Synthesis Of Fructooligosaccharides Research Articles

Boosting Fructosyl Transferase's Thermostability and Catalytic Performance for Highly Efficient Fructooligosaccharides (FOS) Production.

Achieving enzymatic food processing at high substrate concentrations can significantly enhance production efficiency; however, related studies are notably insufficient. This study focused on the enzymatic synthesis of fructooligosaccharides (FOS) at high temperature and high substrate concentration. Results revealed that increased viscosity and limited substrate solubility in high-concentration systems could be alleviated by raising the reaction temperature, provided it aligned with the enzyme's thermostability. Further analysis of enzyme thermostability in real sucrose solutions demonstrates that the enzyme's thermostability was remarkedly improved at higher sucrose concentrations, evidenced by a 10.3 °C increase in melting temperature (Tm) in an 800 g/L sucrose solution. Building upon these findings, we developed a novel method for enzymatic FOS synthesis at elevated temperatures and high sucrose concentrations. Compared to existing commercial methods, the initial transglycosylation rate and volumetric productivity for FOS synthesis increased by 155.9% and 113.5%, respectively, at 65 °C in an 800 g/L sucrose solution. This study underscores the pivotal role of substrate concentration, incubation temperature, and the enzyme's actual status in advancing enzyme-catalyzed processes and demonstrates the potential of enzymatic applications in enhancing food processing technologies, providing innovative strategies for the food industry.

Short-chain fructooligosaccharides synthesis using a commercial enzyme complex from Aspergillus sp. and anti-cancer activity on HCT116 and HT-29 cell lines

Over the last years, short-chain fructooligosaccharides (FOS) have gained significant attention as valuable food and dietary supplement components. Apart from implications such as dietary fibres, sweeteners, and humectants, FOS are hailed for their prebiotic properties. Moreover, much research is dedicated to their role as anti-cancer agents. Despite the ongoing interest, both technical aspects, challenges of FOS production, and their anti-cancer role necessitate more in-depth investigations to understand their healthcare role better. This study aimed to optimise critical FOS synthesis parameters using a commercial enzyme, Viscozyme® L, to obtain a preparation with a high FOS content. A central composite design and response surface methodology evaluated the effect of pH, temperature, synthesis time, substrate, and enzyme load on multiple responses, chosen as independent variables, were utilised to define optimal conditions. Under the optimal conditions (temperature 50 °C, pH 5.5, sucrose concentration 352 g/L, enzyme concentration 2.5% v/v, and 5.5 h of reaction), a preparation with a total FOS content of 58.8% was obtained. The FOS preparation showed minimal in vitro antioxidant capacity in the ORAC, CUPRAC, and ABTS*+assays. In addition, the cytotoxicity of FOS against tested cell lines was evaluated. FOS did not reduce HT-29 cell viability even at the concentration of 2.5 mg/mL, whereas FOS inhibited the proliferation of HCT116 cells by 50% at 2.35 mg/mL. Finally, the sensitisation effect of FOS on HCT116 cell treatment with doxorubicin has been revealed.

Assessment of different kinetic models for the production of fructo‐oligosaccharides through enzymatic synthesis

AbstractMathematical modeling is a great tool to understand the mechanism of fructo‐oligosaccharides (FOS) synthesis. The objective in this work is to adapt different models designed for FOS synthesis. Two approaches for the reaction mechanism, chain reaction and ping‐pong, were included in the analysis and genetic algorithm was implemented to find the best fit. Both approaches offered good results, chain reaction approach showed lower overall relative errors (below 8.5%) while ping‐pong mechanism best fit presented an overall error of 10.5%. No matter the approach used, the models with best fit involved the concept of glucose inhibition for the transfructosylation reaction. Different objective functions can be tested to reduce the relative errors when the model struggled to describe the behavior of one component. The sensitive analysis helps to evaluate all the variables such as reaction rate, time, productivity, and FOS yield, to find an optimal reaction condition.Practical applicationsThis research aims to describe and help understand the reaction mechanism involved in the enzymatic synthesis of fructo‐oligosaccharides, through the study of the kinetics using different mathematical models found in the consulted bibliography. This way, the operational conditions (substrate type and concentration, enzyme source [bacteria or fungi], enzyme configuration [purified or whole cells, free or immobilized], enzyme/substrate ratio, reaction time, temperature, and pH, batch or continuous mode) can be adapted, ensuring the attainment of the desired final product.

