Initial Lactose Concentration Research Articles

In Situ Galacto-Oligosaccharides Synthesis in Whey Powder Fortified Milk by a Modified β-Galactosidase and Its Effect on the Techno-Functional Characteristics of Yogurt.

In situ galacto-oligosaccharide (GOS) synthesis in milk using β-galactosidases is an effective method for developing prebiotic dairy products. However, the low lactose concentration in milk (∼4.6%, w/w) reduces the GOS yield. In this study, a modified β-galactosidase from Bacillus circulans (mBgaD-D) with enhanced transglycosylation activity at low lactose concentration was developed through directed evolution and saturation mutagenesis. The GOS yield by mBgaD-D increased from 22.8% (wild type) to 30.8% in 50 g/L lactose (phosphate buffer). Pmgut was a strong sorbitol-inducible promoter from Bacillus subtilis. The expression of mBgaD-D in B. subtilis, coupled with the Pmgut promoter, resulted in a 6.4-fold increase (compared to the P43 promoter) in extracellular enzyme activity. Additionally, adding whey powder to boost the initial lactose concentration further improved the GOS yield, which reached 43% under the optimized conditions. Combining mBgaD-D and whey powder enhanced milk sweetness, producing no sugar-added, GOS-enriched yogurt (GOSY). The GOS content in GOSY was 4.1/100 g, providing an appropriate level of sweetness and yielding a yogurt that is elastic as well as firm. GOSY also increased the population of Bifidobacterium spp. during a 24 h in vitro fecal fermentation. Thus, fortifying yogurt with mBgaD-D and whey powder can enhance its technological properties and health benefits.

One-pot production of tagatose using l-arabinose isomerase from Thermotoga maritima and β-galactosidase from Aspergillus oryzae

The one-pot production of tagatose from lactose using recombinant l-arabinose isomerase (L-AI) from Thermotoga maritima and commercial β-galactosidase (β-gal) from Aspergillus oryzae was evaluated. The β-gal and L-AI ratio (R β-gal/L-AI), initial lactose concentration and temperature were chosen as study variables. Maximum values of tagatose yield (YTag), specific tagatose productivity (πTag) and sugars ratio (RTag/Gal) obtained were 23 %, 0.202 ± 0.03 mmol Tag/(g prot⋅h) and 1, respectively. The composition of the product on a dry basis was as follows (g/g): 0.23 tagatose, 0.232 galactose, 0.454 glucose, 0.043 fructose, 0.029 lactose and 0.012 GOS. The one-pot production was superior compared with a sequential operation, where values of 16.5 %, 0.11 mmol Tag/(g prot⋅h) and 0.59 were obtained for YTag, πTag and RTag/Gal, respectively.

Evaluation of transgalactosylation activity of commercial β-galactosidase from Bifidobacterium bifidum for synthesis of prebiotic oligosaccharides

Galacto-oligosaccharides (GOS) are products of transgalactosylation reaction of β-galactosidase when lactose is used as the substrate. These carbohydrates are considered prebiotics which stimulate beneficial effects to human health. In the present work, Saphera, a commercial preparation of β-galactosidase from Bifidobacterium bifidum, was biochemically characterised for production of GOS. Using o-nitrophenyl-β-D-galactopyranoside (oNPG) as the substrate, optimal activity for the enzyme was found to be at pH 6.0 and at 45°C. Ten (10) mM each of either Na+ or K+ enhanced enzyme activity by 10%, while Cu2+, Zn2+, Fe2+, and EDTA showed inhibitory effect on the enzyme activity. When incubated in 50 mM sodium phosphate and pH 6.5, the enzyme was found to have half-life time of 136 ± 6 and 2.1 ± 0.2 h at 30 and 50°C, respectively. The hydrolysis activity of the enzyme predominated when the initial lactose concentration used was 5% (w/v). When initial lactose concentration was increased to 20% (w/v), maximum GOS yield obtained was 10% (w/w) achieved at 86% lactose conversion. Analysis using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed that the major oligosaccharides produced by the enzyme were 3’-galactosylglucose, 3’-galactosyllactose, 3’-galactobiose, and allolactose, thus showing that this enzyme would prefer to form β-(13)- and β-(16)-linked GOS. Despite its predominantly hydrolytic activity, Saphera synthesised prebiotic GOS which could be interesting to dairy industry.

