A method for stabilizing nattokinase under acidic conditions

Abstract

The present application relates to the field of functional food and biochemistry, and discloses a method for keeping natto kinase stable under acidic conditions. The method comprises the following steps: performing directional enzymolysis and grading purification on konjac glucomannan to obtain low-molecular-weight konjac polysaccharide with a molecular weight of 6000-10000, and performing carboxymethylation on the low-molecular-weight konjac polysaccharide under alkaline conditions to obtain carboxymethyl low-molecular-weight konjac polysaccharide; constructing a weak cross-linking pH response network by combining sodium tripolyphosphate and soluble calcium salt under the condition of pH 6.8-7.2, then diffusing and loading natto kinase into the network and slightly cross-linking and fixing the natto kinase, and finally performing spray drying or freeze drying to obtain capsules or tablets. The carrier shrinks in gastric acid to block acid and protease, and expands in the intestinal tract to release natto kinase. Simulation gastrointestinal tests show that the preparation can significantly improve the residual enzyme activity of natto kinase under acidic conditions, and achieve high-proportion release in the intestinal tract.

Description

Technical Field

[0001] This invention relates to the fields of functional foods and biochemistry, specifically a method for stabilizing nattokinase under acidic conditions. Background Technology

[0002] Nattokinase is a serine protease isolated from natto. It possesses strong fibrinolytic activity and good oral safety, and is widely used in functional foods and health supplements to improve blood rheology and aid in the prevention of thrombosis-related diseases. For ease of administration and long-term stability, common nattokinase formulations on the market include powders, capsules, tablets, and beverages.

[0003] However, nattokinase is essentially a protein enzyme preparation, which is highly susceptible to conformational destruction and hydrolytic inactivation in the acidic environment of the stomach (pH 1.2) and the presence of pepsin. If it is simply encapsulated or tableted with ordinary excipients, most of the enzyme activity will be destroyed in the stomach, resulting in a limited amount of effective enzyme entering the small intestine, which seriously affects the bioavailability and functional efficacy of oral formulations. To improve its acid resistance, existing technologies often use enteric-coated capsules or enteric-coated tablets, coating the tablet core or capsule with a pH-sensitive polymer film to minimize disintegration in the stomach and re-dissolve under the pH conditions of the small intestine. While this approach improves the direct exposure of the enzyme to gastric acid to some extent, it has drawbacks such as the brittleness of the coating film, its susceptibility to cracking under mechanical stress, difficulty in uniformly controlling film thickness, significant individual variations in dissolution time, and the need for organic solvents in some processes. Therefore, its protection of the activity of temperature- and environment-sensitive proteases remains limited.

[0004] For example, Chinese invention application CN 119924506 A discloses an anti-acid natto powder and its preparation method. It discloses that by adding a high concentration of salt ions to the nattokinase fermentation broth and combining it with hydrophobic polymers such as sodium polyacrylate to form an encapsulation system, the anti-acidity of nattokinase can be improved. However, this type of technology mainly relies on the hydrophobic aggregation of high salt-synthetic polymers and does not make fine designs for the molecular weight window, carboxyl substitution degree and reversible pH response network structure of edible natural polysaccharides. It is difficult to achieve efficient and controllable release in the intestinal segment while ensuring gastric protection.

[0005] Besides enteric coating, some technologies have explored using natural polysaccharides such as alginate, chitosan, starch, and pectin to construct microcapsules or gel carriers, aiming to improve the gastric acid stability of nattokinase through physical barrier and pH-responsive properties. Some literature also discusses the use of konjac glucomannan as dietary fiber or thickener in food or pharmaceutical preparations. Traditional konjac polysaccharides are typically high molecular weight, broadly distributed systems with high solution viscosity, easily forming strong gels or highly viscous systems. While they possess certain film-forming and encapsulation capabilities, practical applications often suffer from: poor solution fluidity, hindering industrial granulation processes such as spray drying; overly dense or heterogeneous gel networks, allowing gradual penetration of acidic gastric media, resulting in limited enzyme activity protection; and incomplete release in the intestines due to excessive cross-linking or network rigidity, making it difficult to simultaneously meet the dual requirements of "gastric protection" and "full intestinal release."

