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    Production of Hypoallergenic Antibacterial Peptides from Defatted Soybean Meal in Membrane Bioreactor: A Bioprocess Engineering Study with Comprehensive Product Characterization

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    Hipoalergeni antibakterijski peptidi male molekularne mase proizvedeni su iz nemasne sojine sačme u membranskom bioreaktoru. U prvom su koraku proteini sojine sačme razgrađeni pomoću tripsina u šaržnom bioreaktoru. Optimalni uvjeti za razgradnju tripsinom bili su: koncentracija sojine sačme od 75 g/L, temperatura od 40 °C i pH=9. Nakon toga su peptidi male molekularne mase pročišćeni pomoću „cross-flow“ membrane (veličina pora 100 μm), a zatim pomoću keramičke cjevaste membrane (veličine pora koja ne propušta molekule veće od 5 kDa). Ispitan je utjecaj transmembranskog pritiska i primjene statičkih promotera turbulencije na smanjenje koncentracijske polarizacije blizu površine ultrafiltracijske membrane, te je potvrđen njihov pozitivan učinak. Transmembranski pritisak od 3•105 Pa i diskontinuirana dijafiltracija provedena u tri koraka bili su optimalni za filtraciju pomoću ultrafiltracijske membrane. Distribucija molekularne mase peptida pročišćenih pomoću ultrafiltracijske membrane utvrđena je pomoću metode LC-ESI-Q-TOF-MS. Više od 96 % peptida (izraženih kao relativna frekvencija) iz permeata ultrafiltracijske membrane imalo je molekularnu masu manju od 1,7 kDa, a najveća je molekularna masa bila 3,1 kDa. Smanjenje alergenih svojstava proteina za više od 99,9 % nakon obrade tripsinom i membranske filtracije utvrđeno je imunoenzimskim testom. Također je zaključeno da peptidi pročišćeni pomoću ultrafiltracijske membrane pozitivno djeluju na rast bakterije Pediococcus acidilactici HA6111-2, te sprečavaju rast bakterije Bacillus cereus.Hypoallergenic antibacterial low-molecular-mass peptides were produced from defatted soybean meal in a membrane bioreactor. In the first step, soybean meal proteins were digested with trypsin in the bioreactor, operated in batch mode. For the tryptic digestion of soybean meal protein, optimum initial soybean meal concentration of 75 g/L, temperature of 40 °C and pH=9.0 were determined. After enzymatic digestion, low-molecular-mass peptides were purified with cross-flow flat sheet membrane (pore size 100 μm) and then with tubular ceramic ultrafiltration membrane (molecular mass cut-off 5 kDa). Effects of transmembrane pressure and the use of a static turbulence promoter to reduce the concentration polarization near the ultrafiltration membrane surface were examined and their positive effects were proven. For the filtration with ultrafiltration membrane, transmembrane pressure of 3·10^5 Pa with 3-stage discontinuous diafiltration was found optimal. The molecular mass distribution of purified peptides using ultrafiltration membrane was determined by a liquid chromatography–electrospray ionization quadrupole time-of-flight mass spectrometry setup. More than 96 % of the peptides (calculated as relative frequency) from the ultrafiltration membrane permeate had the molecular mass M≤1.7 kDa and the highest molecular mass was found to be 3.1 kDa. The decrease of allergenic property due to the tryptic digestion and membrane filtration was determined by an enzyme-linked immunosorbent assay and it was found to exceed 99.9 %. It was also found that the peptides purifi d in the ultrafi tration membrane promoted the growth of Pediococcus acidilactici HA6111-2 and they possessed antibacterial activity against Bacillus cereus
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