lactide-Nisin solid dispersion and its preparation
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- LUOYANG QIHONG BIOTECH
- Filing Date
- 2023-08-04
- Publication Date
- 2026-05-26
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing lactide-Nisin solid dispersion. Background Technology
[0002] Nisin, a lactic acid spore-forming enzyme, exhibits significant inhibitory effects against most Gram-positive bacteria and Bacillus spores. Therefore, Nisin has become a renowned natural biological preservative known for its high efficiency and safety, and is widely used in the preservation of various foods, including dairy and meat products. Nisin is sensitive to proteases, pH, and temperature. It readily reacts with food components, altering its structure. In particular, food proteases can efficiently hydrolyze Nisin, leading to a reduction or even complete loss of its antibacterial activity. Adding protease inhibitors and immobilization techniques can reduce protease damage to Nisin. The pH value of the solvent is a crucial factor affecting Nisin's solubility, thermal stability, and biological activity. Heat treatment is a common method in food processing, but it reduces the biological activity of Nisin in non-acidic environments. Currently, methods to improve Nisin's thermal stability mainly involve adding protective substances and immobilization techniques. The presence of proteases and other food components, along with heat treatment processes, often destroys Nisin's activity, limiting its application in food preservation.
[0003] Lactose is an organic compound that can be classified into three types: L-lactide, D-lactide, and meso-lactide. It can be dispersed in organic solvents. Lactose is commonly used in the production of polylactic acid and as a cyclic esterifying agent. There are occasional reports of its use in food, but research on this is limited. Whether it can stabilize Nisin, improve Nisin potency, or enhance antibacterial effects has not yet been reported. Summary of the Invention
[0004] Objective of the Invention: The objective of this invention is to provide a Nisin-based antibacterial agent with good antibacterial effects. This invention reduces the sensitivity of Nisin to solvent pH and neutral proteases by combining Nisin with lactide, thereby enhancing the thermal stability of Nisin and achieving a long-lasting antibacterial effect.
[0005] Technical solution:
[0006] This invention provides a method for preparing lactide-Nisin solid dispersion, comprising the following steps:
[0007] (1) Prepare a mixture of lactide and a hydrophobic solvent;
[0008] (2) Nisin was added to the mixture and dispersed by stirring to obtain a multi-component mixture;
[0009] (3) The multi-component mixture is cold-treated to obtain a solid mixture containing lactide and Nisin; the mixture can be used as the lactide-Nisin solid dispersion of the present invention, and optionally the hydrophobic solvent in the solid mixture is further removed.
[0010] The mass ratio of Nisin to lactide used in this method can be 1:1-4, such as 1:2-3; the lactide can be D-type, L-type, and / or meso-type. The hydrophobic solvent can be ethyl acetate. Further removal of the hydrophobic solvent can be performed by vacuum distillation. As a specific example, the preparation steps of this invention include:
[0011] (1) Take lactide and ethyl acetate and heat them together. The lactide is dispersed in ethyl acetate to prepare a liquid mixture of lactide and ethyl acetate.
[0012] (2) Nisin was added under heating conditions and stirred to dissolve, resulting in a multi-component mixture;
[0013] (3) Preparation of solid lactide-Nisin mixture: The multi-component mixture was rapidly transferred from heating conditions to freezing conditions to obtain solid lactide-Nisin mixture;
[0014] (4) Removal of ethyl acetate: The solid lactide-Nisin mixture was subjected to vacuum distillation to remove ethyl acetate from the mixture;
[0015] (5) Preparation of lactide-Nisin solid dispersion: Pulverize the solid lactide-Nisin mixture and sieve it to obtain lactide-Nisin solid dispersion powder.
[0016] Furthermore, in step (1), the mass ratio of lactide to hydrophobic solvent is 5:1-2:1.
[0017] Furthermore, in step (1), the temperature conditions for mixing lactide and hydrophobic solvent are 30-70℃, and the time is 1-30min.
[0018] Furthermore, the temperature conditions for the dispersion operation in step (2) are 30-70℃, and the time is 1-30min.
