Application of protocatechuic acid-zinc composite material in inhibition of enteritis serotype of enterosalmonella subsp. Subsp.
Nanoparticles produced by the composite material of protocatechic acid and zinc acetate effectively inhibit the growth and killing of Salmonella enteric, solve the problems of food poisoning and drug-resistant bacteria infection, and achieve food safety and reduced risk of in-hospital infection.
Patent Information
- Application Number
- CN202411291963.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2024-12-27
AI Technical Summary
The prior art is difficult to effectively inhibit the contamination and infection of Salmonella enteritidis, especially in food, and the effect of antibacterial agents on drug-resistant bacteria is limited, resulting in an increased risk of food poisoning and hospital cross-infection.
Procatechic acid and zinc acetate composite materials are used to form nanoparticles through coordination reactions to form procatechic acid-zinc composites, which are used to inhibit the growth and killing of Salmonella enteric and are used as a drug or food additive.
Protocatechic acid-zinc composite material can effectively inhibit the growth and killing of Salmonella enterogenic bacteria, have low-cost, efficient and safe antibacterial effects, and also has certain inhibitory effects on drug-resistant bacteria, reducing food poisoning and cross-infection in hospitals. risk.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food and medicine, and relates to the application of a protocatechuic acid-zinc composite material in the preparation of a preservative and a medical antibacterial agent. Background Art
[0002] Food safety is an important issue facing the world and has a significant impact on public health. Pathogenic microorganisms cause food to spoil and deteriorate, posing a potential hazard to human health. The application of antimicrobial agents in the process of food preservation is crucial to controlling the growth of pathogenic microorganisms and ensuring food safety and human health.
[0003] salmonella( Salmonella ) is a member of the Enterobacteriaceae family and is a facultative anaerobic, Gram-negative, oxidase-negative rod-shaped bacterium. Salmonella can cause a variety of gastrointestinal diseases and can colonize the intestines of humans and animals. Salmonella typhimurium ) and Salmonella Enteritidis ( Salmonella enterica ) is the most common Salmonella serotype that causes gastroenteritis in humans. Salmonella Enteritidis has been identified as one of the major foodborne bacterial pathogens worldwide and has become a major public health risk. Infection with Salmonella Enteritidis will cause diseases in humans and animal hosts, such as acute enteritis or bacteremia, systemic infection, etc., especially for children, there is a risk of cross-infection in the hospital, which may aggravate the disease (see: Ten-year changes in pathogens and drug resistance in children with sepsis. Journal of Clinical Pediatrics, 2014, 32 (07): 629-632.; Surveillance and clinical research on antimicrobial resistance in children in Kunming. Yunnan Province, Kunming Children's Hospital, 2016-01-26. etc.). Salmonella Enteritidis mainly causes disease through contaminated food and water sources, and is most common in egg and meat products in poultry and livestock. During poultry and livestock breeding and subsequent food processing and storage, Enteritidis Salmonella often contaminates food through pathogenic factors and forms biofilms on contact surfaces such as the slaughtering process, which is the main cause of food contamination and safety problems.
[0004] Protocatechuic acid (PCA) is a natural water-soluble organic compound with a molecular formula of C7H6O4 and a molecular weight of 154.12; CAS number is 99-50-3. PCA is widely distributed in vegetables, fruits, cereals and tea leaves, and is also present in a variety of Chinese medicinal materials, such as Salvia miltiorrhiza, Acanthopanax quinquefolius, Eucommia ulmoides, and Holly leaves. Studies have shown that PCA has a wide range of biological activities, such as antioxidant, anti-inflammatory, antibacterial, antiviral, anti-aging, anticancer, anti-tumor and nerve protection. Zinc acetate, also known as zinc acetate, is an organic salt with a chemical formula of Zn(CH3COO)2. Drugs containing 30% zinc acetate are often used as zinc supplements. In addition, zinc acetate tablets can also be used in the clinical treatment of genetic diseases.
