Synthesis Of Galacto-oligosaccharides Research Articles

Non-carrier immobilization of yeast cells by genipin crosslinking for the synthesis of prebiotic galactooligosaccharides from plant-derived galactose

Galactooligosaccharides (GOS), as mimics of human milk oligosaccharides, are important prebiotics for modulating the ecological balance of intestinal microbiota. A novel carrier-free cell immobilization method was established using genipin to cross-link Kluyveromyces lactis CGMCC 2.1494, which produced β-galactosidase, an enzyme essential for GOS synthesis. The resulting immobilized cells were characterized as stable by thermogravimetric analysis and confirmed to be crosslinked through scanning electron microscopy analysis (SEM) and Fourier transform infrared spectroscopy (FTIR). The Km and Vmax values of β-galactosidase in immobilized cells towards o-nitrophenyl β-D-galactoside were determined to be 3.446 mM and 2210 μmol min−1 g−1, respectively. The enzyme in the immobilized showed higher thermal and organic solvent tolerance compared to that in free cells. The immobilized cells were subsequently employed for GOS synthesis using plant-derived galactose as the substrate. The synthetic reaction conditions were optimized through both single-factor experiments and response surface methodology, resulting in a high yield of 49.1 %. Moreover, the immobilized cells showed good reusability and could be reused for at least 20 batches of GOS synthesis, with the enzyme activity remaining above 70 % at 35 °C.

A Novel β-Galactosidase from Kluyvera intermedia and its Potential for Hydrolyzing Lactose in Milk

ABSTRACT A new β-galactosidase (EC 3.2.1.23) discovered in Kluyvera intermedia (β-gal-Kli) was recombinantly produced in Escherichia coli BL21 (DE3). The bioreactor cultivation resulted in a maximum β-gal-Kli activity of 124.6 ± 25.5 μkat oNPGal,37 °C/Lculture. The β-gal-Kli was purified 4.5fold to a specific activity of 172.3 μkat oNPGal,37 °C/gprotein. The β-gal-Kli showed maximum activity at pH 6.5 and 50°C and was sufficiently active and stable at an industrially relevant temperature of 8°C (half-life of 63 days). The β-gal-Kli was compared to a commercially available β-galactosidase (opti-lactase LX2) for lactose hydrolysis in milk (45.3 ± 0.9 glactose/L) at 8°C. With the activity of 2.7 nkatlactose/mLmilk, “lactose-free” (lactose <0.1 g/L) was realized by β-gal-Kli after 72 h, while 192 h was needed for opti-lactase LX2. Additionally, the galactooligosaccharide synthesis was observed with both these two enzymes. In conclusion, the β-gal-Kli showed its potential for the production of “lactose-free” dairy products.

Biochemical characterization of a novel β-galactosidase from Pedobacter sp. with strong transglycosylation activity at low lactose concentration.

A novel β-galactosidase gene (PbBgal35A) from Pedobacter sp. CAUYN2 was cloned and expressed in Escherichia coli. The gene had an open reading frame of 1917bp, encoding 638 amino acids with a predicted molecular mass of 62.3kDa. The deduced amino acid sequence of the gene shared the highest identity of 41% with a glycoside hydrolase family 35 β-galactosidase from Xanthomonas campestris pv. campestris (AAP86763.1). The recombinant β-galactosidase (PbBgal35A) was purified to homogeneity with a specific activity of 65.9 U/mg. PbBgal35A was optimally active at pH 5.0 and 50°C, respectively, and it was stable within pH 4.5‒7.0 and up to 45°C. PbBgal35A efficiently synthesized galacto-oligosaccharides from lactose with a conversion ratio of 32% (w/w) and fructosyl-galacto-oligosaccharides from lactulose with a conversion ratio of 21.9% (w/w). Moreover, the enzyme catalyzed the synthesis of galacto-oligosaccharides from low-content lactose in fresh milk, and the GOS conversion ratios of 17.1% (w/w) and 7.8% (w/w) were obtained when the reactions were performed at 45 and 4°C, respectively. These properties make PbBgal35A an ideal candidate for commercial use in the manufacturing of GOS-enriched dairy products.

