Production Of Fructooligosaccharides Research Articles

An Exploration of various Fructooligosaccharides production methods using novel engineered enzymes with innovative core-shell chitosan beads

This study investigates Fructooligosaccharides (FOSs) production as prebiotics to promote gut health and overall well-being. Various production methods for FOSs were explored, leveraging novel engineered inulosucrase (IN) and levansucrase (LV) enzymes. The methodologies included batch stirred reactors with free enzymes and fixed-bed reactors utilizing enzymes immobilized on hydrogel chitosan beads (HGBs) and core-shell chitosan beads (CSBs). The study found that free enzymes exhibited the highest sucrose consumption rates, with IN achieving 142.8 g/L∙h and LV achieving 82.9 g/L∙h. However, CSB-immobilized enzymes demonstrated substantial enzyme savings (55% and 41% reduction in IN and LV consumption, respectively) through reuse across multiple cycles, while delivering superior FOSs yields. CSB-immobilized enzymes outperformed HGB-immobilized enzymes in terms of immobilization efficiency, activity recovery, sucrose conversion, FOSs yields, and repeatability. A novel reactor setup involving interconnected fixed-bed reactors with different enzymes in series produced promising results, generating novel FOSs species alongside expected I-FOSs and L-FOSs. Consumable cost analysis indicated that while free enzyme usage is initially cost-effective (5.09–7.06 USD/kg FOSs), significant increases in LV prices could make CSB immobilization a cost-competitive alternative. This study highlights the potential of innovative enzyme immobilization strategies and reactor designs for efficient FOSs production, considering both performance and economic feasibility.

IMMOBILIZATION OF A MICROBIAL FRUCTOSYLTRANSFERASE ON BIOCHAR AND BIOCHAR FUNCTIONALIZED WITH GLUTARALDEHYDE AIMING AT FRUCTOOLIGOSSACHARIDES PRODUCTION

Fructooligosaccharides (FOS) are a group of carbohydrates that have fructose units, with a glucose molecule at the end. These are structures that can be found in a wide variety of vegetables, being also produced chemically by the enzymatic transfructosylation reaction of sucrose. The present work aimed at the immobilization of the extracellular FTase enzyme from Aspergillus oryzae-IPT-301 on Eucalyptus biochar and Eucalyptus biochar functionalized with glutaraldehyde, for the enzymatic conversion of sucrose into FOS. Immobilization and reuse tests were carried out and immobilization yield and recovered activity calculated. The immobilization yield (IY) and recovered activity (RA) of the FTase immobilized on biochar were, 10.92% and 77.67%, respectively. The IY and RA of the FTase immobilized on the functionalized biochar were, 22.44% and 96.51%, respectively. The enzyme immobilized on functionalized biochar demonstrated transfructosylation activity for more cycles than the enzyme immobilized on biochar. The use of glutaraldehyde allowed 48% of initial enzymatic activity in the third cycle. Analyzes were conducted up to the sixth cycle, however, only 7% of initial enzymatic activity was achieved in the final cycle. These results suggest that functionalized biochar stands out as a support material for FTase immobilization aiming at FOS production catalyzed on heterogeneous biocatalysts.

Tailored chitosan integration in diatomaceous earth particles as a scaffold for fructosyltransferase immobilization in fructo-oligosaccharide production.

