A method for preparing a high-bioactivity lactase nanoparticle with pH response ability and high vmax

Abstract

The present application belongs to the technical field of lactase, and particularly relates to a preparation method of high-bioactivity lactase nanoparticles with pH response capability and high vmax, comprising the following steps: (1) preparing a chitosan solution; (2) preparing a hyaluronic acid solution; (3) nanoparticle formation: firstly, dissolving lactase in the chitosan solution, slowly adding a trisodium phosphate solution, and performing ion crosslinking to form chitosan nanoparticles; then, suspending the formed chitosan nanoparticles in the hyaluronic acid solution, and stirring to form a double-layer composite coating; and (4) particle characterization detection: determining the particle size of the nanoparticles by dynamic light scattering, and the target is 100-120 nm; and observing the morphology thereof by transmission electron microscopy, so as to ensure that a uniform double-layer structure is formed. The present application provides a lactase raw material treated by nanoparticle coating, so that the lactase is ensured not to be decomposed by gastric acid and can reach the small intestine, and is quickly released, efficiently combined and decomposed in the small intestine.

Description

Technical Field

[0001] This invention belongs to the field of lactase technology, and specifically relates to a method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness. Background Technology

[0002] Lactase, also known as β-galactosidase, is a key enzyme widely used in dairy processing. Its main function is to hydrolyze lactose (a disaccharide with low sweetness and poor solubility) into glucose and galactose. This not only effectively improves the sweetness and solubility of dairy products but also reduces the risk of lactose crystallization. It is particularly suitable for products such as ice cream, condensed milk, and concentrated milk, improving their taste and texture.

[0003] However, due to insufficient or absent lactase secretion in the human body, some people are unable to digest lactose properly, leading to lactose intolerance. Lactose intolerance is caused by a lack of lactase in the body, making it unable to break down lactose, resulting in a series of uncomfortable symptoms such as bloating, diarrhea, flatulence, and acute abdominal pain. According to international research data, the incidence of lactose intolerance is relatively high globally, especially in Asia, where the proportion of adults with lactose malabsorption exceeds 80%, and lactose intolerance is particularly severe. This problem not only affects the body's absorption of important nutrients such as protein and calcium, but may also adversely affect the intelligence and growth and development of infants and young children. Therefore, some countries internationally have included lactase as an important component of infant formula to alleviate digestive problems caused by lactose intolerance.

[0004] Despite the immense potential of lactase in the dairy industry, its large-scale industrial production still faces numerous challenges. Currently, lactase products are primarily derived from intracellular enzymes, requiring cell wall disruption for enzyme release. This not only leads to complex processes and high purification costs, but also results in poor temperature stability and heat resistance, making it susceptible to contamination by other microorganisms during production, further limiting its industrial applications. Therefore, increasing lactase yield, optimizing extraction and purification processes, and improving enzyme stability and applicability have become key issues driving the development and industrial application of lactase technology.

[0005] Lactose intolerance is a common digestive disorder caused by a lack of sufficient lactase, an enzyme that prevents the effective breakdown of lactose, leading to symptoms such as indigestion. To address this issue, lactase supplements have emerged on the market. However, because lactase is a protein, it is easily degraded in the acidic environment of the stomach, resulting in reduced biological activity and thus affecting its effectiveness. Therefore, effectively protecting lactase to ensure its stable presence and accurate release in the gastrointestinal tract is crucial for resolving lactose intolerance.

[0006] Traditional lactase delivery systems often employ encapsulation with biomaterials such as chitosan, but these systems still have some drawbacks, such as insufficient stability in the stomach or difficulty in achieving precise release in specific regions of the small intestine. Therefore, a new technological solution is urgently needed that can precisely control the release of lactase in the small intestine while ensuring its stability.

[0007] Proper encapsulation of lactase can effectively improve its thermal stability and shelf life.

[0008] Dairy products such as milk contain lactose. When dairy products are consumed, most people break down lactose in their bodies using lactase, which is present in the small intestine, and the lactose is then absorbed. However, in some people, there is insufficient lactase in the small intestine, so even if dairy products are consumed, proper absorption in the small intestine does not occur, resulting in symptoms such as indigestion, diarrhea, and abdominal bloating. This condition is called lactose intolerance. To suppress the symptoms of lactose intolerance, it is commonly known to take lactase preparations. Therefore, lactase preparations are manufactured and sold in many countries. On the other hand, it is known that if lactase is taken before consuming dairy products, its effectiveness is reduced or becomes ineffective. Therefore, a patent document discloses a technology that, by simultaneously containing lactase and an antacid selected from at least one of trisodium citrate, sodium potassium tartrate, calcium carbonate, sodium carbonate, or calcium hydrogen phosphate, ensures that the lactase is not inactivated and breaks down lactose in the body even when taken before consuming foods or drinks containing lactose. In order to make unstable bioactive substances into stable products, it is recommended to dry them together with excipients that are easy to vitrify and then seal them in water-soluble glass.

