A 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone against gram-positive bacteria and a preparation method thereof

Abstract

The application discloses a 3'-geranyl-4,2',4',6'-tetrahydroxy dihydrochalcone against gram-positive bacteria, and a chemical structural formula of the dihydrochalcone is shown as formula (I). The application further provides a preparation method and application of the dihydrochalcone derivative. The 3'-geranyl-4,2',4',6'-tetrahydroxy dihydrochalcone has significant antibacterial activity against gram-positive bacteria, MRSA retention bacteria activity, and can significantly inhibit the formation of gram-positive bacterial biofilm.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial compounds, specifically relating to a 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone that is effective against Gram-positive bacteria and its preparation method. Background Technology

[0002] Staphylococcus aureus is a common Gram-positive pathogen that can cause a variety of infections, from minor skin infections to life-threatening pneumonia and sepsis. In recent years, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become a major challenge in global public health due to the widespread use and overuse of antibiotics. MRSA's resistance to β-lactam antibiotics, including methicillin, complicates the treatment of its infections. Even more challenging is the ability of MRSA to form metabolically quiescent "persistent bacteria" within the host. Persistent bacteria are a non-proliferating but drug-resistant cell subset that can survive antibiotic treatment and cause chronic or recurrent infections. Furthermore, MRSA can form biofilms, which not only provide a physical barrier for the bacteria but also enhance their resistance and the survival of persistent bacteria. These characteristics limit the effectiveness of traditional antibiotics in clearing MRSA infections, highlighting the urgent need to develop innovative therapeutic strategies targeting persistent MRSA bacteria and biofilms.

[0003] Natural products, due to their diverse structures and unique mechanisms of action, have become an important source for antibacterial drug development. Among them, flavonoids have attracted much attention due to their broad range of biological activities. However, most antibacterial research on flavonoids has focused on rapidly proliferating bacteria, with relatively few studies on their effects on persistent bacteria and biofilms. Summary of the Invention

[0004] One objective of this invention is to address the above-mentioned technical problems by providing a compound with excellent antibacterial effects, which not only inhibits bacteria but also significantly inhibits persistent bacteria and biofilms.

[0005] Another object of the present invention is to provide a method for preparing the compound.

[0006] Another object of the present invention is to provide applications of the said compound.

[0007] To achieve the above-mentioned objectives, the present invention provides a 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone for resisting Gram-positive bacteria, the chemical structural formula of which is shown in the following formula (I):

[0008]

[0009] On the other hand, the present invention also provides a method for preparing the 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone, which is effective against Gram-positive bacteria, the method comprising the following steps:

[0010] (1) Dissolve 0.5-3g of phloretin in 30-50mL of anhydrous dimethylformamide, add 0.5-1g of anhydrous lithium carbonate and 0.76-4.56mL of geraniol bromide, and heat under reflux at 135-165℃ for 12 hours.

[0011] (2) Add an equal volume of deionized water to the reaction solution to terminate the reaction;

[0012] (3) Filter the reaction solution and extract it with ethyl acetate 3-6 times, retaining the organic phase.

[0013] (4) Add an equal volume of anhydrous sodium sulfate to the organic phase, filter, concentrate under vacuum, and separate and purify to obtain 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone.

[0014] On the other hand, the present invention also provides the application of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone, which is effective against Gram-positive bacteria, in the fight against Gram-positive bacteria.

[0015] On the other hand, the present invention also provides the application of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone, which is effective against Gram-positive bacteria, in enhancing the antibacterial effect of antibiotics.

[0016] Preferably, the antibiotics include, but are not limited to, meropenem, levofloxacin, ofloxacin, vancomycin, norfloxacin, chloramphenicol, gentamicin, azithromycin, and kanamycin.

[0017] On the other hand, the present invention also provides the application of the aforementioned 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone, which is effective against Gram-positive bacteria, in inhibiting the formation of biofilms by Gram-positive bacteria.

[0018] On the other hand, the present invention also provides the use of the aforementioned anti-Gram-positive bacteria 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone in the preparation of anti-Gram-positive bacteria pharmaceuticals, health products or skin care products.

[0019] Preferably, the Gram-positive bacteria include Gram-positive bacilli and Gram-positive cocci. More preferably, the Gram-positive bacteria include, but are not limited to, Staphylococcus aureus, Bacillus subtilis, Streptococcus, Streptococcus pneumoniae, Clostridium tetani, Enterococcus, Corynebacterium diphtheriae, Listeria, etc. More preferably, the Gram-positive bacteria include, but are not limited to, Staphylococcus aureus 29213, Staphylococcus aureus RN4220, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, Streptococcus, Streptococcus pneumoniae, Clostridium tetani, etc.