Fructo-oligosaccharide (FOS) belongs to the group of short inulin-type fructans and is one of the most important non-digestible bifid-oligosaccharides capable of biotransforming sucrose using fructosyltransferase (FTase). However, there are no immobilized FTase products that can be successfully used industrially. In this study, diatomite was subjected to extrusion, sintering and granulation to form diatomaceous earth particles that were further modified via chitosan aminomethylation for modification. FTase derived from Aspergillus oryzae was successfully immobilized on the modified support via covalent binding. The immobilized enzyme activity was 503 IU g-1 at an enzyme concentration of 0.6 mg mL-1, immobilization pH of 7.0 and contact time of 3 h. Additionally, the immobilization yield was 56.91%. Notably, the immobilized enzyme was more stable under acidic conditions. Moreover, the half-life of the immobilized enzyme was 20.80 and 10.96 times as long as that of the free enzyme at 45 and 60 °C, respectively. The results show good reusability, as evidenced by the 84.77% retention of original enzyme activity after eight cycles. Additionally, the column transit time of the substrate was 35.56 min when the immobilized enzyme was applied in a packed-bed reactor. Furthermore, a consistently high FOS production yield of 60.68% was achieved and maintained over the 15-day monitoring period. Our results suggest that immobilized FTase is a viable candidate for continuous FOS production on an industrial scale. © 2024 Society of Chemical Industry.

Cross-linked whole cells for the sucrose transfructosylation reaction in a continuous reactor

Fructooligosaccharides (FOS) are fructose oligomers beneficial to human health and nutrition for prebiotic sugars. Their production occurs by a transfructosylation reaction in sucrose molecules catalyzed by fructosyltransferase enzymes (FTase, E.C.2.4.1.9) adhered to microbial cells. The purpose of this work was to study the preparation, enzymatic activity, and stability of glutaraldehyde-crosslinked Aspergillus oryzae IPT-301 cells used as a biocatalyst for the transfructosylation reaction of sucrose in a packed bed reactor (PBR), aiming at FOS production. The highest transfructosylation activity (AT) was presented by the biocatalyst prepared by cross-linking at 200 rpm and 45 min. The highest AT in the PBR was obtained at 50 ?C, with flow rates from 3 mL min-1 to 5 mL min-1 and sucrose concentrations of 473 g L-1 and 500 g L-1. The enzymatic kinetics was described using the Michaelis-Menten model. Finally, the biocatalyst showed constant AT of approximately 75 U g-1 and 300 U g-1 for 12 h of reaction in the PBR operating in continuous and discontinuous flow, respectively. These results demonstrate a high potential of glutaraldehyde-crosslinked A. oryzae IPT-301 cells as heterogeneous biocatalysts for the continuous production of FOS in PBR reactors.

Rational design and immobilization of a recombinant sucrose: Sucrose 1-fructosyltransferase on Sepabeads® and ReliZyme™ supports for short-chain fructooligosaccharides production

The recombinant sucrose:sucrose 1-fructosyltransferase from Schedonorus arundinaceus (Sa1-SSTrec) produces short-chainfructooligosaccharides (scFOS). In this work, Sa1-SSTrec was covalently or hydrophobic immobilized on Sepabeads® and ReliZyme™ supports. The possible clusters and the parameters related to the immobilization process were predicted by rational design of immobilized derivatives (RDID). Immobilization was performed at pH 7.0 and the three immobilized derivatives with higher experimental enzymatic activity (one of each epoxy-activated, amino-activated and hydrophobic supports: Sepabeads® EC-HFA/S, ReliZyme™ EA403/M and Sepabeads® EC-BU/M, respectively) were selected. The mismatch between some predictions and experimental results were explained by probable limitations of the RDID strategy. The thermodynamic parameters determined at 40, 50 and 60ºC, under not reactive conditions (i.e. in absence of sucrose), showed high thermal stability of Sa1-SSTrec after immobilization on the Sepabeads®EC-HFA/S. Batch experiments with this support, at 45ºC reached similar total scFOS (1-kestotriose and 1,1-kestotetraose) than the soluble enzyme, but scFOS productivity was higher for the immobilized biocatalyst. Operational stability of the immobilized biocatalyst after 4 cycles dropped to about 60% and 25% of its initial value at 30 and 45ºC, respectively, probably by denaturation at these temperatures. The obtained results suggest that the immobilized enzyme could be effectively exploited for industrial production of 1-kestotriose.

