OF BREWER’S YEAST HYDROLYSATE AND HYDROPHOBIC AMINO ACID CONTENT

Brewer’s yeast spent, obtained after the main fermentation stage, is a rich- in-protein source(protein content accounts for 48 - 50 % dry matter). In order to use efficiently this source, it washydrolysed by different methods. Protein hydrolysate products are normally mixtures of peptidesand amino acids. Protein hydrolysates have a wide range of applications in food. It can be usedas emulsifying agents in a number of applications such as salad dressings, spreads, ice cream,coffee whitener, cracker, and meat products like sausages. However, bitterness in hydrolysates isone of the major undesirable aspects for various applications in food processing. In this study,we used enzymatic mixture alcalase and flavourzyme, yeast treatment methods to hydrolysebrewer’s yeast. The hydrolysate and fractions of protein hydrolysate obtained after filtration with10 kDa and 3 kDa filters were used for determination of bitterness and hydrophobic amino acidscontent. The bitter taste of hydrolysate was determined by sensory method (using quininestandard) and amino acid content was analysed by HPLC method. The result showed the closerelationship between bitter taste and hydrophobic amino acid content. The bitter taste of proteinhydrolysate was reduced as the hydrophobic amino acid content decreased. When the bitter taste(equivalent to quinine concentration) decreased from 16.25 μmol/l to 3.59 μmol/l, the totalcontent of hydrophobic amino acids in protein hydrolysate reduced from 1653 μg/ml to 932μg/ml.

Presence of free amino acids in protein hydrolysate during electroseparation of peptides: Impact on system efficiency and membrane physicochemical properties

Membranes, more particularly ion-exchange membranes (IEMs), are vulnerable to fouling by peptides and amino acids present in protein hydrolysate. In this context, IEM and ultrafiltration membranes (UFMs) staked in an electrodialysis with filtration membrane (EDFM) system were characterized during and after 6 successive peptide fractionations. The peptide concentration in the recovery compartment decreased by more than 22% and 32% for fifth and sixth batches, respectively. In addition, analysis of total nitrogen content of used membranes and FTIR confirmed that AEM and both cation-exchange membranes (CEMs) were fouled by peptides and/or free amino acids. Consequently, their electrical conductivity, IEC and water content decreased. In addition to the fouling, the cathode (diluate) side of AEM, where the dissociation of water molecules occurs, was deteriorated radically, possibly due to chemical reactions with OH− ions produced. Consequently, the surface roughness of both IEMs was found to increase considerably. The IEM fouling noticed in the present study was never observed before in an EDFM treatment. This fouling would be mainly due to the presence of amino acids residues (100% free) in the SCBH, and able to migrate through the AEM (Phe) and the CEM (Arg and Lys).

Production of chitin and bioactive materials from Black tiger shrimp (Penaeus monodon) shell waste by the treatment of bacterial protease cocktail.

The main objective of this study was to obtain chitin in pure form from a new crustacean waste material for industrial applications. Black tiger shrimp shell wastes are a rich source of protein and valuable bioactive carbohydrate polymers such as chitin. After removal of carotenoid, Black tiger shrimp shell wastes (BTSHWs) were treated with chemicals and protease enzyme to extract chitin. Box–Behnken response surface methodology was applied to optimize the deproteinization process to obtain chitin. At optimal pH (8.82), temperature (50.05 °C), agitation speed (100.98 rpm), enzyme substrate ratio of 1:8 (wv−1) and 72 h of incubation with Paenibacillus woosongensis TKB2 crude protease cocktail, 80 % deproteinization was found along with 77.28 % recovery of chitin. The valuable oligopeptides were determined by MALDI-TOF analysis and analysis of adequate amount of free amino acids in protein hydrolysate from BTSHW, indicating a high nutritional value used for food, feed or as a nitrogen source in growth medium for microorganisms. The chitin obtained was compared with the commercial chitin using scanning electron microscopy, Fourier transform infrared spectrometer, X-ray diffraction and 13C CP/MAS-NMR. Chitin obtained from crude protease treatment showed comparable physicochemical and structural properties to those of the commercial chitin. The carotenoid obtained after treatment can be used for medicinal purpose.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-014-0245-6) contains supplementary material, which is available to authorized users.

