Biocatalytic plate based on immobilized polyelectrolyte microcapsules with encapsulated beta-galactosidase for lactose removal

Abstract

FIELD: food industry.SUBSTANCE: invention relates to biotechnological methods for processing dairy products and concerns methods for the enzymatic hydrolysis of lactose using immobilized enzyme systems. The invention is a biocatalytic plate for removing lactose, designed to remove lactose from dairy products by hydrolysis with β-galactosidase. The biocatalytic plate is a polyelectrolyte microcapsule obtained by the method of alternate adsorption of oppositely charged polyelectrolytes with β-galactosidase and fixed on a glass surface.EFFECT: stable, safe and reusable plate for lactose hydrolysis.3 cl, 10 dwg, 1 tbl

Description

[0001] Technical field:

[0002] The invention relates to the food industry, in particular to biotechnological methods for processing dairy products, and concerns methods for the enzymatic hydrolysis of lactose using immobilized enzyme systems.

[0003] Technique level:

[0004] Milk and dairy products are a vital component of a balanced diet due to their high nutritional value, as they contain high-quality proteins, vitamins, minerals, bioactive peptides, prebiotics, and the unique milk fat globule membrane, which is involved in lipid metabolism, neuronal development, and the prevention of diseases such as osteoporosis [1,2]. Although these components are crucial for physiological functions, approximately 68% of the world's adult population suffers from lactose malabsorption [3]. The clinical manifestations of this condition arise from the inability to hydrolyze the disaccharide lactose into monosaccharides (glucose and galactose) in the small intestine, resulting in symptoms such as bloating, diarrhea, and pain [4,5].Current therapeutic strategies typically involve dietary modifications, including limiting or eliminating dairy products, consuming lactose-free alternatives, or using lactase enzyme supplements to improve digestion [6–8]. However, the adverse health effects associated with dairy exclusion [9], coupled with the limited effectiveness of existing enzyme replacement therapies [8], highlight the potential of lactose-free products as a promising alternative for improving nutritional status and minimizing gastrointestinal symptoms.

[0005] In the production of lactose-free products, which are produced using technologies aimed at the preliminary removal of lactose from milk, a number of methods are used: physical (ultrafiltration

[10] , membrane separation

[11] ), chemical (acid hydrolysis

[12] ) and biocatalytic

[13] . Physical methods, despite their environmental safety, require significant energy costs and can reduce the content of low molecular weight nutrients

[10] . Chemical methods, on the other hand, are associated with the risk of protein denaturation, changes in the organoleptic properties of the product and the need to neutralize residual reagents

[13] . Biocatalytic methods using β-galactosidase demonstrate high specificity and preservation of organoleptic characteristics.However, the high cost of recombinant enzymes, their susceptibility to denaturation, and the inability to reuse β-galactosidase significantly increase production costs (US$1.23 billion

[14] ), limiting their industrial scalability.

[0006] To overcome these limitations, methods for β-galactosidase immobilization aimed at stabilizing the enzymatic activity and ensuring the possibility of its repeated use are being actively studied. The most promising technologies are covalent binding to carriers [15,16], adsorption on porous matrices [17,18] and encapsulation in gel systems

[19] . Each approach has unique advantages, such as increased thermal stability, increased duration of catalytic cycles and reduced product contamination [20,21]. However, the search for optimal carriers and immobilization methods remains an important task, since each system has inherent disadvantages: low long-term matrix stability, susceptibility to degradation under the influence of temperature and pH [22,23]. The use of polyelectrolyte microcapsules (PEMC) for enzyme immobilization can eliminate the above-mentioned limitations.Polyelectrolyte microcapsules are modular spherical structures formed by layer-by-layer (LBL) assembly of oppositely charged polyelectrolytes on a solid or porous core, which subsequently dissolves, leaving a hollow capsule

