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Fructose is a carbohydrate with essential applications in the food industry, mainly due to its high sweetness and low cost. The present investigation focused on optimising fructose production from commercial inulin using the enzymatic immobilisation method and applying the response surface methodology in a 12-run central composite design. The independent variables evaluated were the pH (−) and temperature (°C). The substrate consisted of a commercial inulin solution at a concentration of 1 g/L, while the catalyst consisted of the enzyme inulinase from Aspergillus niger (EC 232-802-3), immobilised in 2% m/v sodium alginate. A stirred vessel reactor was used for 90 min at 120 rpm, and quantification of reducing sugars was determined using DNS colorimetric and UV–Vis spectrophotometric methods at a 540 nm wavelength. After applying the response surface methodology, it was determined that the catalytic activity using the immobilisation method allows for a maximum total productivity of 16.4 mg/h under pH and temperature of 3.9 and 37 °C, respectively, with an efficiency of 96.4%. The immobilised enzymes' reusability and stability compared to free enzymes were evaluated, obtaining activity up to the fifth reuse cycle and showing significant advantages over the free catalyst. [ABSTRACT FROM AUTHOR]

The present research was carried out with the objectives of isolating fungal strains capable of exhibiting inulinase activity from a novel source, then optimizing solid state fermentation for the inulinase production and characterizing crude enzyme. Production of inulinase is first reported in this study with a novel substrate, banana plant shoot powder, using newly isolated fungal strain Nothophoma anigozanthi JAM through solid state fermentation. The fungal strain was isolated from the root soil of a white Jamun (Syzygium cumini) tree. Optimization of the solid state fermentation was carried out by central composite design for determining the interaction and impact of the influential process on inulinase production. A mathematical model was developed based on the statistical analysis and recommended optimal process conditions of 2 g of substrate, 5 mL of moistening media, 5 mL of inoculum volume, and 96 h of fermentation time for enhanced production (2.578 U/gds). Crude inulinase enzyme exhibited an 8.0-fold increase in activity after optimization, in comparison with the activity under unoptimized conditions. The optimum pH (5.0), temperature (60 °C) for the enzyme activity, and Km (0.205 mM) and Vmax (0.333 µM/min) were determined based on the biochemical and inulin hydrolytic properties of the crude inulinase enzyme. The production of inulinase was further validated by using TLC, osazone, and Seliwanoff tests. Hence, the inulinase enzyme produced by Nothophoma anigozanthi JAM can be successfully utilized for the production of edible sugar fructose from non-edible polysaccharide inulin, present in agricultural residues such as banana shoot by solid state fermentation. [ABSTRACT FROM AUTHOR]

The food enzyme inulinase (1‐β‐d‐fructan fructanohydrolase; EC 3.2.1.7) is produced with the non‐genetically modified Aspergillus welwitschiae strain NZYM‐KF by Novozymes A/S. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of fructo‐polysaccharides for the production of fructo‐oligosaccharides. Since residual amounts of total organic solids (TOS) are removed during the food manufacturing process, toxicological studies other than allergenicity were considered unnecessary and dietary exposure was not calculated. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with tomato allergens were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to tomato, cannot be excluded, but is expected not to exceed that of tomato. As the prevalence of allergic reactions to tomato is low, also the likelihood of such reactions to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Background: Luffa cylindrica has numerous domestic and industrial uses. For example, this natural fiber supports immobilization through the covalent bonding of an inulinase. In addition, fructose syrup from crude inulin obtained from prickly pear (Opuntia ficus-indica) can be obtained in a plug-flow mini reactor with this immobilized inulinase on L. cylindrica. Results: A central composite design of experiments was used to maximize the enzymatic fructose production from crude inulin obtained from Opuntia ficus-indica in a plug-flow mini reactor. The experiments explore temperature (between 45 and 55°C), pH (4.0–5.0), and feed flow (0.1–0.2 ml/min). After verifying the adequacy of the quadratic model for productivity, it was maximized to find the optimal condition. It was at 49.97°C, 4.6 and 0.20 ml/min for the temperature, pH, and flow, respectively. Under the optimal condition, the quadratic model suggested a productivity of 2.456 ± 0.015 mg/h. Three validation experiments confirmed the validity of the model. Conclusions: The results confirmed the suitability of L. cylindrica as support for the immobilization of inulinase.How to cite: Lara-Fiallos MV, Montalvo-Villacreses DT, Espín-Valladares RC, et al. Optimization of fructose-rich syrups production from Opuntia ficus-indica inulin using immobilized inulinase on Luffa cylindrical. Electron J Biotechnol 2023;66. https://doi.org/10.1016/j.ejbt.2023.03.007.

Background: Luffa cylindrica has numerous domestic and industrial uses. For example, this natural fiber supports immobilization through the covalent bonding of an inulinase. In addition, fructose syrup from crude inulin obtained from prickly pear (Opuntia ficus-indica) can be obtained in a plug-flow mini reactor with this immobilized inulinase on L. cylindrica. Results: A central composite design of experiments was used to maximize the enzymatic fructose production from crude inulin obtained from Opuntia ficus-indica in a plug-flow mini reactor. The experiments explore temperature (between 45 and 55°C), pH (4.0-5.0), and feed flow (0.1-0.2 ml/min). After verifying the adequacy of the quadratic model for productivity, it was maximized to find the optimal condition. It was at 49.97°C, 4.6 and 0.20 ml/min for the temperature, pH, and flow, respectively. Under the optimal condition, the quadratic model suggested a productivity of 2.456 ± 0.015 mg/h. Three validation experiments confirmed the validity of the model. Conclusions: The results confirmed the suitability of L. cylindrica as support for the immobilization of inulinase. [ABSTRACT FROM AUTHOR]

Inulinases are enzymes of great interest in the food industry, especially due to their application in the synthesis of fructose and fructo-oligosaccharides. Moreover, some inulinases (I) also present invertase activity (S), making them useful for sucrose hydrolysis processes. In the present study, the production of inulinase by Aspergillus niger URM5741 was evaluated and optimized using two statistical approaches. First, the composition of the cultivation medium was determined through a simplex centroid mixture design, followed by the selection of optimal fermentation conditions using the Box–Behnken design. Based on these experimental designs, the maximum activities of inulinase (16.68 U mL−1) and invertase (27.80 U mL−1) were achieved using a mixture of wheat, soy, and oat brans (5 g), along with 2.5% inulin and 40% moisture. The inulinase exhibited optimum temperature and pH of 60 °C and 4.0, respectively, displayed a high affinity for both substrates, as evidenced by very-low Michaelis constant values (1.07–1.54 mM). A relative thermostability was observed at 55–60 °C as indicated by half-lives values (I: 169.06–137.27 min; S: 173.29–141.52 min) and D-values (I: 561.61–456.00 min; S: 575.65–470.11 min) which were further confirmed by the high activation energy (123.01 and 143.29 kJ mol−1). The enzyme demonstrated favorable results in terms of inulin and sucrose hydrolysis, being a maximum release of reducing sugars of 6.04 and 15.80 g L−1, respectively. These results indicate that the sequential statistical approach proved to be beneficial to produce inulinase by A. niger URM5741, with the obtained enzyme considered promising for long-term industrial applications. [ABSTRACT FROM AUTHOR]