[0019] Furthermore, the temperature conditions for the cold treatment in step (3) are -20 to 0°C, and the time can be 12 to 48 hours, such as 24 hours.
[0020] Furthermore, the vacuum distillation conditions are 0.08-0.1 MPa, the temperature can be 30-60℃, and the time can be 25-300 min.
[0021] Furthermore, the sieving condition in step (5) is a 100-mesh sieve.
[0022] The present invention also discloses the lactide-Nisin solid dispersion prepared by the above method, which is confirmed by FTIR infrared spectroscopy to contain characteristic peaks of lactide and Nisin.
[0023] The present invention also discloses the method and the obtained lactide-Nisin solid dispersion for antibacterial, food preservation and other applications.
[0024] Beneficial effects: This invention has the following advantages:
[0025] 1. This invention improves the mixing process of lactide and Nisin, thereby protecting nisin, reducing the activity loss caused by proteases and other substances, improving the bioavailability of Nisin, and prolonging the antibacterial effect.
[0026] 2. This invention improves the solubility, thermal stability and bioactivity of Nisin.
[0027] 3. This invention reduces the activity loss of Nisin during processing.
[0028] 4. This invention fully utilizes the binding properties of lactide and Nisin, effectively enhancing the stability of Nisin in complex environments, further expanding the application potential of the prepared antibacterial agent, and providing a new approach for developing multifunctional green food preservatives. Attached Figure Description
[0029] Figure 1 Infrared comparison of Nisin, lactide, and the lactide-Nisin solid dispersion of the present invention;
[0030] Figure 2 This is a comparison of the cumulative in vitro dissolution rates of Nisin products from each example. Detailed Implementation
[0031] The following experiment uses L-type lactide as an example. Unless otherwise specified, all reagents and instruments used are conventional products.
[0032] Example 1 describes a method for preparing a lactide-Nisin solid dispersion, comprising the following steps:
[0033] 1. Weigh lactide, mix it with ethyl acetate in a mass ratio of 2:1, seal the mixture, and heat it in a water bath at 70°C for 20 minutes to prepare a liquid mixture of lactide and ethyl acetate.
[0034] 2. Weigh out nisin and add it to the liquid lactide and ethyl acetate mixture. In this example, the mass ratio of lactide to nisin is set to 1:1. Stir at 70°C for 5 minutes.
[0035] 3. The stirred mixture was rapidly transferred from a 70°C water bath to a -20°C freezer for 24 hours to obtain a solid lactide-Nisin mixture;
[0036] 4. Transfer the solid lactide-Nisin mixture to a 60°C water bath and distill under reduced pressure of 0.09 MPa for 25 min to remove ethyl acetate; 5. Take the distilled solid lactide-Nisin mixture, pulverize it, and pass it through a 100-mesh sieve to obtain lactide-Nisin solid dispersion powder.
[0037] Example 2 describes a method for preparing a lactide-Nisin solid dispersion, comprising the following steps:
[0038] 1. Weigh lactide, mix it with ethyl acetate in a mass ratio of 2:1, seal the mixture, and heat it in a water bath at 70°C for 20 minutes to prepare a liquid mixture of lactide and ethyl acetate.
[0039] 2. Weigh out nisin and add it to the liquid lactide and ethyl acetate mixture. In this example, the mass ratio of lactide to nisin is set to 2:1. Stir at 70°C for 5 minutes.
[0040] 3. The stirred mixture was rapidly transferred from a 70°C water bath to a -20°C freezer for 24 hours to obtain a solid lactide-Nisin mixture;
[0041] 4. Transfer the solid lactide-Nisin mixture to a 60°C water bath and distill under reduced pressure of 0.09 MPa for 25 min to remove ethyl acetate; 5. Take the distilled solid lactide-Nisin mixture, pulverize it, and pass it through a 100-mesh sieve to obtain lactide-Nisin solid dispersion powder.
[0042] Example 3 describes a method for preparing a lactide-Nisin solid dispersion, comprising the following steps:
[0043] 1. Weigh lactide, mix it with ethyl acetate in a mass ratio of 2:1, seal the mixture, and heat it in a water bath at 70°C for 20 minutes to prepare a liquid mixture of lactide and ethyl acetate.
