The Analysis Techniques of Amino Acid and Protein in Food and Agricultural Products
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INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 10, OCTOBER 2020 ISSN 2277-8616 The Analysis Techniques Of Amino Acid And Protein In Food And Agricultural Products Edy Subroto, Elazmanawati Lembong, Fitry Filianty, Rossi Indiarto, Gisella Primalia, Miswa Salza Kirana Zaenal Putri, Hanna Christy Theodora, Salsabila Junar Abstract: The protein content in food and agricultural products affects the physicochemical and nutritional properties of these products. This review aims to discuss the analysis techniques of protein and amino acid in food and agricultural products. The qualitative analysis can be conducted using the Hopkins-Cole, Xanthoproteic, Millon, Nitroprusside, and Sakaguchi test. In contrast, the quantitative analysis of proteins can use the Kjehldahl, Biuret, Lowry, UV Spectrophotometry, and Turbidimetry. It also discussed the immunohistochemical techniques to identify cellular or tissue constituents (antigens) by staining techniques, while Formol titration measures the hydrolysis of proteins and N-amino quickly. The amino acids can be analyzed by microbiological methods, colorimetric, high-performance liquid chromatography (HPLC), and gravimetric techniques. These methods/techniques can be chosen according to the type of sample and the purpose of the analysis so that the results can be obtained accurately. Index Terms: Protein, amino acid, analysis technique, food, agricultural product —————————— —————————— 1. INTRODUCTION specifically associated with forming proteins [2]. Based on the Protein is one of the main macromolecular components that source, protein can be divided into two groups, namely composed of amino acids through peptide bonds in specific vegetable protein and animal protein [9]. Vegetable proteins sequences and types. Amino acids contained several main are the protein derived from vegetable materials, such as nuts, elements, such as C, H, O, and N. In addition, proteins also soybean, and other cereals. Animal proteins are the protein contain other elements such as sulfur, phosphorus, iron, and derived from animal products such as milk, eggs, meat, and copper [1]. Amino acids contained in proteins are linked by a fishery products. Animal protein has a complete and high- bond called a peptide bond [2]. Protein functions to form body quality protein because it contains essential amino acids, tissue, growth, and form various bioactive compounds [3]. especially amino acids that contain sulfur [10]. Both vegetable Specifically, proteins contain carbon atoms (50-55%), oxygen and animal proteins can also be modified into bioactive (20-23%), nitrogen (12-19%), hydrogen (6-7%), and sulfur peptides that are beneficial both in the field of food and (0.2-3%) [4]. The amino acid has two functional groups, a pharmaceutical technology [11], [12]. To determine the content namely amino group and a carboxyl group [5]. Amino groups of protein and amino acids in food and agricultural products, a provide alkaline properties, whereas carboxyl groups provide quantitative or qualitative analysis must be conducted. acidic properties. In liquid or solution form, amino acids have Therefore, an appropriate analysis method is needed to amphoteric properties, which tend to become acidic when in identify the levels of protein and amino acids in food materials. the base solution, and turn into the base when in acidic This review discusses various analytical methods to determine solution. This is due to amino acids being able to become the protein and amino acid content, both qualitative and zwitterions [6]. The characteristics of a protein are determined quantitative analysis. The qualitative analysis includes several by the type of amino acids and their sequence in polypeptides. reactions, including Xanthoproteic reaction, Hopkins-Cole Proteins have types of structures, which include the structure reaction, Millon reaction, Nitroprusside reaction, and of primary, secondary, tertiary, and quaternary [7]. The primary Sakaguchi reaction. Quantitative analysis includes several structure shows the amount and sequence of amino acids in a methods, namely the Kjeldahl, Lowry, Biuret, and polypeptide. The structure of secondary shows the structural Spectrophotometry, Turbidimetry, and Formol titration content of a polypeptide which is influenced by the hydrogen methods. Analysis of amino acid levels can be determined by bonding between oxygen (O) of the carbonyl group (C=O) and several methods, namely the Colorimetric, Gravimetric, hydrogen (H) of the amino group (N-H) of the peptide chain Chromatographic, and Microbiological methods. framework [8]. The tertiary structure of proteins is formed by additional bonds between R groups in amino acids. Disulfide 2 QUALITATIVE ANALYSIS OF PROTEINS bonds are the only covalent bonds involved in tertiary Qualitative analysis proteins consisted of the Hopkins Cole structures, formed by oxidation of sulfhydryl groups from two test, Millon test, Xanthoproteic test, Nitroprusside test, and cysteinyl residues. The quarter structure