The aim of this study was to analyze the features of the spatial organization of the endoinulinase molecule from Aspergillus ficuum after its binding to mono-, di-, and polysaccharides. This study examined changes in volume and number of internal cavities upon binding of inulinase to mono- (glucose, fructose), di- (sucrose, mannose), and polysaccharides (inulin). Transformations in the quantity and length of tunnels and pores were described, and the reorganization of the composition and localization of charged and hydrophobic amino acid residues clusters on the surface of the enzyme molecule was analyzed. It was shown that the models of inulinase in the complex with sucrose (an alternative substrate) and mannose (an activator) exhibit the same types of internal structures. A similar pattern was found in the formation of complexes with fructose (a reaction product) and glucose (an inhibitor). In addition, it was established that both charged and hydrophobic clusters do not undergo significant changes in chemical composition after the binding of inulinase to mono-, di-, and polysaccharides, i.e., the interaction between inulinase and carbohydrates mentioned above primarily affects the internal structures of the enzyme. The specificity of the binding of inulinases to various ligands should be taken into account while developing modern industrial biocatalysts based on inulinase. [ABSTRACT FROM AUTHOR]

Fructose is a carbohydrate with essential applications in the food industry, mainly due to its high sweetness and low cost. The present investigation focused on optimising fructose production from commercial inulin using the enzymatic immobilisation method and applying the response surface methodology in a 12-run central composite design. The independent variables evaluated were the pH (−) and temperature (°C). The substrate consisted of a commercial inulin solution at a concentration of 1 g/L, while the catalyst consisted of the enzyme inulinase from Aspergillus niger (EC 232-802-3), immobilised in 2% m/v sodium alginate. A stirred vessel reactor was used for 90 min at 120 rpm, and quantification of reducing sugars was determined using DNS colorimetric and UV–Vis spectrophotometric methods at a 540 nm wavelength. After applying the response surface methodology, it was determined that the catalytic activity using the immobilisation method allows for a maximum total productivity of 16.4 mg/h under pH and temperature of 3.9 and 37 °C, respectively, with an efficiency of 96.4%. The immobilised enzymes’ reusability and stability compared to free enzymes were evaluated, obtaining activity up to the fifth reuse cycle and showing significant advantages over the free catalyst.

The glycoside hydrolase family contains enzymes that break the glycosidic bonds of carbohydrates by hydrolysis. Inulinase is one of the most important industrial enzymes in the family of Glycoside Hydrolases 32 (GH32). In this study, to identify and classify bacterial inulinases initially, 16,002 protein sequences belonging to the GH32 family were obtained using various databases. The inulin‐effective enzymes (endoinulinase and exoinulinase) were identified. Eight endoinulinases (EC 3.2.1.7) and 4318 exoinulinases (EC 3.2.1.80) were found. Then, the localization of endoinulinase and exoinulinase enzymes in the cell was predicted. Among them, two extracellular endoinulinases and 1232 extracellular exoinulinases were found. The biochemical properties of 363 enzymes of the genus Arthrobacter, Bacillus, and Streptomyces (most abundant) showed that exoinulinases have an acid isoelectric point up to the neutral range due to their amino acid length. That is, the smaller the protein (336 aa), the more acidic the pI (4.39), and the larger the protein (1207 aa), the pI is in the neutral range (8.84). Also, a negative gravitational index indicates the hydrophilicity of exoinulinases. Finally, considering the biochemical properties affecting protein stability and post‐translational changes studies, one enzyme for endoinulinase and 40 enzymes with desirable characteristics were selected to identify their enzyme production sources. To screen and isolate enzyme‐containing strains, now with the expansion of databases and the development of bioinformatics tools, it is possible to classify, review and analyze a lot of data related to different enzyme‐producing strains. Although, in laboratory studies, a maximum of 20 to 30 strains can be examined. Therefore, when more strains are examined, finally, strains with more stable and efficient enzymes were selected and introduced for laboratory activities. The findings of this study can help researchers to select the appropriate gene source from introduced strains for cloning and expression heterologous inulinase, or to extract native inulinase from introduced strains. [ABSTRACT FROM AUTHOR]

The food enzyme inulinase (1‐β‐d‐fructan fructanohydrolase; EC 3.2.1.7) is produced with the genetically modified Aspergillus oryzae strain MUCL 44346 by PURATOS NV. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the production of fructo‐oligosaccharides (FOS) from inulin extracted from chicory roots. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.01 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 100 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 10,000. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found with tomato allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to tomato, cannot be excluded. However, the likelihood of allergic reactions is expected not to exceed the likelihood of allergic reactions to tomato. As the prevalence of allergic reactions to tomato is low, also the likelihood of such reactions to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

This study examined the effect of lactulose, galactooligosaccharide, fructooligosaccharide, inulin, and β-glucan on the probiotic strain Lactobacillus delbrueckii subsp. bulgaricus L14, cultivated in an in vitro gastrointestinal system model. We analyzed the degree of hydrolysis of the studied prebiotic oligosaccharides in condition of simulated gastric fluid. The results showed that lactulose had the highest resistance, galactooligosaccharide underwent hydrolysis, and fructooligosaccharide was the most sensitive. Among the polysaccharides, fructose was released from inulin and glucose from β-glucan. Short-chain oligosaccharides and metabolites derived from studied prebiotic oligosaccharides and polysaccharides, supported the growth of probiotic strain L14, which showed the highest growth with fructooligosaccharides and β-glucan as carbohydrate sources. The profile of the activated enzymes secreted by the probiotic strain L14, indicated their inducible character. Beta-galactosidase was activated in the presence of lactulose and GalOS, inulinase was activated in the presence of inulin and fructooligosaccharides, and β-glucosidase was activated in the presence of β-glucan fragments. Analysis of the produced organic and short-chain fatty acids showed that the typical representative of the homofermentative lactobacilli Lb. delbrueckii subsp. bulgaricus L14 changes its metabolism from a homofermentative to a heterofermentative type, best expressed in the presence of lactulose, galactooligosaccharide, and β-glucan.

