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.

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.

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.

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.

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.

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.

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.

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.

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.

Transglycosylation of Steviol Glycosides and Rebaudioside A: Synthesis Optimization, Structural Analysis and Sensory Profiles.

To improve flavor profiles, three cyclodextrin glucosyltransferases (CGTases) from different bacteriological sources, Paenibacillus macerans, Geobacillus sp. and Thermoanaerobacter sp., were used with an extract of steviol glycosides (SVglys) and rebaudioside A (RebA) as acceptor substrates in two parallel sets of reactions. A central composite experimental design was employed to maximize the concentration of glucosylated species synthesized, considering temperature, pH, time of reaction, enzymatic activity, maltodextrin concentration and SVglys/RebA concentration as experimental factors, together with their interactions. Liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-mass spectrometry (LC-ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to characterize and identify the chemical structures obtained along the optimization. To assess the impact on the sensory properties, a sensory analysis was carried out with a group of panelists that evaluated up to 16 sensorial attributes. CGTase transglucosylation of the C-13 and/or C-19 led to the addition of up to 11 glucose units to the steviol aglycone, which meant the achievement of enhanced sensory profiles due to a diminution of bitterness and licorice appreciations. The outcome herein obtained supposes the development of new potential alternatives to replace free sugars with low-calorie sweeteners with added health benefits.

Prebiotic Properties of Non-Fructosylated α-Galactooligosaccharides from PEA (Pisum sativum L.) Using Infant Fecal Slurries.

The interest for naturally-occurring oligosaccharides from plant origin having prebiotic properties is growing, with special focus being paid to supplemented products for infants. Currently, non-fructosylated α-galactooligosaccharides (α-GOS) from peas have peaked interest as a result of their prebiotic activity in adults and their mitigated side-effects on gas production from colonic bacterial fermentation. In this study, commercially available non-fructosylated α-GOS from peas and ß-galactooligosaccharides (ß-GOS) derived from lactose were fermented using fecal slurries from children aged 11 to 24 months old during 6 and 24 h. The modulatory effect of both GOS on different bacterial groups and bifidobacteria species was assessed; non-fructosylated α-GOS consumption was monitored throughout the fermentation process and the amounts of lactic acid and short-chain fatty acids (SCFA) generated were analyzed. Non-fructosylated α-GOS, composed mainly of manninotriose and verbascotetraose and small amounts of melibiose, were fully metabolized and presented remarkable bifidogenic activity, similar to that obtained with ß-GOS. Furthermore, non-fructosylated α-GOS selectively caused an increase on the population of Bifidobacterium longum subsp. longum and Bifidobacterium catenulatum/pseudo-catenulatum. In conclusion, non-fructosylated α-GOS could be used as potential ingredient in infant formula supplemented with prebiotic oligosaccharides.

Evaluation of the impact of a rat small intestinal extract on the digestion of four different functional fibers.

The degree of digestion, modulated by rat small intestinal extract on different functional fibers was investigated. In general, inulin-type fructans and fructooligosaccharides were the most resistant to the enzymatic digestion. Results evidenced the high-resistance of fructosyl-fructose bonds. This fits well with the concept of prebiotic carbohydrates. However, the mixture of melibiose, manninotriose and verbascotetraose (α-GOS) from peas, with a considerably lower molecular weight (0.6 kDa) than the fructans studied, were highly digested (61.2%). Interestingly, the Gal-(1 → 6)-Gal bonds present into the manninotriose and verbascotetraose were more prone to be hydrolyzed than Gal-(1 → 6)-Glc (melibiose). However, when melibiose was the only disaccharide present in the reaction mixture, the hydrolysis was also high (67.7%). The use of small intestinal enzymatic preparations is a realistic approximation to evaluate the digestion of different carbohydrates, thus, showing that recognized non-digestible carbohydrates can also be partially digested.

Kinetic study on the digestibility of lactose and lactulose using small intestinal glycosidases.

Lactose is mostly hydrolysed in the small intestine, whereas lactulose, recognised prebiotic carbohydrate, reaches the colon to be fermented by the intestinal microbiota. Digestibility of these substrates was investigated by an in vitro digestion model using a Rat Small Intestine Extract (RSIE). A kinetic study of lactose digestion showed levels of hydrolysis (82.8%) at 0.2 mg*mL-1 and the highest hydrolysis rate constant (kobt). Considering these conditions, lactulose showed high resistance to intestinal digestion by RSIE, resulting in low hydrolysis degrees (20.4%) after 5 h reaction. These results underline the suitability of these intestinal extracts under the studied conditions, as a reliable tool to evaluate carbohydrate digestion and support the evidences towards the higher resistance of galactosyl-fructose linkages during its intestinal degradation.

