Application of immobilized lactase in lactose-free dairy product production: A review

Introduction

Lactose (4-O-β-D-galactopyranosyl-D-glucopyranose) is the predominant carbohydrate in mammalian milk, constituting approximately 4.5-5.0% (w/v) of bovine milk and providing nearly 30% of its energy content. Its digestion in the small intestine requires the brush-border enzyme lactase-phlorizin hydrolase (lactase; β-galactosidase; EC 3.2.1.23), which cleaves the β-1,4-glycosidic bond to yield glucose and galactose. Individuals with insufficient intestinal lactase activity are unable to hydrolyze lactose adequately, undigested lactose passes into the colon, where resident microbiota ferment it to short-chain fatty acids, hydrogen, carbon dioxide, and methane, producing characteristic symptoms of bloating, flatulence, diarrhoea, and abdominal cramps collectively termed lactose intolerance (Li et al., 2023) [10] . The condition is remarkably prevalent. A systematic review standardized for country size estimated the global prevalence of lactose malabsorption at 68% with regional variation ranging from approximately 28% in Western Europe to over 70% in East Asia and the Middle East (Storhaug et al., 2017) [16] . In the Indian subcontinent and Southeast Asia, rates exceed 80-90%, making lactose intolerance a major nutritional concern in countries with high dairy consumption. Lactase activity typically declines after weaning a phenomenon termed lactase non-persistence as a result of epigenetic silencing of the LCT gene encoding lactase, with the onset varying by ethnicity and geography. Demand for lactose-reduced and lactose-free dairy products has consequently surged globally. The global lactose-free dairy market was valued at USD 12.87 billion in 2024 and is projected to expand at a compound annual growth rate (CAGR) of 8.6%, reaching USD 13.98 billion in 2026 and an estimated USD 19.37 billion by 2029 (The Business Research Company, 2025) [17] . This growth is driven by rising health consciousness, increased diagnosis of lactose intolerance, product innovation, and expansion of retail and e-commerce channels. Three principal industrial strategies have been deployed for lactose removal or reduction: (i) membrane-based separation (nanofiltration and chromatography), (ii) microbial fermentation International Journal of Advanced Biochemistry Research https://www.biochemjournal.com using high-lactose-fermenting strains, and (iii) enzymatic hydrolysis using lactase. Of these, enzymatic hydrolysis is the most commercially dominant because it is cost-effective, highly specific, preserves the nutritional matrix of milk, and yields a product with improved sensory properties due to the higher sweetness of glucose and galactose compared to lactose. However, free-enzyme systems are limited by poor thermal stability, sensitivity to inhibitory products (glucose and galactose feedback inhibition), non-recoverability, and potential carryover into the final product where continued enzymatic activity may impair shelf life (Deng et al., 2024) [6] . Immobilization, the restriction of enzyme molecules to a solid support or within a matrix while retaining catalytic functionality overcomes these limitations by enabling enzyme reuse, enhancing operational stability, and facilitating integration into continuous flow reactors. Since the first commercial immobilized lactase systems were deployed in the 1980s, significant advances in support materials, immobilization chemistry, and reactor design have transformed the technology. This review synthesizes current knowledge on immobilized lactase systems for lactose-free dairy production, with emphasis on recent advances in nanomaterial supports, multi-enzyme systems, and process optimization.

Lactase (β-Galactosidase): Biochemistry and Industrial Sources 2.1 Catalytic Mechanism

Lactase catalyzes the hydrolysis of lactose according to the reaction: Lactose + H₂O → D-Glucose + D-Galactose The enzyme operates via a double-displacement (retaining) mechanism involving two catalytic glutamate residues. Nucleophilic attack by the first glutamate on the anomeric carbon of the galactose moiety forms a covalent galactosylenzyme intermediate, releasing glucose. Hydrolysis by water (or transglycosylation with lactose) then regenerates the free enzyme and yields galactose (or galactooligosaccharides, GOSs). The relative rates of hydrolysis and transglycosylation depend on lactose concentration, temperature, and pH, and are exploited industrially for GOS production, prebiotic oligosaccharides increasingly valued for gut health (Chalella Mazzocato & Jacquier, 2024) [5] .