The objective of this study was to create a fermentation process for the production of inulinase, an important enzyme with numerous applications in the food and pharmaceutical industries, using low-cost agricultural waste as substrates for Rhizopus oryzae NRRL 3563. High titer inulinase production in chicory roots by Rhizopus oryzae in a submerged culture was accomplished using a statistical experimental design. A two-level Plackett–Burman design followed by a three-level Box–Behnken design producing a high inulinase titer of 1085.11 U/mL, 2.83-fold the maximum level, was obtained in the screening experiment. The optimal levels were as follows: chicory root, 10 g/L; NaNO3, 5 g/L; and KCl, 0.2 g/L. The produced inulinase enzyme was purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.63-fold purification with 60% activity recovery. The enzyme had a molecular weight of approximately 130 KDa. The purified enzyme showed optimum activity at 50 °C and pH 6.0. The pH stability range was three to six and the temperature stability was up 70 °C. The purified inulinase could hydrolyze inulin and sucrose, but not cellobiose or soluble starch. Km and Vmax for inulin were determined to be 0.8 mg/mL and 50,000 U/mg, respectively. The two-level Plackett–Burman design was applied followed by a Box–Behnken model for optimization of fermentation conditions. Accordingly, the optimal combination of fermentation was a reaction time of seven hours, a temperature of 60 °C, and an enzyme concentration of 40,000 U/mL, which resulted in a 58.07% saccharification yield. The characteristics of the enzyme and its kinetic parameters suggested that it was highly effective in the fermentation of inulin and inulin-containing substrates. Additionally, it raises the potential of using inulinase enzymes in pharmaceutical and food industries.

This study aimed to establish a new method for simultaneous determination of sucrase and inulinase activity using ion chromatography. Sucrose was used as the substrate and enzymatic hydrolysised by sucrase. Sodium borohydride solution was applied for converting glucose and fructose to mannitol and sorbitol. Afterwards, sucrase was inactivated at high temperature, and inulinase was added to get the mixed solution containing mannitol, sorbitol, glucose and fructose. To determine the concentration of four compounds in the mixed solution, chromatographic separation was carried out using CarboPaCTM PA1 250 mm×2 mm column with water, sodium hydroxide and sodium acetate as gradient eluents (the flow rate of 1.0 mL/min and the column temperature of 30 ℃). Finally, sucrase activity was calculated based on the total amount of mannitol and sorbitol, and inulinase activity was calculated according to the total amount of glucose and fructose. The results showed that mannitol, sorbitol, glucose and fructose could be determined within 20 min. All the linear correlation coefficients were all above 0.9995, and resolutions were above 1.0. Thus, sucrose could be used as substrate for the determination of inulinase activity. Glucose and fructose could be completely converted into mannitol and sorbitol after adding 1.2 mL of 10 mg/mL sodium borohydride solution in a water bath at 60 °C for 30 min. The activities of sucrase and inulinase determined by this method did not show significantly differences compared to 3,5-dinitrosalicylic acid colorimetry determination method (DNS method) (P>0.05). The relative standard deviation of sucrase activity and inulinase activity determined were 2.0% and 1.7% respectively. The method did not involve toxic and harmful reagents used in traditional methods. Therefore, this method could be used to determine the activities of sucrase and inulinase simultaneously, and would provide a new idea for the determination of other enzyme activities.

Inulin is a renewable and cheap carbon source used in microbial fermentations. Bacillus licheniformis 24 is known as an excellent 2,3-butanediol (2,3-BD) producer from fructose; therefore, the cloning and expression of a robust heterologous inulinase could enhance its 2,3-BD production from inulin. The inu gene of Lacticaseibacillus paracasei DSM 23505 encoding fructan-β-fructosidase (EC 3.2.1.80) was chosen for the purpose. PCR fragments containing the complete inu (3.6 kb) and its truncated variant inu-tr (2.2 kb, lacking Big3 cell wall attachment domains) were cloned into Escherichia coli StellarTM and B. licheniformis 24. The high quality of the recombinant constructs was confirmed by restriction analysis, PCR, sequencing, and phenotypic tests. The results showed that the inulinase activity of B. licheniformis cells harboring the full-length inu variant (T26) was eightfold higher compared to the wild type, retaining cell wall attachment in the B. licheniformis host. In contrast, the truncated variant inu-tr (T14) showed mostly extracellular but weak activity, thus suggesting that the Big3 domains are also important for the enzyme's function. During flask-batch fermentation of 100 g/L raw chicory flour (containing 90% inulin), T26 produced acetoin and 2,3-BD from inulin. Contrariwise, T14 and the wild type formed products only from the mono- and disaccharides naturally found in the chicory flour. In the fermenter, from 200 g/L of raw chicory flour, the recombinant T26 degraded approximately 140 g/L of the inulin. However, the final concentrations of the produced 2,3-BD and acetoin were 18.5 g/L and 8.2 g/L, respectively, because of the accumulation of unconverted sucrose. To conclude, further strain improvement is necessary to make the process efficient for obtaining 2,3-BD from inulin by simultaneous saccharification and fermentation (SSF). [ABSTRACT FROM AUTHOR]

In this study, the effect of pH control and aeration on the production of Aspergillus niger inulinase from sugarbeet molasses in the bioreactor was investigated. The inulinase fermentations were performed at 30 °C temperature in 4°Bx sugarbeet molasses with a 2% (v/v) inoculation rate. The fermentation medium was only supplemented with 4.2% (w/v) yeast extract. In fermentations with aeration (1 vvm), the agitation speed was gradually increased from 200 to 600 rpm. Based on the results, it was determined that the pH-(un)controlled and non-aeration inulinase fermentations caused inadequate fungal growth and thus low inulinase and invertase-type activities, specific inulinase and invertase-type activities, and low maximum production and consumption rates. On the other hand, when the fermentation broth was aerated with 1 vvm, the inulinase and invertase-type activities, specific inulinase and invertase-type activities, and maximum production and consumption rates increased significantly under pH-controlled (5.0) and pH-uncontrolled conditions. In the fermentation with aeration, the highest inulinase and invertase-type activities were yielded as 1825.38 and 1537.88 U/mL under pH-controlled and pH-uncontrolled conditions, respectively. Also, maximum specific inulinase and invertase-type activities and maximum specific inulinase production rate were found as 5361.45 and 4371.58 U/mg and 316.71 U/mL/day with pH-uncontrolled and aerated fermentation. It was found that in both non-aeration and aeration fermentation broths, the pH control had no significant effect on enzyme production (p > 0.05). The inulinase activity/invertase-type activity ratios also varied between 0.95 and 1.56, showing that the enzyme produced is inulinase since the ratios > 10−2. Besides, with aerated fermentations, excessive and irregular fungal growth was also observed. In conclusion, aeration played a crucial role in increasing the production of inulinase and fungal growth (p < 0.05). Therefore, the best fermentation was performed under pH-uncontrolled and aeration (1 vvm) conditions (200–600 rpm agitation speed, 10 days fermentation duration, 2% (v/v) inoculum level, 30 °C temperature, 4°Bx sugarbeet molasses, and 4.2% (w/v) yeast extract). At these conditions, inulinase and invertase-type activities were 1810.76 and 1537.88 U/mL, respectively. [ABSTRACT FROM AUTHOR]