Hydrolysis and transgalactosylation catalysed by ß-galactosidase from brush border membrane vesicles isolated from pig small intestine: A study using lactulose and its mixtures with lactose or galactose as substrates.

Enzymatic transgalactosylation, in different concentrated carbohydrate solutions, was investigated using brush border membrane vesicles (BBMV) from the pig small intestine. When lactulose was incubated with BBMV, the hydrolytic activity of the enzyme towards the disaccharide was observed to be very low compared to that towards the lactose, but the linkage specificity ß-(1 â†’ 3), previously observed in lactose solutions, was not significantly affected. As in the case of lactose, lactulose transgalactosylation by BBMV synthesizes the corresponding 3'-galactosyl derivative (ß-Gal-(1 â†’ 3)-ß-Gal-(1 â†’ 4)-ß-Fru). Fructose released during lactulose hydrolysis was found to be good acceptor for the transgalactosylation reaction, giving rise to the synthesis of the disaccharide ß-Gal-(1 â†’ 5)-Fru. When incubating an 80/20 mixture of lactulose/galactose, the presence of galactose did not affect the qualitative composition of the transglycosylated substrate but enhanced the synthesis of ß-Gal-(1 â†’ 5)-Fru and decreased the synthesis of ß-(1 â†’ 3) glycosidic bonds. The marked tendency for synthesizing this linkage indicates that under hydrolytic conditions, ß-Gal-(1 â†’ 3)-Gal- and ß-Gal-(1 â†’ 5)-Fru glycosidic bonds would be preferentially digested.

Unravelling the carbohydrate specificity of MelA from Lactobacillus plantarum WCFS1: An α-galactosidase displaying regioselective transgalactosylation.

This comprehensive work addresses, for the first time, the heterologous production, purification, biochemical characterization and carbohydrate specificity of MelA, a cold-active α-galactosidase belonging to the Glycoside Hydrolase family 36, from the probiotic organism Lactobacillus plantarum WCFS1. The hydrolytic activity of MelA α-galactosidase on a wide range of p-nitrophenyl glycoside derivatives and carbohydrates of different molecular-weights showed its high selectivity and efficiency towards the α(1 â†’ 6) glycosidic bonds involving the anomeric carbon of galactose and the C6-hydroxyl group of galactose or glucose units. MelA α-galactosidase also presented a high regioselectivity, efficiency and diversity in accommodating donor and acceptor substrates for the synthesis of α-GOS through transgalactosylation reactions. The catalytic mechanism of MelA for the production of α-GOS was elucidated, revealing its great preference for the transfer of galactosyl residues to the C6-hydroxyl group of galactose units to elongate the chain of α-GOS having either a terminal sucrose (raffinose family oligosaccharides, RFOS) or a terminal glucose (melibiose, manninotriose and verbascotetraose). Our findings indicate the feasibility of using MelA α-galactosidase from Lactobacillus plantarum WCFS1 in the hydrolysis of RFOS and in the efficient and versatile synthesis of α-GOS with appealing functional properties in the context of food and nutraceutical applications.

In vitro Gastrointestinal Models for Prebiotic Carbohydrates: A Critical Review.

BACKGROUND: In the last decade, various consortia and companies have created standardized digestion protocols and gastrointestinal simulators, such as the protocol proposed by the INFOGEST Consortium, the simulator SHIME, the simulator simgi®, the TIM, etc. Most of them claim to simulate the entire human gastrointestinal tract. However, few results have been reported on the use of these systems with potential prebiotic carbohydrates. METHODS: This critical review addresses the existing data on the analysis of prebiotic carbohydrates by different in vitro gastrointestinal simulators, the lack of parameters that could affect the results, and recommendations for their enhancement. RESULTS: According to the reviewed data, there is a lack of a realistic approximation of the small intestinal conditions, mainly because of the absence of hydrolytic conditions, such as the presence of small intestinal brush border carbohydrases that can affect the digestibility of different carbohydrates, including prebiotics. CONCLUSION: There is a necessity to standardize and enhance the small intestine simulators to study the in vitro digestibility of carbohydrates.