Industrial Sources

Commercial lactase preparations are predominantly of microbial origin. Three sources dominate industrial application: Kluyveromyces lactis (optimal pH ~7.0; suitable for neutral milk pH), Kluyveromyces marxianus (thermotolerant; active at 40-50 °C), and Aspergillus oryzae/Aspergillus niger (optimal pH 4.0-5.0; better suited to acidic whey systems). Each source confers distinct biochemical properties relevant to the dairy substrate. Bacterial sources such as Bacillus spp. and Streptococcus spp. and recombinant cold-active lactases have also been explored, particularly for cold-pasteurized milk processing where low-temperature activity minimizes undesirable side reactions (Zhou et al., 2024) [20] . Key parameters governing lactase selection for immobilization include optimal temperature and pH, inhibition by product monosaccharides, molecular mass (which affects diffusion within immobilization matrices), and glycosylation pattern (which influences surface chemistry and immobilization efficiency). Genetic engineering approaches, including rational mutagenesis and directed evolution, are increasingly deployed to customize lactases for improved thermal stability, reduced product inhibition, and enhanced compatibility with specific immobilization chemistries.

Principles and Methods of Lactase Immobilization 3.1 Rationale for Immobilization

Enzyme immobilization is defined as the confinement of enzyme molecules to or within a support phase, restricting their free movement while retaining catalytic activity accessible to substrates in the continuous phase. For industrial lactase applications, the primary drivers for immobilization are:  Enzyme reusability across multiple operational cycles, reducing per-unit enzyme cost by 40-60%  Improved thermal and pH stability due to conformational rigidity conferred by multi-point attachment  Easy removal of the biocatalyst from the product, eliminating enzyme carry-over concerns  Compatibility with continuous flow reactor configurations enabling high-throughput processing  Potential for simultaneous lactose hydrolysis and GOS synthesis in a single reactor (Silva Lopes et al., 2025) [14] .

The optimal immobilization strategy for a given application must balance activity retention (activity of immobilized enzyme relative to free enzyme), binding strength (resistance to leakage), operational stability (activity halflife under process conditions), and practical considerations including cost, food-grade status of the support, and scaleup feasibility. [2] .

Immobilization

Covalent Binding

Covalent attachment involves the formation of stable covalent bonds between amino acid residues on the enzyme surface (typically lysine ε-amino groups, cysteine thiols, or carboxyl groups) and reactive functional groups on the support (epoxides, aldehydes, carbodiimides, isocyanates). Glutaraldehyde is a widely used bifunctional cross-linker that bridges enzyme amino groups to support amino groups. Eupergit C (epoxide-activated polyacrylamide beads) has been a commercial standard for dairy lactase immobilization. Covalent binding minimizes enzyme leakage and confers high operational stability, though partial activity loss (10-40%) is common due to active site involvement or conformational restriction. Multi-point covalent attachment substantially enhances thermal stability by reducing conformational flexibility (Alzahrani et al., 2024) [2] .

Entrapment

Entrapment involves physically enclosing lactase molecules within a polymeric matrix (hydrogel) or porous structure. Calcium alginate, carrageenan, polyacrylamide, and polyvinyl alcohol are the most studied matrices. The enzyme is not chemically modified, preserving activity, but diffusion of substrate to and from the enzyme is restricted, creating mass transfer limitations particularly for large substrates. Alginate bead entrapment is inexpensive and food-grade, making it attractive for scaled applications. Diffusion can be mitigated by using thin matrix films, optimizing bead size, and employing macroporous supports.