Effective synthesis of high-content fructooligosaccharides in engineered Aspergillus niger

BackgroundAspergillus niger ATCC 20611 is an industrially important fructooligosaccharides (FOS) producer since it produces the β-fructofuranosidase with superior transglycosylation activity, which is responsible for the conversion of sucrose to FOS accompanied by the by-product (glucose) generation. This study aims to consume glucose to enhance the content of FOS by heterologously expressing glucose oxidase and peroxidase in engineered A. niger.ResultsGlucose oxidase was successfully expressed and co-localized with β-fructofuranosidase in mycelia. These mycelia were applied to synthesis of FOS, which possessed an increased purity of 60.63% from 52.07%. Furthermore, peroxidase was expressed in A. niger and reached 7.70 U/g, which could remove the potential inhibitor of glucose oxidase to facilitate the FOS synthesis. Finally, the glucose oxidase-expressing strain and the peroxidase-expressing strain were jointly used to synthesize FOS, which content achieved 71.00%.ConclusionsThis strategy allows for obtaining high-content FOS by the multiple enzymes expressed in the industrial fungus, avoiding additional purification processes used in the production of oligosaccharides. This study not only facilitated the high-purity FOS synthesis, but also demonstrated the potential of A. niger ATCC 20611 as an enzyme-producing cell factory.

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.

Optimization strategy for inulinase production by Aspergillus niger URM5741 and its biochemical characterization, kinetic/thermodynamic study, and application on inulin and sucrose hydrolysis.

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 (5g), 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 (1.07-1.54mM). A relative thermostability was observed at 55-60°C as indicated by half-lives values (I: 169.06-137.27min; S: 173.29-141.52min) and D-values (I: 561.61-456.00min; S: 575.65-470.11min) which were further confirmed by the high activation energy (123.01 and 143.29kJmol-1). The enzyme demonstrated favorable results in terms of inulin and sucrose hydrolysis, being a maximum release of reducing sugars of 6.04 and 15.80g 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.

Co-production of Gluconic Acid and Fructo-oligosaccharides by Aureobasidium pullulans from Sugarcane Molasses: Effect of Oxygen Transfer Rate in Stirred Tank and Airlift Bioreactors

Aureobasidium pullulans NCYC 4012 was used to produce gluconic acid (GA) and fructo-oligosaccharides (FOS) from sugarcane molasses (ScM). For the first time, three types of bioreactors operating at different conditions of oxygenation—a stirred tank reactor (STR) operating under atmospheric pressure varying stirring rate, a pressurized STR under increased air pressure up to 4 bar of air, and an airlift reactor under atmospheric pressure varying aeration rate—were employed for GA and FOS biosynthesis by A. pullulans. The highest oxygenation conditions favored GA production in the three bioreactors, whereas the synthesis of FOS was not favored by high oxygen availability. GA yields close to 1 g·g−1 (grams of GA produced per grams of glucose consumed) were achieved in bioreactors under atmospheric pressure, and the highest titer of GA of 75 g·L−1 was obtained in the airlift bioreactor. In this last, the highest concentration of total FOS of 65 g·L−1 and yield of FOS of 0.58 g·g−1 were obtained.

Energy- and evolution-based design of inulosucrase for enhanced thermostability and inulin production.

Inulosucrase from Lactobacillus reuteri 121 (LrInu) exhibits promise in the synthesis of prebiotic inulin and fructooligosaccharides. However, for its use in industry, LrInu's thermostability is a crucial consideration. In this study, the computational program FireProt was used to predict the thermostable variants of LrInu. Using rational criteria, nine variants were selected for protein expression and characterization. The G237P variant was determined to be the greatest designed candidate due to its greatly enhanced stability and activity in comparison to the wild-type enzyme. The optimum temperature of G237P increased from 50 to 60°C, with an over 5-fold increase in the half-life. Spectroscopy studies revealed that the G237P mutation could prevent the structural change in LrInu caused by heat or urea treatment. Molecular dynamics (MD) simulations showed that the enhanced thermostability of the G237P variant resulted from an increase in structural rigidity and the number of native contacts within the protein molecule. In addition, G237P variant synthesizes inulin with greater efficiency than WT. KEY POINTS: • Thermostable inulosucrase variant(s) were designed by Fireprot server. • G237P variant showed significantly improved thermostability compared to the wild type. • Inulin is synthesized more efficiently by G237P variant.

Short-Chain Fructooligosaccharide Synthesis from Sugarcane Syrup with Commercial Enzyme Preparations and Some Physical and Antioxidation Properties of the Syrup and Syrup Powder.