VALIDATION OF AN ANALYTICAL METHOD FOR THE QUANTIFICATION OF 1-KESTOSE, GLUCOSE AND SUCROSE IN THE REACTION PRODUCTS OF A FRUCTOSYLTRANSFERASE

The enzymatic synthesis of fructooligosaccharides (FOS) from sucrose by the action of fructosyltransferases produces a mixture of different carbohydrates. The concentration of each carbohydrate involved must be quantified to follow its variation as the kinetics of the reaction progresses. This requires a method such as high-performance liquid chromatography (HPLC) with refractive index detector (IR). The aim of this work was to validate the HPLC-IR analytical method for the quantification of total and individual carbohydrates in mixtures of 1-kestose, sucrose and glucose. For this study, the validation parameters recommended by the FDA (Food and Drug Administration), the ICH (International Council for Harmonisation) and the EMEA (European Medicines Agency) were followed. The method was able to quantify carbohydrates with adequate levels of precision, accuracy and linearity in ranges of 1.6-8.7, 4.3-22.0 and 7.0-41.0 mg/mL for glucose, 1-kestose and sucrose, respectively. The lowest limit of detection was for glucose (0.2 mg/mL) and the highest for sucrose (0.8 mg/mL), while the lowest and highest limit of quantification were also for glucose (0.6 mg/mL) and sucrose (1.8 mg/mL). The detection (0.7 mg/mL) and quantification (1.4 mg/mL) limits for 1-kestose were intermediate. The HPLC-IR method was used to calculate the concentration of the carbohydrates involved in the reaction kinetics of a recombinant fructosyltransferase (Sa1-SSTrec). This analytical technique can be implemented for the analysis of samples from the FOS production process at both laboratory and industrial scale, but also the technique is useful to detect 1-kestose in functional foods based in fruit juices.

Fructooligosaccharides (FOS) Production by Microorganisms with Fructosyltransferase Activity

Fructans are fructose-based polymers, defined as fructooligosaccharides (FOS), when they possess a short chain. These molecules are highly appreciated in the food and pharmaceutical international market and have an increasing demand worldwide, mainly for their prebiotic activity and, therefore, for all their health benefits to those who consume them constantly. Thus, new natural or alternative FOS production systems of industrial scale are needed. In this regard, microorganisms (prokaryotes and eukaryotes) have the potential to produce them through a wide and diverse number of enzymes with fructosyltransferase activity, which add a fructosyl group to sucrose or FOS molecules to elongate their chain. Microbial fructosyltransferases are preferred in the industry because of their high FOS production yields. Some of these enzymes include levansucrases, inulosucrases, and β-fructofuranosidases obtained and used through biotechnological tools to enhance their fructosyltransferase activity. In addition, characterizing new microorganisms with fructosyltransferase activity and modifying them could help to increase the production of FOS with a specific degree of polymerization and reduce the FOS production time, thus easing FOS obtention. Therefore, the aim of this review is to compile, discuss, and propose new perspectives about the microbial potential for FOS production through enzymes with fructosyltransferase activity and describe the modulation of FOS production yields by exogenous stimuli and endogenous modifications.

Bioprocess development for fructooligosaccharides and carotenoids production using Agave mead

Fructooligosaccharides (FOS) and carotenoids are bioactive compounds of interest to the food industry due to the multiple benefits associated with their daily intake. One of the strategies that can be used to obtain said products of interest is successive fermentation, in which a compound of interest is produced in an initial fermentation, and the same resulting medium can be used to be fermented by another microorganism to generate another metabolite of interest. The successive fermentation strategy was used to produce FOS (first fermentation) and carotenoids (successive fermentation) using Agave mead as fermentation media. First, fermentation was evaluated for 120 h to determine the best fermentation time in terms of FOS productivity (g FOS/L*h) and carotenoids (mg/L*h). Once the best fermentation time was selected, the effect of different variables was evaluated. A Box Hunter & Hunter experimental design was used, considering five independent variables (pH, inoculum concentration, agitation, temperature, and light) at two levels (−1, +1) that influence total FOS (g/L) and total carotenoids (mg/L). Selected variables were later evaluated in a third-order experimental design (Box Behnken) to optimize total FOS (g/L) and total carotenoids (mg/L) in successive fermentation. As a result, it was possible to take advantage of the sugars present in the Agave mead to produce FOS and successively carotenoids in the same fermentation medium. In addition, through the experimental design, it was possible to obtain a concentration of 33.33 ± 1.39 g/L of total FOS and 5.11 ± 0.25 mg/L of total carotenoids in 48 h of fermentation.

