Lactose Hydrolysis Process Research Articles

Determining patterns of lactose hydrolysis in liquid concentrates of demineralized whey

The object of this study was the process of milk sugar hydrolysis in liquid whey concentrates. The problem solved was the intensification of the lactose hydrolysis reaction in liquid whey concentrates through the use of a combination of leavening and enzyme preparations. Patterns in the lactose hydrolysis process in liquid concentrates of demineralized whey were studied. It was established that the use of an enzyme preparation for liquid whey concentrates does not make it possible to achieve a degree of lactose hydrolysis higher than 75–77 % within 10 h. The simultaneous use of enzyme and leavening preparations for 6 hours ensures the conversion of more than 95 % of lactose for concentrates with a mass fraction of dry substances of 10–30 % and more than 90 % for a 40 % concentrate. The rational duration of lactose hydrolysis in demineralized whey concentrates for the combination of preparations is 6 hours for 10 and 20 % concentrates, and 8 h for 30 and 40 % concentrates, which enables lactose hydrolysis at the level of 96.8–100 %. The rheological properties of concentrates with a solids content of 30 and 40 % indicate that these systems have a high ability to restore the structure. The dynamics of monosaccharide formation during hydrolysis are similar for 10 and 20 % concentrates, in which galactose slightly predominates the systems. Data on the increase in glucose content in fermented 30 and 40 % concentrates contradict the known data regarding the consumption of glucose by the acidophilic bacillus and its conversion to galactose. This may indicate suppression of the activity of the acidophilic bacillus under conditions of increased osmotic pressure in the concentrates. The results of the work could be used in the technology of whey ice cream, as well as dairy products that require adjustment of the chemical composition, primarily in terms of protein and lactose content

The quality indicators of ice cream upon the enzymatic hydrolysis of lactose

Ice cream is a popular type of dairy foods containing up to 6% of lactose. In connection with the lactose intolerance by many consumers and the possibility of its crystallization during storage of finished products, there is a need for a decrease in the content of this nutrient in the composition of ice cream. The aim of the research was to study an effect of the lactose hydrolysis process in ice cream with the fat mass fraction of 15% and different mass fractions of dry skim milk residue on technologically significant and sensory properties of its quality. The main objects of the study were samples of ice cream subjected to lactose hydrolysis at the stage of mixture maturation. The fat mass fraction in the samples was 15%, mass fractions of dry skim milk residue were 7, 10, 12 and 15%. The composition of sugars was determined by high-performance liquid chromatography, the dynamic viscosity of mixes and the consistency of ice cream by rheological methods and the dispersion of structural elements by microcopy. The effects of the mass fraction of dry skim milk residue and lactose hydrolysis on quality indicators of mixtures and ice cream were determined. As the mass fraction of dry skim milk residue rose from 7 to 15%, the residual content of lactose increased from 0.2 to 1.1%, while the dynamic viscosity of the mixture increased by 1.3 times. A decrease in the freezing point by 0.6–0.8 °C and an increase in melting resistance were also observed. The mass fraction of melt after 2 hours of holding decreased to 4.7–0.7%. Also, indicators of consistency (hardness, adhesiveness, adhesion force and rigidity) decreased by 1.1–1.7 times (upon a mass fraction of dry skim milk residue of 7 and 10%). The samples of ice cream subjected to lactose hydrolysis were characterized by a high dispersion of structural elements typical for a traditional product, and by improved texture and increased sensation of sweetness. The complex of investigations to study quality indicators of ice cream with the fat mass fraction of 15% showed that a change in the mass fraction of dry skim milk residue upon enzymatic hydrolysis of lactose results in formation of different structural-mechanical and sensory indicators that should be considered during the creation of assortment and development of formulations of low-lactose products.

Fourier transform infrared spectroscopy analysis of Lactose hydrolysis by beta-galactosidase from Lactiplantibacillus plantarum GV54 and Lactiplantibacillus sp. GV66

