An efficient process for obtaining prebiotic oligosaccharides derived from lactulose using isomerized and purified whey permeate.

BACKGROUND: One of the most promising uses of whey permeate (WP) is the synthesis of prebiotic oligosaccharides. Herein, commercial WP was submitted to chemical isomerization catalysed by sodium borate at an alkaline pH and subsequent purification using anion-exchange resins to remove boron. Subsequently, purified mixtures were used to synthesize prebiotic oligosaccharides using ß-galactosidase from Bacillus circulans. RESULTS: Isomerization of concentrated WP (200 g L-1 lactose) gave rise to levels of lactulose up to 155.5 g L-1 after 30 min of reaction (molar ratio of boron/lactose, 1/1; pH 12; 70 °C). Boron was removed from the isomerized WP (IWP) using the combination of a strong acid (IR-120, H+ ) and a weak base (IRA-743) anion-exchange resins, reducing its level to <1 ppm, without loss of lactulose. During the transglycosylation reaction of purified IWP (lactose/lactulose ratio, 1/2.4) maximum content of prebiotic compounds was achieved, i.e. 690 g kg-1 WP after 3 h of reaction. CONCLUSION: This study shows that combined chemical-enzymatic reactions together with the purification of IWP results in an efficient synthesis of prebiotic oligosaccharides. © 2017 Society of Chemical Industry.

Analysis, structural characterization, and bioactivity of oligosaccharides derived from lactose.

The increasing interest for prebiotic carbohydrates as functional food ingredients has promoted the synthesis of galactooligosaccharides and new lactose derivatives. This review provides a comprehensive overview on the chromatographic analysis, structural characterization, and bioactivity studies of lactose-derived oligosaccharides. The most common chromatographic techniques used for the separation and structural characterization of this type of oligosaccharides, including GC and HPLC in different operational modes, coupled to various detectors are discussed. Insights on oligosaccharide MS fragmentation patterns, using different ionization sources and mass analyzers, as well as data on structural analysis by NMR spectroscopy are also described. Finally, this article deals with the bioactive effects of galacto oligosaccharides and oligosaccharides derived from lactulose on the gastrointestinal and immune systems, which support their consumption to provide significant health benefits.

Synthesis of prebiotic carbohydrates derived from cheese whey permeate by a combined process of isomerisation and transgalactosylation.

BACKGROUND: Lactose from cheese whey permeate (WP) was efficiently isomerised to lactulose using egg shell, a food-grade catalyst, and the subsequent transgalactosylation reaction of this mixture with ß-galactosidase from Bacillus circulans gave rise to a wide array of prebiotic carbohydrates derived from lactose and lactulose. RESULTS: Lactulose obtained by efficient isomerisation of WP (16.1% by weight with respect to the initial amount of lactose) showed great resistance to the hydrolytic action of ß-galactosidase from B. circulans, which preferentially hydrolysed lactose, acting as a galactosyl donor and acceptor. Lactulose had capacity as an acceptor, leading to the formation of lactulose-derived oligosaccharides. The enzymatic synthesis was optimised by studying reaction conditions such as pH, temperature, time, enzyme concentration and carbohydrate concentration. The maximum formation of galactooligosaccharides with degrees of polymerisation from 2 to 4 was achieved after 5 h of reaction at pH 6.5 and 50 °C with 300 g kg(-1) carbohydrates and 3 U mL(-1) ß-galactosidase. CONCLUSION: These findings indicate that the transgalactosylation of isomerised WP with ß-galactosidase from B. circulans could be a new and efficient method to obtain a mixture with 50% of potentially prebiotic carbohydrates composed of lactulose, and galactooligosaccharides derived from lactose and lactulose.

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.

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.

GC-MS characterisation of novel artichoke (Cynara scolymus) pectic-oligosaccharides mixtures by the application of machine learning algorithms and competitive fragmentation modelling.

Novel artichoke pectic-oligosaccharides (POS) mixtures have been obtained by enzymatic hydrolysis using four commercial enzyme preparations: Glucanex®200G, Pentopan®Mono-BG, Pectinex®Ultra-Olio and Cellulase from Aspergillus niger. Analysis by HPAEC-PAD showed that Cellulase from A. niger produced the greatest amount of POS (310.6 mg g-1 pectin), while the lowest amount was produced by Pentopan®Mono-BG (45.7 mg g-1 pectin). To determine structural differences depending on the origin of the enzyme, GC-MS spectra of di- and trisaccharides have been studied employing three machine learning algorithms: multilayer perceptron, random forest and boosted logistic regression. Machine learning models allowed characteristic m/z ions patterns to be established for each enzyme based on their GC-MS spectra with high prediction rates (above 95% on the test set). Possible chemical structures were given for some m/z ions having a decisive influence on these classifications. Finally, it was observed that several ions could be formed from specific POS structures.

