A deamidated cottonseed protein stabilized lipase pickering emulsion and a method of making the same

Abstract

The application discloses a Pickering emulsion of deamidated cottonseed protein stabilizing lipase, which is composed of an aqueous phase, an oil phase, a surfactant and a lipase; the oil phase is medium-chain triglyceride, and the volume ratio is 5-10%; the surfactant is deamidated cottonseed protein, and the concentration in the Pickering emulsion is 0.1-10 mg / mL. The preparation method is as follows: the deamidated cottonseed protein is added into deionized water, and is stirred and dissolved on a magnetic stirrer to prepare a uniform aqueous phase dispersion; the medium-chain triglyceride is added into the aqueous phase dispersion, and is subjected to shearing homogenization treatment to obtain a primary emulsion; the lipase liquid is added into the primary emulsion after the homogenization treatment, and is subjected to shearing homogenization emulsification to obtain the Pickering emulsion of deamidated cottonseed protein stabilizing lipase. The application has the advantages of simple preparation process, low requirement on instruments, easy operation and scale-up, green and safe raw materials, and wide application prospect of the prepared Pickering emulsion in the fields of targeted delivery of nutrients, modification of food structure and function, and biological catalytic reaction carrier.

Description

Technical Field

[0001] This invention belongs to the field of emulsion preparation technology, and more specifically, this invention relates to a Pickering emulsion of deamidated cottonseed protein-stabilized lipase and its preparation method. Background Technology

[0002] Lipases (EC 3.1.1.3) are highly efficient biocatalysts for the hydrolysis of triglycerides, possessing advantages such as high substrate specificity, mild reaction conditions, and environmental friendliness. They are widely used in food processing, bioenergy, and pharmaceutical synthesis, and market demand continues to grow. Their molecular structure contains a catalytic triplet and exhibits a unique "interfacial activation" characteristic—the active site in the aqueous system is sealed by a hydrophobic "cap," which opens upon contact with the oil-water interface, significantly enhancing catalytic activity. This characteristic provides a key approach for optimizing their performance through interfacial modulation.

[0003] However, the practical application of lipases is severely limited by stability issues: protein structures are easily affected by external factors such as temperature, pH, and organic solvents, leading to conformational changes or aggregation and inactivation; free lipases have poor dispersibility in systems, making it difficult to fully contact the oil-water interface, thus hindering effective interfacial activation, and are also difficult to recover and costly to use. Therefore, developing carrier systems that can stabilize lipase structures and promote interfacial activation has become an urgent problem to be solved in this field.

[0004] Pickering emulsions stabilize using solid particles instead of traditional chemical surfactants, offering significant advantages: the solid particles irreversibly adsorb onto the oil-water interface, forming a physical barrier that imparts excellent stability to the emulsion; the particles at the interface can serve as immobilization carriers for lipases, promoting interface activation and protecting the enzyme structure through steric hindrance, thereby enhancing its catalytic activity and storage stability; moreover, the preparation process is simple and environmentally friendly.

[0005] Choosing the right solid particles is key to preparing high-performance Pickering emulsions. Traditional chemical surfactants have poor biocompatibility and are prone to residue, which does not align with the trend towards natural and healthy products. Natural biomolecules are preferred due to their wide availability, safety, and biodegradability. Deamided cottonseed protein, as a modified product of cottonseed protein, exhibits significant advantages: cottonseed protein is a byproduct of cotton processing, with a crude protein content ≥50% (dry basis), wide availability, and controllable cost. After deamide modification, the hydrophilic-hydrophobic ratio and molecular conformation are optimized, resulting in significantly improved surface activity, solubility, and emulsification.

[0006] Compared to other natural surfactants, deamided cottonseed protein has the advantages of being a green and environmentally friendly raw material, having good biocompatibility, strong surface activity, and low cost. Its macromolecular structure can work synergistically with lipase, effectively solving the pain points of poor stability and low catalytic efficiency of lipase. Summary of the Invention

[0007] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0008] To achieve these objectives and other advantages of the present invention, a Pickering emulsion of deamidated cottonseed protein-stabilized lipase is provided, comprising an aqueous phase, an oil phase, a surfactant, and a lipase. The oil phase is a medium-chain triglyceride, and the oil phase accounts for 5-10% of the volume of the Pickering emulsion containing deamidated cottonseed protein-stabilized lipase. The surfactant is deamidated cottonseed protein, and the concentration of the surfactant in the Pickering emulsion of deamidated cottonseed protein-stabilized lipase is 0.1~10 mg / mL.