[0005] At present, nano zinc oxide is often used for antibacterial applications of zinc, and PCA has a certain antibacterial effect against Escherichia coli, etc. However, there are few reports on the use of composite materials prepared by reacting it with metal salts to inhibit or kill Salmonella enteritidis, as well as its application in related food preservatives and new medical antibacterial agents. Summary of the invention
[0006] The purpose of the present invention is to provide an application of a protocatechuic acid-zinc composite material in inhibiting Salmonella enterica subspecies enteritidis serotype, so as to solve the problem of food poisoning and illness caused by contamination or infection with Salmonella enterica subspecies enteritidis serotype and alleviate the risk of severe illness caused by cross-infection with drug-resistant bacteria.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, a protocatechuic acid-zinc composite material is provided for use in preparing a drug for inhibiting foodborne pathogens, wherein the protocatechuic acid-zinc composite material inhibits the growth of Salmonella enteritidis (i.e., the protocatechuic acid-zinc composite material is an effective pharmaceutical ingredient that has an inhibitory effect on foodborne pathogens such as Salmonella enteritidis).
[0008] Preferably, the Salmonella Enteritidis is an isolated strain of Salmonella Enteritidis subspecies Enteritidis serotype that exists in food and eventually causes food poisoning.
[0009] Preferably, the minimum inhibitory concentration of the protocatechuic acid-zinc composite material against Salmonella enterica subspecies enteritidis serotype CICC 21513 is 0.2 mmol / L.
[0010] Preferably, the protocatechuic acid-zinc composite material exerts an inhibitory and killing effect on Salmonella enterica subspecies enterica serotype by causing depolarization of the membrane potential of Salmonella enterica subspecies enterica serotype and causing damage to the cell membrane; that is, the protocatechuic acid-zinc composite material can be used as the main active ingredient of a drug to exert antibacterial and bactericidal effects.
[0011] Preferably, the protocatechuic acid-zinc composite material destroys the bacterial morphology of Salmonella enterica subspecies enteritidis serotype, such as bacterial deformation (caused by surface morphology changes), shrinkage, and accelerates bacterial death as cell membrane damage.
[0012] Preferably, the drug further comprises other components having anti-Salmonella enterica subspecies enteritidis serotype activity (ie, other active ingredients of the drug) that are compatible with the protocatechuic acid-zinc composite material.
[0013] Preferably, the medicine further comprises preparation excipients.
[0014] Preferably, the protocatechuic acid-zinc composite material is prepared by complexing protocatechuic acid with zinc ions via a coordination reaction.
[0015] Preferably, the compounding specifically comprises the following steps: under weak alkaline conditions (such as pH=8.0~8.2), by continuous heating and stirring, protocatechuic acid and zinc acetate added in a certain ratio (such as the chemical reaction molar ratio of protocatechuic acid and zinc acetate is 1:0.5~2) are reacted in a solvent (such as water) (such as reacting at 50~55°C for 3~4 hours), and a protocatechuic acid-zinc complex with a corresponding coordination ratio dispersed in the reaction system can be obtained (in the reaction, zinc acetate dissociates to form acetate and zinc ions, and the zinc ions formed after the dissociation of zinc acetate are combined with the protocatechuic acid after the hydrogen ions are removed through coordination bonds, and the generated nanoparticles are protocatechuic acid-zinc complexes).
[0016] In a second aspect, the invention provides the use of the above-mentioned protocatechuic acid-zinc composite material in the preparation of food additives (such as preservatives).
[0017] In a third aspect, the invention provides the use of the above-mentioned protocatechuic acid-zinc composite material in the preparation of feed additives (such as preservatives).
[0018] Preferably, the food additive or feed additive is used to inhibit the reproduction of Salmonella enteritidis (such as Salmonella enteritidis subspecies enteritidis serotype) on the surface of food or feed.
[0019] The beneficial effects of the present invention are embodied in: In the pharmacological and related drug toxicity experiments conducted around the use of a composite product of protocatechuic acid and zinc acetate to inhibit foodborne pathogens such as Salmonella enterica serovar Enteritidis (such as Salmonella enterica subspecies enterica serovar Enteritidis), the present invention discovered and verified an effective ingredient that can exert antibacterial activity against such foodborne pathogens, that is, the above-mentioned protocatechuic acid-zinc composite material is used in the preparation of pharmaceutical antibacterial agents and food preservatives to prevent food poisoning and related infectious diseases caused by contamination of Salmonella enterica serovar Enteritidis, and to a certain extent avoid the occurrence of serious symptoms caused by possible nosocomial cross-infection (such as MRSA). In addition, the protocatechuic acid-zinc composite material has the advantages of low cost, high efficiency and safety in antibacterial applications (such as food preservation, storage and clinical anti-infection).
