Application of licoflavone compound in resisting food-borne pathogenic bacteria and dairy cow mastitis pathogenic bacteria

Abstract

The invention discloses application of a licoflavone compound in resisting food-borne pathogenic bacteria and dairy cow mastitis pathogenic bacteria. The licoflavone compound has the advantages that the licoflavone compound can be used for treating food-borne bacterial diseases caused by food-borne bacteria such as escherichia coli, bacillus cereus, listeria monocytogenes and salmonella enteritidis; food-borne fungal diseases caused by food-borne fungi such as aspergillus flavus, penicillium expansum and monilinia coerulea; the licochalcone A shows certain inhibitory activity on dairy cow mastitis pathogenic bacteria caused by streptococcus agalactiae, streptococcus dysgalactiae, escherichia coli and staphylococcus aureus, and particularly has an obvious inhibitory effect on food-borne pathogenic bacteria and dairy cow mastitis pathogenic bacteria by three licochalcone A with different contents. The compound disclosed by the invention is simple in structure and relatively high in safety, and is expected to be developed into a novel antibacterial drug.

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry and relates to the application of licorice flavonoid compounds in resisting foodborne pathogens and cow mastitis pathogens. Background Art

[0002] In recent years, the incidence of foodborne diseases has shown an upward trend worldwide. A survey by the World Health Organization found that about 600 million cases of foodborne diseases occur worldwide each year, and 420,000 people die as a result. Once infected with foodborne bacteria, vomiting and diarrhea may occur at the mildest, and life may be endangered at the worst. Foodborne pathogens have become the culprit of foodborne diseases today and are the primary cause of foodborne diseases. Food poisoning and mad cow disease caused by Salmonella, Listeria, Escherichia coli O157: H7, etc. are the most representative foodborne diseases. In addition, fruits and cereals caused by fungi such as stone fruit brown rot, Aspergillus flavus, and Penicillium expansum pose a great threat to agriculture and human health. On the other hand, as one of the most important economic industries in animal husbandry, the common disease of dairy cow mastitis has a serious impact on the quality and output of dairy products. The causes of mastitis include pathogen infection, poor environmental management, and individual differences in dairy cows, among which microbial infection is the main cause, which will lead to reduced milk production, reduced quality of dairy products, increased costs, and even the elimination of dairy cows, causing significant economic losses. Therefore, effective prevention and control of food preservation and dairy cow mastitis are of great significance for ensuring the output and quality of dairy products, protecting human health and economic development.

[0003] At present, fungicides are still one of the main means of preventing and controlling microbial pathogens, but the long-term use and abuse of traditional fungicides have led to the enhancement of pathogen resistance and potential dangers to human health, crop growth, and the ecological environment. With the development of modern agriculture and the improvement of human awareness of environmental protection, green and pollution-free biological control has been widely favored by humans. Therefore, it is imperative to develop new, efficient and low-toxic green fungicides. Finding active ingredients from natural products and optimizing their active structures is one of the main ways to create new pesticides.

[0004] Licorice has the effects of tonifying the spleen and replenishing qi, removing phlegm and relieving cough, clearing away heat and detoxifying, and harmonizing various medicines. As one of the major Chinese medicinal materials in my country, licorice has multiple pharmacological activities. Its rich licorice flavonoids (LF) compounds are mainly found in licorice roots and are one of the most important physiologically active substances in licorice. They have anti-inflammatory, antioxidant, antiviral, anti-tumor, analgesic and other effects. The main active ingredients are liquiritigenin, liquiritigenin, isoliquiritigenin, glabridin, licochalcone A, etc. The present invention tests the activity of a series of licorice flavonoid compounds against foodborne pathogens and bovine mastitis pathogens. The results show that licorice flavonoid compounds have excellent antibacterial activity against bovine mastitis pathogens and foodborne bacteria. Summary of the invention

[0005] The invention aims to provide a new use of licorice flavonoids in resisting foodborne pathogens and bovine mastitis pathogens, and is used for preventing and treating various diseases caused by foodborne pathogens and bovine mastitis pathogens.

