Date of Award
Master of Science
Food Science and Technology
M.R. Johnston, J.T. Smith
This experiment was designed to study the use of a commercial proteolytic enzyme preparation for the partial hydrolysis of proteinaceous vegetable materials so that they may be utilized for the purpose of child feeding practices. The effect of pH, temperature, time, denaturation period, concentrations of enzyme and substrate, and sodium chloride content were factors of the investigation from which optimum conditions for proteolytic activity could be ascertained. Another objective was the evaluation of protein quality by estimating the amounts of limiting essential amino acids of the individual raw materials after enzyme treatment. The raw materials were chick peas, whole wheat flour, sunflower seed press cake and a mixture consisting of 44 percent sunflower seed press cake, 28.5 percent chick peas, and 27.5 percent whole wheat flour.
Effect of pH, temperature, time, denaturation period, concentration of enzyme and substrate, and sodium chloride content on enzymatic solubilization of proteins and production of sugars from the individual sources were determined.
The macro-Kjeldahl method (N x 6.25) was used to express the proteolytic activity of Rhozyme-41. The estimated amount of free amino groups in terms of total ninhydrin positive material CNPM) was determined by the ninhydrin-colorimetric method. The amount of glucose was determined by the Fermco-test S.F.G. The amount of maltose was determined by the A.O.A.C. maltose method. The quantities of limiting amino acids were estimated by one dimensional thin-layer chromatography. The quality of protein for each protein source was evaluated by the chemical score.
Maximum protein solubilization occurred in chick peas between pH 7.5 and 8.5, in whole wheat flour at pH 8.0 and in sunflower seed press cake and the mixture at 8.5. The greatest release of free amino groups in terms of ninhydrin positive material was found between pH 8.0 and 8.5 for individual sources. There was a trend for a reduction in the amount of reducing sugars above pH 6.0 for each protein source.
High levels of soluble protein, ninhydrin positive material, glucose and maltose were observed at 50°C for the individual proteinaceous materials.
The maximum enzymatic hydrolysis was completed within 6 hours for each protein source. However, there was a trend for an increase in NPM content until reaction time was extended to 8 hours or beyond. High maltose contents were observed at 6 hours while no change was observed in glucose level after 2 hours digestion for each plant protein source.
All four protein sources required denaturation of proteins prior to enzyme treatment. The cooking time was found to be 20 minutes for chick peas and 15 minutes for whole wheat flour, sunflower press cake and the mixture for optimum solubilization. Higher amounts of NPM were observed at 20 minutes denaturation period followed by a decrease at a 25 minute cooking period. Glucose and maltose were not affected by the length of denaturation.
For maximum solubility of proteins, whole wheat flour, chick peas, sunflower press cake and the mixture required 1.5, 2.0, 2.5 and 2.5 enzyme preparation, respectively. Except for the mixture, high NPM concentrations occurred at the 2.5 percent enzyme level for the individual protein sources.
There were no significant changes in soluble protein and free amino acid content as the amount of enzyme added reached the 3.0 percent level of each protein source. The amount of sugar increased directly with the amount of enzyme added.
The amount of soluble protein increased only slightly as the substrate was increased from 5 grams to 9 grams.
The maximum protein solubilization of chick pea, whole wheat flour and the mixture occurred between the 0.2 and 0.3 M concentrations of sodium chloride, while the maximum protein solubilization occurred at 0.1 M sodium chloride in sunflower seed press cake. There were no significant changes in free amino acid and sugar content for each protein source.
The soluble protein content of the enzyme hydrolyzates from chick pea, whole wheat flour, sunflower seed press cake and the mixture were 187.7, 111.2, 412.0, and 257.7 mg/g, respectively. These corresponded to 134, 94.8, 387 and 235.6 micromoles of NPM per gram of protein source.
The protein scores based on the quantity of limiting amino acid, determined by thin-layer chromatography, were found to be: 46 percent in relation to reference protein for chick pea, which was deficient in methionine; 73 percent in relation to reference protein for whole wheat flour, which was deficient in lysine; and 84 percent in relation to reference protein for sunflower seed press cake, which is deficient in lysine. The protein score for the mixture was 75 percent.
Burklin, Sevim Caglar, "Enzymatic modification of proteinaceous vegetable materials for infant food in Turkey. " Master's Thesis, University of Tennessee, 1973.