[0020] Furthermore, the present invention determines the minimum inhibitory concentration (MIC) of the protocatechuic acid-zinc composite material against Salmonella enterica subspecies enteritidis serotype by a mixed co-culture plate coating method, which not only provides a basis for the protocatechuic acid-zinc composite material as an antibacterial agent, but also provides a reference for the application research of related products.
[0021] Furthermore, the present invention has found through experiments that as the concentration of the protocatechuic acid-zinc composite material increases, it can destroy the integrity of the cell membrane of the Salmonella enterica subspecies enteritidis serotype strain (mainly referring to the morphological deformation and shrinkage) and the stability of the cell membrane surface potential (i.e., membrane potential), not only inhibiting the growth of the Salmonella enterica subspecies enteritidis serotype, but also having a certain killing effect on the Salmonella enterica subspecies enteritidis serotype. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1a This is a scanning electron microscope image of the material: protocatechuic acid-zinc composite material after washing and drying.
[0023] Figure 1b This is the EDS spectrum of the material: protocatechuic acid-zinc composite material after washing and drying (At%: C 53.8%; Zn 23.9%; O 22.3%).
[0024] Figure 2 This is a dot plate diagram of the inhibition experiment of protocatechuic acid-zinc composite material, protocatechuic acid, and zinc acetate on Salmonella enterica subspecies enteritidis serotype (CK is the blank control).
[0025] Figure 3 This is a picture of the bacterial solution coated on the plate after co-culture with different drug concentrations using the co-culture plate coating method (CK is the blank control; protocatechuic acid-zinc: protocatechuic acid-zinc composite material).
[0026] Figure 4 These are scanning electron micrographs of the effects of different concentrations of protocatechuic acid, zinc acetate, and protocatechuic acid-zinc composite materials on Salmonella enterica subspecies Enteritidis serotype strains (CK is the blank control).
[0027] Figure 5 The statistical diagram of the changes in membrane potential of Salmonella enterica subspecies Enteritidis serotype after the action of different concentrations (0, 0.10, 0.20, 0.40, 0.80 mM) of protocatechuic acid-zinc composite material (abbreviated as PCA-Zn), PCA, and zinc acetate (represented by Zn) (** P <0.05).
[0028] Figure 6 This is a plate diagram of the MRSA inhibition experiment of protocatechuic acid, zinc acetate, protocatechuic acid-zinc composite material, and protocatechuic acid and zinc acetate mixture (CK is the blank control).
[0029] Figure 7 The results of H&E staining (CK is blank control; protocatechuic acid-zinc: protocatechuic acid-zinc composite material). DETAILED DESCRIPTION
[0030] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only for explanation of the present invention, but not for limiting the protection scope of the present invention.
[0031] (I) Preparation of protocatechuic acid-zinc composite materials Using protocatechuic acid and zinc acetate as basic raw materials, a protocatechuic acid-zinc composite material with a spherical nanostructure is obtained by stirring and heating in a weakly alkaline environment. The specific description is as follows: After adding analytically pure reaction raw materials zinc acetate (12.98 mM) and protocatechuic acid (the chemical reaction molar ratio of zinc acetate: protocatechuic acid, i.e., the molar ratio of substance is 1:1) to deionized water, the reaction environment pH was adjusted to 8.2 using 1 mol / L NaOH solution. After adjusting the pH, the reaction temperature was heated to 50°C and stirred vigorously (speed was 550 r / min) for 4 h. After the reaction was completed, the heating and stirring were stopped, and the reaction system (in the form of a suspension) was centrifuged at 8000 r / min (room temperature) for 10 min. The solids initially separated after the reaction were washed alternately with anhydrous ethanol and deionized water for 4 times, and finally dried in a vacuum drying oven at 50°C to obtain a solid powder.
[0032] See also Figure 1a The obtained solid powder was sampled by electron microscopy, and a large number of spherical nanoparticles of protocatechuic acid-zinc complex were observed by scanning electron microscopy.
[0033] See also Figure 1b The obtained solid powder was detected by EDS energy dispersive spectrometer, and the results verified that the protocatechuic acid-zinc composite material was formed by the complex of protocatechuic acid and zinc ions.
