A review of the state of sweeteners science: the natural versus artificial non-caloric sweeteners debate. Stevia rebaudiana and Siraitia grosvenorii into the spotlight.

The rapid increase in the worldwide prevalence of obesity and certain non-communicable diseases (NCDs), such as: cardiovascular diseases, cancers, chronic respiratory diseases, and diabetes, has been mainly attributed to an excess of sugar consumption. Although the potential benefits of the synergetic use of sweeteners have been known for many years, recent development based on synthesis strategies to produce sucrose-like taste profiles is emerging where biocatalyst approaches may be preferred to produce and supply specific sweetener compounds. From a nutritional standpoint, high-intensity sweeteners have fewer calories than sugars while providing a major sweet potency, placing them in the spotlight as valuable alternatives to sugar. Due to the modern world awareness and incidence of metabolic diseases, both food research and growing markets have focused on two generally regarded as safe (GRAS) groups of compounds: the sweet diterpenoid glycosides present on the leaves of Stevia rebaudiana and, more recently, on the cucurbitane triterpene glycosides present on the fruits of Siraitia grosvenorii. In spite of their flavor advantages, biological benefits, including: antidiabetic, anticancer, and cardiovascular properties, have been elucidated. The present bibliographical review dips into the state-of-the-art of sweeteners and their role in human health as sugar replacements, as well as the biotransformation methods for steviol gylcosides and mogrosides apropos of enzymatic technology to update and locate the discoveries to date in the scientific literature to help boost the continuity of research efforts of the ongoing sweeteners.

In vitro and in vivo digestibility of prebiotic galactooligosacharides synthesized by ß-galactosidase from Lactobacillus delbruecki subsp. bulgaricus CRL450.

BACKGROUND: The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non-digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by ß-galactosidase (ß-gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450-ß-gal) from lactose, lactulose and lactitol. RESULTS: In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450-ß-gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush-border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose-oligosaccharides were the most resistant to the physiological conditions of mice. In general ß (1→6) linked products showed higher resistance compared to ß (1→3) oligosaccharides. CONCLUSION: In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1.

BACKGROUND: α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included. RESULTS: In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases. CONCLUSIONS: The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.

Identification and determination of 3-deoxyglucosone and glucosone in carbohydrate-rich foods.

BACKGROUND: α-Dicarbonyl compounds (α-DCs) such as 3-deoxyglucosone (3-DG) and glucosone are markers of both Maillard and degradation reactions of sugars and also of certain enzymatic processes. However, quantitation of these compounds is not straightforward when more abundant carbohydrates are present in real samples. Therefore in this work a GC/MS method was developed to separate monosaccharides, 3-DG and glucosone and applied to analyze them in carbohydrate-rich food products. Difructose anhydrides (DFAs), known markers of sugar degradation, were also determined. The effect of time and temperature in the production and storage of these compounds was also evaluated. RESULTS: Under optimized conditions, good separation between monosaccharides and α-DCs was achieved. Must syrups showed the highest concentrations of 3-DG and glucosone (average values 9.2 and 5.8 mg g(-1) respectively). Coffee substitutes based on carob, chicory and blends showed the highest content of DFAs. Heating and storage assays proved that production of 3-DG was influenced by temperature, while glucosone was more affected by storage time. CONCLUSION: The proposed method allows the rapid quantitation of 3-DG and glucosone along with carbohydrates and DFAs in different food products, which is essential to determine their degradation level. Moreover, the α-DC content in several foods is reported for the first time.

Exploring the modulatory effect of trehalose-derived galactooligosaccharides on key gut microbiota groups.

Trehalose (α-d-glucopyranosyl-(1-1)-α-D-glucopyranoside) has found applications in diverse food products as a sweetener, stabilizer, and humectant. Recent attention has focused on trehalose due to its contradictory effects on the virulence of Clostridium difficile. In this study, we investigate the impact of novel trehalose-derived galactooligosaccharides (Treh-GOS) on the human gut microbiota using in vitro fecal fermentation models. Distinct Treh-GOS structures elicit varying taxonomic responses. For instance, ß-Gal-(1-4)-trehalose [DP3(1-4)] leads to an increase of Bifidobacterium, comparable to results observed with commercial GOS. Conversely, ß-Gal-(1-6)-trehalose [DP3(1-6)] prompts an increase in Lactobacillus. Notably, both of these trisaccharides yield the highest concentrations of butyric acid across all samples. On the other hand, Treh-GOS tetrasaccharide mixture (DP4), featuring a novel trehalose galactosylation in both glucose units, fosters the growth of Parabacteroides. Our findings underscore the capacity of novel Treh-GOS to modulate the human gut microbiota. Consequently, these innovative galactooligosaccharides emerge as promising candidates for novel prebiotic applications.

