Galacto-oligosaccharides Yield Research Articles

Kinetics and products of Thermotoga maritima β-glucosidase with lactose and cellobiose

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother’s milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose.Key points• β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield.• Thermotoga maritima β-glucosidase has high activity and high thermostability.• Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages.Graphical

Potential and Scale-Up of Pore-Through-Flow Membrane Reactors for the Production of Prebiotic Galacto-Oligosaccharides with Immobilized β-Galactosidase

The production of prebiotics like galacto-oligosaccharides (GOS) on industrial scale is becoming more important due to increased demand. GOS are synthesized in batch reactors from bovine lactose using the cost intensive enzyme β-galactosidase (β-gal). Thus, the development of sustainable and more efficient production strategies, like enzyme immobilization in membrane reactors are a promising option. Activated methacrylatic monoliths were characterized as support for covalent immobilized β-gal to produce GOS. The macroporous monoliths act as immobilized pore-through-flow membrane reactors (PTFR) and reduce the influence of mass-transfer limitations by a dominating convective pore flow. Monolithic designs in the form of disks (0.34 mL) and for scale-up cylindric columns (1, 8 and 80 mL) in three different reactor operation configurations (semi-continuous, continuous and continuous with recirculation) were studied experimentally and compared to the free enzyme system. Kinetic data, immobilization efficiency, space-time-yield and long-term stability were determined for the immobilized enzyme. Furthermore, simulation studies were conducted to identify optimal operation conditions for further scale-up. Thus, the GOS yield could be increased by up to 60% in the immobilized PTFRs in semi-continuous operation compared to the free enzyme system. The enzyme activity and long-time stability was studied for more than nine months of intensive use.

Production and identification of galacto-oligosaccharides from lactose using β-D-galactosidases from Lactobacillus leichmannii 313

Crude β-D-galactosidase from Lactobacillus leichmannii 313 was used to synthesise galacto-oligosaccharides (GOS) from lactose. Using an initial lactose concentration of 170 g/L, the yield of GOS was 52 g/L (i.e. 62% lactose conversion) after 2 h of enzymatic reaction. The highest number of unique individual GOS were generated after 48 h of reaction. ESI-MS and HPAEC analysis of the GOS enriched fractions obtained from 48 h mixtures showed the presence of sugars with degrees of polymerization (DP) ranging from 2 to 6. Further analysis (by NMR spectroscopy) of GOS in fraction 3 (containing ~ 96% w/w GOS) showed non-reducing terminals that were mostly Galp; and reducing ends that were →4)-Glcp, and →6)-Glcp. The most abundant internal linkages in fraction 3 were deduced to be →3)-Galp-(1→, and →6)-Galp-(1→. The predominance of β-D-(1→3, or 6) linkages in GOS fractions suggests that β-D-galactosidase from LL313 is suitable for generating unique GOS from lactose.

Immobilization of \u03b2-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain

Enzyme immobilization is a powerful tool not only as a protective agent against harsh reaction conditions but also for the enhancement of enzyme activity, stability, reusability, and for the improvement of enzyme properties as well. Herein, immobilization of β-glucosidase from Thermotoga maritima (Tm-β-Glu) on magnetic nanoparticles (MNPs) functionalized with chitin (Ch) was investigated. This technology showed a novel thermostable chitin-binding domain (Tt-ChBD), which is more desirable in a wide range of large-scale applications. This exclusive approach was fabricated to improve the Galacto-oligosaccharide (GOS) production from a cheap and abundant by-product such as lactose through a novel green synthesis route. Additionally, SDS-PAGE, enzyme activity kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) revealed that among the immobilization strategies for Thermotoga maritime-β-Glucosidase thermostable chitin-binding domain (Tm-β-Glu-Tt-ChBD) on the attractive substrate; Ch-MNPs had the highest enzyme binding capacity and GOS production ratio when compared to the native enzyme. More interestingly, a magnetic separation technique was successfully employed in recycling the immobilized Tm-β-Glu for repetitive batch-wise GOS without significant loss or reduction of enzyme activity. This immobilization system displayed an operative stability status under various parameters, for instance, temperature, pH, thermal conditions, storage stabilities, and enzyme kinetics when compared with the native enzyme. Conclusively, the GOS yield and residual activity of the immobilized enzyme after the 10th cycles were 31.23% and 66%, respectively. Whereas the GOS yield from native enzyme synthesis was just 25% after 12 h in the first batch. This study recommends applying Tt-ChBD in the immobilization process of Tm-β-Glu on Ch-MNPs to produce a low-cost GOS as a new eco-friendly process besides increasing the biostability and efficiency of the immobilized enzyme.