[0034] (II) Experiment on the inhibitory effect of protocatechuic acid-zinc composite material on Salmonella enterica subspecies enteritidis serotype The minimum inhibitory concentration of the protocatechuic acid-zinc composite material against the enteric subspecies enteritidis serotype strain of Salmonella enterica serotype Enteritidis was determined by the co-culture plate coating method, and the morphological changes of the enteric subspecies enteritidis serotype strain of Salmonella enterica serotype Enteritidis treated with the protocatechuic acid-zinc composite material were further observed by scanning electron microscopy; the specific instructions are as follows.
[0035] 2.1 Experimental strains Salmonella enterica subspecies enteritidis serotype ( Salmonella enterica subsp.enterica serovar Enteritidis The strain number is CICC 21513 and was purchased from China Industrial Microbiological Culture Collection Center.
[0036] 2.2 Culture medium LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, and sodium chloride (NaCl) 10 g / L; pH was adjusted to 7.4 and sterilized at 121°C for 20 min.
[0037] LB solid medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, and agar 15 g / L; pH was adjusted to 7.4 and sterilized at 121°C for 20 min.
[0038] 2.3 Determination of the antibacterial properties of protocatechuic acid, zinc acetate, and protocatechuic acid-zinc composite materials by mixed co-culture bacterial solution spot plate method The thawed Salmonella enterica subspecies enteritidis serotype strain was inoculated onto LB solid culture medium by streaking and incubated in an incubator at 37°C for 18 h. Then, a single colony was picked and inoculated into LB liquid culture medium and incubated in a shaking incubator at 37°C for 12-18 h to activate the first generation of bacteria. Subsequently, 100 μL of the first generation of bacteria was inoculated into 30 mL of LB liquid culture medium and incubated in a shaking incubator at 37°C until the bacterial concentration reached 1×10 8 CFU / mL. Pipette 1 mL of bacterial solution into a centrifuge tube, centrifuge at 8000 r / min (room temperature) for 5 min to remove the culture medium, wash the bacterial precipitate with sterilized saline for 2-3 times, and resuspend the bacterial precipitate with 1 mL of protocatechuic acid, zinc acetate, and protocatechuic acid-zinc composite drug solution of the same concentration; resuspend the bacterial precipitate with 1 mL of saline as a blank control. After co-cultivation at 37°C for 4 h, aspirate 3 μL of bacterial solution treated with different drug solutions and spot plate on LB solid culture medium, and spot the blank control bacterial solution at the same time, then invert the plate in a 37°C incubator for 10 h to observe the growth of the colonies. The preparation of the above-mentioned drug solutions is described as follows (the drug solution concentrations are shown in Table 1): a protocatechuic acid solution, a zinc acetate solution and a protocatechuic acid-zinc composite material suspension are prepared using physiological saline.
[0039] Table 1. Protocatechuic acid, zinc acetate, and protocatechuic acid-zinc composite solution gradients
[0040] Note: The medicinal solutions in Table 1 are all prepared with normal saline as solvent; the "protocatechuic acid-zinc composite material" specifically adopts the protocatechuic acid-zinc composite material prepared in (I).
[0041] from Figure 2It can be seen that the protocatechuic acid-zinc composite material has good antibacterial properties against Salmonella enterica subspecies enteritidis serotype CICC21513, and is significantly better than protocatechuic acid and zinc acetate.
[0042] 2.4 Determination of the minimum inhibitory concentration (MIC) of protocatechuic acid-zinc composite materials against Salmonella enterica subspecies enteritidis serotype MIC is defined as the minimum inhibitory concentration that completely inhibits the growth of microorganisms. The inhibitory effect of protocatechuic acid-zinc composite material on Salmonella enterica subspecies enteritidis serotype was detected by co-culture plate coating method. The bacterial solution of Salmonella enterica subspecies enteritidis serotype strain was obtained by culture according to the method described in 2.3. 1 mL of bacterial solution was centrifuged at 8000 r / min (room temperature) for 5 min to remove the culture medium, and the bacterial precipitate was washed 2 to 3 times with sterile saline. 1 mL of different concentrations of protocatechuic acid, zinc acetate, and protocatechuic acid-zinc composite material were added to resuspend the bacterial precipitate (the concentration of each drug solution was as follows: Figure 3 As shown, sterile saline was used as the solvent), wherein the protocatechuic acid-zinc composite material was prepared according to the method in (I), and 1 mL of saline was used to resuspend the bacterial precipitate as a blank control. After 4 h of co-culture (37°C shaker), 50 μL of bacterial solution was respectively applied to LB solid culture medium, and inverted culture was carried out in a 37°C incubator for 10 h to observe the growth of the colonies.