[24] . These microcapsules are widely used for encapsulation of proteins and enzymes due to their ability to protect sensitive substances and provide stability under various environmental conditions [25–27]. We previously demonstrated the feasibility of encapsulating β-galactosidase in polyelectrolyte microcapsules

[28] . This study showed that encapsulated β-galactosidase retained 10% of its activity with reduced substrate affinity (Km = 96 mM versus 36 mM for the free enzyme) and was stable to acidity for 2 h (residual enzymatic activity 18% pH 2), and also reduced the negative impact of ionic strength.However, the current use of polyelectrolyte microcapsules containing encapsulated β-galactosidase is impractical, as their free movement in solution raises concerns about potential contamination of the final product (e.g., milk or dairy products) with residual capsule particles. To address this limitation, our study aimed to immobilize these enzyme-loaded microcapsules onto a solid substrate and evaluate the effectiveness of the immobilized system for removing lactose from the incubation medium.

[0007] The aim of this study was to develop a stable and safe system for lactose removal from dairy products by immobilizing polyelectrolyte microcapsules containing encapsulated β-galactosidase onto a solid substrate. The study also evaluated the system's performance under various conditions (temperature, number of microcapsules, surface area), its reusability, and long-term stability.

[0008] Disclosure of invention:

[0009] To create a reusable system for removing lactose from dairy products, it is necessary to immobilize β-galactosidase (β-Gal) encapsulated in polyelectrolyte microcapsules (PEC) on a solid surface. A modified method of charge-controlled adhesion of microcapsules to surfaces structured by a layer-by-layer method was used as a method for immobilizing PMC [31-34]. A schematic illustration of this method is shown in Figure 1 (Immobilization of β-galactosidase encapsulated in polyelectrolyte microcapsules (PEC) on a solid surface). A glass plate (GP) (stage I) with a working surface area of ​​2 cm was sequentially coated with alternating layers of polycation and polyanion by adsorption (stages II and III), followed by the application of a layer of polyelectrolyte microcapsules containing encapsulated β-galactosidase (stage IV). At the final stage, additional layers of polycations and polyanions were applied to the capsules (stage V).Polystyrene sulfonate (PSS) was used as the polyanion, as described in the method above. However, either polyallylamine (PAA) or polydiallyldimethylammonium chloride (PDADMAC) was used as the polycation. Unlike PAA, PDADMAC is classified as a strong polyelectrolyte. When interacting with PSS (which is also a strong polyelectrolyte), their electrostatic charges neutralize each other, which can affect the stability of the system and its catalytic performance. Therefore, this study also included a comparison of all investigated characteristics for the two variants: SP-β-Gal. ПДАДМАХ and SP-β-Gal ПАА , where PDADMAC or PAA was used as a polycation for capsule immobilization. The image of a glass plate coated with PLA is shown in Figure 2 (Image of glass plates of SP-β-Gal ПДАДМАХ (A) and SP-β-Gal РАА(B) coated with PLA encapsulating β-galactosidase. A: PDADMAC was used as a polycation for capsule immobilization; B: PAA was used as a polycation for capsule immobilization)

[0010] To evaluate the differences in lactose hydrolysis efficiency between SP-β-Gal systems ПДАДМАХ and SP-β-Gal РАН The samples were incubated for 24 hours in a lactose solution (concentration: 50 mg / ml). The resulting glucose was quantitatively analyzed using a colorimetric method. The percentage of hydrolyzed lactose relative to the initial amount was calculated based on the glucose yield, and the results are presented in Figure 3 (The amount of hydrolyzed lactose in SP-β-Gal systems ПДАДМАХ (A) and SP-β-Gal ПАА (B))

[0011] As shown in Figure 3, the amount of hydrolyzed lactose does not differ between the samples studied, regardless of the type of system used. Therefore, it can be concluded that both variants, SP-β-GalПДАДМАХ and SP-β-Gal ПАА , can be used for lactose hydrolysis.

