Application of schizonepeta essential oil in bacteriostasis

Abstract

The invention belongs to the technical field of natural products, and particularly relates to application of herba schizonepetae essential oil in bacteriostasis. The herba schizonepetae essential oil is used for inhibiting the activity of bacteria or fungi; an extraction method of the schizonepeta essential oil comprises the following steps: extracting a schizonepeta medicinal material by adopting a water distillation method to obtain the schizonepeta essential oil. The herba schizonepetae essential oil provided by the invention has an obvious inhibition effect on bacillus subtilis, escherichia coli, aspergillus niger, mucor, cladosporium cladosporium, aspergillus flavus, ash tree colletotrichum gloeosporioides, fusarium oxysporum and fusarium oxysporum.

Description

Technical Field

[0001] The invention belongs to the technical field of natural products, and particularly relates to application of schizonepeta essential oil in antibacterial treatment. Background Art

[0002] Nepeta, formerly known as Pseudospermum, belongs to the genus Nepeta of the Lamiaceae family, and its essential oil is an important physiologically active substance in Nepeta. Essential oil is one of the main effective material bases of Nepeta. It has a unique aroma of Nepeta spike, a bitter and spicy taste, and a yellow or light yellow oily liquid appearance, which is easy to identify. Modern pharmacological research and clinical application show that Nepeta essential oil has anti-inflammatory, antipyretic, expectorant, antiasthmatic, analgesic, antiviral, and antiallergic effects.

[0003] At present, studies have shown that schizonepeta essential oil has antibacterial and anti-inflammatory effects. For example, in the experimental study on the extraction and antibacterial properties of volatile oil from Jinan schizonepeta conducted by Ren Yun and others, the results showed that the essential oil had a strong inhibitory effect on Staphylococcus aureus, but a relatively weak inhibitory effect on Escherichia coli. However, at present, there is no product use of schizonepeta essential oil that can simultaneously inhibit bacteria and plant pathogenic fungi. Summary of the invention

[0004] In order to solve the problem that there is no product use of schizonepeta essential oil for simultaneously inhibiting bacteria and plant pathogenic fungi in the prior art, the present invention aims to provide an application of schizonepeta essential oil in the preparation of an antibacterial product. To achieve the above object, the present invention adopts the following technical solution:

[0005] The present invention analyzes and studies the extraction technology of schizonepeta essential oil, explores the best extraction process of schizonepeta essential oil, and provides scientific experimental support for the extraction method of schizonepeta essential oil and its further development and application. Antibacterial detection is an important means to evaluate the antibacterial effect of schizonepeta essential oil. Antibacterial experiments are helpful to screen out schizonepeta essential oil with high antibacterial activity, and provide ideas and directions for the development of schizonepeta natural antibacterial agents and preservatives, which conforms to the future development trend of food safety and green, and further expands its application in grape pest control and preservation, as well as wine production, processing and preservation.

[0006] Based on this, the present invention provides an application of schizonepeta essential oil in antibacterial treatment, wherein the schizonepeta essential oil is used to inhibit the activity of bacteria or plant pathogenic fungi.

[0007] Wherein, the bacteria include Bacillus subtilis and / or Escherichia coli. Bacillus subtilis and Escherichia coli are common contaminating bacteria in food production and agricultural product storage, and are also common indicator bacteria in antibacterial experiments. Therefore, based on this, the present invention provides the use of catmint essential oil for inhibiting the activity of Bacillus subtilis and Escherichia coli.

[0008] Bacillus subtilis is a type of Bacillus, widely distributed in soil and corrupted organic matter, and is named for its ability to reproduce easily in the juice of blight. Bacillus subtilis undergoes spore promotion and microencapsulation treatment on special bacteria, and has good stability in the spore state and is resistant to oxidation; it is resistant to extrusion; it is resistant to high temperatures, can withstand high temperatures of 60°C for a long time, and can survive for 20 minutes at 120°C; it is resistant to acid and alkali, can maintain activity in the acidic stomach environment, can withstand the attack of saliva and bile, and is a live bacteria among microorganisms that can 100% directly reach the large and small intestines.

[0009] Escherichia coli is also called Escherichia coli. It is a species of the genus Escherichia in the family Enterobacteriaceae. Escherichia coli is a conditional pathogen that can cause gastrointestinal infections or urinary tract infections in humans and various animals under certain conditions.

[0010] The plant pathogenic fungi include any one or more of Aspergillus niger, Mucor, Cladosporium, Aspergillus flavus, Colletotrichum ashii, Fusarium spp., and Fusarium spp. Among them, Aspergillus niger, Mucor, Cladosporium spp., Aspergillus flavus, and Fusarium spp. are common air fungi that may cause plant diseases, and Colletotrichum ashii and Fusarium spp. are pathogenic fungi of the roots of Polygonatum cyrtonema.

[0011] Aspergillus niger is a common species in the subphylum Ascomycota, class Hyphomycetes, orders Hyphomycetales, family Hyphomycetaceae, genus Aspergillus. Aspergillus niger is widely distributed in grains, plant products and soils around the world. It is an important fermentation industrial strain that can produce amylase, acid protease, cellulase, pectinase, glucose oxidase, citric acid, gluconic acid and gallic acid. Some strains can also convert hydroxyprogesterone into androstenone. The optimum growth temperature is about 37°C and the minimum relative humidity is 88%. It can cause mildew in grains with high moisture content and mildew in other industrial equipment.

[0012] Mucor is also called black mold and long hair mold. Mucor is a large genus of fungi in the order Mucorales, family Mucoraceae, in the subphylum Zygomycetes. It reproduces by sporangiospores and zygospores. Mucor exists in soil, feces, grass and air. It grows well under high temperature, high humidity and poor ventilation.