Introduction and Aim: The enzyme beta-galactosidase is extremely important in the food and pharmaceutical industries. The goal of this work was to improve the lactose hydrolysis process employing beta-galactosidase to convert glucose and galactose for alleviating lactose intolerance. Methods: Intracellular beta-galactosidase was extracted from Lactiplantibacillus spp. (GV54 and GV66) by cell permeabilized method using toluene/acetone solvents. Lactose hydrolysis was performed using 50 g/L of lactose with partially purified beta-galactosidase enzyme at different time intervals 2, 4, 6 and 12 h. Fourier transforms infrared spectroscopy (FTIR) was used to evaluate the degree of hydrolysis at different time intervals and lactose hydrolysis was high at 6 h of incubation. Results: The FTIR peak of lactose molecules hydrolysis by beta-galactosidase (GV54 and GV66) with different time intervals (2h, 4h, 6h, and 12h) indicates similarities in bond lengths and vibrational modes. The amide I group of beta-galactosidase is uniformly bound to lactose at 1634 per cm and C-H group of glucose and galactose was observed at 1386 per cm. In the range of 2000-800 per cm, we could identify variations in the shape and strength of these peaks, compared with control (lactose solution). Conclusion: The precise and direct measurement of individual carbohydrates such as glucose and galactose in lactose solution hydrolyzed by beta-galactosidase enzyme was successfully accomplished using FTIR spectrometry. Comparing FTIR analysis to traditional procedures, the time required is drastically decreased. These findings have the analytical power to follow the dynamics of in vitro enzymatic activity of naturally occurring substrates rich in natural carbohydrate polymers.

Development of a biocomposite based on alginate/gelatin crosslinked with genipin for β-galactosidase immobilization: Performance and characteristics

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for β-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized β-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.

Ultrasonic analysis of effects of varying temperature, pH, and proteolytic enzymes on hydrolysis of lactose by neutral lactase formulations in infant milk and in the infant digestive system

Lactose intolerance in infants can be overcome by adding lactase enzyme formulations to milk, hydrolysing the lactose present. Commonly, such formulations require a significant incubation period after addition to milk to allow time for hydrolysis to take place at desired temperature. Such incubation is often problematic for household conditions, and therefore, formulations with short or, ideally, no incubation period are needed. Development of such formulations requires understanding of the complex process of lactose hydrolysis within the variable temperature and pH conditions in the milk bottle and in the gastrointestinal tract. The current paper describes the application of high-resolution ultrasonic spectroscopy for detailed characterisation of the effect of pH, temperature, and proteases present in the digestive tract on hydrolysis of lactose. The results were employed in the development of a predictive model of lactose hydrolysis within conditions of the infant digestive system. The model was applied for analysis of the whole infant feeding process: from hydrolysis in the bottle, to the stomach and small intestine, and for assessment of the impact of enzyme dosage, the incubation period, and bottle temperature profile on the levels of lactose and galacto-oligosaccharides (produced during hydrolysis), released from the stomach and reaching the large intestine where symptoms of intolerance arise. The results showed that the activity of Ha-Lactase in the stomach and small intestine assists significantly in the reduction of lactose load to 10 % or less of the amount of lactose in the feed, depending on conditions.

Lactose quantification in UHT milk by high-performance liquid chromatography and cryoscopy (freezing point depression)

Due to the large number of people with lactose maldigestion, the dairy industries have increased production and diversity of low lactose and lactose-free foods. Consequently, the need to control the lactose hydrolysis process has also risen. This study aimed to correlate freezing point depression (cryoscopy) and lactose concentration, quantified by high-performance liquid chromatography (HPLC), in UHT milk. To accomplish this, UHT milk samples were subjected to seven lactose hydrolysis treatments, using lactase enzyme, resulting in different lactose concentrations. All samples were subjected to HPLC analysis and freezing point measurement, using a cryoscope. The results were plotted on a graph and a linear regression was performed. There was a strong correlation between lactose concentration and freezing point (R = 0,9973) and the coefficient of determination (R2) was 0,9946, which means that 99,46% of the variability of the response data is explained by the linear regression model. Therefore, the results point to the feasibility of estimating the lactose concentration in milk during the hydrolysis process for the production of low lactose milk, by cryoscopy, a quick analysis, with lower cost compared to HPLC and that is already among the analyses commonly performed in dairy industries.

Development of low-lactose curd product with papaya

Today, low-lactose (LL) and lactose-free (LF) dairy products have widespread health benefits throughout the world. To obtain LL and LF of curd products, the modern market of LL and LF products was studied, based on the analysis of which the composition of raw materials and methods for obtaining LL of a curd product were substantiated using the example of LL curd paste with papaya puree. The process of lactose hydrolysis was studied and the optimal modes of its hydrolysis were determined. The development of LL curd products with papaya will help reduce vitamin A deficiency and protein-energy malnutrition (PEM) in the population in Vietnam. The new product will expand the range of functional foods, increase the consumption of fermented milk products among the population of all ages, provide the body with many important nutrients, and strengthen the immune system.

