Synthesis of Lactulose in Continuous Stirred Tank Reactor With β-Galactosidase of Apergillus oryzae Immobilized in Monofunctional Glyoxyl Agarose Support (original) (raw)
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Commercial-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-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.
International Dairy Journal, 2014
In this study, lactulose production with b-galactosidase from Kluyveromyces lactis in aqueous and nonconventional media was studied. The optimisation of the rate of enzymatic lactulose production in aqueous media was carried out using factorial design methodology. The results showed a statistically significant effect of both lactose and fructose levels e as well as their interaction e on the rate of lactulose production. Maximum lactulose production rate was obtained at fructose and lactose concentrations of 25e30% (w/w) and 9e12% (w/w), respectively. The addition of acetone had a negative effect on lactulose production, whereas the incorporation of triethyl phosphate up to a concentration of 30% (w/w) caused an increase of up to 20% in the lactulose production.
Food Chemistry, 2008
A study on optimisation of the conditions for galactooligosaccharide (GOS) formation during lactose hydrolysis, produced by Lactozym 3000 L HP G, was carried out. The synthesis was performed during times up to 300 min at 40, 50 and 60°C, pH 5.5, 6.5 and 7.5, lactose concentration 150, 250 and 350 mg/mL and enzyme concentration 3, 6 and 9 U/mL. The product mixtures were analysed by highperformance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). During the hydrolysis of lactose, besides glucose and galactose, galactobiose, allolactose and 6 0 galactosyl lactose were also formed as a result of transgalactosylation catalysed by the enzyme. The effect of the reaction conditions was different in the formation of di-and the trisaccharide. Thus, the optimal conditions for galactobiose and allolactose synthesis were 50°C, pH 6.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 300 min, whereas the best reaction conditions for 6 0 galactosyl lactose production were 40°C, pH 7.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 120 min. These results show the possibility to obtain reaction mixtures with Lactozym 3000 L HP G, with different composition, depending on the assayed conditions.
The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae p-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased ~25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40°C and 48 days at 50oC. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40°C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported.
Bioresource technology, 2017
Lactulose synthesis was done in repeated-batch mode with Aspergillus oryzae β-galactosidase immobilized in glyoxyl-agarose (GA-βG), amino-glyoxyl-agarose (Am-GA-βG) and chelate-glyoxyl-agarose (Che-GA-βG), at fructose/lactose molar ratios of 4, 12 and 20. Highest yields of lactulose in batch were obtained with Che-GA-βG (0.21, 0.29 and 0.32g·g(-1)) for 4, 12 and 20 fructose/lactose molar ratios respectively; when operating in 10 repeated batches highest product to biocatalyst mass ratios were obtained with Am-GA-βG (1.82, 2.52 and 2.7g·mg(-1)), while the lowest were obtained with Che-GA-βG (0.25, 0.33 and 0.39g·mg(-1)). Operational stability of Am-GA-βG was higher than GA-βG and Che-GA-βG and much higher than that of the free enzyme, at all fructose/lactose molar ratios evaluated. Efficiency of biocatalyst use for GA-βG were 64.4, 35.5 and 18.4kglactulose/gprotein, for fructose/lactose molar ratios of 4, 12 and 20 respectively, while for Che-GA-βG were 1.46, 1.05 and 0.96kglactulose...
Fed-batch operation for the synthesis of lactulose with β-galactosidase of Aspergillus oryzae
Bioresource Technology, 2017
Fed-batch synthesis of lactulose from lactose and fructose with Aspergillus oryzae βgalactosidase was evaluated, obtaining a concentration of 40.4 g⋅L-1 , which is 20 % higher than obtained in batch, while the concentration of transgalactosylated oligosaccharides (TOS) was reduced by 98%. Therefore, selectivity of lactulose synthesis can be significantly higher by operating in fed-batch mode. The enzyme-limiting substrate mass ratio (E/S) is a critical variable in fed-batch operation. Higher values favor lactose hydrolysis over transgalactosylation, being 400 IU/g the limit for proper lactulose synthesis in fed-batch operation. Selectivity of lactulose synthesis increased with E/S being quite high at 800 IU H ⋅g-1 or higher. However, this increase was obtained at the expense of lactulose yield. Lactulose synthesis in fed-batch operation was a better option than conventional batch synthesis, since higher product concentration and selectivity of lactulose over TOS synthesis were obtained.