Design Optimization of a Novel Catalytic Approach for Transglucosylated Isomaltooligosaccharides into Dietary Polyols Structures by Leuconostoc mesenteroides Dextransucrase.

Polyols, or sugar alcohols, are widely used in the industry as sweeteners and food formulation ingredients, aiming to combat the incidence of diet-related Non-Communicable Diseases. Given the attractive use of Generally Regarded As Safe (GRAS) enzymes in both academia and industry, this study reports on an optimized process to achieve polyols transglucosylation using a dextransucrase enzyme derived from Leuconostoc mesenteroides. These enzyme modifications could lead to the creation of a new generation of glucosylated polyols with isomalto-oligosaccharides (IMOS) structures, potentially offering added functionalities such as prebiotic effects. These reactions were guided by a design of experiment framework, aimed at maximizing the yields of potential new sweeteners. Under the optimized conditions, dextransucrase first cleared the glycosidic bond of sucrose, releasing fructose with the formation of an enzyme-glucosyl covalent intermediate complex. Then, the acceptor substrate (i.e., polyols) is bound to the enzyme-glucosyl intermediate, resulting in the transfer of glucosyl unit to the tested polyols. Structural insights into the reaction products were obtained through nuclear maneic resonance (NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analyses, which revealed the presence of linear α(1 → 6) glycosidic linkages attached to the polyols, yielding oligosaccharide structures containing from 4 to 10 glucose residues. These new polyols-based oligosaccharides hold promise as innovative prebiotic sweeteners, potentially offering valuable health benefits.

Tailoring the natural rare sugars D-tagatose and L-sorbose to produce novel functional carbohydrates.

This multidisciplinary study details the biosynthesis of novel non-digestible oligosaccharides derived from rare sugars, achieved through transfructosylation of D-tagatose and L-sorbose by levansucrase from Bacillus subtilis CECT 39 (SacB). The characterization of these carbohydrates using NMR and molecular docking was instrumental in elucidating the catalytic mechanism and substrate preference of SacB. Tagatose-based oligosaccharides were higher in abundance than L-sorbose-based oligosaccharides, with the most representative structures being: ß-D-Fru-(2→6)-ß-D-Fru-(2→1)-D-Tag and ß-D-Fru-(2→1)-D-Tag. In vitro studies demonstrated the resistance of tagatose-based oligosaccharides to intestinal digestion and their prebiotic properties, providing insights into their structure-function relationship. ß-D-Fru-(2→1)-D-Tag was the most resistant structure to small-intestinal digestion after three hours (99.8% remained unaltered). This disaccharide and the commercial FOS clustered in similar branches, indicating comparable modulatory properties on human fecal microbiota, and exerted a higher bifidogenic effect than unmodified tagatose. The bioconversion of selected rare sugars into ß-fructosylated species with a higher degree of polymerization emerges as an efficient strategy to enhance the bioavailability of these carbohydrates and promote their interaction with the gut microbiota. These findings open up new opportunities for tailoring natural rare sugars, like D-tagatose and L-sorbose, to produce novel biosynthesized carbohydrates with functional and structural properties desirable for use as emerging prebiotics and low-calorie sweeteners.

Macromolecular characterization of high ß-glucan oat lines.