Due to the limited natural resources, the tendency toward using agro-industrial wastes as the main substrate in the biotechnological productions has increased. In order to obtain biologically available compounds out of these wastes, various extraction processes need to be applied. However, during these processes, some other inhibitory compounds such as furfural and hydroxymethylfurfural (HMF) also occur. Therefore, evaluation of these inhibitor effects on the biological systems before using these wastes as substrate is crucial for the overall success of the process. This study was undertaken to investigate the effect of various concentrations of furfural and HMF on the Aspergillus niger A42 (ATCC 204,447) inulinase activity, invertase activity, biomass production, and sugar consumption in sucrose-based medium and to model the production outcomes. The results showed that furfural had higher inhibitory effect on the process than HMF at the same concentrations. However, fungi were able to tolerate up to 3 g/l of HMF or 0.5 g/l of furfural in the production medium with minor changes in the yield. The highest inulinase activities were measured as 268.737 and 197.350 U/ml in the presence of 1 g/l of HMF and 0.1 g/l of furfural, respectively. In these conditions, inulinase productions were successfully represented by Baranyi and Weibull models. This study clearly showed that inulinase production with Aspergillus niger A42 can be successfully performed in the presence of a certain amount of furfural and HMF, which indicates the strength of potential usage of lignocellulosic materials as production substrate. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme inulinase (1‐β‐d‐fructan fructanohydrolase; EC 3.2.1.7) is produced with the genetically modified Aspergillus oryzae strain MUCL 44346 by PURATOS NV. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the production of fructo‐oligosaccharides (FOS) from inulin extracted from chicory roots. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.01 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 100 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 10,000. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found with tomato allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to tomato, cannot be excluded. However, the likelihood of allergic reactions is expected not to exceed the likelihood of allergic reactions to tomato. As the prevalence of allergic reactions to tomato is low, also the likelihood of such reactions to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

The objective of this study was to create a fermentation process for the production of inulinase, an important enzyme with numerous applications in the food and pharmaceutical industries, using low-cost agricultural waste as substrates for Rhizopus oryzae NRRL 3563. High titer inulinase production in chicory roots by Rhizopus oryzae in a submerged culture was accomplished using a statistical experimental design. A two-level Plackett–Burman design followed by a three-level Box–Behnken design producing a high inulinase titer of 1085.11 U/mL, 2.83-fold the maximum level, was obtained in the screening experiment. The optimal levels were as follows: chicory root, 10 g/L; NaNO3, 5 g/L; and KCl, 0.2 g/L. The produced inulinase enzyme was purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.63-fold purification with 60% activity recovery. The enzyme had a molecular weight of approximately 130 KDa. The purified enzyme showed optimum activity at 50 °C and pH 6.0. The pH stability range was three to six and the temperature stability was up 70 °C. The purified inulinase could hydrolyze inulin and sucrose, but not cellobiose or soluble starch. Km and Vmax for inulin were determined to be 0.8 mg/mL and 50,000 U/mg, respectively. The two-level Plackett–Burman design was applied followed by a Box–Behnken model for optimization of fermentation conditions. Accordingly, the optimal combination of fermentation was a reaction time of seven hours, a temperature of 60 °C, and an enzyme concentration of 40,000 U/mL, which resulted in a 58.07% saccharification yield. The characteristics of the enzyme and its kinetic parameters suggested that it was highly effective in the fermentation of inulin and inulin-containing substrates. Additionally, it raises the potential of using inulinase enzymes in pharmaceutical and food industries. [ABSTRACT FROM AUTHOR]

This study examined the effect of lactulose, galactooligosaccharide, fructooligosaccharide, inulin, and β-glucan on the probiotic strain Lactobacillus delbrueckii subsp. bulgaricus L14, cultivated in an in vitro gastrointestinal system model. We analyzed the degree of hydrolysis of the studied prebiotic oligosaccharides in condition of simulated gastric fluid. The results showed that lactulose had the highest resistance, galactooligosaccharide underwent hydrolysis, and fructooligosaccharide was the most sensitive. Among the polysaccharides, fructose was released from inulin and glucose from β-glucan. Short-chain oligosaccharides and metabolites derived from studied prebiotic oligosaccharides and polysaccharides, supported the growth of probiotic strain L14, which showed the highest growth with fructooligosaccharides and β-glucan as carbohydrate sources. The profile of the activated enzymes secreted by the probiotic strain L14, indicated their inducible character. Beta-galactosidase was activated in the presence of lactulose and GalOS, inulinase was activated in the presence of inulin and fructooligosaccharides, and β-glucosidase was activated in the presence of β-glucan fragments. Analysis of the produced organic and short-chain fatty acids showed that the typical representative of the homofermentative lactobacilli Lb. delbrueckii subsp. bulgaricus L14 changes its metabolism from a homofermentative to a heterofermentative type, best expressed in the presence of lactulose, galactooligosaccharide, and β-glucan. [ABSTRACT FROM AUTHOR]

Research background. Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production. Experimental approach. Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production. Results and conclusions. The most common species were Aspergillus tubingensis (51.2%), followed by A. niger (23.1%), A. welwitschiae (23.1%) and A. uvarum (2.6%). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production. Novelty and scientific contribution. The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally.

The enriched nutritional and functional properties of inulinase with wide attention are considered commercial/industrial food enzymes. It can be produced by many microorganisms such as yeasts, fungi, and bacteria. Nocardiopsis is a genus under Actinomycetes, which has biotechnologically important microorganisms. This study aims to isolate and identify marine Actinomycetes Nocardiopsis species and to evaluate the antibacterial potential of the inulinase enzyme obtained from it. Marine actinobacteria (Nocardiopsis sp.) were isolated from sediment samples on YM agar. The isolate was identified by biochemical analysis of cell walls (amino acid and sugar). Enzyme screening assay was performed with temperature and pH influence in the production inulinase enzyme production. Antibacterial activity and minimal inhibitory activity of inulinase enzyme were performed with Staphylococcus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Antimicrobial testing revealed that with higher concentrations of inulinase enzyme, the zone of inhibition of bacterial growth increased, and the minimum inhibitory concentration of inulinase enzyme that prevented the growth of bacteria was close to the standard tetracycline. Inulinase enzyme obtained from Nocardiopsis species shows good antibacterial activity against Staphylococcus aureus, K. pneumoniae, and P. aeruginosa in comparison to the standard, tetracycline.

