Recent advances on prebiotic lactulose production

Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic. The production of lactulose is important in the dairy industry, as it is regarded as a high value-added derivative of whey or lactose. The industrial production of lactulose is still mainly done by chemical isomerization. Due to concerns on the environmental and tedious separation processes, the enzymatic-based lactulose synthesis has been regarded as an interesting alternative. This work aims at comparing chemical and enzyme-catalyzed lactulose synthesis. With an emphasis on the latter one, this review discusses the influences of the critical operating conditions and the suited operation mode on the transgalactosylation of lactulose using microbial enzymes. As an update and supplement to other previous reviews, this work also summarizes the recent reports that highlighted the enzymatic isomerization of lactose using cellobiose 2-epimerase to produce lactulose at elevated yields.     

Lactulose biosynthesis by β-galactosidase from a newly isolated <Emphasis Type="Italic">Arthrobacter</Emphasis> sp.

Lactulose, a ketose disaccharide, is used in both pharmaceutical and food industries. This study was undertaken to screen and isolate potent β-galactosidase-producing bacteria and to evaluate their enzymatic production of lactulose. Soil samples from fruit gardens were collected. One isolate designated LAS was identified whose cell extract could convert lactose and fructose into lactulose. The 16S rDNA gene analysis of LAS revealed its phylogenetic relatedness to Arthrobacter sp. The β-galactosidase produced by LAS was purified 15.7-fold by ammonium sulfate precipitation and subsequent Phenyl-Sepharose hydrophobic chromatography. The optimum pH and temperature for lactulose synthesis by this β-galactosidase were 6.0 and 20°C, respectively. The low optimum temperature of this enzyme compared to the currently used ones for lactulose production has the advantage of reducing the nonenzymatic browning in biotransformations. The results indicated that Arthrobacter could be used as a novel bacterial β-galactosidase source for lactulose production.     

β-Galactosidase production using glycerol and byproducts: Whey and residual glycerin

The present study aimed to evaluate β-galactosidase production by liquid-state fermentation using an experimental design and response surface methodology. A culture medium containing lactose and analytical grade glycerol was formulated to maximize β-galactosidase production. The effects of the pH, lactose, and glycerol concentration on the enzyme production were studied using a Central Composite Design (CCD; 2³ plus central points), followed by a Central Composite Rotatable Design (CCRD; 2³ plus axial and central points). The conditions that maximized β-galactosidase production were: lactose concentration of 20?g?L^?1, glycerol concentration of 60?g?L^?1, and pH 5.0. Under these conditions, the highest enzymatic activity was 40.7?U?mL^?1. Glycerol and lactose were replaced by residual glycerin and whey respectively, according to the best condition obtained in CCRD, reaching enzymatic values of 31.8?U?mL^?1, and thus demonstrating to be great alternative sources for β-galactosidase production.     

Practical Preparation of Epilactose Produced with Cellobiose 2-Epimerase from Ruminococcus albus NE1

A practical purification method for a non-digestible disaccharide, epilactose (4-O-β-galactosyl-D-mannose), was established. Epilactose was synthesized from lactose with cellobiose 2-epimerase and purified by the following procedure: (i) removal of lactose by crystallization, (ii) hydrolysis of lactose by β-galactosidase, (iii) digestion of monosaccharides by yeast, and (iv) column chromatography with Na-form cation exchange resin. Epilactose of 91.1% purity was recovered at 42.5% yield.     

Detailed kinetic model describing new oligosaccharides synthesis using different β-galactosidases

The production of prebiotic galactooligosaccharides (GOS) from lactose has been widely studied whereas the synthesis of new prebiotic oligosaccharides with improved properties as those derived from lactulose is receiving an increasing interest. Understanding the mechanism of enzymatic oligosaccharides synthesis from lactulose would help to improve the quality of the products in a rational way as well as to increase the production efficiency by optimally selecting the operating conditions. A detailed kinetic model describing the enzymatic transgalactosylation reaction during lactulose hydrolysis is presented here for the first time. The model was calibrated with the experimental data obtained in batch assays with two different β-galactosidases at various temperatures and concentrations of substrate. A complete system identification loop, including model selection, robust estimation of the parameters by means of a global optimization method and computation of confidence intervals was performed. The kinetic model showed a good agreement between experimental data and predictions for lactulose conversion and provided important insights into the mechanism of formation of new oligosaccharides with potential prebiotic properties.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

Background

Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β-1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization.

Results

Using superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used.

Conclusion

These results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.