[0009] Preferably, the aqueous phase is deionized water and the lipase is a lipase solution.

[0010] A method for preparing a Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described above includes the following steps: Step 1: Add deamided cottonseed protein to deionized water and stir to dissolve on a magnetic stirrer to prepare a uniform aqueous dispersion, namely deamided cottonseed protein solution. Step 2: Add medium-chain triglycerides to the deamidated cottonseed protein solution, and then perform shearing and homogenization to obtain the primary emulsion; Step 3: Add the lipase solution to the homogenized primary emulsion, then shear and homogenize to obtain the Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0011] Preferably, in step one, the mass-to-volume ratio of deamided cottonseed protein to deionized water is 0.1~10 mg:1 mL.

[0012] Preferably, in step one, the stirring speed is 200~600 rpm / min and the stirring time is 20~40 min.

[0013] Preferably, step one is replaced by: dispersing the modified deamidated cottonseed protein in deionized water, stirring it on a magnetic stirrer at a speed of 200-600 rpm / min for 20-40 min, and then sonicating it at a power of 200-400W for 10-20 min to prepare a modified deamidated cottonseed protein dispersion.

[0014] Preferably, the modified deamidated cottonseed protein is prepared as follows: 10-20 parts by weight of deamidated cottonseed protein are dissolved in 100-200 parts by weight of deionized water to obtain a deamidated cottonseed protein solution. The pH is adjusted to 7.8 with 1 mol / L NaOH solution. A maleic anhydride-anhydrous ethanol solution containing 0.5-2 parts by weight of maleic anhydride and 5-10 parts by weight of anhydrous ethanol is slowly added to maintain the pH at 7.8 ± 0.1. The solution is then stirred at 400-600 rpm / min at room temperature for 0.5-2 hours. The solution is then placed in a dialysis bag and dialyzed at 2-8°C for 4-8 hours. 1-2 parts by weight of sodium trimetaphosphate are added to the dialyzed solution to adjust the pH to 8.5. The reaction is allowed to proceed for 2-6 hours. The pH is then adjusted to 4.0-4.5 with 1 mol / L hydrochloric acid solution. After sufficient precipitation, the solution is filtered and the precipitate is vacuum dried at 40-60°C for 4-12 hours to obtain the modified deamidated cottonseed protein.

[0015] Preferably, in step two, the volume ratio of deamidated cottonseed protein solution to medium-chain triglycerides is 15-25:1-3.

[0016] Preferably, in step two, the rotational speed of the shearing homogenizer is 8000~12000 rpm / min, and the shearing homogenizer time is 2~5 min.

[0017] Preferably, in step three, the lipase solution is produced by using Aspergillus niger as the production strain and employing a liquid deep fermentation process; the volume ratio of the lipase solution to the colostrum is 1:8~12.

[0018] Preferably, in step three, the rotational speed of the shearing homogenizer is 8000~12000 rpm / min, and the shearing homogenizer time is 2~5 min.

[0019] More preferably, the medium-chain triglycerides account for 10% of the volume of the Pickering emulsion containing deamidated cottonseed protein-stabilized lipase.

[0020] More preferably, the concentration of the deamided cottonseed protein in the Pickering emulsion of deamided cottonseed protein-stabilized lipase is 5 mg / mL.

[0021] Applications of a Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described above, or a Pickering emulsion of deamidated cottonseed protein-stabilized lipase prepared by the preparation method described above, in the fields of targeted nutrient delivery, food structure and function modification, or biocatalytic reaction carriers.