Short-chain fructooligosaccharides (sc-FOS) are prebiotics beneficial to human health, which can be synthesized from raw material containing a high sucrose content. Sugarcane syrup (SS) without molasses removal contains sucrose as a major sugar and is a rich source of several bioactive compounds. The aim of this study is to investigate factors affecting sc-FOS synthesis from SS using commercial enzyme preparations containing fructosyltransferase activity as biocatalysts. sc-FOS content increased significantly as the sucrose concentration of SS in the reaction mixture increased up to 40% (w/v). Changes in carbohydrate compositions during the transfructosylating reaction of a pure sucrose solution and SS prepared from the two sugarcane cultivars Khon Kaen 3 and Suphanburi 50, catalyzed by Pectinex Ultra SP-L and Viscozyme L, showed similar profiles. Both enzymes showed a high ability to transfer fructosyl moieties to produce sc-FOS and a plateau of the total sc-FOS concentration was observed after 4 h of reaction time. For the pure sucrose solution and SS (Suphanburi 50), Viscozyme showed a superior ability to convert sucrose to Pectinex, with a higher sc-FOS yield (g FOS/100 g of initial sucrose), GF2 or 1-kestose yield (g GF2/g of initial sucrose) and GF3 or nystose yield (g GF3/g of initial sucrose). sc-FOS syrup (FOS SS) and the foam-mat-dried syrup powder prepared from SS and FOS SS, respectively, contained a high total phenolic content and possessed higher antioxidant activities than those prepared from pure sucrose, but contained lower calories.

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.

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.

Development of a functional prebiotic strawberry preparation by in situ enzymatic conversion of sucrose into fructo-oligosaccharides

Food industry has been pressed to develop products with reduced sugar and low caloric value, while maintaining unchanged their rheological and physicochemical properties. The development of a strawberry preparation for the dairy industry, with prebiotic functionality, was herein investigated by in situ conversion of its intrinsic sucrose content into prebiotic fructo-oligosaccharides (FOS). Two commercial enzymatic complexes, Viscozyme® L and Pectinex® Ultra SP-L, were evaluated for the synthesis of FOS. Operational parameters such as temperature, pH, and enzyme:substrate ratio (E:S) were optimized to maximize FOS yield. The rheological and physicochemical properties of the obtained strawberry preparation were evaluated. For functional analysis, the resistance of FOS to the harsh conditions of the gastro-intestinal digestion was evaluated by applying the standardized INFOGEST static protocol. At optimal conditions (60 ℃, pH 5.0), Pectinex® produced 265 ± 3 g·L−1 FOS, yielding 0.57 ± 0.01 gFOS·ginitial sucrose−1 after 7 h reaction (E:S:1:40); and Viscozyme® produced 295 ± 1 g·L−1 FOS, yielding 0.66 ± 0.00 gFOS·ginitial sucrose−1 after 5 h (E:S:1:30). The obtained strawberry preparations contained more than 50 %(w/w) prebiotic FOS incorporated (DP 3–5), with 80 % reduction of its sucrose content. The caloric value was therefore reduced by 26–31 %. FOS showed resistance to gastrointestinal digestion being only slightly hydrolysed (<10 %). 1F-Fructofuranosylnystose was not digested at any phase of the digestion. Although the physicochemical properties of the prebiotic preparations were different from the original one, parameters such as the lower °Brix, water activity, consistency and viscosity, and its different color, may be easily adjusted. Results indicate that in situ synthesis strategies are efficient alternatives in the manufacture of reduced sugar and low-caloric food products with prebiotic potential.

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.

Upcycling of carrot discards into prebiotics (fructooligosaccharides) as high value food ingredients

A great amount of carrots is discarded daily in the word because they do not meet the quality standards imposed by consumer market. The aim of this work was to evaluate the feasibility of using carrot discards as substrate of solid-state fermentation to produce β-fructofuranosidase by Aspergillus niger and its subsequent use to catalyze the synthesis of fructooligosaccharides (prebiotics) using carrot juice. Results indicated that carrot bagasse has adequate nutrients for the growth of A. niger, but supplementation with a source of nitrogen is required to obtain a high production of β-fructofuranosidase (90.82 U/mL). Maximum specific activity (251 U/mg) was obtained after 3 days of fermentation at 30 ºC, using an inoculum of 106 conidia/ mL. β-fructofuranosidase was successfully used to synthesize fructooligosaccharides using carrot juice as substrate, raising the possibility for future development of a natural functional beverage. Tweetable abstractA great amount of carrots is discarded daily. This paper evaluates the use of carrot bagasse to produce β-fructofuranosidase and its subsequent application in carrot juice to produce a natural beverage with prebiotics.