The present work aimed to model Aspergillus niger inulinase fermentation performed in the medium using sigmoidal functions, validate the selected models using an independent set of the experimental values, and perform a sensitivity analysis of the selected models. Based on the results, the selected models were Stannard and Fitzhugh models for substrate consumption (R2 = 0.9976 and 0.9974, respectively), Huang model for inulinase production (R2 = 0.9967), Weibull model for invertase‐type production (R2 = 0.9963), and modified logistic model for invertase‐type activity/inulinase activity ratio (R2 = 0.9292) with high R2 values (>0.90). Kinetics predicted by particularly selected models mentioned above fit well with the experimental kinetic results. Besides, validation of the selected models with an independent set of the experimental data indicated that they gave satisfying results with high R2 values for consumption and production (R2 > 0.90). Sensitivity analysis of the selected models showed that the yielded R2 values (R2 ≥ 0.9775) were in good agreement with those obtained from the selected models. Consequently, A. niger inulinase fermentation was successfully modeled and the selected models were successfully validated with an independent set of the observed data. Besides, the sensitivity analysis also verified the reliability of the selected models. Those models can serve as universal equations to describe the A. niger inulinase fermentation. [ABSTRACT FROM AUTHOR]

The enriched nutritional and functional properties of inulinase with wide attention are considered commercial/industrial food enzymes. It can be produced by many microorganisms such as yeasts, fungi, and bacteria. Nocardiopsis is a genus under Actinomycetes, which has biotechnologically important microorganisms. This study aims to isolate and identify marine Actinomycetes Nocardiopsis species and to evaluate the antibacterial potential of the inulinase enzyme obtained from it. Marine actinobacteria (Nocardiopsis sp.) were isolated from sediment samples on YM agar. The isolate was identified by biochemical analysis of cell walls (amino acid and sugar). Enzyme screening assay was performed with temperature and pH influence in the production inulinase enzyme production. Antibacterial activity and minimal inhibitory activity of inulinase enzyme were performed with Staphylococcus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Antimicrobial testing revealed that with higher concentrations of inulinase enzyme, the zone of inhibition of bacterial growth increased, and the minimum inhibitory concentration of inulinase enzyme that prevented the growth of bacteria was close to the standard tetracycline. Inulinase enzyme obtained from Nocardiopsis species shows good antibacterial activity against Staphylococcus aureus, K. pneumoniae, and P. aeruginosa in comparison to the standard, tetracycline. [ABSTRACT FROM AUTHOR]

Inulinases are enzymes involved in the hydrolysis of inulin, which can be used in the food industry to produce high-fructose syrups and fructo-oligosaccharides. For this purpose, different Aspergillus strains and substrates were tested for inulinase production by solid-state fermentation, among which Aspergillus terreus URM4658 grown on wheat bran showed the highest activity (15.08 U mL−1). The inulinase produced by this strain exhibited optimum activity at 60 °C and pH 4.0. A detailed kinetic/thermodynamic study was performed on the inulin hydrolysis reaction and enzyme thermal inactivation. Inulinase was shown to have a high affinity for substrate evidenced by very-low Michaelis constant values (0.78–2.02 mM), which together with a low activation energy (19.59 kJ mol−1), indicates good enzyme catalytic potential. Moreover, its long half-life (t1/2 = 519.86 min) and very high D-value (1726.94 min) at 60 °C suggested great thermostability, which was confirmed by the thermodynamic parameters of its thermal denaturation, namely the activation energy of thermal denaturation (E*d = 182.18 kJ mol−1) and Gibbs free energy (106.18 ≤ ΔG*d ≤ 111.56 kJ mol−1). These results indicate that A. terreus URM4658 inulinase is a promising and efficient biocatalyst, which could be fruitfully exploited in long-term industrial applications. [ABSTRACT FROM AUTHOR]

Research background. Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production. Experimental approach. Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production. Results and conclusions. The most common species were Aspergillus tubingensis (51.2 %), followed by A. niger (23.1 %), A. welwitschiae (23.1 %) and A. uvarum (2.6 %). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production. Novelty and scientific contribution. The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally. [ABSTRACT FROM AUTHOR]

Background: Inulinase is widely found in microorganisms and higher plants. It catalyzes the reaction of the hydrolysis of inulin, which is present in the tubers and roots of many plants, to fructose and a small amount of glucose. Inulin is one of several plant-based polysaccharides that contain glucose or fructose. It is used as a substrate in industrial fermentation processes and in the food industry due to its relatively cheap and abundant source for the microbiological production of high fructose juices, ethanol, acetone and butanol. Inulin-derived oligosaccharides are also used in the medical and food sectors. Inulinase which produced from the yeast K. marxianus at levels close to commercial use. This indicates that inulinase can be used in the production of fructose and fructose syrups. So our main object is to study the effect of different temperatures on the conformation of the Kluyveromyces marxianus inulinase macromolecule, to provide optimal conditions for aniline hydrolysis by inulinase. Materials and method: Method of isolation and purification of the inulinase enzyme from Kluyveromyces marxianus, as well as the determination of the protein content and enzyme activity as mentioned in the context of the research. Experiments were carried out to study the thermal stability of enzyme. For this, an enzyme solution at a concentration of 5•10-5 mol / l was incubated in a time interval of 10-60 min at different temperatures, followed by determination of the catalytic activity. It is known that different types of bonds and interactions participate in the formation of the molecular structure of enzymes, which are covalent bonds, hydrogen bonds, salt bridges, hydrophobic interactions, so our main goal is to study the effect of different temperatures on the formation of the enzyme molecule in order to provide optimal conditions for aniline hydrolysis. by enzyme. Results: of this study revealed that the residual activity of the enzyme after 60 minutes of incubation at 50 °C was 10% of the initial activity at 60 °C 7%. As for the Rate Constants of thermal inactivation of Inulinase at 70 °C were 3.45. Conclusions: Results of this study were important and useful in determining parameters of inulinase enzyme. Based on the shape of the curves of the dependence of the catalytic activity of Kluyveromyces marxianus inulinase on the time of thermal inactivation in the temperature range of 20-80 ° C.

