2.3. Production of Pea Protein Isolate

The isolation of pea protein was performed according to Tian and Kyle [26] following an alkaline extraction with isoelectric precipitation (AE-IEP) with some changes. An aqueous alkaline extract of the pea flour was prepared in deionized (DI) water at a ratio of 1:5 (w/w) at pH 8.0 using 3.0 mol/L NaOH, which was stirred for 60 min. The protein extract was sieved (0.8 mm) after centrifugation at 8000× g for 20 min at 15 °C (8K, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). For isoelectric precipitation, the protein extract was adjusted to pH 4.5 using 3.0 mol/L HCl and left overnight at 4 °C. The precipitated proteins were separated by centrifugation at 8000× g for 20 min at 15 °C and the protein isolate was dispersed in DI water to a dry matter content of 8%. After neutralization to pH 7.0, the isolate dispersion was homogenized at 11,000 rpm for 2 min using an Ultraturrax (IKA^®-Werke GmbH and Co. KG, Staufen, Germany) prior to spray drying. The spray drying was performed using a Mini Spray Dryer B-191 (BUCHI Labortechnik GmbH, Essen, Germany) at inlet and outlet temperatures of 180 °C and 80 °C, respectively, as well as with a 95% aspirator output. The spray-dried isolates were used for further analysis. The protein yield was calculated as grams of protein per kilogram of seeds. Due to the limited amounts of pea seeds, the protein extractions and spray drying were performed once. We assumed that the protein extraction and yield values are representative of the process, as other studies have shown low standard deviations in their own extractions [17,18].