[0013] Cladosporium is a fungus that can produce conidia and belongs to the Deuteromyces. It is a common fungus both indoors and outdoors. It is dark green in color, so it is often confused with Aspergillus. However, unlike Penicillium and Aspergillus, Cladosporium has colored hyphae, and the colony is dark in color.

[0014] Aspergillus flavus, also known as yellow fungus, Aspergillus flavus, etc., belongs to the Fungi, Aspergillus subphylum, Combretaceae, Aspergillus genus, and is a common saprophytic mold widely distributed throughout the world. Among them, 30% to 60% of the strains can produce aflatoxins; most non-pathogenic species are often used as koji species in the fermentation industry, mainly in the fermentation production of some organic acids. For example, Aspergillus flavus H-98 can be used for the fermentation production of L-malic acid.

[0015] Colletotrichum fraxini can cause anthracnose in ash trees. Anthracnose is a parasitic disease caused by anthracnose. Black spots of varying sizes will appear on diseased ash trees, which gradually expand and turn into round anthracnose sores. Anthracnose is very harmful to ash trees, causing a reduction in leaves and early leaf fall, affecting the ornamental value of ash trees.

[0016] Fusarium is a type of fungus distributed worldwide. It can not only overwinter and oversummer in the soil, but also infect a variety of plants: food crops, cash crops, medicinal plants and ornamental plants, causing a variety of plant diseases such as root rot, stem rot, stem base rot, flower rot and ear rot. It has more than 100 host plants, infects the host plant vascular system, destroys the plant's conductive tissue vascular bundle, and produces toxins during the growth, development and metabolism process that harm crops, causing crops to wilt and die, affecting yield and quality. It is one of the most difficult and important diseases to prevent and control in production.

[0017] Fusarium spp. can cause root rot in plants.

[0018] The present invention has found through experimental studies that the schizonepeta essential oil provided in the present application has different degrees of antibacterial effects on the above two bacteria and seven fungi.

[0019] The extraction method of the schizonepeta essential oil provided by the present invention is as follows:

[0020] The herbal medicine Nepeta is extracted by water distillation to obtain Nepeta essential oil.

[0021] Preferably, the Nepeta medicinal material is first crushed to obtain Nepeta powder, distilled water is added to the Nepeta powder, volatile oil is extracted by water distillation, and the upper oily layer obtained by extraction is collected to obtain the Nepeta essential oil.

[0022] The solid-liquid ratio of the schizonepeta powder to the distilled water is 1 g: 10 mL to 100 mL. The solid-liquid ratio is the result of a single factor experiment on the extraction method of schizonepeta essential oil in the early stage.

[0023] Preferably, the extraction time is 2 to 4 hours. This extraction time is the result of a single factor experiment on the extraction method of schizonepeta essential oil in the early stage.

[0024] The research results of the present invention prove that schizonepeta essential oil has the potential to be developed as a natural antibacterial agent. Based on this, the present application provides an antibacterial product. Wherein, the antibacterial product includes any one of an antibacterial agent, a preservative, and a surface disinfectant.

[0025] Preferably, the antibacterial agent is prepared by using the schizonepeta essential oil as a raw material and supplemented with auxiliary materials acceptable in food science.

[0026] Auxiliary materials: any one or more of liquid auxiliary materials and solid auxiliary materials.

[0027] Preferably, the solid excipient comprises β-cyclodextrin.

[0028] Preferably, the liquid auxiliary material includes any one or more of propylene glycol, glycerol, liquid paraffin, Tween and butylene glycol.

[0029] The antibacterial products provided by the present invention can be used for antibacterial treatment of food.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention provides an application of schizone essential oil in the preparation of antibacterial products. The present invention uses the inhibition zone method and the mycelium growth rate inhibition method to study the inhibitory effects of schizone essential oil mother liquor diluted 500 times and 1000 times on two common bacteria, four air fungi and three plant pathogenic fungi in life. The research results show that the optimal extraction process conditions of schizone essential oil are: soaking time 1h, solid-liquid ratio 1:10, and extraction time 3h. The antibacterial effects of schizone essential oil on bacteria and fungi were obtained by measuring the diameter of the inhibition zone and calculating the antibacterial rate. The results show that schizone essential oil has different degrees of antibacterial effects on two bacteria and seven fungi, indicating that schizone essential oil has the potential to be developed as a natural antibacterial agent.

[0032] 2. The present invention uses schizonepeta as a test material, and adopts a single factor test method to examine the effects of soaking time, material-liquid ratio and extraction time on the extraction amount of essential oil, determines the optimal extraction process of essential oil by water distillation, and provides a method for extracting schizonepeta essential oil and its application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of the inhibition of Aspergillus niger by the essential oil solutions of different dilution multiples in the present invention, wherein: Figure 1 Figure A in the figure shows the antibacterial results of the control group; Figure 1 Figure B shows the antibacterial results of Nepeta tenuifolia essential oil diluted 500 times; Figure 1 C in the figure is the antibacterial result when Nepeta tenuifolia essential oil was diluted 1000 times.

[0034] Figure 2The results of the inhibition of essential oil solutions of different dilution ratios on Mucor sp. are shown in FIG. 1 , wherein: Figure 2 Figure A in the figure shows the antibacterial results of the control group; Figure 2 Figure B shows the antibacterial results of Nepeta tenuifolia essential oil diluted 500 times; Figure 2 C in the figure is the antibacterial result when Nepeta tenuifolia essential oil was diluted 1000 times.

[0035] Figure 3 The results of the antibacterial activity of the essential oil solutions at different dilution ratios against Cladosporium cladosporioides in the present invention are shown in FIG. Figure 3 Figure A in the figure shows the antibacterial results of the control group; Figure 3 Figure B shows the antibacterial results of Nepeta tenuifolia essential oil diluted 500 times; Figure 3 C in the figure is the antibacterial result when Nepeta tenuifolia essential oil was diluted 1000 times.

