Galacto-oligosaccharide synthesis from lactose solution or skim milk using the β-galactosidase from Bacillus circulans

The synthesis of galacto-oligosaccharides (GOS) catalyzed by a novel commercial preparation of β-galactosidase from Bacillus circulans (Biolactase) was studied, and the products were characterized by MS and NMR. Using 400 g/L lactose and 1.5 enzyme units per milliliter, the maximum GOS yield, measured by HPAEC-PAD analysis, was 165 g/L (41% w/w of total carbohydrates in the mixture). The major transgalactosylation products were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The GOS yield increased to 198 g/L (49.4% w/w of total carbohydrates) using a higher enzyme concentration (15 U/mL), which minimized the enzyme inactivation under reaction conditions. Using skim milk (with a lactose concentration of 46 g/L), the enzyme also displayed transgalactosylation activity: maximum GOS yield accounted for 15.4% (7.1 g/L), which was obtained at 50% lactose conversion.     

Enzymatic synthesis of galacto-oligosaccharides in an organic-aqueous biphasic system by a novel β-galactosidase from a metagenomic library

Prebiotic galacto-oligosaccharides (GOS) were effectively synthesized from lactose in organic-aqueous biphasic media by a novel metagenome-derived β-galactosidase BgaP412. A maximum GOS yield of 46.6% (w/w) was achieved with 75.4% lactose conversion rate in the cyclohexane/buffer system [95:5 (v/v) cyclohexane/buffer] under the optimum reaction conditions (initial lactose concentration = 30% (w/v), T = 50 °C, pH 7.0, and t = 8 h). The corresponding productivity of GOS was approximately 17.5 g L(-1) h(-1). The GOS mixture consisted of tri-, tetra-, and pentasaccharides. Trisaccharides were the chief component of reaction products. These experimental results showed that a low water content, a high initial lactose concentration, and an elevated reaction temperature could significantly promote the transgalactosylation activity of β-galactosidase BgaP412; at the same time, the enhanced GOS yield in an organic-aqueous biphasic system is because of the fact that thermodynamic equilibrium can be shifted to the synthetic direction by reversing the normal hydrolysis.     

Isomerization of lactose-derived oligosaccharides: a case study using sodium aluminate

Galactooligosaccharides (GOS) obtained during the enzymatic hydrolysis of lactose contain large amounts of glucose, galactose, and unreacted lactose, which do not have prebiotic properties and increase the calorific value of the product. In this work, the isomerization of the GOS mixture by the action of sodium aluminate has been studied. During the reaction, lactose, glucose, and galactose were isomerized to lactulose, fructose, and tagatose, respectively, and in addition allolactose, 6-galactobiose, and 6'-galactosyl-lactose were also converted to the corresponding keto-sugars. The effect of time, temperature, and aluminate/initial lactose ratio has been studied. After 9 h at 40 degrees C and molar ratio aluminate/lactose 3:1, the isomerization yield was >60%, and the amount of final carbohydrates was close to 90% of the initial product. This process considerably decreases the amount of lactose, glucose, and galactose.     

Comparison between discontinuous and continuous lactose conversion processes for the production of prebiotic galacto-oligosaccharides using beta-galactosidase from Lactobacillus reuteri

Galacto-oligosaccharide (GOS) formation from lactose in discontinuous and continuous modes of conversion was investigated using beta-galactosidase (beta-gal) from Lactobacillus reuteri. A continuous stirred tank reactor (CSTR) with an external crossflow membrane was set up, and continuous GOS production was analyzed and compared to the batchwise formed GOS product. Marked differences were detected for the two reactor setups. Above 65% lactose conversion, the GOS yield was lower for the CSTR due to a lower content of tri- and tetrasaccharides in the reaction mixture. In the CSTR, beta-gal from L. reuteri showed up to 2-fold higher specificity toward the formation of beta-(1-->6)-linked GOS, with beta-D-Galp-(1-->6)-D-Glc and beta-D-Galp-(1-->6)-D-Gal being the main GOS components formed under these conditions. This could be used to synthesize more defined GOS products.     