[0022] This invention offers at least the following advantages: First, it utilizes deamided cottonseed protein (CPH) as a solid particulate surfactant to stabilize lipase, constructing a Pickering emulsion system. The resulting emulsion exhibits uniform particle size, good dispersibility, long-term storage stability, and significantly promotes lipase interfacial activation, enhancing catalytic activity and structural stability. Second, the deamided cottonseed protein used in the emulsion preparation is derived from cotton processing byproducts, making it a green, environmentally friendly, biocompatible, non-toxic, and biodegradable raw material. It can replace traditional chemical surfactants, avoiding residual risks and aligning with the trend towards natural and healthy development. Third, this invention requires minimal equipment; the preparation process only requires dissolution and two shearing homogenization steps, making the method simple, convenient, and cost-effective, facilitating large-scale production. Fourth, this invention provides a novel technical pathway for the efficient and stable application of lipase, with broad application prospects in areas such as targeted nutrient delivery, food structure and function modification, and biocatalytic reaction carriers. It also achieves high-value utilization of agricultural byproducts, aligning with the concept of a circular economy.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1 The images show the appearance of the emulsions prepared in Examples 1-8 and Comparative Examples 1-7 of the present invention after 1 day and 60 days of storage. Figure 2 The Zeta potential diagrams are of the emulsions prepared in Examples 1-4 and Comparative Example 1 of this invention. Figure 3 The Zeta potential diagrams are for the emulsions prepared in Examples 5-8 and Comparative Example 2 of this invention. Figure 4 The Zeta potential diagrams are of the emulsions prepared in Comparative Examples 3-7 of this invention. Figure 5 The average particle size diagrams are of the emulsions prepared in Examples 1-4 and Comparative Example 1 of this invention. Figure 6 The average particle size diagrams are for the emulsions prepared in Examples 5-8 and Comparative Example 2 of this invention. Figure 7 The average particle size diagrams are for the emulsions prepared in Comparative Examples 3-7 of this invention. Figure 8 The surface tension diagrams are for deionized water, lipase, 1 mg / mL CPH solution containing lipase, 2 mg / mL CPH solution containing lipase, 5 mg / mL CPH solution containing lipase, and 10 mg / mL CPH solution containing lipase used in this invention. Figure 9 The enzyme activity retention rate of the emulsions prepared in Examples 5-8 and Comparative Example 2 of this invention; Figure 10 The enzyme activity retention rate is the enzyme activity retention rate of the emulsions prepared in Examples 9-12 and Comparative Example 2 of this invention. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0027] In this invention, the preparation method of deamided cottonseed protein is as follows: 40g of cottonseed protein powder is dispersed in 800ml of 70% anhydrous ethanol aqueous solution, 16g of NaOH is added and stirred evenly, and the reaction is carried out at a constant temperature of 37℃ for 72h; after the reaction is completed, the pH of the system is adjusted to neutral (pH=7) with 5mol / L hydrochloric acid solution, and then spray-dried to obtain deamided cottonseed protein.

[0028] The lipase solution was provided by Mianyang Heben Bioengineering Co., Ltd. It is produced using *Aspergillus niger* as the production strain and a liquid submerged fermentation process. The liquid submerged fermentation process specifically includes the following steps: (1) Shake flask seed culture: Take the Aspergillus niger suspension stored in the glycerol tube and inoculate it into 5 500mL shake flasks containing 200mL seed culture medium at an inoculation volume of 1mL per flask. The seed culture medium formula is as follows: 5g glucose, 2.5g yeast extract, 2.5g peptone, 20g corn steep liquor, 5g soybean meal, 15g potassium dihydrogen phosphate, 1g citric acid, 0.2g calcium chloride, 1000mL water, and adjust the initial pH to 6.0. After inoculation, place the flasks at 34℃ and 200 rpm / min for shaking culture for 40h. (2) Primary seed culture: Combine the five shake flask seed cultures and inoculate them into a 500 L primary seed tank containing 300 L of seed culture medium. Culture at 34℃ and 200 rpm / min for 30 h with shaking. (3) Secondary seed culture: All of the primary seed culture was transferred into a 6T secondary seed tank containing 3T seed culture medium and cultured under shaking at 34℃ and 140rpm / min for 18 h; (4) Fermentation production: All the secondary seed liquid was transferred to a 60 T fermenter containing 30 T seed culture medium. The fermentation temperature was controlled at 34℃, pH at 4.5, and dissolved oxygen at 20-30%. The fermentation culture was carried out for 140-160 h to obtain lipase fermentation broth with an enzyme activity of 60,000 U / mL and a protein content of 20 mg / mL. (5) Post-processing: The fermentation broth is separated into solid and liquid by a plate and frame filter press, the filtrate is collected, and then concentrated by an ultrafiltration system with a molecular weight cutoff of 10 kDa until the enzyme activity of the concentrated enzyme solution is not less than 300,000 U / mL, thus obtaining the lipase solution.