[0036] Figure 4 The results of the inhibition of Aspergillus flavus by the essential oil solutions of different dilution multiples in the present invention, wherein: Figure 4 Figure A in the figure shows the antibacterial results of the control group; Figure 4 Figure B shows the antibacterial results of Nepeta tenuifolia essential oil diluted 500 times; Figure 4 C in the figure is the antibacterial result when Nepeta tenuifolia essential oil was diluted 1000 times.

[0037] Figure 5 The results of the inhibition of essential oil solutions of different dilution multiples on Colletotrichum spaethianum are shown in the present invention, wherein: Figure 5 Figure A in the figure shows the antibacterial results of the control group; Figure 5 Figure B shows the antibacterial results of Nepeta tenuifolia essential oil diluted 500 times; Figure 5 C in the figure is the antibacterial result when Nepeta tenuifolia essential oil was diluted 1000 times.

[0038] Figure 6 The results of the inhibition of essential oil solutions of different dilution ratios on Fusarium solani are shown in the present invention, wherein: Figure 6 Figure A in the figure shows the antibacterial results of the control group; Figure 6 Figure B shows the antibacterial results of Nepeta tenuifolia essential oil diluted 500 times; Figure 6 C in the figure is the antibacterial result when Nepeta tenuifolia essential oil was diluted 1000 times.

[0039] Figure 7 The results of the inhibition of essential oil solutions of different dilution ratios on Fusarium proliferatum in the present invention are shown in FIG. Figure 7 Figure A in the figure shows the antibacterial results of the control group; Figure 7 Figure B shows the antibacterial results of Nepeta tenuifolia essential oil diluted 500 times; Figure 7 C in the figure is the antibacterial result when Nepeta tenuifolia essential oil was diluted 1000 times. DETAILED DESCRIPTION

[0040] The following will be combined with the specific implementation of the present invention to clearly and completely describe the technical solution in the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0042] Materials and Apparatus:

[0043] 1. Materials

[0044] The Chinese herbal medicines for testing were purchased from Anhui Bozhou Safeng Pharmaceutical Co., Ltd. and were identified as Nepeta tenuifolia by Dr. Zhang Ximei of Taishan University.

[0045] Main utensils and tools: disposable culture dishes, centrifuge tubes, 250mL Erlenmeyer flasks, 500mL Erlenmeyer flasks, punchers, inoculation needles, tweezers, and applicators.

[0046] Potato dextrose agar medium: weigh 12.03g of the finished product and 300mL of distilled water. Sterilize with high pressure steam at 121℃ for 20min. Potato dextrose agar medium is also called PDA medium.

[0047] Beef extract peptone agar medium: weigh 12.0g of the finished product and 300mL of distilled water. Sterilize with high pressure steam at 121℃ for 20min. Beef extract peptone agar medium is also called LB medium.

[0048] Beef extract peptone medium: peptone 5.0g, yeast extract powder 2.5g, NaCl 5.0g, distilled water 500mL, pH 7.2. Sterilize with high-pressure steam at 121℃ for 20min.

[0049] The strains used in the following examples are shown in Tables 1 and 2, all of which were stored in the Traditional Chinese Medicine Quality Inspection Laboratory of Taishan University.

[0050] Table 1 Seven fungal types

[0051]

[0052] Table 2 Two types of bacteria

[0053]

[0054] 2. Equipment and Instruments

[0055] Digital constant temperature water bath: HH-4, Jintan Xinhang Instrument Factory.

[0056] Vertical pressure steam sterilizer: YXQ-LS-100SⅡ, Shanghai Boxun Industrial Co., Ltd.

[0057] High-speed multifunctional crusher: KX-150, Wuyi Dingzang Daily Metal Products Factory.

[0058] Clean bench: SW-CJ-2FD, Suzhou Antai Air Technology Co., Ltd.

[0059] Digital constant temperature electric heating mantle: DRT-1000S, Shanghai Leigu Instrument Co., Ltd.

[0060] Constant temperature incubator: Model DHP-9272, Shanghai Yiheng Technology Co., Ltd.

[0061] Illumination incubator: GZX-250BS-III, Shanghai Xinmiao Medical Equipment Manufacturing Co., Ltd.

[0062] Full temperature culture shaker: QYC-2102C, Shanghai Xinmiao Medical Equipment Manufacturing Co., Ltd.

[0063] Electronic balance: FA214, Shanghai Mettler-Toledo Instrument Co., Ltd.

[0064] Essential oil extraction device: water distillation extraction device, purchased from Tai'an Yizhan Experimental Equipment Company.

[0065] 3. Main reagents and solutions

[0066] Yeast extract powder: OXOID.

[0067] Peptone: OXOID.

[0068] Agar powder: Nanjing Maojie Microbiological Technology Co., Ltd.

[0069] Sodium chloride: Tianjin Kaitong Chemical Reagent Co., Ltd.

[0070] Sodium hydroxide: Tianjin Kaitong Chemical Reagent Co., Ltd.

[0071] Beef extract peptone agar medium: Shanghai Bowei Microbiology Technology Co., Ltd.

[0072] Potato dextrose agar medium: Shanghai Bowei Microbiology Technology Co., Ltd.

[0073] Example 1

[0074] The present invention adopts a water distillation method and a heating reflux method to extract schizonepeta essential oil, and the extracted light yellow oily substance is the extracted schizonepeta essential oil. The specific method is as follows:

[0075] S1. Take 200 g of Nepeta herb and crush it to obtain Nepeta powder. Weigh the obtained Nepeta powder and put it into a ziplock bag for later use.

[0076] S2. Put 50g of Nepeta powder in a 1000mL round-bottom flask, add 500mL of distilled water at a solid-liquid ratio of 1:10, and add several glass beads to prevent violent boiling. Install the essential oil extraction device, start the condensation reflux water, adjust the temperature to 260℃, and start the heating distillation extraction process. Start timing when the distilled water starts to boil, and keep the extraction in a slightly boiling state for 3h.

