Application of peanut red skin procyanidine nano-liposome in inhibition of salmonella typhimurium
By encapsulating peanut red skin proanthocyanidins using nanoliposome technology, the problems of insufficient targeting and poor stability were solved, achieving highly efficient inhibition of Salmonella typhimurium and food preservation effects, thus enhancing the application value of peanut resources.
Patent Information
- Application Number
- CN202511354369.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-22
- Publication Date
- 2025-12-30
AI Technical Summary
Peanut red skin proanthocyanidins have problems such as insufficient targeting, poor stability and low bioavailability when inhibiting Salmonella typhimurium, and there is also a risk of aflatoxin contamination, which limits their application in food preservation.
Peanut red skin proanthocyanidins were encapsulated using nanoliposome technology and prepared via an ultrasonic-microwave-reverse evaporation synergistic method to enhance their targeting and stability. The targeted delivery and synergistic antibacterial effect of the liposomes significantly inhibited the growth of Salmonella typhimurium.
It improves the stability and targeting of peanut red skin proanthocyanidins, significantly inhibits the growth of Salmonella typhimurium, extends the shelf life of food, and enhances the added value of peanut resources.
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Figure CN121220533A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of food safety, and relates to application of peanut red skin procyanidins nanoliposomes in inhibition of Salmonella typhimurium. BACKGROUND
[0002] Pathogenic microorganisms cause food spoilage and pose potential harm to human health. Salmonella typhimurium (S. typhimurium) is an important foodborne pathogenic bacterium that can cause serious infection through contaminated food, and its multi-drug resistance problem is increasingly serious. Studies have shown that the drug resistance rate of the bacterium to antibiotics such as cephalosporins and fluoroquinolones is as high as 17.6-81.6%, and the spread of drug resistance genes (such as blaCTX-M and gyrA mutations) further exacerbates the difficulty of treatment. Traditional antibiotics can inhibit bacterial proliferation, but they have problems such as disrupting the balance of intestinal flora and easily inducing drug resistance, so it is urgent to develop new antibacterial strategies.
[0003] Peanut red skin is a byproduct of peanut processing and is rich in procyanidins (PSPc), which are condensed from catechins or epicatechins and have significant antioxidant, anti-inflammatory and broad-spectrum antibacterial activities. In vitro studies have shown that PSPc can inhibit gram-negative bacteria (including S. typhimurium) by damaging the integrity of bacterial cell membranes and inhibiting DNA synthesis. However, PSPc faces challenges in practical applications such as insufficient targeting, poor water solubility, easy oxidation and degradation, and low bioavailability, and the peanut red skin raw material may be contaminated with aflatoxins, which limits its direct application.
[0004] Nanoliposome technology provides an innovative solution to improve the stability and delivery efficiency of PSPc. Liposomes encapsulate active ingredients through a phospholipid bilayer, which can protect PSPc from environmental factors such as light and oxidation, and enhance the penetration of bacterial biofilms through surface modification such as TPGS and antibody targeting ligands. In addition, the sustained-release properties of nanoliposomes can maintain an effective antibacterial concentration, reduce the frequency of administration and reduce toxicity. Therefore, if PSPc is combined with nanoliposomes, it is expected to improve the application value of procyanidins in food preservation. SUMMARY
[0005] The present application aims to solve the technical problems of insufficient targeting, poor stability and low bioavailability of peanut red skin procyanidins, and provides application of peanut red skin procyanidins nanoliposomes in inhibition of Salmonella typhimurium. PSPc coated with nanoliposomes can overcome the physicochemical defects of PSPc, and also take advantage of the targeted delivery and synergistic antibacterial effect of liposomes to significantly inhibit the growth of Salmonella typhimurium.
[0006] To achieve the above object, the application adopts the following technical solutions:
[0007] The application provides application of peanut red skin proanthocyanidin nanoliposomes in inhibition of salmonella typhimurium, wherein the peanut red skin proanthocyanidin nanoliposomes cause permeability change of cell membrane and cell wall of salmonella typhimurium, and then cause death of the bacteria.
[0008] In the technical solution, the content of peanut red skin proanthocyanidin in the peanut red skin proanthocyanidin nanoliposomes is not less than 75%.
[0009] In the technical solution, the minimum bacteriostatic concentration of the peanut red skin proanthocyanidin nanoliposomes on salmonella typhimurium is 250 μg / mL.
[0010] In the technical solution, the peanut red skin proanthocyanidin nanoliposomes are prepared by a microwave-ultrasonic-reverse evaporation synergistic method.
