Preparation method and antibacterial application of alpinia oxyphylla essential oil
By extracting volatile oil from tutoring, the threat of food-borne pathogenic bacteria to food safety is solved, and effective antibacterial treatment to a variety of pathogenic bacteria is achieved, and the extraction process is safe and efficient.
Patent Information
- Application Number
- CN202411514339.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art is difficult to effectively solve the threat of foodborne pathogenic bacteria to food safety, especially in the development of natural plant preservatives and antibacterial agents.
The educational essential oil is prepared by extracting volatile oil ingredients from the educational system, and extracting it by freezing treatment, defrosting treatment and steam distillation to prepare educational essential oils with antibacterial effects.
Zhizhi essential oil has significant antibacterial effects on a variety of foodborne pathogenic bacteria, including Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, etc., with a high extraction rate, simple operation, safe and efficient.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial medicine, and in particular to a preparation method of lily of the valley essential oil and its antibacterial application. Background Art
[0002] Alpinia oxyphylla Miq, also known as Alpinia oxyphylla, Alpinia oxyphylla, is a perennial herb of the genus Alpinia. Alpinia oxyphylla is one of the four major southern medicines in my country. It can not only be used as a Chinese herbal medicine, but also has a wide range of pharmacological effects. At present, many components have been isolated from Alpinia oxyphylla at home and abroad, such as terpenoids, sterols, diphenylheptanes, flavonoids and phenolic compounds. Natural active ingredients are an important part of the modern extraction of traditional Chinese medicine. At present, the market for health foods and medicines with natural products as the main raw materials is quite large.
[0003] A series of food safety problems caused by foodborne pathogens seriously endanger human health. Foodborne pathogens include Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas fragi, etc. They are common pathogens of foodborne diseases, and the food poisoning caused by them seriously endangers human health. According to statistics from the World Health Organization, about 10% of consumers worldwide will get sick from eating spoiled food every year, and food poisoning caused by foodborne pathogens is becoming increasingly serious and common. Food preservatives are divided into two categories: plant natural preservatives and chemical synthetic preservatives. With the green barrier problem faced by agricultural products in export requirements, plant natural preservatives have received more and more attention. Therefore, it is particularly important to develop plant natural preservatives and antibacterial agents. Summary of the invention
[0004] In view of the above-mentioned defects of the prior art, the present invention proposes a preparation method of an essential oil for improving intelligence and its antibacterial application to solve the problems raised by the above-mentioned background technology.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] The invention discloses an application of Alpinia oxyphylla essential oil in preparing an antibacterial agent capable of preventing and controlling foodborne pathogenic bacteria contamination. The Alpinia oxyphylla essential oil is a volatile oil component extracted from the traditional Chinese medicine Alpinia oxyphylla, and the foodborne pathogenic bacteria are Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas fragariae.
[0007] Preferably, the preparation method of the essential oil comprises the following steps:
[0008] (1) Freezing treatment: taking ripe fruits of Alpinia oxyphylla and freezing them at a freezing temperature of -10°C to -2°C for 2 to 5 hours to obtain frozen Alpinia oxyphylla;
[0009] (2) thawing treatment: adding water at a temperature of 70° C. to 90° C. to the frozen Alpinia oxyphylla obtained in step (1), soaking for 1 to 3 hours, draining the water, and obtaining thawing Alpinia oxyphylla;
[0010] (3) Final treatment: crush and sieve the thawed Alpinia oxyphylla obtained in step (2) to obtain powder; mix the powder with water and let it stand and soak for 2.2 to 2.5 hours, then place it in water vapor for distillation for 5 to 7 hours, collect the upper oily liquid, add anhydrous sodium sulfate to dry, and obtain Alpinia oxyphylla essential oil; wherein the amount of anhydrous sodium sulfate added per liter of oily liquid is 10 to 30 g.
[0011] Preferably, the freezing temperature in step (1) is -5°C.
[0012] Preferably, the freezing treatment time in step (1) is 2.5 hours.
[0013] Preferably, the mass ratio of the powder to water mixed in step (3) is 1:(10.5-11.5).