Production of prebiotic galacto-oligosaccharides from lactose using beta-galactosidases from Lactobacillus reuteri

The beta-galactosidases (beta-Gals) of Lactobacillus reuteri L103 and L461 proved to be suitable biocatalysts for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. Maximum GOS yields were 38% when using an initial lactose concentration of 205 g/L and at approximately 80% lactose conversion. The product mixtures were analyzed by capillary electrophoresis (CE) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Disaccharides other than lactose and trisaccharides made up the vast majority of GOS formed. The main products were identified as beta-d-Galp-(1-->6)-d-Glc (allolactose), beta-d-Galp-(1-->6)-d-Gal, beta-d-Galp-(1-->3)-d-Gal, beta-d-Galp-(1-->6)-Lac, and beta-d-Galp-(1-->3)-Lac. There were no major products with beta1-->4 linkages formed. Both intermolecular and intramolecular transgalactosylation were observed. d-Galactose proved to be a very efficient galactosyl acceptor; thus, a relatively large amount of galactobioses was formed. Monosaccharides could be conveniently separated from the mixture by chromatography using a strong cation-exchange resin.     

Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose

The enzymatic transgalactosylation from lactose to fructose leading to the prebiotic disaccharide lactulose was investigated using the beta-galactosidase from Aspergillus oryzae and the hyperthermostable beta-glycosidase from Pyrococcus furiosus (CelB). The conditions for highest lactulose yields relative to the initial lactose concentration were established on a 1 mL scale. Dependent on the initial molar ratio of lactose to fructose, more or fewer oligosaccharides other than lactulose were generated. Bioconversions on a 30 mL scale in a stirred glass reactor were performed, and lactulose yields of 46 mmol/L (44% relative to lactose) for CelB and 30 mmol/L (30% relative to lactose) for A. oryzae beta-galactosidase were achieved. Only <5% of other oligosaccharides were detectable. The corresponding productivities were 24 and 16 mmol/L/h, respectively. The molecular structure of lactulose was investigated in detail and confirmed after purification of the reaction solution by LC-MS and 1D and 2D NMR. Lactulose (4-O-beta-D-galactopyranosyl-D-fructose) was unambiguously proved to be the major transglycosylation disaccharide.     

Characterization of a heterodimeric GH2 β-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.     

Production of Galacto-oligosaccharides by the β-Galactosidase from Kluyveromyces lactis : comparative analysis of permeabilized cells versus soluble enzyme

The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-β(1→6)-Gal] and allolactose [Gal-β(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-β(1→3)-Glc, was carried out. GOS yield obtained with soluble β-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of β-galactosidase when permeabilized whole cells were used.     

Evaluation of oligosaccharide synthesis from lactose and lactulose using β-galactosidases from Kluyveromyces isolated from artisanal cheeses

The β-galactosidase activity of 15 Kluyveromyces strains isolated from cheese belonging to Kluyveromyces lactis and Kluyveromyces marxianus species was tested for the production of oligosaccharides derived from lactose (GOS) and lactulose (OsLu). All Kluyveromyces crude cell extracts (CEEs) produced GOS, such as 6-galactobiose and 3'-, 4'-, and 6'-galactosyl-lactose. At 4 h of reaction, the main trisaccharide formed was 6'-galactosyl-lactose (20 g/100 g of total carbohydrates). The formation of OsLu was also observed by all CEEs tested, with 6-galactobiose, 6'-galactosyl-lactulose, and 1-galactosyl-lactulose being found in all of the reaction mixtures. The synthesis of trisaccharides predominated over other oligosaccharides. K. marxianus strain O3 produced the highest yields of GOS and OsLu after 4 h of reaction, reaching 42 g/100 g of total carbohydrates (corresponding to 80% lactose hydrolysis) and 45 g/100 g of total carbohydrates (corresponding to 87% lactulose hydrolysis), respectively. Therefore, the present study contributes to a better insight into dairy Kluyveromyces β-galactosidases and shows the feasibility of these enzymes to transglycosylate lactose and lactulose, producing high yields of prebiotic oligosaccharides.     

Mutagenicity of heated sugar-casein systems: effect of the Maillard reaction

The formation of mutagens after the heating of sugar-casein model systems at 120 degrees C was examined by the Ames test, using Salmonella typhimurium strain TA100. Several sugars (glucose, fructose, galactose, tagatose, lactose, and lactulose) were compared in their mutagenicities. Mutagenicity could be fully ascribed to Maillard reaction products and strongly varied with the kind of sugar. The differences in mutagenicity among the sugar-casein systems were caused by a difference in reaction rate and a difference in reaction mechanism. Sugars with a comparable reaction mechanism (glucose and galactose) showed a higher mutagenic activity corresponding with a higher Maillard reactivity. Disaccharides showed no mutagenic activity (lactose) or a lower mutagenic activity (lactulose) than their corresponding monosaccharides. Ketose sugars (fructose and tagatose) showed a remarkably higher mutagenicity compared with their aldose isomers (glucose and galactose), which was due to a difference in reaction mechanism.     

