Preparation method of pH cycle induced alpha-lactalbumin-thymol nanoparticles
The pH cycle induction method was used to construct thymeol in the hollow nanocage structure of α-lactal albumin, which solved the problem of insufficient antibacterial activity of thymeol in food, and achieved efficient antibacterial effect and safety.
Patent Information
- Application Number
- CN202510414580.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art is difficult to effectively deliver and protect thymeol, resulting in insufficient antibacterial activity in food and unable to effectively resist the threat of drug-resistant bacteria.
The pH cycle induction method was used to construct the hollow nanocage structure of α-lactal albumin. By accurately controlling the hydrophobic exposure and refolding process of the protein, it was loaded with thymeol to form efficient antibacterial nanoparticles.
The loading rate and encapsulation rate of thymeol are significantly improved, the inhibitory effect on pathogenic bacteria is enhanced, the antibacterial activity is increased by more than 50%, and the nanoparticles are low in cytotoxicity and high safety.
Smart Images
Figure CN120021656A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food processing, and relates to a method for preparing pH cycle-induced alpha-lactalbumin-thymol nanoparticles, which is expected to be used as a natural food antibacterial agent for food preservation. Background Art
[0002] According to the World Health Organization, the threat from drug-resistant bacteria is becoming more and more serious, and the arrival of the post-antibiotic era is becoming more and more real. In particular, food spoilage and foodborne diseases caused by microbial contamination of food have attracted widespread attention from consumers. Due to the diversity of types of dangerous bacteria and the environments in which they are found, a variety of antimicrobial agents are needed to combat bacterial resistance. However, long-term consumption of foods containing excessive amounts of chemical and artificial preservatives may also be harmful to human health. Therefore, exploring non-toxic and effective natural antibacterial compounds as alternatives is an urgent issue to be addressed.
[0003] Thymol is a natural compound extracted from thyme. my country's "Sanitary Standards for the Use of Food Additives" states that thymol is allowed to be added to food as a spice. However, the use of thymol is not limited to spices. It is also a natural antibacterial agent and preservative with broad-spectrum antibacterial properties, especially for Gram-positive and Gram-negative bacteria. However, because thymol is highly volatile and has poor hydrophobicity, it is not easy to disperse in water and is easily degraded by oxygen, ultraviolet rays, moisture and heat. Therefore, how to deliver and protect thymol and improve its antibacterial activity in food is a technical key to breaking through the widespread application of natural antibacterial agents in the food field.
[0004] α-Lactalbumin is the second most abundant small molecule protein in whey protein and is widely present in the milk of mammals. α-Lactalbumin can be used to increase the water solubility of hydrophobic substances, thereby improving their functional activity. α-Lactalbumin has been widely reported as a nano-delivery carrier for transporting hydrophobic active substances. This indicates that the interaction between α-lactalbumin and hydrophobic bioactive compounds can be used to break through the application barriers of hydrophobic active substances in the food field.
[0005] The present invention uses α-lactalbumin as a carrier to load thymol, and innovatively adopts the three-stage cycle process of "alkaline swelling-acidic folding-neutral reconstruction". By precisely controlling the exposure and refolding process of the hydrophobic domain of the protein, α-lactalbumin forms a hollow nanocage structure. Compared with the traditional single pH adjustment method, the loading amount is increased from 12% to 28-32% (w / w), and the encapsulation rate is >95%, which can achieve effective loading of thymol and prepare a natural antibacterial nanoparticle; compared with free thymol, the present invention can increase the inhibition rate of pathogenic bacteria by more than 50%. Therefore, the pH cycle-induced α-lactalbumin-thymol nanoparticles proposed in the present invention have obvious advantages, which will make the antibacterial agents based on natural products more diversified and can expand the application range of thymol in the food industry. Summary of the invention
[0006] The technical problem to be solved by the present invention is to provide a method for preparing pH cycle-induced alpha-lactalbumin-thymol nanoparticles.