[0020] The 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone provided by this invention has good antibacterial ability, especially against Gram-positive bacteria, particularly Staphylococcus aureus Sa29213, Staphylococcus aureus RN4220, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, Streptococcus, Streptococcus pneumoniae, Clostridium tetani, and their persistent bacteria. In addition, it can also eliminate MRSA persistent bacteria that cannot be eliminated by common antibiotics such as meropenem, levofloxacin, ofloxacin, vancomycin, norfloxacin, chloramphenicol, gentamicin, azithromycin, and kanamycin, and can eliminate persistent bacteria formed by the above common antibiotics. At the same time, it can significantly inhibit the formation of MRSA persistent bacterial biofilms. Compared to traditional antibiotics, this compound not only effectively kills metabolically quiescent resident bacteria but also inhibits biofilm formation, providing a novel approach to the treatment of MRSA infections. It can be used alone or in combination with other antibiotics for antibacterial effects and can be used in the preparation of pharmaceuticals, health products, or skincare products. This characteristic will help address the problems of chronic and recurrent MRSA-related infections, improve infection cure rates, and reduce dependence on high-dose antibiotics, thereby mitigating the risk of further drug resistance due to antibiotic abuse. Furthermore, this compound exhibits good safety and low toxicity, showing potential for the prevention and treatment of clinically refractory MRSA infections, particularly in complex conditions such as medical device-related infections and chronic wound infections. This invention not only broadens the field of antimicrobial drug development but also provides new candidate molecules for global antimicrobial drug development, possessing significant academic value and clinical translational potential. Attached Figure Description

[0021] Figure 1 The results of the antibacterial experiments on Gram-positive bacteria by the compounds of the present invention are shown.

[0022] Figure 2 The results of the compounds of the present invention acting as synergistic antibiotics to inhibit bacterial growth are shown.

[0023] Figure 3 The results of the compound of the present invention inhibiting the formation of persistent bacterial biofilms are shown.

[0024] Figure 4 The results of the antibacterial experiments on Gram-negative bacteria by the compounds of the present invention are shown. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] Unless otherwise specified, the reagents and instruments used in the embodiments of the present invention are all commonly known reagents and instruments in the art, and can be purchased through commercial channels.

[0027] Example 1

[0028] 1. Preparation of compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone

[0029] (1) Dissolve 1g of phloretin in a three-necked round-bottom flask containing 30mL of anhydrous dimethylformamide, add 0.7mg of anhydrous lithium carbonate and 2.86mL of gerany bromide, and heat under reflux at 165℃ for 18 hours.

[0030] (2) Add an equal volume of deionized water to the reaction solution to terminate the reaction;

[0031] (3) Filter the reaction solution and extract it 6 times with ethyl acetate, retaining the organic phase.

[0032] (4) Add an equal volume of anhydrous sodium sulfate to the organic phase, filter, and then perform vacuum concentration and separation purification.

[0033] The yield of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone obtained by this method was 200 mg / g, with a purity of 98%.

[0034] 2. Structural identification of compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone

[0035] Mass spectrometry results show that the molecular weight of this compound is 410.

[0036] The compound 1 H NMR (500MHz, CD3OD) and 13 The chemical shifts of the C NMR (125MHz, CD3OD) spectrum are shown in Table 1.

[0037] Table 1: 3'-Germanyl-4,2',4',6'-Tetrahydroxydihydrochalcone 13 C and 1 H-spectral chemical shift

[0038]

[0039] The chemical structure of this compound is shown in formula (I), classifying it as a dihydrochalcone derivative. This compound is readily soluble in methanol. It is named 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone.

[0040]

[0041] Example 2

[0042] 2. Preparation of compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone

[0043] (1) Dissolve 3g of phloretin in a three-necked round-bottom flask containing 50mL of anhydrous dimethylformamide, add 1g of anhydrous lithium carbonate and 4.56mL of geraniol bromide, and heat under reflux at 145℃ for 12 hours.

[0044] (2) Add an equal volume of deionized water to the reaction solution to terminate the reaction;

[0045] (3) Filter the reaction solution and extract it 6 times with ethyl acetate, retaining the organic phase.