Oat (Avena sativa) is a cereal grain rich in fibers, proteins, vitamins and minerals. Oats have been linked to several health benefits, such as lowering blood cholesterol levels, counteracting cardiovascular disease and regulating blood sugar levels. This study aimed to characterize two new oat lines with high ß-glucan content emanating from ethyl methyl sulphonate mutagenesis on the Lantmännen elite variety Belinda. Two of the mutated lines, and the mother variety Belinda, were profiled for ß-glucan, arabinoxylan, total dietary fiber and starch composition. In addition, total lipid and protein content, amino acid composition and ß-glucan molecular weights were analyzed. The high levels of ß-glucan resulted in a significant increase in total dietary fiber, but no correlation could be established between higher or lower levels of the assayed macromolecules, i.e., between arabinoxylan-, starch-, lipid- or protein levels in the mutated lines compared to the reference. The results indicate separate biosynthetic pathways for ß-glucans and other macromolecules and an independent regulation of the different polysaccharides studied. Therefore, ethyl methyl sulphonate mutagenesis can be used to increase levels of multiple macromolecules in the same line.

Transglycosylation catalysed by Caco-2 membrane disaccharidases: A new approach to understand carbohydrates digestibility.

Under appropriate experimental conditions, some glycoside hydrolases can catalyze transglycosylation reactions; a hypothesis associated with this is that the glycosidic linkages formed will be preferentially hydrolyzed under optimal conditions. Therefore, the hydrolytic and transglycosylation activities of isolated membranes from differentiated Caco-2 cells on sucrose, maltose and isomaltulose were evaluated. After the enzymatic reactions, the di- and trisaccharides obtained were identified by gas chromatography coupled to a mass spectrometer. Differentiated Caco-2 cell membranes exerted hydrolytic and transglycosylation activities towards the studied disaccharides. The obtained di- and trisaccharides were detected for the first time using human cell models. Due to the absence of maltase-glucoamylase complex (MGAM) in Caco-2 cells, and the known hydrolytic activity of sucrase-isomaltase (SI) towards sucrose, maltose and isomaltulose, it is plausible that the glycosidic linkages obtained after the transglycosylation reaction, mainly α-glucosyl-fructoses and α-glucosyl-glucoses, were carried out by SI complex. This approach can be used as a model to explain carbohydrate digestibility in the small intestine and as a tool to design new oligosaccharides with low intestinal digestibility.

Potentially Health-Promoting Spaghetti-Type Pastas Based on Doubly Modified Corn Starch: Starch Oxidation via Wet Chemistry Followed by Organocatalytic Butyrylation Using Reactive Extrusion.

Extruded spaghetti-type pasta systems were obtained separately either from native or oxidized starch prepared via wet chemistry with the aim of evaluating the effect of oxidation modification of starch. In addition to this, the butyrylation reaction (butyrate (Bu) esterification-short-chain fatty acid) using native or oxidized starch was analyzed under reactive extrusion (REx) conditions with and without the addition of a green food-grade organocatalyst (l(+)-tartaric acid) with the purpose of developing potentially health-promoting spaghetti-type pasta systems in terms of increasing its resistant starch (RS) values. These would be due to obtaining organocatalytic butyrylated starch or not, or the manufacture of a doubly modified starch (oxidized-butyrylated-starch oxidation followed by organocatalytic butyrylation) or not. To this end, six pasta systems were developed and characterized by solid-state 13C cross-polarization magic angle spinning nuclear magnetic resonance (CP MAS NMR) spectroscopy, degree of substitution (DS), attenuated total reflectance Fourier transform infrared (ATR/FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), pancreatic digestion, free Bu content analysis and in vitro starch digestibility. The results obtained here suggest that starch oxidation hydrolytically degrades starch chains, making them more susceptible to enzymatic degradation by α-amylase. However, the oxidized starch-based pasta systems, once esterified by Bu mainly on the amylose molecules (doubly modified pasta systems) increased their RS values, and this was more pronounced with the addition of the organocatalyst (maximum RS value = ~8%). Interestingly, despite the checked chemical changes that took place on the molecular structure of starch upon butyrylation or oxidation reactions in corn starch-based spaghetti-type pasta systems, and their incidence on starch digestibility, the orthorhombic crystalline structure (A-type starch) of starch remained unchanged.

Monomer and linkage type of galacto-oligosaccharides affect their resistance to ileal digestion and prebiotic properties in rats.

