Initial Lactose Concentration Research Articles

Simultaneous enzymatic hydrolysis and lactic fermentation to obtain a yogurt with low lactose content

In a single-stage process with the simultaneous addition of β-galactosidase and lactic culture, the lactose conversion, the processing time, viscosity and syneresis were evaluated. Fermentation was promoted by lactic culture containing two probiotic microorganisms, Bifidobacterium animalis and Lactobacillus acidophilus, associated with the typical microorganisms of yogurt. An enzymatic preparation containing β-galactosidases from Kluyveromyces lactis and Aspergillus niger was used. A central composite design (2³ trials plus three central points) was proposed in order to evaluate the effects of initial lactose concentration, enzyme concentration and the time of addition of the enzyme. The following conditions were established: initial lactose concentration of 91 g L-1, enzyme concentration of 0.5 g L-1 and enzyme addition at the beginning of fermentation. In these conditions, a decrease in processing time (from 4.55 to 3.68 h) and an increase in lactose conversion (from 15.2 to 97.9%) were observed in relation to the fermentation without enzyme addition, and no detrimental changes in some physical properties of yogurt (viscosity, and syneresis).

Kinetics and design relation for enzymatic conversion of lactose into galacto-oligosaccharides using commercial grade β-galactosidase

The enzymatic synthesis of galacto-oligosaccharides (GOS) from lactose was studied using commercial grade β-galactosidase (Biolacta FN5) from Bacillus circulans. The reaction was carried out under free enzyme condition varying initial lactose concentration (ILC: 55-525 g/L), enzyme concentration (0.05-1.575 g/L), temperature (30-50°C) and pH (5.0-6.0). Reaction mixture compositions were analyzed utilizing high performance liquid chromatography (HPLC). A maximum GOS formation of 39% (dry basis) was achieved at an ILC of 525 g/L converting 60% of the lactose fed. Tri-saccharides were the major types of GOS formed, accounting approximately 24%; whereas, tetra-saccharides and penta-saccharides account approximately 12% and 3%, respectively. Design correlation was developed in order to observe the quantitative effect of operating parameters on GOS yield. Further, based on Michaelis-Menten model, four-step reaction pathways were considered for simplistic understanding of the kinetics. Apart from predicting the reaction mixture composition, the approach also provided kinetic parameters though simulation using COPASI 4.7®. Excellent agreements were observed between simulated and experimental results.

Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis

The enzymatic transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis (KlβGal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(β-d-galactopyranosyl-(1→1)-d-fructose) with two main isomers in D2O was identified to be the major transgalactosylation product while lactulose(β-d-galactopyranosyl-(1→4)-d-fructose) turned out to be the minor one, indicating that KlβGal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4g/l, respectively, at 66.5% lactose conversion (200g/l initial lactose concentration).

Enzymatic Synthesis of Galacto-oligosaccharides in an Organic–Aqueous Biphasic System by a Novel β-Galactosidase from a Metagenomic Library

Prebiotic galacto-oligosaccharides (GOS) were effectively synthesized from lactose in organic-aqueous biphasic media by a novel metagenome-derived β-galactosidase BgaP412. A maximum GOS yield of 46.6% (w/w) was achieved with 75.4% lactose conversion rate in the cyclohexane/buffer system [95:5 (v/v) cyclohexane/buffer] under the optimum reaction conditions (initial lactose concentration = 30% (w/v), T = 50 °C, pH 7.0, and t = 8 h). The corresponding productivity of GOS was approximately 17.5 g L(-1) h(-1). The GOS mixture consisted of tri-, tetra-, and pentasaccharides. Trisaccharides were the chief component of reaction products. These experimental results showed that a low water content, a high initial lactose concentration, and an elevated reaction temperature could significantly promote the transgalactosylation activity of β-galactosidase BgaP412; at the same time, the enhanced GOS yield in an organic-aqueous biphasic system is because of the fact that thermodynamic equilibrium can be shifted to the synthetic direction by reversing the normal hydrolysis.