Immobilization of β-galactosidase on chitosan-coated magnetic nanoparticles and its application for synthesis of lactulose-based galactooligosaccharides

Abstract β-Galactosidase was covalently immobilized on the chitosan-coated magnetic nanoparticles activated with glutaraldehyde. The effect of three factors including protein content, pH and time on the yield of immobilization was investigated, and the optimum values were 0.45 mg/ml, 4.9 and 4.9 h, respectively. The maximum yield of immobilization reached 98.8% in term of enzyme activity. The stability of β-galactosidase significantly was improved after immobilization from 2.5 days to over 6 days of half-life at 60 °C and was active in wider ranges of temperature (45–60 °C), and pH (4.0–5.5) compared to soluble form. The activity of immobilized enzyme remained 85% of origin after eight recycles (at 60 °C, pH 4.5 for 4 h). Different initial lactulose concentrations (0.58–2.34 M) were applied for synthesis of galactooligosaccharides. The maximum yield of galactooligosaccharides was 17% mol/mol at the initial lactulose concentration of 2.34 M at 36 h of reaction. It showed that both hydrolytic and trans-galactosylation activities of β-galactosidase remained well after immobilization. The kinetic model of immobilized enzyme was determined. These results revealed that immobilized enzyme on chitosan-coated magnetic nanoparticles has application potentials in the production of prebiotic lactulose-based galactooligosaccharides.

Production Optimization of an Active β-Galactosidase of Bifidobacterium animalis in Heterologous Expression Systems.

β-Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The β-galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a β-galactosidase gene (bg42-106) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0–8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of β-galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.

Rational design of the beta-galactosidase from Aspergillus oryzae to improve galactooligosaccharide production

The β-galactosidase from Aspergillus oryzae produces galactooligosaccharides with beneficial prebiotic effects through the transglycosylation of lactose. However, its low galactooligosaccharide yield greatly limits its use in industrial applications. Rational design and site-directed mutagenesis were used to produce three mutants of A. oryzae β-galactosidase: N140C, W806F, and N140C/W806F. The galactooligosaccharide yields of N140C (50.7%), W806F (49.3%), and N140C/W806F (59.8%) represent substantial improvements over that of the wild-type (35.7%). The galactooligosaccharide yield of N140C/W806F is the highest reported to date. Our rational design approach suggests novel strategies for further study of the β-galactosidase reaction mechanism and has produced mutants may be more useful in industrial applications than wild-type A. oryzae β-galactosidase.

A Method of Early Phase Selection of Carrier for Aspergillus Oryzae β-Galactosidase Immobilization for Galactooligosaccharides Production.

Early phase development of industrial immobilized biocatalysts has to address the selection of the best candidates from dozens of available carriers and binding methods. This work presents a simple selection method for the immobilization of industrial-grade Aspergillus oryzae β-galactosidase suitable for the production of galactooligosaccharides. Immobilization efficiency and yield of a variety of immobilized biocatalysts are evaluated using simple activity measurements and mathematical modeling of intraparticle kinetics and mass transfer. Activated carriers and some ion-exchange carriers provided highly active biocatalysts and are therefore selected for the investigation of GOS production in the batch reactor. Biocatalysts exhibited significant differences in the yield of galactooligosaccharides and productivity of GOS formation. Based on these process performance criteria, cost considerations are made that allow further selection of the best biocatalyst for future process optimization. The presented method of biocatalyst selection can be useful for other industrial enzymes applications.

Production and partial purification of galacto-oligosaccharides by sequential fermentation

Galacto-oligosaccharides are compounds that are synthesized from lactose containing two to five galactose units and a terminal glucose residue. They are formed from lactose by the transgalactosylation activity of the enzyme β-galactosidase. During the galacto-oligosaccharides synthesis are formed significant amounts of glucose and galactose, products that are considered reaction inhibitors. This work aims to produce and partly purify galacto-oligosaccharides using sequential fermentation. Galacto-oligosaccharides were produced using Aspergillus oryzae ATCC 11488 by solid-state fermentation (first part); Saccharomyces cerevisiae was used for the selective removal of the monosaccharides from the GOS mixture, (second part). The substrate for the solid-state fermentation was moistened wheat bran with a solution of 2% (w/v) lactose whey permeate. For the production of the galacto-oligosaccharides, a solution of 25 % (w/v) lactose whey permeate was used. Enzymatic activity of β-galactosidase was determined using o- nitrophenyl-galactopyranoside as substrate. β-galactosidase maximum activity was 0,43 U mL-1, obtained in 96 hours of solid-state fermentation. Galacto-oligosaccharide yield was 13,7%, area percentage of glucose and galactose was 13.76%, and 7.93% respectively, after 24 hours of fermentation at 40 °C and pH 5.9. After sequential fermentation, 85% of glucose was removed in 48 hours of fermentation and a 63% increase in the area percentage of galacto-oligosaccharides after 60 hours of fermentation. It was shown that sequential fermentation can be used in the partial purification of mixtures of carbohydrates, which facilitates the subsequent separation stages.