Karakteristik mutu karbohidrat dan evaluasi mutu sensoris minuman fungsional berbasis FOS dan inulin

Introduction: Fructooligosaccharides (FOS) and inulin are some of the many bioactive elements that are often used in functional food products. FOS and inulin compounds have various benefits that can be used as low-calorie food products and as raw materials for making fructose syrup. So that FOS and inulin compounds have the potential to be developed into functional drinks. The purpose of this study was to evaluate the quality characteristics of carbohydrates in functional drinks FOS and inulin, as well as to determine the level of preference or feasibility of a product so that it can be accepted by panelists (consumers). Method: The research was conducted in two stages. The first stage was the preliminary stage which includes the production of FOS with PDA solid media, extraction of inulin from dahlia tubers, and the formulation of FOS and inulin functional drinks. The second stage was the main research by conducting chemical quality characteristics in the form of reducing sugar content, fructose, sucrose, glucose, inulin, soluble fiber, and organoleptic tests. Results: Based on the observations, several characteristics of the carbohydrate quality of functional drinks based on FOS and inulin were obtained, namely reducing sugar levels ranging from 0.22 to 5.60%. Fructose and sucrose levels of functional drinks based on FOS and inulin ranged from 1-2%, while glucose levels were between 0.1-2%. The levels of inulin and soluble fiber in functional drinks based on FOS and inulin were 55-86% and 2-5%, respectively. Also, the pH value of functional drinks based on FOS and inulin ranged from 5-7. Conclusion: The results of the organoleptic test showed that the best functional drink based on FOS and inulin was in the AD treatment, namely the addition of 7 grams of inulin and 50 ml of fructooligosaccharides.

Safety evaluation of the food enzyme inulinase from the genetically modified Aspergillus oryzae strain MUCL 44346.

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.

Inulinase and fructooligosaccharide production from carob using Aspergillus niger A42 (ATCC 204447) under solid-state fermentation conditions

This study aimed to the production of inulinase and fructooligosaccharides (FOSs) from carob under the solid-state fermentation (SSF) conditions by using Plackett-Burman Design (PBD). Based on the results the maximum inulinase and specific inulinase activities were 249.98 U/mL and 318.29 U/mg protein, respectively. When the fructooligosaccharide (FOS) results were evaluated, the maximum values of 1,1,1-Kestopentaose, 1,1-Kestotetraose, and 1-Kestose were 182.01, 506.16, 132.16 ppm while the lowest and highest total FOS values were 179.35 and 516.66 ppm, respectively. On the other hand, it was observed that the maximum inulinase activity was found at the center points of the design. Therefore, validation fermentations were carried out at center point conditions. Subsequently, the yielded bulk enzyme extracts were partially purified using Spin-X UF membranes with 10, 30, and 50 kDa cut-off values. After purification, the maximum inulinase activity was 247.30 U/mg using a 50 kDa cut-off value. Followed by this process, the purified enzyme was used to produce FOSs and the results indicated that the maximum total FOS amount was 28,712.70 ppm. Consequently, this study successfully demonstrates that Aspergillus niger A42 inulinase produced from carob under the SSF conditions can be used in FOSs production.