Assessment of enzymatic hydrolysis of lactose in lactose-free milk production - A comparative study using capillary zone electrophoresis and cryoscopy

A comparison of the capillary zone electrophoresis under indirect detection at ultraviolet region (CZE-UV) method with the traditional cryoscopic method for lactose hydrolysis process monitoring is proposed. Two different lactose enzymatic hydrolysis processes (in batch and sterile) were assessed. The hydrolysis levels obtained by the CZE-UV method differed statistically from those obtained by cryoscopy, at 95% confidence, showing variations up to 20% at the same time of processing. During sterile hydrolysis, 90% of lactose reduction was detected after 1 h by CZE-UV and after 3 h by cryoscopy. For in batch hydrolysis, the CZE-UV method indicated 50% of lactose hydrolysis after 3 h of processing, while the cryoscopic method did not identify such lactose reduction during the process simulation. Our results demonstrated that CZE-UV method is precise and reliable analytical method, acting as a good alternative for lactose hydrolysis process monitoring.

Research of lactose hydrolysis depending on the type of the enzyme

In order to produce low-lactose dairy products the process of lactose hydrolysis is studied and optimal process parameters depending on the type of enzyme samples are determined. By the example of two types of enzymatic samples of lactase (β-galactosidase): “Lacta-free” TM “Lactoferm ECO”, obtained from mushrooms Aspergillus oryzae, is produced by Biochem srl. (Italy) and “Maxilact-2000”, obtained from Kluyveromyces lactis yeast, is produced by “DSM Food Specialties” (Netherlands) and the most effective samples has been tested. Optimal parameters for “Lacta-free”: at temperature (40±2)°C, duration of hydrolysis process (5.0±0.5) hours; for “Maxilact-2000”: at temperature (37±2)°C, duration of hydrolysis process (4.0±0.5) hours.

Effects of enzymatic lactose hydrolysis on thermal markers in lactose-free UHT milk.

Lactose-free products are more susceptible to chemical and physical modifications during heating and storage, due to the release of glucose and galactose during enzymatic processing, both more reactive than lactose. The present study demonstrates the effect of enzymatic lactose hydrolysis on 5-hydroxymethylfurfural (HMF), whey protein nitrogen index (WPNI) and lactulose used as thermal markers for UHT milk process monitoring. Six milk leading brands which provided regular and lactose-free UHT milk were selected, giving a total of 12 UHT milk samples analyzed in authentic duplicates. All lactose-free samples showed high levels of HMF index (42.15µmol L-1, against 13.11µmol L-1 for regular samples) and low lactulose contents (13.03mg 100mL-1, against 35.59mg 100mL-1 of regular ones). High variations in HMF (55-85%) and lactulose (42-91%) intra-brand analysis indicated that both markers are influenced by the lactose hydrolysis process. The paired t test indicated there was no difference among WPNI indexes of regular and lactose-free milks suggesting that this thermal marker is suitable to infer about heat damage in lactose-free dairy matrices.

3D enzymatic preparations with graphene oxide flakes and hydrogel to obtain lactose-free products

Graphene oxide (GO), due to its properties, such as large specific surface area and the presence of numerous active groups on its surface, can be used as a carrier in enzyme immobilization. The procedure of its chemical activation before enzyme molecules binding was elaborated. β-galactosidase, the enzyme used in obtaining lactose-free products, was immobilized after GO activation with divinyl sulfone. The preparation was active, stable, but troublesome for separation. Thus, the innovative solution – 3D preparations were proposed. GO with immobilized enzyme was encapsulated in alginate gel. The 3D preparations have been physically stable in milk by at least 30 days, and their separation by sedimentation or filtration was very easy. Substrate diffusion was fast thus the process ran in a kinetic regime, and the Michaelis–Menten equation was applied. A continuous process of lactose hydrolysis was performed in a column filled with the 3D preparations. By controlling the residence time, it was possible to obtain low lactose concentration in the effluent stream at very low temperature, 6 °C, which corresponds to refrigerated milk storage conditions.

Ultrasound assisted acid catalyzed lactose hydrolysis: Understanding into effect of operating parameters and scale up studies