Functionalized electrospun nanofibers to enhance β-Galactosidase immobilization and catalytic activity for efficient galactooligosaccharide synthesis

This study aimed to immobilize β-galactosidase (β-GAL) into enhanced polystyrene (PS) electrospun nanofiber membranes (ENMs) with functionalized graphene oxide (GO). Initially, GO sheets were functionalized by salinization with 3-aminopropyl triethoxysilane (APTES). Then the ENMs (PS, PS/GO, and PS/GO-APTES) were prepared and characterized. Then, the β-GAL was immobilized in the different ENMs to produce the β-GAL-bound nanocomposites (PS-GAL, PS/GO-GAL, and PS/GO-APTES-GAL). Immobilization of β-GAL into PS/GO-APTES significantly improved enzyme adsorption by up to 87 %. Also, PS/GO-APTES-GAL improved the enzyme activity, where the highest enzyme activity was obtained at enzyme concentrations of 4 mg/L, 50 °C, and pH 4.5. Likewise, the storage stability and reusability of immobilized β-GAL were improved. Furthermore, this process led to enhanced catalytic behavior and transgalactosylation efficiency, where GOS synthesis (72 %) and lactose conversion (81 %) increased significantly compared to the free enzyme. Overall, the immobilized β-GAL produced in this study showed potential as an effective biocatalyst in the food industry.

Enzymatic synthesis of prebiotic galactooligosaccharides from galactose derived from gum arabic.

A novel enzymatic process was established for galactooligosaccharides (GOS) synthesis by using plant-derived galactose as substrate, without producing any byproducts. The galactose was prepared from the acid hydrolysate of gum arabic. The yeast Kluyveromyces lactis producing β-galactosidase capable of catalyzing GOS synthesis from galactose was screened out. The synthesis conditions using the yeast cells as enzyme source were optimized by both single-factor experiment and response surface methodology, with the highest GOS yield reached 45%. The composition of reaction mixture contained only GOS and unreacted galactose, which could be easily separated by the cation exchange resin column. The structures of major GOS products were identified as Gal-β-D-(1→6)-Gal, Gal-β-D-(1→3)-Gal, and Gal-β-D-(1→6)-Gal-β-D-(1→6)-Gal by MS and NMR spectra. Moreover, the β-galactosidase-containing cells can be recycled for at least 30 batches of GOS synthesis at 35°C, with the enzyme activity remaining above 60%.

Ultrasound-assisted enzymatic synthesis of galacto-oligosaccharides using native whey with two commercial β-galactosidases: Aspergillus oryzae and Kluyveromyces var lactis.

Native whey obtained during casein micelle microfiltration was used as a novel source to produce galacto-oligosaccharides (GOS). Since the presence of macromolecules and other interferers reduces biocatalyst performance, this work evaluated the effect of different ultrasound processing conditions on GOS synthesis using concentrated native whey. Ultrasonic intensities (UI) below 11W/cm2 tended to increase the activity in the enzyme from Aspergillus oryzae for several minutes but accelerated the inactivation in that from Kluyveromyces lactis. At 40°C, 40% w/w native whey, 70% wave amplitude, and 0.6s/s duty-cycle, a UI of 30W/cm2 was achieved, and the increased specific enzyme productivity was similar to the values obtained with pure lactose (∼0.136g GOS/h/mgE). This strategy allows for obtaining a product containing prebiotics with the healthy and functional properties of whey proteins, avoiding the required purification steps used in the production of food-grade lactose.