Endophytic bacteria refer to bacteria which promote plant growth via direct and indirect mechanisms. Three endophytic bacteria isolated from Jerusalem artichoke exhibited plant growth induction and inulin production. These bacteria had functions of fructan degradation and synthesis from inulinase and levansucrase, respectively. Rossellomorea aquimaris 3.13 and Priestia megaterium 3.5 obtained inulinase/levanase enzyme with inulin and levan as substrates; enzyme production showed the optimum conditions in 1% inulin medium of 35 °C, pH 7.0. Bacillus velezensis 5.18 and Priestia megaterium 3.5 had inulosucrase/levansucrase enzyme with sucrose as a major carbon source; the enzyme had optimum temperature and pH conditions of 30 °C and pH 7.0, respectively. A combination of carbon sources had effect on decreasing enzyme activity; in addition, co-inoculation of bacteria showed a slight difference in enzyme production compared with single inoculation. The inulosucrase/levansucrase was produced earlier in co-culture containing bacteria with inulinase activity. Plant fructan synthesis was involved in 1-SST and 1-FFT, while 1-FEH encoded inulin degradation; these genes were evaluated in Jerusalem artichoke inoculated with the endophytic bacteria to quantify gene expression level using qPCR. All genes expressed in low levels at early stage of growth, responding to all endophytic bacteria. Significantly, Bacillus velezensis 5.18 induced all genes of the plant at 65 days of inoculation; Rossellomorea aquimaris 3.13 induced 1-FFT while Priestia megaterium 3.5 induced 1-SST. [ABSTRACT FROM AUTHOR]

Inulinase enzyme (EC 3.2.1.80) is an enzyme capable of hydrolyzing inulin and is used as a catalyst in the production of High Fructose Syrup (HFS) and fructooligosaccharides (FOS). Inulinase enzymes can be produced by yeasts found in waste such as kepok banana peels (Musa acuminata x balbisiana). The aims of this study were to obtain the inulinase enzymeproducing yeast from kepok banana peels and to determine the effect of commercial inulin concentration and incubation time on enzyme activity. This research was conducted experimentally using a factorial completely randomized design with factors being inulin concentration of 1% (K1), 3% (K2) and 5% (K3) and incubation time of 6th (W1), 12th (W2), 18th (W3), and 24th hours (W4). The results showed that there were 4 isolates of inulinolytic yeasts obtained from kepok banana peels, namely YP1, YP2, YP4, and YP6. However, the most potential isolate to be used in inulinase production was YP2 isolate because it had the highest inulinolytic ratio value of 1.96. The results of inulinase activity showed that only inulin substrate with concentration of 5% (K3) had a significant or significant effect on inulinase production. [ABSTRACT FROM AUTHOR]

Inulinases are commonly used for the production of high-fructose syrup, fructooligosaccharides, and inulooligosaccharides and also in the food and pharmaceutical industries. Besides, inulinases are also utilized for the fabrication of various high-added-value products such as bioethanol, lactic acid, citric acid, sugar alcohols, single-cell oils and proteins, 2,3-butanediol, gluconic acid, and butanol. Besides, before using an enzyme in a bioprocess, the determination of its properties is very essential. Therefore, this study evaluated the thermostability (30–80 °C for 0.5–6 h) of Aspergillus niger inulinase produced from sugar beet molasses in the shake flask fermentation and determined its kinetic and thermodynamic features. Based on the thermostability test of inulinase, it was stable at 30 °C and 40 °C and activity retained approximately 78.5% and 21.8% at 50 °C and 60 °C at the end of 6-h incubation time. Besides, half-life and D-value of enzyme reduced with increasing temperature, but increased with increasing incubation time at a fixed temperature. Q10-values were generally below one and reduced with increasing temperature. Nevertheless, inactivation energy and Z-value of inulinase were between 203.8 and 293.2 kJ/mol and between 17.05 and 20.99 °C to varying incubation times. Enthalpy and entropy of inulinase decreased with increasing temperature. The free energy of inulinase was unstable with increasing temperature depending on its inactivation energy. Consequently, inulinase can be used for biocatalytic strategies in various bioprocesses at relatively high temperatures. [ABSTRACT FROM AUTHOR]

Heterogeneous expression of enzymes allows large-scale production with reduced costs. Changes in glycosylation often occur due to changes in the expression host. In the study, the catalytic and biochemical properties of Aspergillus awamori exo-inulinase 1 are compared for A. awamori and Penicillium verruculosum expression hosts. The tertiary structure contains seven sites of N -glycosylation, with two of them located near the active center. If expressed in P. verruculosum , the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in k cat. These substrates with a short chain of 2–4 monosaccharide units were used to characterize the interaction of the substrate with the amino acid residues in the active center while preventing the interaction of the substrate with N -linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of N -linked glycans. The fluctuation of the residues N40 and Q57, which interact with the hydroxyl group O5 of the fructose unit in the −1 subsite of the active center, was increased by 1.6 times. The fluctuation of the residue W335, which interacts with the hydroxyl group O1 of the fructose unit together with the catalytic residue D41 and affects the torsion angle geometry of the substrate molecules, was increased by 1.5 times. The residue R188, which analogously to W335 affects the torsion angle geometry of the substrate molecules, was also among the affected residues with a 1.2-fold increase in the fluctuation. [Display omitted] • Recombinant and native forms of A. awamori exo-inulinase differed in properties. • If expressed in Penicillium verruculosum , the enzyme was four times less glycosylated. • Recombinant enzyme was two times more active toward sucrose, raffinose, and stachyose. • A decrease in glycosylation causes a decrease in fluctuation of the active center. [ABSTRACT FROM AUTHOR]

Inulin is the rich water-soluble storage polysaccharide after starch in nature, and utilization of inulin through hydrolysis of exo-inulinases has attracted much attention. Thermo-halo-alcohol tolerance is essential for exo-inulinase applications, while no report reveals the molecular basis involved in halo-alcohol tolerance of exo-inulinases via experimental data. In this study, two loops of exo-inulinase InuAMN8, including the loop built with 360GHVRLGPQP368 linking domains of Glyco_hydro_32N and Glyco_hydro_32C and another loop built with 169GGAG172 in the catalytic domain, were deleted to generate mutants MutG360Δ9 and MutG169Δ4, respectively. After heterologous expression, purification, and dialysis, InuAMN8, MutG169Δ4, and MutG360Δ9 showed half-lives of 144, 151, and 7 min at 50°C, respectively. InuAMN8 and MutG169Δ4 were very stable, while MutG360Δ9 showed a half-life of approximately 60 min in 5.0% (w/v) NaCl, and they showed half-lives of approximately 60 min in 25.0, 25.0, and 5.0% (w/v) ethanol, respectively. Structural analysis indicated that two cation-π bonds, which contributed to thermal properties of InuAMN8 at high temperatures, broke in MutG360Δ9. Four basic amino acid residues were exposed to the structural surface of MutG360Δ9 and formed positive and neutral electrostatic potential that caused detrimental effects on halo-alcohol tolerance. The study may provide a better understanding of the loop-function relationships that are involved in thermo-halo-alcohol adaptation of enzymes in extreme environment. [ABSTRACT FROM AUTHOR]