Effect of purification of galactooligosaccharides derived from lactulose with Saccharomyces cerevisiae on their capacity to bind immune cell receptor Dectin-2.

Lactulose-derived oligosaccharides (OsLu) are prebiotic galactooligosaccharides (GOS) beneficial for human health including immunomodulatory properties; however, the molecular mechanism is unclear. OsLu produced by enzymatic synthesis can be purified with Saccharomyces cerevisiae (OsLu-Sc). We show that this purification introduces yeast-derived proteins reactive to Dectin-2, an innate immune receptor for fungal polysaccharides. Using a cell-based bioassay, we tested the binding of OsLu and GOS samples to Dectin-2. While OsLu purified with active charcoal and commercial GOS failed to bind to Dectin-2, we found OsLu-Sc bound to this receptor. The carbohydrate-binding incompetent mutant of Dectin-2 failed to bind to OsLu-Sc. These data suggest that OsLu-Sc introduced carbohydrate ligands for Dectin-2. In accordance with this, proteomic analysis revealed OsLu-Sc contained S. cerevisiae-derived mannoproteins. Therefore, our data highlight the importance of the purification method for OsLu, which may positively affect the bioactivity of OsLu. Data are available via ProteomeXchange with identifier PXD010495.

Trans-ß-galactosidase activity of pig enzymes embedded in the small intestinal brush border membrane vesicles.

This work highlights the utility of brush border membrane vesicles (BBMV) from the pig small intestine as a reliable model for gathering information about the reaction mechanisms involved in the human digestion of dietary carbohydrates. Concretely, the elucidation of the transgalactosylation mechanism of pig BBMV to synthesize prebiotic galacto-oligosaccharides (GOS) is provided, unravelling the catalytic activity of mammalian small intestinal ß-galactosidase towards the hydrolysis of GOS. This study reveals that pig BBMV preferably synthesizes GOS linked by ß-(1 → 3) bonds, since major tri- and disaccharide were produced by the transfer of a galactose unit to the C-3 of the non-reducing moiety of lactose and to the C-3 of glucose, respectively. Therefore, these results point out that dietary GOS having ß-(1 → 3) as predominant glycosidic linkages could be more prone to hydrolysis by mammalian intestinal digestive enzymes as compared to those linked by ß-(1 → 2), ß-(1 → 4), ß-(1 ↔ 1) or ß-(1 → 6). Given that these data are the first evidence on the transglycosylation activity of mammalian small intestinal glycosidases, findings contained in this work could be crucial for future studies investigating the structure-small intestinal digestibility relationship of a great variety of available prebiotics, as well as for designing tailored fully non-digestible GOS.

Mass spectrometric characterization of glycated beta-lactoglobulin peptides derived from galacto-oligosaccharides surviving the in vitro gastrointestinal digestion.

A mass spectrometric study has been carried out to elucidate the structures of glycated peptides obtained after in vitro gastrointestinal digestion of bovine beta-lactoglobulin (beta-LG) glycated with prebiotic galacto-oligosaccharides (GOS). The digests of both native and glycated beta-LG were analyzed by MALDI-MS, LC-ESI-MS, and LC-ESI-MS/MS. MALDI-MS profiles showed marked differences mainly related to the lower intensity of ions corresponding to the digest of glycated beta-LG. Overall, 58 and 23 unglycated peptides covering 97% and 63% of the mature beta-LG sequence could be identified in the digests of native and glycated samples, respectively. The LC-ESI-MS analyses corroborated the MALDI-MS results regarding the unglycated peptides but they also enabled an extensive investigation into the digest of glycated beta-LG. Thus, a total of 19 peptides glycated with GOS from two to seven hexose units could be identified. The tandem mass spectra of glycated peptides were mostly characterized by two neutral losses of 1026/1056, 864/894, 702/732, 540/570, 378/408, and 216/246 u, corresponding to the formation of the furylium ion and its subsequent "CHOH" loss, indicative of the peptide glycation with hepta-, hexa-, penta-, tetra-, tri-, and disaccharides, respectively. Also, other minor ionic species containing the furylium ring linked to different galactose units could be also detected, showing the diversity of the fragmentation pattern of peptides glycated with larger size carbohydrates. Finally, the putative GOS glycation sites could be determined at the NH(2)-terminal Leu residue and at Lys residues located in positions 14, 47, 75, 77, 83, 91, 100, 135, and 138.

