A natural antibacterial agent and uses thereof

Abstract

The present application relates to the technical field of antibacterial technology, and particularly relates to a natural antibacterial agent and application thereof. The present application provides a method for preparing a composite antibacterial preparation by using Chinese medicine pomegranate peel and Bacillus subtilis AC-1 as raw materials. The composite antibacterial agent has the characteristics of strong resistance to gram-negative bacteria Escherichia coli, gram-negative bacteria Staphylococcus aureus, Staphylococcus pseudintermedius, pathogenic fungus Gymnoascus ruber, Microsporum canis and Microsporum gypseum.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibacterial technology, in particular to a natural antibacterial agent and its use. BACKGROUND

[0002] Pathogenic bacteria and pathogenic fungi are harmful microorganisms that harm humans, animals, plants and the environment. In order to solve the problem of pathogenic bacterial infection, various antibacterial agents have been developed for different microorganisms, such as various antibiotics, biological pesticides, alcohol disinfectants, chlorine-containing disinfectants, etc., which have made great contributions to killing harmful microorganisms that infect humans, animals, plants and environmental health. However, most of the above-mentioned microorganism-killing products are chemical preparations, and natural antibacterial agents are relatively rare. At present, there is no report on natural antibacterial agents that can simultaneously have strong resistance to gram-negative bacteria, gram-positive bacteria and fungi.

[0003] Traditional Chinese medicine is a treasure trove of natural antibacterial agents. Many traditional Chinese medicines have certain antibacterial effects, but their antibacterial spectrum and antibacterial activity differ greatly, and most of them do not have application value as antibacterial agents. It is necessary to screen a large number of different traditional Chinese medicines to select those with strong antibacterial activity and broad antibacterial spectrum, and to develop the preparation process of antibacterial agents.

[0004] The extraction method has a great influence on the antibacterial effect of traditional Chinese medicine. At present, most of the extraction of traditional Chinese medicine is by decoction, which may cause the decomposition of heat-sensitive antibacterial components and the volatilization of volatile antibacterial components. Improving the extraction method of traditional Chinese medicine to enhance its antibacterial activity is the focus of scientists.

[0005] Probiotics are also a source of natural antibacterial agents. Many probiotics have certain antibacterial effects, but their antibacterial spectrum and antibacterial activity differ greatly, and most of them do not have application value as antibacterial agents. It is necessary to screen a large number of different types of probiotics and different strains of the same type of probiotics to select those with strong antibacterial activity and broad antibacterial spectrum, and to develop antibacterial agents with better effects. SUMMARY

[0006] The present application provides a natural antibacterial agent and its use, and obtains a complex bacterial agent with strong antibacterial activity and broad antibacterial spectrum.

[0007] The present application provides a preparation method of a traditional Chinese medicine preparation, which comprises digesting traditional Chinese medicine powder by digestion reflux technology; the digestion temperature is 120-140℃, and the digestion time is 20-40 minutes.

[0008] The Chinese medicine is one or several of pomegranate peel and wodowia floribunda. The Chinese medicine preparation is a bacteriostatic preparation, and the bacteriostatic preparation prepared by the preparation method has better effect. The method for preparing the antibacterial preparation by using the Chinese medicine pomegranate peel as a raw material has the characteristics of strong resistance to gram-negative bacteria Escherichia coli and gram-negative bacteria Staphylococcus aureus and Staphylococcus pseudintermedius, and the method makes the MIC of pomegranate peel against Escherichia coli and Staphylococcus aureus as low as 0.78 mg / ml and 0.39 mg / ml (corresponding to the weight of crude drug per milliliter volume).

[0009] According to the preparation method of the Chinese medicine preparation, the Chinese medicine powder is first soaked in water for 1.5-2.5 hours and then digested.

[0010] Preferably, the Chinese medicine powder is obtained by crushing the Chinese medicine and then passing through a 70-90 mesh sieve.

[0011] Preferably, the supernatant is obtained after centrifugation of the digestion mixture.

[0012] Further preferably, centrifugation is performed at room temperature, the rotation speed of the centrifugation is 3500-4500 rpm, and the centrifugation time is 10-20 min.