[0014] Preferably, the soaking time in step (3) is 2.4 hours.
[0015] Preferably, the distillation temperature in step (3) is 215-240°C.
[0016] Preferably, the distillation temperature in step (3) is 230°C.
[0017] Preferably, the distillation time in step (3) is 5.5 h.
[0018] Preferably, the essential oil is used directly or prepared into a bacterial agent for use.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] (1) The obtained Yizhi essential oil of the present invention shows a certain sensitivity to foodborne pathogens, among which the diameter of the inhibition zone of Bacillus cereus is 13.11±0.44cm, the MIC reaches 0.29mg / mL, and the MBC reaches 1.17mg / mL; the diameter of the inhibition zone of Bacillus subtilis is 13.05±0.36cm, the MIC reaches 0.29mg / mL, and the MBC reaches 0.59mg / mL; the diameter of the inhibition zone of Staphylococcus aureus is 22.29±0.92cm, the MIC reaches 0.29mg / mL, and the MBC reaches 4.69mg / mL; the diameter of the inhibition zone of Staphylococcus epidermidis is 19.74±0.15cm, the MIC reaches 2.34mg / mL, and the MBC reaches 18.75mg / mL; and the MIC of Pseudomonas fragariae reaches 2.34mg / mL, and the MBC reaches 2.34mg / mL.
[0021] (2) The present invention provides a method for preparing the essential oil of Alpinia oxyphylla by steam distillation. The freezing treatment of the mature fruits of Alpinia oxyphylla in the early stage can quickly reduce the respiratory metabolism of the fruits and control the transpiration water loss of the fruits. At the same time, the pre-cooling treatment helps to keep the peel intact and prevent the volatilization of volatile oil. After the thawing treatment, the cell structure is destroyed, the Alpinia oxyphylla volatile oil collected has many components, and the extraction rate of the essential oil of Alpinia oxyphylla is above 1.93-2.10%;
[0022] (3) The extraction method of the present invention is simple to operate, does not require large-scale extraction equipment to efficiently extract Alpinia oxyphylla, and does not require the use of a large amount of chemical reagents during the preparation process, which is safe and efficient. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The results of the antibacterial zone test of the Yizhi essential oil prepared in Example 3 on different strains are shown in FIG. Figure 1 A is the antibacterial effect of Yiqi essential oil, ampicillin and sterile water on golden yellow grapes; Figure 1 B is the antibacterial effect of Yiqi essential oil, levofloxacin and sterile water on Staphylococcus epidermidis; Figure 1 C is the antibacterial effect of Yiqi essential oil, levofloxacin and sterile water on Bacillus subtilis; Figure 1 D is the antibacterial effect of Yiqi essential oil, levofloxacin and sterile water on Bacillus cereus; Figure 1 E is the antibacterial effect of Yiqi essential oil, levofloxacin and sterile water on Pseudomonas fragrans; Figure 1 F is the antibacterial effect of Yiqi essential oil, levofloxacin and sterile water on Pseudomonas aeruginosa; Figure 1 G is the antibacterial effect of Yiqi essential oil, levofloxacin and sterile water on Escherichia coli; Figure 1 H is the antibacterial effect of Yiqi essential oil, levofloxacin and sterile water on Salmonella enterica; Figure 1 I is the antibacterial effect of Yiqi essential oil, levofloxacin and sterile water on Shigella flexneri. DETAILED DESCRIPTION
[0024] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.