[0077] S3. After the extraction is completed, the temperature of the distillation device in the essential oil extraction device is allowed to cool to room temperature by standing for 30 min, the lower layer of water extract in the essential oil extraction device is separated and sealed for storage, the upper oily layer is accurately read and collected, and the schizonepeta essential oil is harvested and placed in a 4°C refrigerator for standby use.

[0078] The present invention below measures the extraction rate of the Nepeta essential oil obtained by the above extraction, and the specific steps are as follows:

[0079] The mass of the obtained Nepeta essential oil was weighed, and the extraction rate of Nepeta essential oil was calculated according to the following formula:

[0080] Essential oil yield = ml of essential oil / weight of Nepeta tenuifolia medicinal material, mL / 100g.

[0081] The extraction rate of Nepeta tenuifolia essential oil was measured to be 0.45 mL / 100 g.

[0082] The present invention uses the process conditions in the above embodiment 1 to extract schizonepeta essential oil from schizonepeta medicinal materials. In addition, the antibacterial properties of the schizonepeta essential oil obtained by the above extraction are studied, and the specific research is as follows:

[0083] 1. Methods

[0084] 1. Preparation of essential oil mother liquor

[0085] In a sterilized 50 mL centrifuge tube, 1 mL of the schizonepeta essential oil prepared in Example 1, 1 mL of a 1% by volume Tween aqueous solution, and 48 mL of sterile water were injected with a pipette to obtain a first essential oil mother solution diluted 50 times. Then, 16 mL of the first essential oil mother solution and 16 mL of sterile water were drawn with a pipette and added to another sterilized 50 mL centrifuge tube to obtain 32 mL of a second essential oil mother solution diluted 100 times.

[0086] 2. Study on antibacterial activity of bacteria

[0087] 2.1 Bacterial Activation

[0088] Prepare beef extract peptone agar medium, sterilize with high pressure steam, pour the medium into a disposable culture dish, wait for the plate to cool and solidify, take out the strains of Escherichia coli and Bacillus subtilis stored in the glycerol tube and inoculate them on the prepared beef extract peptone agar medium by streaking method, and incubate them in a 37°C biochemical incubator to activate them. After clones grow on the culture medium, pick the bacteria with better growth and inoculate them into the prepared and sterilized LB liquid culture medium, put them into a shaker for expansion culture, set the conditions to 37°C, 180r / min, and culture for 16h.

[0089] 2.2 Preparation of drug-sensitive paper

[0090] Use a hole puncher to punch out circular paper pieces with a diameter of 5 mm on the filter paper, put them in a glass culture dish, sterilize them with high-pressure steam at 121°C for 0 min, and then dry them for later use.

[0091] 2.3 Preparation of test plates

[0092] Pour the prepared and autoclaved LB solid medium into a disposable culture dish, wait for the agar surface of the plate to solidify and the water to dry, and then use the spread plate method to inoculate. The specific inoculation operation is: take 20 μL of the bacterial solution of Escherichia coli and Bacillus subtilis that have been expanded and cultured in a shaker, drop it in the center of the agar plate, and evenly spread the entire plate with a sterilized spreader to make a bacterial plate. Repeat each group of treatments 3 times.

[0093] 2.4 Inhibition zone method test

[0094] The antibacterial zone method test of bacteria adopts the paper diffusion method, and the specific method is as follows:

[0095] Use a pipette to take 20 μL of the first essential oil mother liquor and the second essential oil mother liquor and drop them into the sterilized filter paper. Add 20 μL of sterile water to the control group. This volume can make it completely infiltrated. Use sterilized tweezers to clamp the filter paper, spread it in the center of the bacterial plate, and place it in a 37°C biochemical incubator for about 18 hours. Observe and measure the diameter of the inhibition zone. Each group of experiments was carried out three times, and the average value was taken as the final inhibition zone diameter. The antibacterial effect of essential oils was evaluated by measuring the diameter of the colony inhibition zone. The larger the diameter, the better the antibacterial effect.

[0096] 3. Determination of fungal antibacterial activity

[0097] 3.1 Bacterial Activation

[0098] Prepare 300 mL of potato dextrose agar medium and sterilize it with high-pressure steam at 121°C for 20 min. Under sterile conditions, pour the medium into a disposable culture dish to make a plate. After the medium cools and solidifies, take out the seven fungi shown in Table 1 that have been subcultured in the laboratory in advance, inoculate them into PDA medium respectively, and put the plate upside down in a light incubator at 28°C for 48 h.

[0099] 3.2 Preparation of test plates

[0100] Prepare potato dextrose agar medium and sterilize it with high pressure steam at 121℃ for 20min. The fungus is grown using the pour plate method. The specific steps are as follows:

[0101] In the clean bench, 0.5 mL of the schizonepeta essential oil prepared in Example 1 was accurately pipetted with a pipette, 0.5% Tween 20 was added for emulsification, and then sterilized water was used to dilute the mother liquor concentration gradients of 50 times and 100 times, which were the third essential oil mother liquor and the fourth essential oil mother liquor, respectively. Among them, the mother liquor diluted to a concentration of 50 times is the third essential oil mother liquor, and the mother liquor diluted to a concentration of 100 times is the fourth essential oil mother liquor.

[0102] The sterilized PDA medium was cooled to 50°C, and the diluted third essential oil mother liquor and fourth essential oil mother liquor were added and mixed thoroughly to dilute the essential oil concentration to 500 times and 1000 times, and the diluted medium was poured onto a plate and cooled for later use. At the same time, another sterilized potato dextrose agar medium was taken and poured onto a plate without any treatment as a control group of the experiment.