[0013] Preferably, the method for extracting the Chinese medicine preparation by using the Chinese medicine pomegranate peel as a raw material comprises digesting the pomegranate peel by using a digestion instrument, the digestion instrument is at 130 DEG C, and the digestion is performed for 30 minutes, and then the temperature is cooled to room temperature. During the digestion process, the reflux device of the digestion instrument ensures that the volatile components are refluxed into the digestion bottle during the digestion process.

[0014] Preferably, the preparation method further comprises a filtering step of activated carbon (special for drug decolorization).

[0015] The application provides an antibacterial agent prepared from pomegranate peel and / or wodowia floribunda.

[0016] Preferably, the preparation method of the Chinese medicine preparation is used.

[0017] The pomegranate peel and / or wodowia floribunda are used in any one or several of the following aspects:

[0018] 1) preparing an antibacterial agent.

[0019] Preferably, the preparation method of the Chinese medicine preparation is used.

[0020] 2) resisting bacteria and / or fungi.

[0021] Preferably, the bacteria are gram-negative bacteria and / or gram-positive bacteria.

[0022] According to the application, the bacteria are one or more of Escherichia coli, Staphylococcus aureus, and Staphylococcus intermedius.

[0023] The application also provides an application of the wormwood preparation in resisting Staphylococcus aureus.

[0024] The application provides a method for extracting an antibacterial preparation from the traditional Chinese medicine Radix Dipsaci.

[0025] The application provides an application of one or more of Bacillus subtilis AC-1, a fermentation liquor of Bacillus subtilis AC-1, and a metabolite of Bacillus subtilis AC-1 in any one or more of the following aspects:

[0026] 1) preparing an antibacterial agent.

[0027] 2) resisting fungi.

[0028] Preferably, the fungi are one or more of Microsporum canis, Trichophyton rubrum, and Microsporum gypseum. The AC-1 fungal resistance preparation has the characteristics of strong resistance to pathogenic fungi such as Trichophyton rubrum, Microsporum canis, and Microsporum gypseum.

[0029] The Bacillus subtilis AC-1 provided by the application is classified and named as Bacillus subtilis, has a preservation number of CGMCC No. 26448, is preserved by the General Microbiological Center of the Chinese Microorganism Bacterium Preservation Management Committee (CGMCC), has a preservation date of January 12, 2023, and has an address of the preservation unit of No. 3, Institute of Microbiology, Chinese Academy of Sciences, West Beichen Road, Chaoyang District, Beijing, China, and a postal code of 100101.

[0030] The application provides a composite antibacterial agent, raw materials of which include Bacillus subtilis AC-1 and pomegranate peel.

[0031] The application provides a preparation method of the composite antibacterial agent, a preparation of pomegranate peel by the method, and preparation of the composite antibacterial agent by the pomegranate peel preparation.

[0032] The application also provides an application of the composite antibacterial agent in any one of the following aspects:

[0033] 1) resisting one or more of Escherichia coli, Staphylococcus aureus, and Staphylococcus intermedius.

[0034] 2) resisting one or more of Microsporum canis, Trichophyton rubrum, and Microsporum gypseum.

[0035] 3) reducing bacteria on pet hair.

[0036] 4) preparing one or several of feed additives, cat litter, pet care products.

[0037] Preferably, the pet care product is a pet cleaning product.

[0038] Preferably, the pet is a cat.

[0039] The present application combines the screened target traditional Chinese medicine and target probiotics to form a new type of composite antibacterial agent with better antibacterial activity, which can broaden the antibacterial spectrum and application range.

[0040] The present application screens the traditional Chinese medicine pomegranate peel and its antibacterial active product preparation method with strong antibacterial activity (including gram-negative bacteria and gram-positive bacteria).

[0041] The present application screens the probiotic Bacillus subtilis AC-1 with strong antifungal activity and its antifungal active product preparation method.

[0042] The present application provides a method for preparing a strong antifungal and bacterial (including gram-negative bacteria and gram-positive bacteria) preparation by combining pomegranate peel and Bacillus subtilis AC-1 and its application.

[0043] The present application uses traditional Chinese medicine and Bacillus subtilis extract to form a natural strong active antibacterial agent (bacteria and fungi). The composite antibacterial agent has strong antibacterial activity against gram-negative bacteria Escherichia coli, gram-negative bacteria Staphylococcus aureus, Staphylococcus pseudintermedius, pathogenic fungi Xerula rubescens, Microsporum canis and Microsporum gypseum.