[0012] In a further study, two modifications of the capsule immobilization method on a glass plate were used: one at room temperature (22°C, method 1) and the other in a dry heating chamber at 40°C (method 2). The use of these two methods can affect the efficiency of lactose hydrolysis in the presented systems. The immobilization procedure involves the application of layers of polyelectrolytes and microcapsules to the surface of a glass substrate, followed by evaporation of the solvent (water). Elevated temperature accelerates the evaporation of the solvent, but can cause denaturation of the encapsulated β-galactosidase enzyme, reducing its catalytic activity. To assess the thermal stability of the system, a comparative study was conducted on the effect of two microcapsule immobilization methods on their ability to catalyze lactose hydrolysis. The results are shown in Figure 4 (The amount of lactose hydrolyzed by the SP-β-Gal systems ПДАДМАХ (A) and SP-β-Gal ПАА(B) depending on the temperature regime)

[0013] As shown in Figure 4, the temperature regime during the PLA immobilization procedure does not significantly affect the amount of hydrolyzed lactose. To confirm this conclusion, all subsequent studies were conducted at these temperature regimes (22°C and 40°C), which also revealed no correlation between the immobilization temperature and the catalytic activity of the system.

[0014] The next step was to investigate the effect of PLA concentration on the efficiency of lactose hydrolysis. Potential application of this method for lactose removal from dairy products will require scale-up of the process, which will necessitate further investigation of this parameter. For this purpose, a multilayer PLA deposition method was developed, as shown in Figure 5 (Methods for multilayer deposition of microcapsules on a glass plate).

[0015] Each layer of microcapsules was separated by an intermediate PSS layer, which provided electrostatic stabilization by compensating for the positive charge on the surface of polyelectrolyte microcapsules formed during the formation of their polyelectrolyte shell

[35] . The number of applied PMC layers was varied to analyze the dependence of the catalytic activity of the system on the number of immobilized capsules. The detailed methodology is described in the Materials and Methods section. The results demonstrating the effect of the amount of PMC on the rate of lactose hydrolysis are presented in Figure 6 (Figure 6. The amount of lactose hydrolyzed by SP-β-Gal systems ПДАДМАХ (A) and SP-β-Gal ПАА (B) depending on the number of microcapsules)

[0016] As shown in Figure 6, the amount of hydrolyzed lactose differs between the SP-β-Gal systems ПДАДМАХ and SP-β-Gal ПАА depending on the number of microcapsules. For SP-β-Gal ПААA gradual increase in the hydrolyzed lactose content was observed with increasing number of immobilized capsules. However, the results show that this increase is not proportional to the increase in the number of capsules and does not differ significantly. This effect may arise due to decreased permeability of the substrate / enzyme reaction product to deeper layers of microcapsules, or the initial diffusion rate of the substrate may be insufficient, leading to complete hydrolysis in the upper layers of the PLA.

[0017] On the contrary, for SP-β-Gal ПДАДМАХThe amount of hydrolyzed lactose does not increase with the amount of immobilized PLA, remaining constant. This suggests that only the upper layer of PLA is active. This behavior may be due to the presence of PDADMAC in the outer layer of the polyelectrolyte coating. The high charge density of PDADMAC and its diffusion mobility ensure the formation of dense layer-by-layer (LbL) membranes in combination with PSS. Excess charges of PDADMAC generate a positively charged surface, enhancing Donnan exclusion and reducing permeability

[36] .