Invertase Immobilization on Magnetite Nanoparticles for Efficient Fructooligosaccharide Generation: A Comprehensive Kinetic Analysis and Reactor Design Strategy

This study investigated the effectiveness of immobilizing Saccharomyces cerevisiae invertase (SInv) on magnetite nanoparticles to produce fructooligosaccharides (FOSs). Based on the existing literature and accompanied by parameter estimation, a modified kinetic model was employed to represent the kinetics of sucrose hydrolysis and transfructosylation using SInv immobilized on magnetite nanoparticle surfaces. This model was utilized to simulate the performance of batch reactors for both free and immobilized enzymes. The maximum FOS concentration for the free enzyme was determined to be 123.1 mM, while the immobilized case achieved a slightly higher concentration of 125.4 mM. Furthermore, a continuous stirred-tank reactor (CSTR) model was developed for the immobilized enzyme, resulting in a maximum FOS concentration of 73.96 mM at the reactor’s outlet and a dilution rate of 14.2 h−1. To examine the impact of glucose inhibition on FOS production, a glucose oxidase reaction mechanism was integrated into the fitted immobilized theoretical model. In a batch reactor, the reduction or elimination of glucose in the reactive media led to a 2.1% increase in FOS production. Immobilizing the biocatalyst enhanced the overall performance of SInv. This enzyme immobilization approach also holds the potential for coupling glucose oxidase onto functionalized nanoparticles to minimize glucose inhibition, thereby improving FOS synthesis and facilitating optimal enzyme recovery and reuse.

Investigation of biocatalytic production of lactosucrose and fructooligosaccharides using levansucrases and dairy by-products as starting materials

Selected levansucrases (LSs) were investigated for their ability to catalyze the transfructosylation of lactose/sucrose into lactosucrose and fructooligosaccharides (FOSs). Additionally, dairy by-products, including whey permeate (WP) and milk permeate (MP), were assessed for their effectiveness as lactose sources. LSs from Gluconobacter oxydans (LS1), Vibrio natriegens (LS2), Novosphingobium aromaticivorans (LS3), and Burkholderia graminis (LS4) were utilized in three transfructosylation reactions that combined sucrose with either lactose, WP, or MP. All LSs demonstrated a higher transfructosylation activity than hydrolytic one, except for V. natriegens LS2 in the presence of sucrose and MP/sucrose. Furthermore, the bioconversion efficiency of lactose/sucrose into lactosucrose and FOSs exhibited varying time courses and end-product profiles. Both the acceptor specificity of LS and the thermodynamic equilibrium of its reaction modulated the end-product profile. V. natriegens LS2 resulted in the highest lactosucrose production of 328 and 251 g/L with lactose/sucrose and WP/sucrose, respectively. Our results revealed the potential of LS-catalyzed transfructosylation for the biocatalytic production of both lactosucrose and FOSs from abundant biomasses.

Techno‐economic comparison of different reactor types used in the manufacture of fructooligosaccharides from sucrose

AbstractFructooligosaccharides, a low‐calorie alternative sweetener with prebiotic effects, can be synthesized from sucrose via enzymatic reactions. Recently, methods for producing novel fructooligosaccharides using free and immobilized Bacillus licheniformis RN‐01 levansucrase were developed at the laboratory scale. However, there was a need to apply engineering knowledge in production process design and scale‐up to develop and evaluate the feasibility of this technology in the industrial scale. In this paper, three different types of reactors for fructooligosaccharides production using B. licheniformis RN‐01 levansucrase were compared in terms of technical and economic aspects. The previously collected experimental data, including enzyme activities, immobilization yields, product yields, and material balances, were used to estimate the capital and operating costs of fructooligosaccharides production using (a) immobilized enzyme in a stirred‐tank reactor, (b) immobilized enzyme in a packed‐bed reactor, and (c) free enzyme in a stirred‐tank reactor. The technical issues in reactor design and operation were also evaluated. The results showed that, in terms of minimum product selling prices, the process using immobilized enzyme had more economic advantages than the process using free enzyme because the cost savings from enzyme purchasing could compensate the additional equipment and chemical costs in the enzyme immobilization process. The results could be a guideline for selecting suitable reactor types for fructooligosaccharides production and provide directions for further reactor design and development.