Cross-linked enzyme aggregates (combi-CLEAs) derived from levansucrase and variant inulosucrase are highly efficient catalysts for the synthesis of levan-type fructooligosaccharides

Efficient and convenient access to short to medium chain length levan-type fructooligosaccharides (LFOS) is needed in order realise the nutritional potential of this class of bioactive oligosaccharides. While LFOS are synthesised by fructansucrase enzymes, these reactions are routinely associated with high molecular weight fructan polymer formation. Recent studies have shown that FOS production can be enhanced by the combination of levansucrase and inulosucrase in a one-pot reaction. In the present study, the novel mixed enzyme cross-linked enzyme aggregates (combi-CLEAs) based on levansucrase (Lev) and N543A variant inulosucrase (Inu) were prepared by ammonium sulfate precipitation, followed by glutaraldehyde cross-linking. The effect of Lev and Inu ratio on the activity of combi-CLEAs was explored. The results showed that >70% of total sucrase activity was recovered after immobilization and that the combi-CLEAs produced high amounts of LFOS (degree of polymerisation 3 to 21), while high molecular weight polysaccharide production was much reduced. Biochemical characterisation indicated that the optimum pH and temperature of combi-CLEAs (pH 5.5 and 50ᵒC, respectively) were comparable to those of free enzyme; however, the stability of the enzyme was improved. In addition, these combi-CLEAs have operational stability for several reaction cycles, which makes them very attractive for biotechnology applications.

Continuous production of fructo-oligosaccharides using selectively immobilized fructosyltransferase from Aspergillus aculeatus onto Purolite® A109

Within this study, the investigation of potential selective immobilization of fructosyltransferase (FTase) derived from Aspergillus aculeatus (from commercial enzyme preparation Pectinex® Ultra SP-L) onto the polystyrenic macroporous resin Purolite® A109 and its further utilization for the synthesis of a highly valuable group of prebiotic compounds named fructo-oligosaccharides (FOS), was performed. The obtained results suggest that partial purification of fructosyltransferase from complex enzyme mixture with predominant pectinase activity (ratio of activity immobilization yields around 20.0) occurred by performing a simple immobilization protocol, provided that immobilization lasted for 5 h using an enzyme concentration of 50 mg g−1 of support under slightly acidic conditions (pH 6.0). Immobilized preparation was thereafter successfully utilized for FOS synthesis (56–59 % FOS of total sugars) in both a batch and air-lift reactor system. Operational stability study proved the exceptionally high potential of immobilized preparation for reproducible and cost-effective application, since 90 % of initial activity was preserved after ten consecutive reaction cycles. Thereafter, a prospectus for continuous production of FOS in the air-lift reactor was assayed. Achieved yields, 53 % FOS of total sugars for at least 9 h, indicated the great suitability of the obtained immobilized preparation for application in large-scale systems.

Engineering thermotolerant Yarrowia lipolytica for sustainable biosynthesis of mannitol and fructooligosaccharides

Yarrowia lipolytica is an important industrial microbe and a commonly used synthetic chassis in synthetic biology. In recent years, it has become increasingly popular in the field of industrial biotechnology to synthesize lipophilic molecules, such as lycopene, β-carotene, docosahexaenoic acid (DHA) and arachidonic acid. It is also a novel chassis for synthesizing erythritol and D-mannitol. Chemical synthesis is currently the most common method for producing mannitol, which has disadvantages, including multi-steps and great risks (under high pressure and high temperature). Based on the unique properties of Y. lipolytica to synthesize erythritol and mannitol simultaneously, this study overexpressed its heat shock protein Hsp90 gene to enhance its heat tolerance and ferment glucose at a higher temperature (34–35 °C) to realize the shift from mainly synthesizing erythritol to mannitol. The relative expression level of genes in the mannitol and erythritol synthesis pathway was detected by qPCR. The shift was achieved, according to preliminary findings, by raising the expression level of genes in the mannitol synthesis route and decreasing the expression level of genes in the erythritol synthesis pathway. To diversify the sugar alcohol profile, fructosyltransferase was displayed on the cell surface. The yeast cells, after mannitol synthesis, can also be used as a whole cell catalyst to catalyze the synthesis of fructooligosaccharides (FOS) from sucrose.