Encapsulation

Encapsulation confines lactase within a semi-permeable membrane (microcapsule or hollow fibre), allowing substrates and products to diffuse through while retaining the enzyme. Microencapsulation protects the enzyme against harsh environmental conditions and immune inactivation in oral delivery applications. In dairy processing, membrane reactors employing hollow fibres with immobilized lactase enable continuous hydrolysis of milk permeate streams. High production cost and membrane fouling remain primary challenges.

Cross-Linking (CLEAs)

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitating the enzyme from solution (using ammonium sulfate, acetone, or ethanol) followed by cross-linking with bifunctional reagents (typically glutaraldehyde), producing carrier-free, highly dense enzyme aggregates. CLEAs exhibit high enzyme loading per unit volume and eliminate the cost of support material. However, they can be mechanically fragile and difficult to recover from reactors.

Composite CLEAs incorporating magnetic nanoparticles (combi-CLEAs) address recovery issues by enabling magnetic separation. A magnetic CLEA of β-galactosidase demonstrated preservation of 80% activity after 10 reaction cycles with complete magnetic separation (Duan et al., 2022) [7] . [11] . Titanium dioxide nanoparticles have also been explored for lactase immobilization due to their photocatalytic properties and OH-rich surface.

Nanomaterials: The Next Generation

Nanomaterial-based supports have attracted intense research interest owing to their exceptionally high surface area-tovolume ratios, tunable surface chemistry, and functional

properties not available in macroscopic supports. Key nanomaterial categories include:

Magnetic iron oxide nanoparticles (Fe₃O₄ MNPs):

Fe₃O₄ MNPs functionalized with chitosan, amino groups, or polymer coatings enable covalent lactase immobilization and magnetic recovery from reaction media, eliminating the need for centrifugation or filtration. Recombinant βgalactosidase immobilized on histidine-nickelfunctionalized MNPs exhibited approximately 50-fold improvement in thermal stability relative to the free enzyme (Gennari et al., 2024) [8] . Chitosan-coated Fe₃O₄ nanocomposites immobilized with Aspergillus oryzae βgalactosidase showed 15.5% GOS yield with superior recyclability (Ansari & Damanhory, 2023) [3] .

Electrospun nanofibers:

Electrospinning generates fibrous polymer mats with high porosity (>80%), extremely large surface areas, and tunable fiber diameter (50 nm-10 μm). Lactase immobilized on electrospun poly (acrylonitrile) or poly (vinyl alcohol) nanofibers has demonstrated enhanced pH and temperature stability relative to free enzyme. A recent study by Stoilova et al. (2025) [15] reported a multi-enzyme system combining lactase with glucose oxidase and catalase on polymer nanofibers, which reduced glucose feedback inhibition and showed prolonged hydrolytic activity. The authors propose a "tea-bag" format for convenient low-cost lactose-free milk production, a concept with strong commercial potential.

Graphene oxide (GO) and carbon nanotubes:

GO provides rich surface functionality (epoxide, carboxyl, and hydroxyl groups) for covalent lactase attachment, while maintaining a two-dimensional support structure with superior electrical and mechanical properties. Multi-walled carbon nanotubes (MWCNTs) have been used for K. lactis β-galactosidase immobilization with enhanced stability and reusability (Ansari et al., 2013) [4] . Regulatory clearance for food contact remains an ongoing challenge for these materials.

Metal-organic frameworks (MOFs):

Porous crystalline materials composed of metal ions coordinated to organic ligands offer ultra-high surface areas, tunable pore chemistry, and unique adsorption properties. A nickel organic framework with amorphous-crystalline phase transition properties was recently employed for efficient βgalactosidase immobilization and magnetic recycling, demonstrating high GOS synthesis capacity (Addai et al., 2024) [1] . MOF-based enzyme supports represent a frontier area with potential for applications requiring precise control of the enzymatic microenvironment. [11] Nanomaterial Fe3O4 magnetic nanoparticles Magnetic recovery; large surface area Recombinant β-Gal: ~50× improved thermal stability; easy magnetic separation (Gennari et al., 2024) [8] Nanomaterial Graphene oxide Large surface area; functional groups; mechanical strength Enhanced lactase loading and catalytic efficiency; emerging food-grade applications Nanomaterial Electrospun nanofibers High porosity; large area; flexible design Multi-enzyme system (lactase + glucose oxidase): prolonged activity; "tea-bag" format potential (Stoilova et al., 2025) [15] 5. Industrial Applications in Lactose-Free Dairy Product Manufacturing