[0006] The present invention provides the use of a licorice flavonoid compound in the preparation of a drug for resisting foodborne pathogenic fungi. The structural formula of the licorice flavonoid compound is: Aspergillus flavus Aspergillus flavus ACCC32601, Penicillium expansum Penicillium expansum ACCC30898, Pathogen of stone fruit brown rot Monilinia fructifera ACCC 36263.

[0007] The present invention also provides the use of licorice flavonoids in the preparation of drugs for resisting foodborne pathogenic bacteria. The structural formula of the licorice flavonoids is: Against foodborne pathogens such as Escherichia coli Escherichia coli O157: H7 ATCC 35150, Bacillus cereus Bacillus cereus BNCC 103930, Listeria monocytogenes Listeria monocytogenes ATCC19115, Salmonella Enteritidis Salmonella enterica subsp. enteric ATCC 14028.

[0008] The present invention provides the use of a licorice flavonoid compound in the preparation of a drug for resisting pathogenic bacteria of bovine mastitis. The structural formula of the licorice flavonoid compound is: Streptococcus agalactiae Streptococcus agalactiae ATCC27956, Streptococcus dysgalactiae Streptococcus dysgalactiae ATCC27957, Escherichia coli Escherichia coli ATCC 25922, Staphylococcus aureus Staphylococcus aureus Newman.

[0009] The present invention has the following beneficial effects: 1) The present invention provides the use of licorice flavonoids in resisting foodborne pathogens and bovine mastitis pathogens, and shows certain inhibitory activity against foodborne bacterial diseases, foodborne fungal diseases, and bovine mastitis pathogens caused by various bacteria and fungi. In particular, three different contents of licorice chalcone A have particularly significant inhibitory effects on foodborne pathogens and bovine mastitis pathogens, providing a new direction for the development of safe, efficient and green prevention and control technology for bovine mastitis.

[0010] 2) The licorice flavonoids in the present invention are derived from licorice, which is both medicinal and edible. They are green, efficient, safe, low-toxic and residue-free in food preservation and prevention and treatment of bovine mastitis, and open up a new path for using natural antibacterial agents to replace food additives. DETAILED DESCRIPTION

[0011] In order to better understand the present invention, the above content of the present invention is further described in detail by the following specific examples. However, this should not be construed as limiting the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified.

[0012] Example 1: Indoor antibacterial activity determination of licorice flavonoids against foodborne pathogenic fungi The foodborne pathogenic fungi used in this experiment were strains stored at 4°C in the laboratory, and the culture medium used for all foodborne pathogenic fungi in the laboratory was potato agar glucose medium (PDA for short). PDA medium formula: 200 g potatoes (peeled), 20 g glucose, 15 g agar, 1000 mL deionized water, sterilized at 121°C with high pressure steam for 20 min and then taken out for use.

[0013] The mycelium growth rate method was used for the indoor antibacterial activity determination. Activation of strains: Culture foodborne pathogenic fungi on PDA plates at 25 °C for 3 to 6 days. Preparation of drug plates: Pour the PDA medium sterilized at 121 °C into a culture dish, cool it to 45-50 °C, and add different concentrations of the test compounds to make drug-containing plates. Inoculation and culture: In the clean bench, use a puncher to punch a bacterial cake (5 mm in diameter) at the edge of the mycelium cultured for 3 to 6 days (the growth conditions should be as consistent as possible), then use an inoculation needle to pick it to the center of the drug plate, and then invert it in an incubator (25 °C) for culture. Result determination: After the mycelium of the blank control group is fully grown, the growth diameter of the mycelium of the drug group is measured by the cross method, and the growth inhibition rate of the mycelium of this type of compound is calculated.

[0014] Three parallel experiments were performed for each concentration to determine the inhibition rate of this type of compound. The results are shown in Table 1.

[0015] The above results show that the licorice flavonoids disclosed in the present invention have a certain antibacterial effect on foodborne pathogenic fungi, especially 98% of licorice chalcone A has the most significant antibacterial effect on Aspergillus flavus, with an inhibition rate of 79.67% at 50 μg / mL, which is better than the positive drug natamycin (70.90%). In addition, different contents of chalcone A have good antibacterial activity against Aspergillus flavus and stone fruit brown rot fungi, and the inhibition rate is above 60% at 50 μg / mL.