Ultra-performance liquid chromatographic analysis of amino acids in protein hydrolysates using an automated pre-column derivatisation method

In the present study, the changeover from the Pico·Tag HPLC method to the AccQ·Tag ultra UPLC method for the analysis of amino acids in casein and bovine serum albumine hydrolysates is described. The total chromatographic run time of the AccQ·Tag ultra UPLC method was only 40% of the time required for the Pico·Tag HPLC method. Quantitative results of both methods for casein and bovine serum albumine hydrolysates compared fairly well. The derivatisation protocol for the formation of AQC derivatives of amino acids was automated using a Gilson Model 215 liquid handler. Comparison of the manual derivatisation protocol with the automated protocol showed lower coefficients of variation for the latter. Combination of the AccQ·Tag ultra UPLC method and automated derivatisation resulted in improved throughput compared to the Pico·Tag HPLC method.

Analysis of underivatized amino acids in protein hydrolysates for cosmetic application by capillary electrophoresis

AbstractIn this work, the use of two chromophores, 3,5‐dinitrobenzoate and phthalate, and the additives α‐cyclodextrin and methanol was investigated with the goal of developing three electrolyte systems for the capillary electrophoretic analysis of free underivatized amino acids in commercially available protein hydrolysates of diverse origin: keratin, silk fiber, milk, soybean, fish tissue, and cattle leather. Sixteen amino acids could be effectively separated by two distinct methods. Several amino acids were identified in the studied hydrolysates and confirmed by spiking techniques. As expected, those products which originated from acidic hydrolysis were richer in free amino acids than those which originated from enzymatic hydrolysis. The quantitative determination of Asp, Glu, Cys, Ser, and Pro in Queratan, a keratin hydrolysate, was also performed and some method validation parameters were established. The coefficient of variation for migration time was better than 1% for all amino acids except Asp, Leu, Lys, and Pro. Overall, peak height repeatability was slightly better than peak area. Calibration curves based on peak heights present a good fit, with coefficients of correlation better than 0.99. The LOD for the amino acids identified in Queratan ranged from 1.0 to 7.0 mg L–1.

Automated amino acid determination by high-performance liquid chromatography with 2-(9-anthryl)ethyl chloroformate as precolumn reagent

A fully automated HPLC method for the determination of amino acids in protein hydrolysates has been developed, using 2-(9-anthryl)ethyl chloroformate and precolumn derivatization. This UV sensitive reagent reacts rapidly with both primary and secondary amines at room temperature to form stable carbamate derivatives. The reaction conditions have been thoroughly investigated. A complete separation of protein hydrolysates is accomplished within 40 min. The results are compared to those of the well known 9-fluorenylmethyl chloroformate method. Detection limits for UV and fluorescence detection were found to be 0.5 pmol and 0.06 pmol, respectively.

High-performance liquid chromatography of amino acids after derivatization with 9-isothiocyanatoacridine

This study describes a reproducible method for the routine determination of amino acids in protein hydrolysates using 9-isothiocyanatoacridine as an HPLC precolumn derivatization reagent. Derivatization is carried out by adding the isothiocyanate dissolved in dry acetonitrile to a buffered amino acid solution without the following extraction of the excess reagent. The resulting N-(9-acridinylthiocarbamoyl)amino acids were separated by one single isocratic HPLC analysis with the UV detection at 280 nm.

High-performance liquid chromatographic separation of phenylthiocarbamyl derivatives of amino acids from protein hydrolysates using a Partisphere C1 8 column

The conditions for the rapid analysis of amino acids in protein hydrolysates are given for the Whatman Partisphere C 18 cartridge column. Amino acids from protein hydrolysates are derivatized to phenylthiocarbamyl (PTC) derivatives and separated within 22 min. The Partisphere column is generally less expensive than specialty columns designed for the PTC derivative. The “void sealing” capability of this column, in addition to its guard column attachment, increases the life span of the column.