Lactose-Free Fluid Milk

The most significant commercial application of immobilized lactase is continuous lactose hydrolysis in fluid milk processing. In packed-bed reactors, milk is pumped through columns packed with immobilized lactase at controlled temperature (typically 5-10 °C for cold processing or 35-40 °C for higher-efficiency warm processing) and flow rate, achieving lactose reduction to below 0.1 g/100 mL, the threshold for lactose-free labelling in most regulatory frameworks. Absence of enzyme in the final product prevents post-processing sweetening, off-flavour development, and Maillard browning during heat treatment.

Operational reusability exceeding 30 cycles has been reported, reducing enzyme costs by approximately 40% compared to batch free-enzyme processes (Deng et al., 2024) [6] . Fluidized-bed reactors and magnetic-enzyme configurations represent important advances. Gennari et al. (2024) [8] demonstrated lactose hydrolysis in both packed-bed and fluidized-bed reactors using a recombinant β-galactosidase immobilized on magnetic core-shell capsules, achieving efficient lactose conversion in skim milk and cheese whey with exceptional operational stability. The magnetic support allowed straightforward enzyme recovery and recycling, addressing a key economic bottleneck of continuous processing.

Yogurt and Fermented Dairy Products

Pre-treatment of milk with immobilized lactase before fermentation reduces the initial lactose load, resulting in products with improved digestibility for lactose-intolerant consumers without compromising the fermentation process. Lower residual lactose at the end of fermentation reduces post-acidification during storage, improving product shelf life. Improved substrate availability for lactic acid bacteria due to glucose and galactose availability can accelerate fermentation kinetics, reducing manufacturing time by up to 25%. Enhanced sweetness of the fermented product may allow reduction in added sugar, aligning with clean-label formulation trends.

Cheese Production

In cheese manufacturing, lactase treatment of cheese milk or whey reduces residual lactose that would otherwise cause undesirable browning (Maillard reactions) during ripening or heat processing. Lactose reduction also benefits the flavour profile of aged cheeses, where incomplete lactose fermentation produces off-flavors. For certain soft cheeses intended for lactose-intolerant consumers, immobilized lactase allows targeted lactose reduction without altering the proteolytic or lipolytic processes central to flavor development. Lactase-treated cheese milk yields products suitable for the rapidly growing lactose-sensitive consumer segment without necessitating reformulation of established cheese recipes.

Ice Cream and Frozen Desserts

Lactose crystallization in ice cream, manifested as the sandy or gritty texture defect is a significant quality and consumer acceptance issue, particularly in high-solids formulations. Enzymatic hydrolysis of lactose prior to freezing converts it to glucose and galactose, which are more soluble and do not crystallize under typical frozen storage conditions. Immobilized lactase treatment of the ice cream mix before freezing effectively eliminates the lactose crystallization problem while enhancing perceived sweetness enabling partial reduction of added sucrose and aligning with sugar reduction targets. Improved overrun properties have also been reported, contributing to a lighter texture (Vallejo Garcia et al., 2024) [18] .

Infant Formula

Low-lactose and lactose-free infant formulas are essential for infants with congenital lactase deficiency or secondary lactase insufficiency following gastrointestinal disease. Immobilized lactase technology offers controlled lactose reduction while preserving the nutritional integrity of the base milk in particular, the heat-sensitive whey proteins and bioactive components that are susceptible to degradation during prolonged heat treatment. Regulatory requirements for infant formula production demand stringent enzyme-free final products, making immobilized systems which are removed prior to finishing inherently superior to freeenzyme batch processes.