Synthesis of Galacto-oligosaccharides in Milk by Using Bifidobacterium bifidum β-galactosidases (Saphera 2600L and Nola Fit 5500) Immobilized on Chitosan Beads

The lactose intolerance—as a limiting factor for dairy milk consumption—has a high prevalence worldwide. Dairy milk and milk-derived products are major sources of multiple inorganic compounds and nutrients and thus are considered to be functional foods. β-galactosidases are able to hydrolyze lactose and are therefore widely applied for the production of lactose-free products. In addition, they are capable of the synthesis of galacto-oligosaccharides (GOSs); thus, the dairy industry has a special interest in applying them for the enrichment of dairy products with prebiotic GOSs. In this work, we studied two commercially available β-galactosidase products: Saphera 2600L and Nola Fit 5500. Both enzyme solutions contain a recombinant β-galactosidase of Bifidobacterium bifidum and have already been authorized for food industrial application, but the information about their hydrolytic and/or synthetic activities is only limited. After immobilization on chitosan beads, the enzymes were used for lactose hydrolysis and simultaneous synthesis of GOSs, by performing the reactions in pasteurized milk (skim milk). Both immobilized β-galactosidase exhibited elevated lactose hydrolysis (vmax increased from ~ 1 to ~ 4 mM/min) and GOS synthesis as compared to the free enzymes. The enzyme-coated beads were efficiently re-used at least 15 cycles; the residual lactose concentration was < 2 mg/ml after each cycle. After treatment, GOSs were present in ≤ 9% of the total sugar content, indicating that the prepared low-lactose milks were enriched in prebiotic GOSs. The application of immobilized Saphera 2600L and Nola Fit 5500 β-galactosidases may be implemented for the large-scale production of GOS-enriched low-lactose milk.Graphical

Measuring β-galactosidase activity in opaque dairy solutions under optimum conditions for galactooligosaccharide synthesis by isothermal titration calorimetry

The dairy industry uses enzymes to make cheese, alter product flavor, and eliminate lactose. The activities of these enzymes have been measured in clear buffered solutions, but because of the limitations of spectrophotometric methods, enzyme activities have not been measured in opaque or colored dairy products where they are used. Isothermal titration calorimetry (ITC) can be used to determine reaction kinetics in opaque and colored solutions by measuring the heat rate (thermal power) from enzyme-catalyzed reactions as a function of time. This study used ITC to measure β-galactosidase activity in opaque solutions of milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate with 2 β-galactosidase (Enzyme Commission number 3.2.1.23) isozymes derived from Aspergillus oryzae and Kluyveromyces lactis. The components of the dairy fluids alter the enzyme kinetics and reaction thermodynamics, and the reactions catalyzed by the 2 homologues differ as shown by differing thermodynamic profiles. The study demonstrates that ITC can be used to measure enzyme activity in opaque and colored dairy fluids and identify reactions by their thermodynamic properties.

Enzymatic Synthesis of Galacto-Oligosaccharides from Concentrated Sweet Whey Permeate and Its Application in a Dairy Product

Valorization of by-products in the dairy industry as a strategy to promote circular economies has become a priority around the globe. Cheese whey and its derivates from filtration technologies offer a source of valuable molecules such as proteins, fat, lactose, and minerals. For this study, after protein concentration and recovery through ultrafiltration, lactose remaining in the permeate was furtherly concentrated with nanofiltration, resulting in a retentate used as substrate for the enzymatic production of galacto-oligosaccharides (GOS). The kinetics of GOS generation with a commercial β-galactosidase, was carried out, quantifying the carbohydrate composition by HPAEC-PAD. Results showed that at 0.5 h, GOS yield reached a maximum of 74% (g GOS/g lactose) with a lactose utilization of 63%. Under these conditions, a GOS syrup (75% soluble solids) was generated and applied in a porridge for blind paired comparison test, including a control without the syrup. No differences were identified in color and odor between porridges; however, flavor and mouthfeel of the GOS-added sample improved according to the comments of panelists. This study presents an alternative process for the valorization of whey permeate to produce GOS ingredients that can be used directly in day-to-day dairy products.