The current work deals with the value addition of lactose by transforming into hydrolyzed lactose syrup containing glucose and galactose in major proportion using the novel approach of ultrasound assisted acid catalyzed lactose hydrolysis. The hydrolysis of lactose was performed in ultrasonic bath (33kHz) at 50% duty cycle at different temperatures as 65°C and 70°C and two different hydrochloric acid (HCl) concentrations as 2.5N and 3N. It was observed that acid concentration, temperature and ultrasonic treatment were the major factors in deciding the time required to achieve ∼90% hydrolysis. The ultrasonic assisted approach resulted in reduction in the reaction time and the extent of intensification was established to be dependent on the temperature, acid concentration and time of ultrasonic exposure. It was observed that the maximum process intensification obtained by introduction of ultrasound in the lactose hydrolysis process performed at 70°C and 3N HCl was reduction in the required time for ∼90% hydrolysis from 4h (without the presence of ultrasound) to 3h. The scale-up study was also performed using an ultrasonic bath with longitudinal horn (36kHz as operating frequency) at 50% duty cycle, optimized temperature of 70°C and acid concentration of 3N. It was observed that the reaction was faster in the presence of ultrasound and stirring by axial impeller at rpm of 225±25. The time required to complete ∼90% of hydrolysis remained almost the same as observed for small scale study on ultrasonic bath (33kHz) at 50% duty cycle. The use of recovered lactose from whey samples instead of pure lactose did not result in any significant changes in the progress of hydrolysis, confirming the efficacy of the selected approach. Overall, the work has presented a novel ultrasound assisted approach for intensified lactose hydrolysis.

Three-dimensional reconstruction of bioactive membranes and pore-scale simulation of enzymatic reactions: The case of lactose hydrolysis

Two-layered hollow-fiber membrane bioreactors containing immobilized enzymes are investigated in this work at mesoscopic and macroscopic scales. Layer-by-layer reconstruction of the membrane in three dimensions is achieved using two different stochastic techniques that draw data only from electron microscopy images of the structure. The reconstructed layers are subsequently used for the prediction of effective transport properties through the numerical solution of the relevant transport equations. The methodology is used for the investigation of hydrolysis of lactose, employing a convective diffusion and reaction macroscopic model. Direct comparison of model predictions with experimental data from the literature reveals a good agreement, provided that one uses the effective transport coefficients that are calculated from the reconstruction process. A parametric investigation of the hydrolysis process and its efficiency was conducted. The three-dimensional reconstructions are also used for the modeling of the hydrolysis of lactose at the pore scale, taking into account mass transport limitations and applying the same hydrolysis kinetics as in the macroscopic model. The agreement was quite satisfactory. Among the advantages of the pore scale approach that is formulated here is its ability to correlate pore structure details with the performance of the lactose hydrolysis process at the inevitable expense of computational resources.

Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases.

This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry.

Novel high-performance metagenome β-galactosidases for lactose hydrolysis in the dairy industry

The industrially utilised β-galactosidases from Kluyveromyces spp. and Aspergillus spp. feature undesirable kinetic properties in praxis, such as an unsatisfactory lactose affinity (KM) and product inhibition (KI) by galactose. In this study, a metagenome library of about 1.3 million clones was investigated with a three-step activity-based screening strategy in order to find new β-galactosidases with more favourable kinetic properties. Six novel metagenome β-galactosidases (M1–M6) were found with an improved lactose hydrolysis performance in original milk when directly compared to the commercial β-galactosidase from Kluyveromyces lactis (GODO-YNL2). The best metagenome candidate, called “M1”, was recombinantly produced in Escherichia coli BL21(DE3) in a bioreactor (volume 35L), resulting in a total β-galactosidase M1 activity of about 1100μkatoNPGal,37°C L−1. Since milk is a sensitive and complex medium, it has to be processed at 5–10°C in the dairy industry. Therefore, the β-galactosidase M1 was tested at 8°C in milk and possessed a good stability (t1/2=21.8d), a desirably low apparent KM,lactose,8°C value of 3.8±0.7mM and a high apparent KI,galactose,8°C value of 196.6±55.5mM. A lactose hydrolysis process (milk, 40 nkatlactose mLmilk,8°C−1) was conducted at a scale of 0.5L to compare the performance of M1 with the commercial β-galactosidase from K. lactis (GODO-YNL2). Lactose was completely (>99.99%) hydrolysed by M1 and to 99.6% (w/v) by K. lactis β-galactosidase after 25h process time. Thus, M1 was able to achieve the limit of <100mg lactose per litre milk, which is recommended for dairy products labelled as “lactose-free”.

Lactose hydrolysis by β-galactosidase enzyme: optimization using response surface methodology

In the present study, it was aimed to optimize the process of lactose hydrolysis using free and immobilized β-galactosidase to produce glucose and galactose. Response surface methodology (RSM) by central composite design (CCD) was employed to optimize the degree of hydrolysis by varying three parameters, temperature (15–45°C), solution pH (5–9) and β-galactosidase enzyme concentration (2–8mg/mL) for free mode of analysis and sodium alginate concentration (2–4%), calcium chloride concentration (3–6%) and enzyme concentration (2–8mg/mL) for immobilized process. Based on plots and variance analysis, the optimum operational conditions for maximizing lactose hydrolysis were found to be temperature (35.5°C), pH (6.7) and enzyme concentration (6.7mg/mL) in free mode and sodium alginate concentration (3%), calcium chloride concentration (5.9%) and enzyme concentration (5.2mg/mL) in immobilized mode.

