Production Of Galacto-oligosaccharides Research Articles

Production of Galacto‐Oligosaccharides by Enzymatic Membrane Reactors Using Free β‐Galactosidase

AbstractPrebiotic galacto‐oligosaccharides (GOSs) are synthesized by transgalactosylation of lactose using β‐galactosidases. Well‐established, large‐scale production of GOS is performed by stirred‐tank reactors (STR) operating in batch fashion. Main manufacturing expenses are attributed to enzyme purchase costs and the downstream operations required for subsequent enzyme inactivation and removal. In the last decades, several studies and patents have appeared on free enzyme membrane reactor (FEMR) as an economic alternative to STR due to enhanced enzyme recovery and reuse. In this review, we attempt to summarize the key design aspects, the catalytic performance and efficiency of FEMR compared to STR technology, and the main concerns related to stable product quality over long‐term operation.

[Investigation on the recommended dose of prebiotic in health food in China in 1996-2022].

To understand the recommended dose distribution of prebiotic-containing health food in China. The overall recommended dose of prebiotic health food was available from the label information of approved prebiotic health food from 1996 to 2022; the recommended dose distribution of prebiotic-containing health food was analyzed from different healthy functions and different ways of addition. There were 174 prebiotic-containing health food products with clear dose information, respectively, involving 5 prebiotics including Fructooligosaccharides, Galactooligosaccharides, Isomaltooligosaccharides, Xylo-oligosaccharides and Polydextrose, and the majority of prebiotics were added in combination, with 159 products. The recommended dose range of prebiotic-containing health food products was wide, and in general, the dose of prebiotic-containing health food products used alone was higher than the dose used in combination. The recommended daily intake range of health food containing Fructooligosaccharides was 5.28-17 500 mg/d, the recommended daily intake range of health food containing Isomaltooligosaccharides was 220-28 000 mg/d, the dose range of health food containing Xylo-oligosaccharides was 8.4-2 800 mg/d, the dose range of health food containing Polydextrose was 4-12 120 mg/d, the number of Galacto-Oligosaccharides products Only two kinds of products were included, with doses of 259.8 mg/d and 3500 mg/d, respectively. The claimed functions of prebiotic health food products were focused on laxative function, immunity enhancement, and regulation of intestinal flora. The application dose of prebiotic health food with different functional compounding additions was close to the overall dose. The recommended dosage range of prebiotics in health food containing prebiotics in China is large, and prebiotics in products are mainly added by compounding.

Cheese and Yogurt By-Products as Valuable Ingredients for the Production of Prebiotic Oligosaccharides

The growing global market of dairy products has led to the need for alternative approaches regarding whey valorization, which is the primary by-product of cheese and strained yogurt production. In this context, prebiotic galactooligosaccharides can be produced enzymatically from whey using commercially available β-galactosidases. A comparative study was conducted to assess the production of galactooligosaccharides from sweet and acid whey, thereby employing two commercial β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis. The study considered the initial lactose content and enzyme load as variables. The maximum yields of galactooligosaccharides in concentrated sweet whey (15% w/v initial lactose) and raw acid whey (3.1% w/v initial lactose) reached 34.4 and 14.7% with lactase from Kluyveromyces lactis (0.13 U/mL), respectively. The corresponding galactooligosaccharide yields for lactase from Aspergillus oryzae were equal to 27.4 and 24.8% in the most concentrated sweet and acid whey, respectively, using enzyme loads of 2 U/mL in sweet whey and 1 U/mL in acid whey. Concerning the profile of the produced galactooligosaccharides, the Kluyveromyces lactis lactase hydrolyzed lactose more rapidly and resulted in higher levels of allolactose and lower levels of 6-galactosyl-lactose, compared to the lactase from Aspergillus oryzae, and achieved in both cases a polymerization degree of up to six.

Safety evaluation of the food enzyme β-galactosidase from the non-genetically modified Papiliotrema terrestris strain AE-BLC.