[0044] The present application provides a composite antibacterial preparation prepared from traditional Chinese medicine pomegranate peel and Bacillus subtilis AC-1 as raw materials. On this basis, the composite antibacterial agent has strong antibacterial activity against gram-negative bacteria Escherichia coli, gram-negative bacteria Staphylococcus aureus and Staphylococcus pseudintermedius, and pathogenic fungi Xerula rubescens, Microsporum canis and Microsporum gypseum.

[0045] The present application provides evidence that the combination of pomegranate peel antibacterial agent and AC-1 antibacterial agent does not affect the antibacterial activity of pomegranate peel antibacterial agent against Escherichia coli, but increases the antibacterial activity of the composite antibacterial agent against Staphylococcus aureus and Staphylococcus pseudintermedius.

[0046] The composite antibacterial agent of the present application is used for pet cat litter, realizes long-acting antibacterial, reduces the number of bacteria in the pet breeding environment, and improves the pet breeding environment.

[0047] The composite antibacterial agent is used for pet cat hair care, reduces the number of bacteria on pet hair, and improves the health of cats and their feeding environment. BRIEF DESCRIPTION OF DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort.

[0049] Figure 1 Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C), Bar = 2 cm.

[0050] Figure 2 Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C).

[0051] Figure 3 Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C).

[0052] Figure 4 Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C).

[0053] Figure 5 Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C).

[0054] Figure 6 Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C).

[0055] Figure 7 Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C).

[0056] Figure 8 Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C).

[0057] Figure 1 is a resistance diagram of the pomegranate peel extract of the present application to Escherichia coli (A), Staphylococcus aureus (B) and Pseudointermediate Staphylococcus (C).

[0058] Figure 9Figure 1 is a chart showing the resistance of the composite antibacterial solution of the present application to Microsporum canis;

[0059] (A) Control, (B) stock solution (1x), (C) diluted 10x, (D) diluted 100x; Bar = 2 cm. DETAILED DESCRIPTION

[0060] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in conjunction with the drawings in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without any creative work fall within the protection scope of the present application.

[0061] Example 1: Screening of Chinese medicines against gram-positive bacteria and gram-negative bacteria

[0062] The present example provides a method for preparing an antibacterial preparation using various Chinese medicines as raw materials, which specifically comprises the following steps:

[0063] 1. Chinese medicine materials

[0064] Pomegranate peel (purchased from Beijing Tong Ren Tang Pharmacy)

[0065] Radix Acanthopanacis Senticosi (purchased from Beijing Tong Ren Tang Pharmacy)

[0066] Artemisia vulgaris (purchased from Beijing Tong Ren Tang Pharmacy)

[0067] Perilla frutescens (purchased from Beijing Tong Ren Tang Pharmacy)

[0068] Pulsatilla chinensis (purchased from Beijing Tong Ren Tang Pharmacy)

[0069] Saposhnikovia divaricata (purchased from Beijing Tong Ren Tang Pharmacy)

[0070] Lonicera japonica (purchased from Beijing Tong Ren Tang Pharmacy)

[0071] Taraxacum mongolicum (purchased from Beijing Tong Ren Tang Pharmacy)

[0072] 2. Chinese medicine digestion

[0073] (1) Chinese medicine crushing: take the above-mentioned appropriate amount of Chinese medicine and put it into a crushing machine (multifunctional crushing machine, model: BJ-200, Deqing Baijie Electrical Appliance Co., Ltd.), fix the cover, and crush for 1 to 3 minutes at a time.

[0074] (2) Sieving: sieve the crushed Chinese medicine with an 80-mesh sieve. Put the sieved powder into a self-sealing bag and store it at 4°C for standby use.

[0075] (3) Digestion: 10 g of traditional Chinese medicine powder was weighed, 90 mL of sterilized deionized water was measured and put into a digestion conical flask for mixing; after 2 hours of normal temperature soaking, digestion was carried out, the digestion instrument (standard COD digester, COD-100W, Qingdao Jinghong Environmental Protection Technology Co., Ltd., Qingdao) was set to 130°C, and digestion was carried out for 30 minutes, and then cooled to room temperature.