involves the Sakaguchi test. The specificity of the qualitative analysis interaction between two or more polypeptide chains that are techniques of protein can be seen in Table 1. Tabel 1. The specificity of the qualitative analysis techniques of protein. Analysis No Analysis specificity ———————————————— techniques Identify the tryptophan based on Indole ring Edy Subroto, Elazmanawati Lembong, Fitry Filianty, Rossi Indiarto, 1 Hopkins Cole test Gisella Primalia, Miswa Salza Kirana Zaenal Putri, Hanna Christy group. Identify the phenolic amino acids such as Theodora, Salsabila Junar: Department of Food Industrial 2 Millon test Technology, Faculty of Agro-Industrial Technology, Universitas tyrosine and its derivatives. Padjadjaran, Jl.Raya Bandung-Sumedang Km. 21, Jatinangor, Identify the proteins and amino acids that Sumedang 40600, Indonesia. E-mail: [email protected] have aromatic rings or benzene groups 3 Xanthoproteic test such as phenylalanine, tyrosine, tryptophan, etc. 29 IJSTR©2020 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 10, OCTOBER 2020 ISSN 2277-8616 Detect the presence of cysteine amino 4 Nitroprusside test appears based on the nitration reaction on the aromatic ring of acids. amino acids [18]. The principle of testing phenylalanine, Identify the amino acid arginine based on 5 Sakaguchi test tyrosine, and tryptophan by the Xanthoproteic test method is guanidine group in its side chain. by homogenizing 1 mL of HNO solution that already contains 3 a 2% protein solution. After that, the sample is heated using a 2.1 Hopkins Cole test water bath for 15-20 minutes or on fire for 2 minutes, then a Hopkins Cole Test is one of the qualitative test methods to change in the color of the solution is observed. The test tube is determine differences in types of proteins and amino acids. incubated at room temperature until the temperature This test is used to identify the presence of the amino acid decreases then NH solution or 20-40% NaOH is added to it, tryptophan, which is the only amino acid that has an indole 3 and a change in the color of the formed solution is observed ring group [13]. Tryptophan belongs to the group of essential again [15]. amino acids. Tryptophan is a precursor of niacin vitamin and an introduction to the serotonin nerve. Tryptophan functions to 2.4 Nitroprusside test maximize the use of vitamin B complex, improve nerve health, Nitroprusside test is a test used to detect the presence of stabilize emotions, increase feelings of calm, prevent cysteine amino acids. This test uses sodium nitroprusside insomnia, and increase the release of growth hormone. One of reagents and ammonia solution. The testing principle is that these amino acids can be found in egg whites. Hopkins Cole the free -SH group called thiol or mercapto, which is owned by reagents which contain glyoxylic acid (C H O ) react with 2 2 3 the amino acid cysteine will react with nitroprusside in the sulfuric acid. Tryptophan in the protein solution will be case of excess ammonia to form red compounds. The S-S- condensed with the aldehyde group on glyoxylic acid with the group in cysteine will give positive results in nitroprusside help of strong oxidizing sulfuric acid. Positive results are testing if it is reduced first. Nitroprusside testing is conducted indicated by the formation of purple rings between 2 separate by entering 0.5 mL of sodium nitroprusside 1% into the test layers [13], [14]. According to Elzagheid [15], the principle of tube then add 2 mL of the sample to be tested, then add 0.5 testing tryptophan by the Hopkins Cole test method is by mL of NH OH solution. The sodium nitroprusside solution used adding 1 mL of Hopkins Cole reagent into a test tube that 4 must be in a new condition and made just before the test [19]. already contains a 2% protein solution, then the concentrated Nitroprusside reactions can be applied in various tests, one of sulfuric acid solution is added as much as 1-2 mL. The which is to detect the content of cysteine in urine clinically [20]. solution is homogeneous with vortex, and then color changes The test was carried out by adding of sodium nitroprusside are formed. 20% (w/v), then the color intensity was measured using a spectrophotometer at a wavelength of 521 nm in 1 minute, and 2.2 Millon test the cysteine concentration was calculated using a standard Millon test is one of the qualitative test methods to determine curve [21]. differences in types of proteins and amino acids, namely the type of phenolic amino or amino acids that have phenol 2.5 Sakaguchi Test groups such as tyrosine and its derivatives [13]. Tyrosine is a The Sakaguchi test is a chemical test that includes a non-essential amino acid that has a phenyl group and is a colorimetric reaction for the identification and quantification of weak acid. One of these amino acids can be found in milk the amino acid arginine. Arginine is an amino acid that has a casein. Millon test is not a specific test, because this test guanidine group in its side chain, which is the C atom that identifies all types of phenol compounds, so to ensure that binds N with a single bond and binds N with a double bond.