[0006] Furthermore, existing polysaccharide-based carrier designs largely focus on "selecting a certain natural polysaccharide + simple cross-linking," with less attention paid to: (1) precise control of the polysaccharide molecular weight and distribution window; (2) the influence of the degree of introduction of carboxyl-based ionic groups on pH-responsive stretching behavior; and (3) the balance between the amount of cross-linking agent and the strength of divalent cations on the network's reversibility and flexibility. For nattokinase, which is extremely sensitive to gastric acid, existing publicly available technologies still lack targeted solutions for how to "embed" the enzyme in a three-dimensional polysaccharide network that can contract and block under acidic conditions and recover and rapidly release under neutral conditions, without using high temperatures and organic solvents.

[0007] Therefore, there is an urgent need to propose a new method for stabilizing oral nattokinase formulations. This method involves selecting edible and safe polysaccharide carriers as raw materials and constructing a network structure with reversible pH-responsive behavior by precisely controlling its molecular weight, degree of substitution, and degree of cross-linking. This allows nattokinase to be fully protected in the acidic environment of the stomach and to be released in a high proportion and in a controlled manner under intestinal pH conditions. At the same time, it takes into account the mildness of the preparation process and the feasibility of industrialization, thereby solving the problems of insufficient acid stability, incomplete release, and poor process adaptability in existing technologies.

[0008] Therefore, this invention proposes a method to stabilize nattokinase under acidic conditions, in order to solve the problems mentioned above. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a method for maintaining the stability of nattokinase under acidic conditions, thereby solving the problems mentioned in the background section.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] S1. Dissolve and enzymatically hydrolyze konjac glucomannan to reduce the number-average molecular weight to a certain level. ;

[0012] S2. The enzymatic hydrolysis products were fractionated and purified to obtain low molecular weight konjac polysaccharide LM-KGM with a number average molecular weight of 6000-10000.

[0013] S3. The LM-KGM obtained in step S2 is subjected to carboxymethylation, and the degree of carboxymethyl substitution is controlled to be 0.15-0.25, preferably 0.18-0.22, to obtain CM-LM-KGM;

[0014] S4. CM-LM-KGM was weakly cross-linked with food-grade polyphosphate under pH conditions of 6.5–7.5;

[0015] S5. Add nattokinase at a temperature not exceeding 20°C and a pH of 6.5–7.5 to allow it to diffuse into the pre-network;

[0016] S6. Add polyphosphate cross-linking agent or edible calcium salt;

[0017] S7. The resulting composite system is dried and prepared into an oral formulation.

[0018] In the above steps, the enzymatic hydrolysis step of konjac glucomannan uses hemicellulase or β-mannanase, with the enzyme dosage being 0.1% to 2.0% of the mass of konjac glucomannan, and the hydrolysis time being 1 to 3 hours.

[0019] In step S2, the fractional purification process involves removing high molecular weight components with 30%–60% ethanol by volume, precipitating the target components with 60%–80% ethanol by volume, and then separating them twice using an ultrafiltration membrane with a molecular weight cutoff of 3–10 kDa.

[0020] The food-grade polyphosphate buffer or citrate buffer in step S4.

[0021] The polyphosphate crosslinking agent mentioned in step S6 is sodium tripolyphosphate, and the edible calcium salt is calcium chloride or calcium lactate.

[0022] In step S4, the molar ratio of food-grade polyphosphate to CM-LM-KGM anhydrous glucose unit is 0.02 to 0.10.

[0023] In step S5, the mass ratio of nattokinase to CM-LM-KGM is 1:3 to 1:8.

[0024] The reaction temperature in step S6 shall not exceed 20°C throughout the entire process, and the pH of the system shall be maintained at 6.5–7.5.

[0025] The drying process employs either spray drying or freeze drying. For spray drying, the inlet air temperature is 80–120°C and the outlet temperature is no higher than 40°C. For freeze drying, the pre-freezing temperature is -40°C to -20°C.

[0026] This invention provides a method for stabilizing nattokinase under acidic conditions. It has the following beneficial effects:

[0027] 1. This invention involves targeted enzymatic hydrolysis of konjac glucomannan, combined with ethanol fractionation and membrane separation, retaining only the low molecular weight range with a number average molecular weight of 6000–10000. Further, carboxymethylation is controlled to maintain the degree of substitution between 0.15 and 0.25, resulting in a CM-LM-KGM carrier with suitable rheological properties and pH response characteristics. This carrier readily shrinks under acidic conditions and expands sufficiently under neutral conditions, thus significantly improving the stability of nattokinase in the acidic environment of the stomach.