Inulin can be hydrolyzed by inulinases to yield inulo-oligosaccharides (IOSs), which have great application potential in the food and nutraceutical industries. However, conventional enzymatic production of IOSs is limited by long hydrolysis times and poor thermo-stability of inulinases. Here, the self-assembling protein scaffold EutM was engineered to co-immobilize exo-inulinase (EXINU) and endo-inulinase (ENINU) for synergistic hydrolysis of inulin to produce IOSs with 3 to 5 monosaccharide units (DP3–5 IOSs). The immobilization of EXINU/ENINU onto the EutM scaffold resulted in an increase of catalytic efficiency, a 65% increase of the V max of ENINU, as well as an increase of thermo-stability, with 4.26-fold higher residual activity of EXINU after 22 h-incubation at 50 °C. After optimization, two efficient production protocols were obtained, in which the yield and productivity of DP3–5 IOSs reached 80.38% and 70.86 g·(L·h)−1, respectively, which were at a high level in similar studies. Overall, this study provides an attractive self-assembling protein platform for the co-immobilization of inulinases, as well as optimized bioprocesses with great promise for the industrial production of DP3–5 IOSs. [Display omitted] • Inulin was efficiently hydrolyzed by synergistic action of EXINU and ENINU to produce DP3-5 IOSs. • Self-assembling protein scaffold EutM was used for the co-immobilization of EXINU/ENINU. • Improved catalytic efficiency and thermo-stability of ENINU/EXINU were obtained by using EutM. • A high level of DP3-5 IOSs productivity of 70.86 g·(L·h)-1 was obtained from 10% long chain inulin. [ABSTRACT FROM AUTHOR]

Bran can enrich snacks with dietary fibre but contains fructans that trigger symptoms in people with irritable bowel syndrome (IBS). This study aimed to investigate the bioprocessing of wheat and amaranth bran for degrading fructans and its application (at 20% flour-based) in 3D-printed snacks. Bran was bioprocessed with Saccharomyces cerevisiae alone or combined with inulinase, Kluyveromyces marxianus, Limosilactobacillus fermentum, or commercial starter LV1 for 24 h. Fructans, fructose, glucose, and mannitol in the bran were analysed enzymatically. Dough rheology, snack printing precision, shrinkage in baking, texture, colour, and sensory attributes were determined. The fructan content of wheat bran was 2.64% dry weight, and in amaranth bran, it was 0.96% dry weight. Bioprocessing reduced fructan content (up to 93%) depending on the bran type and bioprocessing agent, while fructose and mannitol remained below the cut-off value for IBS patients. Bran bioprocessing increased the complex viscosity and yield stress of dough (by up to 43 and 183%, respectively) in addition to printing precision (by up to 13%), while it lessened shrinkage in baking (by 20–69%) and the hardness of the snacks (by 20%). The intensity of snack sensory attributes depended on the bran type and bioprocessing agent, but the liking ("neither like nor dislike") was similar between samples. In conclusion, snacks can be enriched with fibre while remaining low in fructans by applying bioprocessed wheat or amaranth bran and 3D printing. [ABSTRACT FROM AUTHOR]

Inulin is the rich water-soluble storage polysaccharide after starch in nature, and utilization of inulin through hydrolysis of exo-inulinases has attracted much attention. Thermo-halo-alcohol tolerance is essential for exo-inulinase applications, while no report reveals the molecular basis involved in halo-alcohol tolerance of exo-inulinases via experimental data. In this study, two loops of exo-inulinase InuAMN8, including the loop built with 360GHVRLGPQP368 linking domains of Glyco_hydro_32N and Glyco_hydro_32C and another loop built with 169GGAG172 in the catalytic domain, were deleted to generate mutants MutG360Δ9 and MutG169Δ4, respectively. After heterologous expression, purification, and dialysis, InuAMN8, MutG169Δ4, and MutG360Δ9 showed half-lives of 144, 151, and 7 min at 50°C, respectively. InuAMN8 and MutG169Δ4 were very stable, while MutG360Δ9 showed a half-life of approximately 60 min in 5.0% (w/v) NaCl, and they showed half-lives of approximately 60 min in 25.0, 25.0, and 5.0% (w/v) ethanol, respectively. Structural analysis indicated that two cation-π bonds, which contributed to thermal properties of InuAMN8 at high temperatures, broke in MutG360Δ9. Four basic amino acid residues were exposed to the structural surface of MutG360Δ9 and formed positive and neutral electrostatic potential that caused detrimental effects on halo-alcohol tolerance. The study may provide a better understanding of the loop-function relationships that are involved in thermo-halo-alcohol adaptation of enzymes in extreme environment.

HFS (High Fructose Syrup) from gembolo tubers is an innovation in to utilize gembolo tubers which so far have not been used optimally. The purpose of this study was to determine the effect of substrate concentration and duration of saccharification on the physicochemical characteristics of HFS (High Fructose Syrup). This study used a completely randomized design (CRD) with 2 factors consisting of 9 treatment levels. The factor I was the substrate concentration (15%, 20% and 25%) and factor II was the length of saccharification (24 hours, 36 hours and 48 hours). Data were analyzed using ANOVA level 5%. If there is a difference proceed with Duncan's Test (DMRT) 5%. Based on the results of the study, the best treatment was a substrate concentration of 25% with a saccharification period of 48 hours which produced HFS with the following characteristics: yield of 18.08%; reducing sugar 41, 28%; total dissolved solids 42.00oBrix; viscosity 58.30 cP; fructose content is 14.68% and organoleptic test got the highest score.

Inulinase was immobilised by entrapment method in polyacrylamide/polyethylene glycol composite and evaluated for its efficiency for short-chain fructooligosaccharides (3–6 degrees of polymerisation) production in batch hydrolysis system. Aqueous two-phase polymerisation technique was used to synthesise the composite, where aqueous polyethylene glycol 1000 containing the enzyme was used as dispersant with ammonium persulfate as initiator. The characteristics of free and immobilised inulinase were investigated and compared, and the results showed shift of pH and temperature optimum and change in stability caused by the immobilisation material. The immobilised preparation retained 50% of its initial activity after 20 successive batch cycles of 1 h each. The conversion degree of highly polymerised inulin to fructooligosaccharides (3–6 degrees of polymerisation) was 36% when using 30% PAA/PEG, w/v. ATPS extracted inulinase was effectively immobilised in PAA/PEG composite. Immobilised enzyme showed good pH and thermal stability. Immobilised catalyst presents good retention of activity after 20 reuses. The entrapped enzyme is effective in producing FOS with the DP from 3 to 6 with proved prebiotic activities. The optimum conditions for batch operating mode were: 2% (w/v) inulin, 30% (w/v) PAA/PEG composite, pH = 4.4 and T = 40ºC. [ABSTRACT FROM AUTHOR]