Bioethanol production from deproteinized cheese whey powder by local strain isolated from soured milk: influence of operating parameters

ABSTRACT In this paper, a local isolated yeast strain was studied for bioethanol production using protein-free cheese whey powder (CWP) in batch alcoholic fermentation experiments. Ethanol tolerance tests were performed with different ethanol concentrations (from 0% to 20%). The maximum ethanol tolerance obtained for our strain was 5% (v/v). The influence of initial lactose concentration, temperature and pH on fermentation kinetic was studied. For initial lactose concentration ranging from 57.5 to 260 gL−1, the highest ethanol production 34.8 gL−1 was obtained at 109 gL−1 initial lactose concentration corresponding to 0.413 gg−1 ethanol yield (YP/S); (While it was noticed less ethanol production, namely 0.178 g g−1) at a higher initial whey sugar concentration of 260 gL−1. The effect of the initial pH on the fermentation was studied at three different values: 3, 4 and 5. The ethanol production reached a maximum of 22 g L−1 with a lactose consumption of 77.62% at pH 4. However, an observed decrease occurred in ethanol production and lactose consumption at pH 3, with values of 10.34 gL-1 and 24.46%, respectively. For the fermentation conducted at the three temperatures of 37°C, 40°C and 43°C, the maximum ethanol production and lactose consumption were 34.82 gL-1 and 80.75%, respectively, achieved at 37°C. The experimental data was analyzed using the Gompertz model to obtain the following kinetic parameters: the maximum ethanol concentration (Pm) was found to be 36.97 gL-1, the maximum ethanol production rate (rp,m) was 0.402 gL−1h−1 and the lag phase (tl) was 10.8 h. The comparison of the isolated Kluyveromyces marxianus TXID4911 strain with the reference strain Kluyveromyces marxianus DSM 5421, showed a relatively close ethanol yield (YP/S) 0.415 and 0.505 gg,−1 respectively.

Estimation of selectivities in transglycosylation systems with multiple transglycosylation products

Transglycosylation can be used to produce oligosaccharides that act as prebiotics. The maximum yield of the oligosaccharides depends on the selectivities for transglycosylation, not only over primary hydrolysis of the original donor molecule, but also over secondary hydrolysis of the oligosaccharides. A recent approach to determining these selectivities only considered systems in which there is a single transglycosylation product. The current work extends this approach, determining selectivities for cases involving multiple transglycosylation products. A model is used to analyze literature data for the production of galactooligosaccharides through the transgalactosylation of lactose, catalyzed by β-galactosidases of Bacillus circulans and Aspergillus oryzae. In the case studies, the selectivity for using oligosaccharides rather than lactose as the galactosyl acceptor ranged from 1.0 (i.e. not selective) to 2.4. Selectivities for transgalactosylating lactose with lactose over primary hydrolysis of lactose ranged from 113 to 919. The selectivities for producing oligosaccharides over secondary hydrolysis of the oligosaccharides decreased with increase in the initial lactose concentration; the values obtained ranged from 6 to 159. Knowledge of these selectivities is useful for guiding the improvement of enzymes for transglycosylation processes.

Microalgal production under mixotrophic conditions using cheese whey as substrate

Microalgae are known for producing various biotechnological products. Moreover, they absorb nutrients from dairy wastewater, grow well, and accumulate valuable compounds faster. In this study, photoautotrophic and mixotrophic cultivation with different initial lactose concentrations present in cheese whey (CW) were established to investigate their effect on cell concentration (Xm, mg L-1), cell productivity (Px, mg L-1day-1), and specific cell growth (µmax, day-1) of Chlorella vulgaris, Dunaliella tertiolecta, and Tetradesmus obliquus. The biomass production of C. vulgaris (Xm= 1,520 ± 30.3 mg L-1, Px = 147 ± 3.00 mg L-1, and µmax = 0.150 ± 0.00 mg L-1) in mixotrophic culture with 10.0 g L-1 of lactose, the main constituent of CW, was notably enhanced by 55% in comparison with their photoautotrophic cultures, whereas a lower effect of these lactose concentrations on cell growth was observed in T. obliquus and D. tertiolecta. Thus, mixotrophic cultivation of C. vulgaris using CW as a carbon and energy source could be considered a feasible alternative to obtain high value-added biomass.