[0103] 3.3 Determination of antibacterial activity by growth rate inhibition method

[0104] The growth rate inhibition method was used to determine the antifungal activity of Nepeta essential oil. To avoid contamination of other fungi, Mucor was inoculated by bacterial suspension injection method. The specific steps were as follows: 1 mL of 0.5% Tween 20 aqueous solution and 1 mL of sterile water were pipetted into the activated plate and repeatedly pipetted to mix to prepare a bacterial suspension, which was counted by direct counting under a microscope and injected into a centrifuge tube for later use. 1 μL of bacterial suspension was pipetted into the center of the solidified 500-fold and 1000-fold essential oil plates and the control plate to complete the inoculation.

[0105] The other 6 fungi were inoculated by the punching method. The specific steps are as follows: select a pre-cultured uniformly grown colony plate, and punch out a bacterial cake with a diameter of about 5 mm along its edge with a sterile puncher. After the culture medium solidifies, use a sterilized inoculation needle to carefully pick up a 5 mm bacterial cake and invert it on the center of the agar surface of the 500-fold, 1000-fold and control plates. After the inoculation is completed, place the culture dish in a 28°C light incubator for about a week. When the colony of the control group is about to fill the culture dish, use a ruler to measure the colony diameter of the treatment group, take the average value, and repeat 3 times.

[0106] 4. Data processing

[0107] The experimental data were processed and analyzed using Microsoft Excel software, and the relative inhibition rate was calculated according to the following formula:

[0108] Bacterial inhibition rate (%) = [(average diameter of control colonies - 5 mm) - (average diameter of treated colonies - 5 mm)] / (average diameter of control colonies - 5 mm) × 100%.

[0109] 2. Results

[0110] 1. Antibacterial activity measurement results and analysis

[0111] The relationship between the inhibition zone and the antibacterial effect is: when the diameter of the inhibition zone exceeds 15mm, the inhibition effect is most significant; when the diameter of the inhibition zone is 12.5±2.5mm, the inhibition effect shows moderate sensitivity; when the diameter of the inhibition zone is 8±1mm, the inhibition effect shows low sensitivity; when there is no inhibition zone, the inhibition effect is insensitive. Take out the cultured test plate, measure the diameter of the inhibition zone with a ruler, and express the size of the inhibition zone in diameter: mm. The diameter of the inhibition zone is positively correlated with the antibacterial activity.

[0112] Among the two tested bacteria, the average diameters of the inhibition zones of the two dilution multiples of essential oil mother liquors: the first essential oil mother liquor and the second essential oil mother liquor against Bacillus subtilis were 7.3 mm and 7.0 mm, respectively, and the average diameters of the inhibition zones against Escherichia coli were 8.4 mm and 7.9 mm, respectively, as shown in Table 3. Therefore, according to the results of the inhibition zone measurement of Nepeta essential oil against Escherichia coli and Bacillus subtilis, the sensitivity of the two bacteria to Nepeta essential oil is Escherichia coli, then Bacillus subtilis.

[0113] Table 3 Diameters of inhibition zones of various tested strains

[0114]

[0115] 2. Antifungal activity measurement results and analysis

[0116] The results of the change in the average diameter of the colony caused by the antibacterial effect of the schizonepeta essential oil solution are shown in Table 4, the calculated relative antibacterial rate is shown in Table 5, and the antifungal effect results are shown in Figure 1 to Figure 7 . Figure 1 to Figure 7 The following are the antibacterial results of essential oil solutions with different dilution multiples on Aspergillus niger, Mucor sp, Cladosporim cladosporioides, Aspergillus flavus, Colletotrichum spaethianum, Fusarium solani, and Fusarium proliferatum. Figure 1 to Figure 7 Figure A in the figure shows the antibacterial results of the control group; Figure 1 to Figure 7 Figure B shows the antibacterial results of Nepeta tenuifolia essential oil diluted 500 times; Figure 1 to Figure 7 C in the figure is the antibacterial result when Nepeta tenuifolia essential oil was diluted 1000 times.

[0117] It can be seen from the table and figure that when the schizonepeta essential oil was diluted 500 times and 1000 times, it had different degrees of inhibitory effect on the 7 kinds of fungi shown in Table 1. It was preliminarily shown that the schizonepeta essential oil also had good antibacterial properties against molds. This may be because the essential oil damaged the morphological structure of mold spores, destroyed the cell membrane and cell wall of the mold, and affected the normal life of the mold.

[0118] Table 4 Average colony diameter of fungi grown for 8 days

[0119]

[0120] Note: “ / ” indicates there is no measurement result here.

[0121] Table 5 Inhibition rate of schizonepeta essential oil on 7 kinds of fungi

[0122]

[0123] From the above results, it can be concluded that when diluted 500 times and 1000 times, Nepeta essential oil has different degrees of inhibitory effects on 7 fungi, and Nepeta essential oil has a broad-spectrum antibacterial property.

[0124] The present invention uses a water distillation method to extract the essential oil of Nepeta, and by analyzing the results of three groups of single-factor tests, the process parameters of the three factors that mainly affect the oil yield of the essential oil: soaking time, material-liquid ratio, and extraction time are optimized, and finally the optimal extraction process conditions of the essential oil are determined as follows: soaking time 1h, material-liquid ratio 1:10, and extraction time 3h. Nepeta essential oil has a light yellow oily liquid appearance, has a unique aroma of Nepeta spike, has high clarity, and is easy to identify.

[0125] A large number of studies have shown that plant essential oils have antibacterial effects. In order to further analyze the antibacterial effect of schizone essential oil, the present invention studies the antibacterial activity of schizone essential oil on bacteria by the inhibition zone method, and determines that schizone essential oil has an inhibitory effect on Bacillus subtilis and Escherichia coli; by calculating the relative inhibition rate of schizone essential oil on fungi, it is known that schizone essential oil also has different degrees of antibacterial effect on 7 test fungi, indicating that schizone essential oil has a broad spectrum of antibacterial properties. Due to limited conditions and time, the chemical components of schizone essential oil were not analyzed and determined. For compounds with antibacterial and antibacterial activities, further separation is required for activity detection.

