exo-Inulinase (Aspergillus niger)

An AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measuring oligofructans and fructan polysaccharide (inulins) in mixed materials and food products. The sample is extracted with hot water, and an aliquot is treated with a mixture of sucrase (a specific sucrose-degrading enzyme), α-amylase, pullulanase, and maltase to hydrolyze sucrose to glucose and fructose, and starch to glucose. These reducing sugars are then reduced to sugar alcohols by treatment with alkaline borohydride solution. The solution is neutralized, and excess borohydride is removed with dilute acetic acid. The fructan is hydrolyzed to fructose and glucose using a mixture of purified exo- and endo-inulinanases (fructanase mixture). The reducing sugars produced (fructose and glucose) are measured with a spectrophotometer after reaction with para-hydroxybenzoic acid hydrazide. The samples analyzed included pure fructan, chocolate, low-fat spread, milk powder, vitamin tablets, onion powder, Jerusalem artichoke flour, wheat stalks, and a sucrose/cellulose control flour. Repeatability relative standard deviations ranged from 2.3 to 7.3%; reproducibility relative standard deviations ranged from 5.0 to 10.8%.

Fructans are defined as any compound in which one or more fructosyl-fructose linkages constitute a majority of the linkages (1). This refers to polymeric material as well as to oligomers as small as disaccharide inulobiose. Fructans are widely distributed in the plant kingdom. They are present in monocotyledons, dicotyledons, and green algae. Fructans differ in molecular structure and in molecular weight. They may be classified into three main types, the inulin type, the levan (previously called phlein) type, and the graminan type (2). The inulin group consists of material that has mostly of exclusively the (2-1) fructosly-fructose linkage. Levan is material that contains mostly or exclusively the (2-6) fructosyl-fructose linkage. The graminan (or branched) type has both (2-1) and (2-6) fructosly-fructose linkages in significant amounts (e.g. graminan from Gramineae). The distribution of fructans in nature, and the production of fructooligosaccharides, such as neosugar, using fructosyltransferase, has been reviewed in a monograph (3). In the context of this article and the analytical procedure described, the term fructan relates only to inulin and graminan. The current analytical procedure has not been evaluated on levan.

A non-instrumental method for the measurement of fructan is described. The method simplifies fructan analysis, is easy to perform, uses standard laboratory equipment, and is accurate, reproducible and specific. The procedure employs highly purified and specific enzymes to hydrolyse sucrose, starch and fructans (inulins and graminan).

Fructans are defined as any compound where one or more fructosyl-fructose linkages constitute a majority of the linkages. This refers to polymeric material as well as oligomers as small as the diasaccharide inulobiose. Material included in this definition may or may not contain attached glucose. The terms oligomer and polymer are used by fructan researchers to distinguish between materials that can be specifically characterised and those that can not. Fructans are widely distributed in the plant kingdom. They are present in monocotyledons, dicotyledons and in green algae.

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin “mining” conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.

The advantages of recombinant enzymes over native include the control in a production environment, product purity and also high yield. The paper presents the determination the optimum temperatures and the activation energies for various origin recombinant exo-inulinases, among others from Aspergillus niger, A. awamori, Kluyveromyces marxianus and K. cicerisporus. The parameters were estimated based on the literature of the activity curves versus temperature for hydrolysis of inulin. It was assumed that both the hydrolysis reaction process and the deactivation process of recombinant exo-inulinase were first-order reactions by the enzyme concentration. A mathematical model describing the effect of temperature on recombinant exo-inulinase activity was used. Based on the comparison analysis, values of the activation energies E_a were in the range from 32.01±7.8032.01±7.80 to 43.83±4.8743.83±4.87 kJ mol⁻¹, the deactivation energies E_a were in the range from 83.93±4.8283.93±4.82 to 352.44±14.26352.44±14.26 kJ mol⁻¹ and the optimum temperature T_opt were obtained in the range from 318.91±318.91± 1.19 to 328.76±0.25328.76±0.25 K.

Gerbera is one of the most important ornamental plants on the cut flower market. The basic reproduction methods of numerous cultivars of this species are in vitro techniques. In in vitro cultures, all plant growth conditions are controlled, including the light (intensity, quality, and duration). In tissue cultures, light quality is the most important factor that influences plant morphogenesis (growth and development). Light emitting diodes (LEDs), in contrast to the commonly used fluorescent lamps, allow for adjusting the light quality to the specific requirements of plants. LEDs are also energy-efficient and contain no harmful substances (e.g., mercury). The aim of the study was to analyze the effect of different light qualities emitted by LEDs during in vitro multiplication of Gerbera on its metabolic and physiological development. We compared endogenous carbohydrate content in the tissues and the condition of the photosynthetic apparatus in plants grown under fluorescent lamps and LED light. The study showed that the use of LEDs did not disturb the secondary metabolism of carbohydrates and the multiplied shoots were of high quality. The mixture of red and blue LED light in a 7:3 proportion is recommended for gerbera micropropagation. This light quality positively influenced the functioning of the photosynthetic apparatus.