A detailed study was performed to compare the in vivo ileal digestibility and modulatory effects in fecal microbiota of novel galacto-oligosaccharides (GOS) derived from lactulose [GOS-Lu; degree of polymerization (DP) ≥2, 14.0% trisaccharides] and commercial GOS derived from lactose (GOS-La; DP ≥3, 35.1% trisaccharides) in growing rats (5 wk old). Rats were fed either a control diet or diets containing 1% (wt:wt) of GOS-Lu or GOS-La for 14 d. Quantitative analysis of carbohydrates from dietary and ileal samples demonstrated that the trisaccharide fraction of GOS-Lu was significantly more resistant to gut digestion than that from GOS-La, as indicated by their ileal digestibility rates of 12.5 ± 2.6% and 52.9 ± 2.7%, respectively, whereas the disaccharide fraction of GOS-Lu was fully resistant to the extreme environment of the upper digestive tract. The low ileal digestibility of GOS-Lu was due to the great resistance of galactosyl-fructoses to mammalian digestive enzymes, highlighting the key role played by the monomer type and linkage involved in the oligosaccharide chain. The partial digestion of GOS-La trisaccharides showed that glycosidic linkages (1→6) and (1→2) between galactose and glucose monomers were significantly more resistant to in vivo gastrointestinal digestion than the linkage (1→4) between galactose units. The absence of GOS-La and GOS-Lu digestion-resistant oligosaccharides in fecal samples indicated that they were readily fermented within the large intestine, enabling both types of GOS to have a potential prebiotic function. Indeed, compared with controls, the GOS-Lu group had significantly more bifidobacteria in fecal samples after 14 d of treatment. The number of Eubacterium rectale also was greater in the GOS-Lu and GOS-La groups than in controls. These novel data support a direct relationship between patterns of resistance to digestion and prebiotic properties of GOS.

Starch determination, amylose content and susceptibility to in vitro amylolysis in flours from the roots of 25 cassava varieties.

BACKGROUND: Cassava cultivars are classified following different criteria, such as cyanogenic glucoside content or starch content. Here, flours from the roots of 25 cassava varieties cultivated simultaneously in a single plantation, were characterized in terms of starch content (SC), amylose content (AC), α-amylolysis index (AI) and gel formation ability. Resistant starch content (RS) was measured in 10 of the samples. RESULTS: Cassava flours exhibited high SC, low AC and low AI values, with differences among varieties. Cluster analysis based on these parameters divided the cultivars in four groups differing mainly in SC and AC. AI and AC were inversely correlated (r = -0.59, P < 0.05) in 18 of the cultivars, suggesting AC as an important factor governing the susceptibility to enzymatic hydrolysis of starch in raw cassava. Differences in susceptibility to amylolysis, assessed by RS, were also recorded in the sample subset analyzed. Most flours yielded pastes or gels upon heating and cooling. Gels differed in their subjective grade of firmness, but none exhibited syneresis, confirming the low retrogradation proclivity of cassava starch. CONCLUSION: Some differences were found among cassava samples, which may be ascribed to inter-cultivar variation. This information may have application in further agronomic studies or for developing industrial uses for this crop.

Deciphering the Myrosinase-like Activity of Lactiplantibacillus plantarum WCFS1 among GH1 Family Glycoside Hydrolases.

The hydrolysis of plant glucosinolates by myrosinases (thioglucosidases) originates metabolites with chemopreventive properties. In this study, the ability to hydrolyze the glucosinolate sinigrin by cultures or protein extracts of Lactiplantibacillus plantarum WCFS1 was assayed. This strain possesses myrosinase-like activity as sinigrin was partly hydrolyzed by induced cultures but not by protein extracts. The 11 glycoside hydrolase GH1 family proteins, annotated as 6-phospho-ß-glucosidases, were the proteins most similar to plant myrosinases. The activity of these proteins was assayed against sinigrin and synthetic glucosides. As expected, none of the proteins assayed possessed myrosinase activity against sinigrin or the synthetic ß-thio-glucoside derivative or against the ß-glucoside. However, all 11 proteins were active on the phosphorylated-ß-glucoside derivative. Moreover, only eight of these proteins were active on phospho-ß-thioglucose. These results supported that, in L. plantarum WCFS1, glucosinolates may undergo previous phosphorylation, and GH1 proteins are the glycosidases involved in the hydrolysis of phosphorylated glucosinolates.