[0046] (4) Add an equal volume of anhydrous sodium sulfate to the organic phase, filter, and then concentrate under vacuum.

[0047] The yield of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone obtained by this method was 143 mg / g, with a purity of 98%.

[0048] The subsequent chemical structure identification steps and results were the same as in Example 1, thereby obtaining the compound and identifying it as 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone.

[0049] Example 3

[0050] 3. Preparation of compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone

[0051] (1) Dissolve 0.5g of phloretin in a three-necked round-bottom flask containing 30mL of anhydrous dimethylformamide, add 0.5g of anhydrous lithium carbonate and 0.76mL of gerany bromide, and heat under reflux at 135℃ for 12 hours.

[0052] (2) Add an equal volume of deionized water to the reaction solution to terminate the reaction;

[0053] (3) Filter the reaction solution and extract it three times with ethyl acetate, retaining the organic phase.

[0054] (4) Add an equal volume of anhydrous sodium sulfate to the organic phase, filter, and then concentrate under vacuum.

[0055] The yield of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone obtained by this method was 43 mg / g, with a purity of 98%.

[0056] The subsequent chemical structure identification steps and results were the same as in Example 1, thereby obtaining the compound and identifying it as 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone.

[0057] Example 4

[0058] 4. Preparation of compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone

[0059] (1) Dissolve 2g of phloretin in a three-necked round-bottom flask containing 40mL of anhydrous dimethylformamide, add 1g of anhydrous lithium carbonate and 3.65mL of gerany bromide, heat at 145℃ under reflux for 12 hours, observe the color change of the reaction solution, and then stop heating.

[0060] (2) Add an equal volume of deionized water to the reaction solution to terminate the reaction;

[0061] (3) Filter the reaction solution and extract it three times with ethyl acetate, retaining the organic phase.

[0062] (4) Add an equal volume of anhydrous sodium sulfate to the organic phase, filter, and then concentrate under vacuum.

[0063] The yield of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone obtained by this method was 164 mg / g, with a purity of 98%.

[0064] The subsequent chemical structure identification steps and results were the same as in Example 1, thereby obtaining the compound and identifying it as 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone.

[0065] Comparative Example

[0066] Testing with 4,2',4',6'-tetrahydroxydihydrochalcone revealed no antibacterial activity against Gram-positive bacteria, including Staphylococcus aureus 29213, Staphylococcus aureus RN4220, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, Dicoccoccus pneumoniae, and Clostridium tetani. This indicates that geranyyl substitution is crucial for the antibacterial activity of dihydrochalcone derivatives.

[0067] Antibacterial effect test

[0068] The antibacterial activity of the compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone prepared in Examples 1-4 was evaluated.

[0069] The experimental bacteria are as follows:

[0070] Gram-positive bacteria: Methicillin-resistant Staphylococcus aureus (MRSA), purchased from Huankai Company; Staphylococcus aureus 29213, from Guangzhou Institute of Respiratory Health; Bacillus subtilis WB600, purchased from Shanghai Dibo; Staphylococcus aureus RN4220, Streptococcus, Streptococcus pneumoniae, Clostridium tetani, Enterococcus, Corynebacterium diphtheriae, Listeria, etc., were all purchased from Beina Biotechnology.

[0071] Gram-negative bacteria: Pseudomonas aeruginosa PAO1 (Beina Biotechnology), Pseudomonas aeruginosa PAO1 eGFP (Beina Biotechnology), Klebsiella pneumoniae KP13883 (Shanghai Fuxiang Biotechnology Co., Ltd.), Acinetobacter baumannii Ab19606 (Beina Biotechnology), Escherichia coli 25922 (Shanghai Weidi Biotechnology Co., Ltd.), Escherichia coli BW25113 (Shanghai Weidi Biotechnology Co., Ltd.).

[0072] 1. Minimum inhibitory concentration (MIC) determination

[0073] The test compound (3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone) or antibacterial drugs (meropenem, chloramphenicol, gentamicin, levofloxacin, mitomycin, azithromycin, kanamycin, ofloxacin) were mixed with Gram-positive bacterial culture medium (bacterial concentration of 10). 8 Mix the bacterial suspension (CFU / mL) to a final concentration of 50 μM and place it in a 96-well plate with a volume of 200 μL per well. Set up a blank control group (LB medium only) and a negative control group (bacterial suspension, without antibiotics or compounds). Incubate at 37°C on a shaker, and measure the optical density at 600 nm hourly using a microplate reader to record the bacterial growth curve. At the end of each treatment period, take an appropriate amount of bacterial suspension from each group of samples and perform 10-fold serial dilutions with physiological saline. Spread the diluted samples onto LB agar plates, with three replicates for each dilution. Incubate the plates at 37°C for 16-24 hours until colonies are clearly visible, and count the colony-forming units (CFU).