Production of substrate for galactose oxidase by depolymerization of an arabinogalactan-peptide from wheat flour

Water extractable arabinogalactan-peptide (WE-AGP) isolated from white wheat flour was depolymerized enzymatically to liberate substrate for a galactose oxidase from Dactylium dendroides. A crude liquid pectolytic preparation from Aspergillus niger (p70) displayed activities capable of converting WE-AGP into a substrate for galactose oxidase. The most favorable substrate was observed when WE-AGP was not fully depolymerized into galactose and arabinose. alpha-L-Arabinofuranosidase B from A. niger was also able to produce substrate from WE-AGP; arabinofuranosidase-treated WE-AGP was a better substrate for galactose oxidase than galactose. Treatment by the crude p70 and purified enzymes showed that alpha-L-arabinofuranosidase was partly responsible for the production of substrate, whereas beta-galactosidase did not result in any substrate production or improve the effect of alpha-L-arabinofuranosidase. However, the positive effect of alpha-L-arabinofuranosidase was increased when p70 was added at the same level of arabinofuranosidase activity, suggesting that additional enzyme activities present in p70 were responsible for production of substrate for galactose oxidase.     

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.     

Chemical indicators of heat treatment in fortified and special milks

Carbohydrate and furosine contents in 12 commercial fortified and special milk samples (pasteurized goat's and ewe's milks; ultrahigh-temperature (UHT) goat's milk, UHT milks fortified with calcium, magnesium, fiber, or royal jelly and honey; and lactose-hydrolyzed milks) were analyzed. Except for lactose-hydrolyzed milks, furosine, lactose, lactulose, galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, and myo-inositol contents were similar to the previously reported values for UHT or pasteurized milk samples. In lactose-hydrolyzed milks, lactulose was not detectable and lactose was present in low amount; high levels of glucose, galactose, fructose, tagatose, and furosine were also detected in this type of milk. Results found in commercial milks were compared to those obtained in laboratory-prepared UHT milks with lactose hydrolyzed prior to heating. Hydrolysis of lactose before thermal treatments promoted elevated accumulation of reducing sugars (galactose and glucose) that could be partially converted to the corresponding isomers (tagatose and fructose) during heating. In addition, the reducing sugars could also react with the amino groups of proteins, giving rise to the corresponding Amadori compound. According to the obtained results, heating prior to hydrolysis of lactose is suggested to avoid a considerable loss of available lysine.     

Purification of lactulose from mixtures with lactose using pressurized liquid extraction with ethanol-water at different temperatures

The viability of the purification of lactulose from a mixture with lactose [70:30 (w/w)] using pressurized liquid extraction (PLE) at 1500 psi for 30 min was studied. Different temperatures (from 40 to 130 degrees C) and proportions of ethanol:water (70:30, 80:20, 90:10, 95:5, and 100:0) as the extraction solvent were assayed. Lactose and lactulose were measured by gas chromatographic analysis as their trimethylsilyl derivatives. Data were fitted through multiple linear regressions to different quadratic models to describe both the extraction yield (in terms of mg of lactulose) and the purity of the lactulose extracted. The optimum extraction conditions provided by the model were as follows: extraction temperature, 40 degrees C; and solvent composition, 70:30 ethanol:water. PLE extraction under the optimized conditions was also applied to purify lactulose from lactose in a synthesis mixture. To our knowledge, this is the first time that PLE has been tested for extraction and purification of lactulose from its mixture with lactose; this technique showed several advantages over classical methods such as the short extraction time and the low solvent consumption.     

Effect of the substrate concentration and water activity on the yield and rate of the transfer reaction of β-galactosidase from Bacillus circulans

Prebiotic galactosyl oligosaccharides (GOS) are produced from lactose by the enzyme β-galactosidase. It is widely reported that the highest GOS levels are achieved when the initial lactose concentration is as high as possible; however, little evidence has been presented to explain this phenomenon. Using a system composed of the commercial β-galactosidase derived from Bacillus circulans known as Biolacta FN5, lactose and sucrose, the relative contribution of water activity, and substrate availability were assessed. Oligosaccharide levels did not appear to be affected by changes in water activity between 1.0 and 0.77 at a constant lactose concentration. The maximum oligosaccharide concentration increased at higher initial concentrations of lactose and sucrose, while initial reaction rates for transfer increased but remained constant for hydrolysis. This suggests that the high oligosaccharide levels achieved at the raised initial saccharide concentration are due to increases in reactions that form oligosaccharides rather than decreases in concurrent reactions, which degrade oligosaccharides. There were different effects from changing the initial concentration of lactose compared to sucrose, suggesting that the ability of lactose to act as a donor saccharide may be more important for increasing maximum oligosaccharide concentrations than the combined ability of both saccharides to act as galactosyl acceptors.     