[0009] In the manufacture of biological agents such as enzymes, existing technologies have been disclosed: carbohydrates are important as excipients. Examples of carbohydrates that are easy to form glass include glycerol, ribose, xylose, sorbitol, mannitol, fructose, glucose, galactose, sucrose, maltose, lactose, trehalose, maltotriose, and raffinose.

[0010] Prior art also discloses that, in the presence of carbohydrate excipients that stabilize enzymes by providing an amorphous or glassy solid matrix, the Maillard reaction occurs during the drying and neutralization of biological material. It has been reported that the degree of staining caused by the Maillard reaction appears to be related to a decrease in enzyme activity. Therefore, a method is disclosed whereby, in order to prevent the Maillard reaction caused by carbohydrate excipients and prolong the lifespan of the dried biological material, the biological material is dried in the presence of a carbohydrate excipient effective in stabilizing the dried biological material, and in the presence of a Maillard reaction inhibitor effective in substantially preventing the Maillard reaction. As a specific example, it is shown that by including lysine, which acts as a Maillard reaction inhibitor, in glucose, a carbohydrate excipient, and then drying alkaline phosphatase, the activity of the enzyme is increased.

[0011] Existing technology has also disclosed that in the processing and manufacturing of foods using enzymes, water content in the starting ingredients of the food can cause Maillard browning. It has been disclosed that if less water is used, the degree of Maillard browning decreases; therefore, a method for controlling Maillard browning is to reduce the water content in the starting ingredients of the food. On the other hand, it has been pointed out that in the processing and manufacturing of foods, drying based on water removal as a method for controlling Maillard browning can cause other problems. Therefore, as a solution to this problem, a method has been disclosed in which an amount of oxidoreductase effective in preventing excessive browning is added to the starting ingredients of the food (whole milk or skim milk) to form an aqueous mixture, followed by drying, and then the food (cheese) is manufactured.

[0012] Current technologies primarily utilize chitosan or its derivatives as drug carriers for the preparation and application of drug-loaded microspheres, with particle sizes ranging from nanometer to micrometer. Drug release is characterized by slow or programmed slow release. However, composite nanoparticles prepared by encapsulating lactase with chitosan are easily broken down by gastric acid and fail to reach the small intestine, resulting in insufficient absorption. Summary of the Invention

[0013] To overcome the shortcomings of existing technologies, this invention provides a method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness. The method employs nanoparticle encapsulation technology combined with a bilayer composite encapsulation scheme of chitosan and hyaluronic acid, effectively solving the problems of lactase stability and release control in the gastrointestinal tract. Through this technology, lactase can be effectively protected in the stomach and rapidly released in the small intestine, ensuring that lactase can efficiently bind to and break down lactose molecules, thereby improving the quality of life for patients with lactose intolerance.

[0014] The technical solution of this invention, by selecting appropriate materials and preparation methods, utilizes the pH-responsive characteristics of chitosan and the hydration and anti-degradation properties of hyaluronic acid to successfully achieve the targeted delivery of lactase in the gastrointestinal tract, exhibiting high stability and targeting, and significantly improving the therapeutic effect of lactase.

[0015] To address the problems mentioned above, this invention provides a method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness, and provides a lactase raw material treated with nano-carrier technology to ensure that the lactase is not decomposed by gastric acid and can reach the small intestine (in vivo stability), and is rapidly released in the small intestine (specific pH response), efficiently binding to and decomposing lactose.

[0016] The specific technical solution is as follows:

[0017] A method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness includes the following steps:

[0018] (1) Prepare chitosan solution;

[0019] (2) Prepare a hyaluronic acid solution;

[0020] (3) Nanoparticle formation: First, lactase is dissolved in chitosan solution, and sodium tripolyphosphate solution is slowly added dropwise to carry out ionic cross-linking to form chitosan nanoparticles;

[0021] Next, the formed chitosan nanoparticles were suspended in a hyaluronic acid solution and stirred to form a double-layer composite coating.