Enhanced thermal stability of the β-galactosidase BgaB from Bacillus circulans by cyclization mediated via SpyTag/SpyCatcher interaction and its use in galacto-oligosaccharides synthesis

Cyclization of proteins using SpyTag/SpyCatcher is a novel approach to increase their thermal stability. In this paper, we test this approach on two β-galactosidases from Bacillus circulans, BgaB and BgaC, and find that BgaB was stabilized while BgaC was not. Wild-type BgaB precipitated completely upon heating above 70 °C, but after SpyRing cyclization, it remained soluble after heating to 90 °C. Similarly, wild-type BgaB retained only 50 % activity after heating at 60 °C for 10 min, but this increased to 80 % after SpyRing cyclization. In contrast, cyclization decreased the stability of BgaC. After SpyRing cyclization, BgaC only retained 2 % activity after 20-min incubation at 55 °C, whereas the wild-type BgaC retained 25 % activity. One reason for the different effect of cyclization may the shorter distance between the N- and C-termini in BgaB (20.2 Å) as compared to BgaC (43.7 Å). The intrinsic fluorescence and circular dichroism spectra suggested that SpyRing cyclization of BgaB did not significantly change its conformation or secondary structure. SpyRing cyclized BgaB yielded similar amounts and compositions of galacto-oligosaccharides using a high initial lactose concentration (40 %, w/v), but a slightly higher amount at low initial lactose concentration (5 %, w/v) suggesting increased transgalactosylation activity.

Kinetic Model for Galacto-Oligosaccharide Synthesis

A new micro-kinetic model is proposed for galacto-oligosaccharide (GOS) synthesis with β-galactosidase from Aspergillus oryzae. Several kinetic models with similar reaction mechanisms, i.e., steps and kinetic constants, have been investigated covering a range of initial lactose (20–40 wt %) and enzyme concentrations (0.5–2 mg/mL). The kinetic parameters were estimated simultaneously for all experimental results using a hybrid genetic algorithm. A detailed analysis of the effect of process parameters on the concentrations of lactose and GOS is performed. The proposed model has a very good quantitative fit of the experimental data, with an average error of 9.34%. More importantly, qualitative trends for the change in the concentration of lactose and GOS are predicted excellently, especially if one takes into account the variation of both concentrations of enzyme and lactose in a range wider than reported in the literature to date. Also, the analysis between the two comparative reactions of transglycosylation and lactose hydrolysis confirmed the positive effect of increasing lactose concentration on the production of GOS. On the other hand, a higher enzyme concentration resulted in a faster production of GOS but with more intensive decomposition at longer reaction times.

Effect of the concentration of L-Phenylalanine and lactose on 2-Phenylethanol production by whey fermentation using the yeasts Kluyveromyces marxianus and Debaryomyces hansenii under co-culture mode

Whey is a by-product generated by the dairy industry which contains lactose and proteins. As a valorization method, fermentation is a biotechnological practice to obtain high value-added biomolecules from whey, such as 2-Phenylethanol (2PE). Sterilization methods were assessed to determine which was more appropriate for whey fermentation using Kluyveromyces marxianus and Debaryomyces hansenii under co-culture mode. Then, the effect of lactose and L-Phenylalanine (L-Phe) initial concentrations on the production of 2PE was studied using a central composite design. The initial concentration of lactose and L-Phe of 40 g/L and 4 g/L respectively, allowed to obtain the highest concentration of 2PE (2.55 ± 0.12 g/L). The main effect of lactose on the production of 2PE was the vigorous induction of yeast growth in 24 h of fermentation. The L-Phe presence in the culture medium promoted the production of 2PE during the yeast's secondary metabolism, corresponding to the stationary growth phase.

Modeling Gene Expression: Lac operon.

Gene regulation is an essential process for cell development, having a profound effect in dictating cell functions. Bacterial genes are often regulated through inducible systems like the Lac operon which plays an important role in cell metabolism. An accurate model of its regulation can reveal the dynamics of gene expression. In this paper, a mathematical model of this system is constructed by focusing on regulation by the Lac repressor. The results show, as expected, that the concentration of lactose approaches zero while glucose concentration approaches the initial concentration of lactose by the action of β-galactosidase, expressed by the Lac operon. Addition of PD control improves stability of the system, with the phase margin increasing from 45° to 90°. Modeling the dynamics of gene expression in inducible operons like Lac operon can be essential for its applications in the production of recombinant proteins and its potential usage in gene therapy.