The food enzyme β-galactosidase (β-d-galactoside galactohydrolase; EC 3.2.1.23) is produced with the non-genetically modified Papiliotrema terrestris strain AE-BLC by Amano Enzyme Inc. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the production of galacto-oligosaccharides (GOS) from lactose. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.441 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1800 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 4082. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Assessing the production of galactooligosaccharides in batch and continuous mode by using β‐galactosidase immobilised on mesoporous silicon dioxide nanoparticles

SummaryThe present study evaluated the production of galactooligosaccharides (GOSs) from lactose in both batch and continuous modes using nano‐immobilised β‐galactosidase. Mesoporous silicon dioxide nanoparticles were used to immobilise β‐galactosidase to enhance the stability and production of GOSs. The change in enzyme‐characteristics was assessed using Fourier‐transform infrared and scanning electron microscope analysis. The nano‐immobilisation increased the enzyme's substrate affinity, but decreased its hydrolytic activity. A significant increase (P < 0.05) in stability was observed across a wide range of temperature (30 °C–70 °C) and pH (5.0–8.0). Both forms of enzymes (free and nano‐immobilised enzymes) followed the zero‐order reaction in all temperatures, while the nano‐immobilised enzyme had the highest activation energy. The free and nano‐immobilised enzymes retained 64.65% and 77.82% of their initial activity after 90 days of storage, respectively. In a batch mode, the nano‐immobilised enzyme produced 2.35 times more GOSs than the free enzyme. The GOSs production remained consistent while the enzyme activity decreased by 12.7% after 5 cycles of operation. The continuous mode of production in a packed bed reactor achieved a GOSs yield of 16.92%. Overall, the nano‐immobilised enzyme showed better GOSs production under batch and continuous mode of operation.

Preparation of nano-immobilised β-galactosidase using mesoporous silicon dioxide nanoparticles and its efficiency in production of galactooligosaccharides

The β-galactosidase (produced from Aspergillus oryzae) was immobilised on epichlorohydrin functionalised mesoporous silicon dioxide nanoparticles to produce galactooligosaccharides (GOS) from lactose. The characteristic amide bonds and increased particle size confirmed the adsorption of enzyme on nanoparticles. Nano-immobilisation resulted in an increase in enzyme affinity towards the substrate and its hydrolytic property. Free and nano-immobilised enzymes retained 58 and 77% of the original enzyme activity up to 90 days of storage, respectively. The nano-immobilised enzyme exhibited greater stability over a broad range of temperature (30–70 °C) and pH (5.0–8.0). The GOS yield obtained from nano-immobilised enzyme was 2.23 times higher than the free enzyme. GOS production was decreased by 90% and enzyme activity was reduced to 10% in a run of 5 cycles. Overall, the developed nano-immobilisation technique has potential to enhance the stability of the enzyme and GOS production.

Modeling and simulation of the enzymatic kinetics for the production of Galactooligosaccharides (GOS) using an Artificial Neural Network hybrid model

Due to the complexity of biochemical systems, the development of traditional phenomenological models is limited if the underlying mechanics are not entirely known. As an alternative, hybrid model frameworks, consisting of data-driven models complemented with first principles models like conservation law, are starting to be used for complex systems. In this work, a comparison of the modeling capabilities between a data-driven model and a hybrid model was developed. The enzymatic production of Galactooligosaccharides (GOS) with the effect of metallic ions was considered as case study. Compared with the experimental results, predictions from data-driven model achieve an R2 of 0.9188 in the best training fold, and the hybrid model an R2 of 0.9696 in the best training fold. Illogical predictions were avoided by including non-phenomenological first-principles constraints into the hybrid model. Finally, an optimization analysis was carried out to find the highest GOS productivity using the hybrid model, optimization results present a deviation of 5.99 % compared to the highest productivity found from experimental data.

Production of Prebiotic Galacto-Oligosaccharides from Acid Whey Catalyzed by a Novel β-Galactosidase from Thermothielavioides terrestris and Commercial Lactases: A Comparative Study

The steadily increasing global popularity of Greek strained yoghurt has necessitated alternative valorization approaches for acid whey, the major straining process effluent. In this context, prebiotic galacto-oligosaccharides can be enzymatically synthesized from acid whey lactose, via either commercial or novel β-galactosidases. A comparative study of galacto-oligosaccharide production from acid whey was carried out, employing two commercial β-galactosidases (from Kluyveromyces lactis and Aspergillus oryzae) and one novel, in-house produced (from Thermothielavioides terrestris), as a function of the initial lactose content and enzyme load. Selected reaction conditions for β-galactosidases from K. lactis, A. oryzae, and T. terrestris were 35 °C at pH 7.2, 45 °C at pH 4.5, and 50 °C at pH 4.0, respectively. Maximum galacto-oligosaccharide yields equal to 23.7, 23.4, and 25.7% were achieved with, respectively, 0.13 U/mL of K. lactis β-galactosidase in non-concentrated acid whey, 4 U/mL of A. oryzae β-galactosidase, and 8 U/mL of T. terrestris β-galactosidase in acid whey concentrated to 20% w/v initial lactose content. The increased galacto-oligosaccharide productivity of the thermophilic β-galactosidase from T. terrestris can be a determining asset in a combined concentration and oligomerization industrial process. This will allow for high galacto-oligosaccharide yields for efficient, cost-effective production of valuable prebiotics from acid whey.