[0076] (4) Centrifugation: the digestion mixture cooled to room temperature was divided into 50 mL centrifuge tubes, 4000 rpm, normal temperature centrifugation for 10-20 min, the residue was discarded, and the supernatant was taken into a new centrifuge tube and stored at 4°C.

[0077] 3. Traditional Chinese medicine decolorization

[0078] (1) Experimental supplies

[0079] Activated carbon: A805346-1 kg (pharmaceutical decolorization, 200 mesh, 1 kg, Shanghai Maikelin Biotechnology Co., Ltd.);

[0080] Qualitative filter paper (Xinxing brand, diameter 11 cm, manufacturer).

[0081] Glass funnel: outer diameter of mouth 10 cm, outer diameter of foot 1 cm, foot length 10 cm, borosilicate glass.

[0082] (2) Decolorization steps

[0083] ①Fold 1 piece of filter paper by 3+1 layers, and to prevent damage, add 2 more pieces of filter paper;

[0084] ②Wet the filter paper with sterile water along the edge of the funnel to make it adhere to the inner wall of the funnel, and then wet the filter paper again with sterile water, and let the sterile water flow out naturally.

[0085] ③Add an appropriate amount of activated carbon (special for pharmaceutical decolorization) to the funnel, so that the edge of the folded filter paper is not more than 2 cm, and if the edge of the filter paper is exceeded, the activated carbon will easily pass through the filter paper into the filtered liquid (8-10 grams of activated carbon are added to the funnel, which can be used to filter about 100 ml of traditional Chinese medicine liquid).

[0086] ④Take 50 ml of digestion traditional Chinese medicine and add it to the activated carbon slowly, and collect the filtrate in a 250 ml triangular flask. (Note: The filtration must be suspended, otherwise the filtration speed will be affected by air pressure and will be very slow.)

[0087] ⑤After the traditional Chinese medicine digestion liquid (dark brown) is filtered, a light brown digestion liquid is obtained, which is the different traditional Chinese medicine antibacterial agent stock solution, 10 g of crude drug, the total volume of the traditional Chinese medicine antibacterial agent stock solution is 100 ml, the concentration is 100 mg / ml (100 mg of crude drug per milliliter), and it is sealed and stored at 4°C for use.

[0088] Example 2 antibacterial activity detection of antibacterial preparations prepared with different traditional Chinese medicines as raw materials

[0089] 1. Strains

[0090] Representative gram-negative bacteria Escherichia coli ATCC25922 (preserved by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences), gram-positive bacteria Staphylococcus aureus ATCC25923 (preserved by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences), and Staphylococcus pseudintermedius ATCC49051 (preserved by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). The above strains are commercially available strains.

[0091] 2. Design of bacteriostatic gradient

[0092] The original solution of the antibacterial agent obtained in Example 1 is set to 1x, and on this basis, it is diluted by 2 times, 5 times, and 10 times to set dilutions of 2x, 5x, and 10x, with three replicates for each group of experiments.

[0093] 3. Experimental steps

[0094] 1) Take single colonies of the three indicator bacteria and use LB liquid medium (50 ml liquid / 125 ml flask) to culture at 37°C and 200 rpm overnight (culture for 12 hours). The bacterial count of the three indicator bacteria is about 10 8 cfu / ml.

[0095] 2) According to the ratio of 1:5000 of bacterial liquid to LB medium, take the liquid of the three indicator bacteria and add it to the melted LB solid medium (50-60°C), mix thoroughly, plate, and dry. This is the indicator bacteria culture medium.

[0096] Note: When mixing the LB solid medium and the indicator bacteria, the temperature should not be too high, as high temperature can cause bacterial death.

[0097] 3) Use a sterile puncher to punch the indicator bacteria culture medium, and place 200 μl of the traditional Chinese medicine antibacterial agent original solution (1x), 2x dilution, 5x dilution, and 10x dilution into each hole.

[0098] 4) Incubate in a 37°C incubator overnight, and the next morning (after 12 hours of culture), measure the diameter of the bacteriostatic ring and take a photo.

[0099] 4. Experimental results

[0100] The antibacterial effects of the pomegranate peel, Radix Euonymi, wormwood, and honeysuckle antibacterial solutions are shown in Figures 1-4 From Figure 1 it can be seen that the pomegranate peel antibacterial solution still has strong resistance to Escherichia coli, Staphylococcus aureus, and Staphylococcus pseudintermedius at a dilution of 10x.