Synthesis of oligosaccharides derived from lactulose and pectinex ultra SP-L.

The beta-galactosidase activity of the commercial enzymatic preparation Pectinex Ultra SP-L derived from Aspergillus aculeatus has been used to hydrolyze and transgalactosylate the prebiotic carbohydrate lactulose. During this reaction, new oligosaccharides derived from lactulose have been detected by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The presence of the trisaccharide 6'-galactosyl-lactulose, the major compound formed, has been confirmed by NMR. In addition, disaccharides and other oligosaccharides with higher retention times have been also detected. The effect of transgalactosylation conditions such as time, temperature, pH, and initial lactulose and enzyme concentrations, as well as product inhibition on oligosaccharide synthesis, has been studied. The optimal conditions for the formation of tri and higher oligosaccharides were 60 degrees C, pH 6.5, 450 g/L lactulose, 16 units/mL of enzyme, and 7 h of reaction. Selective formation of disaccharides was achieved under the same conditions with the exception of pH (4.5). The present work provides additional knowledge on the synthesis of new oligosaccharides with potential prebiotic properties.

Structural characterization of bovine beta-lactoglobulin-galactose/tagatose Maillard complexes by electrophoretic, chromatographic, and spectroscopic methods.

To investigate the influence of the type of carbonyl group of the sugar on the structural changes of proteins during glycation, an exhaustive structural characterization of glycated beta-lactoglobulin with galactose (aldose) and tagatose (ketose) has been carried out. Conjugates were prepared via Maillard reaction at 40 and 50 degrees C, pH 7, and a w = 0.44. The progress of the Maillard reaction was followed by indirect formation of Amadori and Heyns compounds, advanced glycation end products, and brown polymers. The structural characterization of glycoconjugates was conducted by using a number of analytical techniques such as RP-HPLC, isoelectric focusing, MALDI-ToF, SDS-PAGE, size exclusion chromatography, and spectrofluorimetry (tryptophan fluorescence). In addition, the surface hydrophobicity of the beta-lactoglobulin glycoconjugates was also assessed. The results showed a higher reactivity of galactose than tagatose to form the glycoconjugates, probably due to the higher electrophilicity of the aldehyde group. At 40 degrees C, more aggregation was produced when beta-lactoglobulin was conjugated with tagatose as compared to galactose. However, at 50 degrees C hardly any difference was observed in the aggregation produced by galactose and tagatose. These results afford more insight into the importance of the functional group of the carbohydrate moiety during the formation of protein-carbohydrate conjugates via Maillard reaction.

Enzymatic synthesis and identification of two trisaccharides produced from lactulose by transgalactosylation.

The enzymatic transgalactosylation during lactulose hydrolysis was studied using the beta-galactosidase from Kluyveromyces lactis and an initial lactulose concentration of 250 g/L. During hydrolysis of lactulose, the formation of two novel trisaccharides was followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A maximum trisaccharide yield of 14.05% was observed at 91.9% of lactulose hydrolysis. The two novel trisaccharides obtained by transglycosylation of lactulose were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignment were carried out using 1D ( 1H, 13C, and 1D TOCSY) and 2D (gCOSY, TOCSY, ROESY, gHSQC, and gHMBC) NMR experiments. The trisaccharides were shown to be lactulose-based structures; the main one has a Gal unit linked to C-6 of the galactose moiety, and the other one has a Gal unit linked to C-1 of the fructose moiety. Transglycosylation of lactulose allows for the obtention of galacto-oligosaccharides with new glycosidic structures and would open new routes to the synthesis of prebiotics.

Enzymatic extraction of pectin from artichoke (Cynara scolymus L.) by-products using Celluclast®1.5L.

The aim of this study was to optimise pectin extraction from artichoke by-products with Celluclast®1.5L using an experimental design analysed by response-surface methodology (RSM). The variables optimised were artichoke by-product powder concentration (2-7%, X1), enzyme dose (2.2-13.3 U g-1, X2) and extraction time (6-24 h, X3). The variables studied were galacturonic acid (GalA) (R2 93.9) and pectic neutral sugars (R2 92.8) content and pectin yield (R2 88.6). In the optimum extraction conditions (X1 = 6.5%; X2 = 10.1 U g-1; X3 = 27.2 h), pectin yield was 176 mgg-1 dry matter (DM). Considering 27.2 h of treatment as the +α value given by the design, the extraction time was increased up to 48 h obtaining a yield of 221 mg g-1 DM. The enzymatic method optimised allows obtaining artichoke pectin with good yield, high GalA (720 mg g-1 DM) and arabinose (127.6mgg-1 DM) contents and degree of methylation of 19.5%.