Whey Processing and Galactooligosaccharide Production

Dairy whey generated in large volumes during cheese and casein manufacture contains 4-5% lactose and represents a significant effluent management challenge as well as a valorization opportunity. Immobilized β-galactosidase in packed-bed or membrane reactors enables continuous lactose hydrolysis of whey permeate, reducing biological oxygen demand and producing glucose-galactose syrups useful as fermentation substrates or sweeteners. At elevated lactose concentrations, the transglycosylation activity of βgalactosidase can be harnessed to synthesize galactooligosaccharides (GOSs), a bifidogenic prebiotic oligosaccharides with demonstrated benefits for gut microbiome composition and immune function. Fe₃O₄chitosan-immobilized β-galactosidase achieved GOS yields of up to 15.5% (w/v) with excellent recyclability in batch systems (Ansari & Damanhory, 2023) [3] . [8] 6. Recent Technological Advances

Nanotechnology-Enabled Immobilization

The convergence of nanotechnology with enzyme engineering has opened new avenues for lactase immobilization with performance characteristics unattainable with macroscopic supports. Nanomaterialbased supports offer surface areas of 100-1,000 m²/g, enabling high enzyme loading; reduced mass transfer limitations due to nanoscale particle size and unique functional properties (magnetism, photocatalysis, tunable surface chemistry). Multiple studies have demonstrated that nanostructured supports improve kinetic parameters notably, an increase in apparent Vmax and decrease in apparent Km for lactose compared to free enzyme under equivalent conditions reflecting both increased enzymes loading and facilitated substrate accessibility (Zhou et al., 2024) [20] .

Multi-Enzyme Cascade Systems

An emerging strategy is the co-immobilization of lactase with auxiliary enzymes on the same support to address cascade reactions or feedback inhibition. A critical limitation of lactase-catalyzed lactose hydrolysis is glucose feedback inhibition, as glucose accumulates, it competitively inhibits lactase, reducing the rate of hydrolysis. Stoilova et al. (2025) [15] addressed this by coimmobilizing lactase with glucose oxidase (GOx) and catalase on polymer nanofibers. GOx oxidizes glucose to gluconic acid, reducing the inhibitory glucose concentration and simultaneously generating H₂O₂, which is then detoxified by catalase. The multi-enzyme systemmaintained lactose hydrolysis activity substantially longer than single-enzyme systems, representing a significant advance in biocatalyst efficiency for continuous dairy processing.

Oriented Immobilization

Conventional immobilization relies on random attachment of the enzyme to the support, which may involve amino acid residues in or near the active site, causing partial or complete loss of catalytic function. Oriented immobilization which directs enzyme attachment away from the active site maximizes activity retention. His tag based oriented immobilization, exploiting specific coordination of the polyhistidine sequence with Ni²⁺functionalized supports, allows recombinant lactases to be immobilized in a defined orientation. Gennari et al. (2024) [8] demonstrated that Histagged recombinant β-galactosidase on Ni²⁺functionalized magnetic nanoparticles showed increased substrate affinity and approximately 50-fold improved thermal stability compared to the free enzyme, with excellent operational performance for lactose hydrolysis in skim milk and cheese whey.

Food-Grade and Sustainable Immobilization Systems

Industrial deployment of immobilized lactase in food systems requires that support materials meet food safety standards, particularly GRAS classification under FDA regulations or equivalent international frameworks. Recent research has focused on developing food-grade immobilization matrices that are safe for incidental food contact, including chitosan, food-grade alginate, carrageenan, gellan gum, and cellulose-based materials. Chalella Mazzocato and Jacquier (2024) [5] provided a comprehensive review of recent advances in food-grade immobilization systems, highlighting that safety, stability, and scalability must be simultaneously addressed for regulatory approval. Sponge-based materials are inexpensive, porous, and mechanically robust have recently been explored as novel supports for β-galactosidase crosslinked enzyme aggregates, offering high surface area and mass transfer efficiency at low cost (LWT, 2024) [12] .