Enzymatic synthesis of fructooligosaccharides: From carrot discards to prebiotic juice

A great volume of carrots is discarded daily worldwide because they do not meet the required shape and size standards. However, they have the same nutritional characteristics as those commercialized, and can be used in different food products. Carrot juice is an excellent matrix for the development of functional foods with prebiotic compounds, such as fructooligosaccharides (FOS). In this work, the production of FOS in situ in carrot juice was evaluated using a fructosyltransferase from Aspergillus niger, produced by solid-state fermentation on carrot bagasse. The enzyme was partially purified 12.5-fold with a total yield of 93 %, and specific activity of 59 U/mg of protein by Sephadex G-105 molecular exclusion chromatography. It was identified by nano LC-MS/MS as a β-fructofuranosidase with a 63.6 kDa MW and it allowed obtaining a FOS yield of 31.6 % in carrot juice. The result was a prebiotic juice with a final concentration of 32.4 mg/mL of FOS. Using the commercial enzyme Viscozyme L a higher yield of FOS (39.8 %) was obtained in carrot juice, corresponding to a total amount of FOS of 54.6 mg/mL. This circular economy scheme allowed the obtention of a functional juice, that may contribute to improve health of consumers.

Production of Fructooligosaccharides Using a Commercial Heterologously Expressed Aspergillus sp. Fructosyltransferase

The catalytic properties of Seqenzym® FT, a fungal fructosyltransferase heterologously expressed in yeasts, were investigated at a temperature of 55 °C and pH 5.5. The initial rate measurements showed that the transfructosylation rate was only slightly inhibited by sucrose above the concentration of 1.5 M. A rather low level of hydrolytic side activity was observed even at sucrose concentrations as low as 0.25 M. In progress curve experiments, the mass yield of fructooligosaccharides (FOS) reached a maximum value of 57% at this sucrose concentration, although it dropped to about 35% later on. At high initial sucrose concentrations up to 2 M, the FOS yield reached a maximum value of approximately 63% at a sucrose conversion of approximately 90%. Although neither the yield nor the conversion changed much later on, the progress of the reaction was manifested by the gradual depletion of shorter chain FOS, 1-kestose and nystose, and the accumulation of 1-β-fructofuranosyl nystose. At initial sucrose concentrations of 2 M, the degree of polymerization expressed through the number of fructosyl units grew from 2.3 at a conversion degree of 87% to 3.1 at a conversion degree of 94%. Compared to other commercial preparations, Seqenzym® FT can better produce FOS with a higher degree of polymerization.

Micro‐kinetic model of fructo‐oligosaccharide synthesis for prebiotic products

AbstractA new micro‐kinetic model of the enzyme‐catalyzed synthesis of fructo‐oligosaccharides (FOS) was developed. A commercial enzyme mixture Pectinex® Ultra SP‐L derived from Aspergillus aculeatus was used. A variety of initial enzyme concentrations (1–5 vol%) and sucrose concentrations (400–600 g/L) were experimentally investigated and included in kinetic modeling. Several variations of kinetic mechanisms and corresponding models have been examined. A hybrid genetic algorithm was used to predict the kinetic parameters simultaneously for all experimental data. The best fitting model has been adopted, and with an average error of 13.34%, it describes the experimental data very well. The influence of initial concentrations on the conversion of sucrose and production of FOS is being carefully investigated. It was shown that the initial sucrose concentration significantly affects the highest level of FOS concentration, but the enzyme concentration controls the time at which maximum is reached as well as the rate of FOS decomposition.

Production, Kinetic/Thermodynamic Study, and Evaluation of the Influence of Static Magnetic Field on Kinetic Parameters of β-Fructofuranosidase from Aspergillus tamarii Kita UCP 1279 Produced by Solid-State Fermentation.

