Pontificia Universidad Catolica de Valparaiso
Facultad De Ingenieria
Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at... more
Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product distribution in terms of prebiotic potential. The selectivity of fermentation of each carbohydrate alone as well as mixtures of both was determined using pH-controlled anaerobic batch cultures with faecal inocu-lum. Within the experimental range considered, lactulose/GOS molar ratio of 4 resulted in the highest selectivity for Bifidobacterium and Lactobacillus/Enterococcus, so this ratio was selected as the target for the synthesis of lactulose from fructose and lactose with Aspergillus oryzae-galactosidase. Synthesis was optimized using response surface methodology, considering temperature, initial concentrations of acceptor sugars and fructose/lactose molar ratio as key variables, with the aim of maximizing lactulose yield at the optimal product distribution in terms of prebiotic potential (lactulose/GOS molar ratio of 4). Under optimal conditions (50 • C, 50%w/w total initial concentrations of sugars and fructose/lactose molar ratio of 6.44), lactulose yield of 0.26 g of lactulose produced per g of initial lactose was obtained at the optimal product distribution.
- by Carlos Vera Vera and +1
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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... more
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.
• Four fouling mechanisms were assessed during enzymatic GOS synthesis in an UF-MBR. • High lactose concentration (468 g/L) and different process conditions were tested. • Strong statistical analyses avoided misinterpretation of the... more
• Four fouling mechanisms were assessed during enzymatic GOS synthesis in an UF-MBR. • High lactose concentration (468 g/L) and different process conditions were tested. • Strong statistical analyses avoided misinterpretation of the fitted model mechanism. • Cake fouling and intermediate blocking were the predominant mechanisms. • Boundary gel layer formation was due to the reaction products rather than the enzyme. a b s t r a c t The flux decay of an ultrafiltration membrane bioreactor for the synthesis of galacto-oligosaccharides was modeled, by varying the processing conditions: temperature (40–60 °C), transmembrane pressure (2.5–4 bar) and cross-flow velocity (3.5–7 m/s) according to a 2 k design. Fouling mechanisms varied according to the operational condition, showing that the predominant mechanism were the intermediate fouling, which was associated to the higher flux decay due to the partial adsorption of the enzyme on the membrane, and the cake fouling mechanism, which was associated to those runs where the flux declined by around 20% because of the high concentration of substrate used (40% w/w). Strong statistical analysis allowed validation or rejection of initial adjustments given by a simple R-square statistical test, showing that misinterpretation of the fouling mechanism can be done under particular conditions, but also revealed that fouling equations have some limitations when drastic flux decay occurs or when this remains virtually unchanged. The experimental design also showed that temperature was the variable having the main positive effect on flux stability by decreasing the solution viscosity; however, the interaction between cross-flow velocity and transmembrane pressure had the main effect on flux decay.
- by Carlos Vera Vera and +1
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Galactosidase from Aspergillus oryzae was immobilized by different methods, being the enzyme immobilized in glyoxyl-agarose selected for the synthesis of galacto-oligosaccharides from lactose at high concentrations. The synthesis was... more
Galactosidase from Aspergillus oryzae was immobilized by different methods, being the enzyme immobilized in glyoxyl-agarose selected for the synthesis of galacto-oligosaccharides from lactose at high concentrations. The synthesis was optimized with respect to lactose concentration and enzyme to sub-strate ratio using lactose conversion into galacto-oligosaccharides as objective function. Optimum values were 54.6% (w/v) lactose and 33 IU g −1 lactose and at such conditions conversion was close to 30%, similar to the one obtained with soluble-galactosidase and comparable with the best reported values for immobilized-galactosidases. In the sequential batch production of galacto-oligosaccharides, bio-catalyst efficiency was increased by 190% with respect to the free enzyme in solution, and 8500 g of galacto-oligosaccharides per gram of enzyme preparation were produced after 10 batches. This amount can be further increased by considering additional batches up to the point of biocatalyst replacement and is significantly higher than the maximum attainable by using the soluble enzyme.
Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by... more
Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by β-galactosidase, named transglycosylation. Lactose is the main constituent of whey permeate (WP), normally wasted output from the cheese industry. Therefore, the main goal of this work was to optimise the synthesis of GOS in WP using β-galatosidase from Aspergillus oryzaea. WP and whey permeate enzymatically treated (WP-GOS) were used as culture media of Lactobacillus plantarum 299v. Lb. plantarum 299v attained the stationary phase in approximately 16 h, reaching 3·6 and 4·1 × 10 8 CFU/ml in WP and WP-GOS, respectively. The in situ synthesised GOS were not consumed during growth. No significant differences were observed in the growth kinetics of microorganisms in both media. After fermentation, microorganisms were dehydrated by freeze-drying and spray-drying and stored. The recovery of microorganisms after fermentation, dehydration and storage at 4 °C for at least 120 d was above 10 8 CFU/g. These studies demonstrated that WP is an appropriate substrate for the synthesis of GOS and the obtained product is also adequate as culture medium of Lb. plantarum 299v. The coexistence of GOS and dehydrated viable probiotic microorganisms, prepared using an effluent as raw material, represents the main achievement of this work, with potential impact in the development of functional foods.
A pseudo steady-state model for the kinetically controlled synthesis of galacto-oligosaccharides (GOS) with Aspergillus oryzae b-galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of... more
A pseudo steady-state model for the kinetically controlled synthesis of galacto-oligosaccharides (GOS) with Aspergillus oryzae b-galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta-oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 558C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl–enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl–enzyme complex is higher than that of the free enzyme.
