An immobilized dual-enzyme complex and its preparation method, low-lactose milk and low-lactose dairy products

Abstract

This invention relates to the field of bioengineering technology, and more particularly to an immobilized dual-enzyme complex and its preparation method, low-lactose milk, and low-lactose dairy products. It comprises: chitosan nanoparticles; lactase covalently linked to the surface of the chitosan nanoparticles using genipin as a cross-linking agent; and glucose oxidase covalently linked to the surface of the chitosan nanoparticles using genipin as a cross-linking agent; the lactase and glucose oxidase are respectively immobilized on the surface of different batches of chitosan nanoparticles. This invention successfully combines lactose degradation with the natural antibacterial system in milk, improving enzyme stability and reusability, simplifying the production process of low-lactose dairy products, and enhancing the antibacterial effect. The immobilized dual-enzyme system constructed by this invention can efficiently prepare low-lactose milk and extend its shelf life. It is green, safe, and reusable, meeting the needs of the food industry development and has significant application value in satisfying the consumption needs of lactose-intolerant populations and promoting the upgrading of the dairy industry.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an immobilized dual-enzyme complex and its preparation method, as well as low-lactose milk and low-lactose dairy products. Background Technology

[0002] Milk is rich in high-quality protein, milk fat, vitamins, and calcium, making it an important source of nutrition for the human body. The average lactose content in milk is approximately 4.8%. Lactose promotes the growth and reproduction of beneficial microorganisms in the intestines, producing organic acids and maintaining a suitable acidic environment. This inhibits the growth of putrefactive bacteria and alkaliphilic microorganisms, reducing the adverse effects of abnormal intestinal fermentation. However, some people have insufficient lactase activity in their intestines, preventing the effective hydrolysis of ingested lactose into glucose and galactose. The undigested lactose is fermented by microorganisms in the intestines, causing symptoms such as bloating, abdominal pain, and diarrhea—a condition known as lactose intolerance. This condition limits the adequate intake of milk and its products for a significant proportion of consumers; therefore, developing low-lactose milk products is of great importance.

[0003] Currently, enzymatic hydrolysis technology is widely used in the production of low-lactose milk. Figure 1 The core of low-lactose milk production is the use of lactase to hydrolyze lactose in milk into glucose and galactose, which are easily absorbed by the human body. Current industrial production processes for low-lactose milk mainly include two routes: aseptic and batch processing. The aseptic method involves ultra-high temperature sterilization of milk, followed by the addition of sterile filtered lactase. After bottling, the lactase gradually hydrolyzes the lactose during the shelf life. The batch method involves adding lactase to the milk, slowly incubating it at low temperatures (usually 4-8°C) with stirring for 24 hours, followed by pasteurization and bottling.

[0004] However, both methods have significant technical shortcomings. The aseptic method requires stringent requirements for production equipment and operating environment, necessitating a professional aseptic filling system and high-efficiency filtration devices, resulting in high equipment investment and operating costs. Furthermore, this method demands highly skilled operators to prevent microbial contamination during enzyme addition. More importantly, after aseptic treatment, lactase remains active in the milk. During storage and transportation, seasonal temperature fluctuations make precise control of lactose hydrolysis difficult, leading to inconsistent lactose residue levels across different batches and poor product quality stability. While the batch method eliminates the need for aseptic filling equipment, the milk has not yet undergone sterilization during the low-temperature incubation stage, requiring continuous low temperatures to prevent microbial growth. Simultaneously, continuous and slow stirring is necessary to prevent milk fat from rising to the surface. This process is lengthy and energy-intensive. To ensure low lactose levels are achieved within limited time and low-temperature conditions, higher doses of lactase are required, increasing enzyme preparation costs.

[0005] In addition to the aforementioned technological shortcomings, current low-lactose milk production generally employs free lactase for hydrolysis. Free lactase has inherent drawbacks such as poor thermal stability and susceptibility to degradation by endogenous proteases in milk. Furthermore, it cannot be recovered and reused after the reaction, further increasing production costs. Moreover, low-lactose milk is rich in nutrients, providing an excellent substrate for microbial growth and reproduction. Even after pasteurization, its shelf life remains limited, and microbial spoilage remains a significant factor affecting product safety and shelf life. Therefore, in the preparation of low-lactose milk, how to achieve efficient lactose hydrolysis while effectively controlling microbial contamination and extending product shelf life is a pressing technical challenge in the dairy processing industry.

[0006] To address the aforementioned issues, researchers have proposed using immobilized lactase to replace free enzymes, thereby enabling enzyme reuse and reducing production costs. As early as the 1980s, some researchers attempted to produce low-lactose milk using immobilized lactase, even conducting pilot-scale trials. However, because milk is a natural microbial culture medium, immobilized enzymes are highly susceptible to microbial contamination during reuse, leading to bacterial growth in the immobilized enzyme bed. This severely impacts the enzyme's reusability and product safety, preventing the industrial-scale application of this technology. Currently, research on lactase immobilization primarily focuses on characterizing the enzymatic properties before and after immobilization, or using lactase as a model enzyme to evaluate the feasibility of novel immobilization methods. Systematic research and effective solutions are scarce regarding the microbial stability issues faced by immobilized lactase in actual milk hydrolysis systems, and how to simultaneously achieve antibacterial and preservative effects during enzymatic hydrolysis.