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.     

Homodimeric β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization

The lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081, encoding a β-galactosidase of the glycoside hydrolase family GH2, was cloned into different inducible lactobacillal expression vectors for overexpression in the host strain Lactobacillus plantarum WCFS1. High expression levels were obtained in laboratory cultivations with yields of approximately 53000 U of β-galactosidase activity per liter of medium, which corresponds to ~170 mg of recombinant protein per liter and β-galactosidase levels amounting to 63% of the total intracellular protein of the host organism. The wild-type (nontagged) and histidine-tagged recombinant enzymes were purified to electrophoretic homogeneity and further characterized. β-Galactosidase from L. bulgaricus was used for lactose conversion and showed very high transgalactosylation activity. The maximum yield of galacto-oligosaccharides (GalOS) was approximately 50% when using an initial concentration of 600 mM lactose, indicating that the enzyme can be of interest for the production of GalOS.     

Nutritional Enhancement of Soy Meal via Aspergillus oryzae Solid‐State Fermentation

Antinutritional factors in soy meal (SM) include trypsin inhibitor, galactooligosaccharides (GOSs), structural polysaccharides, and large‐molecular‐weight protein. These antinutritional factors limit the usage of SM for young monogastric animals. Aspergillus oryzae solid‐state fermentation was applied to eliminate these factors, and changes in physical and chemical characteristics of SM were investigated. A. oryzae–treated SM was more nutrient dependent than oxygen dependent, which was illustrated by scanning electron microscopy. After 36 h of fermentation, the concentration of GOSs (raffinose, stachyose, and verbascose) and trypsin inhibitor decreased from an initial value of 9.48 mmol/100 g to a nondetectable level. Structural polysaccharides decreased by 59% (w/w), and the degree of hydrolysis of SM protein increased from an initial value of 0.9 to 7% (w/w) through the seven‐day fermentation. Fermentation also modified nutritional factors. Protein content increased from 50.47 to 58.93% (w/w) after 36 h of fermentation. Amino acid contents were significantly enhanced. The research thoroughly studied the A. oryzae solid‐state fermentation of SM, and the resulting premium product could provide a better protein source for monogastric animals.     

In vitro study of microencapsulated isoflavone and beta-galactosidase

This study was designed to find the optimum conditions for isoflavone or beta-galactosidase microencapsulation and to examine the release efficiency of microcapsules in simulated gastrointestinal conditions. Coating materials were either medium-chain triacylglycerol (MCT) or polyglycerol monostearate (PGMS). The highest rate of microencapsulation was found at 15:1 (w/w) ratio of MCT to isoflavone or beta-galactosidase as 70.2 or 75.4%, respectively. When PGMS was used as the coating material, 91.5% beta-galactosidase was microencapsulated with 15:1 mixture (w/w). In vitro study, less than 6.3-9.3% of isoflavone was released in simulated gastric fluid (pH 2-5) during 1 h incubation. Comparatively, isoflavone release increased dramatically to 87.8% at pH 8 for 1 h incubation in simulated intestinal fluid and was maintained thereafter. The release of beta-galactosidase showed a similar trend to that of isoflavone. It appeared in the range of 12.3-15.2% at pH 2-5; however, it increased significantly to 80.6% as the highest value at pH 8. Among the released isoflavones, 53.5% was converted into the aglycone form of isoflavone at pH 8 for 3 h incubation. The present study indicated that isoflavone or beta-galactosidase could be microencapsulated with fatty acid esters and released effectively in simulated intestinal condition.     

In vitro fermentation of lactulose-derived oligosaccharides by mixed fecal microbiota

Fermentation properties of oligosaccharides derived from lactulose (OsLu) and lactose (GOS) have been assessed in pH-controlled anaerobic batch cultures using lactulose and Vivinal-GOS as reference carbohydrates. Changes in gut bacterial populations and their metabolic activities were monitored over 24 h by fluorescent in situ hybridization (FISH) and by measurement of short-chain fatty acid (SCFA) production. Lactulose-derived oligosaccharides were selectively fermented by Bifidobacterium and lactic acid bacterial populations producing higher SCFA concentrations compared to GOS. The highest total SCFA production was from Vivinal-GOS > lactulose > OsLu > GOS. Longer incubation periods produced a selective fermentation of OsLu when they were used as a carbon source reaching the highest selective index scores. The new oligosaccharides may constitute a good alternative to lactulose, and they could belong to a new generation of prebiotics to be used as a functional ingredient for improving the composition of gut microflora.