[0074] Figure 1The results of the antibacterial experiment on persistent MRSA bacteria by the compound of the present invention are shown. The concentrations are sequentially diluted 10-fold from left to right. As shown in the figure, the compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone of the present invention exhibits excellent antibacterial effects against persistent MRSA bacteria. The above antibacterial experiments were also conducted on other types of Gram-positive bacteria (including Staphylococcus aureus 29213, Staphylococcus aureus RN4220, Bacillus subtilis WB600, Streptococcus, Streptococcus pneumoniae, Clostridium tetani, Enterococcus, Corynebacterium diphtheriae, Listeria, etc.). The results show that the compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone of the present invention also exhibits significant and excellent antibacterial effects against these Gram-positive bacteria.

[0075] In addition, inhibition tests were conducted against different Gram-negative bacteria (including *Pseudomonas aeruginosa* PAO1, *Pseudomonas aeruginosa* PAO1eGFP, *Klebsiella pneumoniae* KP13883, *Acinetobacter baumannii* Ab19606, *Escherichia coli* 25922, and *Escherichia coli* BW25113). The experimental conditions were the same as those for the Gram-positive bacteria experiments, except that the bacterial species were replaced with various Gram-negative bacteria. The results of the inhibition tests are as follows: Figure 4 As shown. By Figure 4 It is evident that the compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone of this invention has no inhibitory effect on Gram-negative bacteria. This may be due to the difference in cell walls between Gram-negative and Gram-positive bacteria.

[0076] 2. Synergistic Antibacterial Analysis of Drugs

[0077] The synergistic antibacterial effect of the compound with different antimicrobial agents was determined using a checkerboard gradient dilution method. The preliminary method was the same as described in "1. Determination of Minimum Inhibitory / Bactericidal Concentration" above, except that 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone was added again to the culture medium already containing the antimicrobial agent (drug concentration 50 μM) for a two-fold gradient dilution (final compound concentration 50 μM). The control group did not contain the compound of this invention or any antimicrobial agent.

[0078] The Fractional Inhibitory Concentration Index (FICI) is an important indicator for evaluating the efficacy of combined use of two or more antimicrobial agents. The FICI is calculated based on the minimum inhibitory concentration (MIC) of each drug when used alone and in combination, using the following formula:

[0079] FICI = (MIC of drug A when used in combination / MIC of drug A when used alone) + (MIC of drug B when used in combination / MIC of drug B when used alone).

[0080] The type of drug interaction can be determined based on the FICI value:

[0081] Synergistic effect: FICI ≤ 0.5;

[0082] Additive effect: 0.5 <FICI≤1;

[0083] Irrelevant function: 1 <FICI≤2;

[0084] Antagonistic effect: FICI>2.

[0085] Experiments were conducted using MRSA bacteria in the logarithmic growth phase.

[0086] Figure 2 The results demonstrate the synergistic antibacterial effects of the compounds of this invention with antibiotics. The results indicate that the compounds can synergistically fight bacteria with other antibacterial drugs, enhancing the antibacterial efficacy of other drugs.

[0087] 3. Biofilm formation inhibition assay

[0088] The formation of biofilm was quantified using crystal violet staining.

[0089] The Gram-positive bacteria were cultured overnight (LB medium), centrifuged, and the supernatant was discarded to collect the bacterial strain. The cells were then resuspended in sterile phosphate buffer, and this washing process was repeated three times to thoroughly remove any residual culture medium. After cell counting, the cell concentration was adjusted to approximately 10-1. 8 CFU / mL was added to the wells of a cell culture microplate (6-well plate), along with antibiotics (meropenem, gentamicin, levofloxacin) or the compound 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone) at 5 times the MIC. Blank control wells (culture medium only, no bacterial suspension) and negative control wells (biofilm growth control, bacterial suspension added, no antibiotics or compounds added) were also provided. The cell culture dishes were incubated at 37°C for 48 hours. The supernatant was collected and the absorbance (OD600) was measured using a microplate reader. The bottom biofilm was washed three times with sterile phosphate buffer, fixed with 1 mL of methanol for 30 min, washed once with sterile phosphate buffer, stained with 0.05% crystal violet for 30 min, washed three times with sterile phosphate buffer, and the cells were lysed with 1 mL of 33% (v / v) acetic acid. The absorbance (OD595) was measured using a microplate reader.