High-performance liquid chromatographic determination of amino acids in protein hydrolysates and in plasma using automated pre-column derivatization with o-phthaldialdehyde/2-mercaptoethanol

A sensitive and reproducible method for the routine determination of amino acids in plasma and in protein hydrolysates based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. The resolution of all amino acids was found to be good. The total time for analysis, including separation and reconditioning, ranged from 38 min for protein hydrolysates to 62 min for 29 physiological amino acids. The precision of hydrolysate analysis was within a relative standard deviation of 0.8-7.3% depending on the use of internal or external standards. The relative standard deviations of peak areas for physiological amino acids (standard) ranged between 1.8 and 5.6%. The relative standard deviations of retention times were less than 0.5% for all amino acids. This method can be used for routine analysis. One single column with 4-microns end-capped C18 material was found to be sufficient for 400-500 successive runs.

High-performance liquid chromatographic method for determination of hypusine and deoxyhypusine

A selective and sensitive reversed-phase high-performance liquid chromatographic method is described for the determination of the amino acid hypusine which occurs ubiquitously in mammalian cells and for the simultaneous measurement of its immediate precursor deoxyhypusine. These amino acids, after their ion-exchange separation from the bulk of other amino acids in protein hydrolysates, are derivatized with o-phthalaldehyde and the fluorescent derivatives are separated by reverse-phase liquid chromatography. The sensitivity of this method allows detection of less than 5 pmol of each of these unusual amino acids. The method is applied to the determination of hypusine and deoxyhypusine in acid hydrolysates of cultured cells and tissues.

Complete amino acid analysis of proteins by dabsyl derivatization and reversed-phase liquid chromatogrphy

A method is described for the quantification of the primary amino acids in protein hydrolysates by dabsylation and high-performance liquid chromatography. Improvements in the established conditions for the formation and storage of amino acid dabsyl derivatives and the use of new reversed-phase columns allow the chromatographic analysis in 25 min of all the proteinogenic amino acids in well resolved peaks of homogenous and highly reproducible size. The method permits the simultaneous quantification of tryptophan residues by previous hydrolysis of the protein with sulphonic acids. The other acid-labile residues, asparagine and glutamine, can also be analysed by previous conversion into diaminopropionic and diaminobutyric acids, respecitvely, by treatment of the protein with [bis(trifluoroacetoxyiodo]-benzene. An extended chromatographic gradient programme allows the separation of many modified amino acids, naturally occurring or produced after chemical modification of proteins. The above characteristics together with a demonstrated high reproducibility (relative standard deviation 2.1%), flexibility, sensitivity (below 100 pmol), and inertness to extraneous chromatographic contamination make this improved method a good alternative to other currently used chromatographic methods for amino acid analysis.

Some problems associated with the analysis of 2-aminoethylphosphonic acid using automated ion-exchange chromatography

Amino acid analysis of 2-aminoethylphosphonic acid (2-AEP) under the conditions normally used for the analysis of acidic amino acids in protein hydrolysates resulted in the elution of 2-AEP as two incompletely resolved peaks. Evidence is provided indicating that this phenomenon was due to the formation of a degradation product or an isomer on the ion-exchange column and that the resin, buffer pH and column temperature were all contributory factors.

Quantitative high-performance liquid chromatographic determination of sulfur amino acids in protein hydrolysates

A rapid, quantitative high-performance liquid chromatographic procedure for the determination of methionine and cystine after oxidation to methionine sulfone and cysteic acid is described. The Dns derivatives of the amino acids are separated by reversed-phase chromatography with a phosphate buffer-acetonitrile gradient and detected by UV absorption at 254 nm. The procedure is validated by confirming the methionine and cystine content of ribonuclease A. The average yields of cysteic acid and methionine sulfone from triplicate analyses of ribonuclease A were 98.1% (±3.3) and 106.1% (±2.4) of the theoretical values, respectively.