[0025] Example 1
[0026] A preparation method of the essential oil of Qiqihar comprises the following steps:
[0027] (1) Freezing treatment: taking ripe Alpinia oxyphylla fruits and freezing them at a freezing temperature of -10°C for 2 hours to obtain frozen Alpinia oxyphylla;
[0028] (2) thawing treatment: adding water at a temperature of 70° C. to the frozen Alpinia oxyphylla obtained in step (1) and soaking for 1 hour, draining the water, and obtaining thawed Alpinia oxyphylla;
[0029] (3) Final treatment: Crush and sieve the thawed Alpinia oxyphylla obtained in step (2) to obtain powder; mix the powder with water in a material-liquid mass ratio of 1:10.5 and let it stand and soak for 2.2 hours, then place it in water vapor at a temperature of 215°C for distillation for 5 hours, collect the upper oily liquid, add anhydrous sodium sulfate and dry it to obtain Alpinia oxyphylla essential oil. According to the "Method A" of the essential oil determination method of the 2020 edition of the "Chinese Pharmacopoeia", the essential oil extraction rate is determined. Essential oil extraction rate (%) = (essential oil volume (mL) / plant powder mass (g)) × 100%, the measured Alpinia oxyphylla essential oil extraction rate is 1.93%
[0030] Example 2
[0031] A preparation method of the essential oil of Qiqihar comprises the following steps:
[0032] (1) Freezing treatment: taking ripe fruits of Alpinia oxyphylla and freezing them at a freezing temperature of -2°C for 5 hours to obtain frozen Alpinia oxyphylla;
[0033] (2) thawing treatment: adding water at a temperature of 90° C. to the frozen Alpinia oxyphylla obtained in step (1), soaking for 3 hours, draining the water, and obtaining thawed Alpinia oxyphylla;
[0034] (3) Final treatment: Crush and sieve the thawed Alpinia oxyphylla obtained in step (2) to obtain powder; mix the powder with water in a material-liquid mass ratio of 1:11.5 and let it stand and soak for 2.5 hours, then place it in water vapor at a temperature of 240°C for distillation for 7 hours, collect the upper oily liquid, add anhydrous sodium sulfate and dry it to obtain Alpinia oxyphylla essential oil. According to the "Method A" of the essential oil determination method of the 2020 edition of the "Chinese Pharmacopoeia", the essential oil extraction rate is determined. Essential oil extraction rate (%) = (essential oil volume (mL) / plant powder mass (g)) × 100%, the measured Alpinia oxyphylla essential oil extraction rate is 1.95%
[0035] Example 3
[0036] A preparation method of the essential oil of Qiqihar comprises the following steps:
[0037] (1) Freezing treatment: taking ripe fruits of Alpinia oxyphylla and freezing them at a freezing temperature of -5°C for 2.5 hours to obtain frozen Alpinia oxyphylla;
[0038] (2) thawing treatment: adding water at a temperature of 90° C. to the frozen Alpinia oxyphylla obtained in step (1), soaking for 3 hours, draining the water, and obtaining thawed Alpinia oxyphylla;
[0039] (3) Final treatment: Crush and sieve the thawed Alpinia oxyphylla obtained in step (2) to obtain powder; mix the powder with water in a material-liquid mass ratio of 1:11 and let it stand and soak for 2.4 hours, then place it in water vapor at a temperature of 230°C for distillation for 5.5 hours, collect the upper oily liquid, add anhydrous sodium sulfate and dry it to obtain Alpinia oxyphylla essential oil. According to the "Method A" of the essential oil determination method of the 2020 edition of the "Chinese Pharmacopoeia", the essential oil extraction rate is determined. Essential oil extraction rate (%) = (essential oil volume (mL) / plant powder mass (g)) × 100%, the measured Alpinia oxyphylla essential oil extraction rate is 2.10%
[0040] Comparative Example 1
[0041] This comparative example differs from Example 3 in that there is no freezing treatment and thawing treatment, a placing step is added, and the other steps and ingredients remain unchanged.
[0042] A preparation method of the essential oil of Qiqihar comprises the following steps:
[0043] The mature fruit of Alpinia oxyphylla was crushed and sieved to obtain powder; the powder was placed at 35±5℃ and relative humidity of 60-70% for 7 days, the powder was mixed with water at a material-liquid mass ratio of 1:10.3 and then allowed to stand and soak for 2.4 hours, then placed in water vapor at a temperature of 230℃ for distillation for 5.5 hours, the upper oily liquid was collected, and anhydrous sodium sulfate was added to dry to obtain Alpinia oxyphylla essential oil. According to the "Method A" of the essential oil determination method of the 2020 edition of the "Chinese Pharmacopoeia", the essential oil extraction rate was determined. Essential oil extraction rate (%) = (essential oil volume (mL) / plant powder mass (g)) × 100%, and the extraction rate of Alpinia oxyphylla essential oil was measured to be 1.71%.