[0029] Example 1 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 20 mg of deamided cottonseed protein to 20 mL of deionized water and stir on a magnetic stirrer at 500 rpm / min for 30 min to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 1 mg / mL. Step 2: Take 17 mL of deamidated cottonseed protein solution, add 1 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0030] Example 2 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 40 mg of deamided cottonseed protein to 20 mL of deionized water and stir at 500 rpm for 30 min on a magnetic stirrer to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 2 mg / mL. Step 2: Take 17 mL of deamidated cottonseed protein solution, add 1 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0031] Example 3 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 100mg of deamided cottonseed protein to 20mL of deionized water and stir at 500rpm / min for 30min on a magnetic stirrer to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 5mg / mL. Step 2: Take 17 mL of deamidated cottonseed protein solution, add 1 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0032] Example 4 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 200 mg of deamided cottonseed protein to 20 mL of deionized water and stir on a magnetic stirrer at 500 rpm / min for 30 min to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 10 mg / mL. Step 2: Take 17 mL of deamidated cottonseed protein solution, add 1 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0033] Example 5 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 20 mg of deamided cottonseed protein to 20 mL of deionized water and stir on a magnetic stirrer at 500 rpm / min for 30 min to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 1 mg / mL. Step 2: Take 16 mL of deamidated cottonseed protein solution, add 2 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0034] Example 6 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 40 mg of deamided cottonseed protein to 20 mL of deionized water and stir at 500 rpm for 30 min on a magnetic stirrer to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 2 mg / mL. Step 2: Take 16 mL of deamidated cottonseed protein solution, add 2 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0035] Example 7 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 100mg of deamided cottonseed protein to 20mL of deionized water and stir at 500rpm / min for 30min on a magnetic stirrer to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 5mg / mL. Step 2: Take 16 mL of deamidated cottonseed protein solution, add 2 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0036] Example 8 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 200 mg of deamided cottonseed protein to 20 mL of deionized water and stir on a magnetic stirrer at 500 rpm / min for 30 min to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 10 mg / mL. Step 2: Take 16 mL of deamidated cottonseed protein solution, add 2 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0037] Example 9 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 20 mg of modified deamidated cottonseed protein to 20 mL of deionized water, stir at 500 rpm / min for 30 min on a magnetic stirrer, and then sonicate at 400 W for 20 min to prepare a modified deamidated cottonseed protein dispersion with a concentration of 1 mg / mL. The modified deamidinated cottonseed protein is prepared as follows: 10 parts by weight of deamidinated cottonseed protein are dissolved in 100 parts of deionized water to obtain a deamidinated cottonseed protein solution. The pH is adjusted to 7.8 with 1 mol / L NaOH solution. A maleic anhydride-anhydrous ethanol solution containing 1 part maleic anhydride and 8 parts anhydrous ethanol is slowly added to maintain the pH at 7.8 ± 0.1. The solution is then stirred at 500 rpm / min for 0.5 h at room temperature. The solution is then placed in a dialysis bag and dialyzed at 8°C for 6 h. 2 parts of sodium trimetaphosphate are added to the dialyzed solution to adjust the pH to 8.5. The reaction is carried out for 3 h. The pH is then adjusted to 4.0-4.5 with 1 mol / L hydrochloric acid solution. After sufficient precipitation, the solution is filtered and the precipitate is vacuum dried at 60°C for 10 h to obtain the modified deamidinated cottonseed protein. Step 2: Take 16 mL of modified deamidated cottonseed protein dispersion, add 2 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0038] Example 10 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 40 mg of modified