Aspergillus welwitschiae inulinase enzyme cocktails obtained on agro-material inducers for the purpose of fructooligosaccharides production

Production of fructooligosaccharides (FOS) is a trending topic due to their prebiotic effect becoming increasingly important for the modern human diet. The most suitable process for FOS production is the one using fungal inulinases. Introduction of new fungal inulinase producers and their implementation in production of inulinase enzymes is therefore gaining interest. This study provides a new approach to FOS synthesis by fungal enzyme complex without prior separation of any specific enzyme. Inulinase enzyme complexes could be used for the synthesis of FOS in two possible ways – hydrolysis of inulin (FOSh) and transfructosylation process of sucrose (FOSs), as demonstrated here. Depending on the fungal growth inducing substrate, a variety of inulinase enzyme complexes was obtained – one of which was most successful in production of FOSh and another one of FOSs. Substrates derived from crops: triticale, wheat bran, Jerusalem artichoke and Aspergillus welwitschiae isolate, previously proven as safe for use in food, were utilized for production of inulinase enzyme cocktails. The highest FOSs production was obtained by enzyme complex rich in β-fructofuranosidase, while the highest FOSh production was obtained by enzyme complex rich in endoinulinase. Both FOSh and FOSs showed antioxidant potential according to ABTS and ORAC, which classifies them as a suitable additive in functional food. Simultaneous zymographic detection of inulinase enzymes, which could contribute to expansion of the knowledge on fungal enzymes, was developed and applied here. It demonstrated the presence of different inulinase isoforms depending on fungal growth substrate. These findings, which rely on the innate ability of fungi to co-produce all inulinases from a cocktail, could be useful as a new, easy approach to FOS production by fungal enzymes without their separation and purification, contributing to cheaper and faster production processes.

Levan-type fructooligosaccharides synthesis by novel levansucrase-inulosucrase fusion enzyme

A novel strategy to enhance the yield of levan-type fructooligosaccharide (LFOS) was recently introduced, whereby levansucrase and inulosucrase reactions were coupled together in one pot. In order to simplify the process, in the present study we report the first example of a recombinant levansucrase-inulosucrase fusion protein and investigate its impact on LFOS production. Sequences for levansucrase from Bacillus amyloliquefaciens KK9 and inulosucrase from Lactobacillus reuteri 121 were fused genetically with a flexible eighteen residue glycine-serine peptide linker. SDS-PAGE analysis showed that the molecular weight of obtained fusion protein is approximately of 120 kDa, corresponding to the expected fusion protein molecular weight. Kinetic analysis revealed that after protein combination, the kinetic parameters of both enzymes are slightly changed. Biochemical characterization revealed that fusion did not affect the optimum pH and temperature for catalysis, but significantly change the stability of the enzyme. HPAEC-PAD analysis and enzymatic hydrolysis assays demonstrated that the fusion enzyme produced the desired higher yield of LFOS compared to those of individual levansucrases.

Optimization of the enzymatic synthesis of fructo-oligosaccharides using fructosyltransferase from aspergillus aculeatus

The obvious benefits of employing prebiotics as functional components in many food and feed products have resulted in higher demand for their industrial production, necessitating the development of more efficient and cost-effective manufacturing procedures. As a result, the goal of this study was to synthesize confirmed prebiotics, namely fructo-oligosaccharides (FOS), using sucrose as a substrate, since it allows the synthesis of oligosaccharides with lower polymerization degree, and consequently, a more pronounced prebiotic effect. Due to its availability, low market price, and high stability under industrial conditions, a commercial enzymatic mixture, Pectinex® Ultra SP-L, is used as a source of enzyme – fructosyltransferase (FTase). By optimizing reaction conditions such as pH, temperature, enzyme and substrate concentrations, as well as the duration of the process, the composition of the FOS mixture can be adjusted to fit the potential applications. It was found that by performing the reaction in an aqueous medium (pH 7), at a temperature of 50 °C using an enzyme concentration of 1% (v/v) and any sucrose concentration in the range of 200-700 g/L, it was possible to achieve maximum FOS yield of 60% of total carbohydrates within a 24 h. The produced syrup with a high content of FOS can be further used as an adequate food additive, or else, optimized proceses should be used for transformation of various food products (such as juices, jams, fillings, candies, cakes, etc.) in which sucrose dominates, creating products with lower caloric and higher functional value.