[0126] The research results of the present invention preliminarily show that catmint essential oil has both aromaticity, antibacterial and antimicrobial activity, therefore, it has great potential to be developed into a natural antibacterial agent, can be directly applied to the field of food preservation, and effectively prolong the shelf life of food. It can also be applied to the pollution of wine body in the production process and preservation of wine, provide consumers with safer and healthier high-quality wine, and provide certain economic benefits for the wine industry. On this basis, the present invention provides a kind of antibacterial product with catmint essential oil as the main active ingredient, which can be used for food preservation. The product includes an antibacterial agent with catmint essential oil as the main active ingredient, a preservative with catmint essential oil as the main active ingredient, a surface disinfectant with catmint essential oil as the main active ingredient, and the remaining conventional products used in food antibacterial. These products can all be added with conventional auxiliary materials when such products are prepared, and are prepared by the conventional preparation method of such products.

[0127] The antibacterial agent is composed of schizonepeta essential oil and auxiliary materials, which are any one or more of liquid auxiliary materials and solid auxiliary materials.

[0128] Wherein, the solid auxiliary material includes β-cyclodextrin.

[0129] The liquid auxiliary material includes any one or more of propylene glycol, glycerol, liquid paraffin, Tween 20 and butylene glycol.

[0130] The preservative is composed of nepeta essential oil and the following auxiliary materials:

[0131] Any one or more of Tween 20, β-cyclodextrin, and propylene glycol.

[0132] The surface disinfectant is composed of nepeta essential oil and the following excipients:

[0133] Any one or more of liquid paraffin, Tween 20, and butanediol.

[0134] The antibacterial agent prepared by the present invention is a solution, which is composed of schizonepeta essential oil and a dispersant; the dispersant includes any one or more of water, an organic solvent and an ionic dispersant. The organic solvent includes any one of ethanol and acetone; the ionic dispersant includes any one of sodium oleate, carboxylates, sulfate ester salts, sulfonates, octadeceneamine acetate, and polyethylene glycol-type polyols.

[0135] Below we take antibacterial agents, preservatives, and surface disinfectants as examples to illustrate their effects. The specific research is as follows:

[0136] Example 2

[0137] An antibacterial agent with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of 1% by volume Tween 20 aqueous solution and 48 mL of sterile water.

[0138] The preparation method of the antibacterial agent comprises the following steps:

[0139] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% Tween 20 aqueous solution and 48 mL of sterile water were injected with a pipette and mixed. The mixture was stored at 4°C.

[0140] When used, 1 mL of the antibacterial agent stock solution was diluted with water to 500 times and applied to the plate inoculated with ash anthracnose. After 72 hours, the inhibition rate of the antibacterial liquid on ash anthracnose was 6%.

[0141] From the above results, it can be seen that the antibacterial agent with schizonepeta essential oil as the main active ingredient provided by the present invention has an antibacterial effect on Colletotrichum fraxinus, and the antibacterial efficiency is 6%.

[0142] Example 3

[0143] An antibacterial agent with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of a 1% by volume beta-cyclodextrin aqueous solution and 48 mL of sterile water.

[0144] The preparation method of the antibacterial agent comprises the following steps:

[0145] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% by volume β-cyclodextrin aqueous solution and 48 mL of sterile water were injected with a pipette and mixed, and the mixture was stored at 4°C.

[0146] When used, 1 mL of the antibacterial agent stock solution was diluted with water to 500 times and applied to the plate inoculated with Fusarium solani. After 72 hours, the inhibition rate of the antibacterial liquid on Fusarium solani was 5.8%.

[0147] From the above results, it can be seen that the antibacterial agent with schizonepeta essential oil as the main active ingredient provided by the present invention has an antibacterial effect on Fusarium solani, and the antibacterial efficiency is 5.8%.

[0148] Example 4

[0149] The invention discloses an antibacterial agent with schizonepeta essential oil as a main active ingredient. The antibacterial agent is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of a 1% by volume propylene glycol aqueous solution and 48 mL of sterile water.

[0150] The preparation method of the antibacterial agent comprises the following steps:

[0151] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% by volume propylene glycol aqueous solution and 48 mL of sterile water were injected with a pipette and mixed. The mixture was stored at 4°C.

[0152] When used, 1 mL of the antibacterial agent stock solution was diluted with water to 500 times and applied to the plate inoculated with Bacillus subtilis. After 72 hours, the inhibition rate of the antibacterial solution on Bacillus subtilis was found to be 6.1%.

[0153] From the above results, it can be seen that the antibacterial agent with schizonepeta essential oil as the main active ingredient provided by the present invention has an antibacterial effect on Bacillus subtilis, and the antibacterial efficiency is 6.1%.

[0154] Example 5

[0155] An antibacterial agent with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of a 1% by volume mixed auxiliary material solution and 48 mL of sterile water.

[0156] The preparation method of the 1% by volume mixed auxiliary material solution is as follows: take 800.3 mL of Tween 20, 0.3 mL of β-cyclodextrin, and 0.4 mL of propylene glycol, add 99 mL of sterile water and mix well.

[0157] The preparation method of the antibacterial agent comprises the following steps:

[0158] In a sterilized 50 mL centrifuge tube, inject 1 mL of nepeta essential oil, 1 mL of 1% by volume mixed auxiliary material solution and 48 mL of sterile water using a pipette, mix and store at 4°C.

[0159] When used, 1 mL of the antibacterial agent mother solution was diluted with water to 500 times and applied to the plate inoculated with Cladosporium. After 72 hours, the inhibition rate of the antibacterial solution on Cladosporium was found to be 6.2%.

[0160] From the above results, it can be seen that the antibacterial agent with schizonepeta essential oil as the main active ingredient provided by the present invention has an antibacterial effect on Cladosporium cladosporium, and the antibacterial efficiency is 6.2%.