[0022] (4) Particle characterization and detection: The particle size of nanoparticles is determined by dynamic light scattering, with a target of 100-120 nm. Their morphology is observed by transmission electron microscopy to ensure the formation of a uniform bilayer structure.

[0023] Preferred:

[0024] The preparation method for step (1) is to prepare a chitosan solution with a w / v of 0.22%, dissolve it in acetic acid with a mass concentration of 0.87%, stir it evenly to form a uniform solution.

[0025] In step (2), a hyaluronic acid solution with a w / v of 0.07% is prepared as the outer coating material.

[0026] In step (3), sodium tripolyphosphate with a mass concentration of 0.08% is added dropwise.

[0027] In this invention, chitosan is used as the main nanoparticle carrier. Chitosan is widely used due to its biocompatibility, biodegradability, and pH-responsive properties (stable in acidic environments and soluble in alkaline environments). It can effectively protect lactase from degradation in the acidic environment of the stomach and allow it to be released in the small intestine.

[0028] In this invention, hyaluronic acid (HA) has excellent hydration and anti-degradation properties, which can protect enzymes from damage by gastric acid and other digestive enzymes, and regulate the hydration state of particles under various environments, thereby increasing the stability and targeting of nanoparticles.

[0029] This invention employs a double-layer nano-encapsulation technology combining hyaluronic acid and chitosan, which offers the following technical advantages compared to existing technologies:

[0030] Traditional chitosan encapsulation technology can effectively protect lactase from gastric acid, but this method, which incorporates hyaluronic acid in a composite encapsulation technique, offers greater advantages in both the protection and controlled release of lactase.

[0031] Double-layer encapsulation design: Hyaluronic acid serves as the outer layer, enhancing the stability of nanoparticles in the stomach and reducing enzyme inactivation caused by gastric acid and pepsin degradation; Chitosan serves as the inner layer, dissolving in the pH 5-5.5 environment of the front of the small intestine, ensuring precise release of the enzyme at the front of the small intestine, where it immediately and rapidly binds to and breaks down lactose molecules.

[0032] Prolonging the half-life in vivo: The hyaluronic acid layer increases the hydration state of the particles, reduces their excretion rate in the digestive tract, and prolongs the enzyme's action time. Simultaneously, its anti-protease degradation properties significantly improve the stability of lactase.

[0033] Targeted release: Hyaluronic acid has the ability to bind to specific cell surface receptors (such as the CD44 receptor), which provides nanoparticles with potential targeted delivery capabilities, especially in the small intestine, enabling lactase to function more effectively. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The present invention also provides the following embodiments:

[0036] A method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness includes the following steps:

[0037] (1) Prepare chitosan solution: Prepare a 0.22% (w / v) chitosan solution, dissolve it in 0.87% acetic acid, stir until uniform, and form a homogeneous solution.

[0038] (2) Prepare hyaluronic acid solution: Prepare 0.07% (w / v) hyaluronic acid solution as the outer coating material.

[0039] (3) Nanoparticle formation: First, lactase was dissolved in chitosan solution, and 0.08% sodium tripolyphosphate (TPP) solution was slowly added dropwise to form chitosan nanoparticles through ionic cross-linking. Then, the formed chitosan nanoparticles were suspended in hyaluronic acid solution and stirred for 1.2 hours to form a double-layer composite coating.

[0040] (4) Particle characterization: The particle size of the nanoparticles was determined by dynamic light scattering (DLS), with a target of 100-120 nm. The morphology was observed by transmission electron microscopy (TEM) to ensure the formation of a uniform bilayer structure.

[0041] The pH-responsive release mechanism of lactase nanoparticles provided by this invention primarily involves gastric protection and intestinal release: In the gastric juice environment at pH 1.2, the outer layer of hyaluronic acid and the inner layer of chitosan work together to provide dual protection, preventing lactase inactivation; in the proximal intestinal environment at pH 5.5 (the part of the small intestine connecting to the stomach), chitosan dissolves, releasing lactase. The hydration properties of hyaluronic acid further ensure the stable function of the enzyme in the small intestine and control its gradual release, ensuring maximum enzyme activity and lactose hydrolysis efficiency.

[0042] This embodiment also includes experiments to verify the technical effectiveness of nano-encapsulated lactase.

[0043] The following is a comparative experiment designed to compare the effects of different treatments on lactase performance. Data simulations demonstrate the significant effects of the technology. The experiment includes a comparison of key performance indicators between conventional lactase and the improved lactase produced by this technology.