The effect of enzyme to substrate ratio, initial lactose concentration and temperature has been studied for the kinetically controlled reaction of lactose transgalactosylation with Aspergillus oryzae-galactosidase, to produce prebiotic galacto-oligosaccharides (GOS). Enzyme to substrate ratio had no significant effect on maximum yield and specific productivity. Galacto-oligosaccharide syntheses at very high lactose concentrations (40, 50 and 60%, w/w, lactose monohydrate) were evaluated at different temperatures (40, 47.5 and 55 • C). Within these ranges, lactose could be found as a supersaturated solution or a heterogeneous system with precipitated lactose, resulting in significant effect on GOS synthesis. An increase in initial lactose concentration produced a slight increase in maximum yield as long as lactose remained dissolved. Increase in temperature produced a slight decrease in maximum yield and an increase in specific productivity when supersaturation of lactose occurred during reaction. Highest yield of 29 g GOS/100 g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5 • C. Highest specific productivity of 0.38 g GOS h −1 mg enzyme −1 was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55 • C, where a maximum yield of 27 g GOS/100 g lactose added was reached. This reflects the complex interplay between temperature and initial lactose concentration on the reaction of synthesis. When lactose precipitation occurred, values of yields and specific productivities lower than 22 g GOS/100 g lactose added and 0.03 g GOS h −1 mg enzyme −1 were obtained, respectively.
Carbohydrate Polymers, 2013
Kluyveromyces lactis was immobilized on glutaraldehyde-activated chitosan and used in a packed-bed reactor for the continuous hydrolysis of lactose and the synthesis of galactooligosaccharides (GOS). The biocatalyst was tested for its optima pH and temperature, thermal stability in the presence of substrate and products, and operational stability. Immobilization increased the range of operational pH and temperature, and the enzyme thermal stability was sharply increased in the presence of lactose. Almost complete lactose hydrolysis was achieved for both milk whey and lactose solution at 37 • C at flow rates up to 2.6 mL min −1. Maximal GOS concentration of 26 g L −1 was obtained at a flow rate of 3.1 mL min −1 , with a productivity of 186 g L −1 h −1. Steady-state operation for 15 days showed the reactor stability concerning lactose hydrolysis.
Enzyme and Microbial Technology, 2012
The effect of enzyme to substrate ratio, initial lactose concentration and temperature has been studied for the kinetically controlled reaction of lactose transgalactosylation with Aspergillus oryzae -galactosidase, to produce prebiotic galacto-oligosaccharides (GOS). Enzyme to substrate ratio had no significant effect on maximum yield and specific productivity. Galacto-oligosaccharide syntheses at very high lactose concentrations (40, 50 and 60%, w/w, lactose monohydrate) were evaluated at different temperatures (40, 47.5 and 55 • C). Within these ranges, lactose could be found as a supersaturated solution or a heterogeneous system with precipitated lactose, resulting in significant effect on GOS synthesis. An increase in initial lactose concentration produced a slight increase in maximum yield as long as lactose remained dissolved. Increase in temperature produced a slight decrease in maximum yield and an increase in specific productivity when supersaturation of lactose occurred during reaction. Highest yield of 29 g GOS/100 g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5 • C. Highest specific productivity of 0.38 g GOS h −1 mg enzyme −1 was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55 • C, where a maximum yield of 27 g GOS/100 g lactose added was reached. This reflects the complex interplay between temperature and initial lactose concentration on the reaction of synthesis. When lactose precipitation occurred, values of yields and specific productivities lower than 22 g GOS/100 g lactose added and 0.03 g GOS h −1 mg enzyme −1 were obtained, respectively.