Invited review: Properties of β-galactosidases derived from Lactobacillaceae species and their capacity for galacto-oligosaccharide production

β-galactosidase (enzymatic class 3.2.1.23) is one of the dairy industry's most important and widely used enzymes. The enzyme is part of a large family known to catalyze hydrolysis and transglycosylation reactions. Its hydrolytic activity is commonly used to decrease lactose content in dairy products, while its transglycosylase activity has recently been used to synthesize galacto-oligosaccharides (GOS). During the past couple of years, researchers have focused on studying β-galactosidase isolated and purified from lactic acid bacteria. This review will focus on β-galactosidase purified and characterized from what used to be the Lactobacillus genera. Furthermore, particular emphasis is given to its kinetics, biochemical characteristics, GOS production, market, and utilization by Lactobacilllaceae species.

Chitosan with modified porosity and crosslinked with genipin: A dynamic system structurally characterized

In this work, a deep structural characterization of the chitosan-genipin complex was performed by techniques like small-angle X-ray scattering, dye absorption, atomic force microscopy, compression strength, nitrogen adsorption/desorption, scanning electronic microscopy, and thermogravimetric analysis to understand the changes in the structure through all steps of the process. For this, chitosan beads were modified with Na2CO3 to change porosity and then crosslinked with genipin. The prepared particles were used as support for enzyme immobilization. Our results demonstrated modification in the porosity of the chitosan beads, changing the number of pores and pore volume. After crosslinking process (pH 9), especially the microstructure of chitosan-genipin beads was modified, enhancing the fractality and, after enzyme immobilization (pH 4.5) the fractality increased at the higher scale (∼100 nm) and reduced at the lower scale (∼1–10 nm). β-galactosidase immobilized on the modified particles presented high stability on the continuous production of galactooligosaccharides for 30 days. Our results indicate that the chitosan-genipin complex can be characterized as a plastic and dynamic system due to its structural modifications, particularly in response to changes in the pH environment, even after the crosslinking process.

Targeted gene manipulation of Leloir pathway genes for the constitutive expression of β-galactosidase and its transgalactosylation product galacto-oligosaccharides from Kluyveromyces lactis GG799 and knockout strains

Galacto-oligosaccharides (GOS) are used as prebiotic ingredients in various food and pharmaceutical industry. At present, production of GOS involves the enzymatic transformation of lactose by transgalactosylation using β-galactosidase. The yeast Kluyveromyces lactis can utilize lactose as its carbon and energy source. In this species lactose is hydrolyzed by an intracellular β-galactosidase (EC 3.2.1.23) which is induced by its substrate and related compounds like galactose. The molecular details of gene regulation in kluyveromyces lactis, we have used multiple knockout approaches to study the constitutive expression by which galactose induces β-galactosidase. The present study involved carrying out to a method of enhancing the constitutive expression of β-galactosidase through galactose induction and its trans-galactosylation reaction for the production of galacto-oligosaccharides (GOS) in Kluyveromyces lactis (K. Lactis) by applying a knockout based approach on Leloir pathway genes based on fusion-overlap extension polymerase chain reaction and transformation into its genome. The k.lactis strain subjected to Leloir pathway genes knockout, resulted in the accumulation of galactose intracellularly and this internal galactose acts as an inducer of galactose regulon for constitutive expression of β-galactosidase at early stationary phase was due to the positive regulatory function of mutant gal1p, gal7p and both. These resulted strains used for trans-galactosylation of lactose by β – galactosidase is characterized for the production of galacto-oligosaccharides. Galactose-induced constitutive expression of β-galactosidase during the early stationary phase of knockout strains was analysed qualitatively & quantitatively. The activity of β-galactosidase of wild type, gal1z, gal7k and gal1z & gal7k strains were 7, 8, 9 and 11 U/ml respectively using high cell density cultivation medium. Based on these expression differences in β-galactosidase, the trans-galactosylation reaction for GOS production and percentage yield of GOS were compared at 25% w/v of lactose. The percentage yield of GOS production of wild type, Δgal1z Lac4+, Δgal7k Lac4++ and Δgal1z Δgal7k Lac4+++mutants strains were 6.3, 13, 17 and 22 U/ml, respectively. Therefore, we propose that the availability of galactose can be used for constitutive over expression of β – galactosidase in Leloir pathway engineering applications and also for GOS production. Further, increased expression of β – galactosidases can be used in dairy industry by-products like whey to produce added value products such as galacto-oligosaccharides.