[0007] Therefore, developing a low-lactose milk preparation technology that can simultaneously achieve efficient lactose hydrolysis, enzyme reuse, and effective inhibition of harmful microorganisms is of great significance for simplifying production processes, reducing production costs, extending product shelf life, and promoting technological progress in the dairy industry. Summary of the Invention

[0008] The purpose of this invention is to provide an immobilized dual-enzyme complex and its preparation method, as well as low-lactose milk and low-lactose dairy products. Since milk itself is an excellent microbial culture medium, immobilized enzymes are easily contaminated by microorganisms during repeated use, preventing the industrial-scale application of this method. Currently, research on lactase immobilization mainly focuses on characterizing the enzymatic properties before and after immobilization, or using lactase as a model enzyme to evaluate the effectiveness of immobilization methods. There is still no systematic research on how to overcome the microbial stability problem of immobilized lactase in actual production.

[0009] This invention constructs a dual-enzyme cascade system by immobilizing lactase and glucose oxidase separately. While lactose is hydrolyzed to produce glucose, glucose oxidase partially oxidizes glucose to gluconic acid and slowly releases trace amounts of H2O2, thereby activating the endogenous lactoperoxidase antibacterial system in cow's milk and achieving the unity of efficient lactose hydrolysis and synergistic inhibition of microorganisms.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an immobilized dual-enzyme complex comprising: chitosan nanoparticles; lactase covalently linked to the surface of the chitosan nanoparticles using genipin as a crosslinking agent; and glucose oxidase covalently linked to the surface of the chitosan nanoparticles using genipin as a crosslinking agent; wherein the lactase and the glucose oxidase are respectively immobilized on the surface of different batches of chitosan nanoparticles.

[0011] Preferably, the chitosan nanoparticles are formed by the self-assembly of chitosan through genipin crosslinking induced by sodium sulfate.

[0012] Preferably, the method for preparing the immobilized dual-enzyme complex includes the following steps: (1) Chitosan was dissolved in acetic acid solution, sodium sulfate solution was added under ultrasonic conditions, and the precipitate was collected by centrifugation after stirring to obtain chitosan nanoparticles; (2) The chitosan nanoparticles were placed in a genipin solution and activated at 50-70°C for 0.5-2 hours. Unreacted genipin was washed away to obtain activated chitosan nanoparticles. (3) The activated chitosan nanoparticles were mixed with lactase solution and glucose oxidase solution respectively, and incubated at 20~30℃ for 6~18h. The immobilized enzyme particles were collected by centrifugation and washed to obtain immobilized lactase and immobilized glucose oxidase respectively. (4) The immobilized lactase and the immobilized glucose oxidase are mixed to obtain the immobilized dual-enzyme complex.

[0013] This invention provides the application of the immobilized dual-enzyme complex in the preparation of low-lactose dairy products.

[0014] This invention provides a low-lactose milk, which is prepared by treating milk with the aforementioned immobilized dual-enzyme complex.

[0015] This invention provides a method for preparing low-lactose milk, comprising the following steps: adding the immobilized dual-enzyme complex to milk for enzymatic hydrolysis; separating and collecting the hydrolyzed milk to obtain low-lactose milk.

[0016] Preferably, the enzymatic hydrolysis reaction is carried out at a temperature of 20~30℃ for a reaction time of 6~24h; the amount of the immobilized dual-enzyme complex added is: the final concentration of immobilized lactase is 0.1~0.3 mg / mL (based on chitosan nanoparticles), and the final concentration of immobilized glucose oxidase is 0.005~0.02 mg / mL (based on chitosan nanoparticles).

[0017] This invention provides a low-lactose dairy product, which is prepared using the aforementioned low-lactose milk as a base material.

[0018] Preferably, the low-lactose dairy product is low-lactose yogurt, low-lactose cheese, low-lactose whipping cream, or low-lactose butter.

[0019] This invention provides a method for preparing low-lactose yogurt, comprising the following steps: sterilizing the low-lactose milk and cooling it to the fermentation temperature; inoculating it with lactic acid bacteria for fermentation; and cooling and ripening it after fermentation to obtain low-lactose yogurt.

[0020] In the technical solution of this invention, chitosan nanoparticles possess advantages such as non-toxicity, good biocompatibility, simple preparation, and a surface rich in amino groups, making them an ideal carrier for enzyme covalent immobilization. Cow's milk itself contains a natural antibacterial system composed of lactoperoxidase, thiocyanate, and hydrogen peroxide; however, the natural concentration of H2O2 is insufficient to activate this system. Directly adding H2O2 can easily lead to excessively high local concentrations, causing lactoperoxidase inactivation and oxidation of proteins and fats in milk, affecting sensory quality and safety. Furthermore, H2O2 itself is unstable, posing safety hazards during storage and transportation. Therefore, using an enzymatic reaction to continuously and controllably generate H2O2 is a superior alternative.

[0021] Based on this principle, this invention immobilizes lactase and glucose oxidase on the surface of chitosan nanoparticles. Lactase hydrolyzes lactose into glucose, and glucose oxidase then oxidizes glucose into gluconic acid, while slowly releasing trace amounts of H₂O₂. This gently and continuously activates the bovine milk peroxidase system, generating hypothiocyanate ions with broad-spectrum antibacterial activity, achieving a synergistic effect of hydrolysis and antibacterial action. The immobilized linker uses genipin, a natural cross-linking agent with a toxicity only one ten-thousandth that of glutaraldehyde, exhibiting excellent biocompatibility and safety for food industry applications.