Prebiotic Potential of a New Sweetener Based on Galactooligosaccharides and Modified Mogrosides.

This study was conducted to investigate the sweetness intensity and the potential fecal microbiome modulation of galactooligosaccharides in combination with enzymatically modified mogrosides (mMV-GOS), both generated through a patented single-pot synthesis. Sweetness intensity was performed in vivo by trained sensory panelists. The impact on the human fecal microbiome was evaluated by in vitro pH-controlled batch fermentation, and bacterial populations and organic acid concentrations were measured by qPCR and GC-FID, respectively. Significant growth (p ≤ 0.05) during the fermentation at 10 h of bacterial populations includes Bifidobacterium (8.49 ± 0.44 CFU/mL), Bacteroides (9.73 ± 0.32 CFU/mL), Enterococcus (8.17 ± 0.42 CFU/mL), and Clostridium coccoides (6.15 ± 0.11 CFU/mL) as compared to the negative control counts for each bacterial group (7.94 ± 0.27, 7.84 ± 1.11, 7.52 ± 0.37, and 5.81 ± 0.08 CFU/mL, respectively) at the same time of fermentation. Likewise, the corresponding significant increase in production of SCFA in mMV-GOS at 10 h of fermentation, mainly seen in acetate (20.32 ± 2.56 mM) and propionate (9.49 ± 1.44 mM) production compared to a negative control at the same time (8.15 ± 1.97 and 1.86 ± 0.24 mM), is in line with a positive control (short-chain fructooligosaccharides; 46.74 ± 12.13 and 6.51 ± 1.91 mM, respectively) revealing a selective fermentation. In conclusion, these substrates could be considered as novel candidate prebiotic sweeteners, foreseeing a feasible and innovative approach targeting the sucrose content reduction in food. This new ingredient could provide health benefits when evaluated in human studies by combining sweetness and prebiotic fiber functionality.

In vitro digestion of polysaccharides: InfoGest protocol and use of small intestinal extract from rat.

Starch, dextran, pectin and modified citrus pectin were subjected to intestinal digestion following InfoGest protocol and a rat small intestine extract (RSIE) treatment. Gastric stage did not show any modification in the structure of the carbohydrates, except for modified pectin. Regarding intestinal phases, starch was hydrolyzed by different ways, resulting in a complementary behavior between InfoGest and RSIE. Contrarily, digestion of dextran was only observed using RSIE. Similar situation occurred in the case of pectins with RSIE, obtaining a partial hydrolysis, especially in the modified citrus pectin. However, citrus pectin was the less prone to hydrolysis by enzymes. The results demonstrated that InfoGest method underestimates the significance of the carbohydrates hydrolysis at the small intestine, thus indicating that RSIE is a very reliable and useful method for a more realistic study of polysaccharides digestion.

Enzymatic Synthesis and Structural Characterization of Novel Trehalose-Based Oligosaccharides.

Trehalose, α-d-glucopyranosyl-(1↔1)-α-d-glucopyranoside, is a disaccharide with multiple effects on the human body. Synthesis of new trehalose derivatives was investigated through transgalactosylation reactions using ß-galactosidase from four different species. ß-galactosidases from Bacillus circulans (B. circulans) and Aspergillus oryzae (A. oryzae) were observed to be the best biocatalysts, using lactose as the donor and trehalose as the acceptor. Galactosyl derivatives of trehalose were characterized using nuclear magnetic resonance spectroscopy. Trisaccharides were the most abundant oligosaccharides obtained followed by the tetrasaccharide fraction (19.5% vs 8.2% carbohydrates). Interestingly, the pentasaccharide [ß-Galp-(1→4)]3-trehalose was characterized for the first time. Greater oligosaccharide production was observed using ß-galactosidase from B. circulans than that obtained from A. oryzae, where the main structures were based on galactose monomers linked by ß-(1→6) and ß-(1→4) bonds with trehalose in the ending. These results indicate the feasibility of commercially available ß-galactosidases for the synthesis of trehalose-derived oligosaccharides, which might have functional properties, excluding the adverse effects of the single trehalose.

Characterization and antioxidant activity of avenanthramides from selected oat lines developed by mutagenesis technique.