Sensory Assessment of Bi-Enzymatic-Treated Glucose-Galactose Syrup

There are a variety of ways to make glucose-galactose syrup (GGS) and other products of lactose hydrolysis; therefore, research is still ongoing and will undoubtedly result in improved methods and lower costs. The aim of the study was to use a two-stage fermentation approach to increase the sweetness of glucose-galactose syrup. Comparing lactose hydrolysis with β-galactosidases, the enzyme Ha-Lactase 5200 (K. lactis) showed the highest hydrolysis yield but NOLA™ Fit5500 (B. licheniformis) and GODO-YNL2 (K. lactis) hydrolysis yields varied. After the two-stage fermentation, the syrups from sweet whey permeate had shown the highest sweet taste intensity scores; the sweetest samples were 1NFS and 1HLS with a score of 9.2 and 9.3, respectively. The presence of fructose in the range of 14 ± 3 to 25 ± 1 %, significantly (p < 0.05) increased the sweetness of the syrups. Obtained syrups from whey permeates using enzymes NOLA™ Fit5500 and Ha-Lactase 5200 contained less than 10% lactose. Additionally, results indicate that hydrolysis of lactose and subsequent enhancement of sweetness through glucose isomerisation may provide additional benefits through the production of galacto-oligosaccharides (GOS) in the range of 2 ± 1 to 34 ± 7%.

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.

In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of Lactobacillus bulgaricus.

β-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of β-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the β-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other β-galactosidases of this species. The molecular model revealed that the new β-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg2+ ions and substrate. The β-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme's temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg2+, Mn2+, and Ca2+ on its activity were observed. From lactose, β-Gal produced a large amount of GOS with DP3 containing β-(1→3) and β-(1→4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of β-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.

Safety evaluation of the use of the non-genetically modified Hamamotoa singularis strain YIT 10047 as a source of β-galactosidase.

This assessment addresses the enzyme β-galactosidase which is not separated from the yeast cells used for its production. The β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) is produced with the non-genetically modified Hamamotoa singularis (formerly Sporobolomyces singularis) strain YIT 10047 by Yakult Pharmaceutical Industry Co., Ltd. The yeast cell suspension contains both live and dead yeast cells. It is intended to be used in the production of galacto-oligosaccharides (GOS). The final GOS products are free of viable cells of the H. singularis. Dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 0.683mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests of the cell suspension did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panelidentified a no observed adverse effect level of 296.25mg TOS/kg bw per day, the highest dose tested. This results in a margin of exposure above 434. A search for the similarity of the amino acid sequence of the β-galactosidase to known allergens was made and no matches were found. The Panelconsidered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the Panelconcluded that this yeast suspension used as a source of β-galactosidase does not give rise to safety concerns under the intended conditions of use.

Enzymatic Synthesis of Galacto-Oligosaccharides from Concentrated Sweet Whey Permeate and Its Application in a Dairy Product

Valorization of by-products in the dairy industry as a strategy to promote circular economies has become a priority around the globe. Cheese whey and its derivates from filtration technologies offer a source of valuable molecules such as proteins, fat, lactose, and minerals. For this study, after protein concentration and recovery through ultrafiltration, lactose remaining in the permeate was furtherly concentrated with nanofiltration, resulting in a retentate used as substrate for the enzymatic production of galacto-oligosaccharides (GOS). The kinetics of GOS generation with a commercial β-galactosidase, was carried out, quantifying the carbohydrate composition by HPAEC-PAD. Results showed that at 0.5 h, GOS yield reached a maximum of 74% (g GOS/g lactose) with a lactose utilization of 63%. Under these conditions, a GOS syrup (75% soluble solids) was generated and applied in a porridge for blind paired comparison test, including a control without the syrup. No differences were identified in color and odor between porridges; however, flavor and mouthfeel of the GOS-added sample improved according to the comments of panelists. This study presents an alternative process for the valorization of whey permeate to produce GOS ingredients that can be used directly in day-to-day dairy products.