[0022] Specifically, this invention employs genipin to covalently immobilize lactase from *Kluyveromyces lactis* and glucose oxidase from *Aspergillus niger* onto the surface of chitosan nanoparticles, constructing a progressive catalytic system: First, the immobilized lactase hydrolyzes lactose to produce glucose and galactose; second, the immobilized glucose oxidase uses glucose as a substrate to generate gluconic acid and H₂O₂; third, H₂O₂ activates the lactoperoxidase system to produce hypothiocyanate, achieving antibacterial and preservation effects. This system combines lactose hydrolysis with a natural antibacterial mechanism, providing a new pathway for the production of low-lactose dairy products. Developing low-lactose yogurt, cheese, and other derivative products using low-lactose milk as a base is an important direction for expanding the low-lactose dairy product market and increasing product added value.

[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention employs an immobilized dual-enzyme system and genipin crosslinking technology to immobilize β-galactosidase (lactase) and glucose oxidase onto the surface of chitosan nanoparticles, constructing a novel dual-enzyme cascade catalytic system. The chitosan nanoparticles before and after enzyme immobilization were systematically characterized. Simultaneously, the enzymatic properties of both the free and immobilized enzymes were comprehensively evaluated. The results show that the enzymes were successfully immobilized on the surface of chitosan nanoparticles, exhibiting an increase in the kinetic particle size and a decrease in the zeta potential after immobilization, while the core morphology of the support remained essentially unchanged.

[0024] Compared to free enzymes, immobilized enzymes exhibit superior pH and thermal stability. At 25°C, this dual-enzyme cascade system achieved over 95% lactose hydrolysis in milk within 12 hours. After 10 reuses, both immobilized enzymes retained over 70% of their initial specific activity, demonstrating excellent reusability. This dual-enzyme cascade system effectively activated the milk's endogenous lactoperoxidase system, achieving 100% inhibition against *Escherichia coli*, *Staphylococcus aureus*, and *Bacillus cereus* within 4 hours. The treated milk's shelf life could be extended to 35 days at 4°C.

[0025] This invention achieves multi-objective synergy in lactose hydrolysis, simultaneous preservation, and derivative product development. It successfully combines lactose degradation with the natural antibacterial system in milk, improving enzyme stability and reusability, and simplifying the production process and antibacterial effect of low-lactose dairy products. The immobilized dual-enzyme system constructed in this invention can efficiently prepare low-lactose milk and extend its shelf life. It is green, safe, and reusable, meeting the needs of the food industry and possessing significant application value in satisfying the consumption needs of lactose-intolerant populations and promoting the upgrading of the dairy industry. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the batch and aseptic processes used to produce lactose-free milk.

[0028] Figure 2 Scanning electron microscope images of (A) chitosan nanoparticles (CSNPs), (B) immobilized lactase (Lac-CSNPs) and (C) immobilized glucose oxidase (GOx-CSNPs).

[0029] Figure 3 The lactose hydrolysis curves of Lac-CSNPs are shown.

[0030] Figure 4 The antibacterial effect of the immobilized dual-enzyme system at 25℃ is shown in Figure 1. (A) Antibacterial effect of Escherichia coli (B) Antibacterial effect of Staphylococcus aureus (C) Antibacterial effect of Bacillus cereus. Black: Free dual-enzyme system; Red: Immobilized dual-enzyme system. Detailed Implementation

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1: Preparation of chitosan nanoparticles

[0033] Under ultrasonic conditions, 5 mL of 1.4 M sodium sulfate aqueous solution was slowly added dropwise to 95 mL of 0.35 M acetic acid solution containing 1% (v / v) Tween 80 and 0.25% (w / v) chitosan. The mixture was stirred continuously at 500 rpm for 2 h. Subsequently, the resulting suspension was centrifuged at 4 °C and 3800 × g for 10 min, and the precipitated particles were collected.

[0034] The obtained chitosan nanoparticles were resuspended in glycine-NaOH buffer (pH 9.0, 0.05 M) to prepare a nanoparticle suspension; then, the nanoparticles were collected by centrifugation at 4℃ and 3800×g for 5 min. After discarding the supernatant, the particles were washed 2-3 times with distilled water, each time by centrifugation at 4℃ and 3800×g for 5 min.

[0035] Finally, the washed chitosan nanoparticles were resuspended in deionized water and stored at 4°C for later use.

[0036] Example 2: Genipin activation, and separate immobilization of lactase and glucose oxidase

[0037] Chitosan nanoparticles (CSNPs, 10 mg / ml, prepared in Example 1) were centrifuged at 3800×g for 5 min at 4 °C, and then placed in 1.5 mg / ml genipin solution (prepared with glycine-NaOH buffer, pH 9.0) and activated at 60 °C and 100 rpm for 1 h. After activation, the CSNPs were subjected to three centrifugation-washing cycles with deionized water to remove unreacted genipin.

[0038] Subsequently, to optimize the enzyme immobilization load, lactase (250 U) and glucose oxidase (120 U) were added to 10 ml of activated CSNP suspension (final carrier concentration 10 mg / ml). After incubation at 25°C and 100 rpm for 12 h, the immobilized enzyme particles were collected by centrifugation, washed with sterile water, resuspended in sterile water, and stored at 4°C for later use.

[0039] Example 3: Surface morphology observation of immobilized lactase (Lac-CSNPs) and immobilized glucose oxidase (GOx-CSNPs)

[0040] The surface morphology of chitosan nanoparticles (CSNPs), immobilized lactase (Lac-CSNPs), and immobilized glucose oxidase (GOx-CSNPs) was observed using field emission scanning electron microscopy (FE-SEM, Quattro S, Thermo Fisher Scientific, USA) with an accelerating voltage of 10 kV.

[0041] CSNPs exhibit a smooth and relatively uniform spherical structure. Lac-CSNPs and GOx-CSNPs maintain their smooth surface morphology and do not show any increase in roughness. Figure 2 This indicates that the genipin activation and subsequent covalent linking of the enzyme did not disrupt the morphology of the vector.