[0043] See also Figure 3 When the concentration of the protocatechuic acid-zinc composite material reaches 0.2 mM (equivalent to the concentration of the drug solution of gradient 7 in Table 1), the protocatechuic acid-zinc composite material has a significant inhibitory effect on the Salmonella enterica subspecies enteritidis serotype CICC 21513. It can be concluded that the MIC value of the protocatechuic acid-zinc composite material against the Salmonella enterica subspecies enteritidis serotype is 0.2 mM.
[0044] 2.5 Observation of the inhibitory effect of protocatechuic acid-zinc composites on Salmonella enterica subspecies enteritidis strains using scanning electron microscopy According to the method described in 2.3, obtain the bacterial suspension of Salmonella enterica subspecies enteritidis serotype strain, take 1 mL of bacterial suspension and centrifuge at 8000 r / min (room temperature) for 5 min, remove the supernatant, wash the bacterial precipitate with sterile saline for 2 to 3 times, add 1 mL of protocatechuic acid-zinc composite material with concentrations of 0.8 mM, 0.4 mM, 0.2 mM, and 0.1 mM respectively to resuspend the bacterial precipitate, and use protocatechuic acid and zinc acetate with the same concentration as the control group, and add physiological saline as the blank control, wherein the protocatechuic acid-zinc composite material is prepared according to the method in (I). The obtained bacterial suspension sample is placed in a 37℃ shaker for 4 h and then centrifuged, the bacterial precipitate is washed with sterile saline for 2 to 3 times, the bacterial precipitate is resuspended with 2.5% glutaraldehyde solution, and fixed in a 4℃ refrigerator for 12 h. The samples were then washed 2 to 3 times with sterile saline and dehydrated for 10 min using ethanol of different concentrations (30%, 50%, 70%, 80%, 90% and 100%). After dehydration, they were resuspended in isoamyl acetate for 30 min. The oven-dried samples were fixed on a stage with conductive glue and sprayed with gold for observation using a scanning electron microscope.
[0045] See also Figure 4 According to the scanning electron microscope images, it can be seen that the blank control Salmonella enterica serotype Enteritidis subspecies has a plump morphology without any wrinkling or deformation. As the concentration of the protocatechuic acid-zinc composite material treatment increases, the corresponding Salmonella enterica serotype Enteritidis subspecies has an increasingly uneven and wrinkled appearance. In addition, the effects of protocatechuic acid and zinc acetate treatments at the same concentration are weaker than those of the protocatechuic acid-zinc composite material treatment.
[0046] (III) Study on the inhibitory mechanism of protocatechuic acid-zinc composite material on Salmonella enterica subspecies enteritidis serotype strains (membrane potential method) The bacterial suspension of Salmonella enterica subspecies enteritidis strain was obtained according to the method described in 2.3, and the cells were collected by centrifugation. After washing the cell pellet with sterile saline for 2-3 times, 1 mL of protocatechuic acid, zinc acetate and protocatechuic acid-zinc composite material with concentrations of 0.8 mM, 0.4 mM, 0.2 mM and 0.1 mM were added to resuspend the cell pellet, and 1 mL of saline was used as a blank control. The protocatechuic acid-zinc composite material was prepared according to the method in (I). After incubation at 37°C in a shaking incubator for 4 h, the cells were collected by centrifugation, washed and resuspended with saline, and 1 mM DiBAC4 (3) membrane potential fluorescent probe (10 μL per 1 mL) was added. The cells were incubated in the dark for 30 min, and 200 μL of the cell suspension was added to a black opaque 96-well plate, with 5 parallels for each group. The fluorescence intensity was detected using a multifunctional microplate reader (excitation wavelength = 492 nm, emission wavelength = 515 nm), and the membrane potential (the potential difference between the inside and outside of the biological cell membrane) was finally characterized.