[0044] 2. Weigh out nisin and add it to the liquid lactide and ethyl acetate mixture. In this example, the mass ratio of lactide to nisin is set to 3:1. Stir at 70°C for 5 minutes.
[0045] 3. The stirred mixture was rapidly transferred from a 70°C water bath to a -20°C freezer for 24 hours to obtain a solid lactide-Nisin mixture;
[0046] 4. Transfer the solid lactide-Nisin mixture to a 60℃ water bath and distill under reduced pressure (0.09 MPa) for 25 min to remove ethyl acetate; 5. Pulverize the distilled solid lactide-Nisin mixture and pass it through a 100-mesh sieve to obtain lactide-Nisin solid dispersion powder. FTIR infrared identification is as follows: Figure 1 As shown, the solid dispersion prepared by this invention contains characteristic peaks of lactide and Nisin.
[0047] Example 4 describes a method for preparing a lactide-Nisin solid dispersion, comprising the following steps:
[0048] 1. Weigh lactide, mix it with ethyl acetate in a mass ratio of 2:1, seal the mixture, and heat it in a water bath at 70°C for 20 minutes to prepare a liquid mixture of lactide and ethyl acetate.
[0049] 2. Weigh out nisin and add it to the liquid lactide and ethyl acetate mixture. In this example, the mass ratio of lactide to nisin is set to 4:1. Stir at 70°C for 5 minutes.
[0050] 3. The stirred mixture was rapidly transferred from a 70°C water bath to a -20°C freezer for 24 hours to obtain a solid lactide-Nisin mixture;
[0051] 4. Transfer the solid lactide-Nisin mixture to a 60°C water bath and distill under reduced pressure of 0.09 MPa for 25 min to remove ethyl acetate; 5. Take the distilled solid lactide-Nisin mixture, pulverize it, and pass it through a 100-mesh sieve to obtain lactide-Nisin solid dispersion powder.
[0052] Experiment I: Performance Testing. Taking the lactide-Nisin solid dispersion prepared in Example 1 as an example, the technical indicators measured by the agar diffusion method are as follows:
[0053] Antimicrobial stability test - agar diffusion method:
[0054] The Nisin titer of lactide-Nisin solid dispersion was determined according to the detection method in GB1886.231-2016. Micrococcus luteus [CMCC(B)28001] was used as the test bacterium, and the colonies were diluted to OD500 with physiological saline. 600The bacterial suspension was prepared at a concentration of 0.33. The test medium (0.8% tryptone, 0.5% sodium chloride, 0.5% yeast extract, 0.2% disodium hydrogen phosphate dodecahydrate, pH 6.7-6.8 after sterilization) was prepared according to QB-2394-99. When the test medium was cooled to 70℃, 3.0% Tween-20 aqueous solution (1:1g / g) was added and thoroughly mixed. The medium was then cooled further to 54℃, at which point 3% bacterial suspension was added. After mixing, 27mL of the medium was weighed and poured into a 90mm sterile petri dish. After the test medium solidified, it was transferred to a 4℃ refrigerator and refrigerated for at least 1 hour. When using the test medium, holes were punched using a 7mm puncher, and 90μL of antibacterial agent was added to each hole. The dish was then covered with a sterile ceramic tile lid and horizontally placed in a 29℃ incubator for 40 hours. The diameter of the inhibition zone was measured.
[0055] Nisin standard was diluted to 50, 100, 200, 400, and 800 IU / mL using a 2-fold dilution method with 0.02 mol hydrochloric acid aqueous solution as solvent. The diameter of the inhibition zone formed by each Nisin solution at different potencies was determined using the aforementioned detection method (agar diffusion method). A linear regression curve was plotted between the diameter of the Nisin inhibition zone and the logarithm of the Nisin biopotency, yielding the linear regression equation y = 7.5767x + 0.6437, R0. 2 =0.9938, this equation is used to calculate the Nisin biopotency of antibacterial agents.