[0028] 2. This invention, under pH 6.5–7.5 conditions, adds food-grade polyphosphates such as sodium tripolyphosphate at a low molar ratio, along with an appropriate amount of edible calcium salt, to form a weakly cross-linked three-dimensional network structure. This structure can shrink under acidic conditions and re-expand under neutral conditions, enabling the resulting CM-LM-KGM carrier to function effectively in the gastric region. It forms a physical barrier with pepsin and re-stretches in the intestinal pH environment to promote the release of nattokinase.

[0029] 2. This invention first constructs a weakly cross-linked pre-network with good fluidity, then adds nattokinase at a temperature not exceeding 20°C and a pH of 6.5–7.5, allowing it to diffuse into the pre-network. Subsequently, a small amount of cross-linking agent or calcium salt is added for mild cross-linking fixation, resulting in a composite dispersion in which nattokinase is uniformly embedded within the network. This method avoids simple surface coating and reduces the damage to enzyme activity caused by localized high shear and pH abrupt changes. Attached Figure Description

[0030] Figure 1 The remaining nattokinase activity of free nattokinase aqueous solution and samples from Examples 1, 2, Comparative Example 1, and Comparative Example 2 after 2 hours in simulated gastric juice;

[0031] Figure 2 The cumulative release of nattokinase is measured in samples from Examples 1, 2, Comparative Examples 1, and 2 of the present invention after being transferred to simulated intestinal fluid 2 hours later in simulated gastric fluid. Detailed Implementation

[0032] To enable those skilled in the art to understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0033] The present invention will now be described in detail with reference to the accompanying drawings:

[0034] Example 1 provides a method for stabilizing nattokinase under acidic conditions, and the specific implementation method is as follows:

[0035] 1. Preparation of low molecular weight konjac polysaccharide LM-KGM

[0036] (1) Raw material dissolution and pretreatment

[0037] 100 g of high molecular weight konjac glucomannan powder was weighed and added to 10 L of deionized water. The mixture was stirred at 50 °C for 2 h to ensure complete dissolution. The pH was then adjusted to 7.0, and the solution was filtered through a 100-mesh sieve to remove insoluble impurities. A homogeneous and transparent aqueous solution of konjac polysaccharide with a concentration of 1% was finally obtained.

[0038] (2) Targeted enzymatic hydrolysis

[0039] β-Mannanase was added to the above solution at a rate of 0.5% of the mass of the konjac glucomannan powder. The reaction was carried out at 50°C and pH 7.0 for 2 h, with stirring maintained and samples taken every 30 min to monitor molecular weight changes. When the number-average molecular weight (Mn) decreased to... The reaction system was heated to 90°C and maintained for 10 min to inactivate the enzyme, followed by cooling to room temperature. This yielded an enzymatically hydrolyzed konjac polysaccharide solution with a significantly reduced number-average molecular weight.

[0040] This step can reduce the molecular weight while maintaining the continuity of the main chain structure, laying the foundation for subsequent fractionation to obtain the target low molecular weight segment.

[0041] (3) Fractional purification

[0042] Ethanol was added to the enzymatic hydrolysis solution to adjust the ethanol volume fraction to 40%. After stirring for 30 min, the mixture was allowed to stand for 1 h, and centrifuged to remove the precipitate and remove high molecular weight components. Ethanol was then added to the supernatant to bring the ethanol volume fraction to 70%, and the mixture was allowed to stand for 1 h before centrifugation to collect the precipitate. The precipitate was washed twice with 70% ethanol and then vacuum dried. The dried product was redissolved in water and ultrafiltered sequentially using 10 kDa and 3 kDa membranes to remove residual high molecular weight components and concentrate the target component.

[0043] Finally, 60 g of low molecular weight konjac polysaccharide LM-KGM was obtained, with a number average molecular weight of 8000 and a narrow molecular weight distribution.

[0044] This step combines ethanol fractionation with membrane separation to precisely extract the target molecular weight range, forming low molecular weight konjac polysaccharides that are easy to construct controllable network units.

[0045] 2. Preparation of carboxymethyl low molecular weight konjac polysaccharide CM-LM-KGM

[0046] Weigh 50 g of konjac polysaccharide LM-KGM and add it to 500 mL of deionized water. After dissolving by stirring, add 15 g of sodium hydroxide and stir at 25 °C for 30 min. Then add 20 g of sodium chloroacetate, maintain the reaction temperature at 25 °C, and continue stirring for 4 h. After the reaction is complete, adjust the pH to 7.0 using dilute hydrochloric acid. Place the solution in a dialysis bag with a molecular weight cutoff of 3 kDa and dialyze for 48 h, changing the deionized water periodically during this period. After dialysis, add 70% ethanol (v / v) to precipitate the target product, collect the precipitate by centrifugation, and vacuum dry at 40 °C.