Continuous Bioreactor Configurations

Packed-bed reactors (PBRs), fluidized-bed reactors (FBRs), and membrane bioreactors (MBRs) are the principal continuous configurations employed for immobilized lactase in dairy processing. PBRs offer high enzyme density and simple operation but are prone to fouling and pressure drop build-up with fat-containing dairy streams; they perform best with clarified whey permeate or skim milk. FBRs provide better mass transfer through fluid-particle contact and are more tolerant of particulate feeds. MBRs particularly hollow fibre configurations decouple enzyme retention from substrate flow, enabling simultaneous hydrolysis and product removal. Spiral-wound membrane bioreactors have been used for whey lactose hydrolysis with extended operational periods and consistent productivity (Vasileva et al., 2016) [19] . Recent computational approaches using computational fluid dynamics (CFD) modeling are improving reactor design by optimizing flow distribution and minimizing enzyme denaturation zones.

Artificial Intelligence and Process Optimization

Artificial intelligence (AI) and machine learning (ML) tools are beginning to transform immobilized enzyme process development. AI algorithms can predict optimal immobilization conditions (pH, temperature, cross-linker concentration, support chemistry) from prior experimental data, substantially reducing the experimental screening burden. In reactor operation, ML-based process control systems monitor substrate conversion in real time and dynamically adjust operating parameters like flow rate, temperature, pH to maintain target lactose hydrolysis levels while maximizing enzyme longevity. Protein engineering guided by ML is enabling the rational design of thermostable, inhibitor-tolerant lactases optimized for specific immobilization platforms, accelerating the development of next-generation biocatalysts (IntechOpen, 2025) [9] . These advances collectively indicate that AIintegrated immobilized enzyme systems represent the future of industrial lactose-free dairy production.

Challenges and Limitations

Despite significant progress across immobilization chemistry, support materials, and reactor design, several technical, economic, and regulatory challenges continue to impede the full potential of immobilized lactase technology at industrial scale. Mass transfer limitations remain the most fundamental technical constraint of immobilized enzyme systems. When lactase is entrapped within a polymeric matrix or confined within a porous support, diffusion of the large lactose molecule (MW 342 Da) to the enzyme active site may be rate-limiting, reducing the apparent catalytic rate. This limitation is particularly pronounced in alginate beads of conventional size (1-3 mm), and necessitates miniaturization of the matrix (nanoparticles, thin films, nanofibers) or adoption of open-pore architectures to restore adequate substrate accessibility. Activity loss during immobilization is inherent to most covalent and cross-linking strategies. When reactive groups are present near the active site of lactase, their modification during immobilization directly reduces catalytic capacity. Multi-point attachment enhances stability but amplifies this effect. Optimizing the orientation of immobilization through his-tag systems or site-directed chemical modification can minimize this problem but adds process complexity and cost. Economic viability at industrial scale remains a significant barrier, particularly for nanostructured and specialty supports. Graphene oxide, electrospun nanofibers, and MOF-based supports offer impressive performance in laboratory studies but their manufacturing costs are substantially higher than conventional Eupergit C or alginate systems. Demonstrating sufficient enzyme loading, reuse cycles, and operational stability to justify the premium cost is essential for commercial adoption. Regulatory compliance, particularly with regard to the use of nanomaterials in food contact applications, is an evolving challenge. Most regulatory frameworks have not yet established clear approval pathways for engineered nanomaterials in direct food processing applications, creating uncertainty for industrial developers. The European Food Safety Authority (EFSA) and the United States FDA are developing guidance frameworks, but approvals remain limited. This regulatory gap constrains the commercial deployment of the most technologically advanced immobilized systems despite their superior performance characteristics (Chalella Mazzocato & Jacquier, 2024) [5] .

