Enzymatic Conversion Of Lactose Research Articles

Potential of Integrated Semi‐Continuous Enzymatic Synthesis and Filtration Processes for Efficiency Enhancement

AbstractGalacto‐oligosaccharides (GOS), increasingly popular prebiotics, are synthesized by enzymatic conversion of lactose. Among others, the total production costs are significantly influenced by the costly enzyme. Therefore, it was investigated if the reuse and full recovery of the enzyme is feasible, followed by the development of a semi‐continuous process in order to maintain a consistent high GOS yield. As a preliminary step, the enzyme‐catalyzed reaction was recorded within the permissible operating parameters. It was successfully shown that steady high GOS yields can be synthesized semi‐continuously within a filtration plant functioning as an enzymatic membrane reactor.

Continuous Production of Galacto-Oligosaccharides by an Enzyme Membrane Reactor Utilizing Free Enzymes.

Galacto-oligosaccharides (GOS) are prebiotic compounds widely used for their health-promoting effects. Conventionally, GOS is produced by the enzymatic conversion of lactose in stirred tank reactors (STR). The high operational costs associated with enzyme inactivation and removal might be reduced by the application of enzyme membrane reactors (EMR). In this study, we aimed to assess the potential of continuous GOS production by EMR using soluble Biolacta N5, a Bacillus circulans-derived commercial enzyme preparation. The steady-state performance of the EMR equipped with an ultrafiltration module was investigated as function of residence time (1.1–2.8 h) and enzyme load (17–190 U·g−1) under fixed operational settings of temperature (50 °C), pH (6.0), lactose feed concentration (300 g·kg−1), and recirculation flow-rate (0.18 m3·h−1). Results indicate that the yield of oligosaccharides with higher degree of polymerization (DP3-6) in STR (approx. 38% on total carbohydrate basis) exceeds that measured in EMR (ranging from 24% to 33%). However, a stable catalytic performance without a significant deterioration in product quality was observed when operating the EMR for an extended period of time (>120 h). Approx. 1.4 kg of DP3-6 was produced per one gram of crude enzyme preparation over the long-term campaigns, indicating that EMR efficiently recovers enzyme activity.

Limitation of Maillard Reactions in Lactose-Reduced UHT Milk via Enzymatic Conversion of Lactose into Galactooligosaccharides during Production.

Lactose-hydrolyzed (LH) ultrahigh temperature (UHT) processed milk is more prone to Maillard reactions and formation of advanced glycation end products (AGEs) during processing and storage than conventional (CON) UHT milk because of the presence of free galactose and glucose. Commercially available β-d-galactosidases with transgalactosylating activity can incorporate galactose into galactooligosaccharides (GOSs) and potentially limit Maillard reactions in this lactose-reduced GOS-containing milk. The aim of this study was to examine the extent of Maillard reactions in a lactose-reduced GOS milk compared to LH and CON milk after UHT processing. The GOS milk had significant lower levels of lysine- and arginine-derived AGEs compared to LH milk, while their concentrations were similar to those found in CON milk. The total concentration of measured Arg-derived AGEs was similar to the total concentration of Lys-derived AGEs in the three types of milk, indicating that Arg is an important source of AGEs in milks. Interestingly, the GOS milk generated threefold higher concentrations (up to 330 ± 6 μM) of 3-deoxyglucosone (3-DG, a C6 α-dicarbonyl). These results demonstrate that GOS milk could be a potential alternative for LH milk for lactose-intolerant individuals, although further studies are needed to understand the increased formation of 3-DG in GOS-containing milk.

Enzymatic conversion of lactose into galacto-oligosaccharides: The effect of process parameters, kinetics, foam architecture, and product characterization

Galacto-oligosaccharides (GOS) synthesized from lactose using Aspergillus oryzae β-galactosidases. We studied the influences of enzyme concentration (0.02–0.1%. w/v), temperature (40–70 °C), pH (4.5–8), lactose concentration (5–40%, w/v) and foam architecture reaction media on formation of GOS. The total GOS increased from about 7 to 23 (wt.% of initial lactose) with an increase in lactose from 5 to 40% (w/v). Trisaccharides were the major GOS accounting for approximately 78% (w/w) of GOS. The developed kinetic model for predicting kinetic parameters using the genetic algorithm of MATLAB Software resulted in good agreement between simulated and experimental data. Conducting the reaction in foam architecture with an initial lactose concentration of 20% (w/v), 0.05% (w/v), 0.05% (w/v) enzyme and 0.02% (w/v) ranaspumin-2 (surfactant) at pH of 4.5 and 45 °C did not have any effect on reaction and conversion efficiency. The structure of the main fractionated trisaccharide determined with NMR was galactose-β (1,6)-galactose-β (1,4)-glucose.

Enzymatic production of lactulose and 1-lactulose: current state and perspectives

Lactulose, a synthetic ketose disaccharide, has been widely used in food and pharmaceutical industries as prebiotic food additives and drugs against constipation and hepatic encephalopathy. Lactulose has, so far, been produced chemically using lactose on a commercial scale. The key problems associated with such chemical process are the cost of removal of the catalyst and colored by-products and the product safety. Enzymatic production of lactulose is safe, environment-friendly, and simpler in comparison to the chemical method. As a promising alternative to the chemical method, enzymatic conversion of lactose into lactulose by β-galactosidase or cellobiose 2-epimerase has recently gained a great deal of attention. This could be considered as a possible route for whey surplus because lactose is the major component of cheese whey. Herein, we summarize recent advances on the enzymatic synthesis of lactulose with emphasis on the selectivity of biocatalysts and their catalytic efficiency in free and immobilized forms. The production of 1-lactulose by enzymatic bioconversion of lactose has also been discussed. Furthermore, future research needs of β-galactosidase and cellobiose 2-epimerase for the enzymatic synthesis of lactulose and 1-lactulose are reviewed.

Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable β-glycosylhydrolase CelB fromPyrococcus Furiosus

The synthesis of glycosides by enzymatic transglycosylation is a kinetically controlled reaction performed in the context of a non-favorable thermodynamic equilibrium. An unreactive organic cosolvent which increases the selectivity of the enzyme for glycosyl transfer to the acceptor nucleophile compared with water (Ksel) could improve maximum product yield. Here we report on the effect of the ionic liquid 1,3-dimethylimidazoliummethylsulfate on hydrolase and transferase activities of the hyperthermostable beta-glycosidase CelB from the archaeon Pyrococcus furiosus. CelB retained full catalytic efficiency for lactose hydrolysis at 80 degrees C in a 50% (by vol.) solution of ionic liquid in sodium citrate buffer, pH 5.5. It was inactive but not irreversibly denatured at 70% ionic liquid. Using lactose (0.15 M) as galactosyl donor, values of Ksel for a representative series of eight acceptor alcohols were determined in kinetic assays at 80 degrees C and found to increase between 1.3-fold (D-xylose) and 3.1-fold (glycerol) in 45% ionic liquid. Enhancement of Ksel was dependent on ionic liquid concentration and higher than expected from the decrease in water activity caused by the cosolvent. Experimental molar ratios of D-glucose and D-galactose produced during enzymatic conversion of lactose (75-150 mM) in the presence of D-xylose (0.5 M) or glycerol (0.5 M) showed excellent agreement with predictions based on Ksel values and confirm a significant, yet moderate effect of 45% ionic liquid on increasing the yield of D-galactoside product, by < or = 10%.

Determination of apparent kinetic parameters for competitive product inhibition in packed-bed immobilized enzyme reactors

In this study, a simple and effective technique for characterizing Michaelis–Menten type kinetics with competitive product inhibition in packed-bed re-circulated immobilized enzyme reactors is presented, where the use of nonlinear regression techniques for multi-parameter estimation are not required. In order to demonstrate the new technique introduced in this work, enzymatic conversion of lactose in a recycling packed-bed reactor is envisaged where β-galactosidase (lactase, EC 3.2.1.23) enzyme is immobilized on a weak base ion exchanger resin (Duolite A 568). For the experimental conditions used in this research, the total competitive inhibition by product (galactose) model is sufficient to represent the lactose hydrolysis kinetics in a packed-bed reactor.

Separation of lactose, lactobionic acid and lactobionolactone by high-performance liquid chromatography

Complete separation of the three title compounds has been achieved for the first time on either a β-cyclodextrinor an aminopropyl-silica gel-bonded-phase HPLC column eluted with acetonitrile-aqueous buffer mixtures. An HPLC system using either of these stationary phases and a refractive index detector directly quantified lactose, lactobionic acid and lactobionolactone at levels between 0.3 and 40 μg of analyte per injection. Simple separations of lactose and lactobionic acid were also readily accomplished on a calcium-form cation-exchange type column eluted with 1.2 m M CaSO 4 solution. These methods are useful for monitoring the chemical or enzymatic conversion of lactose into the valuable derivatives, lactobionic acid and lactobionolactone.

Direct Enzymatic Conversion of Lactose in Milk to Acid

Commercial production of acidified dairy products depends on a fermentative conversion of lactose to lactic acid, a multi-enzyme process. This slow and complicated biological process naturally tends to have disadvantages such as susceptibility to contamination and variation in acid formation; and production difficulties like starter preparation, inflexible equipment requirements, and scheduling. In 1965, we originally suggested that some of these problems in milk acidification might be overcome by using an oxidoreductase enzyme (6). At the time several enzymes were known with the capability of converting monoand disaccharides in the presence of oxygen to a lactone, followed bv spontaneous hydrolysis to the corresponding aldobionic acid. Thus, it appeared feasible that enzymatic conversion of lactose in milk directly to acid could provide an alternative method for acidification. Enzymes capable of oxidizing lactose, galactose, or glucose have been isolated and studied, but applications to milk acidification have not been considered. For example, hexose oxidase (EC 1.1.3.5) was found in the marine red alga lridophycus flaccidum (1); a glucose dehydrogenase (EC 1.1.99.10) was isolated from Bacterium anitratum (3); a lactose dehydrogenase (EC 1.1.99) was separated from lactose-adapted cells of Pseudomonas graveolens (5); and glucose oxidase (EC 1.1.8.4) from Aspergillus niger has been available as a commercial food-grade enzyme for over 20 yr (8). Our initial studies with enzymatic conversion of lactose to acid indicated that the hydrolyzed sugar, as glucose and galactose, could be utilized to acidify milk to pH 4.5 to 5.0 (7). However, a source of oxygen to provide uniform distribution throughout milk was an essential factor to provide a uniform rate of reaction. The combination of hydrogen peroxide

The enzymatic conversion of lactose into galactosyl oligosaccharides.

The enzymatic conversion of lactose into galactosyl oligosaccharides.