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The objective of present research was to develop an easy, precise and accurate HPTLC densitometry method for quantification of fructooligosaccharides (FOSs) from inulin hydrolysate. The chromatographic separation of FOSs was performed on pre-coated silica gel (60, F 254 ) TLC plates using a mobile phase (butanol:ethanol:water, 60:24:16), and densitometry evaluation of FOSs was performed at A 500 . Both kestose and nystose were successfully resolved with R f value of 0.43 and 0.34, respectively. The accuracy, reliability and reproducibility of developed method was assessed by percent relative standard deviation of kestose and nystose for instrument precision (1.43% and 1.50%), repeatability (1.48% and 1.56%), intra-day precision (1.60% and 1.63%), inter-day precision (1.62% and 1.66%), limit of detection (4.58 ng/spot and 4.58 ng/spot), limit of quantification (13.87 ng/spot and 13.89 ng/spot) and recovery (98.81% and 98.69%). Moreover, overlapping spectra of test sample with standard confirms the specificity of developed method, which was validated as per ICH guidelines., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Statistical optimization of hydrolytic conditions for the production of fructooligosaccharides (FOSs) from pure inulin using Aspergillus tritici endoinulinase was carried out in a batch system. FOSs yield 99.19% was obtained under the optimized hydrolytic conditions i.e. inulin concentration (7.3%), enzyme load (65 IU), hydrolysis time (13 h) and agitation (100 rpm). The closeness of value of co-efficient of determination (R 2 ) to 1, good agreement between model's predicted and experimental values, low percentage error (<5%), high adequate precision (>4%) and F value (11,634.32), and low Lack of fit (0.60) of the designed model authenticates its fitness. High substrate concentration, low enzyme load and short hydrolysis span justifies efficiency of developed process for the preparation of FOSs from inulin using fungal endoinulinase. TLC chromatographic and densitometry studies confirmed the synthesis of short-chain length FOSs. FOSs preparation contained 33.85% GF2 (ketose), 24.50% GF3 (nystose), 7.26% GF4 (fructofuranosylnystose) and 33.58% FOSs of DP5-9., Competing Interests: Declaration of competing interest No conflict of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Three-step purification technique (isopropanol precipitation, ion-exchange and size-exclusion chromatography) was used for the purification of an endoinulinase from the culture broth of Aspergillus tritici BGPUP6. The molecular mass of purified endoinulinase was found to be 53.45 kDa and 53.70 kDa by denatured protein gel (SDS-PAGE) and size-exclusion (Sephadex G-100) chromatographic analysis, respectively. Higher K m (0.90 mM), V max (19.60 mM/min·mg), K cat (1.3 × 10 -3 /min) and V max /K m ratio (21.77/min·mg) of purified endoinulinase for inulin than stachyose depicts its higher affinity towards inulin. Purified enzyme was found stable in a pH range 4.0-7.0 with an optimal pH 5.5. The optimal temperature of purified biocatalyst was 55 °C with thermostability in the range of 50-70 °C. D-value and Z-value for endoinulinase at 55 °C was found to be 100.08 h and 11.62 °C, respectively. Thermodynamics inactivation parameters (ΔG, ΔH and ΔS) of endoinulinase shows its wide range thermal stability. Endoinulinase activity was enhanced by CaCl 2 and MnSO 4 , while CuSO 4 , CoCl 2 , AgNO 3 , CdCl 2 , NiCl 2 , ZnSO 4 , BaCl 2 , HgCl 2 and EDTA inhibited the activity of enzyme. Purified endoinulinase was successfully used for the production of fructooligosaccharides from inulin., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2018. Published by Elsevier B.V.)