[0047] 55 g of carboxymethyl low molecular weight konjac polysaccharide CM-LM-KGM was obtained, and its degree of carboxymethyl substitution was determined to be DS=0.20.

[0048] This step introduces an appropriate amount of carboxymethyl side groups onto the low molecular weight backbone, making CM-LM-KGM easy to coil under acidic conditions and easy to stretch under neutral conditions, thus providing a structural basis for constructing a pH-responsive network.

[0049] 3. Construction of pH-responsive pre-network

[0050] 10 g of CM-LM-KGM was weighed and dissolved in 500 mL of pH 7.0 phosphate buffer to prepare a 2.0% (w / w) solution. The solution was stirred at room temperature for 1 h to ensure complete dissolution. Then, an aqueous solution of sodium tripolyphosphate (STPP) was added to achieve a molar ratio of STPP to anhydrous glucose units of CM-LM-KGM of 0.05. The solution was stirred at 20 °C for 20 min. During this time, the viscosity of the system increased slightly but remained fluid. A weakly cross-linked CM-LM-KGM pre-network solution was obtained.

[0051] This step constructs a pre-network with a certain three-dimensional structure but low cross-linking degree, creating a suitable space for the subsequent diffusion and embedding of nattokinase.

[0052] 4. Diffusion loading and mosaic fixation of nattokinase

[0053] (1) Diffusion loading

[0054] The weakly cross-linked CM-LM-KGM pre-network solution was cooled to 10°C, the pH was adjusted to 7.0, and 10000 FU / g of lyophilized nattokinase powder was added, maintaining a mass ratio of nattokinase to the weakly cross-linked CM-LM-KGM pre-network solution of 1:5. The solution was stirred at 10°C for 30 min and then allowed to stand for 30 min to allow nattokinase to fully diffuse into the pre-network. A nattokinase-pre-network composite solution was obtained.

[0055] This step enables the diffusion of nattokinase into the polysaccharide network under low-temperature, neutral, and organic solvent-free conditions, avoiding simple surface coating and reducing enzyme activity loss.

[0056] (2) Weak crosslinking fixation

[0057] STPP aqueous solution was added to the above nattokinase-pre-network complex solution, and then further STPP aqueous solution was added to increase the molar ratio of STPP to CM-LM-KGM anhydrous glucose units from 0.05 to 0.08, and 0.2% of the solution was added. The stock solution makes the system The final concentration was 2 mmol / L. The mixture was stirred at 10℃ for 20 min, at which point the system exhibited a distinct three-dimensional network characteristic while still maintaining a sprayable rheological state. A nattokinase-CM-LM-KGM composite dispersion was obtained, in which nattokinase was embedded and immobilized within a weakly cross-linked pH-responsive network.

[0058] This step locks nattokinase in a reversibly coiled / stretched network structure through weak cross-linking, providing a basis for subsequent gastric acid protection and intestinal release.

[0059] 5. Drying and capsule preparation

[0060] The above-mentioned nattokinase-CM-LM-KGM composite dispersion was spray-dried with an inlet air temperature of 100°C and an outlet air temperature of 38°C, and a spray pressure of 0.6 MPa. The resulting powder was collected. The collected powder was mixed evenly with trehalose and microcrystalline cellulose at a mass ratio of 70:15:15, and then filled into No. 0 hard capsules under standard conditions. Each capsule contained 2000 FU of nattokinase activity. This yielded the nattokinase-CM-LM-KGM oral capsule formulation. This step facilitates oral administration and industrial production while maintaining the internal pH-responsive mosaic network structure.

[0061] Example 2 provides a method for stabilizing nattokinase under acidic conditions, and the specific implementation method is as follows:

[0062] This embodiment has the same overall steps as Example 1, including: preparation of low molecular weight konjac polysaccharide LM-KGM → preparation of carboxymethyl low molecular weight konjac polysaccharide CM-LM-KGM → construction of pH-responsive pre-network → diffusion loading and embedding of nattokinase → drying and preparation.

[0063] Except for the parameter adjustments described below, the other operating conditions are the same as in Example 1.