[0007] A method for preparing pH cycle-induced α-lactalbumin-thymol nanoparticles is characterized by comprising the following steps:
[0008] (1) Preparation and pretreatment of protein solution: α-lactalbumin was dissolved in deionized water and stirred magnetically (500 rpm, 25° C.) until completely dissolved to prepare a protein solution with a concentration of 5% (w / v);
[0009] (2) pH cycle induction stage 1: The pH of the protein solution was adjusted to 8.0-10.0 using 1 M sodium hydroxide solution, and the temperature was raised to 40 °C and kept for 30 min to allow the protein structure to fully unfold;
[0010] (3) pH cycle induction stage 2: The pH of the protein solution in (2) was adjusted to 3.0-5.0 using 1 M hydrochloric acid solution, and the solution was gradually cooled to 25°C and allowed to stand for 30 min;
[0011] (4) pH cycle induction stage 3: After adjusting the pH of the protein solution in (3) to 7.0 using 1 M sodium hydroxide solution, thymol was added, with the mass ratio of α-lactalbumin to thymol being (7:1, 6:1, 5:1), and ultrasonic treatment (20 kHz, 200 W, 5 min) was performed to evenly disperse the thymol;
[0012] (5) The mixed solution in (4) was dialyzed in deionized water at 4° C. for 48 h through a 1 kDa cellulose dialysis membrane to remove unbound thymol, and the dialyzed composite solution was freeze-dried to obtain nanoparticles.
[0013] Features of the method: The present invention innovatively uses the pH cycle induction method to construct a cage-type structure of α-lactalbumin. Compared with the traditional single pH adjustment method, it has a stronger loading capacity and can effectively prepare a natural α-lactalbumin-thymol antibacterial nanoparticle. Compared with free thymol, the present invention can increase the inhibition rate of pathogenic bacteria by more than 50%. The operation method is simple and can prepare a class of natural foodborne antibacterial agents that can be used in the field of food processing. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 The scanning electron microscopic image of the antibacterial nanoparticles prepared by the present invention acting on the strains of Staphylococcus aureus and Escherichia coli;
[0015] Figure 2 This is a laser confocal scanning electron microscope image of the antibacterial nanoparticles prepared in the present invention acting on cervical cancer cells. DETAILED DESCRIPTION
[0016] The present invention will be further described below in conjunction with specific examples. It should be understood that the examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
[0017] Example 1
[0018] (1) α-lactalbumin was dissolved in deionized water and stirred magnetically (500 rpm, 25° C.) until completely dissolved to prepare a protein solution with a concentration of 5% (w / v).
[0019] (2) Use 1M sodium hydroxide solution to adjust the pH to 8.0, place the protein solution in a water bath, raise the temperature to 40°C, keep warm for 30 minutes, use 1M hydrochloric acid solution to adjust the pH of the solution to 3.5, gradually cool the solution to 25°C, and let it stand for 30 minutes. Use 1M sodium hydroxide solution to adjust the pH of the solution to 7.0 again.
[0020] (3) Thymol was added, with the mass ratio of α-lactalbumin to thymol being 7:1, and ultrasonic treatment was performed (20 kHz, 200 W, 5 min) to uniformly disperse the thymol.
[0021] Similarly, a comparative experiment was conducted in which the pH of the α-lactalbumin protein solution in (1) was adjusted to 7.0 using a single pH adjustment method and then mixed with thymol according to step (3).
[0022] (4) The α-lactalbumin-thymol nanoparticle solutions prepared by different treatment methods were dialyzed in deionized water at 4°C for 48 h through a 1 kDa cellulose dialysis membrane to remove unbound thymol. The dialyzed composite solution was freeze-dried to obtain two types of nanoparticles and the loading rate and encapsulation efficiency were tested.
[0023] Weigh thymol standard (purity ≥ 98%), use high performance liquid chromatography (C18 column, mobile phase acetonitrile: water = 70:30, flow rate 1mL / min) to measure the absorbance of thymol of different concentrations at 280nm, and draw a concentration-peak area standard curve (R2>0.999). Take 10mg of freeze-dried nanoparticles, dissolve in 5mL of methanol, ultrasonically crush, centrifuge and take the supernatant for detection, and calculate the loading rate and encapsulation efficiency according to the formula:
[0024]
[0025] Results: The loading rate of thymol in α-lactalbumin-thymol nanoparticles induced by pH cycling was 31.7±1.2%, and the encapsulation efficiency was 96.3±0.8% (P<0.05); the loading rate of thymol in single pH adjustment method was 11.3±0.9%, and the encapsulation efficiency was 90.6±1.1% (P<0.05). This shows that the pH cycling induction method can effectively change the configuration of α-lactalbumin and increase its loading rate for thymol, which has significant advantages.
[0026] Example 2
[0027] (1) α-lactalbumin was dissolved in deionized water and stirred magnetically (500 rpm, 25° C.) until completely dissolved to prepare a protein solution with a concentration of 5% (w / v).
[0028] (2) Use 1M sodium hydroxide solution to adjust the pH to 9.0, place the protein solution in a water bath, raise the temperature to 40°C, keep warm for 30 minutes, use 1M hydrochloric acid solution to adjust the pH of the solution to 4.5, gradually cool the solution to 25°C, and let it stand for 30 minutes. Use 1M sodium hydroxide solution to adjust the pH of the solution to 7.0 again.