Scale-up from laboratory to pilot to industrial scale frequently reveals engineering challenges including pressure drop, channeling, fouling by milk fat and proteins, microbial contamination, and inadequate heat management-that are not apparent at bench scale. Dairy streams containing fat and casein micelles are particularly challenging substrates for packed-bed systems, often requiring pre-clarification or membrane pre-filtration steps that add process complexity and cost.

Future Perspectives

The trajectory of immobilized lactase research points toward several convergent developments that are likely to define the next generation of lactose-free dairy processing technology. Smart and responsive immobilization materials supports that can modulate enzyme activity in response to external stimuli (temperature, pH, magnetic field, or light) represent an exciting frontier. Thermoresponsive polymer coatings (e.g., poly-N-isopropylacrylamide) can be engineered to alter porosity and substrate accessibility in response to temperature, enabling "on-demand" enzyme activation and inhibition that could be coupled with temperature cycling in process control systems. Magnetic enzyme recovery systems, incorporating superparamagnetic nanoparticle cores, are expected to become increasingly commercially viable as scalable nanoparticle synthesis methods reduce costs. These systems eliminate the need for centrifugation or filtration and enable continuous enzyme recycling in stirred-tank reactors, a configuration more amenable to whole milk processing than packed-bed systems (Gennari et al., 2024) [8] . Three-dimensional (3D) printing of enzyme reactor components using food-compatible materials opens opportunities for the fabrication of geometrically optimized reactor structures including customized flow channels, integrated heat exchangers, and enzyme-loaded structured packings that are unattainable through conventional manufacturing. 3D-printed alginate-lactase constructs have been demonstrated at laboratory scale, and scale-up is anticipated as 3D bioprinting technology matures. AI-assisted directed evolution of lactase is expected to accelerate the development of enzyme variants with dramatically improved stability, reduced product inhibition, and optimized compatibility with specific immobilization chemistries. By training ML models on sequence-functionstability datasets from large enzyme libraries, it is possible to predict beneficial mutations without exhaustive experimental screening. Such customized lactase variants could be engineered in tandem with their intended support material, creating a co-designed enzyme-support system optimized from the ground up for industrial dairy processing conditions. Integrated biotransformation platforms combining lactose hydrolysis with GOS synthesis, milk sugar isomerization (lactulose production), or valorization of hydrolysis products into high-value metabolites within a single immobilized multi-enzyme reactor represent the long-term vision for sustainable dairy bioprocessing converting a potential waste stream (lactose) into multiple value-added products simultaneously.

Conclusion

Immobilized lactase technology has matured from a laboratory concept to a cornerstone of industrial lactose-free dairy production, driven by the global imperative to address the digestive needs of the estimated 68-70% of adults worldwide affected by lactose malabsorption. By enabling enzyme reuse, improving operational stability, eliminating enzyme carry-over into products, and facilitating continuous manufacturing, immobilized lactase systems offer compelling economic and technical advantages over freeenzyme processes across the full spectrum of dairy applications from fluid milk and yogurt to cheese, ice cream, infant formula, and whey valorization. Advances in nanomaterial-based supports, including magnetic nanoparticles, electrospun nanofibers, and MOFbased carriers, are redefining the performance benchmarks for enzyme stability, loading, and recovery. Multi-enzyme cascade systems that address the glucose feedback inhibition problem, oriented immobilization strategies that maximize activity retention, and AI-guided process optimization are collectively advancing the technology toward its full industrial potential. Significant challenges remain principally in mass transfer efficiency, scale-up engineering, economic viability of advanced supports, and regulatory pathways for