Functional foods are essential food products that possess health-promoting properties for the treatment of infectious diseases. In addition, they provide energy and nutrients, which are required for growth and survival. They occur as prebiotics or dietary supplements, including oligosaccharides, processed foods, and herbal products. However, oligosaccharides are more efficiently recognized and utilized, as they play a fundamental role as functional ingredients with great potential to improve health in comparison to other dietary supplements. They are low molecular weight carbohydrates with a low degree of polymerization. They occur as fructooligosaccharide (FOS), inulooligosaccharadie (IOS), and xylooligosaccahride (XOS), depending on their monosaccharide units. Oligosaccharides are produced by acid or chemical hydrolysis. However, this technique is liable to several drawbacks, including inulin precipitation, high processing temperature, low yields, and high production costs. As a consequence, the application of microbial enzymes for oligosaccharide production is recognized as a promising strategy. Microbial enzymatic production of FOS and IOS occurs by submerged or solid-state fermentation in the presence of suitable substrates (sucrose, inulin) and catalyzed by fructosyltransferases and inulinases. Incorporation of FOS and IOS enriches the rheological and physiological characteristics of foods. They are used as low cariogenic sugar substitutes, suitable for diabetics, and as prebiotics, probiotics and nutraceutical compounds. In addition, these oligosaccharides are employed as anticancer, antioxidant agents and aid in mineral absorption, lipid metabolism, immune regulation etc. This review, therefore, focuses on the occurrence, physico-chemical characteristics, and microbial enzymatic synthesis of FOS and IOS from coprophilous fungi. In addition, the potential health benefits of these oligosaccharides were discussed in detail.

Biomass has recently been extensively exploited as an attractive alternative to the scarce fossil fuels and has been widely used for the production of fine chemicals. The present work defines an integrated approach for high yield of 5-hydroxymethylfurfural (HMF) from inulin via a two-step process. Initially, insolubilization of inulinase from Aspergillus niger on chitosan-coated magnetic nanoparticles (cMNP) was achieved, and under optimal conditions, hydrolysis of 5% inulin by immobilized inulinase (2.5 U/mg) led to fructose release of 35.8 g/L at pH 6.0 and temperature 60 °C in 3 h. Subsequently, the fructose was dehydrated to HMF in the presence of TiO2-magnetic silica spheres (TiO2-MSS). Upon optimizing the process parameters, HMF yield of 96.58% was achieved with 15% fructose in 15 min at 140 °C with a TiO2-MSS load of 10% (w/w). Concerning the operational stability of the immobilized biocatalyst/catalyst, the immobilized inulinase was recycled up to 10 cycles of inulin hydrolysis with 9.2 g/L of fructose released at the 10th cycle. TiO2-MSS also showed high operational stability and were recycled up to 10 cycles of HMF production with 5.4 g/L of HMF produced at 10th cycle. The mass balance for inulin to HMF was performed which showed the efficiency of the process. [ABSTRACT FROM AUTHOR]

Introduction: Klebsiella pneumoniae are Gram-negative which cause many diseases such as urinary tract infections, respiratory tract infections and septicemia. Inulinase is an enzyme used in food manufacture and pharmaceuticals. Inulinase is used in decreasing lipid ratio and, cholesterol in blood and considered as a prebiotic factor inside intestine. Many microorganisms can produce inulinase, such as yeast, fungi and bacteria; among such bacteria: Bacillus spp., Arthrobacter spp., and Pseudomonas spp. but there are no studies about inulinase production by K. pneumoniae have been reported. So the current study aims at investing the ability of producing and purification inulinase by K. pneumoniae. Method: K. pneumoniae were isolated from many hospitals and screened for the production of inulinase. Isolation percentage was 32%. A combination between the enzyme and the ceftazidime were assayed for detecting the antibacterial activity agonist Gram positive and Gram negative bacteria were done. Results: It is found that K. pneumoniae K4 isolate is the best producer of this enzyme. Inulinase, purified with ammonium sulfate at 70% saturation with specific activity 7.01 U/mg protein. As well, it's found that inulinase had increased the activity of ceftazidime against bacteria when combination between this enzyme and the antibiotic had done. Conclusion: This study proves for the first time that K. pneumoniae can produce inulinase which can be used in tremendous applications and also proves the broad spectrum bioactivity of inulinase against microbial pathogens. Ceftazidime antimicrobial activity against bacteria, is increased when a combination between inulinase and ceftazidime had done.

Inulinases are enzymes involved in the hydrolysis of inulin, which can be used in the food industry to produce high-fructose syrups and fructo-oligosaccharides. For this purpose, different Aspergillus strains and substrates were tested for inulinase production by solid-state fermentation, among which Aspergillus terreus URM4658 grown on wheat bran showed the highest activity (15.08 U mL−1). The inulinase produced by this strain exhibited optimum activity at 60 °C and pH 4.0. A detailed kinetic/thermodynamic study was performed on the inulin hydrolysis reaction and enzyme thermal inactivation. Inulinase was shown to have a high affinity for substrate evidenced by very-low Michaelis constant values (0.78–2.02 mM), which together with a low activation energy (19.59 kJ mol−1), indicates good enzyme catalytic potential. Moreover, its long half-life (t1/2 = 519.86 min) and very high D-value (1726.94 min) at 60 °C suggested great thermostability, which was confirmed by the thermodynamic parameters of its thermal denaturation, namely the activation energy of thermal denaturation (E*d = 182.18 kJ mol−1) and Gibbs free energy (106.18 ≤ ΔG*d ≤ 111.56 kJ mol−1). These results indicate that A. terreus URM4658 inulinase is a promising and efficient biocatalyst, which could be fruitfully exploited in long-term industrial applications.

Inulin is a kind of polysaccharide that can be obtained various biomass. Inulooligosaccharides (IOS), a kind of oligosaccharides that can be obtained from inulin by enzymatic hydrolysis using inulinases, have been regarded as the functional food ingredients. Commercially available inulinases produced by natural Aspergillus niger contained both endo- and exo-inulinase activities. For IOS production from inulin, it is desirable to use only endo-inulinase as exo-inulinase would produce mainly the monosacchairde fructose from inulin. In the present study, a simple inulin-mediated ethanol precipitation method was developed to separate endo- and exo-inulinases present in natural inulinases. IOS production from inulin using the enriched endo-inulinase was then optimized in process conditions including pH and temperature, achieving a high yield of ∼94%. The resultant IOS products had a degree of polymerization ranging from 2 to 7. The study demonstrated a novel method for obtaining partially purified or enriched endo-inulinase for IOS production from inulin in an efficient process.