A Galacto-Oligosaccharides Preparation Derived From Lactulose Protects Against Colorectal Cancer Development in an Animal Model.

Colorectal cancer (CRC) is one of the most common neoplasias worldwide, and its incidence is increasing. Consumption of prebiotics is a useful strategy in order to prevent this important disease. These nutraceutical compounds might exert protective biological functions as antitumors. In order to test the chemopreventive effect of GOS-Lu (galacto-oligosaccharides derived from lactulose) prebiotic preparation against this cancer, an animal model (Rattus norvegicus F344) was used. In this model, two doses of azoxymethane (10 mg/kg) and two treatments with dextran sodium sulfate (DSS) were administered to the animals. Animals were fed for 20 weeks, and either control drinking water or drinking water containing 10% (w/w) GOS-Lu prebiotic preparation was provided to them. Animals were sacrificed after those 20 weeks, and their digestive tract tissues were analyzed. The results revealed a statistically significant reduction in the number of colon tumors in the GOS-Lu cohort with respect to control animals. Metagenomics sequencing was used for studying colon microbiota populations, revealing significant reductions in populations of pro-inflammatory bacteria families and species, and significant increases in interesting beneficial populations, such as Bifidobacterium. Therefore, oral administration of the prebiotic GOS-Lu preparation may be an effective strategy for preventing CRC.

2-Furoylmethyl amino acids as indicators of Maillard reaction during the elaboration of black garlic.

This study reports the formation of 2-furomethyl-amino acids (2-FM-AA) as indicators of Maillard reaction (MR) in black garlic elaboration, followed by the determination of furosine by ion-pair RP-HPLC-UV. The method was assessed for accuracy, repeatability and detection and quantitation limits indicating its adequacy. Traditional procedure of black garlic obtainment and the inclusion of convective drying (CDP) and ohmic heating (OHP) were assayed. For comparison purposes, three commercial black garlic samples were used. Together with furosine (2-FM-lysine), 2-furoylmethyl-γ-aminobutyric acid and 2-FM-arginine were detected. Levels of furosine were higher in CDP (46.6-110.1mg/100g protein) than in OHP (13.7-42.0mg/100g protein) samples, probably due to the most severe processing conditions used in the former. These results highlight the suitability of 2-FM-AA as chemical indicators to monitor the process of black garlic elaboration in order to obtain high quality products.

A GC-FID method for analysis of lysinoalanine.

Lysinoalanine (LAL) is an unwanted byproduct, which is formed during the processing of protein and protein-containing foods and feeds. A GC method for the quantitative analysis of LAL under conventional chromatographic conditions has been developed. The method was applied to the analysis of pure standard substances, boiled eggs, commercial caseinates, fresh cheese, fresh cheese made from milk supplemented with caseinate, and fresh cheeses adulterated with caseinate after cheese making process. Results demonstrated the reliability of the GC capillary chromatography for the analysis of LAL in protein containing foods. LOD and LOQ of 50 and 152 ppm of LAL in protein, respectively, were achieved. Range of linearity, precision, and accuracy of the method, measured using diaminopimelic acid as internal standard, were satisfactory for quantification purpose. The method might also be suitable for the quantitative analysis of other amino acids such as lysine and arginine. Results also indicated the utility of this methodology for detecting protein quality of egg products and caseinates as well as fresh cheese adulterations.

Characterization and in vitro digestibility of bovine beta-lactoglobulin glycated with galactooligosaccharides.

Galactooligosaccharides (GOS) are well-known prebiotic ingredients which can form the basis of new functional dairy products. In this work, the production and characterization of glycated beta-lactoglobulin (beta-LG) with prebiotic GOS through the Maillard reaction under controlled conditions ( a(w) = 0.44, 40 degrees C for 23 days) have been studied. The extent of glycation of beta-LG was evaluated by formation of furosine which progressively increased with storage for up to 16 days, suggesting that the formation of Amadori compounds prevailed over their degradation. RP-HPLC-UV, SDS-PAGE, and IEF profiles of beta-LG were modified as a consequence of its glycation. MALDI-ToF mass spectra of glycated beta-LG showed an increase of up to approximately 21% in its average molecular mass after storage for 23 days. Moreover, a decrease in unconjugated GOS (one tri-, two tetra-, and one pentasaccharide) was observed by HPAEC-PAD upon glycation. These results were confirmed by ESI MS. The stability of the glycated beta-LG to in vitro simulated gastrointestinal digestion was also described and compared with that of the unglycated protein. The yield of digestion products of glycated beta-LG was lower than that observed for the unglycated protein. The conjugation of prebiotic carbohydrates to stable proteins and peptides could open new routes of research in the study of functional ingredients.