[0101] From Figure 2It can be seen that Huangdu antibacterial solution still has strong resistance to Escherichia coli and Staphylococcus pseudointermediate at a dilution of 2×, but its antibacterial activity is lost at a dilution of 5×. It still has some resistance to Staphylococcus aureus at a dilution of 5×.

[0102] from Figure 3 It can be seen that the antibacterial solution of Artemisia argyi has almost no obvious resistance to Escherichia coli, still has some resistance to Staphylococcus aureus at a dilution of 5×, and only weak resistance to Staphylococcus pseudointermedia at a dilution of 1×.

[0103] from Figure 4 As can be seen, honeysuckle antibacterial solution at various concentrations did not show significant resistance to Escherichia coli, Staphylococcus aureus, and Staphylococcus pseudointermediate.

[0104] Other Chinese herbs tested, including Perilla frutescens, Pulsatilla chinensis, Saposhnikovia divaricata, Lonicera japonica, and Taraxacum mongolicum, showed almost no or weak resistance to Escherichia coli, Staphylococcus aureus, and Staphylococcus pseudointermediate at various concentrations (images not provided).

[0105] Example 3: The minimum inhibitory concentration of the pomegranate peel antibacterial preparation obtained by digestion method was significantly reduced.

[0106] 1. Antibacterial preparation of pomegranate peel obtained by digestion method

[0107] Following the method of Example 1, an antibacterial preparation of pomegranate peel was obtained, with a concentration of 100 mg / ml.

[0108] 2. Determination of the minimum inhibitory concentration of pomegranate peel antibacterial preparation

[0109] The bacteria used were the representative Gram-negative Escherichia coli standard strain ATCC25922 (preserved by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) and the Gram-positive Staphylococcus aureus standard strain ATCC25923 (preserved by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences).

[0110] For the experiment, 96-well plates were used. First, 100 μL of LB broth was added to wells 1-16 in every two rows. Then, in wells 1-14 in every two rows, the extraction solution was added using a 2-fold serial dilution method (initial drug concentration 100 mg / ml, 100 μL added to the first well, half the liquid from the previous well added to the next well). Any excess 100 μL from well 14 in each row was discarded. Finally, 100 μL of diluted 1.0 × 10⁻⁶ LB broth was added to wells 1-15 in every two rows. 6CFU / mL of E. coli or S. aureus bacteria solution. The 15th well without drug was the positive control group, and the 16th well without bacteria solution and supplemented with 100 μL of LB broth was the negative control group. After gentle mixing, the 96-well plate was incubated in a 37°C incubator for 24 h, and then detected. In a clean bench, 5 μL of TTC (5%) and sodium succinate (2%) solutions were added to each well of the 96-well plate containing the liquid, and then the plate was incubated in a 37°C incubator for 1 h and 2 h, and the test results were observed. Based on the test results of the negative control well and the positive control well as the reference object, the test results of the 1st to 14th wells were determined. If the results of the 1st to 14th wells were the same as those of the positive control group, the wells were pink, indicating bacterial growth; if the results of the 1st to 14th wells were the same as those of the negative control group, the wells were clear, indicating no bacterial growth; the lowest concentration of the drug with no bacterial growth was the MIC of the Mongolian medicine. The MIC was calculated in milligrams of crude drug per milliliter.

[0111] 3Results

[0112] According to the above method, the MIC of the pomegranate peel antibacterial agent obtained by the digestion reflux method against E. coli was 0.78 mg / ml, and the MIC against S. aureus and S. intermedius was 0.39 mg / ml.

[0113] In another independent experiment, the MIC of the pomegranate peel antibacterial agent extracted by the water decoction method reported in the literature against E. coli was 31.25 mg / ml, which was consistent with the previous report (Liu L et al., Chinese Journal of Animal Husbandry and Veterinary Medicine, 2023, 50(1):328-340). The MIC of the pomegranate peel antibacterial agent extracted by the water decoction method against S. aureus was 3.92 mg / ml.