Enhanced production of thrombinase by Streptomyces venezuelae: Kinetic studies on growth and enzyme production of mutant strain

This investigation provides the enhanced production of thrombinase, a fibrinolytic enzyme using mutant Streptomyces venezuelae. Initially the mutagenesis of the marine isolate was done by UV and Ethyl methane sulfonate (EMS) and their mutational efficiencies were compared. The mutants were selected based on their high thrombinase activity and used for further studies. The mutant was found to be more halo and thermo tolerant comparing to wild. The effect of Dissolved oxygen level was also determined and the mutant offered the maximum specific growth rate as 0.2404 (h−1). The mutant showed high resistance to higher initial lactose concentration and the inhibition concentration was found to be 155.1mg/mL. The effect of S0/X0 ratio on specific substrate consumption and production rate were also investigated. Both mutant and wild showed increase in specific substrate consumption and production rate at higher S0/X0 ratio but the mutant showed better values than the wild strain.

Optimization Of Ethanol Production From Cheese Whey Fermentation In A Batch-Airlift Bioreactor

In this work, Kluyveromyces fragilis yeast was used for bio-ethanol production from cheese-whey in batch fermentation. The present study consisted of two steps: The first was a central composite design (CCD) for evaluating of important factors including: pH, initial lactose concentration (L), yeast cells concentrations (Y) and temperature (T). In order to optimize the fermentation process, response surface methodology (RSM) was used in this stage. The best operating conditions were found to be pH = 5.3, L = 41.8 g/l, Y = 0.57 g/l and T= 30.8°C. The second step was to determine the effect of aeration rate on the fermentation process in an airlift bioreactor. The best conditions were the aeration rate of 0.4 vvm with 89.28% of ethanol production yield. In this research, the concentrated cheese whey was also used for obtaining a bio-ethanol fermentation product.

Synthesis of galacto-oligosaccharides by β-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose

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 β-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 29g GOS/100g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5°C. Highest specific productivity of 0.38g GOSh−1mgenzyme−1 was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55°C, where a maximum yield of 27g GOS/100g 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 22g GOS/100g lactose added and 0.03gGOSh−1mgenzyme−1were obtained, respectively.

Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system

A spectrophotometric assay was developed for the quantification of lactose in aqueous solution via a one-pot enzymatic cascade reaction at 25 °C and pH 7.2. Lactose (0.2-1.8 mM), E. coli β-galactosidase (β-Gal), Aspergillus niger glucose oxidase (GOD), horseradish peroxidase (HRP) and o-phenylenediamine (OPD) were incubated, and the increase in absorbance at 417 nm (A (417)) due to the formation of DAP (2,3-diaminophenazine), the dimeric oxidation product of OPD, was followed. The increase in A (417) was found to depend linearly on the initial lactose concentration via three consecutive but simultaneously occurring enzymatic reaction steps catalyzed by β-Gal, GOD, and HRP. No pre-incubation of lactose with β-Gal is needed with this simple lactose assay.

Hybrid modeling of xanthan gum bioproduction in batch bioreactor

This work is focused on hybrid modeling of xanthan gum bioproduction process by Xanthomonas campestris pv. mangiferaeindicae. Experiments were carried out to evaluate the effects of stirred speed and superficial gas velocity on the kinetics of cell growth, lactose consumption and xanthan gum production in a batch bioreactor using cheese whey as substrate. A hybrid model was employed to simulate the bio-process making use of an artificial neural network (ANN) as a kinetic parameter estimator for the phenomenological model. The hybrid modeling of the process provided a satisfactory fitting quality of the experimental data, since this approach makes possible the incorporation of the effects of operational variables on model parameters. The applicability of the validated model was investigated, using the model as a process simulator to evaluate the effects of initial cell and lactose concentration in the xanthan gum production.

Production of lactobionic acid and sorbitol from lactose/fructose substrate using GFOR/ GL enzymes from Zymomonas mobilis cells: A kinetic study