[0018] To evaluate the scalability of this technology, the effect of working surface area on the efficiency of lactose hydrolysis by SP-β-Gal systems was investigated. ПДАДМАХ and SP-β-Gal ПАА Samples were incubated for 24 h in lactose solution (50 mg / ml) using systems with a working surface area of ​​2 cm 2 , 4 cm 2 and 8 cm 2These results are presented in Figure 7 (The amount of hydrolyzed lactose by SP-β-Gal systems ПДАДМАХ (A) and SP-β-Gal ПАА (B) depending on the area of ​​the working surface)

[0019] As shown in Figure 7, increasing the working surface area leads to an increase in the amount of hydrolyzed lactose. Therefore, it can be concluded that increasing the working surface area improves the efficiency of both systems, SP-β-Gal ПДАДМАХ and SP-β-Gal РАН . For SP-β-Gal ПАА Increasing the number of microcapsules immobilized on the surface using the method described above also proves effective.

[0020] To develop a reusable lactose removal system based on SP-β-Gal ПДАДМАХ and SP-β-Gall ПАА For dairy products, it is important to evaluate their reuse potential. For this purpose, the same SP-β-Gal tablets were used for lactose hydrolysis. ПДАДМАХ and SP-β-Gal РАН, with the test solution replaced every 24 hours with an identical lactose solution (concentration: 50 mg / ml). These results will be presented in Figure 8 (Amount of hydrolyzed lactose in SP-β-Gal systems ПДАДМАХ (A) and SP-β-Gal ПАА (B) depending on the number of applications)

[0021] As shown in Figure 8, both systems can withstand at least 4 applications with a total incubation time of approximately 120 h. This number of applications represents a fivefold increase in reusability compared to the free enzyme currently used in industrial production

[11] .

[0022] The final stage of evaluating the effectiveness of these systems involved determining the degree of lactose hydrolysis over time without replacing the incubation solution. For this purpose, SP-β-Gal ПАА incubated in a lactose solution (50 mg / ml). Since the SP-β-Gal system ПААcan be modified by increasing the number of microcapsule layers (thereby increasing its efficiency), in the last experiment this system was used exclusively. These results are presented in Figure 9 (The amount of hydrolyzed lactose using the SP-β-Gal system ПАА (depending on the incubation time)

[0023] As shown in Figure 9, a significant decrease in enzyme activity is observed after 150 hours of incubation, and the amount of hydrolyzed lactose stops increasing over time. During this period, SP-β-Gal РАН (8 million PMC) hydrolyzed approximately 50% of lactose. According to our previous study of the catalytic properties of encapsulated β-galactosidase

[28] , at this concentration of lactose (70 mM) in solution, the enzyme activity decreases more than threefold, with almost complete loss of activity observed at concentrations of approximately 20 mM.

[0024] In the current study, the activity of SP-β-Gal ПААmay be even lower, and a decrease in activity may occur at higher substrate concentrations, since additional polyelectrolyte layers on top of the PLA may reduce permeability, artificially increasing the Km of the enzymatic system

[37] . For the SP-β-Gal system ПАА (32 million PMC) also shows a decrease in activity with increasing incubation time. However, in this case, the enzyme concentration is four times higher, which is sufficient for the production of lactose-free (100 μg / ml) or low-lactose (10 mg / ml) dairy products

[38] .

[0025] Further studies were conducted on the system's ability to hydrolyze lactose in cow's milk. For this purpose, a plate coated with β-galactosidase (SP-β-Gal) ПАА), encapsulated in polyelectrolyte microcapsules, was placed in a solution of ultra-pasteurized cow's milk with 3.2% fat content and an initial lactose content of 4.7%. The mixture was incubated for 2 hours at room temperature. The amount of hydrolyzed lactose was determined by the accumulated reaction product, glucose. Glucose was determined by the standard glucose determination method using the commercial KT Glucose kit from Diakon. The results of the study are shown in Fig. 10 (The amount of hydrolyzed lactose using the SP-β-Gal system ПАА in milk (1) and in an aqueous solution of glucose (2))