[0114] The water decoction method used to prepare the pomegranate peel traditional Chinese medicine water extract is as follows: 30 g of traditional Chinese medicine is weighed and soaked in 300 ml of (45±5) °C warm water for 1 h, then heated to boiling with a strong fire, and then changed to a weak fire for 1 h of decoction. The filtrate (1) is collected by filtering through 4 layers of gauze. Another 300 ml of warm water is added to the residue and soaked for 1 h, and the decoction operation is repeated once. The filtrate (2) is collected. The filtrate (1) and (2) are combined and centrifuged at 5000 rpm for 5 min. The supernatant is filtered, and the filtrate is concentrated to 30 ml on a weak fire. 0.3% sodium benzoate is added as a preservative. At this time, the concentration of each liquid is equivalent to 1 g / ml of crude drug. The liquid is stored at 4°C for standby use. (Liu L et al., Chinese Journal of Animal Husbandry and Veterinary Medicine, 2023, 50(1):328-340).

[0115] Example 4 Preparation of Bacillus subtilis AC-1 antifungal preparation

[0116] Bacillus subtilis AC-1 (Preservation No. CGMCC No. 26448, Preservation Unit: China General Microbiological Culture Collection Center, CGMCC, Institute of Microbiology, Chinese Academy of Sciences).

[0117] Glycerol-piped Bacillus subtilis AC-1 was streaked on LB solid medium and incubated at 37°C overnight. The next day, a single colony was picked and inoculated into a 100 mL LB liquid medium in a flask (250 mL scale) and incubated at 37°C, 200 rpm for 12 h. Then, 2% of the inoculum was inoculated into a 100 mL Endo-KNO3 medium in a flask (250 mL scale) and incubated at 35°C, 200 rpm for 48 h. The fermentation broth was centrifuged at 10,000-11,000 rpm for 10 min, and the supernatant was filtered 1-2 times with a 0.22 μm sterile filter on a clean bench. The obtained liquid was an AC-1 antifungal preparation (concentration marked as 1 mL sterile bacterial liquid / mL, containing 1 mL of sterile bacterial liquid per mL of volume). The vacuum concentration method was used to concentrate 5 times (concentration of 5 mL / mL, indicating that 5 mL of sterile bacterial liquid was contained per mL of volume), and the concentrated liquid was stored at 4°C. When needed, it could be diluted in proportion.

[0118] Example 5 Resistance of Bacillus subtilis AC-1 antifungal preparation to fungi and bacteria

[0119] 1) Microsporum canis ATCC36299 (from the Institute of Microbiology, Chinese Academy of Sciences).

[0120] 2) Trichophyton rubrum ATCC10218 (from the Institute of Microbiology, Chinese Academy of Sciences).

[0121] 3) Microsporum gypseum ATCC14683 (from the Institute of Microbiology, Chinese Academy of Sciences).

[0122] 4) Escherichia coli ATCC25922 (preserved by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences).

[0123] 6) Staphylococcus pseudintermedius ATCC49051 (preserved by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences).

[0124] 2. AC-1 antifungal preparation used: the antifungal preparation obtained in Example 3 (concentration of 1 mL of sterile bacterial liquid / mL).

[0125] 3 Resistance of Bacillus subtilis AC-1 antifungal preparation to fungi and bacteria

[0126] Resistance test to fungi: plate confrontation method: mark the center point on the bottom of PDA solid medium plate, then inoculate the pathogenic fungus with a diameter of 0.6 cm, and then punch two holes with a puncher at a distance of 3 cm in two directions perpendicular to the center point, and then add 100 μL (1 ml / ml) of AC-1 antifungal preparation into the holes. The sample without punching holes and without adding any reagent is used as a control.

[0127] Resistance test to bacteria: see Example 2

[0128] 4. Test results

[0129] Test of pathogenic fungi: the test results are shown in Figure 5 It can be seen from Figure 5 that the Bacillus subtilis AC-1 antifungal preparation has strong resistance to Microsporum canis, Trichophyton rubrum and Microsporum gypseum.

[0130] Resistance test to bacteria:

[0131] The AC-1 antibacterial preparation has no resistance to Escherichia coli and has weak resistance to Staphylococcus aureus and Staphylococcus pseudintermedius.

[0132] The representative results of Staphylococcus aureus are shown in Figure 6 . Other result figures are not given. In two independent experiments, the AC-1 antifungal preparation has no resistance to Escherichia coli, Staphylococcus aureus and Staphylococcus pseudintermedius.