In this work, we have investigated the kinetics of the biotechnological production of lactobionic acid ( LBA) and sorbitol by the catalytic action of glucose–fructose oxidoreductase ( GFOR) and glucono-δ-lactonase ( GL) enzymes. The cells of bacterium Zymomonas mobilis ATCC 29191 containing this enzymatic complex were submitted to permeabilization and reticulation procedures. The effect of the concentration of substrates on the rate of product formation using a mobilized cell system was investigated. The application of higher fructose concentration seems to not affect the initial rate of formation of the bionic acid. Under conditions of low initial concentration of lactose, the experimental kinetic data of the bi-substrate reaction were modelled by assuming a rate equation of the classical ping-pong mechanism. The found kinetic parameters displayed a low affinity of the GFOR enzyme for both substrates. The enzymatic system did not exhibit normal Michaelis–Menten kinetics in response to a change of concentration of lactose, when fructose was held constant, presenting a sigmoid relationship between initial velocity and substrate concentration. A rate equation based on Hill kinetics was used to describe the kinetic behaviour of this enzyme-substituted reaction at higher lactose concentrations. The results from batch experiments using immobilized cells within Ca-alginate beads revealed that there is no pronounced occurrence of mass transfer limitations on LBA production for beads with 1.2 mm in average diameter. This discussion aids for defining the best operating conditions to maximize the productivity for LBA and sorbitol in this bioconversion and also for reducing the complexity of downstream separation processes.

β-Galactosidase of Aspergillus oryzae immobilized in an ion exchange resin combining the ionic-binding and crosslinking methods: Kinetics and stability during the hydrolysis of lactose

The strong dependence of the stability of the immobilized enzyme on the temperature. ► The use of ionic exchange resins for immobilization is used food industry. ► Glutaraldehyde activation of the carriers is one technique to immobilize enzymes. ► β-galactosidase e immobilized on the ionic exchange resin followed by crosslinking. ► The hydrolysis kinetics of lactose by β-galactosidase immobilized. ► The influence of temperature in the enzymatic activity of immobilized enzyme. In this work, the hydrolysis kinetics of lactose by Aspergillus oryzae β-galactosidase was studied using the ionic exchange resin Duolite A568 as a carrier. The enzyme was immobilized using a β-galactosidase concentration of 16 g/L in pH 4.5 acetate buffer and an immobilization time of 12 h at 25 ± 0.5 °C. Next, the immobilized β-galactosidase was crosslinked using glutaraldehyde concentration of 3.5 g/L for 1.5 h. The influence of lactose concentration was studied for a range of 5–140 g/L, and the Michaelis–Menten model was fitted well to the experimental results with V m and K m values of 0.71 U and 35.30 mM, respectively. The influence of the product galactose as an inhibitor on the hydrolysis reaction was studied. The model that was best fitted to the experimental results was the competitive inhibition by galactose with V m , K m and K i values of 0.77 U, 35.30 mM and 27.44 mM, respectively. The influence of temperature on the enzymatic activity of the immobilized enzyme was studied in the range of 10–80 °C, in which the temperature of the maximum activity was 60 °C, with an activation energy of 5.32 kcal/mol of lactose, using an initial concentration of lactose of 50 g/L in a pH 4.5 sodium acetate buffer solution. The thermal stability of the immobilized biocatalyst was determined to be in the range 55–65 °C. The first-order model described well the kinetics of thermal deactivation for all the temperatures studied. The activation energy of thermal deactivation from immobilized biocatalyst was 66.48 kcal/mol with a half-life of 8.9 h at 55 °C.

Effect of the Substrate Concentration and Water Activity on the Yield and Rate of the Transfer Reaction of β-Galactosidase from Bacillus circulans

Prebiotic galactosyl oligosaccharides (GOS) are produced from lactose by the enzyme β-galactosidase. It is widely reported that the highest GOS levels are achieved when the initial lactose concentration is as high as possible; however, little evidence has been presented to explain this phenomenon. Using a system composed of the commercial β-galactosidase derived from Bacillus circulans known as Biolacta FN5, lactose and sucrose, the relative contribution of water activity, and substrate availability were assessed. Oligosaccharide levels did not appear to be affected by changes in water activity between 1.0 and 0.77 at a constant lactose concentration. The maximum oligosaccharide concentration increased at higher initial concentrations of lactose and sucrose, while initial reaction rates for transfer increased but remained constant for hydrolysis. This suggests that the high oligosaccharide levels achieved at the raised initial saccharide concentration are due to increases in reactions that form oligosaccharides rather than decreases in concurrent reactions, which degrade oligosaccharides. There were different effects from changing the initial concentration of lactose compared to sucrose, suggesting that the ability of lactose to act as a donor saccharide may be more important for increasing maximum oligosaccharide concentrations than the combined ability of both saccharides to act as galactosyl acceptors.