[0056] (1) Sensitivity test of lactide-Nisin solid dispersion to solvent pH. The pH of the solvent is an important factor affecting the bioavailability of Nisin. The environment provided by food often cannot exert the best antibacterial performance of Nisin, so solving the pH sensitivity of Nisin is of great significance for its application in the food industry. The antibacterial agent prepared in Example 1 and the control group were dissolved in aqueous solutions with pH values of 3, 7 and 10 respectively (pH value adjusted with HCl and NaOH) to prepare an antibacterial solution of 16.7 ppm. After standing for 12 h, the Nisin potency of the antibacterial agent was determined by agar diffusion method.
[0057] Table 1. Antibacterial activity of antibacterial agents dissolved in solvents of different pH values.
[0058]
[0059] Note: Data in the table is presented as mean ± standard deviation (n=3). Different uppercase letters in the same row indicate significant differences, different lowercase letters in the same column indicate significant differences (P<0.05), and different uppercase letters in the same column indicate significant differences (P<0.01).
[0060] As shown in the table, under high pH conditions, the antibacterial activity of Example 1 was higher than that of the control group antibacterial agent, indicating that Example 1 was less sensitive to solvent pH and that Nisin had enhanced antibacterial activity in an alkaline environment.
[0061] (2) Protease sensitivity test
[0062] Nisin itself can bind to or react with various components in food, leading to changes in its structure and reduced biological activity. The most significant factor affecting Nisin activity in food is protease, which can effectively hydrolyze Nisin, causing it to completely lose its biological activity. The most effective way to protect Nisin from protease hydrolysis is to add protease inhibitors and perform heat treatment. The sample prepared in Example 1 and the control group antimicrobial agent were dissolved in a 1 ppm neutral protease (200 U / mg) aqueous solution at pH 3.0 for determination.
[0063] Table 2
[0064] Note: Data in the table is presented as mean ± standard deviation (n=3). Different uppercase letters in the same row indicate significant differences, different lowercase letters in the same column indicate significant differences (P<0.05), and different uppercase letters in the same column indicate significant differences (P<0.01).
[0065]
[0066] Note: Data in the table is presented as mean ± standard deviation (n=3). Different uppercase letters in the same row indicate significant differences, different lowercase letters in the same column indicate significant differences (P<0.05), and different uppercase letters in the same column indicate significant differences (P<0.01).
[0067] As shown in Table 2, the antibacterial effect of Example 1 treated with 0.1 ppm protease was significantly higher than that of the control group. Even at a protease concentration of 0.01 ppm, the antibacterial effect of Example 1 was still slightly higher than that of the control group. This indicates that Example 1 showed reduced sensitivity to protease, demonstrating that the method of the present invention can effectively improve the biostability of Nisin.
[0068] (3) Thermal sensitivity test
[0069] The sample prepared in Example 1 (Nisin and lactide in a mass ratio of 1:1) and the control group antibacterial agent (Nisin and sodium chloride mixed in a 1:1 ratio) were dissolved in an aqueous solution at pH 7.5 to prepare an antibacterial solution of 33.3 ppm. The solutions were heated at 60℃, 85℃ and 100℃ for 15 min respectively, and after standing for 12 h, the potency of Nisin was determined by agar diffusion method.
[0070] Table 3. Antibacterial activity of antibacterial agent solutions after treatment at different temperatures.
[0071]
[0072] Note: Data in the table is presented as mean ± standard deviation (n=3). Different uppercase letters in the same row indicate significant differences, different lowercase letters in the same column indicate significant differences (P<0.05), and different uppercase letters in the same column indicate significant differences (P<0.01).
[0073] As shown in Table 3, after heat treatment at different temperatures, the antibacterial activity of Example 1 was higher than that of the control group antibacterial agent, indicating that the antibacterial agent of Example 1 has stronger thermal stability.
[0074] Experiment II - Performance Comparison:
[0075] I. Thermal Sensitivity of Each Embodiment
[0076] Referring to the aforementioned heat sensitivity test, the Nisin potency of the products of each example and the control group antibacterial agent (Nisin and sodium chloride mixed in corresponding proportions) under room temperature conditions was compared. The results are shown in the table below:
[0077] Table 4
[0078]
[0079] Note: Data in the table is presented as mean ± standard deviation (n=3). Different uppercase letters in the same row indicate significant differences, different lowercase letters in the same column indicate significant differences (P<0.05), and different uppercase letters in the same column indicate significant differences (P<0.01).