References

1. Addai FP, Wu J, Liu Y, Ma X, Han J, Lin F, et al. Amorphous-crystalline phase transition and intrinsic magnetic property of nickel organic framework for easy immobilization and recycling of β-galactosidase. Int J Biol Macromol. 2024;254:127901.
2. Alzahrani F, Akanbi TO, Scarlett CJ, Aryee AN. The use of immobilised enzymes for lipid and dairy processing and their waste products: A review of current progress. Processes. 2024;12(4):634.
3. Ansari SA, Damanhory AA. Biotechnological application of Aspergillus oryzae β-galactosidase immobilized on glutaraldehyde modified zinc oxide nanoparticles. Heliyon. 2023;9:e13089.
4. Ansari SA, Satar R, Chibber S, Khan MJ. Enhanced stability of Kluyveromyces lactis β-galactosidase immobilized on glutaraldehyde modified multiwalled carbon nanotubes. J Mol Catal B Enzym. 2013;97:258263.
5. Chalella Mazzocato M, Jacquier JC. Recent advances and perspectives on food-grade immobilisation systems for enzymes. Foods. 2024;13(13):2127.
6. Deng Z, Deng Q, Li B, Li J, Jung S, Cho NJ, et al. Strategies for lactase immobilization and delivery to relieve lactose intolerance. Trends Food Sci Technol. 2024;143:104244.
7. Duan F, Sun T, Zhang J, Wang K, Wen Y, Lu L. Recent innovations in immobilization of βgalactosidases for industrial and therapeutic applications. Biotechnol Adv. 2022;61:108053.
8. Gennari A, Simon R, Renard G, et al. Lactose hydrolysis in packed-and fluidized-bed reactors using a recombinant β-galactosidase immobilized on magnetic core-shell capsules. Bioprocess Biosyst Eng. 2024;47:263-273.
9. IntechOpen. Optimizing enzymes for dairy processing: A protein engineering perspective. 2025.
10. Li A, Zheng J, Han X, Yang S, Cheng S, Zhao J, et al. Advances in low-lactose/lactose-free dairy products and their production. Foods. 2023;12(13):2553.
11. Li Z, Shi H, Yang Y, Liang H. Lactose-free milk preparation by immobilized lactase in glass microsphere bed reactor. Food Biophys. 2018;13:216224.
12. Immobilization of β-galactosidase within sponge: Hydrolysis of lactose in milk. LWT Food Sci Technol. 2024;215:115779.
13. Robescu MS, Bavaro T. A comprehensive guide to enzyme immobilization: All you need to know. Molecules. 2025;30(4):939.
14. Silva Lopes PH, Nelson DL, Damasceno SM. Enzyme immobilization: Advancements, techniques, and industrial applications. Curr Enzyme Inhib. 2025.
15. Stoilova O, Ignatova M, Manolova N. Immobilization of enzymes for synergy in polymers to produce lactose free milk. Sci Rep. 2025;15:17402.
16. Storhaug CL, Fosse SK, Fadnes LT. Country, regional, and global estimates for lactose malabsorption in adults: A systematic review and meta-analysis. Lancet Gastroenterol Hepatol. 2017;2(10):738-746.
17. The Business Research Company. Lactose-free dairy global market report. 2025.
18. Vallejo García JLAU, Cutillo Foraster A, Arnáiz Alonso AAU, Vallejos Calzada SAU, García Pérez JMAU, Muñoz Santamaría MAA, et al. Hydrolysis of lactose: Conventional techniques and enzyme immobilization strategies on polymeric supports. In: Milk Proteins-Technological Innovations, Nutrition, Sustainability and Novel Applications. 2024. p. 45-89.
19. Vasileva N, Ivanov Y, Damyanova S, Kostova I, Godjevargova T. Hydrolysis of whey lactose by immobilized β-galactosidase in a bioreactor with a spirally wound membrane. Int J Biol Macromol. 2016;82:339-346.
20. Zhou M, Chen X, Zheng J, Li S. β-Galactosidase: Insights into source variability, genetic engineering, immobilisation and diverse applications in food, industry and medicine. Int J Dairy Technol. 2024;77(4):812-835.