From a mutagenized oat population, produced by ethyl methanesulfonate mutagenesis, hulled grains from 17 lines with elevated avenanthramide (AVN) content were selected and their AVN structures, concentrations and antioxidant potentials were determined by HPLC-MS2 and HPLC equipped with an on-line ABTS+ antioxidant detection system. The data obtained showed qualitative and quantitative differences in the synthesis of AVNs in the different lines, with a total AVN concentration up to 227.5 µg/g oat seed flour in the highest line, compared with 78.2 µg/g seed in the commercial line, SW Belinda. In total, 25 different AVNs were identified with avenanthramide B structures being among the most abundant, and AVN C structures having the highest antioxidant activity. The findings indicate the potential of oat mutagenesis in combination with a high precision biochemical selection method for the generation of stable mutagenized lines with a high concentration of total and/or individual AVNs in the oat seed grain.

Hydrolysis and transglycosylation activities of glycosidases from small intestine brush-border membrane vesicles.

In order to know the catalytic activities of the disaccharidases expressed in the mammalian small intestinal brush-border membrane vesicles (BBMV) high concentrated solutions of sucrose, maltose, isomaltulose, trehalose and the mixture sucrose:lactose were incubated with pig small intestine disaccharidases. The hydrolysis and transglycosylation reactions generated new di- and trisaccharides, characterized and quantified by GC-MS and NMR, except for trehalose where only hydrolysis was detected. In general, α-glucosyl-glucoses and α-glucosyl-fructoses were the most abundant structures, whereas no fructosyl-fructoses or fructosyl-glucoses were found. The in-depth structural characterization of the obtained carbohydrates represents a new alternative to understand the potential catalytic activities of pig small intestinal disaccharidases. The hypothesis that the oligosaccharides synthesized by glycoside hydrolases could be also hydrolysed by the same enzymes was confirmed. This information could be extremely useful in the design of new non-digestible or partially digestible oligosaccharides with potential prebiotic properties.

High-Yield Synthesis of Transglycosylated Mogrosides Improves the Flavor Profile of Monk Fruit Extract Sweeteners.

Luo Han Guo fruit extract (Siraitia grosvenorii), mainly composed of mogroside V (50%), could be considered a suitable alternative to free sugars; however, its commercial applications are limited by its unpleasant off-notes. In the present work, a central composite design method was employed to optimize the transglycosylation of a mogroside extract using cyclodextrin glucosyltransferases (CGTases) from three different bacteriological sources (Paenibacillus macerans, Geobacillus sp., and Thermoanaerobacter sp.) considering various experimental parameters such as maltodextrin and mogroside concentration, temperature, time of reaction, enzymatic activity, and pH. Product structures were determined by liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sensory analysis of glucosylated mogrosides showed an improvement in flavor attributes relevant to licorice flavor and aftereffect. Consequently, an optimum methodology was developed to produce new modified mogrosides more suitable when formulating food products as free sugar substitutes.

Bifidobacterial ß-Galactosidase-Mediated Production of Galacto-Oligosaccharides: Structural and Preliminary Functional Assessments.

In the current study the ability of four previously characterized bifidobacterial ß-galactosidases (designated here as BgaA, BgaC, BgaD, and BgaE) to produce galacto-oligosaccharides (GOS) was optimized. Of these enzymes, BgaA and BgaE were found to be promising candidates for GOS production (and the corresponding GOS mixtures were called GOS-A and GOS-E, respectively) with a GOS concentration of 19.0 and 40.3% (of the initial lactose), respectively. GOS-A and GOS-E were partially purified and structurally characterized. NMR analysis revealed that the predominant (non-lactose) disaccharide was allo-lactose in both purified GOS preparations. The predominant trisaccharide in GOS-A and GOS-E was shown to be 3'-galactosyllactose, with lower levels of 6'-galactosyllactose and 4'-galactosyllactose. These three oligosaccharides have also been reported to occur in human milk. Purified GOS-A and GOS-E were shown to be able to support bifidobacterial growth similar to a commercially available GOS. In addition, GOS-E and the commercially available GOS were shown to be capable of reducing Escherichia coli adhesion to a C2BBe1 cell line. Both in vitro bifidogenic activity and reduced E. coli adhesion support the prebiotic potential of GOS-E and GOS-A.