Optimiranje proizvodnje inulinaze s Kluyveromyces bulgaricus

The present work is based on observation of the effects of pH and temperature of fermentation on the production of microbial enzyme inulinase by Kluyveromyces marxianus var. bulgaricus. Inulinase hydrolyzes inulin, a polysaccharide which can be isolated from plants such as Jerusalem artichoke, chicory or dahlia, and transformed into pure fructose or fructooligosaccharides. Fructooligosaccharides have great potential in food industry because they can be used as calorie-reduced compounds and noncariogenic sweeteners as well as soluble fibre and prebiotic compounds. Fructose formation from inulin is a single step enzymatic reaction and yields are up to 95 % the fructose. On the contrary, conventional fructose production from starch needs at least three enzymatic steps, yielding only 45 % of fructose. The process of inulinase production was optimised by using experimental design method. pH value of the cultivation medium showed to be the most significant variable and it should be maintained at optimum value of 3.6. The effect of temperature was slightly lower and optimal values were between 30 and 33 °C. At a low pH value of the cultivation medium, the microorganism was not able to producem enough enzyme and enzyme activities were low. Similar effect was caused by high temperature. The highest values of enzyme activities were achieved at optimal fermentation conditions and the values were: 100.16–124.36 IU/mL (with sucrose as substrate for determination of enzyme activity) or 8.6–11.6 IU/mL (with inulin as substrate), respectively. The method of factorial design and response surface analysis makes it possible to study several factors simultaneously, to quantify the individual effect of each factor and to investigate their possible interactions. As a comparison to this method, optimisation of a physiological enzyme activity model depending on pH and temperature was also studied.Ovaj se rad temelji na ispitivanju utjecaja pH i temperature na proces biosinteze enzima inulinaze s kvascem Kluyveromyces bulgaricus (prije Kluyveromyces marxianus). Mikrobni enzim inulinaza hidrolizira inulin, polisaharid izoliran najčešće iz biljaka (jeruzalemska artičoka, cikorija i dalija), do fruktoze ili fruktooligosaharida. Fruktooligosaharidi se uvelike primjenjuju u prehrambenoj industriji jer se mogu koristiti kao niskokalorični i zaslađivači koji sprečavaju karijes, te kao topljiva vlakna i prebiotički spojevi. Potpuna je hidroliza inulina jednostupanjska enzimska reakcija koju katalizira inulinaza uz 95 %–tni prinos fruktoze. Nasuprot tome, konvencionalna proizvodnja fruktoze iz škroba zahtijeva barem tri stupnja, s tri različita enzima, a prinos je tek 45 % fruktoze. Proces proizvodnje inulinaze optimiran je metodom planiranja pokusa (»experimental design«). pH-vrijednost hranjive podloge pokazala se kao najva`niji parametar u proizvodnji inulinaze i optimalna je vrijednost iznosila 3,6. Temperatura je imala nešto slabiji utjecaj, a optimalne su vrijednosti bile između 30 i 33 °C. Pri niskoj pH-vrijednosti podloge mikroorganizam nije uspijevao proizvesti dovoljnu količinu enzima, pa su i enzimske aktivnosti male. Slično djelovanje uzrokovano je i pri visokim temperaturama uzgoja. Najviše vrijednosti enzimske aktivnosti, postignute pri optimalnim uvjetima, iznosile su: 100,16 do 124,36 U/mL (sa saharozom kao supstratom za određivanje enzimske aktivnosti) ili 8,6 do 11,6 U/mL (s inulinom kao supstratom). Metode planiranja pokusa i analize odzivnih ploha omogućuju promatranje utjecaja nekoliko parametara istodobno, te utvrđivanje intenziteta tih utjecaja, a i njihovih mogućih interakcija. Usporedo je provedeno optimiranje fiziološkog modela enzimske aktivnosti ovisno o pH i temperaturi