Assessment of in Vitro Digestibility of Dietary Carbohydrates Using Rat Small Intestinal Extract.

There are few studies on the assessment of digestibility of nondigestible carbohydrates, despite their increasingly important role in human health. In vitro digestibility of a range of dietary carbohydrates classified as digestible (maltose, sucrose, and lactose), well-recognized (lactulose, fructooligosaccharides (FOS), and two types of galactooligosaccharides (GOS) differing in the predominant glycosidic linkage), and potential (lactosucrose and GOS from lactulose, OsLu) prebiotics using a rat small intestinal extract (RSIE) under physiological conditions of temperature and pH is described. Recognized and potential prebiotics were highly resistant to RSIE digestion although partial hydrolysis at different extents was observed. FOS and lactulose were the most resistant to digestion, followed closely by OsLu and more distantly by both types of GOS and lactosucrose. In GOS, ß(1 → 6) linkages were more resistant to digestion than ß(1 → 4) bonds. The reported in vitro digestion model is a useful, simple, and cost-effective tool to evaluate the digestibility of dietary oligosaccharides.

Uptake of 2S albumin allergens, Ber e 1 and Ses i 1, across human intestinal epithelial Caco-2 cell monolayers.

We have investigated the absorption rates of two purified major allergen 2S albumins, Ber e 1 from Brazil nuts (Bertholletia excelsa Humb. & Bonpl.) and Ses i 1 from white sesame seeds (Sesamum indicum L.), across human intestinal epithelial Caco-2 cell monolayers following gastrointestinal digestion in vitro. The transport from apical to basolateral side in cell monolayers was evaluated by RP-HPLC-UV and indirect competitive ELISA methods, being confirmed by western-blotting analysis. Significant amounts (approximately 15-25 nmol micromol(-1) initial amount/h) of intact Ber e 1 and Ses i 1 were found in the basolateral side. The absorption rates of both plant allergens through the cell monolayer were shown to be constant during the whole incubation period (4 h at 37 degrees C), verifying that the permeability of the membrane was not altered by the allergen digests. Our findings revealed that both purified 2S albumin allergens may be able to survive in immunologically reactive forms to the simulated harsh conditions of the gastrointestinal tract to be transported across the Caco-2 cell monolayers, so that they would be able to sensitize the mucosal immune system and/or elicit an allergic response.

Selective recovery of tagatose from mixtures with galactose by direct extraction with supercritical CO2 and different cosolvents.

A selective fractionation method of carbohydrate mixtures of galactose/tagatose, using supercritical CO(2) and isopropanol as cosolvent, has been evaluated. Optimization was carried out using a central composite face design and considering as factors the extraction pressure (from 100 to 300 bar), the extraction temperature (from 60 to 100 degrees C), and the modifier flow rate (from 0.2 to 0.4 mL/min, which corresponded to a total cosolvent percentage ranging from 4 to 18% vol). The responses evaluated were the amount (milligrams) of tagatose and galactose extracted and their recoveries (percent). The statistical analysis of the results provided mathematical models for each response variable. The corresponding parameters were estimated by multiple linear regression, and high determination coefficients (>0.96) were obtained. The optimum conditions of the extraction process to get the maximum recovery of tagatose (37%) were 300 bar, 60 degrees C, and 0.4 mL/min of cosolvent. The predicted value was 24.37 mg of tagatose, whereas the experimental value was 26.34 mg, which is a 7% error from the predicted value. Cosolvent polarity effects on tagatose extraction from mixtures of galactose/tagatose were also studied using different alcohols and their mixtures with water. Although a remarkable increase of the amount of total carbohydrate extracted with polarity was found, selective extraction of tagatose decreased with increase of polarity of assayed cosolvents. To improve the recovery of extracted tagatose, additional experiments outside the experimental domain were carried out (300 bar, 80 degrees C, and 0.6 mL/min of isopropanol); recoveries >75% of tagatose with purity >90% were obtained.