Enhanced thermal stability of the β-galactosidase BgaB from Bacillus circulans by cyclization mediated via SpyTag/SpyCatcher interaction and its use in galacto-oligosaccharides synthesis

Cyclization of proteins using SpyTag/SpyCatcher is a novel approach to increase their thermal stability. In this paper, we test this approach on two β-galactosidases from Bacillus circulans, BgaB and BgaC, and find that BgaB was stabilized while BgaC was not. Wild-type BgaB precipitated completely upon heating above 70 °C, but after SpyRing cyclization, it remained soluble after heating to 90 °C. Similarly, wild-type BgaB retained only 50 % activity after heating at 60 °C for 10 min, but this increased to 80 % after SpyRing cyclization. In contrast, cyclization decreased the stability of BgaC. After SpyRing cyclization, BgaC only retained 2 % activity after 20-min incubation at 55 °C, whereas the wild-type BgaC retained 25 % activity. One reason for the different effect of cyclization may the shorter distance between the N- and C-termini in BgaB (20.2 Å) as compared to BgaC (43.7 Å). The intrinsic fluorescence and circular dichroism spectra suggested that SpyRing cyclization of BgaB did not significantly change its conformation or secondary structure. SpyRing cyclized BgaB yielded similar amounts and compositions of galacto-oligosaccharides using a high initial lactose concentration (40 %, w/v), but a slightly higher amount at low initial lactose concentration (5 %, w/v) suggesting increased transgalactosylation activity.

Kinetic Model for Galacto-Oligosaccharide Synthesis

A new micro-kinetic model is proposed for galacto-oligosaccharide (GOS) synthesis with β-galactosidase from Aspergillus oryzae. Several kinetic models with similar reaction mechanisms, i.e., steps and kinetic constants, have been investigated covering a range of initial lactose (20–40 wt %) and enzyme concentrations (0.5–2 mg/mL). The kinetic parameters were estimated simultaneously for all experimental results using a hybrid genetic algorithm. A detailed analysis of the effect of process parameters on the concentrations of lactose and GOS is performed. The proposed model has a very good quantitative fit of the experimental data, with an average error of 9.34%. More importantly, qualitative trends for the change in the concentration of lactose and GOS are predicted excellently, especially if one takes into account the variation of both concentrations of enzyme and lactose in a range wider than reported in the literature to date. Also, the analysis between the two comparative reactions of transglycosylation and lactose hydrolysis confirmed the positive effect of increasing lactose concentration on the production of GOS. On the other hand, a higher enzyme concentration resulted in a faster production of GOS but with more intensive decomposition at longer reaction times.

Molecular dynamics simulation guided distal mutation of Thermotoga naphthophila β-glucosidase for significantly enhanced synthesis of galactooligosaccharides and expanded product scope

For efficient enzymatic production of health-beneficial galactooligosaccharides (GOSs), a glycone (−1)/aglycone (+2) subsite mutation strategy to engineer a thermophilic GH1 β-glucosidase (Tn0602) from Thermotoga naphthophila RKU-10 was introduced. Six single mutation variants (F226G, N246G, N246E, N222F, N222Y, G224T) and two double mutants (F226GF414S, F226GF414Y) were designed. The +2-subsite variant F226G produced 136 mM galactooligosaccharide 1.2-fold more than the wild type (115 mM). More significantly, a superimposed mutation of the −1/+2 subsites F226G/F414S gave a total GOS production of 314 mM (82.16% lactose conversion), 2.7-fold higher than the total GOS production of the wild type. Furthermore, the variant F226GF414S was profiled 241 mM of trisaccharide (galβ (1 → 3)/(1 → 4) lactose) and 73 mM tetrasaccharide (galβ (1 → 3)/(1 → 4) galβ (1 → 3)/(1 → 4) lactose). According to a 300-ns molecular dynamic simulation, the superimposed mutation increased GOS productivity and expanded the scope of products by changing the structural flexibility and reducing the steric hindrance of the substrate tunnel. Overall, our study successfully demonstrated that a − 1/+2 subsite mutagenesis method could be used in β-glucosidases Tn0602 to improve enzyme productivity and expand product scope, which could be a potential route to evolve retaining glycosidases towards the desired direction.