[0011] In the technical solution, the preparation method of the peanut red skin proanthocyanidin nanoliposomes comprises the following steps: after soybean lecithin and cholesterol powder are mixed, peanut red skin proanthocyanidin solution is added, ethanol is added to constant volume, ultrasonic treatment is performed, microwave treatment is performed under ice bath condition, and a mixture is formed; the mixture is subjected to low-temperature and reduced-pressure rotary evaporation to remove ethanol and become a gel state, preheated distilled water is added, hydration reaction is performed, finally ultrasonic treatment is performed, and peanut red skin proanthocyanidin nanoliposome suspension is obtained, which is stored in a refrigerator at 4 ℃.
[0012] In the technical solution, the temperature of the low-temperature and reduced-pressure rotary evaporation is 40-50 ℃, and the vacuum degree is 45 kPa.
[0013] In the technical solution, the A-type PC dimer accounts for 35-40% in the peanut red skin proanthocyanidin, and the purity is more than 98%.
[0014] Compared with the prior art, the application has the beneficial effects that:
[0015] The application overcomes the inherent limitations of natural bacteriostatic substance PSPc by using the high-efficiency delivery system of nanoliposomes, improves the stability and targeting of the PSPc, makes the activity of the PSPc in inhibiting salmonella typhimurium fully play, durably maintain and more efficient and safe, has application prospect in food preservation, and improves the value-added utilization of peanut resources. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 The PSPc total ion chromatogram used in the embodiment of the application.
[0017] Figure 2 The transmission electron microscope image of the PSPc nanoliposomes prepared in the embodiment 1 of the application.
[0018] Figure 3 The minimum inhibitory concentration of the PSPc nano-liposome of the application on Salmonella typhimurium was determined. Note: NC is a negative control, and CK is a blank control.
[0019] Figure 4 The influence of the PSPc nano-liposome of the application on the permeability of the cell membrane and cell wall of Salmonella typhimurium. Note: A is the determination of alkaline phosphatase activity, B is the determination of cell membrane potential, C is the determination of conductivity, D is the determination of Na + content, E is the determination of K + content, and F is the determination of Mg 2+ content.
[0020] Figure 5 The antibacterial effect of the PSPc nano-liposome of the application on Salmonella typhimurium in cooked chicken was evaluated. A is the 4℃ refrigeration condition, and B is the 25℃ normal temperature condition.
[0021] Figure 6 The influence of the PSPc nano-liposome of the application on the total number of colonies in the storage period of cooked chicken. A is the 4℃ refrigeration condition, and B is the 25℃ normal temperature condition. DETAILED DESCRIPTION
[0022] The following examples are used to illustrate the application, but are not used to limit the protection scope of the application. If not specifically indicated, the technical means used in the examples is the conventional means familiar to those skilled in the art. The test methods in the following examples are conventional methods, unless otherwise specified.
[0023] The PSPc used in the following examples was prepared by the method described in CN 114524793A, and the components of the PSPc were analyzed and identified by high performance liquid chromatography tandem mass spectrometry (UPLC-QTOF-MS / MS). The results are shown in Figure 1 and Table 1, and the purity of the PSPc is more than 98%, and the proportion of type A PC dimer is 37%.
[0024] Table 1 Mass spectrometry information of main compounds in PSPc
[0025]
[0026] Example 1 Preparation of PSPc nano-liposome
[0027] Soy lecithin and cholesterol powder were mixed at 7:1, then 0.4 mg / mL PSPc solution was added, and anhydrous ethanol was added to 20 mL. After 10 min of 200 W ultrasonic treatment, the mixture was completely dissolved to form a mixture. Then the mixture was treated by microwave (power 200 W, time 100 s) under ice bath condition. After 10 min of rotary evaporation at 50°C and 45 kPa, ethanol was removed to become a gel state. 30 mL of distilled water preheated to 50°C was added, and the mixture was hydrated for 15 min and then ultrasonicated for 5 min to obtain a procyanidine nanoliposome suspension. Then 5% trehalose was added, and the solid powder was obtained by freeze-drying. The nanoparticle of PSPc liposome was measured, and the particle size was 170-180 nm, and the encapsulation rate was 80.6%.
[0028] Example 2: Preparation of PSPc nanoliposome
[0029] Soy lecithin and cholesterol powder were mixed at 7:1, then 0.4 mg / mL PSPc solution was added, and anhydrous ethanol was added to 20 mL. After 10 min of 200 W ultrasonic treatment, the mixture was completely dissolved to form a mixture. Then the mixture was treated by microwave (power 200 W, time 100 s) under ice bath condition. After 10 min of rotary evaporation at 50°C and 45 kPa, ethanol was removed to become a gel state. 30 mL of distilled water preheated to 50°C was added, and the mixture was hydrated for 15 min and then ultrasonicated for 5 min to obtain a procyanidine nanoliposome suspension. Then 5% trehalose was added, and the solid powder was obtained by freeze-drying.