β-Galactosidase activity of commercial lactase samples in raw and pasteurized milk at refrigerated temperatures

Many consumers are unable to enjoy the benefits of milk due to lactose intolerance. Lactose-free milk is available but at about 2 times the cost of regular milk or greater, it may be difficult for consumers to afford. The high cost of lactose-free milk is due in part to the added cost of the lactose hydrolysis process. Hydrolysis at refrigerated temperatures, possibly in the bulk tank or package, could increase the flexibility of the process and potentially reduce the cost. A rapid β-galactosidase assay was used to determine the relative activity of commercially available lactase samples at different temperatures. Four enzymes exhibited low-temperature activity and were added to refrigerated raw and pasteurized milk at various concentrations and allowed to react for various lengths of time. The degree of lactose hydrolysis by each of the enzymes as a function of time and enzyme concentration was determined by HPLC. The 2 most active enzymes, as determined by the β-galactosidase assay, hydrolyzed over 98% of the lactose in 24h at 2°C using the supplier's recommended dosage. The other 2 enzymes hydrolyzed over 95% of the lactose in 24h at twice the supplier's recommended dosage at 2°C. Results were consistent in all milk types tested. The results show that it is feasible to hydrolyze lactose during refrigerated storage of milk using currently available enzymes.

Mathematical modeling and simulation of steady state plug flow for lactose-lactase hydrolysis in fixed bed

A detailed mathematical model for evaluating lactose hydrolysis with immobilized enzyme in a packed bed tubular reactor is presented. The model accounts for axial and radial dispersion effects, chemical reaction and external mass transfer resistances but is void of significant internal diffusion resistances of the particles. The comprehensive model was then simplified to a plug flow model for lactose-lactose hydrolysis in fixed bed. The resulting plug flow model was solved by using Runge-Kutta-Gill method via employing different kinetics for lactose hydrolysis. The reliability of model simulations was tested using experimental data from a laboratory packed bed column, where the β-galactosidase of Kluyveromyces fragilis was immobilized on spherical chitosan beads. Comparison of the simulated results with experimental exit conversion show that the plug flow model incorporating Michaelis-Menten kinetics with competitive product (galactose) inhibition are appropriate to interpret the experimental results and simulate the process of lactose hydrolysis in a fixed bed when the mass transfer resistance was reduced by a factor of 34.5.

A modelling study on milk lactose hydrolysis and β-galactosidase stability under sonication

In the present study, it was aimed to investigate the effect of ultrasonication as an alternative method on the kinetics of lactose hydrolysis process. The effects of the duty cycle and acoustic power of sonifier and enzyme concentration on hydrolysis of lactose and as well as on enzyme stability were studied. The lactose hydrolysis reactions were carried out using a commercial β-galactosidase produced from Kluyveromyces marxianus. Ultrasonic treatment resulted by the lactose hydrolysis degree of 90% and the residual enzyme activity of 75% at the optimum operational conditions; acoustic power of 20 W, duty cycle rate of 10% and enzyme concentration of 1 mL/L. These results show that the sonication was beneficial for lactose hydrolysis in milk. Mathematical models were also derived for residual lactose concentration and residual enzyme activity depending on the operating conditions. The predicted models have been confirmed with the experimental results.

Hydrolysis of Lactose in a Fluidized Bed of Zeolite Pellets Supporting Adsorbed ß - Galactosidase

The purpose of this work is to verify the possibility of using a simple physical adsorption technique to improve enzyme efficiency in performing lactose hydrolysis. Immobilization experiments of ß -galactosidase on zeolite pellets showed that this process is fairly slow and can be accomplished with contact times shorter than few hours. Comparison between homogeneous and heterogeneous hydrolysis experiments in mixed batch reactors indicates that the enzyme distribution within the zeolite pellets is concentrated in a thin shell under the pellet surface. Continuous conversion experiments were carried out on a purposely-built fluidization column. Results were fitted with a mathematical model for fluidized bed conversion without the use of any further adjustable parameter than those used in the heterogeneous batch conversion. Furthermore, no change of the parameter values was necessary. Scale-up predictions given by the model showed that fluidized columns few meters high of pellets supporting b-galactosidase are sufficient for an industrially suitable process of lactose hydrolysis. which can be performed on columns of few meters and, therefore, these are applicable to relatively small process scales as those found in traditional Italian cheese making factories.