[0016] Example 2: Indoor antibacterial activity determination of licorice flavonoids against foodborne pathogenic bacteria The indoor antibacterial activity was determined by the micro-broth dilution method. The strain used in this experiment was a strain frozen at -80°C in the laboratory with 30% glycerol. The frozen strains were taken out and streaked on MH solid culture medium (sterilized at 121°C for 20 min), and cultured at a constant temperature of 37°C until single colonies grew. Single colonies on the solid culture medium were picked and transferred to MH liquid culture medium (sterilized at 121°C for 20 min), and cultured on a constant temperature shaker at 37°C and 180 rpm until the logarithmic growth phase. The strains in the logarithmic growth phase were diluted to about 10 with MH liquid culture medium. 6 Dissolve the compounds in DMSO, add them to the liquid culture medium, mix them evenly, and prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. Take 50 μL of the drug-containing culture medium and the same volume of the drug-containing medium containing about 10 6 CFU / mL bacterial culture was added to the wells of a 96-well plate, and the final drug concentration was 100 μg / mL. 100 μL of bacterial solution of the same concentration containing an equal amount of DMSO was used as a control. The 96-well plate was cultured in a 37°C constant temperature incubator for 12 h until the control group bacterial solution grew, and the OD value (OD 600 ). In addition, the OD values ​​of 100 μL of liquid culture medium and the drug with a concentration of 100 μg / mL were measured to correct the OD values ​​caused by the culture medium and the drug itself. The calculation formula for the corrected OD value and inhibition rate is as follows: Corrected OD 600 = OD of culture medium containing bacteria 600 - Axenic culture OD 600 ; All experiments were repeated three times to determine the inhibitory activity of licorice flavonoids against four foodborne pathogenic bacteria. The results are shown in Table 2.

[0017] The compound-containing liquid culture medium was diluted by two-fold dilution in a 96-well plate to obtain 50 μL of a series of concentrations of the drug-containing culture medium, and then the inhibition rate corresponding to the series of concentrations was determined according to the experimental method in this example, and the lowest concentration with an inhibition rate greater than 90% was defined as MIC (μg / mL). From the above determination results, it can be seen that licorice chalcone A and isoliquiritigenin have the most significant inhibitory activity against foodborne bacteria. The MIC activity data obtained by the determination are shown in Table 3.

[0018] Note: “-” indicates that the MIC value of the compound was not tested.

[0019] From the experimental results in Table 2 and Table 3, it can be seen that the licorice flavonoids involved in the present invention have good antibacterial activity against the tested foodborne pathogenic bacteria. It is worth noting that 98% licorice chalcone A has very excellent antibacterial activity, and the MIC values ​​for Escherichia coli ATCC 35150, Bacillus cereus BNCC 103930, Listeria monocytogenes ATCC 19115, and Salmonella enteritidis ATCC 14028 can reach 0.78~1.56 μg / mL, while the activity of 20% and 40% licorice chalcone A can also reach 3.12~12.5 μg / mL, and the antibacterial activity is better than that of isoliquiritigenin, while the MIC of the positive control drug ε-polylysine only reaches 100μg / mL and 25 μg / mL.

[0020] Example 3: Indoor antibacterial activity determination of licorice flavonoids against pathogenic bacteria of bovine mastitis The strains used in this experiment were frozen in the laboratory at -80℃ with 30% glycerol. The frozen Streptococcus agalactiae and Streptococcus dysgalactiae strains were taken out and streaked on sheep blood agar plates respectively, and cultured at 37℃ until single colonies grew. Single colonies were picked and added to brain heart infusion broth (BHI) liquid culture medium (tryptone: 10.0 g, beef heart extract powder: 17.5 g, sodium chloride: 5.0 g, glucose: 2.0 g, disodium hydrogen phosphate (12H2O): 2.5 g, distilled water: 1 L; sterilized at 121℃ for 20 min), and cultured at 37℃, 140 rpm constant temperature shaker until the logarithmic growth phase. Similarly, the frozen strains of Escherichia coli ATCC25922 and Staphylococcus aureus were taken out and streaked on MH solid medium (beef powder: 2.0g, soluble starch: 1.5g, acid hydrolyzed casein: 17.5g, agar: 20g, distilled water: 1L; sterilized at 121℃ for 20min), and cultured at 37℃ until single colonies grew. Single colonies were picked to MH broth medium (beef powder: 2.0g, soluble starch: 1.5g, acid hydrolyzed casein: 17.5g, distilled water: 1L; sterilized at 121℃ for 20min) liquid medium. The remaining experimental operation methods and calculation formulas are the same as those in Example 2. All experiments were set up with three replicates to determine the inhibition rate of licorice flavonoids on four bovine mastitis pathogenic bacteria, and the results are shown in Table 4.

