Study of amino acid profile, solubility and antioxidant properties of quinoa protein hydrolysates

Quinoa with scientific name "Chenopodium quinoa Willd", unlike real grains that belong to the family Poaceae, is a pseudocereal belonging to the Amaranthaceae family of dicotyledonous or dual-leaf vegetables, and Its origin is in South America. Quinoa seeds also contain a variety of bioactive components such as polyphenols, carotenoids, and oleic acid, all of them are beneficial to human health. Quinoa, with its essential amino acids, protein content, and high protein bioavailability, can be a good alternative for plant and animal proteins in patients with celiac disease. Seventeen bioactive peptides with potential properties were isolated and identified from quinoa proteins. In recent years, much research has been done on use of quinoa and its bioactive peptides for its functional properties. Nowak et al., (2016) in study of nutrients in quinoa expressed that; Quinoa is an ancient agricultural product and can play an important role in food safety around the world. Fischer et al., (2017). In present study, amino acid sequence is extracted and quinoa protein is hydrolyzed using pancreatin enzyme and its functional properties such as emulsifying, foaming, solubility, antioxidant activity, etc. are investigated, so it can be used as a bioactive compound with nutritional and functional properties in food systems.
MATERIAL AND METHODS
Quinoa seeds purchased from National Salinity Research Center dependent to Yazd Agricultural investigation organization (Yazd, Iran), Pancreatic enzyme (active at pH of 8.0 and temperature of 37 °C ) and DPPH free radical prepared from Sigma Aldrich (Steinheim, Germany).
Chemical analysis, preparation of sample for protein hydrolysis
Flour preparation from quinoa seeds and defatting by hot method and solvent extraction Whole seeds were washed for 4-5 times until there was no foam in the solution that was the sense of saponins, then seeds oven-dried at 45+1 ˚C until being dry, then whole seeds were ground into flour using Miller (Proctor Silex model EI60, UPC) with a sixty-mesh screen (Elsohaimy et al., 2015). Defatting from quinoa flour was performed with Soxhlet technique and by hot solvent of hexan (normal) at a raito of 1:4 seed flour to solvent in 9 hr. (Sánchez-Vioque et al., 1999).
Amino acids compositions
Protein samples hydrolyzed with HCI 6 N in time of 24 hours at 110 ˚C. The excitation wavelength was 330 nm and the emission spectra were recorded at 480 nm, The analysis was carried out with a gas flow rate of 1.3 ml/min at separation temperature of 35 °C.
Obtaining of quinoa protein concentrate
At first, defatted Quinoa flour was suspended in distilled water in ratio of 1:10. Then pH of solution was adjusted to 10.0 using NaOH at a concentration of 1 N and resulted solution was thoroughly stirred at room temperature for 60 min. During this time interval, pH was kept constant at set value to maximize proteins dissolution. The mixture was then mixed for 30 minutes at 9000 rpm at 4°C in a centrifugal refrigerator (K241R, Pro-Research, Centurion Scientific Ltd, UK), The solid phase was then separated and pH of the supernatant was reduced to 5 using 1 N hydrochloric acid to precipitate quinoa proteins. same centrifuge operation, with above conditions was repeated again. The centrifuge precipitate, which is protein concentrate, was lyophilized with freeze dryer (Christ, Germany). And were stored in freezer at -18 °C for subsequent experiments )Živanović et al., 2011).
Preparation of protein hydrolyzate from quinoa protein concentrate
To complete enzymatic hydrolysis process, first, protein isolate sample was dispersed and dissolved in 0.01 M phosphate buffer with pH = 7.4 for 30 minutes at a concentration of 5% (w/v). And constant stirring at ambient temperature allowed it to be completely hydrated. Then, initial solution of pancreatin enzyme was prepared in 0.01 M phosphate buffer, This solution was added to the protein isolated solution in ratio of enzyme to protein substrate equal to 2.5% (w/w). Reaction temperature for pancreatin enzyme was 40 °C and continuous stirring was performed at 200 rpm for 4 hours. After completion of enzymatic hydrolysis process, sample reaction medium was placed in a 95 °C water bath for 15 minutes to inactivate enzyme and stop reaction. After that solution was cooled to ambient temperature, centrifugation was performed for 15 minutes at 9000 rpm, then supernatant solution was separated and lyophilized at a temperature of -20 °C with an approximate pressure of 0.1 mB, and then stored at -20 °C until use .
resuults
The results were in a completely randomized design with three replications and a significance level of 5% with a moisture content of 9.36, ash 2.29, crude fiber 4.6, protein 12.51, fat 5.36 and carbohydrate 71.48%. Results of chemical properties, shows quinoa seeds as an excellent potential food source with functional properties and this is due to quinoa essential nutrients content (such as proteins, carbohydrate, lipid and fiber). Result of present study is in agreement with (James, 2009), which in case quinoa seeds had about 11.2% moisture, 13.2% protein, 9% crude fiber, 1.2% total ash, and about 48.2% carbohydrate, however, this amount of carbohydrate was relatively less compared to present study. previous studies have shown that average protein content in quinoa seeds varies between 12% to 23%. The highest percentage of quinoa amino acids were glutamic acid and lysine, but there was a shortage of sulfur amino acids. The highest degree of hydrolysis (19.17%) was obtained after 180 minutes. Quinoa peptides had the lowest solubility in the isoelectric pH range and their solubility was increased in pH values below and above the isoelectric range. Quinoa bioactive peptides significantly reduced DPPH radical reduction and had high antioxidant activity (67.8% after 6 hours of hydrolysis and decreased to 59.8% after 8 hours). Quinoa with high percentage of protein has favorable physicochemical, functional and antioxidant properties and the resulting peptides can be used as bioactive food sources in pragmatic products.