D-Tagatose: A Rare Sugar with Functional Properties and Antimicrobial Potential against Oral Species.

Carbohydrates have a dietary role, but excessive consumption of high-calorie sugars can contribute to an increased incidence of metabolic diseases and dental caries. Recently, carbohydrates with sweetening properties and low caloric value, such as D-tagatose, have been investigated as alternative sugars. D-tagatose is a rare sugar that has nutritional and functional properties of great interest for health. This literature review presents an approach to the biological effects of D-tagatose, emphasizing its benefits for oral health. Studies report that D-tagatose has antioxidant and prebiotic effects, low digestibility, reduced glycemic and insulinemic responses, and the potential to improve the lipid profile, constituting an alternative for diabetes mellitus and obesity. It can also be observed that D-tagatose has an antioxidant action, favoring the elimination of free radicals and, consequently, causing a reduction in cellular oxidative stress. Furthermore, it also has antibacterial potential against oral species. Regarding oral health, studies have shown that D-tagatose efficiently reversed bacterial coaggregations, including periodontopathogenic species, and impaired the activity and growth of cariogenic bacteria, such as S. mutans. D-tagatose significantly inhibited biofilm formation, pH decrease and insoluble glucan synthesis in S. mutans cultures. Salivary S. mutans counts were also significantly reduced by the consumption of chewing gum containing D-tagatose and xylitol. In addition, there is evidence that tagatose is effective as an air-polishing powder for biofilm decontamination. The literature indicates that D-tagatose can contribute to the prevention of systemic diseases, also constituting a promising agent to improve oral health.

Alternative Heterologous Expression of L-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 By Residual Whey Lactose Induction.

This study reports an alternative strategy for the expression of a recombinant L-AI from Enterococcus faecium DBFIQ E36 by auto-induction using glucose and glycerol as carbon sources and residual whey lactose as inducer agent. Commercial lactose and isopropyl ß-D-1-thiogalactopyranoside (IPTG) were also evaluated as inducers for comparison of enzyme expression levels. The enzymatic extracts were purified by affinity chromatography, characterized, and applied in the bioconversion of D-galactose into D-tagatose. L-AI presented a catalytic activity of 1.67 ± 0.14, 1.52 ± 0.01, and 0.7 ± 0.04 U/mL, when expressed using commercial lactose, lactose from whey, and IPTG, respectively. Higher activities could be obtained by changing the protocol of enzyme extraction and, for instance, the enzymatic extract produced with whey presented a catalytic activity of 3.8 U/mL. The specific activity of the enzyme extracts produced using lactose (commercial or residual whey) after enzyme purification was also higher when compared to the enzyme expressed with IPTG. Best results were achieved when enzyme expression was conducted using 4 g/L of residual whey lactose for 11 h. These results proved the efficacy of an alternative and economic protocol for the effective expression of a recombinant L-AI aiming its high-scale production.

Modeling of lactose enzymatic hydrolysis using Monte Carlo method

Background: Mathematical modeling is useful in the analysis, prediction, and optimization of an enzymatic process. Unlike the conventional modeling methods, Monte Carlo method has special advantages in providing representations of the molecule's spatial distribution. However, thus far, Monte Carlo modeling of enzymatic system is namely based on unimolecular basis, not suitable for practical applications. In this research, Monte Carlo modeling is performed for enzymatic hydrolysis of lactose for the purpose of real-time applications. Results: The enzyme hydrolysis of lactose, which is conformed to Michaelis­Menten kinetics, is modeled using the Monte Carlo modeling method, and the simulation results prove that the model predicts the reaction kinetics very well. Conclusions: Monte Carlo modeling method can be used to model enzymatic reactions in a simple way for real-time applications.

Biochemical Characterization of Heat-Tolerant Recombinant L-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 Strain with Feasible Applications in D-Tagatose Production.