[0064] 1. Preparation of low molecular weight konjac polysaccharide LM-KGM

[0065] The basic process route in this step is the same as in Example 1, which includes three sub-steps: "raw material dissolution and pretreatment – ​​directional enzymatic hydrolysis – fractional purification". The only adjustment is to the specific conditions as follows:

[0066] (1) Raw material dissolution and pretreatment

[0067] The procedure for this step is the same as in Example 1, except that the solvent volume and dissolution conditions are adjusted. Weigh 100 g of high molecular weight konjac glucomannan powder, add it to 8 L of deionized water, stir at 45°C for 3 h to fully dissolve it, then adjust the pH to 7.0, and filter it with a 100-mesh sieve to remove insoluble impurities, finally obtaining a konjac polysaccharide aqueous solution with a concentration of 1.25%.

[0068] (2) Targeted enzymatic hydrolysis

[0069] The enzymatic hydrolysis method in this step is the same as in Example 1, only the amount of enzyme added and the reaction conditions are adjusted. β-mannanase is added to the above solution at an amount of 0.8% of the konjac polysaccharide mass; the reaction is carried out at 48℃ and pH 7.0 for 1.5 h, with stirring maintained and the molecular weight change monitored regularly; when the number average molecular weight Mn drops to The reaction system was heated to 90°C and maintained for 10 min to inactivate the enzyme, and then cooled to room temperature to obtain an enzymatic hydrolysate of konjac polysaccharide.

[0070] (3) Fractional purification

[0071] The grading process is the same as in Example 1, including ethanol precipitation and membrane separation, only the ethanol volume fraction and membrane retention conditions are adjusted: ethanol is added to the enzymatic hydrolyzed konjac polysaccharide solution, the ethanol volume fraction is adjusted to 50%, the mixture is stirred for 30 min and then allowed to stand for 1 h, and the precipitate is removed by centrifugation to remove the high molecular weight components.

[0072] Ethanol was added to the supernatant until the ethanol volume fraction reached 75%. After standing for 1 hour, the precipitate was collected by centrifugation. The precipitate was washed twice with 75% ethanol and then vacuum dried. The dried product was dissolved in water and then subjected to ultrafiltration using 5 kDa and 3 kDa membranes to remove residual high molecular weight components and concentrate the target component. The low molecular weight konjac polysaccharide LM-KGM was finally obtained, with a number-average molecular weight (Mn) of 7500.

[0073] This step, by adjusting the enzymatic hydrolysis intensity and fractionation conditions, yielded LM-KGM with a slightly lower molecular weight compared to Example 1, making it easier to examine the effect of molecular weight on the performance of the carrier network.

[0074] 2. Preparation of carboxymethyl low molecular weight konjac polysaccharide CM-LM-KGM

[0075] The carboxymethylation reaction process in this step is the same as in Example 1, except that the amount of raw materials and the target degree of substitution are adjusted: 40 g of konjac polysaccharide LM-KGM is weighed and added to 400 mL of deionized water. After stirring and dissolving, 12 g of solid sodium hydroxide is added and stirred at 20°C for 30 min. Then, sodium chloroacetate is added in the same manner as in Example 1, with an addition amount of 16 g, and the carboxymethylation reaction is carried out. By adjusting the amount of sodium chloroacetate added, the final degree of carboxymethyl substitution (DS) is 0.18. After the reaction, the pH is adjusted to 7.0 using dilute hydrochloric acid, and the solution is dialyzed for 48 h in a dialysis bag with a molecular weight cutoff of 3 kDa. The water exchange conditions are the same as in Example 1. Then, 70% ethanol is added to precipitate the target product, which is then centrifuged and vacuum dried at 40°C. Carboxymethyl low molecular weight konjac polysaccharide CM-LM-KGM is obtained with a DS of 0.18.

[0076] This step, by reducing the degree of substitution (DS=0.20 in Example 1), yields a polysaccharide carrier with a slightly different pH response, which is used to verify the effect of the degree of substitution window on the curl-stretch behavior.

[0077] 3. Construction of pH-responsive pre-network and loading of nattokinase

[0078] The overall approach of this step is the same as in Example 1, except that the buffer system, degree of crosslinking, and type and concentration of calcium salt are adjusted.

[0079] 10 g of CM-LM-KGM was dissolved in citrate buffer at pH 7 to prepare a 1.5% solution. The solution was stirred at room temperature for 1 h to ensure complete dissolution. An aqueous STPP solution was added to make the molar ratio of STPP to anhydrous glucose units of CM-LM-KGM 0.04. The solution was stirred at 20 °C for 20 min to obtain a weakly cross-linked CM-LM-KGM pre-network solution.