Optimization and comparison of the production of galactooligosaccharides using free or immobilized Aspergillus oryzae β-galactosidase, followed by purification using silica gel

The aim of this study was to optimize and compare the production of galactooligosaccharides (GOSs) by free and cotton cloth-immobilized Aspergillus oryzae β-galactosidase, and perform economical evaluation of production of GOSs (100%) between them. Using the response surface method, the optimal reaction time (3.9 h), initial lactose concentration (57.13%), and enzyme to lactose ratio (44.81 U/g) were obtained for the free enzyme, which provided a GOSs yield of 32.62%. For the immobilized enzyme, the optimal yield of GOSs (32.48%) was obtained under reaction time (3.09 h), initial lactose concentration (52.74%), and temperature (50.0 ℃). And it showed desirable reusability during five successive enzymatic reactions. The recovery rate of GOSs (100%) is 65% using silica gel filtration chromatography. The economical evaluation showed almost no difference in the manufacturing cost for the GOSs (100%) between these two systems, and that the recovery rate had a great impact on the cost.

Identification of a lactose-oxidizing enzyme in Escherichia coli and improvement of lactobionic acid production by recombinant expression of a quinoprotein glucose dehydrogenase from Pseudomonas taetrolens

Lactobionic acid (LBA), an aldonic acid prepared by oxidation of the free aldehyde group of lactose, has been broadly used in cosmetic, food, and pharmaceutical industries. Although Escherichia coli is unable to produce LBA naturally, a wild-type E. coli strain successfully produced LBA from lactose upon pyrroloquinoline quinone (PQQ) supplementation, indicating that E. coli contains at least one lactose-oxidizing enzyme as an apo-form. By inactivating the candidate genes in the E. coli chromosome, we found that the lactose-oxidizing enzyme of E. coli was the quinoprotein glucose dehydrogenase (GCD). To improve the LBA production ability of the E. coli strain, quinoprotein glucose dehydrogenase (GDH) from Pseudomonas taetrolens was recombinantly expressed and culture conditions such as growth temperature, initial lactose concentration, PQQ concentration, and isopropyl-β-D-1-thiogalactopyranoside induction concentration were optimized. We performed batch fermentation using a 5-L bioreactor under the optimized culture conditions determined in flask culture experiments. After batch fermentation, the LBA production titer, yield, and productivity of the recombinant E. coli strain were 200 g/L, 100 %, and 1.28 g/L/h, respectively. To the best our knowledge, this is the first report to identify the lactose-oxidizing enzyme of E. coli and to produce LBA using a recombinant E. coli strain as the production host. Because E. coli is one of the most easily genetically manipulated bacteria, our result provides the groundwork to further enhance LBA production by metabolic engineering of LBA-producing E. coli.

Valorisation of sweet whey by fermentation with mixed yoghurt starter cultures with focus on galactooligosaccharide synthesis

Sweet whey was fermented with yoghurt starter cultures (YO-MIX 101 containing Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium lactis and YO-MIX 532 containing Lactobacillus delbrueckii subsp. bulgaricus and S. thermophilus) as well as enzyme extracts thereof to synthesise galactooligosaccharides from lactose. The influence of fermentation temperature and initial lactose concentration was studied. The highest yield in sweet whey (34.2% including disaccharide GOS, 25.4% excluding disaccharide GOS) was obtained with the YO-MIX 532 culture at 50 °C and 100 g L−1 initial lactose. With YO-MIX 101, yields were considerably lower, but transgalactosylation was favoured in sweet whey compared with buffer (23.4% versus 8.5% total GOS at 100 g L−1 initial lactose). Of the isolated strains derived from the mixed cultures, only Lactobacillus bulgaricus and both strains of S. thermophilus showed notable GOS synthesis. Up-scaling to a stirred tank reactor system yielded 26.3% total GOS within 24 h of fermentation.