[0029] (3) Thymol was added, with the mass ratio of α-lactalbumin to thymol being 6:1, and ultrasonic treatment was performed (20 kHz, 200 W, 5 min) to uniformly disperse the thymol.
[0030] Similarly, a comparative experiment was conducted in which the pH of the α-lactalbumin protein solution in (1) was adjusted to 7.0 using a single pH adjustment method and then mixed with thymol according to step (3).
[0031] (4) The α-lactalbumin-thymol nanoparticle solutions prepared by different treatment methods were dialyzed in deionized water at 4°C for 48 h through a 1 kDa cellulose dialysis membrane to remove unbound thymol. The dialyzed composite solution was freeze-dried to obtain two types of nanoparticles and subjected to in vitro release experimental analysis.
[0032] Simulated gastric fluid release: the nanoparticles were dispersed in a pH 1.2 hydrochloric acid solution (containing 0.32% pepsin), shaken at 37°C (100 rpm), and samples were taken at 0.5, 1, and 2 h. The release of thymol was detected by HPLC. Simulated intestinal fluid release: the gastric fluid-treated samples were transferred to pH 7.4 PBS (containing 1% pancreatin), and the release of thymol was continued to be detected by HPLC for 6 h.
[0033] Results: The cumulative release of thymol from α-lactalbumin-thymol nanoparticles induced by pH cycling was 4.8±0.3% (P<0.05) 2 hours after gastric juice digestion; after 30 minutes of intestinal juice treatment, the release rate of thymol reached 91.2±2.1%. The cumulative release of thymol from α-lactalbumin-thymol nanoparticles prepared by single pH adjustment method was 6.8±0.5% (P<0.05) 2 hours after gastric juice digestion; after 30 minutes of intestinal juice treatment, the release rate of thymol reached 79.3±0.9%. This indicates that the pH cycling induction method can effectively protect the loaded thymol, make it gastric juice tolerant (2h release rate <5%), and intestinal targeted release (30min release rate >90%), which has significant advantages.
[0034] Example 3
[0035] (1) α-lactalbumin was dissolved in deionized water and stirred magnetically (500 rpm, 25° C.) until completely dissolved to prepare a protein solution with a concentration of 5% (w / v).
[0036] (2) Use 1M sodium hydroxide solution to adjust the pH to 10.0, place the protein solution in a water bath, raise the temperature to 40°C, keep warm for 30 minutes, use 1M hydrochloric acid solution to adjust the pH of the solution to 5.0, gradually cool the solution to 25°C, and let it stand for 30 minutes. Use 1M sodium hydroxide solution to adjust the pH of the solution to 7.0 again.
[0037] (3) Thymol was added in a mass ratio of α-lactalbumin to thymol of 5:1, and ultrasonic treatment was performed (20 kHz, 200 W, 5 min) to uniformly disperse the thymol.
[0038] Similarly, a comparative experiment was conducted in which the pH of the α-lactalbumin protein solution in (1) was adjusted to 7.0 using a single pH adjustment method and then mixed with thymol according to step (3).
[0039] (4) The α-lactalbumin-thymol nanoparticle solutions prepared by different treatment methods were dialyzed in deionized water at 4°C for 48 h through a 1 kDa cellulose dialysis membrane to remove unbound thymol. The dialyzed composite solution was freeze-dried to obtain two types of nanoparticles and their antibacterial activity was analyzed.
[0040] The antibacterial activity of the nanoparticles was evaluated by the microbroth dilution method (Staphylococcus aureus ATCC 29213, Escherichia coli ATCC o157:h7), and the minimum inhibitory concentration (MIC) of the nanoparticles prepared by free thymol, pH cycle induction method and single pH adjustment method was compared. The plate diffusion method was used to continuously dilute the 1 / 2MIC sample with physiological saline until the appropriate concentration was reached, and 100 μL of the sample bacterial suspension was spread on the agar plate. The inhibition rate (IR) was calculated by the following formula.
[0041] IR(%)=(N 0 -N)×100 / N 0
[0042] In the formula, N 0 and N are the colony forming units (CFU) of the control group and the experimental group, respectively.
[0043] Results: The MIC values of different samples are shown in Table 1. This indicates that compared with the single pH adjustment method, the α-lactalbumin-thymol nanoparticles prepared by the pH cycle induction method have better antibacterial activity.