[0047] Depend on Figure 5 It can be seen that compared with the blank control (0 mM), the membrane potential of the Salmonella enterica serotype Enteritidis strain treated with the protocatechuic acid-zinc composite material for 4 h was significantly increased (when the treatment concentration reached 0.2 mM or above; protocatechuic acid and zinc acetate treatments required higher concentrations); among them, the fluorescence intensity of the blank control was -47012.33, the fluorescence intensity of the 0.1 mM treatment group was -36296.67, the fluorescence intensity of the 0.2 mM treatment group was -22703, the fluorescence intensity of the 0.4 mM treatment group was -1933.67, and the fluorescence intensity of the 0.8 mM treatment group was 18460, which increased by 22.79%, 51.71%, 95.89%, and 139.27%, respectively; this indicates that the protocatechuic acid-zinc composite material caused the depolarization of the membrane potential of the Salmonella enterica serotype Enteritidis strain at a lower concentration. Abnormal changes in the cell membrane surface potential of Salmonella enterica serotype Enteritidis strains may change the activity of sodium ion channels, leading to increased influx, interfering with the normal life activities of the bacteria and causing damage to the cell membrane.
[0048] 4. Antibacterial test of drug-resistant bacteria The mixed co-culture plate coating method was used to compare the growth of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) treated with protocatechuic acid, zinc acetate, a mixture of protocatechuic acid and zinc acetate, and a protocatechuic acid-zinc composite material to determine the antibacterial effect of the protocatechuic acid-zinc composite material on drug-resistant bacteria. The preparation instructions of each solution are as follows: protocatechuic acid solution (0.81 mM), zinc acetate solution (0.81 mM), a mixed solution of protocatechuic acid (0.81 mM) and zinc acetate (0.81 mM), and a protocatechuic acid-zinc composite material suspension (0.81 mM) were prepared with physiological saline; the protocatechuic acid-zinc composite material was prepared according to the method in (I).
[0049] The results are as follows Figure 6 As shown, only the experimental group treated with protocatechuic acid-zinc composite material did not see colony growth, which was significantly different from the blank control (normal saline). The above results show that the protocatechuic acid-zinc composite material has good antibacterial properties against common drug-resistant bacteria (i.e. MRSA), and is significantly better than protocatechuic acid, zinc acetate, and a mixture of protocatechuic acid and zinc acetate.
[0050] (V) Hepato-renal toxicity test of protocatechuic acid-zinc composite material SPF grade 6-week-old male BLAB / C mice were selected, and the treatment of all animals in the experiment complied with the "Guidelines for the Care and Use of Laboratory Animals": ISBN-10:0-309-15396-4 (Eighth Edition) and "Regulations on Laboratory Animal Management of Shaanxi University of Science and Technology".
[0051] 20 SPF grade 6-week-old female BLAB / C mice, weighing 18-20 g, were fed and drunk freely at 25°C. The bedding was changed every two days to ensure that the cages were dry and clean. The cages were wiped and disinfected with 75% anhydrous ethanol. The light was on for 12 h per day, and 5 mice were fed in each cage. Before the experiment, the mice were fed adaptively for 7 days. The 20 mice were randomly divided into cages and divided into two groups. The body weight was measured every day during the adaptation period. The above 20 mice were divided into a protocatechuic acid-zinc composite material hepatotoxicity test group and a blank test group. According to the daily weight of the mice, the protocatechuic acid-zinc composite material hepatotoxicity test group was set to be gavaged with a 0.4 mM concentration of the drug solution, and the blank test group was gavaged with normal saline for 7 days. The protocatechuic acid-zinc composite material was prepared according to the method in (I).
[0052] On the 8th day of the experiment, mice were anesthetized by intraperitoneal injection of 3.5% chloral hydrate, and blood was collected from the eye sockets of the mice. The mice were then killed by cervical dislocation, and the liver, kidney, spleen, jejunum, ileum, and colon of the mice were removed. The required parts were cut and added to 8 mL of 4% paraformaldehyde fixative and placed at 4°C for use. The obtained samples were paraffin-embedded and sliced, and the paraffin sections were dewaxed and stained using the H&E staining kit (the specific process was: staining with hematoxylin staining solution for 10 min, differentiation solution for 30 s, soaking in tap water for 15 min, staining with eosin staining solution for 1 min, soaking in tap water for 3 min, and drying naturally). The remaining tissues were fixed in 4% paraformaldehyde solution for more than 24 h, and all the remaining tissues were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for use. The blood from each group was divided into two portions, one was placed in an anticoagulant tube for physiological and biochemical tests, and the other was centrifuged at 4°C and 3000 r / min for 10 min to separate the serum, discard the insoluble matter, transfer the serum to a clean test tube, and divide it into small portions and store it at -80°C for use.