deamidated cottonseed protein to 20 mL of deionized water, stir at 500 rpm / min for 30 min on a magnetic stirrer, and then sonicate at 400 W for 20 min to prepare a modified deamidated cottonseed protein dispersion with a concentration of 2 mg / mL. The modified deamidinated cottonseed protein is prepared as follows: 10 parts by weight of deamidinated cottonseed protein are dissolved in 100 parts of deionized water to obtain a deamidinated cottonseed protein solution. The pH is adjusted to 7.8 with 1 mol / L NaOH solution. A maleic anhydride-anhydrous ethanol solution containing 1 part maleic anhydride and 8 parts anhydrous ethanol is slowly added to maintain the pH at 7.8 ± 0.1. The solution is then stirred at 500 rpm / min for 0.5 h at room temperature. The solution is then placed in a dialysis bag and dialyzed at 8°C for 6 h. 2 parts of sodium trimetaphosphate are added to the dialyzed solution to adjust the pH to 8.5. The reaction is carried out for 3 h. The pH is then adjusted to 4.0-4.5 with 1 mol / L hydrochloric acid solution. After sufficient precipitation, the solution is filtered and the precipitate is vacuum dried at 60°C for 10 h to obtain the modified deamidinated cottonseed protein. Step 2: Take 16 mL of modified deamidated cottonseed protein dispersion, add 2 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0039] Example 11 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 100 mg of modified deamidated cottonseed protein to 20 mL of deionized water, stir at 500 rpm / min for 30 min on a magnetic stirrer, and then sonicate at 400 W for 20 min to prepare a modified deamidated cottonseed protein dispersion with a concentration of 5 mg / mL. The modified deamidinated cottonseed protein is prepared as follows: 10 parts by weight of deamidinated cottonseed protein are dissolved in 100 parts of deionized water to obtain a deamidinated cottonseed protein solution. The pH is adjusted to 7.8 with 1 mol / L NaOH solution. A maleic anhydride-anhydrous ethanol solution containing 1 part maleic anhydride and 8 parts anhydrous ethanol is slowly added to maintain the pH at 7.8 ± 0.1. The solution is then stirred at 500 rpm / min for 0.5 h at room temperature. The solution is then placed in a dialysis bag and dialyzed at 8°C for 6 h. 2 parts of sodium trimetaphosphate are added to the dialyzed solution to adjust the pH to 8.5. The reaction is carried out for 3 h. The pH is then adjusted to 4.0-4.5 with 1 mol / L hydrochloric acid solution. After sufficient precipitation, the solution is filtered and the precipitate is vacuum dried at 60°C for 10 h to obtain the modified deamidinated cottonseed protein. Step 2: Take 16 mL of modified deamidated cottonseed protein dispersion, add 2 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0040] Example 12 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 200 mg of modified deamidated cottonseed protein to 20 mL of deionized water, stir at 500 rpm / min for 30 min on a magnetic stirrer, and then sonicate at 400 W for 20 min to prepare a modified deamidated cottonseed protein dispersion with a concentration of 10 mg / mL. The modified deamidinated cottonseed protein is prepared as follows: 10 parts by weight of deamidinated cottonseed protein are dissolved in 100 parts of deionized water to obtain a deamidinated cottonseed protein solution. The pH is adjusted to 7.8 with 1 mol / L NaOH solution. A maleic anhydride-anhydrous ethanol solution containing 1 part maleic anhydride and 8 parts anhydrous ethanol is slowly added to maintain the pH at 7.8 ± 0.1. The solution is then stirred at 500 rpm / min for 0.5 h at room temperature. The solution is then placed in a dialysis bag and dialyzed at 8°C for 6 h. 2 parts of sodium trimetaphosphate are added to the dialyzed solution to adjust the pH to 8.5. The reaction is carried out for 3 h. The pH is then adjusted to 4.0-4.5 with 1 mol / L hydrochloric acid solution. After sufficient precipitation, the solution is filtered and the precipitate is vacuum dried at 60°C for 10 h to obtain the modified deamidinated cottonseed protein. Step 2: Take 16 mL of modified deamidated cottonseed protein dispersion, add 2 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0041] Comparative Example 1 A method for preparing a lipase emulsion includes the following steps: Step 1: Take 17 mL of deionized water, add 1 mL of medium-chain triglycerides, and homogenize by shearing at 10000 r / min for 3 min to obtain the primary emulsion. Step 2: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain lipase emulsion.