Galacto-Oligosaccharide (GOS) Synthesis during Enzymatic Lactose-Free Milk Production: State of the Art and Emerging Opportunities

Much attention has recently been paid to β-Galactosidases (β-D-galactoside galactohidrolase; EC 3.2.1.23), commonly known as lactases, due to the lactose intolerance of the human population and the importance of dairy products in the human diet. This enzyme, produced by microorganisms, is being used in the dairy industry for hydrolyzing the lactose found in milk to produce lactose-free milk (LFM). Conventionally, β-galactosidases catalyze the hydrolysis of lactose to produce glucose and galactose in LFM; however, they can also catalyze transgalactosylation reactions that produce a wide range of galactooligosaccharides (GOS), which are functional prebiotic molecules that confer health benefits to human health. In this field, different works aims to identify novel microbial sources of β-galactosidase for removing lactose from milk with the relative GOS production. Lactase extracted from thermophilic microorganisms seems to be more suitable for the transgalactosylation process at relatively high temperatures, as it inhibits microbial contamination. Different immobilization methods, such as adsorption, covalent attachment, chemical aggregation, entrapment and micro-encapsulation, have been used to synthesize lactose-derived oligosaccharides with immobilized β-galactosidases. In this mini-review, particular emphasis has been given to the immobilization techniques and bioreactor configurations developed for GOS synthesis in milk, in order to provide a more detailed overview of the biocatalytic production of milk oligosaccharides at industrial level.

Membrane Bioreactor for Simultaneous Synthesis and Fractionation of Oligosaccharides.

Galacto-oligosaccharides (GOS) are prebiotic sugars obtained enzymatically from lactose and used in food industry due to their nutritional advantages or technological properties. Selective mass transport and enzymatic synthesis were integrated and followed using a membrane bioreactor, so that selective removal of reaction products may lead to increased conversions of product-inhibited or thermodynamically unfavorable reactions. GOS syntheses were conducted on lactose solutions (150 g·L−1) at 40 °C and 10 Uβ-galactosidase.mL−1, and sugar fractionation was performed by cellulose acetate membranes. Effects of pressure (20; 24 bar) and crossflow velocity (1.7; 2.0; 2.4 m·s−1) on bioreactor performance were studied. Simultaneous GOS synthesis and production fractionation increased GOS production by 60%, in comparison to the same reactions promoted without permeation. The presence of a high-molecular-weight solute, the enzyme, in association with high total sugar concentration, leads to complex selective mass transfer characteristics. Without the enzyme, the membrane presented tight ultrafiltration characteristics, permeating mono- and disaccharides and retaining just 25% of trisaccharides. During simultaneous synthesis and fractionation, GOS-3 were totally retained, and GOS-2 and monosaccharides were retained at 80% and 40%, respectively. GOS synthesis—hydrolysis evolution was strongly dependent on crossflow velocity at 20 bar but became fairly independent at 24 bar.

Batch synthesis of galactooligosaccharides from co-products of milk processing using immobilized β-galactosidase from Bacillus circulans

Milk whey (MW) and milk whey permeate (MWP) constitute the main part of co-products derived from milk processing. The use of the transgalactosylation activity of β-galactosidases is an excellent alternative to add value to these co-products since this is the route for the synthesis of galactooligosaccharides (GOS), a valuable prebiotic sugar. Hereby, we immobilized a β-galactosidase from Bacillus circulans onto glutaraldehyde-activated chitosan beads. The immobilized enzyme was characterized and the parameters affecting GOS synthesis (pH, temperature, and concentration of MW and MWP) were evaluated. The optimum pH and temperature of activity for the immobilized β-galactosidase were 50 °C and pH 6.0, respectively. The immobilization improves enzyme thermal stability by 2.9 times. In the batch process, the immobilized enzyme exhibited excellent operational stability at 40 °C, being able to hydrolyze 91% lactose after 17 cycles of reuse. Regarding GOS synthesis, the immobilized enzyme could be reused for 10 consecutive cycles retaining 74% of the initial activity at 50 °C. The maximum concentration of GOS achieved, using MW and MWP, was 159.4 g L−1 and 168.8 g L−1, respectively. Therefore, these results proved that it was possible to obtain, in high concentration, a useful and valuable prebiotic sugar from co-products of milk processing.