Catalytic and kinetic study of the liquid-phase oxidation of lactose over Au/Al2O3 nanostructured catalysts in a monolithic stirrer reactor

The liquid-phase oxidation of lactose (LA) to lactobionic acid (LB) was studied over Au(2 %)/Al2O3 nanostructured catalyst. Catalytic tests were carried out at 65 °C, varying O2 partial pressures in the range of 0.21−1 bar and LA initial concentrations between 0.007 and 0.089 M. In all of the cases, the selectivity to LB was always 100 %. It was observed that the trends for the LA conversion (XLA) with time depends on the ratio of catalyst weight to initial LA moles W/nLA0 and the initial LA concentration CLA0. Experimental data were interpreted by kinetic modelling applying pseudo-homogeneous and Langmuir-Hinshelwood-Hougen-Watson (LHHW) models. It was found that irreversible surface oxidation of LA rules the reaction rate when W/nLA0= 30 g mol−1. At the early stages, the reaction rates did not depend on the LA concentration, and thus the reaction order with respect to LA was zero. In this case, an LHHW model considering total coverage of the active sites gives a very good fitting of the experimental data. As LA was consumed, the progress of conversion with time is different for each case and the reaction order changes to positive. Now, a better fitting is obtained with an LHHW model in which partial coverage of the active sites is considered. When liquid phase was diluted in LA and W/nLA0= 95 g mol−1, the LA chemisorption becomes the controlling step. Therefore, the results obtained by kinetic modelling helped to explain the dependence of LA oxidation rate with CLA0 and W/nLA0.

Production and identification of galacto-oligosaccharides from lactose using β-D-galactosidases from Lactobacillus leichmannii 313

Crude β-D-galactosidase from Lactobacillus leichmannii 313 was used to synthesise galacto-oligosaccharides (GOS) from lactose. Using an initial lactose concentration of 170 g/L, the yield of GOS was 52 g/L (i.e. 62% lactose conversion) after 2 h of enzymatic reaction. The highest number of unique individual GOS were generated after 48 h of reaction. ESI-MS and HPAEC analysis of the GOS enriched fractions obtained from 48 h mixtures showed the presence of sugars with degrees of polymerization (DP) ranging from 2 to 6. Further analysis (by NMR spectroscopy) of GOS in fraction 3 (containing ~ 96% w/w GOS) showed non-reducing terminals that were mostly Galp; and reducing ends that were →4)-Glcp, and →6)-Glcp. The most abundant internal linkages in fraction 3 were deduced to be →3)-Galp-(1→, and →6)-Galp-(1→. The predominance of β-D-(1→3, or 6) linkages in GOS fractions suggests that β-D-galactosidase from LL313 is suitable for generating unique GOS from lactose.

β-galactosidase GALA from Bacillus circulans with high transgalactosylation activity

ABSTRACT β-galactosidase catalyzes lactose hydrolysis and transfers reactions to produce prebiotics such as galacto-oligosaccharides (GOS) with potential applications in the food industry and pharmaceuticals. However, there is still a need for improved transgalactosylation activity of β-galactosidases and reaction conditions of GOS production in order to maximize GOS output and reduce production costs. In this study, a β-galactosidase gene, galA, from Bacillus circulans was expressed in Pichia pastoris, which not only hydrolyzed lactose but also had strong transgalactosylation activity to produce GOS. Response surface methodology was adopted to investigate the effects of temperature, enzyme concentration, pH, initial lactose concentration, and reaction time on the production of GOS and optimize the reaction conditions for GOS. The optimal pH for the enzyme was 6.0 and remained stable under neutral and basic conditions. Meanwhile, GALA showed most activity at 50°C and retained considerable activity at a lower temperature 30–40°C, indicating this enzyme could work under mild conditions. The enzyme concentration and temperature were found to be the critical parameters affecting the transgalactosylation activity. Response surface methodology showed that the optimal enzyme concentration, initial lactose concentration, temperature, pH, and reaction time were 3.03 U/mL, 500 g/L, 30°C, 5.08, and 4 h, respectively. Under such conditions, the maximum yield of GOS was 252.8 g/L, accounting for approximately 50.56% of the total sugar. This yield can be considered relatively high compared to those obtained from other sources of β-galactosidases, implying a great potential for GALA in the industrial production and application of GOS.

Characterization of Dairy Waste Whey and Its’ Utilization for the Production of Ethanol

The objective of this study was characterization of dairy waste whey and its’ utilization for the production of ethanol. Whey is a byproduct and not considered as a resource in many milk processing factories. Usually, it is discharged to rivers and surface water. The physiochemical characteristics of whey was determined by Lactoscan Milk Analyzer, Refractometer and Titration Methods. Dairy whey consisted of biological oxygen demand and chemical oxygen demand. It represents largely disaccharide sugar content which is known as lactose (0. 36 – 0. 48) g/mL and it could highly pollute the environment or water bodies. However, this whey waste was changed into ethanol by yeast strain, kluyveromyces delphensis. The experimental design was studied with central composite design to investigate the effects of significant factors including initial lactose concentration, yeast cell concentration, temperature and pH value on fermentation process. In the present work, the best operating conditions were found at 6.15g/L (initial lactose conc.), 10g/L (yeast cell conc.), 27oC (temperature) and 5.5 pH value respectively. The maximum ethanol yield obtained from cheese whey was 40.4 % (2.5g/L). Functionally, ethanol was determined by Fourier Transmission Infrared with the help of Infrared correlation charts and moreover, its characteristic was investigated. As a result, cheese whey is a good resource for a production of ethanol.