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus

β-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000U of β-galactosidase activity per liter of culture corresponding to approximately 170mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205gL−1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.

Engineering a thermostable Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis.

Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining β-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a β-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (−1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent kcat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower kcat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

Comparison of the galacto-oligosaccharide forming activity of different β-galactosidases

Galacto-oligosaccharides (GOS) were synthesised by enzymatic transgalactosylation in UF-skimmed milk permeate fortified with lactose (40% w/w) using β-galactosidases (β-GAL) derived from Aspergillus aculeatus, Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. GOS yields were assessed arithmetically after photometric determination of the sugar profile. GOS composition was characterised by HPAEC-PAD/MS analysis. The GOS yields, expressed as a percentage of the initial lactose content, were 41%, 21%, 13% and 11% with β-GALs from B. circulans, A. oryzae, A. aculeatus and K. lactis, respectively, under optimal conditions. Thus, the B. circulans enzyme revealed the highest affinity to transgalactosylation relative to hydrolysis. HPAEC-PAD/MS results showed similarities in GOS composition, especially with β-GAL from A. oryzae, A. aculeatus and K. lactis with a preference towards β-galactosyl residues β(1–6). GOS generated are mainly di-, tri- and a few tetra-saccharides. Only the B. circulans β-GAL formed above all penta-saccharides, mainly via β1-4 glycosidic linkages.

Process engineering studies of free and micro-encapsulated β-galactosidase in batch and packed bed bioreactors for production of galactooligosaccharides

In the present investigation, an attempt has been made to produce galactooligosaccharides (GOS)11GOS – Galactooligosaccharides. from de-proteinated casein whey using free and immobilized β-galactosidase. An extensive investigation on β-galactosidase immobilization by micro-encapsulation on calcium alginate has been carried out to evaluate the optimum process parameters namely sodium alginate to enzyme ratio (SER)22SER – Sodium alginate to enzyme ratio. (100:3) and calcium chloride concentration (0.3M) with an intention to get maximum enzyme loading and retention in activity. Investigations on GOS formation in mechanically agitated batch bioreactor using free enzyme indicate that the overall observed rate can be well explained by conventional Michaelis–Menten model (Km=195.2kgm−3, kcat=5.37min−1). Effects of different operating parameters (temperature, initial substrate concentration) on reaction rate and GOS yield during batch processing have been investigated. Studies on GOS production in packed bed bioreactor using immobilized β-galactosidase indicate that although the observed rate can be well explained by Michaelis–Menten model (K′m=200.2 kg m−3, K′cat=1.39 min−1), enzyme activity is partially blocked due to immobilization. A comparison of activation energies for both systems indicates that the immobilized enzyme system requires more active enzyme compared to its free counterpart for same GOS yield.

Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation

A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest immobilization yield and stability being selected for the optimization of immobilization conditions and application in GOS synthesis. A central composite rotatable design was conducted to optimize contacted protein and immobilization time, using maximum catalytic potential as the objective function. Optimal conditions of immobilization were 28.9mg/g and 36.4h of contact, resulting in a biocatalyst with 595IU/g and a half-life 89-fold higher than soluble enzyme. Immobilization process did not alter the synthetic capacity of β-galactosidase, obtaining the same GOS yield and product profile than the free enzyme. GOS yield and productivity remained unchanged along 10 repeated batches, with values of 39% (w/w) and 5.7g GOS/g of biocatalyst·batch. Total product obtained after 10 batches of reaction was 56.5g GOS/g of biocatalyst (1956g GOS/g protein). Cumulative productivity in terms of mass of contacted protein was higher for the immobilized enzyme than for its soluble counterpart from the second batch of synthesis onwards.

Kinetics and design relation for enzymatic conversion of lactose into galacto-oligosaccharides using commercial grade β-galactosidase

The enzymatic synthesis of galacto-oligosaccharides (GOS) from lactose was studied using commercial grade β-galactosidase (Biolacta FN5) from Bacillus circulans. The reaction was carried out under free enzyme condition varying initial lactose concentration (ILC: 55-525 g/L), enzyme concentration (0.05-1.575 g/L), temperature (30-50°C) and pH (5.0-6.0). Reaction mixture compositions were analyzed utilizing high performance liquid chromatography (HPLC). A maximum GOS formation of 39% (dry basis) was achieved at an ILC of 525 g/L converting 60% of the lactose fed. Tri-saccharides were the major types of GOS formed, accounting approximately 24%; whereas, tetra-saccharides and penta-saccharides account approximately 12% and 3%, respectively. Design correlation was developed in order to observe the quantitative effect of operating parameters on GOS yield. Further, based on Michaelis-Menten model, four-step reaction pathways were considered for simplistic understanding of the kinetics. Apart from predicting the reaction mixture composition, the approach also provided kinetic parameters though simulation using COPASI 4.7®. Excellent agreements were observed between simulated and experimental results.