The catalytic potential of b-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... more
The catalytic potential of b-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 b-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 b-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 b-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.
in Wiley Online Library (wileyonlinelibrary.com) Fed-batch synthesis of galacto-oligosaccharides (GOS) from lactose with b-galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal... more
in Wiley Online Library (wileyonlinelibrary.com) Fed-batch synthesis of galacto-oligosaccharides (GOS) from lactose with b-galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed-batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L 21 as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40 C). Simulations predicted a GOS yield of 32.5 g g 21 in fed-batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g 21). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS-5 and GOS-6) was observed in the fed-batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis.
- by Carlos Vera Vera and +1
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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.... more
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.
- by Carlos Vera Vera and +1
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- Bioengineering, Biotechnology
Description Enzyme biocatalysis is a fast-growing area in process biotechnology that has expanded from the traditional elds of foods, detergents, and leather applications to more sophisticated uses in the pharmaceutical and ne-chemicals... more
Description Enzyme biocatalysis is a fast-growing area in process biotechnology that has expanded from the traditional elds of foods, detergents, and leather applications to more sophisticated uses in the pharmaceutical and ne-chemicals sectors and environmental management. Conventional applications of industrial enzymes are expected to grow, with major opportunities in the detergent and animal feed sectors, and new uses in biofuel production and human and animal therapy. In order to design more efcient enzyme reactors and evaluate performance properly, sound mathematical expressions must be developed which consider enzyme kinetics, material balances, and eventual mass transfer limitations. With a focus on problem solving, each chapter provides abridged coverage of the subject, followed by a number of solved problems illustrating resolution procedures and the main concepts underlying them, plus supplementary questions and answers.
Batch synthesis of fructosyl-galacto-oligosaccharides from lactulose was performed with commercial b-galactosidase preparations from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. The enzyme from A. oryzae produced the... more
Batch synthesis of fructosyl-galacto-oligosaccharides from lactulose was performed with commercial b-galactosidase preparations from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. The enzyme from A. oryzae produced the highest yield and specific productivity of synthesis, being selected for further studies. Optimization of fructosyl-galacto-oligosaccharides synthesis was carried out using response surface methodology, considering temperature and initial sugar concentration as variables and yield and specific productivity as response parameters. Maximum yield of 0.41 g g À1 fructosyl-galacto-oligosaccharides was obtained at 70 °C and 60% w/w lactulose concentration, while maximum specific productivity of 1.2 g h À1 mg À1 was obtained at 70 °C and 40% w/w lactulose concentration.
- by Carlos Vera Vera and +1
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- Bioengineering, Biotechnology
Performance of an ultrafiltration membrane bioreactor (UF-MBR) as a processing strategy for the synthesis of galacto-oligosaccharides at high substrate concentrations. a b s t r a c t The performance of an ultrafiltration membrane... more
Performance of an ultrafiltration membrane bioreactor (UF-MBR) as a processing strategy for the synthesis of galacto-oligosaccharides at high substrate concentrations. a b s t r a c t The performance of an ultrafiltration membrane bioreactor for galacto-oligosaccharides (GOS) synthesis using high lactose concentrations (470 g/L) and-galactosidase from Aspergillus oryzae was assessed. Tested processing variables were: transmembrane-pressure (PT), crossflow-velocity (CFV) and temperature. Results showed that processing variables had significant effect on the yield, the enzyme productivity and the flux but did not on GOS concentration and reaction conversion obtained. As expected, the use of high turbulences improved mass transfer and reduced the membrane fouling, but the use of very high crossflow-velocities caused operational instability due to vortex formation and lactose precipitation. The use of a desirability function allowed determining optimal conditions which were: PT (4.38 bar), CFV (7.35 m/s) and temperature (53.1 • C), optimizing simultaneously flux and specific enzyme productivity Under these optimal processing conditions, shear-stress and temperature did not affect the enzyme but long-term operation was limited by flux decay. In comparison to a conventional batch system, at 12.5 h of processing time, the continuous GOS synthesis in the UF-MBR increased significantly the amount of processed substrate and a 2.44-fold increase in the amount of GOS produced per unit mass of catalyst was obtained with respect to a conventional batch system. Furthermore, these results can be improved by far by tuning the membrane area /reaction volume ratio, showing that the use of an UF-MBR is an attractive alternative for the GOS synthesis at very high lactose concentrations.
- by Carlos Vera Vera
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Galacto-oligosaccharides (GOS) were synthesised from 50% (w/w) lactose with b- Aspergillus oryzae b-galactosidase. Reacted medium without nutrient supplementation (raw GOS) was fermented with Kluyveromyces marxianus cells obtaining GOS of... more
Galacto-oligosaccharides (GOS) were synthesised from 50% (w/w) lactose with b- Aspergillus oryzae b-galactosidase. Reacted medium without nutrient supplementation (raw GOS) was fermented with Kluyveromyces marxianus cells obtaining GOS of 95% purity containing mostly tri- and tetrasaccharides with total recovery of GOS after 24 h. The effect of the initial concentration of carbohydrate was assessed in the range of 10 to 50% (w/w), such purity being obtained even at 50% (w/w) (undiluted raw GOS) though being strongly influenced by the dry cell to total carbohydrate mass ratio (RCC). The process of purification by selective fermentation of raw GOS at 20% (w/w) was optimised in terms of the specific productivity of unwanted carbohydrate removal, considering temperature, pH and RCC as operational variables. According to the experimental design, maximum specific productivity predicted was 0.16 g g-1 h-1, obtained at the optimal operating conditions of 40°C, pH 3.5 and RCC 1.55 g g-1
- by Carlos Vera Vera
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