Enzymatic synthesis and membrane purification of fructo‐oligosaccharides

[M. B. Veljković]

VI,180 str.

Tehnološko inženjerstvo - Biohemijsko inženjerstvo i biotehnologija / Technological engineering - Biochemical engineering and biotechnology

okviru ove doktorske disertacije razvijeni su postupci sinteze frukto-oligosaharida (FOS) primenom slobodne i imobilisane fruktoziltransferaze (FT) iz industrijskog preparata Pectinex® Ultra SP-L. Primenom slobodne FT i izborom optimalnih uslova dobijena je smeša koja sadrži 60,2 % FOS i sporedne proizvode poput saharoze i monosaharida. Radi razvijanja efikasnijih postupaka proizvodnje FOS koji podrazumevaju primenu imobilisanih preparata, FT je imobilisana na nosače različitih funkcionalnih karakteristika putem adsorpcije ili kovalentnog vezivanja. Posebno pogodnim pokazali su se nemodifikovani i epoksi- modifikovani Purolite® A109 nosač čijom je primenom omogućena selektivna imobilizacija FT, a time i delimično prečišćavanje preparata od drugih enzima. Imobilisani preparati su upotrebljeni za proizvodnju FOS u šaržnom i pneumatskom reaktorskom sistemu. S obzirom na njihovu izuzetnu operativnu stabilnost, razvijeni su i kontinualni procesi proizvodnje FOS. Dobijena smeša FOS podvrgnuta je i prečišćavanju primenom nanofiltracije sa dijafiltracijom. Izvođenjem ovog procesa pri izabranim optimalnim uslovima, dobijena je smeša čistoće 87,2 % FOS. Radi dodatnog unapređenja procesa prečišćavanja, primenjen je i membranski reaktor gde je simultanim izvođenjem sinteze FOS i prečišćavanja, nakon 9 h dobijena smeša sa 91,8 % FOS. Visokoprečišćena smeša FOS testirana je u in vitro TIM-2 sistemu koji uključuje primenu ljudske crevne mikrobiote i simulira procese varenja u debelom crevu, gde rezultati potvrđuju postojanje prebiotskog potencijala. Primenom stečenih znanja, razvijen je i funkcionalni proizvod izvođenjem in situ biokonverzije saharoze u FOS primenom FT i komercijalnog javorovog sirupa. Ovim pristupom dobijen je javorov sirup sa dodatom vrednošću, smanjenog kalorijskog sadržaja i poboljšanih funkcionalnih karakteristika uz zadržavanje standardnih parametara kvaliteta.

As part of this doctoral dissertation, procedures for the synthesis of fructo-oligosaccharides (FOS) were developed using free and immobilized fructosyltransferase (FT) from the industrial preparation Pectinex® Ultra SP-L. By applying free FT under selected optimal conditions, a mixture containing 60.2 % FOS and by-products such as sucrose and monosaccharides. To develop a more efficient FOS production process that involves immobilized preparations, FT is immobilized on supports with different functional characteristics via adsorption or covalent binding. The unmodified and epoxy-modified Purolite® A109 support are considered particularly suitable supports, which enabled selective immobilization of FT, and thus partial purification of the preparation from other enzymes. The immobilized preparations were used for the FOS production in batch and air-lift reactor system. Considering their exceptional operational stability, continuous FOS production processes have been developed. The synthesized FOS mixture was subjected to purification using nanofiltration with diafiltration. By performing this process under the selected optimal conditions, a mixture with a purity of 87.2 % FOS was obtained. To improve the purification process, a membrane reactor was used, where the simultaneous execution of the FOS synthesis and purification resulted in a mixture with 91.8 % FOS after 9 h. The highly purified FOS mixture was tested in the in vitro TIM-2 system, which includes the application of human gut microbiota and simulates digestion processes in the large intestine, where the results confirm the existence of prebiotic potential. By applying the acquired knowledge, a functional product was developed by performing in situ bioconversion of sucrose into FOS using FT and commercial maple syrup. This approach resulted in a value-added maple syrup with reduced caloric content and improved functional characteristics while maintaining standard quality parameters.

frukto-oligosaharidi; prebiotici; fruktoziltransferaza; imobilizacija; nanofiltracija; reaktorski sistemi; javorov sirup; funkcionalni proizvodi

fructo-oligosaccharides; prebiotics; fructosyltransferase; immobilization; nanofiltration; reactor systems; maple syrup; functional products

604.4:577.15(043.3)

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