Hereus multifuge x3

Water was heated to a temperature of 60 °C, then HTAA and XBG enzymes were added at the range of 0.1% by volume each. The flour and enzymes ratio was based on earlier authors’ experiments. Room temperature oat flour was added at the ratio 1:10 by weight continuously stirring by Promix (Phillips, Hungary) hand mixer. The obtained mixture was periodically kept stirred (30 s for each 3 min) by Promix (Phillips, Hungary) hand mixer for 20 min at the temperature of 60 °C. Then, temperature was raised to 75 °C for 20 min, while stirring remained at the same interval rate, 30 s for each 3 min. The obtained hydrolysate was cooled down to 25 °C and passed separation by centrifuge Hereus Multifuge X3 (Thermo Fisher Scientific, Langenselbold, Germany), at the G-force 4400, for 4 min. The obtained biomass was washed by water at the ratio 1:10 and repeatedly passed separation, wherein separation parameters remained the same as previous. The washed biomass was dried in the hot air oven at the temperature 60 °C for 24 h. Dried protein concentrate was cooled down to room temperature and passed through the hammer mill LM 3100 Perten Instruments (Perkin Elmer, Waltham, MA, USA), sieve 0.8 mm. A milled sample of oat protein concentrate (OC1) was collected in a plastic sealed container and kept at room temperature for further analysis and processing.

Oat flakes were mixed with previously heated water at the temperature of 80 ± 1 °C, wherein HTAA was added at the amount of 0.05% by volume. Then, while continuously stirring, oat flakes (room temperature) were added at the ratio of 1:10 by weight. The mixture was stirred periodically at intervals of about 30 s every 3 min using the hand mixer Promix (Phillips, Hungary) for 30 min while the temperature of hydrolysis was kept within the range of 75–80 °C. The hydrolysate was then separated by Hereus Multifuge X3 (Thermo Fisher Scientific, Osterode am Harz, Germany) at the G-force 900 for 1 s to separate the fiber. The obtained clarified hydrolysate was then separated at G-force 4800 for 5 min. The extracted protein biomass was washed with water at a ratio of 1 to 4 by weight. The washed protein biomass passed separation by the aforementioned centrifuge at G-force 4800 for 5 min and then dried in a 65 ± 2 °C hot air oven B5745-5-M (AEG, Germany) for 24 h. The dried oat protein was milled by hammer mill LM 3100 Perten Instruments (Perkin Elmer, USA) and a sieve of 0.8 mm. The separated fiber was dried in a 65 ± 2 °C hot air oven for 24 h. Obtained samples were coded as A1 for protein and AF1 for fiber.

For alkaline hydrolysis the isolated AGPs from Echinacea and Zostera were dissolved in 0.44 M sodium hydroxide solution in a screw-cap tube (Pyrotube C vial; Associates of Cape Cod Inc., East Falmouth, MA, USA) resulting in a final concentration of 6.7 mg/mL. The solutions were hydrolyzed for 20 h at 105 °C in a heating block (Bioblock Scientific, Thermolyne Corp., Ramsey, MN, USA). Afterwards the solution was neutralized with diluted hydrochloric acid and added to the four-fold volume of absolute ethanol and cooled overnight at 4 °C. Subsequently the precipitate was separated by centrifugation at 19,000× g for 30 min (Hereus Multifuge X3, Thermo Fisher Scientific Corp., Waltham, MA, USA) and additional washing with 80% (v/v) ethanol two times. The washed residue was redissolved in double-distilled water and freeze-dried (Christ Alpha 1–4 LSC, Martin Christ GmbH, Osterode, Germany).
A total of 20 mg of the dried sample was dissolved in 2 mL of 12.5 mM oxalic acid in a screw-cap tube and hydrolyzed for 5 h at 100 °C in the heating block. Precipitation and further treatment were performed as stated above.