D-Tagatose is a ketohexose, which presents unique properties as a low-calorie functional sweetener possessing a sweet flavor profile similar to D-sucrose and having no aftertaste. Considered a generally recognized as safe (GRAS) substance by FAO/WHO, D-tagatose can be used as an intermediate for the synthesis of other optically active compounds as well as an additive in detergent, cosmetic, and pharmaceutical formulations. This study reports important features for L-arabinose isomerase (EC 5.3.1.4) (L-AI) use in industry. We describe arabinose (araA) gene virulence analysis, gene isolation, sequencing, cloning, and heterologous overexpression of L-AI from the food-grade GRAS bacterium Enterococcus faecium DBFIQ E36 in Escherichia coli and assess biochemical properties of this recombinant enzyme. Recombinant L-AI (rL-AI) was one-step purified to homogeneity by Ni2+-agarose resin affinity chromatography and biochemical characterization revealed low identity with both thermophilic and mesophilic L-AIs but high degree of conservation in residues involved in substrate recognition. Optimal conditions for rL-AI activity were 50 °C, pH 5.5, and 0.3 mM Mn2+, exhibiting a low cofactor concentration requirement and an acidic optimum pH. Half-life at 45 °C and 50 °C were 1427 h and 11 h, respectively, and 21.5 h and 39.5 h at pH 4.5 and 5.6, respectively, showing the high stability of the enzyme in the presence of a metallic cofactor. Bioconversion yield for D-tagatose biosynthesis was 45% at 50 °C after 48 h. These properties highlight the technological potential of E. faecium rL-AI as biocatalyst for D-tagatose production.

One-Step Immobilization and Stabilization of a Recombinant Enterococcus faecium DBFIQ E36 L-Arabinose Isomerase for D-Tagatose Synthesis.

A recombinant L-arabinose isomerase from Enterococcus faecium DBFIQ E36 was immobilized onto multifunctional epoxide supports by chemical adsorption and onto a chelate-activated support via polyhistidine-tag, located on the N-terminal (N-His-L-AI) or on the C-terminal (C-His-L-AI) sequence, followed by covalent bonding between the enzyme and the support. The results were compared to reversible L-AI immobilization by adsorption onto charged agarose supports with improved stability. All the derivatives presented immobilization yields of above 75%. The ionic interaction established between agarose gels containing monoaminoethyl-N-aminoethyl structures (MANAE) and the enzyme was the most suitable strategy for L-AI immobilization in comparison to the chelate-activated agarose. In addition, the immobilized biocatalysts by ionic interaction in MANAE showed to be the most stable, retaining up to 100% of enzyme activity for 60 min at 60 °C and with Km values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively.

Efectos de la ingesta de los edulcorantes estevia y D-tagatosa sobre el metabolismo de la glucosa, ácido úrico y apetito-saciedad / Effects of stevia and D-tagatose sweeteners intake om glucose, uric acid metabolism and appetite-satiety

INTRODUCCIÓN: Si bien, los edulcorantes no nutritivos (ENN) estevia y D-tagatosa han sido reportados como seguros, han demostrado tener algunos efectos metabólicos tras su ingesta. OBJETIVO: Describir los efectos de la ingesta de estevia y D-tagatosa sobre el metabolismo de la glucosa y ácido úrico, y del apetito-saciedad, a partir de la evidencia disponible. MÉTODOS: Revisión descriptiva. Se realizó búsqueda en PubMed utilizando los siguientes términos y palabras clave: "stevia rebaudiana", "tagatose", "D-tagatose", "blood glucose", "insulin", "metabolic processes", "uric acid", "hyperuricemia", "appetite" o "satiety". El análisis de los estudios seleccionados fue discrecional. RESULTADOS: Existen estudios que demuestran efectos beneficiosos tras el consumo de estevia o D-tagatosa sobre el control glicémico, apetito y saciedad tanto en sujetos sanos como con alteraciones en el metabolismo de la glucosa. Por otra parte, un número importante de estudios que evalúan la ingesta de estevia reportan efectos nulos sobre dichos parámetros. En relación al ácido úrico, solo un estudio en sujetos con enfermedad renal crónica reporta aumento en la concentración de ácido úrico plasmático tras la ingesta de 500 mg/día de estevia. Pocos estudios han evaluado el efecto de la ingesta de D-tagatosa sobre uricemia, en sujetos sanos y diabéticos, reportando un aumento transitorio y significativo en los niveles de ácido úrico sérico, sin embargo, no se ha logrado demostrar un efecto hiperuricémico asociado. Es importante destacar que la metodología de los estudios revisados es heterogénea, especialmente en relación al tamaño muestral, tiempo, dosis y vía de adminitración del edulcorante. CONCLUSIÓN: La ingesta de estevia y D-tagatosa ha demostrado efectos beneficiosos sobre el metabolismo de la glucosa, el apetito y la saciedad. El efecto del consumo de D-tagatosa sobre ácido úrico sérico requiere mayor evidencia para demostrar su significancia clínica.