[0030] Figure 2 The transmission electron microscopy of the solid powder was observed by transmission electron microscopy, and the nanoparticle of PSPc liposome was observed, with a particle size of 180-190 nm and an encapsulation rate of 94.84%.
[0031] Example 3: Preparation of PSPc nanoliposome
[0032] Soy lecithin and cholesterol powder were mixed at 7:1, then 0.4 mg / mL PSPc solution was added, and anhydrous ethanol was added to 20 mL. After 10 min of 200 W ultrasonic treatment, the mixture was completely dissolved to form a mixture. Then the mixture was treated by microwave (power 200 W, time 100 s) under ice bath condition. After 10 min of rotary evaporation at 50°C and 45 kPa, ethanol was removed to become a gel state. 30 mL of distilled water preheated to 50°C was added, and the mixture was hydrated for 15 min and then ultrasonicated for 5 min to obtain a procyanidine nanoliposome suspension. Then 5% trehalose was added, and the solid powder was obtained by freeze-drying. The nanoparticle of PSPc liposome was measured, and the particle size was 170-180 nm, and the encapsulation rate was 80.6%.
[0033] Example 4
[0034] S. typhimurium was diluted to 1 x 10 6 CFU / mL with LB liquid medium, and the PSPc nanoliposome powder prepared in Example 2 was dissolved and diluted with LB liquid medium to a series of concentration gradients, with final concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906 μg / mL, respectively. Meanwhile, a blank control, a negative control and a positive control were set. The blank control was sterile water, the negative control was LB liquid medium, and the positive control was PSPc. The above-prepared PSPc nanoliposome containing different concentrations was added to a 96-well cell culture plate, and then the diluted bacterial suspension was added to each well, with a final volume of 200 μL, and 3 parallel groups were prepared for each group. After incubation at 37°C in the dark for 24 h, the absorbance value of each well at 600 nm was recorded, and the lowest concentration corresponding to the sterile growth well was taken as the MIC. The antibacterial rate (X) was calculated using Formula (1) as follows:
[0035]
[0036] Wherein: A is the absorbance value of the positive control group; B is the absorbance value of the experimental group; and C is the absorbance value of the negative control.
[0037] The results are shown in Figure 3 Table 2. The minimum inhibitory concentration (MIC) of PSPc nanoliposome against S. typhimurium was determined to be 250 μg / mL by the doubling dilution method, and the antibacterial rate thereof could be as high as 95.63% at this concentration. The PSPc without nanoliposome coating also had an antibacterial effect, but the effect was significantly reduced.
[0038] Table 2. Antibacterial rate (%) of PSPc nanoliposome against S. typhimurium
[0039]
[0040] Further, the effects of PSPc nanoliposome on the damage of S. typhimurium cells and cell membranes were determined by measuring the alkaline phosphatase (AKP) activity, cell membrane potential, conductivity and Na + , K + , Mg 2+ leakage of S. typhimurium. The results are shown in Figure 4 It can be seen that when the concentration of PSPc nanoliposome was at MIC, the extracellular alkaline phosphatase content of S. typhimurium, the conductivity in the culture solution, the Na + content, the K + content, and the Mg 2+The content significantly increased and the cell membrane potential significantly decreased (P<0.01), indicating that the PSPc nanoliposome can cause the death of the bacteria by changing the permeability of the cell wall and cell membrane of the Salmonella typhimurium.
[0041] Example 6
[0042] In order to verify the sensitivity of the PSPc nanoliposome to different bacteria, other common bacteria in food, such as Staphylococcus aureus, Listeria monocytogenes and Escherichia coli, were selected for antibacterial experiments, and the steps were referred to Example 4. The antibacterial rate results are shown in Table 3. It can be seen that under the same concentration of PSPc nanoliposome, the PSPc nanoliposome exhibits excellent antibacterial effect on Salmonella typhimurium and has higher sensitivity.