[0133] Example 6 Preparation of a composite antibacterial and antifungal preparation

[0134] According to the method of Example 1, 10 g of pomegranate peel was weighed, crushed, added with 100 ml of deionized water, soaked for 2 hours, then digested with a digestion instrument (the plate temperature was adjusted to 130°C) for 30 minutes, cooled to room temperature, and then centrifuged at 4000 rpm for 10-20 min at room temperature. The residue was discarded, and the supernatant was transferred to a new centrifuge tube, filtered through activated carbon (A805346-1 kg, 200 mesh, 1 kg, Shanghai Maikelin Biotechnology Co., Ltd.) and qualitative filter paper (Xinxing brand, diameter 11 cm, manufacturer), and then diluted to 100 ml. This was the pomegranate peel antibacterial agent stock solution, which was sealed, stored at 4°C, and used as the pomegranate peel antibacterial stock solution.

[0135] The Bacillus subtilis AC-1 antibacterial stock solution obtained in Example 3 (concentrated 5 times stock solution, 5 ml / ml) was recorded as the AC-1 antibacterial high-concentration stock solution.

[0136] Take the above Punica granatum L. antibacterial stock solution 10 ml, add AC-1 antibacterial high concentration stock solution 10 ml, add deionized water 30 ml, constant volume to 50 ml (can be scaled up according to the proportion), obtain the composite antibacterial stock solution (composite antibacterial, antifungal and antifungal preparations), the composite solution contains Punica granatum L. fungicide concentration is 20 mg / ml, contains AC-1 antifungal fungicide 1 ml / ml.

[0137] Example 7 Determination of antibacterial activity of composite antibacterial preparation

[0138] The above composite antibacterial stock solution (1x), diluted 2 times (2x), diluted 10 times (10x), using the punching method (see examples 2 and 4), detect the antibacterial activity of Escherichia coli, Staphylococcus intermedius, Staphylococcus aureus, using the plate confrontation method to detect the resistance of the red hairy fungus, Microsporum canis, Gypsum-like Microsporum canis.

[0139] The test results are shown in Figures 7-9 .

[0140] From Figure 7 It can be seen that the prepared composite antibacterial stock solution (1x), diluted 2 times (2x), has strong antibacterial activity, and diluted 10 times (10x) still has weak antibacterial activity.

[0141] In another independent experiment, the MIC of the above composite antibacterial stock solution on Escherichia coli, Staphylococcus aureus and Staphylococcus intermedius was determined. The MIC of the composite antibacterial agent against Escherichia coli was 0.78 mg / ml (the mass of crude drug Punica granatum L. per ml), the MIC against Staphylococcus aureus was 0.195 mg / mL (the mass of crude drug Punica granatum L. per ml), and the MIC against Staphylococcus intermedius was 0.195 mg / mL (the mass of crude drug Punica granatum L. per ml).

[0142] It is shown that the combination of Punica granatum L. antibacterial agent and AC-1 antibacterial agent does not affect the antibacterial activity of Punica granatum L. antibacterial agent against Escherichia coli, but can increase the antibacterial activity of Punica granatum L. antibacterial agent against Staphylococcus aureus and Staphylococcus intermedius.

[0143] Figure 9 The results show that the composite antibacterial solution has strong resistance to Microsporum canis, and still has strong resistance after being diluted 10 times (10 times), and still has certain activity after being diluted 100 times (100 times) (the confrontation effect is not obvious, but the colony diameter is smaller).

[0144] At the same time, in another independent experiment, the composite antibacterial stock solution and 10x diluted solution were detected for their resistance to the red hairy fungus and the Gypsum-like Microsporum canis by the above method, and the results showed that the composite antibacterial stock solution and 10x diluted solution also had strong resistance to the red hairy fungus (the picture is not given) and the Gypsum-like Microsporum canis Figure 9).

[0145] Example 8 Long-term antibacterial effect of the composite antibacterial agent on pet cat litter

[0146] The composite antibacterial agent prepared in Example 5 was mixed with cat litter at a ratio of 1:25 (40 ml of the composite antibacterial agent, 1 kg of cat litter, cat litter purchased from Hebei Demu Pet Food Co., Ltd.) to serve as new cat litter for 10 cats (Beijing Xinnianyin Biological Technology Co., Ltd., English short, 1 year old, male cats), 1 kg of cat litter for each cat. The control group of 10 cats (Beijing Xinnianyin Biological Technology Co., Ltd., English short, 1 year old, male cats) was not added with the composite antibacterial agent, 1 kg of cat litter for each cat. After 72 hours, the cat litter (surface 2-5 mm cat litter) was taken, 3 portions for each cat, each portion about 1-2 g. The LB plate method (refer to the experimental method of GB / T 26428-2010) was used to determine the bacterial content of the cat litter, and the results are shown in Table 1.