[0080] Compared to Example 1, this step reduces the polysaccharide concentration and the amount of crosslinking agent, which is beneficial for forming a softer pre-network structure.

[0081] 4. Diffusion loading and mosaic fixation of nattokinase

[0082] (1) Diffusion loading

[0083] The pre-network solution was cooled to 10°C and the pH was adjusted to 7. Nattokinase lyophilized powder with an activity of 10000 FU / g was added to make the mass ratio of nattokinase to CM-LM-KGM NK:CM-LM-KGM = 1:3. The solution was stirred at 10°C for 30 min and then allowed to stand for 30 min to allow the nattokinase to fully diffuse into the pre-network, thus obtaining the nattokinase-pre-network composite solution.

[0084] Compared to Example 1, this step increases the ratio of nattokinase to the carrier, making it easier to examine the effect of increased drug loading on release behavior and stability.

[0085] (2) Weak crosslinking fixation

[0086] In the nattokinase-pre-network composite solution, STPP aqueous solution was added to increase the total molar ratio of STPP to anhydrous glucose units of CM-LM-KGM from 0.04 to 0.07; at the same time, 0.2 mol / L calcium lactate stock solution was added to make the final concentration of calcium lactate in the system 1 mmol / L; the mixture was stirred at 10℃ for 20 min to form a nattokinase-CM-LM-KGM composite dispersion with three-dimensional network characteristics and still flowable.

[0087] Compared to Example 1, this step uses a lower degree of crosslinking and a lower concentration of calcium salt, and changes the calcium source from calcium chloride to calcium lactate to obtain a carrier structure with a softer network and slightly different expansion behavior.

[0088] 5. Freeze-drying and tableting

[0089] This step corresponds to "drying and capsule preparation" in Example 1, but uses freeze-drying and tablet forming processes:

[0090] (1) Freeze-drying

[0091] The nattokinase-CM-LM-KGM composite dispersion was dispensed into flat-bottomed trays with a liquid layer thickness of 10 mm. It was pre-frozen at -40℃ for 4 h to completely freeze the dispersion. Then it was placed in a freeze dryer and sublimated under a vacuum of <100 Pa. During the drying process, the plate temperature was gradually increased from -20℃ to 20℃, and the total drying time was 36 h. The resulting porous composite freeze-dried block was then removed.

[0092] (2) Crushing and mixing

[0093] The freeze-dried blocks were crushed in a pulverizer and passed through a 60-mesh sieve. The crushed freeze-dried blocks, soluble starch and glycine were mixed evenly at a mass ratio of 70:20:10. Magnesium stearate, accounting for 0.5% of the total powder mass, was added as a lubricant and the mixture was continued to be mixed evenly.

[0094] (3) Tableting

[0095] The mixed powder is compressed into tablets using a rotary tablet press. The compression pressure is adjusted according to the tablet hardness requirements. Each tablet contains 2000 FU of nattokinase activity to obtain nattokinase-CM-LM-KGM oral tablets.

[0096] This step preserves the porous structure of the composite network through freeze-drying and uses tablets instead of capsules, demonstrating that the method of the present invention still has good process adaptability when carrier parameters and drying methods are changed.

[0097] Comparative Example 1: This comparative example provides a formulation that was not prepared by controlling the molecular weight to a low molecular weight range through "directional enzymatic hydrolysis + fractional purification", and the resulting formulation had significantly poor acid stability.

[0098] This comparative example omits steps (2) "directional enzymatic hydrolysis" and (3) "gradual purification" in step 1 of Example 1, does not control the molecular weight, and directly treats the high molecular weight konjac polysaccharide after raw material dissolution and pretreatment according to the carboxymethylation conditions in step 2 of Example 1 to obtain high molecular weight carboxymethyl konjac polysaccharide CM-KGM, with Mn in the hundreds of thousands or more.

[0099] The high molecular weight carboxymethyl konjac polysaccharide CM-KGM was dissolved in a pH 7.0 buffer solution to a mass concentration of 1.0%. Due to the high viscosity of the solution, only slow stirring was possible. STPP and [other ingredients] were added according to the conditions of Example 1. A network was constructed, and nattokinase was added at 10°C, controlling the mass ratio of NK:CM-KGM to 1:5. Crosslinking was then continued to obtain a nattokinase-CM-KGM dispersion. Capsules were prepared using the same spray-drying conditions as in Example 1.

