Effect of pH Changes on Functional Properties of Fenugreek Protein Isolate

Plant proteins play a significant role in the human diet, and among which legumes are excellent foods with several nutritional and functional advantages and low price. Among legumes, fenugreek (Trigonella foenum graecum) seed, belonging to the Fabaceae family, is a great source of plant proteins, with a total protein content of about 25.00 – 38.60%. Functional properties are defined as physical and chemical properties, which could be altered due to the protein source and procedures used for flour preparation, protein concentrate and isolate extraction. Physiochemical parameters such as pH, temperature, salt and ionic strength can also highly affect proteins, functional properties.Therefore, in this study color parameters and functional properties of fenugreek protein isolate (FPI) were investigated. Also, the effect of pH changes on protein solubility, foaming capacity (FC) and stability (FS), emulsifying capacity (EC) and stability (ES) was studied.
Fenugreek seeds were obtained from a retail market in Isfahan, Iran. All chemicals were of analytical grade, and were purchased from Sigma Chemical Co. (St Louis, MO, USA), and Merck KGaA (Darmstadt, Germany).
Protein Isolation: Protein from 10g defatted fenugreek flour (DFF) was extracted with 0.33M sodium chloride solution; the ratio of DFF to NaCl solution was set at 1:20 (w/v) and pH was adjusted to 9.25. The stirring period was 2 h, during which pH was also adjusted. Aliquots were then centrifuged and supernatants were adjusted to pH 4.5 (isoelectric pH of fenugreek protein). Again, the protein solution was centrifuged in order to precipitate the protein. Protein precipitate was washed twice with deionized water, followed by centrifugation and resolubiliztion by adjusting the pH to 7.2. The extraction process was carried out at room temperature (25°C).
Color parameters: A digital colorimeter was used to measure the color of full fat and defatted fenugreek flours, and FPI samples. Color parameters were recorded in L*a*b* space.
Protein solubility: Protein solubility of FPI at pH 2-10 was determined. A 1.5% (w/v) solution of sample was made in deionized water for each pH. The solutions were treated with either 0.5M HCl or 0.5M NaOH to obtain certain pH (2-10), while stirring. The slurry was centrifuged to separate the supernatant. Protein content was determined by the Biuret method using spectrophotometer at 540nm wavelength. Calibration was done using bovine serum albumin (BSA) as external standard.
Viscosity: A suspension of 5% (w/v) FPI in distilled water was prepared. Viscosity of sample was determined using a programmable Brookfeildrheometer, using spindle ULA at room temperature.
Coagulated protein: Percent of coagulated protein was measured by preparing 0.2g of FPI in 10mL of 0.025M citrate-phosphate buffer (pH 7.0). The solution was stirred, followed by centrifugation. The remaining supernatant was heated for 15min in a 100°C water bath, after cooling the same procedure was repeated. The absorbance of both samples, before heating (A1) and after heating (A2), was measured at 540nm according to Biuret method. The percent of coagulated protein was calculated by the following equation: Coagulated protein = (A1 – A2 / A1) × 100 (1)
Foaming capacity and stability: FC and FS were determined using 2% (w/v) FPI solution in distilled water. For each sample solution, the pH was adjusted to desired level (2, 3, 4.5, 6, 8, and 10). The solution was stirred slightly for 5min and then was whipped vigorously by a disperser. The volume of solution before whipping, also the total volume of solution and foam after whipping were recorded as V0 and V1, respectively. The foam capacity percentage was calculated as: FC (%) = (V1- V0/V0) × 100 (2)
Foam stability was determined as the total volume of solution and foam at 30, 60, 90 and 120 min at room temperature and it was recorded as V2. Foam stability (FS) was calculated as: FS (%) = (V2 - V0) × 100/ (V1 - V0) (3)
Emulsifying capacity and stability:For EC and ES, a 1% (w/v) FPI solution in distilled water was prepared. The pH was adjusted to desirable level (2, 3, 4.5, 6, 8, and 10) followed by stirring for 5min, then sunflower oil was added. The mixture was homogenized to make emulsion, followed by centrifugation. Then the height of both emulsified layer after centrifugation (H1) and the total height of content before centrifugation (H0) were measured. Emulsion capacity was calculated as: EC (%) = (H1/H0) × 100 (4)
The ES (%) was calculated at 0, 30, 60, 90 and 120 min by measuring height changes (H2).
ES (%) = (H2/ H1) × 100 (5)
FPI with 89.1% protein content had significantly (p