[0161] Example 6

[0162] A preservative with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of 1% by volume glycerol aqueous solution and 48 mL of sterile water.

[0163] The preparation method of the preservative comprises the following steps:

[0164] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% by volume glycerol aqueous solution and 48 mL of sterile water were injected with a pipette and mixed. The mixture was stored at 4°C.

[0165] When using, take 1 mL of the preservative mother solution and dilute it with water to 500 times, apply it to the snow pear fruit inoculated with Mucor, then dry the snow pear fruit, place it in a plastic frame, and pack it in a polyethylene bag, and store it at 20°C and 90% relative humidity. After one week of storage, the rot rate of the snow pear fruit was counted. Sterile water was used as a control.

[0166] The results showed that the decay rate of snow pear fruits treated with preservatives decreased by 22.1% compared with the control.

[0167] The above results show that the preservative has a preservative effect on snow pear fruits infected with Mucor, effectively delays the decline of fruit hardness and water content, and can be used for the preservation of fruits and vegetables.

[0168] Example 7

[0169] A preservative with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of a 1% by volume beta-cyclodextrin aqueous solution and 48 mL of sterile water.

[0170] The preparation method of the preservative comprises the following steps:

[0171] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% by volume β-cyclodextrin aqueous solution and 48 mL of sterile water were injected with a pipette and mixed, and the mixture was stored at 4°C.

[0172] When using, take 1 mL of the preservative mother solution and dilute it with water to 500 times, apply it to the corn fruit inoculated with Aspergillus flavus, then dry the corn fruit and place it in a plastic frame, and pack it in a polyethylene bag, and store it at 20°C and 90% relative humidity. After one week of storage, the rot rate of corn fruit was counted. Sterile water was used as a control.

[0173] The results showed that the rot rate of corn fruits treated with preservatives decreased by 24.5% compared with the control.

[0174] The above results show that the preservative has a preservative effect on corn fruits infected with Aspergillus flavus, effectively delays the decline in fruit hardness and water content, and can be used for the preservation of fruits and vegetables.

[0175] Example 8

[0176] A preservative with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of 1% by volume Tween 20 aqueous solution and 48 mL of sterile water.

[0177] The preparation method of the preservative comprises the following steps:

[0178] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% Tween 20 aqueous solution and 48 mL of sterile water were injected with a pipette and mixed. The mixture was stored at 4°C.

[0179] When using, take 1 mL of the preservative mother solution and dilute it with water to 500 times, apply it to the corn fruit inoculated with Aspergillus flavus, then dry the corn fruit and place it in a plastic frame, and pack it in a polyethylene bag, and store it at 20°C and 90% relative humidity. After one week of storage, the rot rate of corn fruit was counted. Sterile water was used as a control.

[0180] The results showed that the rot rate of corn fruits treated with preservatives decreased by 23.3% compared with the control.

[0181] The above results show that the preservative has a preservative effect on corn fruits infected with Aspergillus flavus, effectively delays the decline in fruit hardness and water content, and can be used for the preservation of fruits and vegetables.

[0182] Example 9

[0183] A preservative with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of a 1% by volume mixed auxiliary material solution, and 48 mL of sterile water.

[0184] The preparation method of the 1% by volume mixed auxiliary material solution is as follows: take 0.5 mL of glycerol and 0.5 mL of β-cyclodextrin, add 99 mL of sterile water and mix well.

[0185] The preparation method of the preservative comprises the following steps:

[0186] In a sterilized 50 mL centrifuge tube, inject 1 mL of nepeta essential oil, 1 mL of 1% by volume mixed auxiliary material solution and 48 mL of sterile water using a pipette, mix and store at 4°C.

[0187] When using, take 1 mL of the preservative mother solution and dilute it with water to 500 times, apply it to the peach fruit inoculated with Mucor, then dry the corn fruit, place it in a plastic frame, and pack it in a polyethylene bag, and store it at 20°C and 90% relative humidity. After one week of storage, the rot rate of the peach fruit was counted. Sterile water was used as a control.

[0188] The results showed that the rot rate of peach fruits treated with preservatives decreased by 22.7% compared with the control.

[0189] The above results show that the preservative has a preservative effect on peach fruits infected with Mucor, effectively delays the decline in fruit hardness and water content, and can be used for the preservation of fruits and vegetables.

[0190] Example 10

[0191] A surface disinfectant with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of 1% by volume liquid paraffin aqueous solution and 48 mL of sterile water.

[0192] The preparation method of the surface disinfectant comprises the following steps:

[0193] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% by volume liquid paraffin aqueous solution and 48 mL of sterile water were injected with a pipette and mixed. The mixture was stored at 4°C.

[0194] When using, take 1 mL of the surface disinfectant stock solution and dilute it with water to 500 times, apply it to the corn fruit inoculated with Aspergillus flavus, then dry the corn fruit and place it in a plastic frame, package it in a polyethylene bag, and store it at 20°C and 90% relative humidity. Count the diameter of the corn fruit lesions every day within one week after storage. Use sterile water as a control.

[0195] The results showed that the diameter of the fungus ring on corn fruits treated with surface disinfectants was significantly reduced.

[0196] The above results show that the surface disinfectant has a significant inhibitory effect on Aspergillus flavus and can be used to prepare fruit and vegetable surface disinfectants.

[0197] Embodiment 11

[0198] A surface disinfectant with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of 1% by volume Tween 20 aqueous solution and 48 mL of sterile water.

[0199] The preparation method of the surface disinfectant comprises the following steps:

[0200] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% Tween 20 aqueous solution and 48 mL of sterile water were injected with a pipette and mixed. The mixture was stored at 4°C.