Evaluation of Prebiotic Properties of Galactooligosaccharides Produced by Transgalactosylation Using Partially Purified β-Galactosidase from Enterobacter aerogenes KCTC2190.

Transgalactosylation reaction is the penultimate step in the production of galactooligosaccharides (GOSs) which has prominent applications in the treatment of disorders. In the present study, partially purified β-galactosidase from Enterobacter aerogenes KCTC2190 was used for the synthesis of prebiotic GOSs. GOSs were produced using lactose as substrate. Structural elucidation of collected fractions of GOSs by liquid chromatography electrospray ionization mass spectrometry exhibited the appearance of major peaks of produced GOSs at m/z 241.20, 481.39, 365.11, 527.17, and 701.51 respectively. GOSs facilitated the growth of potential probiotic strains (Lactobacillus delbrueckii ssp. helveticus, Bifidobacterium bifidum, and Lactiplantibacillus plantarum) and liberated propionate and butyrate as principal short-chain fatty acids which established its prebiotic potency. Synbiotic combinations exhibited good antioxidant activities. Synbiotic combinations also exhibited antimicrobial activities against pathogenic microorganisms namely Staphylococcus aureus and Escherichia coli. Synbiotic combinations of GOSs and the respective probiotic microorganisms were able to decrease viable human bone cancer cells (MG-63).

Recent Advances in the Production, Analysis, and Application of Galacto-Oligosaccharides

Galacto-oligosaccharides (GOS) are the prebiotic constituents with various functional qualities that promote the optimal proliferation of gut microbiota and are consequently beneficial to human health. GOS are not absorbed by humans and animals, but they play numerous health-related positive roles in the human body. Currently, their production has occupied much attention due to the increasing demand for functional foods. It could be produced by enzymatic and chemical approaches. Many microbial enzymes have been utilized for GOS production but the main source of these enzymes is lactose. Moreover, characterization of GOS structures is also a necessary and critical effort for understanding their mechanism of action. Hence, this review is written to understand the extensive but related features for the synthesis of GOS which includes: the enzymatic production of GOS through various enzymes and the types of enzymes such as free, immobilized, recombinant, crude, and purified enzymes that are involved in their synthesis, the techniques used in the purification of GOS, the analysis and quantification of GOS through various practices, and the functional properties of GOS are also discussed in this review. It also aims to combine new findings with current information in order to improve understanding of GOS formation and other factors related to its functionality.

Production of galactooligosaccharides: alternative for the use of whey. A review

En el mundo la producción de suero de leche está en aumento y su aprovechamiento insuficiente genera impactos ambientales y económicos que se reflejan en el deterioro del medio ambiente y la pérdida de recursos económicos para las comunidades. El objetivo de la presente revisión bibliográfica fue consultar en la literatura científica y técnica los diferentes usos que se le está dando actualmente al lactosuero desde las proteínas y la lactosa. Se identificó que el lactosuero sigue siendo utilizado por lo general para obtener lactosueros en polvo y concentrados y aislados de proteína, entre otros productos. El procesamiento del lactosuero genera residuos como la lactosa que representa un serio problema ambiental y económico por la posible contaminación a fuentes hídricas y el alto costo que tiene su tratamiento (mano de obra e insumos). En este sentido, el proceso de producción de galactooligosacáridos (compuestos prebióticos de alto interés) puede ser una alternativa de inversión y desarrollo tecnológico para el sector lácteo de Colombia que busca nuevas formas de valorizar los residuos y disminuir el impacto ambiental de estos procesos.

Effect of process parameters on the β-galactosidase hydrolysis of lactose and galactooligosaccharide formation in concentrated skim milk

The study aimed at evaluation of β-galactosidase activity for lactose hydrolysis (DH) and galactooligosaccharide (GOS) formation at 7 °C. β-galactosidase derived from K. lactis was more effective than B. lichenformis for DH and GOS formation in 16% lactose solution. β-galactosidase from K. lactis exhibited 96.61% DH and 7.28% GOS production after 12 h of reaction and hence was utilized for lactose hydrolysis in concentrated skim milk (40% total solids). Use of 9.53 U/mL enzyme resulted in significantly high DH (97.06%) after 12 h with 4.90 g/L of residual lactose. However, maximum GOS formation of 12.01% with 94.74% DH was obtained after 4 h. Further increase in reaction time up to 12 h resulted in breakdown of tri and tetrasaccharide GOS, thereby, reducing GOS content. Hence, reaction time of 12 h was finalized to obtain maximum DH along with additional benefit of GOS formation.