[0044] Comparative Example 2
[0045] The difference between this comparative example and Example 3 is that there is no freezing treatment and thawing treatment, and the other steps and ingredients remain unchanged.
[0046] A preparation method of the essential oil of Qiqihar comprises the following steps:
[0047] The mature fruit of Alpinia oxyphylla was crushed and sieved to obtain powder; the powder was mixed with water at a material-liquid mass ratio of 1:10.3 and then allowed to stand and soak for 2.4 hours, then placed in water vapor at a temperature of 230°C for distillation for 5.5 hours, the upper oily liquid was collected, and anhydrous sodium sulfate was added to dry to obtain Alpinia oxyphylla essential oil. According to the "Method A" of the essential oil determination method of the 2020 edition of the "Chinese Pharmacopoeia", the essential oil extraction rate was determined. Essential oil extraction rate (%) = (essential oil volume (mL) / plant powder mass (g)) × 100%. The extraction rate of Alpinia oxyphylla essential oil was measured to be 1.45%.
[0048] Experimental Example 1 Antibacterial Activity Test
[0049] 1.1 Materials, strains, and reagents
[0050] Materials: the essential oils of Alpinia oxyphylla prepared in Examples 1 to 3 and Comparative Examples 1 to 2;
[0051] Bacteria: Bacillus cereus, Staphylococcus epidermidis, Pseudomonas fragi, Shigella flexneri, Salmonella enteritidis, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were purchased from Beijing Beina Chuanglian Biotechnology Research Institute;
[0052] Reagents: NA medium, MHA medium, LA medium, NB liquid medium, MHB liquid medium, LB liquid medium, ampicillin (AM), levofloxacin (LEV), etc. were purchased from Beijing Solebao Technology Co., Ltd.
[0053] 1.2 Experimental methods
[0054] 1.2.1 Preparation of bacterial suspension
[0055] Bacillus cereus, Staphylococcus epidermidis, Pseudomonas fragi, Shigella flexneri and Salmonella enteritidis preserved in glycerol were inoculated into NA medium, Bacillus subtilis, Staphylococcus aureus and Escherichia coli were inoculated into MHA medium, Pseudomonas aeruginosa was inoculated into LA medium, Bacillus cereus and Pseudomonas fragi were cultured at 30°C for 24 h, and the other seven strains were cultured at 37°C for 24 h. Single colonies were picked and inoculated into sterilized liquid culture medium for activation. NB liquid medium was used for Bacillus cereus, Staphylococcus epidermidis, Pseudomonas fragariae, Shigella flexneri and Salmonella enterica, MHB liquid medium was used for Bacillus subtilis, Staphylococcus aureus and Escherichia coli, and LB liquid medium was used for Pseudomonas aeruginosa. Then 200 μL of bacterial solution was inoculated into the sterilized liquid medium. The culture was placed in a constant temperature incubator at 30°C and 37°C and a speed of 90 r / min until the stable logarithmic growth phase. The concentration of the bacterial suspension was calculated using a turbidometer, and then the bacterial suspension was diluted to 1×10 6 ~2×10 6 cfu / mL bacterial suspension for later use.
