Optimization Of The Enzymatic Production Of High Fructose Inulin Syrup (Hfis) And Application Of Inulin As Fat Substitute

The production of fructose has been increasing steadily in recent years due to its sweetness,favorable properties and the drastic increase in sugar prices. Fructose is commercially producedfrom corn starch. This process involves the hydrolysis of the starch into glucose followed byisomerization of glucose to fructose. This process produces syrups containing about 42% fructose.The fructose concentration in the syrup can be enhanced (to 55% and 90% High Fructose CornSyrups, HFCS) by selective removal of glucose or by applying multistage chromatographicseparation methods. These techniques are costly, which reflect on the price of the product thuslimiting its use as a common sweetener.A potentially promising process for fructose production is based on hydrolyzing the fructan-richplants to fructose. The main purpose of this study was first to determine the properties andcharacteristics of Fructozyme, a commercial enzyme which was donated by Novozymes in China.Then, considering the previously determined properties, the optimal hydrolysis conditionsallowing the optimization of high fructose syrups production either from Chicory Inulin (CHI) orJAI (Jerusalem Artichoke Inulin) were investigated.The pH profile of Fructozyme displayed an optimum pH around 4.5. Concerning the pH stability,Fructozyme activity was found to be quite stable for 12 h in pH value ranging from 4.0 to 6.0 atroom temperature. The temperature profile of Fructozyme displayed an optimum temperaturearound 60°C. In terms of thermal stability, Fructozyme activity remained stable after 6h ofexposure at 60°C, with no significant loss of its initial activity. Furthermore, Fructozyme revealeditself to be among the most thermostable inulinases. In fact, after 12 h of heat exposure, only 20%of Fructozyme activity was lost.These properties are favorable in view of large-scale inulinaseapplication for pure fructose production. In fact, high operation temperatures would avoidmicrobial contamination of reactors and would allow the use of high inulin-substrateconcentrations, a factor which was limiting in obtaining high conversion ratios. In addition, theremarkably low pH optimum prevents color formation and undesirable chemical side reactions.The shaking speed was found to have an impact on Fructozyme activity. In fact, an optimumshaking speed could be detected at 150 rpm. However, for practical reasons, it is suggested tooperate with a shaking speed ranging from 150 to 200 rpm.Fructozyme activity varies markedly according to the composition or type of buffer, with sodiumacetate buffer giving the best results. Furthermore, the effect of buffer concentration onFructozyme activity was investigated. Concentrations up to 100 mM, did not influence noticeablythe activity of Fructozyme, whereas concentrations above 100 mM, influenced markedlyFructozyme activity. A complete inhibition was observed for 500 mM.The effects of chemicals and metallic ions on Fructozyme activity were studied. In fact,Fructozyme activity is markedly inhibited by Ag⁺, Hg⁺, Cu²⁺, Mg²⁺ and Fe³⁺, partially inhibitedby Zn²⁺, Fe²⁺, Na+, Pb²⁺, Li⁺, EDTA, 2-Mercapto-Ethanol, and almost unaffected by Ca²⁺, Ba²⁺,K⁺, Al³⁺. However, Mn²⁺and Co²⁺ appeared to enhance Fructozyme activity.By using High Performance Gel Filtration Chromatography (HPGFC), JAI exhibited a molecularweight of approximately 940 g, which might correspond to a DP comprise between 3 and 4, whileCHI displayed a molecular weight of about 2029 g, which should correspond to a DP between 9and 10. This is an indication that both JAI and CHI are not polymers but oligomers of fructose.Substrate specificity revealed that Fructozyme is active on inulin, sucrose, and raffinose. Thissuggested that Fructozyme might contain a certain amount of exo-acting inulinase. In addition,Fructozyme displayed an S/I ratio of 1.79, which corresponds also to an I/S ratio of 0.56. This S/Iratio was found to be relatively low, indicating that Fructozyme is not an invertase, but a trueinulinase. Substrate affinity of Fructozyme was also investigated and kinetic parameters of thisenzyme were determined by using Lineweaver-Burk plots. The Km and Vmax values for sucrosehydrolysis were found to be 0.34 mM and 1.65 mM L-1 min-1, respectively. However, when inulinwas used as substrate, the Km and Vmax values were found to be slightly lower: 0.16 mM and1.32 mM L-1 min-1, respectively.SDS-PAGE indicated that Fructozyme is not a homogeneous and/or pure enzyme, given that nosingle band was observed. Nevertheless, the main band exhibited an active-region whichcorresponds to an apparent Mr of approximately 66 