[0100] Comparative Example 2 is used to illustrate that if the degree of cross-linking is not controlled and the degree of network cross-linking is set too high, the characteristics of the present invention will be destroyed and the intestinal release will be affected.

[0101] Carrier preparation: The same carboxymethyl low molecular weight konjac polysaccharide CM-LM-KGM was prepared according to steps 1 and 2 of Example 1, with Mn=8000 and DS=0.20.

[0102] The pH-responsive pre-network was constructed, and then CM-LM-KGM was dissolved in pH 7.0 phosphate buffer to prepare a 2.0% (w / w) solution. STPP was added at room temperature to achieve a molar ratio of STPP to anhydrous glucose units of CM-LM-KGM of 0.20, and 0.2% (w / w) of [unspecified ingredient] was added. The stock solution makes the system The final concentration was 2 mmol / L, and crosslinking was performed for 40 min. The system rapidly formed a relatively rigid gel block, which was difficult to maintain fluidity. After mechanically dispersing the gel block, 10000 FU / g of lyophilized nattokinase powder was added at 10 °C, controlling the mass ratio of nattokinase to gel block at 1:5, and the mixture was shaken for a short time.

[0103] A dispersion system in which nattokinase is partially coated on the surface of highly cross-linked CM-LM-KGM gel particles or has limited access to the network was obtained, and then capsules were prepared by spray drying according to the method of Example 1.

[0104] To verify the effectiveness of the method for stabilizing nattokinase under acidic conditions according to the present invention, the following samples were selected for comparative evaluation in this experiment, with reference to... Figure 1 and Figure 2 :

[0105] Example 1: Nattokinase-CM-LM-KGM oral capsules;

[0106] Example 2: Nattokinase-CM-LM-KGM oral tablets;

[0107] Comparative Example 1: Nattokinase-High Molecular Weight CM-KGM Oral Capsules;

[0108] Comparative Example 2: Nattokinase-highly cross-linked CM-LM-KGM oral capsules;

[0109] Control: Free nattokinase aqueous solution.

[0110] Each sample was taken according to the dosage indicated on the formulation, so that the nattokinase activity in each sample was 2000 FU.

[0111] 2. Test Methods

[0112] (1) Simulated gastric juice stage

[0113] Each sample was added to simulated gastric fluid at pH 1.2 and incubated at 37°C with shaking for 2 hours. During this period, samples were taken to measure the remaining enzyme activity of nattokinase. Free nattokinase aqueous solution served as a control and was treated under the same conditions. The percentage of remaining enzyme activity of each sample after 2 hours under acidic gastric conditions was recorded. This was used to compare the protective ability of different carrier structures against nattokinase in an acidic environment.

[0114] (2) Simulated intestinal fluid stage

[0115] After incubation for 2 hours, each sample system was transferred to simulated intestinal fluid at pH 6.8 and incubated with shaking at 37°C for another 3–4 hours. Samples were taken at preset time points of 1 hour, 2 hours, 3 hours, and 4 hours to determine the nattokinase activity in the supernatant and calculate the percentage of cumulative release relative to the theoretical total. Release curves for each sample under neutral conditions and the cumulative release percentage at 3 or 4 hours were obtained. This was used to compare the ability of different carrier network structures to release nattokinase in the intestinal environment and whether effective release could be achieved after acid protection.

[0116] The results are shown in the table below:

[0117]

[0118] Note: "Remaining enzyme activity / %" means: measured enzyme activity of the sample after incubation in simulated gastric fluid for 2 h / initial enzyme activity before incubation × 100%.

[0119] As shown in Table 1, the formulations prepared in Examples 1 and 2 retained 80%–88% of their nattokinase activity after 2 hours in simulated gastric fluid, significantly better than Comparative Examples 1 and 2, while free nattokinase only retained 5%. This indicates that the low molecular weight, narrowly distributed carboxymethyl konjac polysaccharide carrier and weakly cross-linked pH-responsive network used in this invention have a significant protective effect on nattokinase under acidic conditions.