[0042] Comparative Example 2 A method for preparing a lipase emulsion includes the following steps: Step 1: Take 16 mL of deionized water, add 2 mL of medium-chain triglycerides, and homogenize by shearing at 10000 r / min for 3 min to obtain the primary emulsion. Step 2: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain lipase emulsion.

[0043] Comparative Example 3 A method for preparing a lipase emulsion includes the following steps: Step 1: Take 15 mL of deionized water, add 3 mL of medium-chain triglycerides, and homogenize by shearing at 10000 r / min for 3 min to obtain the primary emulsion. Step 2: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain lipase emulsion.

[0044] Comparative Example 4 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 20 mg of deamided cottonseed protein to 20 mL of deionized water and stir on a magnetic stirrer at 500 rpm / min for 30 min to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 1 mg / mL. Step 2: Take 15 mL of deamidated cottonseed protein solution, add 3 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0045] Comparative Example 5 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 40 mg of deamided cottonseed protein to 20 mL of deionized water and stir at 500 rpm for 30 min on a magnetic stirrer to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 2 mg / mL. Step 2: Take 15 mL of deamidated cottonseed protein solution, add 3 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0046] Comparative Example 6 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 100mg of deamided cottonseed protein to 20mL of deionized water and stir at 500rpm / min for 30min on a magnetic stirrer to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 5mg / mL. Step 2: Take 15 mL of deamidated cottonseed protein solution, add 3 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0047] Comparative Example 7 A method for preparing a Pickering emulsion containing deamidated cottonseed protein-stabilized lipase includes the following steps: Step 1: Add 200 mg of deamided cottonseed protein to 20 mL of deionized water and stir on a magnetic stirrer at 500 rpm / min for 30 min to dissolve it, thus preparing a uniform aqueous dispersion, i.e., a deamided cottonseed protein solution with a concentration of 10 mg / mL. Step 2: Take 15 mL of deamidated cottonseed protein solution, add 3 mL of medium-chain triglycerides, and perform shear homogenization at 10000 r / min for 3 min to obtain the primary emulsion; Step 3: Add 2 mL of lipase solution to the homogenized primary emulsion, and then perform shear homogenization emulsification at 10000 r / min for 3 min to obtain Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

[0048] Figure 1 The images show the appearance of the emulsions prepared in Examples 1-8 and Comparative Examples 1-7 after 1 day and 60 days of storage. The type of emulsion prepared in this invention is determined by observing its dispersion when dropped into water and oil. When the emulsions prepared in Examples 1-8 and Comparative Examples 4-7 are dropped into water, they are found to disperse quickly, but when dropped into oil, they clump together, indicating that the emulsions prepared in Examples 1-8 and Comparative Examples 4-7 are oil-in-water emulsions. Figure 1 The images showing the appearance of the emulsions after 1 day and 60 days of storage demonstrate that the Pickering emulsions prepared in Examples 1 to 8 exhibit strong stability and did not separate after 60 days at room temperature. In contrast, the emulsions prepared in Comparative Examples 2 to 7 show poor stability, separating after 1 day at room temperature, and separating after 60 days at room temperature in Comparative Examples 1 to 7.

[0049] Figure 2 The figures show the Zeta potential diagrams of the emulsions prepared in Examples 1-4 and Comparative Example 1 of this invention. The Zeta potentials of the freshly prepared emulsions were measured using a laser particle size analyzer (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). The results are as follows: Figure 2 As shown, the Zeta potentials of the emulsions prepared in Examples 1-4 and Comparative Example 1 are -1.66, -13.23, -20.15, -20.07 and -15.85, respectively.

[0050] Figure 3 The Zeta potential diagrams are shown for the emulsions prepared in Examples 5-8 and Comparative Example 2 of this invention. The Zeta potentials of the freshly prepared emulsions were measured using a laser particle size analyzer (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). The results are as follows: Figure 3 As shown, the Zeta potentials of the emulsions prepared in Examples 5-8 and Comparative Example 2 are -3.76, -1.29, -16.87, -20.68 and -3.07, respectively.

[0051] Figure 4 The Zeta potential diagrams are shown for the emulsions prepared in Comparative Examples 3-7 of this invention. The Zeta potentials of the freshly prepared emulsions were measured using a laser particle size analyzer (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). The results are as follows. Figure 3 As shown, the Zeta potentials of the emulsions prepared in Comparative Examples 3 to 7 were -15.97, -16.35, -1.75, -1.68, and -4.71, respectively.

[0052] Figure 5 The average particle size distribution of the emulsions prepared in Examples 1-4 and Comparative Example 1 of this invention is shown in the diagram. The particle size of the freshly prepared emulsions was determined using a laser particle size analyzer (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). The results are as follows. Figure 5 As shown, the particle size distributions of the emulsions prepared in Examples 1-4 and Comparative Example 1 are 81 nm, 24 nm, 158 nm, 184 nm and 187 nm, respectively.