[0026] The developed system for immobilizing β-galactosidase in polyelectrolyte microcapsules on glass plates demonstrates competitive advantages, despite the initially higher requirements for enzyme quantity and hydrolysis time compared to free lactase. In modern industrial processes, ~4000 U of free enzyme are used to completely hydrolyze lactose in 1 liter of milk within 12-24 hours. Our SP-β-Gal system РАНUsing 32 million PMCs (equivalent to ~480,000 U per 1 L), it takes 150 hours to achieve lactose-free standards. Although this implies a 20-fold increase in enzyme quantity and a 6-fold increase in incubation time, the key advantage lies in the system's reusability and exceptional stability. Unlike free enzyme, which is discarded after a single use, immobilized PMCs retain activity for at least 120 hours of continuous operation (5 cycles), and according to literature data, up to 6 months, significantly reducing operating costs. When scaled up to process 1 ton of milk, the predicted enzyme savings will be 8.3 times (480,000 U versus 4 million U for free enzymes).

[0027] The obtained data show that during the first two hours, the activity of encapsulated β-galactosidase (lactase) immobilized on a plate in milk does not differ from the activity in a lactose solution.

[0028] Comparison with modern analogues (Table 1) confirms the advantage of this enzyme immobilization system in terms of long-term stability.

[0029]

[0030] Under normalized conditions (volume, temperature), the hydrolysis efficiency is comparable to analogs, but the SP-β-Gal system РАН , surpasses them in terms of stability of operation (>120 h without loss of activity). In this study, no decrease in the activity of SP-β-Gal was observed throughout the experiment. ПАА In addition, according to literary sources, enzymes encapsulated in polyelectrolyte microcapsules can retain activity for at least 6 months [43,44].

[0031] Implementation of the invention:

[0032] To create a biocatalytic plate based on polyelectrolyte microcapsules containing β-galactosidase, CaCO3 microspherulites were formed by mixing 0.33 M Na2CO3 and 0.33 M CaCl2 with constant stirring for 30 s in the presence of a β-galactosidase solution. The resulting suspension was maintained until the formed particles completely precipitated. The microspherulite maturation process was monitored using a light microscope. The supernatant was then decanted, the precipitate was washed with water, and used to prepare the PMC. The average microparticle diameter was 4.5 ± 1 μm.

[0033] Polyelectrolyte microcapsules were obtained by layer-by-layer adsorption of negatively or positively charged polyelectrolytes on CaCO3 microspherulites, followed by dissolution of the CaCO3. Layer-by-layer adsorption of PAA and PSS on the surface of CaCO3 microspherulites was carried out in polyelectrolyte solutions (concentration 2 mg / ml + 0.5 M NaCl). After each adsorption, CaCO3 particles with adsorbed polyelectrolytes were washed three times with a 0.5 M NaCl solution, which was necessary to remove unadsorbed polymer molecules. The particles were separated from the supernatant by centrifugation. After applying the required number of layers, the carbonate cores were dissolved in a 0.2 M EDTA solution for 12 h. The resulting capsules were washed three times with water to remove core decomposition products. Microcapsules with an average diameter of 4.5±1 μm were obtained. The size and number of microcapsules were measured by dynamic light scattering using a Zetasizer nano ZS instrument (Malvern, UK).

[0034] Then a glass plate was prepared by covering the non-working surface with several layers of paraffin film to determine the working area of ​​4 cm 2 (2 cm each) 2on each side), unless otherwise stated in the study. To immobilize polyelectrolyte microcapsules (PEMCs) on a glass plate, polyelectrolytes were sequentially adsorbed onto the surface: either RAS / PSS / RAN / PSS or PDADMAC / PSS / PDADMAC / PSS. For this, the working surface of the glass plate was immersed in a polyelectrolyte solution (2 mg / mL in 0.5 M NaCl), followed by evaporation of the solvent. This procedure was repeated with subsequent polyelectrolytes until the fourth layer (PSS) was deposited. After the fourth layer, PMC (4 million particles) was deposited on the surface, and the solvent was allowed to evaporate. The plate was then turned over, and another 4 million PMCs were deposited on the opposite side. After deposition of PMC, additional polyelectrolyte layers were deposited: either PSS / RAA / PSS / RAA or PSS / PDADMAC / PSS / PDADMAC. This resulted in two different systems:

[0035] 1. PAA (1st layer) / PSS / PAA / PSS / PML / PSS / PAA / PSS / PAA - designated as SP-β-Gal ПАА .