Synthesis of galactooligosaccharides by CBD fusion β-galactosidase immobilized on cellulose

The β-galactosidase gene (bgaL3) was cloned from Lactobacillus bulgaricus L3 and fused with cellulose binding domain (CBD) using pET-35b (+) vector in Escherichia coli. The resulting fusion protein (CBD-BgaL3) was directly adsorbed onto microcrystalline cellulose with a high immobilization efficiency of 61%. A gram of cellulose was found to absorb 97.6U of enzyme in the solution containing 100mM NaCl (pH 5.8) at room temperature for 20min. The enzymatic and transglycosylation characteristics of the immobilized CBD-BgaL3 were similar to the free form. Using the immobilized enzyme as the catalyst, the yield of galactooligosaccharides (GOS) reached a maximum of 49% (w/w) from 400g/L lactose (pH 7.6) at 45°C for 75min, with a high productivity of 156.8g/L/h. Reusability assay was subsequently performed under the same reaction conditions. The immobilized enzyme could retain over 85% activity after twenty batches with the GOS yields all above 40%.

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.

BbgIV Is an Important Bifidobacterium β-Galactosidase for the Synthesis of Prebiotic Galactooligosaccharides at High Temperatures

The individual contributions of four β-galactosidases present in Bifidobacterium bifidum NCIMB 41171 toward galactooligosaccharide (GOS) synthesis were investigated. Although the β-galactosidase activity of the whole cells significantly decreased as a function of temperature (40-75 °C), GOS yield was at its maximum at 65 °C. Native-PAGE of the whole cells showed that the contributions of BbgIII and BbgIV to GOS synthesis increased as the temperature increased. Moreover, BbgIII and BbgIV were found to be more temperature stable and to produce a higher GOS yield than BbgI and BbgII, when used in their free form. The GOS yield using BbgIV was 54.8% (percent of total carbohydrates) and 63.9% (percent lactose converted to GOS) at 65 °C from 43% w/w lactose. It was shown that BbgIV is the most important β-galactosidase in B. bifidum NCIMB 41171 and can be used for GOS synthesis at elevated temperatures.

Influence of the ionic form of a cation-exchange adsorbent on chromatographic separation of galactooligosaccharides

AbstractSeparation performance of sodium, lithium, potassium and hydrogen ionic forms of a commercial cation exchanger, Diaion UBK 530, based on a styrene-divinylbenzene copolymer resin support was studied. The adsorbents were applied to the separation of galactooligosaccharides from their mixture with lactose, glucose and galactose. Separation performance of the hydrogen ionic form of the adsorbent was superior to that of other adsorbent forms. A much higher yield of galactooligosaccharides of the requested purity was achieved for this adsorbent form owing to its better kinetic properties. Adsorption properties of individual adsorbents were discussed in relation to possible adsorption mechanisms.

Galactooligosaccharide Synthesis by Active β-Galactosidase Inclusion Bodies-Containing Escherichia coli Cells

In this study, galactooligosaccharide (GOS) was synthesized using active β-galactosidase (beta-gal) inclusion bodies (IBs)- containing Escherichia coli (E. coli) cells. Analysis by MALDI-TOF (matrix-assisted laser desorption/ionizationtime of flight) mass spectrometry revealed that a trisaccharide was the major constituent of the synthesized GOS mixture. Additionally, the optimal pH, lactose concentration, amounts of E. coli β-gal IBs, and temperature for GOS synthesis were 7.5, 500 g/l, 3.2 U/ml, and 37 °C, respectively. The total GOS yield from 500 g/l of lactose under these optimal conditions was about 32%, which corresponded to 160.4 g/l of GOS. Western blot analyses revealed that β-gal IBs were gradually destroyed during the reaction. In addition, when both the reaction mixture and E. coli β-gal hydrolysate were analyzed by high-performance thin-layer chromatography (HP-TLC), the trisaccharide was determined to be galactosyl lactose, indicating that a galactose moiety was most likely transferred to a lactose molecule during GOS synthesis. This GOS synthesis system might be useful for the synthesis of galactosylated drugs, which have recently received significant attention owing to the ability of the galactose molecules to improve the drugs solubility while decreasing their toxicity. β-Gal IB utilization is potentially a more convenient and economic approach to enzymatic GOS synthesis, since no enzyme purification steps after the transgalactosylation reaction would be required.