[0042] Example 4: Particle size and zeta potential analysis of CSNPs, Lac-CSNPs and GOx-CSNPs

[0043] The particle size, polydispersity index, and zeta potential of CSNPs, Lac-CSNPs, and GOx-CSNPs were determined using a dynamic light scattering instrument (Zetasizer Nano ZS90, Malvern Instruments). Samples were appropriately diluted before testing, and measurements were performed at 25℃. The experimental results showed that the particle size and potential of CSNPs were 582.03±25.35 nm and 13.43±0.68 mV, respectively; the particle size and potential of Lac-CSNPs were 654.97±11.89 nm and 6.81±0.44 mV, respectively; and the particle size and potential of GOx-CSNPs were 607.70±23.05 mV and 10.23±0.66 mV, respectively.

[0044] Table 1 Comparison of Dynamic Light Scattering (DLS) Analysis Results

[0045] Example 5: Determination of specific activity of Lac-CSNPs

[0046] Free lactase assay: The reaction system consisted of 875 μL of 0.2 M, pH 7.0 PBS buffer, 25 μL of 50 mM o-nitrophenyl-β-D-galactopyranoside (ONPG) substrate solution, and 100 μL of lactase solution with a concentration of 79.88 μg / mL. The initial reaction rate was determined by monitoring the change in absorbance at 410 nm over 1 min.

[0047] The method for determining Lac-CSNPs activity is essentially the same as that for determining free enzyme activity, except that 100 μL of free lactase solution is replaced with 100 μL of 1 mg / mL Lac-CSNPs suspension. After the reaction proceeds for 1 min, the reaction system is heated in a boiling water bath for 3 min to terminate the reaction. The supernatant is then collected by centrifugation, and the absorbance is measured at 410 nm (the reaction system without added enzyme serves as a blank control).

[0048] Lactase activity unit (U) is defined as the amount of enzyme required to catalyze the release of 1 μmol of o-nitrophenol (ONP) per minute under conditions of 25°C and 0.2 M PBS (pH 7.0).

[0049] ONP concentration is calculated using the following formula: c=A / εb Where: c: ONP concentration (mol / L); A: absorbance measured at 410 nm; ε: molar absorptivity of ONP, 3500 M - ¹·cm - ¹; b: Optical path length is 1 cm.

[0050] Specific activity of enzymes is expressed as the activity per unit amount of protein, i.e., U / mg protein. Protein concentration was determined using the 280 nm UV absorbance method and calculated using a bovine serum albumin (BSA) standard curve. The specific activity of free lactase was approximately 25.38 ± 0.15 U / mg, and the specific activity of Lac-CSNPs was approximately 24.31 ± 0.39 U / mg. The specific activity of immobilized lactase did not change significantly from that of the free enzyme; the slight decrease may be due to mass transfer limitations of lactose molecules.

[0051] Example 6: Determination of Specific Activity of GOx-CSNPs

[0052] 0.1 g of o-anisidine was dissolved in 10 mL of methanol to prepare a stock solution. 0.1 mL of the stock solution was added to 12 mL of 0.2 M, pH 6.0 PBS solution, and air was bubbled through for 30 min to ensure the solution reached oxygen saturation. Free glucose oxidase assay: 100 μL of 180 g / L glucose solution was added to 830 μL of working solution. Then, 35 μL of 0.03% (w / v) horseradish peroxidase (HRP) solution and 35 μL of 58.73 μg / mL glucose oxidase solution were added sequentially to initiate the enzymatic reaction. The absorbance at 460 nm was continuously monitored over 1 min during the reaction to calculate the initial reaction rate.

[0053] The method for determining the activity of GOx-CSNPs is basically the same as that for the free enzyme, except that a 35 μL suspension of GOx-CSNPs at a concentration of 1 mg / mL is used instead of the free enzyme solution to initiate the reaction. The reaction system is incubated with shaking at 25°C for 1 min, then heated in a 100°C water bath for 3 min to terminate the reaction; after centrifugation at 3800×g for 5 min, the supernatant is collected and its absorbance is measured at 460 nm.

[0054] The glucose oxidase activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of oxidized o-anisidine per minute under the conditions of 25°C and 0.2 M phosphate buffer (pH 6.0).

[0055] The molar extinction coefficient of oxidized o-anisidine at 460 nm is: ε = 11300 M -1 ·cm -1 Therefore, the concentration of the generated product and the corresponding enzyme activity can be calculated according to the Beer-Lambert law.

[0056] The specific activity of free glucose oxidase was measured to be approximately 22.63 ± 1.79 U / mg, and the specific activity of GOx-CSNPs was approximately 21.00 ± 0.37 U / mg. The specific activity of glucose oxidase after immobilization did not change significantly from that of the free enzyme; the slight decrease may be due to mass transfer limitations of glucose molecules.

[0057] Example 7: pH stability determination

[0058] Free enzyme assay method: 300 μL of free lactase solution with a concentration of 47.93 mg / mL was added to 2700 μL of buffer solution with different pH values ​​(pH 2.2~10.0), mixed well and incubated for 2 h; at the same time, 15 mg of free glucose oxidase was dissolved in 3 mL of buffer solution with different pH ranges (pH 2.2~10.0), mixed well and incubated for 2 h.