β-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and can be used in the production of invert sugar and fructo-oligosaccharides (FOS). This last is an important prebiotic extensively used in the food industry. In the present study, the FFase production by Aspergillus tamarii Kita UCP 1279 was assessed by solid-state fermentation using a mixture of wheat and soy brans as substrate. The FFase presents optimum pH and temperature at 5.0-7.0 and 60 °C, respectively. According to the kinetic/thermodynamic study, the FFase was relatively stable at 50 °C, a temperature frequently used in industrial FOS synthesis, using sucrose as substrate, evidenced by the parameters half-life (115.52 min) and D-value (383.76 min) and confirmed by thermodynamic parameters evaluated. The influence of static magnetic field with a 1450 G magnetic flux density presented a positive impact on FFase kinetic parameters evidenced by an increase of affinity of enzyme by substrate after exposition, observed by a decrease of 149.70 to 81.73 mM on Km. The results obtained indicate that FFases present suitable characteristics for further use in food industry applications. Moreover, the positive influence of a magnetic field is an indicator for further developments of bioprocesses with the presence of a magnetic field.

Use of Tunable Copolymers in Aqueous Biphasic Systems for Extractive Bioconversion Aimed at Continuous Fructooligosaccharide Production

Aqueous biphasic systems (ABSs) based on sodium polyacrylate (NaPA), ethylene oxide/propylene oxide (EO/PO) polymers, and (EO)x-(PO)y-(EO)x triblock copolymers were prepared and applied aiming at continuous fructooligosaccharide (FOS) production and separation. EO/PO hydrophilicity/hydrophobicity balance had a significant effect on ABS formation. To develop an integrated process including the continuous enzymatic (levansucrase) production of FOSs and their purification while improving the production yield by further glucose separation, the potential of these novel polymer-based ABSs as alternative platforms was investigated. They were used for the partitioning of different carbohydrates (FOS, sucrose, d-fructose, and d-glucose) and levansucrase. Results revealed a highly polymer-dependent partition of carbohydrates and a poorly dependent one of the enzymes. Changing EO/PO and copolymers, FOS was purified with high yields (72.94–100.0%). Using polypropylene glycol 400 + NaPA 8000-based ABS, the FOS was precipitated in the interphase and separated from the other components. Pluronic PE-6800 + NaPA 8000 was identified as the best ABS for FOS continuous production and in situ purification, while minimizing levansucrase inhibition by d-glucose. This system allowed selective partition of FOSs and d-glucose toward the top phase and that of levansucrase and its substrates toward the bottom one. COnductor-like Screening MOdel for Real Solvent (COSMO-RS) suggested that ABS formation may have been due to NaPA and polymer/copolymer competition to form hydrogen bonds with water molecules. Moreover, the partition of FOSs and sugar may have been the result of a subtle balance between hydrogen bonding of sugar and polymer/copolymer and electrostatic misfit of solute with NaPA. Finally, two integrated processes were proposed to be applied with real FOS extracts obtained by chemical or enzymatic hydrolysis of inulin or by transfructosylation of concentrated sucrose solutions using bacterial levansucrases.

HPTLC-based fingerprinting: An alternative approach for fructooligosaccharides metabolism profiling

Fructans are categorized as fructose-based metabolites with no more than one glucose in their structure. Agave species possess a mixture of linear and ramified fructans with different degrees of polymerization. Among them, fructooligosaccharides are fructans with low degree of polymerization which might be approachable by high performance thin layer chromatography (HPTLC). Thus, this study used two emblematic Agave species collected at different ages as models to explore the feasibility of HPTLC-based fingerprinting to characterize fructooligosaccharides (FOS) production, accumulation, and behavior through time. To do so, high performance anion exchange was also used as analytical reference to determine the goodness and robustness of HPTLC data. The multivariate data analysis showed separation of samples dictated by species and age effects detected by both techniques. Moreover, linear correlations between the increase of the age in agave and their carbohydrate fraction was established in both species by both techniques. Oligosaccharides found to be correlated to species and age factors, these suggest changes in specific carbohydrate metabolism enzymes. Thus, HPTLC was proven as a complementary or stand-alone fingerprinting platform for fructooligosaccharides characterization in biological mixtures. However, the type of derivatizing reagent and the extraction color channel determined the goodness of the model used to scrutinize agavin fructooligosaccharides (aFOS).