[0043] Table 3 Antibacterial rate (%) of PSPc nanoliposome to different bacteria
[0044]
[0045] Example 7
[0046] Fresh and clean chicken breast meat was taken and cooked in a pressure cooker, cut into chicken pieces with a mass of 1±0.02 g, quickly placed in a biological safety cabinet and cooled to room temperature, and then prepared for use. The bacterial liquid was diluted to 1×10 4 CFU / mL, the PSPc nanoliposome powder was fully dissolved in LB liquid medium, and after sterilization with a 0.22 μm filter, it was diluted to concentrations of 1000, 500 and 250 μg / mL, respectively. The high-pressure cooked chicken pieces were soaked in different concentrations of PSPc nanoliposome diluent for 10 s, taken out and drained, and then the drained chicken samples were placed in the prepared Salmonella typhimurium bacterial suspension, dried and then placed in sterile culture dishes according to the grouping, and then placed in a 4℃ and 25℃ incubator. The samples were taken out at fixed time points, crushed and gradient diluted with sterile water, and 100 μL of the sample was taken and spread on LB solid medium, and then incubated in a 37℃ constant temperature incubator for 24 h before plate counting. Three parallel operations were set for each concentration, and the group without PSPc nanoliposome treatment was used as a negative control.
[0047] Referring to Figure 5 It was found by measuring the growth curve that the PSPc nanoliposome has good inhibitory effect on Salmonella typhimurium in cooked chicken at 4℃ and 25℃, and the final growth amount of Salmonella typhimurium can be lower under the 4℃ cold storage condition.
[0048] Fresh and clean chicken breast meat was cooked in a pressure cooker, cut into 1±0.02 g pieces, quickly placed in a biological safety cabinet and cooled to room temperature, and then used. The bacterial liquid was diluted to 1×10 4 CFU / mL using LB liquid medium, and PSPc nanoliposome powder was dissolved in LB liquid medium, sterilized with a 0.22 μm filter, and then diluted to concentrations of 1000, 500, and 250 μg / mL. The high-pressure cooked chicken pieces were soaked in different concentrations of PSPc nanoliposome diluent for 10 s, then taken out and drained. The drained chicken samples were placed in sterile petri dishes and incubated at 4°C and 25°C, respectively. The samples were taken out at fixed time points, crushed, and gradient diluted with sterile water. 100 μL of the sample was taken and spread on LB solid medium, and then incubated in a 37°C incubator for 24 h before plate counting. Three parallel operations were set for each concentration, and the group without PSPc nanoliposome treatment was used as a negative control.
[0049] Referring to Figure 6 It was found that PSPc nanoliposomes had a certain inhibitory effect on the total number of colonies in cooked chicken at 4°C and 25°C by determining the total number of colonies. Under 4°C refrigeration conditions, 250-1000 μg / mL of PSPc nanoliposomes can extend the shelf life of cooked chicken by at least 2 days, and under 25°C normal temperature conditions, 500-1000 μg / mL of PSPc nanoliposomes can extend the shelf life of cooked chicken by at least 6 h.
[0050] The above-described embodiments are only preferred embodiments of the present application, merely used to explain the present application, and are not intended to limit the scope of the present application. For those skilled in the art, of course, other embodiments can be easily made by substitution or change based on the technical content disclosed in the present specification, and therefore, any changes and improvements made on the principles of the present application should be included in the scope of the present application.
Claims
1. Use of peanut procyanidin nanoliposomes in inhibiting Salmonella typhimurium, characterized in that, The peanut red skin procyanidin nanoliposome causes the permeability of the cell membrane and cell wall of Salmonella typhimurium to change, and further causes the death of the bacteria.
2. Use according to claim 1, characterized in that, The content of peanut red skin procyanidin in the peanut red skin procyanidin nanoliposome is not less than 75%.
3. Use according to claim 1, characterized in that, The minimum inhibitory concentration of the peanut red skin procyanidin nanoliposome on Salmonella typhimurium is 250 μg / mL.
4. Use according to claim 1, characterized in that, The peanut red skin procyanidin nanoliposome is prepared by a microwave-ultrasonic-reverse evaporation synergistic method.
5. Use according to claim 1 or 4, characterized in that, The preparation method of the peanut red skin procyanidin nanoliposome comprises the following steps: after soybean lecithin and cholesterol powder are mixed, peanut red skin procyanidin solution is added, ethanol is added to constant volume, ultrasonic treatment is performed, and microwave treatment is performed under ice bath conditions to form a mixture; the mixture is subjected to low-temperature and reduced-pressure rotary evaporation to remove ethanol and become a gel state, preheated distilled water is added, hydration reaction is performed, and finally ultrasonic treatment is performed to obtain peanut red skin procyanidin nanoliposome suspension, which is stored in a refrigerator at 4 DEG C.
6. Use according to claim 5, characterized in that, The temperature of the low-temperature and reduced-pressure rotary evaporation is 40-50 DEG C, and the vacuum degree is 45 kPa.
7. Use according to claim 5, characterized in that, The A-type PC dimer in the peanut red skin procyanidin accounts for 35-40%, and the purity is more than 98%.