INTRODUCTION: No-nutritive sweeteners stevia and D-tagatose have been reported as safe according to their acceptable daily intake, however, they have been shown to have metabolic effects after their ingestion. OBJECTIVE: To describe the effects of stevia and D-tagatose intake on parameters associated to glucose, uric acid metabolism and on appetite-satiety, considering the available evidence. METHODS: Descriptive review. PubMed search was carried out to identify the totality of the published articles. The following terms and key words were used: "stevia rebaudiana", "tagatose", "D-tagatose", "blood glucose", "insulin", "metabolic processes", "uric acid", "hyperuricemia", "appetite" o "satiety". The analysis of the selected studies was discretionary. RESULTS: studies have shown beneficial effects of stevia and D-tagatose consumption on glycemic control, appetite and satiety in healthy subjects as well as subjects with impairment glucose metabolism. On the other hand, a significant number of studies evaluating estevia intake report null effects on these parameters. In relation to uric acid, only one study in subjects with chronic kidney disease reported an increase in plasmatic uric acid concentration after the intake of 500 mg/day of stevia. Several studies have evaluated the effect of D-tagatose intake on plasmatic uric acid, in healthy and diabetic subjects, reporting a transient and significant increase in serum uric acid levels, however, has not been able to demonstrate an associated hyperuricemic effect. It is important to highlight that the methodology of the studies reviewed is heterogeneous, especially in relation to sample size, dose administered, time and route of exposure to the sweetener. CONCLUSION: Stevia and D-tagatose intake has shown beneficial effects on glucose metabolism, appetite and satiety. The effects of the consumption of both sweeteners on uric acid require further study to demonstrate their clinic significance.

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg-1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.

Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose.

The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, ß-galactosidase from Bacillus circulans, l-arabinose (d-galactose) isomerase from Enterococcus faecium, and d-xylose (d-glucose) isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing l-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and d-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system.

Chemical improvement of chitosan-modified beads for the immobilization of Enterococcus faecium DBFIQ E36 L-arabinose isomerase through multipoint covalent attachment approach.

D-tagatose is produced from D-galactose by the enzyme L-arabinose isomerase (L-AI) in a commercially viable bioprocess. An active and stable biocatalyst was obtained by modifying chitosan gel structure through reaction with TNBS, D-fructose or DMF, among others. This led to a significant improvement in L-AI immobilization via multipoint covalent attachment approach. Synthetized derivatives were compared with commercial supports such as Eupergit(®) C250L and glyoxal-agarose. The best chitosan derivative for L-AI immobilization was achieved by reacting 4 % (w/v) D-fructose with 3 % (w/v) chitosan at 50 °C for 4 h. When compared to the free enzyme, the glutaraldehyde-activated chitosan biocatalyst showed an apparent activity of 88.4 U g (gel) (-1) with a 211-fold stabilization factor while the glyoxal-agarose biocatalyst gave an apparent activity of 161.8 U g (gel) (-1) with an 85-fold stabilization factor. Hence, chitosan derivatives were comparable to commercial resins, thus becoming a viable low-cost strategy to obtain high active L-AI insolubilized derivatives.