The main objective of the present work was to modify multiwalled carbon nanotubes (MWCNTs) using 3-aminopropyl-triethoxysilane (APTES) to generate amino-terminated surfaces for inulinase immobilization, which can be further used for fructose production. CCRD of response surface methodology was used for optimization of inulinase immobilization on MWCNTs. At optimized parameters (APTES concentration 4%; sonication time 4 h; enzyme coupling time 1.5 h and enzyme load 15 IU), maximal inulinase activity and immobilization yield was 60.7% and 74.4%, respectively. Immobilized inulinase showed same pH optima of free enzyme, while an elevation in temperature optima to 60 °C was observed after its immobilization. Immobilized inulinase also shown enhancement in pH stability and thermostability. Overall, 4.54-fold rise in half-life of inulinase was detected after immobilization at 60 °C. K m and V max of inulinase decreased after immobilization. Immobilized inulinase preserved 28% of its residual activity after 10 consecutive batch cycles of inulin hydrolysis for the production of fructose., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Two exoinulinases, Exo-I and Exo-II from the culture broth of Penicillium oxalicum BGPUP-4 was purified using three-step purification method i.e., isopropanol precipitation, Q-Sepharose and Sephadex G-100 column chromatography. The molecular weight of Exo-I and Exo-II was determined to be 64.85 kDa and 32.54 kDa, respectively using MALDI-TOF. Exo-I and Exo-II showed high specificity for inulin and their respective V max /K m ratio was 3.74 and 7.20. Besides, both the inulinases also displayed specificity for lactose, sucrose and raffinose. Exo-I and Exo-II were stable at a pH range of 4.0-8.0 with pH optima 5.0. Optimal temperature for both the inulinases was 55 °C, and both the isoforms retained approximately 50% of their activity up to 70 °C. Ag + , Hg 2+ , Ba 2+ , Cu 2+ and Ca 2+ ions shown stimulatory effect on inulinases activity, while Fe 2+ , Mn 2+ , Co 2+ and EDTA completely inhibited enzyme activity. Purified enzyme was successfully used for the preparation of high fructose syrup from inulin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An extracellular inulinase was partially purified by ethanol precipitation and gel exclusion chromatography from a cell free extract of Kluyveromyces marxianus. Partially purified inulinase exhibited 420 IU/mg specific activity and it was immobilized on chitosan beads. Activity yield of immobilized inulinase was optimized with glutaraldehyde concentration (1-5%), glutaraldehyde treatment time (30-240min), enzyme coupling-time (2-16h) and enzyme loading (5-30 IU) as functions. Under the optimized conditions maximum yield 65.5% of immobilized inulinase was obtained. Maximum hydrolysis of inulin 84.5% and 78.2% was observed at 125rpm after 4h by immobilized and free enzyme, respectively. A retention-time of 4h and 5h was found optimal for the hydrolysis of inulin under agitation (125rpm) by free and immobilized enzyme, respectively. The recycling of the developed immobilized biocatalyst was carried out after 5h of inulin hydrolysis in a batch system. The developed immobilized biocatalyst was successfully used for the hydrolysis of inulin for 14 batches. This is the first report on the immobilization of yeast inulinase on chitosan beads for the hydrolysis of inulin in a batch system., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Endoinulinase is an inulolytic enzyme which is used for the production of fructooligosaccharides from inulin. A new endoinulinase producing fungal strain BGPUP6 was identified as Aspergillus tritici on the basis of its molecular characterization. Response surface methodology was used to optimize the endoinulinase production at shake-flask level from Aspergillus tritici BGPUP6 using raw Asparagus inulin as carbon source. Four independent variables (raw inulin, 2-4%; peptone, 0.3-0.7%; (NH 4 )H 2 PO 4 , 0.3-0.7% and pH, 4.5-6.5) were selected for the study based on the CCRD model of RSM. The other media supplements (FeSO 4 ·7H 2 O, 0.001%; MgSO 4 ·7H 2 O, 0.05% and KCl, 0.02%) were kept constant in the production medium, throughout the study. Endoinulinase production and biomass yield were selected as dependent responses. The optimal combination of media ingredients obtained from the study was 3% raw inulin, 0.5% peptone, 0.5% (NH 4 )H 2 PO 4 and pH 5.5. Using the optimized media constituents, maximum endoinulinase production (25.01 IU/mL) and biomass yield (0.514g dry weight/50 mL) obtained were in good agreement with the predicted values. Crude enzyme produced was also used for the hydrolysis of inulin. The hydrolysate showed the presence of a mixture of fructooligosaccharides with varied degree of polymerization. This is the first report on the production of an endoinulinase from Aspergillus tritici., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the past few years, people are paying more attention to their dietary habits, and functional foods are playing a key role in maintaining the health of man. Prebiotics are considered as a main component of the functional foods which are usually composed of short chains of carbohydrates. Fructooligosaccharides (FOSs) are considered as one of the main group of prebiotics which have recognisable bifidogenic properties. FOSs are obtained either by extraction from various plant materials or by enzymatic synthesis from different substrates. Enzymatically, these can be obtained either from sucrose using fructosyltransferase or from inulin by endoinulinase. Inulin is a potent substrate for the enzymatic production of FOSs. This review article will provide an overview on the inulin as potent substrate, microbial sources of endoinulinases, enzymatic synthesis of FOSs from inulin, commercial status of FOSs, and their future perspectives., (Copyright © 2016 Elsevier B.V. All rights reserved.)