[0044] Experimental objective: To verify the superior performance of lactase encapsulated using nanotechnology in the following aspects:

[0045] In vivo stability: the rate of activity protection in gastric acid.

[0046] Specific pH release: Release efficiency in the small intestinal environment.

[0047] Dynamic parameters: Km and Vmax values.

[0048] Lactose hydrolysis efficiency: the rate of decomposition in a simulated lactose environment.

[0049] Half-life: The duration of action in the body.

[0050] Experimental Design:

[0051] 1. Grouping

[0052] Control group A: Traditionally extracted and purified free lactase.

[0053] Control group B: Traditionally extracted nano-encapsulated lactase (monolayer chitosan).

[0054] Experimental Group C: This technology improves the nano-encapsulation of lactase (hyaluronic acid + chitosan bilayer).

[0055] Control conditions: Tests were conducted under simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 5.5, pH 6.8) conditions.

[0056] 2. Test indicators and methods:

[0057] In vivo stability: activity retention rate of lactase in simulated gastric juice (%).

[0058] pH release performance: Release rate (%) of lactase under different pH conditions.

[0059] Km and Vmax: Affinity and catalytic efficiency of lactase (μmol / min / mg enzyme).

[0060] Lactose breakdown rate: The percentage of lactose broken down within 60 minutes in a simulated lactose environment.

[0061] Half-life: The duration (in hours) of lactase activity measured under in vivo conditions.

[0062] Table 1. Simulation Experiment Results

[0063]

[0064] Data Analysis:

[0065] (1) In vivo stability: The lactase in experimental group C significantly protected lactase activity in the gastric acid environment, with an activity retention rate of up to 92%, which is much higher than that of traditional free lactase and monolayer-encapsulated lactase.

[0066] (2) pH-responsive release: The release efficiency reaches 95% at the front end of the small intestine, ensuring that lactase can be released and act in a timely manner.

[0067] (3) Kinetic optimization: The experimental group C had the lowest Km value and the highest Vmax value, indicating that its affinity for lactose and catalytic efficiency were significantly enhanced.

[0068] (4) Lactose hydrolysis efficiency: The lactose decomposition rate reaches 98%, almost completely hydrolyzing lactose, which is suitable for the treatment of lactose intolerance.

[0069] (5) Extended half-life: The double-layer encapsulation technology extends the action time of lactase to 6 hours, greatly improving its bioavailability.

[0070] Functional validation for lactose intolerance

[0071] Objective: To detect the functional effects of lactase nanoparticles in a lactose intolerance model.

[0072] Experimental methods:

[0073] • Mouse model of lactose intolerance: administer lactose solution and measure the water content of stool (indicating lactose breakdown efficiency).

[0074] • Comparison samples: uncoated lactase, chitosan-coated lactase, chitosan + hyaluronic acid lactase.

[0075] Expected data:

[0076]

[0077] Summarize:

[0078] Experimental results show that the method provided by this invention has significant performance improvements compared to traditional methods, especially in terms of lactase stability, targeting, catalytic efficiency, and reaction time. The synergistic optimization of the double-layer encapsulation technology of nanoparticles and the innovative extraction process is key to this performance improvement.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness, characterized in that, The process includes the following: (1) Prepare chitosan solution; (2) Prepare a hyaluronic acid solution; (3) Nanoparticle formation: First, lactase is dissolved in chitosan solution, and sodium tripolyphosphate solution is slowly added dropwise to carry out ionic cross-linking to form chitosan nanoparticles; Next, the formed chitosan nanoparticles were suspended in a hyaluronic acid solution and stirred to form a double-layer composite coating. (4) Particle characterization and detection: The particle size of the nanoparticles is determined by dynamic light scattering, with a target of 100-120 nm. The morphology is observed by transmission electron microscopy to ensure the formation of a uniform bilayer structure.

2. The method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness according to claim 1, characterized in that, The preparation method for step (1) is to prepare a chitosan solution with a w / v of 0.22%, dissolve it in acetic acid with a mass concentration of 0.87%, stir it evenly to form a uniform solution.

3. The method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness according to claim 1, characterized in that, In step (2), a hyaluronic acid solution with a w / v of 0.07% is prepared as the outer coating material.

4. The method for preparing highly bioactive lactase nanoparticles with high vmax and pH responsiveness according to claim 1, characterized in that, In step (3), sodium tripolyphosphate with a mass concentration of 0.08% is added dropwise.

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