Optimal fermentation conditions for maximizing the ethanol production by Kluyveromyces fragilis from cheese whey powder

Cheese whey powder (CWP) is an attractive raw material for ethanol production since it is a dried and concentrated form of CW and contains lactose in addition to nitrogen, phosphate and other essential nutrients. In the present work, deproteinized CWP was utilized as fermentation medium for ethanol production by Kluyveromyces fragilis. The individual and combined effects of initial lactose concentration (50–150 kg m−3), temperature (25–35 °C) and inoculum concentration (1–3 kg m−3) were investigated through a 23 full-factorial central composite design, and the optimal conditions for maximizing the ethanol production were determined. According to the statistical analysis, in the studied range of values, only the initial lactose concentration had a significant effect on ethanol production, resulting in higher product formation as the initial substrate concentration was increased. Assays with initial lactose concentration varying from 150 to 250 kg m−3 were thus performed and revealed that the use of 200 kg m−3 initial lactose concentration, inoculum concentration of 1 kg m−3 and temperature of 35 °C were the best conditions for maximizing the ethanol production from CWP solution. Under these conditions, 80.95 kg m−3 of ethanol was obtained after 44 h of fermentation.

Effect of process parameters on crystal size and morphology of lactose in ultrasound‐assisted crystallization

Abstractα‐lactose monohydrate is widely used as a pharmaceutical excipient. Drug delivery system requires the excipient to be of narrow particle size distribution with regular particle shape. Application of ultrasound is known to increase or decrease the growth rate of certain crystal faces and controls the crystal size distribution. In the present paper, effect of process parameters such as sonication time, anti‐solvent concentration, initial lactose concentration and initial pH of sample on lactose crystal size, shape and thermal transition temperature was studied. The parameters were set according to the L9‐orthogonal array method at three levels and recovered lactose from whey by sonocrystallization. The recovered lactose was analyzed by particle size analyzer, scanning electron microscopy and differential scanning calorimeter. It was found that the morphology of lactose crystal was rod/needle like shape. Crystal size distribution of lactose was observed to be influenced by different process parameters. From the results of analysis of variance, the sonication time interval was found to be the most significant parameter affecting the volume median diameter of lactose with the highest percentage contribution (74.28%) among other parameters. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Immobilized β-galactosidase onto magnetic particles coated with polyaniline: Support characterization and galactooligosaccharides production

Magnetite particles (<100m) obtained by coprecipitation of Fe 2+ and Fe 3+ and coated with polyaniline (MAG-PANI) were used to immobilize Aspergillus oryzae -galactosidase via glutaraldehyde. The amount of -galactosidase immobilized onto MAG-PANI was ca. 2.04 mg/g support. This magnetic enzymatic derivative was capable to act on lactose and to produce tri and tetragalactosides (transgalactosylation) and the catalytic properties were similar to the soluble enzyme. For lactose concentrations up to 100 g/L, no differences were observed in the enzyme specific activity between free and immobilized forms (100% of specific activity retention), but for higher lactose concentrations the initial specific reaction rate of the immobilized form was affected by increasing lactose concentrations. The Activation Energy values for both free and immobilized forms were similar, around 16 ± 1.4 KJ/mol. The tri and tetra-galactosides production by both soluble and immobilized enzyme was not affected by temperature in the range of 30-60 ◦C. As the initial lactose concentration increased from 5% to 50%, the maximum GOS content in the product increased from 11.2% (at 35% conversion) to 26.1% (at 56% conversion) for the free enzyme and from 10.8% (at 33% conversion) to 26.0% (at 52% conversion) for the immobilized enzyme. The MAG- PANI was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, elemental analyzer, scanning electronic microscope, differential scanning calorimeter, thermogravimetric analyzer, vibrat- ing sample magnetometer and thermomagnetization. These analysis showed rhombohedra particles presenting good magnetic response, evidences for the PANI coating and protein immobilization and magnetite as the predominant component. This magnetic -galactosidase derivative presents the fol- lowing advantages: simple synthesis using low cost reagents, catalytic properties similar to the soluble enzyme and easy removal from the reaction mixture by a magnetic field and reuse.