[0021] The drug-containing liquid culture medium of the compound was diluted by a two-fold dilution method in a 96-well plate to obtain 50 μL of drug-containing culture medium of a series of concentrations. Then, the inhibition rate corresponding to the series of concentrations was determined according to the same test method in Example 2. The lowest concentration with an inhibition rate greater than 90% was defined as MIC (μg / mL). The activity data obtained by the determination are shown in Table 5.

[0022] From the results in Table 4 and Table 5, it can be seen that the 20% licorice chalcone A, 40% licorice chalcone A, and 98% licorice chalcone A involved in the present invention have significant antibacterial activity against Streptococcus agalactiae ATCC 27956 and Streptococcus agalactiae ATCC 27957, and have good inhibitory activity against Staphylococcus aureus, which is close to or equivalent to the inhibitory activity of the positive drug norfloxacin. In addition, this type of compound has only weak inhibitory activity against Escherichia coli.

[0023] In summary, the effects of licorice flavonoids on foodborne bacteria Escherichia coli Escherichia coli O157: H7ATCC 35150, Bacillus cereus Bacillus cereus BNCC 103930, Listeria monocytogenes Listeria monocytogenes ATCC 19115, Salmonella Enteritidis Salmonella enterica subsp. enteric Foodborne bacterial diseases caused by ATCC14028; foodborne fungi Aspergillus flavus Aspergillus flavus ACCC32601, Penicillium expansum Penicillium expansum ACCC30898, Pathogen of stone fruit brown rot Monilinia fructifera ACCC 36263 Foodborne fungal diseases caused by Streptococcus agalactiae Streptococcus agalactiae ATCC27956, Streptococcus dysgalactiae Streptococcus dysgalactiae ATCC27957, Escherichia coli Escherichia coli ATCC 25922, Staphylococcus aureus Staphylococcus aureus Newman caused bovine mastitis pathogens showed certain inhibitory activity. Therefore, licorice flavonoids showed certain antibacterial activity against 7 foodborne pathogens and 4 bovine mastitis pathogens, especially different contents of licorice chalcone A showed excellent and broad-spectrum antibacterial effect, which is worth further research and development in food preservation and prevention and treatment of bovine mastitis.

Claims

1. The use of licorice flavonoids in the preparation of drugs for resisting foodborne pathogenic fungi, characterized in that: The structural formula of licorice flavonoids is: 。 2. The use of the licorice flavonoid compound according to claim 1 in the preparation of a drug for resisting foodborne pathogenic fungi, characterized in that: Foodborne pathogenic fungi are Aspergillus flavus, Penicillium expansum, and drupe brown rot fungus.

3. The use of licorice flavonoids in the preparation of drugs for resisting foodborne pathogenic bacteria, characterized in that: The structural formula of licorice flavonoids is: 。 4. The use of the licorice flavonoid compound according to claim 3 in the preparation of a drug for resisting foodborne pathogenic bacteria, characterized in that: The food-borne pathogens it fights are Escherichia coli, Bacillus cereus, Listeria monocytogenes, and Salmonella Enteritidis.

5. The use of licorice flavonoids in the preparation of drugs for resisting bovine mastitis pathogens, characterized in that: The structural formula of licorice flavonoids is: 。 6. The use of the licorice flavonoid compound according to claim 5 in the preparation of a drug for resisting pathogenic bacteria of bovine mastitis, characterized in that: The pathogenic bacteria of bovine mastitis are Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, and Staphylococcus aureus.

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