[0090] Figure 3The results of the present invention's compounds inhibiting the formation of Gram-positive bacterial biofilms are shown. The results indicate that the 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone provided by the present invention can eliminate Gram-positive bacterial biofilms induced by antibiotics such as meropenem, gentamicin, and levofloxacin (methicillin-resistant Staphylococcus aureus MRSA, Staphylococcus aureus 29213, and Staphylococcus aureus RN4220), inhibiting the formation of Gram-positive bacterial biofilms and demonstrating the ability to synergistically eliminate Gram-positive bacterial biofilms with other antibiotics.

[0091] Experimental results showed that 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone could eliminate persistent Gram-positive bacteria (including Staphylococcus aureus 29213, Staphylococcus aureus RN4220, methicillin-resistant Staphylococcus aureus MRSA, Bacillus subtilis, Streptococcus, Streptococcus pneumoniae, and Clostridium tetani) that were resistant to antibiotics such as meropenem and levofloxacin, with a MIC < 1 μM.

[0092] Similar experimental results against other antibiotics also indicate that the 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone provided by this invention can synergistically enhance the antibacterial (especially anti-Gram-positive bacteria) effects of antibiotics such as ofloxacin, vancomycin, norfloxacin, chloramphenicol, azithromycin, and kanamycin.

[0093] As can be seen, the 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone provided by this invention has excellent antibacterial properties, which are significantly better than common antibiotics such as meropenem, levofloxacin, ofloxacin, vancomycin, norfloxacin, chloramphenicol, gentamicin, azithromycin, kanamycin, etc. It can be used alone or in combination with other antibiotics for antibacterial purposes and can be used to prepare pharmaceuticals, health products or skin care products with antibacterial properties.

[0094] In vivo LD50 results showed that the compound of the present invention, 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone, has an LD50 value greater than 1 mg / kg. At a non-toxic dose (100 mg / kg / day), it can reduce the number of lung colonies and lung index after infection with persistent MRSA bacteria, and directly reduce the accumulation of bacteria in the lungs.

[0095] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone for resisting Gram-positive bacteria, characterized in that, The chemical structural formula is shown in the following formula (I):

2. The method for preparing 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone according to claim 1, characterized in that... Includes the following steps: (1) Dissolve 0.5-3g of phloretin in 30-50mL of anhydrous dimethylformamide, add 0.5-1g of anhydrous lithium carbonate and 0.76-4.56mL of geraniol bromide, and heat under reflux at 135-165℃ for 12 hours. (2) Add an equal volume of deionized water to the reaction solution to terminate the reaction; (3) Filter the reaction solution and extract it with ethyl acetate 3-6 times, retaining the organic phase. (4) Add an equal volume of anhydrous sodium sulfate to the organic phase, filter, concentrate under vacuum, and separate and purify to obtain 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone.

3. The use of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone as described in claim 1 in the treatment of Gram-positive bacteria.

4. The use of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone as described in claim 1 in the fight against Gram-positive bacteria.

5. The use of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone as described in claim 1 in enhancing the antibacterial effect of antibiotics.

6. The application according to claim 5, characterized in that, The antibiotic is any one or more of meropenem, levofloxacin, ofloxacin, vancomycin, norfloxacin, chloramphenicol, gentamicin, azithromycin, and kanamycin.

7. The application of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone as described in claim 1 in inhibiting the formation of biofilms by Gram-positive bacteria.

8. The use of 3'-geranyl-4,2',4',6'-tetrahydroxydihydrochalcone as described in claim 1 in the preparation of pharmaceuticals, health products or skin care products that are effective against Gram-positive bacteria.

9. The application according to any one of claims 3, 4, 7, and 8, characterized in that, The Gram-positive bacteria are one or both of Gram-positive bacilli and Gram-positive cocci.

10. The application according to claim 9, characterized in that, The Gram-positive bacteria are any one or more of Staphylococcus aureus, Bacillus subtilis, Streptococcus, Streptococcus pneumoniae, Clostridium tetani, Enterococcus, Corynebacterium diphtheriae, and Listeria.

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