     

Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases

Commercial β-galactosidase preparations from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae were evaluated as catalysts for the synthesis of lactulose. Among them, the enzyme from A. oryzae was selected for further studies. The effect of reaction conditions was then studied on product composition during the kinetically controlled synthesis of lactulose by transgalactosylation with A. oryzae β-galactosidase. Product composition was not affected by pH, temperature, total initial concentration of sugar (lactose plus fructose) and enzyme to substrate ratio within the ranges studied. However, lactose to fructose ratio strongly influenced product composition being then possible to control the lactulose to galacto-oligosaccharide ratio within ample margins. Maximum lactulose yield (0.282 g of lactulose per g initial lactose) was obtained using 1/8 lactose to fructose molar ratio, 50% (w/w) total initial sugars, 40 °C, pH 4.5 and enzyme to initial lactose ratio equivalent to 200 IU/g.     

Evaluation of the cold-active Pseudoalteromonas haloplanktis β-galactosidase enzyme for lactose hydrolysis in whey permeate as primary step of d-tagatose production

d-Tagatose is an innovative natural low-calorie bulk sweetener with a broad potential for low-calorie and low-glycaemic foods and drinks. Production of this healthy sweetener is realized through enzymatic d-galactose isomerization. d-Galactose needs to be produced in situ due to its limited availability. Whey permeate contains a substantial amount of lactose, which is an interesting source for d-galactose production through enzymatic lactose hydrolysis. In this context, the cold-active β-galactosidase from the psychrophile Pseudoalteromonas haloplanktis was studied. Optimal parameters for efficient lactose hydrolysis in whey permeate have been deduced, viz. optimal incubation temperature, pH and lactose concentration. Hydrolysis efficiencies above 96.0% were realized within 24 h at 23 °C and pH 7.0 in whey permeate with a maximum dry matter content of 10.0% (w/w). In addition, the effect of the presence of d-glucose and d-galactose was investigated up to concentrations of 100 g l⁻¹. d-Glucose inhibited lactose hydrolysis more strongly compared to d-galactose. Also, the operational stability of the cold-active β-galactosidase was studied. Hydrolysis efficiencies above 90.0% were maintained during 7 subsequent hydrolysis cycles.     

An enzymatic glycosylation of nucleoside analogues using β-galactosidase from Escherichia coli

A new enzymatic method for the synthesis of β-galactosides of nucleosides and acyclic nucleoside analogues has been developed, using β-galactosidase from Escherichia coli as a catalyst and lactose as a sugar donor. The method is very rapid, feasible and last but not least inexpensive. Its applicability has been proven for a broad variety of possible substrates with respect to its scaling up for preparative use. Five new compounds from a series of nucleoside and acyclic nucleoside analogues have been prepared on a scale of several hundred milligrams, in all cases revealing very good results of the method concerning the reproducibility of the reaction yields and simplicity of the purification process.     

Lactulose production by β-galactosidase in permeabilized cells of<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis>

Lactulose production from lactose and fructose was investigated with several commercial -galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the -galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l^–1; concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60^°C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l^–1 lactulose in 3 h with a lactulose productivity of 6.8 g l^–1 h^–1. These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.     

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.     

Current studies on physiological functions and biological production of lactosucrose

Lactosucrose (O-β-d-galactopyranosyl-(1,4)-O-α-d-glucopyranosyl-(1,2)-β-d-fructofuranoside) is a trisaccharide formed from lactose and sucrose by enzymatic transglycosylation. This rare trisaccharide is a kind of indigestible carbohydrate, has good prebiotic effect, and promotes intestinal mineral absorption. It has been used as a functional ingredient in a range of food products which are approved as foods for specified health uses in Japan. Using lactose and sucrose as substrates, lactosucrose can be produced through transfructosylation by β-fructofuranosidase from Arthrobacter sp. K-1 or a range of levansucrases, or through transgalactosylation by β-galactosidase from Bacillus circulans. This article presented a review of recent studies on the physiological functions of lactosucrose and the biological production from lactose and sucrose by different enzymes.     

Characterization of a recombinant cellobiose 2-epimerase from Dictyoglomus turgidum that epimerizes and isomerizes β-1,4- and α-1,4-gluco-oligosaccharides

A recombinant putative N-acyl-d-glucosamine 2-epimerase from Dictyoglomus turgidum was identified as a cellobiose 2-epimerase by evaluating its substrate specificity. The purified enzyme was a 46?kDa monomer with a specific activity of 16.8?μmol?min^?1?mg^?1 for cellobiose. The epimerization activity was maximal at pH 7.0 and 70?°C with a half-life of 55?h. The isomerization of the glucose at the reducing end of β-1,4- and α-1,4-linked gluco-oligosaccharides to a fructose moiety by the enzyme took place after the epimerization of the glucose to a mannose moiety. The enzyme converted cellobiose to 12.8?% 4-O-β-d-glucopyranosyl-d-mannose and 54.6?% 4-O-β-d-glucopyranosyl-d-fructose as an equilibrium and converted lactose to 12.8?% epilactose and 54.3?% lactulose.     