CHARACTERIZATION OF THERMOSTABLE BETA-1,4-GALACTANASE AND ITS APPLICATION IN HYDROLYSIS OF PECTIN FROM SWEET POTATO (Ipomoea batatas (L.) Lam) PEELS

Galactooligosaccharides (GOS) synthesis has received much attention due to its prebiotic function. Beta-1,4-galactanase responsible for the hydrolysis of galactan plays an important role in producing GOS from biodegradation of this pectin component. In this study, beta-1,4-galactanase (BgcGC) from a thermophilic Geobacillus mahadii Geo-05 was heterologously expressed in Escherichia coli (E. coli) and characterized. The optimum temperature of BgcGC was at 60°C and stable from 20-60°C while optimum pH was at 6 and stable from pH 4-10. BgcGC showed high catalytic efficiency towards potato galactan (873.8 ml mg-1 s-1) and lupin galactan (1694.4 ml mg-1 s-1). The activity of BgcGC was not significantly affected with the presence of 100 mM K+, Tween-20 and 2-mercaptoethanol. Application of BgcGC towards pectin-containing galactan oligomer extracted from sweet potato peels resulted in galactose and GOS synthesis as revealed by high performance liquid chromatography analysis. Thus, this enzyme has a potential to be one of the enzyme candidates involves in pectin complex degradation to produce GOS.

Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of β-Galactosidase.

Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from β-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected β-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.

Integrated Utilization of Dairy Whey in Probiotic β-Galactosidase Production and Enzymatic Synthesis of Galacto-Oligosaccharides

This work established an integrated utilization of dairy whey in β-galactosidase production from Lactobacillus bulgaricus and prebiotics synthesis by the probiotic enzyme. A cost-effective whey-based medium was newly developed for culturing Lactobacillus bulgaricus to produce β-galactosidase. The medium was optimized through response surface methodology (RSM) involving a series of statistical designs, such as the Plackett–Burman design, steepest ascent experiment, and central composite design. Under the optimized medium, the β-galactosidase activity of L. bulgaricus reached 2034 U/L, which was twice that produced from the traditional MRS medium. The cells of L. bulgaricus harvested from the whey-based medium were subsequently treated with lysozyme. The resulting crude enzyme was used as an efficient catalyst, which catalyzed the synthesis of the prebiotic galacto-oligosaccharides (GOS) in a high yield of 44.7% by using whey (200 g/L) as the substrate. The sugar mixture was further purified by activated charcoal adsorption, thereby yielding a high-purity level of 77.6% GOS.

Valorisation of sweet whey by fermentation with mixed yoghurt starter cultures with focus on galactooligosaccharide synthesis

Sweet whey was fermented with yoghurt starter cultures (YO-MIX 101 containing Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium lactis and YO-MIX 532 containing Lactobacillus delbrueckii subsp. bulgaricus and S. thermophilus) as well as enzyme extracts thereof to synthesise galactooligosaccharides from lactose. The influence of fermentation temperature and initial lactose concentration was studied. The highest yield in sweet whey (34.2% including disaccharide GOS, 25.4% excluding disaccharide GOS) was obtained with the YO-MIX 532 culture at 50 °C and 100 g L−1 initial lactose. With YO-MIX 101, yields were considerably lower, but transgalactosylation was favoured in sweet whey compared with buffer (23.4% versus 8.5% total GOS at 100 g L−1 initial lactose). Of the isolated strains derived from the mixed cultures, only Lactobacillus bulgaricus and both strains of S. thermophilus showed notable GOS synthesis. Up-scaling to a stirred tank reactor system yielded 26.3% total GOS within 24 h of fermentation.

Immobilization of β‐Galactosidase on Monolithic Discs for the Production of Prebiotics Galacto‐oligosaccharides

AbstractFour different activated methacrylatic monoliths, acting as pore‐through‐flow membrane reactors in order to reduce enzyme costs and mass‐transfer limitations, were characterized as support for immobilization of β‐galactosidase to produce galacto‐oligosaccharides (GOS). GOS synthesis was studied in different operation modes. A higher affinity of the immobilized enzyme towards the transgalactosylation reaction could be obtained, which results in desired GOS with higher degree of polymerisation. Kinetic models were successfully parametrized forming a basis for process modeling and optimization.