| Abstract | Zbog brojnih fizioloških i funkcionalnih svojstava proteina sirutke te nedostatka metoda industrijskih razmjera za frakcioniranje istih, cilj ovoga rada je optimirati procese selektivne enzimske hidrolize proteina sirutke u svrhu izolacije α-laktalbumina (α-La) ili β-laktoglobulina (β-Lg) te odrediti selektivnost enzima prema pojedinoj proteinskoj frakciji. Kao supstrat za hidrolizu korišten je izolat proteina sirutke, a upotrijebljeni enzimi su: α-kimotripsin, pepsin, proteaza N, kisela proteaza A, proteza A, proteaza M, Neutrase® i Flavourzyme®. Eksperimenti su provedeni pri različitim koncentracijama supstrata (5 ili 10 % w/v), temperaturama od 25, 30, 35, 37, 40, 45 i 50 °C, pH vrijednostima 1,0; 1,5; 2,0; 2,5; 3,0; 3,5; 4,0; 5,0, 7,0; 7,8; 8,0 8,5 te koncentracijama enzima prema supstratu (E/S) od 0,1; 0,5, 1,0, 1,5 i 2,0%. Nehidrolizirani proteini su određivani pomoću tekućinske kromatografije visoke djelotvornosti sa reverznom fazom (RPHPLC) i elektroforezom (SDS-PAGE). Također je praćena i kinetika reakcija. Za izolaciju čistog i nativnog α-La kao najbolji se pokazao α-kimotripsin, gdje nakon hidrolize zaostaje 81% proteina. Prema dostupnoj literaturi, po prvi puta je prikazan mehanizam hidrolize izolata proteina sirutke α-kimotripsinom, pri čemu dolazi do određene selektivnosti prema proteinima. Isto tako, po prvi puta je ustanovljena sličnost sa tripsinom, a vezana je uz redoslijed hidrolize proteina. Za izolaciju β-Lg kao najbolji su se pokazali pepsin, kisela proteaza A i proteaza M, gdje nakon hidrolize zaostaje čak do 100% nativnog i nehidroliziranog proteina. Rezultati pokazuju da korišteni enzimi imaju veliki potencijal pri proizvodnji proteina i bioaktivnih peptida iz sirutke pri čemu dolazi do minimalne denaturacije proteina, uz smanjenje troškova, u odnosu na dosadašnje metode te moguću primjenu u industrijskom mjerilu. |
| Abstract (english) | Due to a numerous physiological and functional properties of whey proteins and a lack of industrial-scale methods for their fractionation, the aim of the present work was optimising of the selective enzymatic hydrolysis of whey protein for possible isolation of α-lactalbumin (α-La) or β-lactoglobulin (β-Lg) as well as determination of the selectivity of the enzyme for a particular protein fraction. Whey protein isolate (WPI) was used as a substrate for hydrolysis as well as following enzymes: α-chymotrypsin, pepsin, protease N, acid protease A, protease A, protease M, Neutrase® and Flavourzyme®. The experiments were performed at different concentrations of the substrate (5 or 10% w/v), the temperatures: 25, 30, 35, 37, 40, 45 and 50 ° C, pH: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 7.0, 7.8, 8.0 and 8.5, enzyme to substrate concentrations (E/S): 0.1, 0.5, 1.0, 1.5 and 2.0%. Residual proteins were determined by reversed phase high performance liquid chromatography (RP-HPLC) and electrophoresis (SDS-PAGE). The reaction kinetics was also accompanied. α-chymotrypsin was the best enzyme for the isolation of pure and native α-La where, after hydrolysis, left 81% unhydrolysed protein. To the best of knowledge, this work is the first attempt that demonstrates the selective susceptibility of WPI to chymotrypsin hydrolysis. Furthermore, it is revealed for the first time that chymotrypsin shares the same hydrolysis similarities with trypsin with regard to the way it attacked the whey proteins. Pepsin, acid protease A and protease M were the best enzymes for the isolation of β-Lg, where after hydrolyses left up to 100% of native and unhydrolysed protein. The results indicate that the used enzymes have great potential in the production of proteins and bioactive peptides from whey. Also, enzymes cause minimal denaturation of proteins as well as reduced costs compared to the existing methods with the possible simple exploitation to an industrial scale. |