[0059] The buffer systems were as follows: 0.05 M glycine-hydrochloric acid buffer (pH 2.2–3.0), 0.1 M acetate buffer (pH 4.0–5.0), 0.2 M phosphate buffer (pH 6.0–8.0), and 0.05 M glycine-NaOH buffer (pH 9.0–10.0). After incubation, 100 μL of lactase samples treated at different pH levels were added to 900 μL of 0.2 M phosphate buffer (pH 7.0); 100 μL of glucose oxidase samples treated at different pH levels were added to 900 μL of 0.2 M phosphate buffer (pH 6.0); incubation was continued for 30 min, and enzyme activity was measured.

[0060] Immobilized enzyme assay: The experimental procedure was the same as for the free enzyme, except that the free enzyme solution was replaced with 100 μL of immobilized enzyme suspension (chitosan nanoparticle concentration of 10 mg / mL), while all other conditions remained the same. pH stability was evaluated by measuring enzyme activity after treatment at different pH values.

[0061] Free enzymes exhibit a relatively narrow stability range, while immobilized enzymes demonstrate a wider stability range. After treatment with pH 2.2 buffer, free lactase and Lac-CSNPs retained 0.67±0.91% and 11.72±1.09% of their initial specific activities, respectively; after treatment with pH 10 buffer, they retained 24.42±4.32% and 50.25±2.92%, respectively.

[0062] Similarly, after treatment with pH 2.2 buffer, free glucose oxidase and GOx-CSNPs retained 22.76±3.93% and 38.27±3.50% of their initial specific activities, respectively; after treatment with pH 10 buffer, they retained 41.39±3.85% and 58.17±3.39%, respectively. The results indicate that both immobilized lactase and immobilized glucose oxidase exhibit higher enzyme activity retention rates under extreme acidic and alkaline conditions.

[0063] Example 8: Thermal stability determination

[0064] Thermostability assay of free enzymes: 300 μL of lactase stock solution (47.93 mg / mL) was added to 2700 μL of 0.2 M phosphate buffer (pH 7.0); 15 mg of glucose oxidase was dissolved in 3000 μL of 0.2 M phosphate buffer (pH 6.0). The lactase and glucose oxidase solutions were then incubated at different temperatures ranging from 25 to 65 °C for 1 h each. After incubation, the samples were equilibrated at 25 °C for 30 min, and their enzyme activity was measured.

[0065] Thermostability assay of immobilized enzymes: Chitosan nanoparticles loaded with enzymes at a concentration of 1 mg / mL were used for evaluation. Specifically, lactase immobilized nanoparticles (Lac-CSNPs) were resuspended in 0.2 M phosphate buffer (pH 7.0); glucose oxidase immobilized nanoparticles (GOx-CSNPs) were resuspended in 0.2 M phosphate buffer (pH 6.0). The immobilized enzyme systems were incubated under the same temperature conditions as the free enzymes, i.e., at 25–65 °C for 1 h. Enzyme activity was measured after treatment. The thermostability of the free and immobilized enzymes was evaluated by comparing the retention rates of enzyme activity after treatment at different temperatures.

[0066] Table 2: Comparison of specific activity retention percentage of free lactase and Lac-CSNPs

[0067] Table 3: Comparison of the percentage of activity retention of free glucose oxidase and GOx-CSNPs

[0068] Within the temperature range of 25-65℃, the activity of free enzymes decreased significantly faster than that of immobilized enzymes as the temperature increased further. After heat treatment at 45℃, free lactase retained 10.26±2.18% of its initial specific activity, while Lac-CSNPs retained 69.67±4.24% of its initial specific activity. After heat treatment at 55℃ and 65℃, the activity of free lactase was almost completely lost, while Lac-CSNPs still retained 34.50±1.09% and 12.57±0.42% of their initial specific activities, respectively (Table 2). Similarly, after heat treatment at 45℃, 55℃, and 65℃, free glucose oxidase retained 64.09±6.10%, 29.36±3.26%, and 0.71±0.88% of its initial specific activity, respectively, while GOx-CSNPs retained 89.01±0.31%, 66.37±1.13%, and 22.92±1.95% of their initial specific activities (Table 3). These results indicate that the immobilized enzyme experiences a more slow loss of activity compared to the free enzyme, demonstrating its superior thermal stability and stronger resistance to heat denaturation.

[0069] Example 9: Reusability Test

[0070] The reusability of Lac-CSNPs and GOx-CSNPs was evaluated by measuring their specific activity in 10 consecutive cycles. In each cycle, the assay system was constructed according to Examples 5 and 6, but data was collected using continuous 180-second monitoring. In each test, the enzyme-loaded chitosan nanoparticles were recovered by centrifugation (3800 × g, 4 °C, 5 min). The specific activity measured in the first cycle was defined as 100%, and the relative residual activity of Lac-CSNPs and GOx-CSNPs after each cycle was calculated using the following formula: Residual relative enzyme activity (%) = (Specific activity in the nth cycle / Specific activity in the first cycle) × 100.

[0071] The reusability of Lac-CSNPs and GOx-CSNPs was evaluated after 10 consecutive rounds of repeated use. Lac-CSNPs and GOx-CSNPs retained 77.64±0.72% and 71.43±0.97% of their initial activity, respectively. This indicates that both Lac-CSNPs and GOx-CSNPs have good operational stability.

[0072] Example 10: Determination of lactose hydrolysis efficiency

[0073] 1850 μL of free lactase solution (0.48 mg / mL) or 1000 μL of Lac-CSNPs suspension (10 mg / mL) was added to 50 mL of pasteurized milk. The amounts of both were adjusted according to enzyme activity to ensure identical initial catalytic activity. The reaction system was incubated at 25 °C with shaking at 160 rpm. Samples were taken at 0, 2, 4, 6, 8, 10, and 12 h, with 100 μL of sample taken from the reaction system each time and centrifuged at 3800 × g for 5 min. The glucose content in the samples was determined using a glucose assay kit.