Modeling carbohydrates oxidation by oxygen catalyzed by bienzyme glucose dehydrogenase/laccase system immobilized into microreactor with carbon nanotubes

This paper presents a mathematical model of a batch stirred tank reactor based on an array of identical spherical porous microbioreactors loaded with non specific glucose dehydrogenase and oxygen reducing enzyme, i.e. laccase. The model was validated by experimental data. The microreactors (MR) are mathematically modeled by a two-compartment model, based on reaction–diffusion equations containing nonlinear terms related to the Michaelis–Menten kinetics of two enzymatic reactions with addition of the mass transport. The dynamics of oxygen concentration change is analysed numerically using the finite difference technique. The transient effectiveness factor and the process duration are investigated at different initial concentration of carbohydate (lactose) as well as at internal and external diffusion resistances. The simulation results show a non-monotonic effect of the initial concentration of lactose and nonlinear effects of the internal and external diffusion limitations on the transient effectiveness.

One-Pot Synthesis of Lactose Derivatives from Whey Permeate.

The simultaneous production of lactulose (LAU), lactobionic acid (LBA), and organic acids from sweet and acid whey permeate (SWP and AWP) via catalytic synthesis (5% Ru/C) was studied in a continuous stirred-tank reactor. At selected conditions (60 °C, 60 bar, and 600 rpm), a maximum conversion of lactose (37 and 34%) was obtained after 90 min for SWP and AWP, respectively. The highest yield calculated with respect to the initial concentration of lactose for LAU was 22.98 ± 0.81 and 15.29 ± 0.81% after only 30 min for SWP, and AWP, respectively. For LBA, a maximum yield was found in SWP (5.23%) after 210 min, while about 2.2% was found in AWP. Six major organic acids (gluconic, pyruvic, lactic, formic, acetic, and citric acid) were quantified during the one-pot synthesis of lactose.

Enzymatic synthesis of prebiotic carbohydrates from lactose: Kinetics and optimization of transgalactosylation activity of β‐galactosidase from Aspergillus oryzae

AbstractThe aim of the study was to analyze galactooligosaccharide (GOS) synthesis catalyzed by β‐galactosidase from Aspergillus oryzae by applying experimental data to optimization and kinetic parameter estimation methods. The apparent kinetic parameters were determined in varying initial lactose concentrations by using six‐step, 11‐parameter GOS synthesis mechanism. The results revealed that GOS yield was mainly dependent on initial lactose concentration; on the other hand, all of the reaction parameters affected the required time to achieve maximum GOS yield (TMGY) significantly. The values of 32 °Brix, 36.8°C, and 8.32 U/g lactose solution were determined as the optimum reaction experimental set which offers 26.73% GOS yield and 369.82 min TMGY. The increased enzyme‐galactose complex dissociation ratio (k3/k−3) with decreasing initial lactose concentration resulted to low GOS productivity although GOS production/GOS dissociation rate ratio (k4/k−4) increased with decreasing initial lactose concentration. This result signified that hydrolysis characteristics dominated the reaction medium at low initial lactose concentration. The role of monosaccharide inhibition became important on GOS synthesis for increasing initial lactose concentration.Practical ApplicationsThe proposed study deals with investigation of the effects of initial lactose concentration, enzyme concentration, and temperature on enzymatic synthesis of galactoligosaccharide (GOS) from lactose by β‐galactosidase from Aspergillus oryzae (βgAO) and estimation of apparent kinetic rate constants of the reaction which is important to design of futuristic strategies improving the GOS yield. Low GOS yield is the main concern of the prebiotic food industry due to thermodynamic and kinetic realities of the reaction. Deeper evaluation of the reaction mechanism will contribute valuable data to the partners in the scope of designing more productive engineering methods.