[0053] Figure 6 The average particle size distribution of the emulsions prepared in Examples 5-8 and Comparative Example 2 of this invention is shown in the diagram. The particle size of the freshly prepared emulsions was determined using a laser particle size analyzer (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). The results are as follows. Figure 6 As shown, the particle size distributions of the emulsions prepared in Examples 5-8 and Comparative Example 2 are 256 nm, 145 nm, 233 nm, 250 nm and 201 nm, respectively.

[0054] Figure 7 The image shows the average particle size distribution of the emulsions prepared in Comparative Examples 3-7 of this invention. The particle size of the freshly prepared emulsions was determined using a laser particle size analyzer (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). The results are as follows. Figure 7 As shown, the particle size distributions of the emulsions prepared in Comparative Examples 3 to 7 are 247 nm, 357 nm, 614 nm, 732 nm and 711 nm, respectively.

[0055] Figure 8This diagram shows the surface tension of deionized water, lipase, 1 mg / mL CPH solution containing lipase, 2 mg / mL CPH solution containing lipase, 5 mg / mL CPH solution containing lipase, and 10 mg / mL CPH solution containing lipase, as described in this invention. The surface tension of these solutions was measured at 25°C. In each of the deamidated cottonseed protein solutions containing lipase, the lipase solution accounted for 10% of the total volume. The results are as follows: Figure 8 As shown in the figure, deamidated cottonseed protein, as a surfactant, can reduce surface tension and stabilize lipase emulsions.

[0056] Figure 9 The enzyme activity retention rate of the emulsions prepared in Examples 5-8 and Comparative Example 2 of this invention is shown. p-Nitrophenyl palmitate (p-NPP) was used as the hydrolysis substrate, and hydrolysis was catalyzed by an interfacial lipase in the emulsion to produce p-nitrophenol (p-NP). The absorbance was measured at a wavelength of 410 nm, and the absorbance value reflects the concentration of p-nitrophenol. The initial enzyme activity and residual enzyme activity after 60 days of storage were measured for the emulsions prepared in Examples 5-8 and Comparative Example 2, respectively, and the enzyme activity retention rate was calculated. The highest catalytic activity was defined as 100%. The results are as follows: Figure 9 As shown, the horizontal axis represents the concentration of the added deamidated cottonseed protein solution, with Examples 5-8 and Comparative Example 2 corresponding to 1 mg / mL CPH, 2 mg / mL CPH, 5 mg / mL CPH, 10 mg / mL CPH, and 0 mg / mL CPH, respectively. The vertical axis represents the enzyme activity retention rate, with the enzyme activity retention rates of the emulsions prepared in Examples 5-8 and Comparative Example 2 being 68%, 81%, 100%, 98%, and 57%, respectively.

[0057] Figure 10 The enzyme activity retention rate of the emulsions prepared in Examples 9-12 and Comparative Example 2 of this invention is shown. p-Nitrophenyl palmitate (p-NPP) was used as the hydrolysis substrate, and hydrolysis was catalyzed by interfacial lipase in the emulsion to produce p-nitrophenol (p-NP). The absorbance was measured at a wavelength of 410 nm, and the absorbance value reflects the concentration of p-nitrophenol. The initial enzyme activity and residual enzyme activity after 60 days were measured for the emulsions containing modified deamidated cottonseed protein (M-CPH) solution prepared in Examples 9-12 and the emulsion prepared in Comparative Example 2, respectively. The enzyme activity retention rate was calculated, and the highest catalytic activity was defined as 100%. The results are as follows: Figure 10As shown, the horizontal axis represents the concentration of the modified deamidated cottonseed protein dispersion added, with Examples 9-12 and Comparative Example 2 corresponding to 1 mg / mL M-CPH, 2 mg / mL M-CPH, 5 mg / mL M-CPH, 10 mg / mL M-CPH, and 0 mg / mL M-CPH, respectively. The vertical axis represents the enzyme activity retention rate, with the enzyme activity retention rates of the emulsions prepared in Examples 9-12 and Comparative Example 2 being 76%, 90%, 100%, 99%, and 57%, respectively. Figure 10 and Figure 9 In comparison, it can be seen that dual modification of deamidated cottonseed protein by acylation and phosphorylation further increases the carboxyl groups and phosphate groups on the surface of deamidated cottonseed protein, reduces its surface hydrophobicity, weakens the hydrophobic binding between deamidated cottonseed protein and lipase, avoids the inactivation of lipase due to adsorption and conformational changes, improves emulsification stability, and further improves enzyme activity retention.