[0074] The lactose hydrolysis rate is calculated using the following formula: Lactose hydrolysis rate (%) = n1 / n0 × 100 Where: n1 is the amount of glucose detected (mol); n0 is the initial amount of lactose in the system (mol).

[0075] During the calculation, lactose is completely converted into glucose and galactose in a 1:1 stoichiometric ratio under the catalysis of lactase, that is: lactose + H2O → glucose + galactose. Therefore, for every 1 mol of glucose produced, 1 mol of lactose is hydrolyzed.

[0076] In the initial stage of the reaction, the lactose hydrolysis rate of Lac-CSNPs was slightly lower than that of free lactase. After treatment at 25℃ for 12 h, the lactose hydrolysis rates of both Lac-CSNPs and free lactase exceeded 95.00%. Figure 3 This meets the requirements for producing low-lactose milk.

[0077] Example 11: Evaluation of the antibacterial kinetics of a dual-enzyme conjugate system

[0078] Escherichia coli, Staphylococcus aureus, and Bacillus cereus were activated to OD values ​​at 25°C. 600 =0.8, the activated bacterial culture was centrifuged at 13,778×g for 10 min, the bacterial pellet was collected and resuspended in 50 mL of sterile milk. Then, serial dilutions were performed with sterile milk to achieve a final bacterial concentration of 1.16–1.44 × 10³ CFU / mL. Untreated milk was used as a control group.

[0079] Free enzymes: Add 1850 μL of lactase solution (0.48 mg / ml) and 1300 μL of glucose oxidase solution (1.76 μg / ml) to 50 ml of bacterial milk.

[0080] Immobilized enzymes: Add 1000 μL of Lac-CSNPs (10 mg / mL) and 50 μL of GOx-CSNPs (1 mg / mL) to 50 mL of bacterial milk. Add 500 μL of 50 mM NaSCN to each type of bacterial milk to bring the final concentration to 0.5 mM.

[0081] Incubate at 25°C. Take 25 μL samples at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h and spread them onto plates. Perform three parallel experiments for each sample group. Incubate at 37°C for 24 h and count colonies using Scan 300. Calculate the sterilization rate at each time point based on the colony count results using the following formula: Sterilization rate (%) = (N) 对照 -N 样品 ) / N 对照 N 对照 This refers to the total bacterial count (CFU) of the control group at the same time point; N 样品 It is the total number of colonies (CFU) in the sample group with the dual-enzyme system added at the same time point.

[0082] Within the initial 2 hours of treatment, the inhibition rates of the immobilized dual-enzyme conjugate systems (Lac-CSNPs and GOx-CSNPs) against *Escherichia coli*, *Staphylococcus aureus*, and *Bacillus cereus* were 66.39±1.42%, 66.23±3.44%, and 60.00±2.96%, respectively, while the inhibition rates of the free dual-enzyme conjugate systems were 78.69±5.12%, 76.16±1.99%, and 66.45±4.47%, respectively. With prolonged treatment, both the free and immobilized dual-enzyme conjugate systems achieved 100% inhibition of all tested strains within 4 hours. Figure 4 ).

[0083] Therefore, based on the lactose hydrolysis curve and antibacterial efficacy test results, the free and immobilized dual-enzyme coupling system can not only achieve a lactose hydrolysis rate of over 95% after 12 h of treatment, but also effectively inhibit microbial contamination during the production process, providing a reliable guarantee for the safe production of low-lactose dairy products.

[0084] Example 12: Determination of lactoperoxidase activity in milk

[0085] The activity of lactoperoxidase (LPO) in milk was determined using a modified ABTS method. In the lactoperoxidase activity assay, the amounts of the free and immobilized dual-enzyme systems were exactly the same as those used in the antimicrobial kinetic assay (Example 11), except that the milk used in the experiment was not inoculated with bacteria, and untreated milk was used as a control group. Each treatment group was incubated at 25°C, and samples were taken at 0, 2, 4, 6, 8, 10, and 12 h, 100 μL of which was diluted 50 times with deionized water and centrifuged at 3800 × g for 5 min. Subsequently, 100 μL of the supernatant was added to a reaction system containing 800 μL of 2 mM ABTS solution [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] and 100 μL of 5 mM H₂O₂ solution. The absorbance was continuously monitored for 120 s at room temperature and a wavelength of 412 nm using a UV-Vis spectrophotometer. Peroxidase activity is expressed in enzyme activity units (U), which is the amount of enzyme required to catalyze the oxidation of 1 μmol ABTS per minute.

[0086] Dynamic monitoring of LPO activity in milk treated with free and immobilized dual-enzyme coupling systems over 12 hours showed that the LPO activity remained essentially consistent with that of untreated raw milk throughout the 12-hour treatment period, staying within the range of 1.73±0.24~2.57±0.13 U / mL (Table 4). Combined with the simultaneous trend of gradual H2O2 generation, it can be inferred that the H2O2 generated by the two dual-enzyme coupling systems can rapidly participate in the catalytic oxidation reaction of LPO in milk and be promptly consumed. During this process, thiocyanate ions (SCN...)... - It is oxidized to generate hypothiocyanate ions (OSCN), which have antibacterial activity. - H2O2 is reduced to water.