The preparation of immobilized lactase and its use in the enzymatic hydrolysis of acid whey

The enzyme β-galactosidase (lactose) obtained from several microbial sources was immobilized on zirconia-coated porous glass particles. The immobilized enzymes were characterized by determining pH profiles, kinetic constants, thermal profiles, and operationalhalf-lives in lactose and whey ultrafiltrate solutions. Studies were carried out on continuous reactor performance, and enzyme requirements for scale-up were estimated. Lactose or whey hydrolyzed by this technique could find use commercially as a sweetener in a number of dairy products.     

Recent advances on physiological functions and biotechnological production of epilactose

Epilactose (4-O-β-d-galactopyranosyl-d-mannose), an epimer of lactose, is a rare disaccharide existing extremely small quantities in heat-treated milk, in which epilactose is produced by non-enzymatic catalysis from lactose. This disaccharide is a kind of non-digestible carbohydrate, has a good prebiotic effect, and promotes intestinal mineral absorption. This article presents a review of recent studies on epilactose formation in food system, qualitative and quantitative analysis, and its physiological functions. In addition, the biochemical properties and kinetic parameters of the epilactose-producing enzyme, cellobiose 2-epimerase, are compared, and the biotechnological production of epilactose from lactose is reviewed.     

Induction and Repression of β-Galactosidase Synthesis by Verticillum albo-atrum

Verticillium albo-atrum grew on lactose-containing culture media only after a prolonged lag phase. The intracellular specific activity of β-galactosidase [EC 3.2.1.23] increased 40–200 times during he lag phase. The β-galactosidase was induced by lactose and to a lesser degree by galactose. The appearance of the enzyme in lactose cultures was decreased by cycloheximide. Glucose and other readily metabolized carbon sources were effective repressors of β-galactosidase production. The production of β-galactosidase therefore appeared under control by lactose induction and catabolite repression.     

Lactulose: production, purification and potential applications

Lactulose a “bifidus factor” is composed of galactose and fructose, which can be produced by the isomerization of lactose. It is a prebiotic carbohydrate which stimulates the growth of health-promoting bacteria in the gastrointestinal tract, such as bifidobacteria and lactobacilli and at the same time inhibits growth of pathogenic bacteria such as Salmonella. It can also be used for the treatment of constipation, hepatic encephalopathy, tumour prevention, and to maintain blood glucose and insulin level. This review provides comprehensive information on the different techniques used for the production of lactulose, purification and analysis. Besides this mechanism of action and its potential applications in food and pharmaceutical industries have also been discussed.     

Determination of the transgalactosylation activity of Aspergillus oryzae β-galactosidase: effect of pH, temperature, and galactose and glucose concentrations

The catalytic potential of β-galactosidase is usually determined by its hydrolytic activity over natural or synthetic substrates. However, this method poorly predicts enzyme behavior when transglycosylation instead of hydrolysis is being performed. A system for determining the transgalactosylation activity of β-galactosidase from Aspergillus oryzae was developed, and its activity was determined under conditions for the synthesis of galacto-oligosaccharides and lactulose. Transgalactosylation activity increased with temperature up to 55 °C while the effect of pH was mild in the range from pH 2.5 to 5.5, decreasing at higher values. The effect of glucose and galactose on transgalactosylation activity was also assessed both in the reactions for the synthesis of galacto-oligosaccharides and lactulose and also in the reaction of hydrolysis of o-nitrophenyl β-d-galactopiranoside. Galactose was a competitive inhibitor and its effect was stronger in the reactions of transgalactosylation than in the reaction of hydrolysis. Glucose was a mild activator of β-galactosidase in the reaction of hydrolysis, but its mechanism of action was more complex in the reactions of transgalactosylation, having this positive effect only at low concentrations while acting as an inhibitor at high concentrations. This information is relevant to properly assess the effect of monosaccharides during the reactions of the synthesis of lactose-derived oligosaccharides, such as galacto-oligosaccharides and lactulose.     

Conventional and non-conventional applications of β-galactosidases

β-Galactosidase is one of the most important industrial enzymes, that has been used for many decades in the dairy industry. The main application of β-galactosidase is related to the production of low-lactose and lactose-free milk and dairy products, which are now common consumer goods in supermarket shelves. This is a well-established market that is expected to keep on growing as these products become more accessible to mid-income people worldwide. However, a fresh air has come into the β-galactosidase business as non-conventional applications arose in recent decades based on its transgalactosylation activity. This capacity is certainly a major asset for a commodity enzyme that can be used now as a catalyst for the upgrading of readily available and cheap lactose into high added-value glycosides in processes of organic synthesis in tune with green chemistry principles within the framework of sustainability. This is a reflection of a paradigm shift, where enzymes are now being considered as apt catalysts for the synthesis of valuable organic compounds. This article reviews the main applications of β-galactosidase, going from its conventional use related to its hydrolytic activity to the ongoing non-conventional applications in the synthesis of high added-value oligosaccharides based on its transgalactosylation activity.