Maximum Galacto-oligosaccharides Concentration Research Articles

Continuous Packed Bed Reactor with Immobilized β-Galactosidase for Production of Galactooligosaccharides (GOS)

The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal) agarose beads and assayed for the continuous production of galactooligosaccharides (GOS) in a packed-bed reactor (PBR). The immobilization was fast (1 h) and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-d-galactopyranoside (ONPG). The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L) was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS 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 PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases.

Galactooligosaccharides formation during enzymatic hydrolysis of lactose: Towards a prebiotic-enriched milk

The formation of galactooligosaccharides (GOS) in skim milk during treatment with several commercial β-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analysed in detail, at 4 and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40–50% with B. circulans and A. oryzae β-galactosidases, and at 95% lactose depletion for K. lactis β-galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis β-galactosidase was achieved in 1 and 5h at 40 and 4°C, respectively. With this enzyme, it was possible to obtain a treated milk with 7.0g/L GOS − the human milk oligosaccharides (HMOs) concentration is between 5 and 15g/L – and with a low content of residual lactose (2.1g/L, compared with 44–46g/L in the initial milk sample). The major GOS synthesised by this enzyme were 6-galactobiose [Gal-β(1→6)-Gal], allolactose [Gal-β(1→6)-Glc] and 6′-O-β-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc].

Enzymatic synthesis of galactooligosaccharides using pressurised fluids as reaction medium

Abstract This work is focussed on the evaluation of enzymatic synthesis of galactooligosaccharides (GOS) catalysed by β-galactosidase from permeabilized cell of Kluyveromyces marxianus in different compressed fluids. The experiments were performed in a batch mode reactor, where the effects of enzyme and lactose concentrations and system pressure were evaluated. Results showed significant differences in the maximum GOS concentration among the experimental conditions, where the lactose concentration and enzyme activity were found to be statistically significant. Comparing the GOS productions in the three reaction systems, the supercritical CO 2 led to the best results, where the maximum production was 83 g l −1 while, for propane and n -butane, values of 63.0 and 75.0 g l −1 were verified, respectively. According to the kinetic results of GOS production in supercritical CO 2 , a promising route is noted for this system, since the competition between synthesis and hydrolysis was less pronounced compared to the other tested fluids.

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.

Synthesis of galacto-oligosaccharides in AOT/isooctane reverse micelles by β-galactosidase

Synthesis of galacto-oligosaccharides (GOS) from lactose by β-galactosidase was investigated in AOT/isooctane reverse micelles where the enzyme was fairly stable. GOS yield increased with initial lactose concentration in both reverse micelles and aqueous system. Transgalactosylation reaction of the enzyme was strongly dependent on the molar ratio of water to surfactant ( w 0) of reverse micelles. At pH 7.0 and 45°C, a maximum GOS concentration from 45% (w/v) lactose solution was 51.2% (w/w) in the reverse micelles with w 0=15, while a maximum of 31% in aqueous media. Effects of solvents on GOS formation were also examined.