[0087] Table 4: Comparison of specific activities of lactoperoxidase in milk

[0088] Example 13: Determination of Hydrogen Peroxide Content in Milk

[0089] Milk itself does not spontaneously produce H2O2; however, the immobilized dual-enzyme cascade system does. The sample collection and processing methods for hydrogen peroxide (H2O2) content determination were the same as in the lactoperoxidase activity assay (Example 12), but the samples were not diluted before measurement. After collecting samples at each time point, the H2O2 concentration in the samples was measured according to the instructions of the hydrogen peroxide detection kit. After incubation at 25°C for 12 h, the H2O2 concentration in the pure milk group was extremely low (0~5.45±3.78 μmol / L), while the H2O2 concentrations in the free and immobilized coupled dual-enzyme system treatment groups significantly accumulated to 207.92±8.18 μmol / L and 188.65±8.23 μmol / L, respectively, fully validating the effectiveness of this dual-enzyme coupled catalytic strategy.

[0090] Example 14: OSCN in milk - Concentration Measurement

[0091] 19.8 mg of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added to 100 mL of an aqueous solution containing 5 mM EDTA and allowed to dissolve completely. Then, 7 μL of β-mercaptoethanol was added, and the mixture was incubated at room temperature for 2 h to ensure complete reduction of DTNB to 5-mercapto-2-nitrobenzoic acid (TNB). - The time points for concentration determination, the blank group, and the sample group settings are the same as those for the lactoperoxidase activity assay, but the samples are not diluted before determination.

[0092] The specific procedure is as follows: Add 0.1 mL of sample to 4.5 mL of TNB solution and mix well. To eliminate the potential interference of residual hydrogen peroxide (H2O2) on the detection results, add 20 μL of catalase solution (1 mg / mL) to the reaction system before spectrophotometry, and measure the absorbance at 412 nm. Calculate the OSCN based on the amount of TNB consumed. - Concentration. The results showed that OSCN generated after 12 h of treatment of milk with the free and immobilized dual-enzyme coupling system... - The concentrations reached 188.48±1.67 μM and 162.56±3.83 μM, respectively, significantly higher than those in the pure milk group without the added dual-enzyme system, confirming that the dual-enzyme cascade strategy can effectively activate the LPO system in milk; at the same treatment time point, the OSCN in the immobilized dual-enzyme cascade system... - The cumulative concentration of [agent] was lower than that of the free enzyme group, possibly due to slight mass transfer restriction of the substrate molecules during the catalytic reaction. However, it was still able to effectively activate the LPO system and continuously generate OSCN with antibacterial activity. - This allows it to exert a good antibacterial effect.

[0093] Example 15: Evaluation of the shelf life of low-lactose pasteurized milk products

[0094] To 9 L of pasteurized milk, 189 mL of a suspension containing two immobilized enzyme chitosan nanoparticles (CSNPs) was added. This suspension consisted of 180 mL of Lac-CSNPs (10 mg / mL) and 9 mL of GOx-CSNPs (1 mg / mL). The mixture was incubated (reacted) at 25°C and 160 rpm with magnetic stirring for 12 h. After pasteurization at 65°C for 30 min, the treated milk was dispensed into 200 mL aseptic bags and stored at 4°C.

[0095] Samples were collected periodically on days 1, 7, 14, 21, 28, and 35. Various physicochemical and microbiological parameters, including fat, protein, non-fat milk solids (NFMS), acidity, total bacterial count, coliforms, Salmonella, and Staphylococcus aureus, were determined according to Chinese national standards (GB 5009.6-2016, GB5009.5-2016, GB 5413.39-2010, GB 5009.239-2016, GB 4789.2-2022, GB 4789.3-2016, GB4789.4-2024, and GB 4789.10-2016).

[0096] Compared to the 15-day shelf life of pasteurized milk, low-lactose milk treated with an immobilized two-enzyme cascade system has a shelf life extended to 35 days. This is mainly attributed to the generation of H2O2 in the milk by the immobilized two-enzyme cascade system, which activates the milk's endogenous lactoperoxidase system. This system produces OSCN, which has antibacterial activity. - Effectively inhibits the growth of microorganisms (Table 5). In addition, throughout the storage period, key physicochemical indicators such as fat, protein, non-fat milk solids (SNF) and acidity all meet the requirements of the National Food Safety Standard for Pasteurized Milk (GB 19645-2010).

[0097] Table 5. Microbiological detection of low-lactose milk prepared using an immobilized dual-enzyme coupling system.

[0098] Example 16: Preparation of low-lactose yogurt

[0099] Take 200 mL of sterile low-lactose milk (prepared using the same method as in Example 15) into a sterile beaker, seal it with sealing film, and place it in a 95°C water bath for 5 min for sterilization. Quickly transfer the beaker to a 4°C refrigerator and cool it to 42°C. In a clean bench, add 0.02% of a compound biological preservative (nisin: natamycin: ε-polylysine = 1:1:1) to the low-lactose milk, mix thoroughly, and then add 400 mg of lactic acid bacteria starter (Beijing Chuanxiu Technology Co., Ltd.). Stir magnetically at 42°C for 30 min, and place it in a constant temperature incubator at 42°C for 12 h of fermentation. After fermentation, transfer it to a 4°C refrigerator for storage to obtain the finished low-lactose yogurt. The finished product has good sensory properties and good quality stability. No Escherichia coli or mold was detected within 14 days. The number of viable lactic acid bacteria decreased naturally with storage time from 2.5 × 10⁻⁶. 8 CFU / mL decreased to 4.7 × 10⁻⁶ 7 CFU / mL.