Chitosan 담체에 고정화된 β-galactosidase에 의한 유당 분해 특성

-Galactosidase was immobilized on chitosan bead by covalent bonding using glutaraldehyde. The characteristics of the immobilized enzyme were investigated. Maximum immobilization yield of 75% was obtained on chitosan bead. Optimum pH and temperature for the immobilized enzyme was 7.0 and , respectively. The immobilized enzyme showed a broader range of pH and temperature compared to a free one. A mathematical model for the operation of the immobilized enzyme in a packed-bed reactor was established and solved numerically. Under different inlet lactose concentrations and feed flow rate conditions, lactose conversion was measured in a packed-bed reactor. The experimental results of continuous operation in a packed-bed reactor were compared to theoretic results using Michaelis-Menten kinetics with competitive product inhibition and external mass transfer resistance. The model predicted the experimental data with errors less than 5%. Process optimization of continuous operation in a packed-bed reactor was also conducted. In a recirculation packed-bed operation, conversion of lactose was 97% in 3 hours. In a continuous packed-bed operation, the effect of flow rate and initial lactose concentration was investigated. Increasing flow rates and initial lactose concentration decreased the conversion of substrate.

Ethanol production from whey permeate in a continuous anaerobic bioreactor by Kluyveromyces marxianus

The possibility of using whey permeate as a raw material for the production of ethanol by continuous fermentation by using Kluyveromyces marxianus was examined. The ethanol formation was investigated as a function of the hydraulic retention time in a UASB reactor. The initial lactose concentration in permeate was 50.0 g L−1. The hydraulic retention time supplied were 12, 24, 48 h and the daily ethanol formations were 4.46, 8.61, 7.73 g L−1, respectively. The yield coefficient of ethanol was 0.089 g ethanol g−1 lactose when the hydraulic retention time was 12 h and raised to 0.325 g g−1 when the hydraulic retention time was as long as 48 h. The results indicated that in ethanol fermentation the hydraulic retention time should be 24 h to obtain high rates of ethanol formation and to avoid product inhibition.

Cloning, purification, and characterization of β-galactosidase from Bacillus licheniformis DSM 13

The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-D: -galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4-42°C) at pH 6.5 for up to 1 month. The K(m) values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na(+) and K(+) in the concentration range of 1-100 mM as well as the divalent metal cations Mg²(+), Mn²(+), and Ca²(+) at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.

A comprehensive investigation of the synthesis of prebiotic galactooligosaccharides by whole cells of Bifidobacterium bifidum NCIMB 41171

The synthesis of galactooligosaccharides (GOS) by whole cells of Bifidobacterium bifidum NCIMB 41171 was investigated by developing a set of mathematical models. These were second order polynomial equations, which described responses related to the production of GOS constituents, the selectivity of lactose conversion into GOS, and the relative composition of the produced GOS mixture, as a function of the amount of biocatalyst, temperature, initial lactose concentration, and time. The synthesis reactions were followed for up to 36 h. Samples were withdrawn every 4 h, tested for β-galactosidase activity, and analysed for their carbohydrate content. GOS synthesis was well explained by the models, which were all significant ( P < 0.001). The GOS yield increased as temperature increased from 40 °C to 60 °C, as transgalactosylation became more pronounced compared to hydrolysis. The relative composition of GOS produced changed significantly with the initial lactose concentration ( P < 0.001); higher ratios of tri-, tetra-, and penta-galactooligosaccharides to transgalactosylated disaccharides were obtained as lactose concentration increased. Time was a critical factor, as a balanced state between GOS synthesis and hydrolysis was roughly attained in most cases between 12 and 20 h, and was followed by more pronounced GOS hydrolysis than synthesis.

Optimization of ricotta cheese whey (RCW) fermentation by response surface methodology

A central composite design (CCD) was performed to evaluate the effects of four factors, i.e. temperature (T), pH, agitation rate (K) and initial lactose concentration (L), on ricotta cheese whey batch fermentation and to optimize the process leading to the formation of bio-ethanol. Anaerobic batch fermentation experiments were carried out by using the yeast Kluyveromyces marxianus. After a preliminary experimental analysis, the values of the chosen factors were 32 and 40 degrees C for T, 4 and 6 for pH, 100 and 300 rpm for K, 40 and 80 g L(-1) for L. Response surface methodology (RSM) was used to optimize the fermentation process and an empirical polynomial model was used to fit the experimental data. The best operating conditions resulted to be T=32.35 degrees C, pH 5.41, K=195.56 rpm and L=40 g L(-1) and the model ensured a good fitting of the observed data.