kDa. Moreover, considering that the apparentMr exhibited by Fructozyme was found to be higher than those displayed by common exo-inulinases, the hypothesis that Fructozyme may be a complex enzyme preparation containing bothexo-and endo-inulinases was confirmed by our results.In order to optimize the production of High Fructose Inulin Syrup (HFIS), the effects of factorssusceptible to influence hydrolysis rate were investigated.Time course revealed that Fructozyme could hydrolyze up to 70% of the inulin during the firsthour of reaction, and 90% of the inulin in 3h. Thereafter, the extent of inulin hydrolysis remainedalmost constant.The effect of Fructozyme dosage indicated that a total yield of HFIS (95.5%) could be achievedeasily within 4h when the initial Fructozyme concentration was 20FU/g inulin. However, thistotal yield could not be reached when the initial Fructozyme concentration was 2FU/g inulin. Itwas concluded that the reaction time was shortened as the enzyme dosage increased.The effect of substrate type and/or composition was investigated also. The rate of JAI hydrolysiswas found to be slower than that observed for CHI. This is confirmed by the lower total yields ofHFIS (68%) achieved during hydrolysis of JAI by Fructozyme, comparatively with the highertotal yields of HFIS (91%) attained during CHI hydrolysis by using the same optimum conditions.This phenomenon may be explained by the fact that CHI is purer than JAI. In fact, the presence ofsome non-inulin substances (impurities) in the crude JAI may have acted as potential inhibitors ofFructozyme activity during hydrolysis. Moreover, CHI was found to have an average molecularweight and/or DP higher than that of JAI.The effect of substrate concentration revealed that although the initial hydrolysis rate of inulintended to be high when the substrate concentration increases, however, there was no significantdifference in the total HFIS yields finally achieved. In fact, by using up to 100g inulin/L, the totalyields of HFIS were found to be almost independent of initial inulin concentration.Thin Layer Chromatography (TLC) analysis of hydrolyzates revealed that the hydrolysis of inulinby Fructozyme yielded mainly fructose. Moreover, as the hydrolysis of inulin proceeded withtime, the amount of produced fructose increased. Given that fructose was the main product ofinulin degradation by Fructozyme, it can be concluded that exo-activity predominates in thiscomplex enzymatic preparation. However, considering that by using High Performance LiquidChromatography (HPLC) a few amounts of sucrose and/or of lower oligomers from inulin couldbe detected also in the hydrolyzates, the co-presence of low levels of endo-acting inulinase inFructozyme can not be excluded.Considering that the calorie of inulin is much lower than that of regular carbohydrates whenmetabolized in human, inulin can be used not only as a functional food or as a dietary fiber, it canalso be used as a fat substitute in various food systems. In this respect, the effects of inulinaddition to skim milk on the quality characteristic (rheology, texture, microstructure, nutritivevalue, and sensory) of Labneh (concentrated yogurt) have been investigated.Hardness and stickiness of full-fat Labneh (Reference Labneh) were observed to be considerablylower than the skim milk-Labneh (Control Labneh). The addition of up to 5% inulin to skim milkreduced the overall hardness and stickiness of Labneh, whilst the addition of inulin above 5%caused a regain in the overall hardness and stickiness of the product. The incorporation of inulinat 3% into skim milk helped to improve the rheological properties of Labneh. Furthermore, thefortification of skim milk with inulin at 5% provided a low-fat Labneh with microstructure andtextural characteristics resembling those of full-fat Labneh (Reference Labneh).Addition of inulin to the skim milk improved the sensory characteristics of Labneh. In fact,considering those attributes influencing consumer acceptability of Labneh, 5% inulin additionseems to produce an acceptable product. However, given that no significant difference was foundbetween Reference Labneh and Inulin-Labneh at concentrations above 3%, fortification of skimmilk with 3% inulin seems to be more economic for the industrial production of low-fat Labneh.Supplementation of skim milk with inulin allowed producing a low-fat and high-protein Labneh.Moreover, the composition in amino acids and minerals of Inulin-Labneh was also improved,comparatively with both Reference Labneh and Control Labneh samples.Furthermore, nutritional benefits associated with soluble dietary fiber fortification bring attractivenovel products, such as low-and/or reduced-fat Labneh, to fulfill market niches.