[0201] When using, take 1 mL of the surface disinfectant stock solution and dilute it with water to 500 times, apply it to the wax tree leaves inoculated with ash anthracnose, then dry the wax tree leaves, place them in a plastic frame, and pack them in polyethylene bags, and store them at 20°C and 90% relative humidity. Count the diameter of the lesions on the wax tree leaves every day within one week after storage. Use sterile water as a control.

[0202] The results showed that the diameter of the fungus circle on the leaves of wax tree treated with surface disinfectant was significantly reduced.

[0203] The above results show that the surface disinfectant has a significant inhibitory effect on ash anthracnose and can be used to prepare fruit and vegetable surface disinfectants.

[0204] Example 12

[0205] A surface disinfectant with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of a 1% by volume butylene glycol aqueous solution and 48 mL of sterile water.

[0206] The preparation method of the surface disinfectant comprises the following steps:

[0207] In a sterilized 50 mL centrifuge tube, 1 mL of nepeta essential oil, 1 mL of 1% by volume butylene glycol aqueous solution and 48 mL of sterile water were injected with a pipette and mixed. The mixture was stored at 4°C.

[0208] When using, take 1 mL of the surface disinfectant stock solution and dilute it with water to 500 times, apply it to soybean plants inoculated with Fusarium, then dry the soybean plants, place them in a plastic frame, and pack them in polyethylene bags, and store them at 20°C and 90% relative humidity. Count the diameter of the lesions of the soybean plants every day within one week after storage. Use sterile water as a control.

[0209] The results showed that the diameter of the bacterial circle of soybean plants treated with surface disinfectants was significantly reduced.

[0210] The above results show that the surface disinfectant has a significant inhibitory effect on Fusarium sphaeroides and can be used to prepare fruit and vegetable surface disinfectants.

[0211] Example 13

[0212] A surface disinfectant with schizonepeta essential oil as a main active ingredient is composed of the following raw materials: 1 mL of schizonepeta essential oil, 1 mL of a 1% by volume mixed auxiliary material solution, and 48 mL of sterile water.

[0213] The preparation method of the 1% by volume mixed auxiliary material solution is as follows: take 0.3 mL of Tween 20, 0.3 mL of liquid paraffin, and 0.4 mL of butanediol, add 99 mL of sterile water and mix well.

[0214] The preparation method of the surface disinfectant comprises the following steps:

[0215] In a sterilized 50 mL centrifuge tube, inject 1 mL of nepeta essential oil, 1 mL of 1% by volume mixed auxiliary material solution and 48 mL of sterile water using a pipette, mix and store at 4°C.

[0216] When using, take 1 mL of the surface disinfectant stock solution and dilute it with water to 500 times and apply it to the wheat fruit inoculated with Aspergillus niger. Then dry the wheat fruit and place it in a plastic frame, package it in a polyethylene bag, and store it at 20°C and 90% relative humidity. Count the diameter of the lesions on the wheat fruit every day within one week after storage. Use sterile water as a control.

[0217] The results showed that the diameter of the bacterial ring of wheat fruits treated with surface disinfectants was significantly reduced.

[0218] The above results show that the surface disinfectant has a significant inhibitory effect on Aspergillus niger and can be used to prepare fruit and vegetable surface disinfectants.

[0219] In summary, the schizonepeta essential oil provided by the present invention can be used to prepare antibacterial products.

[0220] The present invention provides an application of schizonepeta essential oil in antibacterial treatment. The research results of the present invention show that schizonepeta essential oil can inhibit the activity of Bacillus subtilis, Escherichia coli, Aspergillus niger, Mucor, Cladosporium cladosporum, Aspergillus flavus, Colletotrichum ash, Fusarium spp., and Fusarium solani, indicating that schizonepeta essential oil has the potential to be developed as a natural antibacterial agent.

[0221] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0222] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An application of Nepeta essential oil in antibacterial treatment, characterized in that: The nepeta essential oil is used to inhibit the activity of bacteria or plant pathogenic fungi; The extraction method of described schizonepeta essential oil is as follows: The herbal medicine Nepeta is extracted by water distillation, and the upper oily layer obtained by extraction is collected to obtain the Nepeta essential oil.

2. The use of schizonepeta essential oil in antibacterial treatment according to claim 1, characterized in that: The bacteria include Bacillus subtilis and / or Escherichia coli; The plant pathogenic fungi include any one or more of Aspergillus niger, Mucor, Cladosporium, Aspergillus flavus, Colletotrichum fraxinus, Fusarium solani, and Fusarium solani.

3. The use of a schizonepeta essential oil in antibacterial treatment according to claim 1, characterized in that: The schizonepeta essential oil is used for preparing an antibacterial product, and the antibacterial product has the schizonepeta essential oil as a main active ingredient.

4. The use of schizonepeta essential oil in antibacterial treatment according to claim 3, characterized in that: The antibacterial product is prepared by using the schizonepeta essential oil as a raw material and supplemented with auxiliary materials acceptable in food science; The auxiliary materials include any one or more of liquid auxiliary materials and solid auxiliary materials.

5. The use of schizonepeta essential oil in antibacterial treatment according to claim 4, characterized in that: The solid auxiliary material includes β-cyclodextrin.

6. The use of schizonepeta essential oil in antibacterial treatment according to claim 4, characterized in that: The liquid auxiliary material includes any one or more of propylene glycol, glycerol, liquid paraffin, Tween and butylene glycol.

7. The use of schizonepeta essential oil in antibacterial treatment according to claim 1, characterized in that: When using the hydrodistillation method for extraction, powdered Nepeta tenuifolia is used; The Nepeta medicinal material powder is obtained by crushing the Nepeta medicinal material; When extracting by water distillation, distilled water is added to the schizonepeta powder, and the schizonepeta essential oil is extracted by water distillation; Wherein, the solid-liquid ratio of the Nepeta powder to the distilled water is 1g:10mL-100mL.

8. The use of schizonepeta essential oil in antibacterial treatment according to claim 1, characterized in that: The extraction time is 2 to 4 hours.

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