[0044] Table 1 MIC values and IR values of different samples against Staphylococcus aureus and Escherichia coli
[0045]
[0046] Based on the above research results, the bacterial morphology after the strain was acted on by α-lactalbumin-thymol nanoparticles prepared by the pH cycle induction method was analyzed. The sample solution was added to the bacterial suspension of Staphylococcus aureus and Escherichia coli activated to the logarithmic growth phase. The bacterial suspension containing different samples was cultured in an incubator at 37°C for 4 hours, and then the acted bacterial suspension was centrifuged and the bacterial precipitate was washed with PBS (0.1M, pH 4.2). After repeating the operation three times, the bacterial precipitate was immersed in 2.5% glutaraldehyde and fixed overnight. The 2.5% glutaraldehyde was removed from the bacterial precipitate, and the bacterial precipitate was dehydrated for 20 minutes using a gradient of ethanol solutions of different concentrations (30%, 50%, 70%, 90% and 100%). The dehydrated bacterial precipitate was pre-frozen at -80°C for more than 6 hours, and after freeze-drying, it was photographed under a scanning electron microscope. The results are as follows Figure 1 shown.
[0047] Results: Compared with free thymol, the bacterial cell membrane surface treated with α-lactalbumin-thymol nanoparticles prepared by pH cycling induction method showed serious damage, and the bacteria were also significantly sunken, indicating that α-lactalbumin-thymol nanoparticles prepared by pH cycling induction method can increase the degree of destruction of the cell membranes of Staphylococcus aureus and Escherichia coli strains, thereby enhancing its ability to inhibit the growth of pathogenic bacteria.
[0048] Finally, the cell live-death staining method was used to compare the cytotoxicity of free thymol and α-lactalbumin-thymol nanoparticles prepared by pH cycle induction method to evaluate the biocompatibility of the composite. Cervical cancer cells were subcultured until the cell morphology was stable and then used. 100 μL of cell suspension was inoculated in a 96-well plate with an inoculation density of 8×10 3 After 24 hours of culture, the original culture medium was discarded and fresh culture medium containing samples of different concentrations was added. After another 12 hours of culture, the culture medium was discarded. Staining was performed according to the instructions of the Calcein-AM / PI live cell / dead cell double staining kit (CA1630, Solebol), and the samples were photographed using a laser confocal microscope. The results are shown in Figure 2 shown.
[0049] Results: The cell viability of all test cells was greater than 95%, and the cell survival rate was very high with no significant difference, indicating that the α-lactalbumin-thymol nanoparticles prepared by pH cycle induction method had no toxicity to cells. This proved that the prepared α-lactalbumin-thymol nanoparticles were safe and had potential and application value in inhibiting bacterial growth.
Claims
1. A method for preparing pH cycle-induced α-lactalbumin-thymol nanoparticles, characterized in that: The steps include: (1) Preparation and pretreatment of protein solution: α-lactalbumin was dissolved in deionized water and stirred magnetically (500 rpm, 25° C.) until completely dissolved to prepare a protein solution with a concentration of 5% (w / v); (2) pH cycle induction stage 1: The pH of the protein solution was adjusted to 8.0-10.0 using 1 M sodium hydroxide solution, and the temperature was raised to 40 °C and kept for 30 min to allow the protein structure to fully unfold; (3) pH cycle induction stage 2: The pH of the protein solution in (2) was adjusted to 3.0-5.0 using 1 M hydrochloric acid solution, and the solution was gradually cooled to 25°C and allowed to stand for 30 min; (4) pH cycle induction stage 3: After adjusting the pH of the protein solution in (3) to 7.0 using 1 M sodium hydroxide solution, thymol was added, with the mass ratio of α-lactalbumin to thymol being (7:1, 6:1, 5:1), and ultrasonic treatment (20 kHz, 200 W, 5 min) was performed to evenly disperse the thymol; (5) The mixed solution in (4) was dialyzed in deionized water at 4° C. for 48 h through a 1 kDa cellulose dialysis membrane to remove unbound thymol, and the dialyzed composite solution was freeze-dried to obtain nanoparticles.
2. The method for preparing a pH cycle-induced α-lactalbumin-thymol nanoparticle according to claim 1, characterized in that The pH range described in (2) is 8.0-10.
0.
3. The method for preparing a pH cycle-induced α-lactalbumin-thymol nanoparticle according to claim 1, characterized in that The pH range described in (3) is 3.0-5.
0.
4. The method for preparing a pH cycle-induced α-lactalbumin-thymol nanoparticle according to claim 1, characterized in that The mass ratio of α-lactalbumin to thymol described in (4) is (7:1, 6:1, 5:1).