[0120] The cumulative release (%) of nattokinase in simulated intestinal fluid is as follows:

[0121]

[0122] As shown in Tables 1 and 2, free nattokinase was almost completely inactivated after incubation in simulated gastric juice at pH 1.2 for 2 h, with only 5% remaining enzyme activity. In contrast, the formulations prepared in Examples 1 and 2 of this invention showed residual enzyme activities of 88% and 85% respectively under the same conditions, significantly higher than 35% in Comparative Example 1 and 28% in Comparative Example 2. This indicates that by controlling the molecular weight of konjac polysaccharide to 6000–10000 and performing moderate carboxymethylation, combined with a weakly cross-linked pH-responsive network structure, the stability of nattokinase can be significantly improved under acidic conditions.

[0123] Furthermore, after incubating the above samples in simulated intestinal fluid at pH 6.8 for 3–4 h, the cumulative release of Examples 1 and 2 reached 80%–86%, while Comparative Examples 1 and 2 only reached 32%–38% and 16%–20%, respectively. This indicates that the carrier network of the present invention can fully expand and release almost all of the protected nattokinase under intestinal pH conditions, while the control network with uncontrolled molecular weight or excessive cross-linking has problems such as insufficient enzyme activity protection or incomplete release.

[0124] Therefore, by comprehensively regulating the molecular weight, carboxymethyl substitution degree, and cross-linking degree of konjac polysaccharide, this invention achieves high stability of nattokinase under gastric acid conditions and high-proportion release in the intestinal environment, obtaining a comprehensive technical effect that is significantly better than the control scheme.

[0125] It should be particularly noted that the various embodiments listed in this specification and accompanying drawings are intended to illustrate the technical solutions and advantages of the present invention, and not to limit the scope of protection of the present invention. Without departing from the core ideas and technical effects of the present invention, those skilled in the art can make any form of improvement, substitution, combination, or modification to the structural arrangement, process parameters, material selection, control logic, etc., of the described embodiments; any obvious changes based on the same concept should be considered equivalent solutions of the present invention and should be included within the scope of protection defined by the claims of the present invention. The actual scope of protection of the present invention is determined by the appended claims and should be correctly understood in conjunction with the specification and accompanying drawings.

Claims

1. A method for stabilizing nattokinase under acidic conditions, characterized in that, Includes the following steps: S1. Dissolve and enzymatically hydrolyze konjac glucomannan to reduce the number-average molecular weight to a certain level. ; S2. The enzymatic hydrolysis products were fractionated and purified to obtain low molecular weight konjac polysaccharide LM-KGM with a number average molecular weight of 6000-10000 Da. S3. The LM-KGM obtained in step S2 is subjected to carboxymethylation, and the degree of carboxymethyl substitution is controlled to be 0.15-0.25 to obtain CM-LM-KGM; S4. CM-LM-KGM is weakly cross-linked with food-grade polyphosphate under pH conditions of 6.5 to 7.5 to form a weakly cross-linked CM-LM-KGM pre-network solution; S5. Add nattokinase at a temperature not exceeding 20°C and a pH of 6.5–7.5 to allow it to diffuse into the pre-network; S6. Add polyphosphate cross-linking agent or edible calcium salt; S7. The obtained composite system is dried and prepared into an oral formulation; The polyphosphate crosslinking agent mentioned in step S6 is sodium tripolyphosphate, and the edible calcium salt is calcium chloride or calcium lactate; In step S4, the molar ratio of food-grade polyphosphate to CM-LM-KGM anhydrous glucose unit is 0.02 to 0.

10. In step S5, the mass ratio of nattokinase to CM-LM-KGM is 1:3 to 1:

8.

2. The method according to claim 1, characterized in that, In the enzymatic hydrolysis of konjac glucomannan, hemicellulase or β-mannanase is used, with the enzyme dosage being 0.1% to 2.0% of the mass of konjac glucomannan, and the hydrolysis time being 1 to 3 hours.

3. The method according to claim 1, characterized in that, In step S2, the fractional purification process involves removing high molecular weight components with 30%–60% ethanol by volume, precipitating the target components with 60%–80% ethanol by volume, and then separating them twice using an ultrafiltration membrane with a molecular weight cutoff of 3–10 kDa.

4. The method according to claim 1, characterized in that, The degree of carboxymethyl substitution is 0.18–0.

22.

5. The method according to claim 1, characterized in that, The reaction temperature in step S6 shall not exceed 20°C throughout the entire process, and the pH of the system shall be maintained at 6.5–7.

5.

6. The method according to claim 1, characterized in that, The drying process employs either spray drying or freeze drying. For spray drying, the inlet air temperature is 80–120°C and the outlet temperature is no higher than 40°C. For freeze drying, the pre-freezing temperature is -40°C to -20°C.

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