[0058] This invention innovatively immobilizes lipase at the emulsion interface of deamidated cottonseed protein, a modified cottonseed protein product, using it as a surfactant. This replaces traditional chemical surfactants for stabilizing lipase emulsions. By controlling the CPH concentration and oil phase ratio, the volume average particle size of the prepared emulsion can be reduced to below 200 nm, exhibiting excellent stability after 60 days of storage. Simultaneously, the deamidated cottonseed protein emulsion system provides good activity protection for lipase, with the highest catalytic activity retention rate at 5 mg / mL deamidated cottonseed protein, significantly superior to emulsion systems without CPH stabilization. Furthermore, the enzyme activity retention rate is further improved through dual modification of the deamidated cottonseed protein using acylation and phosphorylation. This invention features a simple preparation process, low instrument requirements, easy scalability, and uses safe and environmentally friendly raw materials. The prepared lipase Pickering emulsion has broad application prospects in areas such as targeted nutrient delivery, food structure and function modification, and biocatalytic reaction carriers.

[0059] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A Pickering emulsion containing deamidated cottonseed protein and stabilized lipase, characterized in that, It consists of an aqueous phase, an oil phase, surfactants, and lipases; The oil phase is a medium-chain triglyceride, and the oil phase accounts for 5-10% of the volume of the Pickering emulsion containing deamidated cottonseed protein-stabilized lipase. The surfactant is deamidated cottonseed protein, and the concentration of the surfactant in the Pickering emulsion of deamidated cottonseed protein-stabilized lipase is 0.1~10 mg / mL.

2. The Pickering emulsion for deamidated cottonseed protein-stabilized lipase as described in claim 1, characterized in that, The aqueous phase is deionized water, and the lipase is a lipase solution.

3. A method for preparing a Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described in claim 2, characterized in that, Includes the following steps: Step 1: Add deamided cottonseed protein to deionized water and stir to dissolve on a magnetic stirrer to prepare a uniform aqueous dispersion, namely deamided cottonseed protein solution. Step 2: Add medium-chain triglycerides to the deamidated cottonseed protein solution, and then perform shearing and homogenization to obtain the primary emulsion; Step 3: Add the lipase solution to the homogenized primary emulsion, then shear and homogenize to obtain the Pickering emulsion with deamidated cottonseed protein-stabilized lipase.

4. The method for preparing Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described in claim 3, characterized in that, In step one, the mass-to-volume ratio of deamided cottonseed protein to deionized water is 0.1~10 mg:1 mL.

5. The method for preparing Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described in claim 3, characterized in that, In step one, the stirring speed is 200~600 rpm / min and the stirring time is 20~40 min.

6. The method for preparing Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described in claim 3, characterized in that, In step two, the volume ratio of deamidated cottonseed protein solution to medium-chain triglycerides is 15-25:1-3.

7. The method for preparing Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described in claim 3, characterized in that, In step two, the rotation speed for shearing and homogenizing is 8000~12000 rpm / min, and the shearing and homogenizing time is 2~5min.

8. The method for preparing Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described in claim 3, characterized in that, In step three, the lipase solution is produced by using Aspergillus niger as the production strain and employing a liquid deep fermentation process; the volume ratio of the lipase solution to the colostrum is 1:8~12.

9. The method for preparing Pickering emulsion of deamidated cottonseed protein-stabilized lipase as described in claim 3, characterized in that, In step three, the rotational speed for shearing and homogenization is 8000~12000 rpm / min, and the shearing and homogenization time is 2~5min.

10. The application of a Pickering emulsion of deamidated cottonseed protein stable lipase as described in any one of claims 1-2 or a Pickering emulsion of deamidated cottonseed protein stable lipase prepared by the preparation method as described in any one of claims 3-9 in the fields of targeted nutrient delivery, food structure and function modification, or biocatalytic reaction carrier.

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