[0080] II. Cumulative in vitro dissolution data for each embodiment
[0081] Liquid phase detection method for lactide-Nisin solid dispersion:
[0082] For the lactide-Nisin solid dispersion, the prepared sample was dissolved in ultrapure water and centrifuged at 3000 rpm for 10 min at 25 °C. The supernatant was collected, the pH was adjusted to 1.8 to 2.2, and then passed through a 0.22 μm membrane. The concentration of Nisin in the supernatant was detected by high performance liquid chromatography. The in vitro cumulative dissolution rate of the lactide-Nisin solid dispersion was calculated by formula (1).
[0083] (1) is:
[0084] Cumulative in vitro dissolution rate (%) = Free nisin content in the dissolution medium / Theoretical total nisin content × 100 (1)
[0085] The cumulative in vitro dissolution data results for each embodiment are as follows: Figure 2As shown in the figure, the proportions represent the mass ratio of Nisin to lactide.
[0086] III. Comparison of the solid dispersion of the present invention with Nisin in application scenarios
[0087] 1) The solid dispersion obtained in Example 3 was directly mixed into apple juice and a Nisin control group was set up. After mixing, the mixture was heated at 70°C for 30 min. The Nisin potency was detected by HPLC method in Section 2. The results are as follows: It can be seen that the solid dispersion can better maintain the Nisin potency when applied to fruit juice.
[0088] Table 6
[0089]
[0090] Note: Data in the table is presented as mean ± standard deviation (n=3). Different uppercase letters in the same row indicate significant differences, different lowercase letters in the same column indicate significant differences (P<0.05), and different uppercase letters in the same column indicate significant differences (P<0.01).
[0091] 2) The solid dispersion obtained in Example 3 was directly mixed into watermelon juice, and a Nisin control group and a blank control group were set up. After mixing, the mixture was heated at 60°C for 15 min, filled under aseptic conditions, and refrigerated at 4°C. The Nisin titer was detected by HPLC according to Section 2, the total bacterial count of the processed food was detected according to GB 4789.2-2022, and the pH value of the processed food was detected according to GB 5009.237-2016. The comparison showed that the solid dispersion of the present invention can better maintain the Nisin titer and effectively improve the antibacterial effect in watermelon juice, and does not significantly change the pH value of watermelon juice. The comparison results on day 0 (directly measured after heating for 15 min and filling) are shown in the table below:
[0092] Table 7
[0093]
Claims
1. A method for preparing lactide-Nisin solid dispersion, comprising the following steps: (1) Preparation of a mixture of lactide and the hydrophobic solvent ethyl acetate; (2) Nisin is dispersed in the mixture obtained in the previous step to obtain a multi-component mixture; (3) The multi-component mixture is cold-treated to obtain a solid mixture containing lactide and Nisin; The mass ratio of Nisin to lactide used in this method is 1:1-4; the temperature of the cold treatment in step (3) is -20-0℃, and the hydrophobic solvent is removed by vacuum distillation at a pressure of 0.08-0.1MPa.
2. The method as described in claim 1, characterized in that, The mass ratio of Nisin to lactide used in this method is 1:2-3.
3. The method as described in claim 1, characterized in that, The mass ratio of lactide to hydrophobic solvent used in step (1) is 5:1-2:
1.
4. The method as described in claim 1, characterized in that, The cooling time in step (3) is 12-48 hours.
5. The method as described in claim 4, characterized in that, The time period is 24 hours.
6. The method as described in claim 1, characterized in that, The lactide is of the D-type, L-type, and / or meso-type.
7. The method as described in claim 1, characterized in that, The vacuum distillation temperature is 25-60℃.
8. The method as described in claim 1, characterized in that, The vacuum distillation time is 25-300 min.
9. A solid dispersion prepared by the method according to any prior claim, comprising lactide and Nisin.