[0100] Table 6. Detection indicators for low-lactose yogurt within 14 days

[0101] Example 17: Preparation of low-lactose cheese

[0102] Take 500 mL of low-lactose milk (prepared using the same method as in Example 15), sterilize it in a 95°C water bath for 5 min, and then quickly transfer it to a 4°C refrigerator to cool to room temperature; add 0.02% of the milk mass of a compound biological preservative (nisin: natamycin: ε-polylysine = 1:1:1) to the milk and prepare it as described in 4.3.2. After thorough mixing, add 1 g of lactic acid bacteria starter in a clean bench, stir for 30 min, and then ferment at 25°C until the pH reaches 6.0-6.4; accurately weigh 4 g of rennet, dissolve it in an appropriate amount of deionized water, and slowly add it to the fermented milk. Incubate at 37°C until curd forms and whey separation begins; cut the curd into uniform sizes and gently stir for 10-15 minutes. To promote whey separation, first drain 1 / 3 to 1 / 2 of the whey, then heat the system to 38°C and stir continuously to drain the remaining whey. The treated curd is then poured into a sterile mold for shaping. After demolding, the cheese base is obtained and stored at 4°C to obtain the finished low-lactose cheese. The finished product exhibits good sensory properties, and no E. coli was detected within 14 days.

[0103] Table 7. Detection indicators for low-lactose cheese within 14 days

[0104] Example 18: Preparation of low-lactose whipping cream

[0105] Take 500 mL of low-lactose milk, sterilize in a 95°C water bath for 5 min, then cool to room temperature; add 0.02% of a compound biological preservative (nisin: natamycin: ε-polylysine = 1:1:1) to the low-lactose milk, mix thoroughly, pour into sterilized centrifuge tubes, centrifuge at 37°C and 6000 r / min for 2 h, scrape the whipped cream into sterile bottles to obtain the finished whipped cream, and simultaneously obtain the byproduct skim milk; pasteurize the whipped cream at 65°C for 30 min, then rapidly cool and store in a low-temperature environment. The low-lactose whipped cream remained stable in quality for 14 days, with a total bacterial count of 0, which was undetectable.

[0106] Table 8. Detection indicators for low-lactose whipping cream within 14 days

[0107] Example 19: Preparation process of low-lactose cream

[0108] The light cream prepared by the method in Example 18 was allowed to stand at 4°C for 15 hours to complete the fat crystallization conversion. After cooling, the light cream was stirred for 60 minutes using a sterilized mixer until cream granules formed and adhered to the sides of the bowl. The finished cream was then stored in a refrigerator at 4°C. The low-lactose cream showed good quality stability within 14 days, with no E. coli or mold detected. The fat content decreased slightly over time, the acidity showed a mild upward trend, and the moisture content increased, but none of these factors significantly affected the product quality.

[0109] Table 9. Detection Indicators for Low-Lactose Cream within 14 Days

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An immobilized dual-enzyme complex, characterized in that, It comprises: chitosan nanoparticles; lactase covalently linked to the surface of the chitosan nanoparticles using genipin as a crosslinking agent; and glucose oxidase covalently linked to the surface of the chitosan nanoparticles using genipin as a crosslinking agent; wherein the lactase and the glucose oxidase are respectively immobilized on the surface of different batches of chitosan nanoparticles.

2. The immobilized dual-enzyme complex according to claim 1, characterized in that, The chitosan nanoparticles are formed by the self-assembly of chitosan through genipin crosslinking induced by sodium sulfate.

3. The immobilized dual-enzyme complex according to claim 1, characterized in that, The method for preparing the immobilized dual-enzyme complex includes the following steps: (1) Chitosan was dissolved in acetic acid solution, sodium sulfate solution was added under ultrasonic conditions, and the precipitate was collected by centrifugation after stirring to obtain chitosan nanoparticles; (2) The chitosan nanoparticles were placed in a genipin solution and activated at 50-70°C for 0.5-2 hours. Unreacted genipin was washed away to obtain activated chitosan nanoparticles. (3) The activated chitosan nanoparticles were mixed with lactase solution and glucose oxidase solution respectively, and incubated at 20~30℃ for 6~18h. The immobilized enzyme particles were collected by centrifugation and washed to obtain immobilized lactase and immobilized glucose oxidase respectively. (4) The immobilized lactase and the immobilized glucose oxidase are mixed to obtain the immobilized dual-enzyme complex.

4. The use of the immobilized dual-enzyme complex according to any one of claims 1 to 3 in the preparation of low-lactose dairy products.

5. A low-lactose milk, characterized in that, It is prepared by treating milk with the immobilized dual-enzyme complex according to any one of claims 1 to 3.

6. The method for preparing low-lactose milk according to claim 5, characterized in that, The process includes the following steps: adding the immobilized dual-enzyme complex according to any one of claims 1 to 3 to milk for enzymatic hydrolysis; separating and collecting the hydrolyzed milk to obtain low-lactose milk.

7. The preparation method according to claim 6, characterized in that, The enzymatic hydrolysis reaction is carried out at a temperature of 20~30℃ for a reaction time of 6~24h; the amount of the immobilized dual enzyme complex added is: the final concentration of immobilized lactase is 0.1~0.3 mg / mL (based on chitosan nanoparticles), and the final concentration of immobilized glucose oxidase is 0.005~0.02 mg / mL (based on chitosan nanoparticles).

8. A low-lactose dairy product, characterized in that, It is prepared using the low-lactose milk described in claim 5 as a base material.

9. The low-lactose dairy product according to claim 8, characterized in that, The low-lactose dairy products are low-lactose yogurt, low-lactose cheese, low-lactose whipping cream, or low-lactose cream.

10. A method for preparing low-lactose yogurt, characterized in that, The process includes the following steps: sterilizing the low-lactose milk as described in claim 5 and cooling it to the fermentation temperature; inoculating it with lactic acid bacteria for fermentation; and cooling and ripening it after fermentation to obtain low-lactose yogurt.

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