Ligand-exchange separation of amino acids : II. Influence of the eluent composition and of the nature of the ion exchanger

The ligand-exchange separation of amino acids in protein hydrolysates has been studied in ammoniacal media. The various factors influencing the chromatographic behaviour have been investigated. The selectivity of the process and the column efficiency depend appreciably on the resin matrix and the nature of the functional groups. The best results have been obtained with the acrylic-type Bio Rex 70 resin, in the copper (II) form. The zinc(II) form of this support has also been tested; the elution order of glycine, alanine and leucine is the same as with the copper(II) form, but the affinities of these molecules for the zinc(II) resin are weaker. The influence of the concentrations of copper(II) and ammonia on the elution volumes obtained for all the amino acids with the copper(II) Bio Rex 70 resin has been studied. The general variation of retention and selectivity with eluent composition confirms the theoretical expressions established previously. Ligand-exchange chromatography has been compared to the conventional ion-exchange process using acidic or basic species. This study reveals analogies and differences between both techniques, and shows that ligand exchange can provide new opportunities for the separation of amino acids.

Amino Acid Profiling of Protein Hydrolysates Using Liquid Chromatography and Fluorescence Detection

Abstract A liquid chromatography procedure is described for separating the amino acids in protein hydrolysates. The proteins are hydrolyzed with hydrochloric acid and an aliquot of the hydrolysate is derivatized with dansyl chloride reagent. The derivatization procedure takes only 2 minutes using a reaction temperature of 100°C. The dansylated amino acids are chromatographed using a reversed-phase C8 column and a multi-step, nonlinear gradient elution solvent program which is readily achieved using a microprocessor-controlled liquid chromatograph. Chromatography is complete in approximately 40 min. The procedure is useful for characterizing proteins and may also be used to analyse intact dansylated polypeptides. Chromatograms showing the amino acid profile of chymotrypsin, albumin and histone are given.

A Simple Chromatographic Method for the Determination of the Methylated Basic Amino Acids in Proteins

A chromatographic method is described for the determination of epsilon-N-monomethyl-epsilon-N, epsilon-N-dimethyl-, and epsilon-N, epsilon-N, epsilon-N-trimethyllysines, 1-methyl-, and 3-methylhistidines, N-G, N-G-dimethyl-, and N-G, N'G-dimethylarginines, ornithine, the diastereoisomers of hydroxylysine, basic amino acids and related compounds, using Durrum type DC-6A 11.0 plus or minus 1.0 mum spherical resin. The complete separation of all of these amino acids as discrete peaks in the Beckman 120B amino acid analyzer was achieved for the first time using a single column (60 times 0.9 cm), one buffer system (0.35 N sodium citrate) adjusted at either pH 5.734 or 5.657 plus or minus 0.002, one buffer flow rate (30 ml/h), and one temperature (28 degrees C). This procedure was used for the analysis of these unusual amino acids in protein hydrolysates of histones and collagen and be applied to the determination of these compounds in hydrolysates of plant and animal tissues.

Quantitative determination of amino acids in protein hydrolyzates by thin-layer chromatography

A micro method for the quantitative determination of amino acids in protein hydrolyzates has been proposed which permits the analysis of two hydrolyzates to be performed in 4–5 h. After the separation of the amino acids by TLC in cellulose, they are determined quantitatively by the colorimetric method using a MKMF-1 microphotocolorimeter. Proline and tryptophan are determined separately with the aid of specific test-tube reactions. The sensitivity of the method permits the analysis of 10–15 µg of aminoacid mixture.

An automated estimation of proline in hydrolyzates

An automated procedure (Technicon AutoAnalyzer) is described for the estimation of proline in the presence of other amino acids in protein hydrolyzates. The method is based on the well-known reaction of proline and ninhydrin at pH 1. The useful ranges of the method are 2 to 20 and 1 to 10 μg proline per milliliter. The method was used on hydrolyzates of several different plant proteins. Under the conditions of the method, other amino acids, hydrochloric acid, and citrate buffer did not interfere. The high sensitivity, speed, and accuracy make this a useful method.

An improved technique for the analysis of amino acids and related compounds on thin layers of cellulose : II. The quantitative determination of amino acids in protein hydrolysates

A simple and accurate method has been devised for the quantitative analysis of protein hydrolysates using thin-layer chromatography. Ninhydrin—cadmium acetate is the staining reagent and, using reflectane densitometry, calibration graphs have been drawn for twenty-two naturally occurring amino acids. The equation of the line for each amino acid has been calculated and used for the determination of the amount of amino acid present in protein hydrolysates. The results obtained agree well with those obtained by the ∝Technicon’ Automatic Amino Acid Analyzer and with those given in the literature.