[0036] 2. PDADMAC (1st layer) / PSS / PDADMACH / PSS / PMC / PDADMACH / PSS / PDADMACH / PSS - designated as SP-β-Gal ПДАДМАХ .

[0037] The evaporation of the solvent (water) was carried out using two methods:

[0038] • Method 1: Evaporation at room temperature (22°C) with the plate fixed in the ambient air (24-28 hours).

[0039] • Method 2: Evaporation at 40°C in a drying oven (4-6 hours). Measurement of the hydrolysis efficiency of SP-β-Gal ПДАДМАХ and SP-β-Gal ППА . Efficiency of hydrolysis of SP-β-Gal ПДАДМАХ and SP-β-Gal ПАА was measured by determining the concentration of glucose in the solution using the glucose oxidase method after incubation with SP-β-Gal ПДАДМАХ and SP-β-Gal ПАА (working surface area: 4 cm 2, unless otherwise stated) for 24 h in a lactose solution (pH 6, T = 22°C). Glucose oxidase catalyzes the oxidation of D-glucose with atmospheric oxygen, forming equimolar amounts of gluconolactone and hydrogen peroxide (pH 7, T = 22°C)

[30] . Peroxidase catalyzes the oxidation of chromogenic substrates with hydrogen peroxide in the presence of phenol, forming a colored compound (pH 7, T = 22°C). The color intensity is directly proportional to the concentration of glucose in the sample and is measured photometrically at a wavelength of 500 nm (480-520 nm).

[0040] To carry out the reaction, 10 μl of the sample was added to 1 ml of the working solution containing: potassium dihydrogen phosphate (KH2PO4) - 250 mmol / L, phenol - 5 mmol / L, 4-aminoantipyrine - 0.5 mmol / L, glucose oxidase - 10,000 U / L and peroxidase - 1000 U / L. The samples were mixed and incubated at room temperature for 20 min. The optical density of the sample was measured compared to the control sample at a wavelength of 500 nm (480-520 nm) using a cuvette with an optical path length of 10 mm.

[0041] Determination of β-galactosidase concentration

[0042] The amount of encapsulated and released β-galactosidase was determined using a spectrophotometric method. The absorbance of β-galactosidase at 280 nm was measured in the supernatant after CaCO3 precipitation (during the preparation of polyelectrolyte microcapsules by co-precipitation (Section 3.2)), in the supernatant after PLA precipitation during the experiments to determine the amount of released enzyme, and in the supernatant after PLA precipitation in the final step of β-galactosidase encapsulation by adsorption. A UNICO 2804 spectrophotometer (Suite E Dayton, NJ, USA) was used for the measurements. The amount of encapsulated protein is 1300 units (U) of enzyme in 5*10 7 polyelectrolyte microcapsules. This result is consistent with the amount of encapsulated enzyme that was encapsulated in our previous study

[28] .

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Claims

1. A biocatalytic plate based on immobilized polyelectrolyte microcapsules with encapsulated β-galactosidase for removing lactose, consisting of polyelectrolyte microcapsules based on polyallylamine and polystyrene sulfonate, obtained by the method of alternate adsorption of oppositely charged polyelectrolytes with β-galactosidase included in them and immobilized on glass plates.

2. A biocatalytic plate according to claim 1, characterized in that the polyelectrolyte microcapsules are stabilized on the surface of the plate by alternating layers of PAA and PSS.

3. A biocatalytic plate according to paragraphs 1 and 2, characterized in that several layers of polyelectrolyte microcapsules are applied to the surface of the glass plate.

Images