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Isolation and Alkaline Hydrolysis of the Protein Gluten

Tiffany Rae Espiritu, Lorenz Rey Esteban, Kathleen Anne Francisco, Ma. Casey Louisse Garcia, Aimee Dianne Hermoso Group 4, 2E Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Gluten is the protein usually found in foods containing wheat or grains. In the experiment, gluten is isolated from wheat flour by continuously washing the dough with water, which removes starch and other water-soluble components. The presence of starch is tested by adding Iodine solution, which will turn blue-violet in color if the solution is positive for starch. The isolated gluten was then subjected to alkaline hydrolysis. The objectives of this experiment are to isolate gluten from wheat flour by difference in solubility and to perform alkaline hydrolysis on the isolated protein and to know the advantages and disadvantages of this type of hydrolysis compared to the other forms of hydrolysis.

Introduction
Proteins are biochemical compounds consisting of polypeptide chains folded in globular or in fibrous form and has a corresponding biological function. For a protein to be biologically active, it must conform its native conformation. These macromolecules are known to have a very large molecular weight, usually greater than 5000. A polypeptide chain is made up of several amino acid residues linked together by peptide bonds. This bond is formed between the - carboxyl group of one amino acid and the -amino group of the next one (Campbell & Farrell, 2012). Among the amino acids, usually 20 are found in proteins. The R-group differentiates each amino acid. One type of protein is gluten. The gluten protein polymeric network plays a pivotal role in determining the end-use quality of wheat in many food products (Shewry & Tatham, 2000). According to Lafiandra, Masci & D'Ovidio (2004), high and low molecular weight glutenin subunits are the most abundant components of gluten and both contribute to the formation of the glutenin polymers. It provides elasticity to the dough, helping it rise and maintain its shape. Isolation of proteins is a process where in a specific protein is separated from a complex mixture. Proteins are separated based on their solubility, molecular size or weight, charged and binding affinity. Some of the methods used are isoelectric precipitation, heat denaturation and saltinduced precipitation. As for gluten, difference in solubility is used; where in starch and other water-soluble components of wheat flour are removed, isolating the insoluble element, which is gluten. Protein hydrolysis on the other hand is the breaking down of protein into small peptide and free amino acids. Hydrolysate is the solution containing the protein; it is the product of hydrolysis. There are different kinds of hydrolysis - acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis. Acid hydrolysis is a process where acids are used to catalyze a cleavage of a chemical bond. Alkaline hydrolysis is a reaction where bases are used to catalyze the cleavage of a chemical bond. While enzymatic hydrolysis is a reaction where enzymes are used to

facilitate the cleavage of the chemical bond.

neutralized sample was then used for another experiment.

MATERIALS AND METHODOLOGIES MATERIALS


A. Isolation of Proteins The materials used in the experiment are wheat flour, handkerchief and I2 solution. B. Alkaline Hydrolysis of the Intact Protein The reagents are NaOH, distilled water and HCl, while the apparatus used are hard glass test tube, autoclave and beaker.

RESULTS AND DISCUSSIONS A. Isolation of Proteins


After the addition of water to the flour, a thick dough was formed. The dough was creamy white and soft in texture. The formed dough was wrapped in cheesecloth and was subjected into running water, the washings were tested with I2 solution, and a blue-violet coloration indicates the presence of starch, so continuous washing was needed. After a few hours of washing, the dough was brown in coloration and has an elastic and gummy-like texture. The washings was again tested with I2 solution, a clear solution indicates the absence of starch. The insoluble material left in the dough is identified to be the crude gluten. The principle involved in this experiment is difference in solubility, since starch is partially soluble in water, it was removed from the dough and the insoluble gluten remained. B. Alkaline Hydrolysis of the Intact Protein After alkaline hydrolysis of the crude gluten obtained from the previous experiment, a brown solution with black-brown precipitate was formed. This solution was placed with drops of 1 M HCl until it was neutralized; no reaction with the litmus paper indicates that it was neutralized. A dirty-white turbid solution was obtained after neutralization.

METHODS
A. Isolation of Proteins The group added water to one cup of wheat flour to make a thick dough. Then wrapped the dough in cheesecloth. Placed it under running water until all starch was removed. The group tested the washings with I2 solution until a negative result was obtained. The insoluble material obtained was the crude gluten. B. Alkaline Hydrolysis of the Intact Protein The group added 10mL of 4 M NaOH to 0.5g isolated proteins in a hard glass test tube and labeled the tube. Then the group placed the cotton as a stopper and the tube was submitted to the instructor for autoclaving (15 psi for 5 hrs.). After autoclaving, the group added 10mL distilled water, and the mixture was transferred into a 250mL beaker. The mixture was then neutralized with 1 M HCl. The

Conclusion
After the experiment the group therefore concludes that gluten was isolated from wheat flour by the method difference in solubility.

REFERENCE/S
Campbell, M, Farrell, S. (2012). Biochemistry. China: China Translation & Printing Services Limited Crisostomo, A., Daya, M., Guia, R., et al. (2010). Laboratory Manual in General Biochemistry. Quezon City: C & E Publishing, Inc. Lafiandra, D., Masci, S., D'Ovidio R. (2004). The Gluten Proteins. UK: Royal Society of Chemistry Shewry, P., Tatham, A. (2000). Wheat Gluten. UK: Royal Society of Chemistry

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