[0147] Table 1 Effect of the composite antibacterial agent on the bacterial content of cat litter

[0148] Group Bacterial content of cat litter (mean ± SD / g) Explanation Control group 3.03 x 10 5 ± 1.05 x 10 4 ***]]> P＜0.01 Experimental group 7.3 x 10 6 ± 3.26 x 10 4 ]]>

[0149] As can be seen from the results in Table 1, the composite antibacterial solution has a significant long-term antibacterial effect (72 hours) on the cat litter being used.

[0150] Example 9 Use of the composite antibacterial agent for pet cat fur care

[0151] 1) The animals were still fed with 10 cats (Beijing Xinnianyin Biological Technology Co., Ltd., English short, 1 year old, male cats) for the experiment, each cat was individually raised.

[0152] 2) Washing and care area: both shoulders.

[0153] 3) Before the experiment, the cat's shoulder area needs to be cleaned with a cat shampoo. Before the experiment, the experimental area and the control area were treated to have similar cleanliness, and were dried.

[0154] 4) Left shoulder (sterile water coating, used as a control group); right shoulder (experimental sample coating, used as an experimental treatment group); coated into the same size area (a square area with a side length of 4 cm), so that the cat fur is saturated and wet, and no water is needed to wash it off after coating. The amount of the composite antibacterial solution is about 6 ml (please check)

[0155] 5) After 72 hours, the cat fur on both shoulders was taken, and the cat fur in the same size area was stored at room temperature.

[0156] 6) 500 mg of cut cat fur (please check) was taken, 50 ml of sterile water was added, and the solution was shaken at 200 rpm for 2 hours at room temperature (25°C). The bacterial content in the solution was determined using LB medium.

[0157] 7) The results are shown in Table 2.

[0158] Table 2 Effect of the composite antibacterial agent on the bacterial content of the pet cat hair

[0159] Group Bacterial content of cat hair (mean ± SD / g) Explanation Control group 8.6 x 10 5 cfu / g ± 1.15 x 10 3 ]]> Experimental group 1.02 x 10 4 ± 2.32 x 10 3 ***]]> P＜0.01

[0160] From the results in Table 2, it can be seen that the composite antibacterial solution has a significant long-acting antibacterial effect (72 hours) on cat hair.

[0161] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art will understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a combination antimicrobial agent for the preparation of an antifungal and / or antibacterial medicament, characterized in that, The raw material is Punica granatum L. antibacterial agent and Bacillus subtilis AC-1; the bacteria is Staphylococcus intermedius and the fungus is one or both of Trichophyton rubrum and Microsporum gypseum; The preparation method of the Punica granatum L. antibacterial agent comprises: digesting Punica granatum L. powder by using digestion reflux technology; the digestion temperature is 120-140 DEG C, and the digestion time is 20-40 min; The preservation number of the Bacillus subtilis AC-1 is CGMCC No.26448.

2. Use according to claim 1, characterized in that, The Punica granatum L. powder is soaked in water for 1.5-2.5 hours before digestion; the material liquid ratio during soaking is 10g:85-95ml.

3. Use according to claim 1 or 2, characterized in that, The Punica granatum L. powder is obtained by crushing Punica granatum L. and then sieving through a 70-90 mesh sieve.

4. Use according to claim 1 or 2, characterized in that, The supernatant is obtained after centrifuging the digestion mixture.

5. Use according to claim 4, characterized in that, The centrifugation speed is 3500-4500 rpm, and the centrifugation time is 10-20 min.

6. The application of the composite antibacterial agent in any one of the applications of claims 1-5 in any one of the following: 1) preparing a product for reducing bacteria on pet hair; 2) preparing one or more of feed additives, cat litter and pet care products.

7. Use according to claim 6, characterized in that, The pet care product is a pet cleaning product.

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