[0053] According to the blood biochemical test, it was found that there was no significant difference in the relevant indicators between the protocatechuic acid-zinc composite material hepato-renal toxicity test group and the blank test group, and all were within the normal range. The H&E staining results showed that the tissue morphology of the organs in the protocatechuic acid-zinc composite material hepato-renal toxicity test group did not change significantly compared with the blank test group. Among them, no obvious damage was found in the liver, kidney, and spleen sections, and the tissue structure was normal ( Figure 7 ). Therefore, it can be concluded that the protocatechuic acid-zinc composite material has no hepatotoxicity and kidney toxicity, and has no effect on animal body weight and blood indicators, and is safe.
[0054] In summary, the protocatechuic acid-zinc composite material proposed by the present invention has the following advantages: (1) Effectively inhibit bacterial growth: The protocatechuic acid-zinc composite material can inhibit the growth of Salmonella enterica subspecies Enteritidis serotype in vitro (with a low MIC), thus achieving the purpose of antibacterial. The protocatechuic acid-zinc complex, as its main component, has a better effect in preventing contamination and infection by Salmonella enterica subspecies Enteritidis serotype.
[0055] (2) Zinc acetate is a common weak acid salt that is easy to obtain, widely available, and inexpensive. Protocatechuic acid is a natural product that can be extracted from plant materials.
[0056] (3) Compared with traditional antibiotics, protocatechuic acid-zinc composite materials have the potential multi-target synergistic effect of zinc acetate and protocatechuic acid in antibacterial performance, are less likely to cause bacteria to develop drug resistance, and have the advantage of low toxicity.
[0057] (4) Other experimental results show that the protocatechuic acid-zinc composite material has a certain inhibitory effect on common drug-resistant bacteria (such as MRSA, etc.) and can alleviate the risk of cross-infection in the hospital.
[0058] The above advantages indicate that protocatechuic acid-zinc composite materials are expected to be used in multiple fields as medical antibacterial agents, food additives, and feed additives, and provide a new drug intervention approach to prevent related bacterial contamination and infection.
Claims
1. Use of a protocatechuic acid-zinc composite material in the preparation of a drug for inhibiting foodborne pathogens, characterized in that: The protocatechuic acid-zinc composite material inhibits the growth of Salmonella enteritidis.
2. The use according to claim 1, characterized in that: The enteritidis salmonella is enteritidis salmonella subspecies enteritidis serotype.
3. The use according to claim 2, characterized in that: The minimum inhibitory concentration of the protocatechuic acid-zinc composite material to Salmonella enterica subspecies enteritidis serotype is 0.2 mmol / L.
4. The use according to claim 2, characterized in that: The protocatechuic acid-zinc composite material causes the membrane potential depolarization of Salmonella enterica subspecies enteritidis serotype and causes cell membrane damage.
5. The use according to claim 2, characterized in that: The medicine also comprises an effective component for resisting enteric Salmonella enterica subspecies enteritis serotype which is compatible with the protocatechuic acid-zinc composite material.
6. The use according to claim 1 or 5, characterized in that: The medicine also includes preparation excipients.
7. Application of the protocatechuic acid-zinc composite material in the preparation of food additives, characterized in that: The protocatechuic acid-zinc composite material inhibits the growth of Salmonella enteritidis.
8. The use according to claim 7, characterized in that: The enteritidis salmonella is enteritidis salmonella subspecies enteritidis serotype.
9. Application of the protocatechuic acid-zinc composite material in the preparation of feed additives, characterized in that: The protocatechuic acid-zinc composite material inhibits the growth of Salmonella enteritidis.
10. The use according to claim 9, characterized in that: The enteritidis salmonella is enteritidis salmonella subspecies enteritidis serotype.