Barley is described to mostly use sucrose for night carbon requirements. To understand how the transient carbon is accumulated and utilized in response to cold, barley plants were grown in a combination of cold days and/or nights. Both daytime and night cold reduced growth. Sucrose was the main carbohydrate supplying growth at night, representing 50–60% of the carbon consumed. Under warm days and nights, starch was the second contributor with 26% and malate the third with 15%. Under cold nights, the contribution of starch was severely reduced, due to an inhibition of its synthesis, including under warm days, and malate was the second contributor to C requirements with 24-28% of the total amount of carbon consumed. We propose that malate plays a critical role as an alternative carbon source to sucrose and starch in barley. Hexoses, malate, and sucrose mobilization and starch accumulation were affected in barley elf3 clock mutants, suggesting a clock regulation of their metabolism, without affecting growth and photosynthesis however. Altogether, our data suggest that the mobilization of sucrose and malate and/or barley growth machinery are sensitive to cold.

The feasibility of operation on a fluidized bed reactor (FBR) with inulinase imbedded in the gelatin alginate microspheres was investigated in order to improve the higher inulin conversion yields and enzyme stability than that of the previously obtained with the packed bed reactors. The operation processes were based on statistical analyses, the operational conditions of immobilized inulinase in a FBR system have been determined for immobilized enzyme load of 18 g, substrate concentration of 80 g/L, expansion ratio of 1.4 and substrate flow rate at 0.5 mL/min. According to the above-mentioned research parameters, the continuous fructose preparation with FBR system was sustainable for 10 days (240 h) and gained the productivity of 86.4 g/Ld. Compared with the previous results of the packed-bed reactor, the immobilized inulinase in the FBR system was applied in the inulin conversion, which appeared more effective. This study suggested that a system for the continuous and efficient enzymatic conversion of inulin in the FBR was founded, which could be potentially applicable for the scale-up production.

Oligosaccharides are known to affect the health of infants. The analysis of these complex molecules in (human) milk samples requires state-of-the-art techniques. This study analyzed the composition and concentration of oligosaccharides in early (day 3) and mature (day 42) human milk as well as in five different infant formula brands. The oligosaccharide content decreased in human milk from 9.15 ± 0.25 g/L at day 3 to 6.38 ± 0.29 g/L at day 42 of lactation. All formulas resulted to be fortified with galacto-oligosaccharides, with one also fortified with polydextrose and another with long-chain fructo-oligosaccharides. About 130 unique oligosaccharide structures were identified in the human milk samples, whereas infant formula contained less diversity of structures. The comparisons indicated that composition and abundance of oligosaccharides unique to human milk are not yet reproduced in infant formulas. The analytical workflow developed is suitable for the determination of prebiotic oligosaccharides in foods that contain diverse carbohydrate structures.

Recently, agavins (branched neo-fructans) of short degree of polymerization have shown beneficial effects on the health of both healthy and overweight individuals. Therefore, the aim of the present work was to investigate the potential use of Agave angustifolia agavins on the generation of branched fructooligosacharides (a-FOS). A. angustifolia agavins were hydrolyzed using exo-, endo-inulinase, and a mixture of both (25 and 75%, respectively). Exo- and the inulinase mixture degraded quickly the agavins in relation to endo-inulinase treatment. Only endo-inulinase and the inulinase mixture generated a-FOS formation. Endo-inulinase degraded 31% of agavins, yielding approximately 20% of a-FOS after 48 h, whereas the inulinase mixture hydrolyzed 33% of agavins in just 90 min, but only yielded 10% of a-FOS. These results suggest that agave plants could be an abundant raw material for a-FOS production, which might have a huge prebiotic potential as new branched fructooligosaccharides with many applications in the alimentary and pharmaceutical industry.

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40°C for the free enzyme and 5.0-5.5 and 45-50°C for the immobilized enzyme. The kinetic parameters of V_max and K_m were 5.24 µmol/min and 57.6 mg/mL for the free inulinase and 4.32 µmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45°C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with β(2-6) and β(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, ¹³C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by ¹³C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55°C, with a drastic decrease at 60°C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.