[0056] 1.2.2 Inhibition zone determination method
[0057] Refer to Akarca's method and modify it. The inhibition zone was determined by agar filter paper diffusion method, that is, 100 μL of bacterial suspension of Bacillus cereus, Staphylococcus epidermidis, Pseudomonas fragariae, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica and Shigella flexneri was added to the cooled and solidified solid culture medium, and then evenly coated and left to stand for 5 minutes. 15 μL of Yiqi essential oil was added to the filter paper, and it was attached to the bacterial plate with sterile tweezers. The filter paper with sterile water was used as a negative control, 20 μg / mL ampicillin (AM) was used as a control drug for Staphylococcus aureus, and levofloxacin (LEV) was used as a control drug for the other 8 strains. Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica and Shigella flexneri were placed at 37°C, Bacillus cereus and Pseudomonas fragaria were placed at 30°C and inverted for 24 hours, and the presence or absence of the inhibition zone and the diameter were observed and recorded as the standard for judging the sensitivity. The diameter of each inhibition zone was measured with a vernier caliper, and 3 parallels were performed in each treatment group, and the average value was taken as the measurement result. The results were judged according to the standards of "Pharmacological Experimental Methodology": the diameter of the inhibition zone was less than 10mm for drug resistance, 10mm for mild sensitivity, 11-15mm for moderate sensitivity, and 16-20mm for high sensitivity.
[0058] 1.2.3 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
[0059] Refer to the method of Kanatt et al. and modify it. Use the micro-broth dilution method to add 100 μL of liquid culture medium of each strain to each well 1 to 11 in each row of a 96-well microplate, then dilute the Alpinia oxyphylla essential oil stock solution to 150 mg / mL with 1% DMSO and 2% Tween 80, add it to the first well, and dilute it to different concentrations in each well. Take 100 μL from the 10th well and discard it. This is the experimental group. 100 μL of the prepared bacterial suspension was added to the 1st to 11th wells in turn and mixed thoroughly. The 1st to 10th wells were the essential oil drug groups, and the essential oil concentrations were 37.50, 18.75, 9.38, 4.69, 2.34, 1.17, 0.59, 0.29, 0.15, and 0.07 mg / mL, respectively. No test drug was added to the 11th well to observe whether the culture medium was suitable for strain growth, which was the growth control group; 200 μL of TSB liquid culture medium was added to the 12th well to observe whether the culture medium was contaminated as the blank control group, and 1% DMSO and 2% Tween were treated in the same way as negative solvent controls. The 96-well plate was then placed in a 37°C constant temperature incubator for 24 hours, and the bacterial solution OD was measured using an enzyme marker after removal. 600nm. According to the American Clinical and Laboratory Standards Association Antimicrobial Susceptibility Test Implementation Standard M100-S25, the sample concentration with an average antibacterial rate of 80% in the experimental group compared with the blank control group is the MIC value of the tested bacteria. Using the agar medium plate method, 10 μL of bacterial suspension greater than MIC is drawn and applied to the corresponding area of the solid culture medium corresponding to each bacterial species. The corresponding culture is carried out for 24 hours, and the growth of the bacterial species is observed. The lowest drug concentration when the colony is ≤5 is the MBC value, and each treatment group has 3 parallels.
[0060] 1.3 Experimental Results
[0061] The experimental results are shown in Tables 1 to 3 and Figure 1 As shown:
[0062] Table 1 Average diameter of inhibition zone of different samples against bacteria
[0063]
[0064] Table 2 MIC of different samples against bacterial species
[0065]
[0066]
[0067] Table 3 MBC of different samples against bacterial species
[0068]
[0069] The results of Tables 1 to 3 show that the fruit essential oil (AOEO) extracted from Alpinia oxyphylla has a good antibacterial effect on foodborne Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, and Bacillus cereus, especially the lowest antibacterial concentration for Bacillus cereus, a foodborne pathogen that seriously endangers human health. Bacillus subtilis can be regarded as a beneficial bacteria, and the other three Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus cereus) are all harmful bacteria, especially Bacillus cereus, which is a foodborne pathogen with strong pathogenicity. However, the essential oil of Alpinia oxyphylla has no sensitivity to the Gram-negative bacteria in the drug sensitivity test. Except for Pseudomonas fraxinus, which has an antibacterial and bactericidal effect within the measured concentration range, the other four Gram-negative bacteria have no antibacterial and bactericidal effect within the measured concentration range. At the same time, it was also found that the effects of the Alpinia oxyphylla essential oils prepared by different preparation methods on various pathogenic bacteria were different. Different preparation methods would lead to different contents and purities of antibacterial components such as cymene, valenciaene, oleic acid ketone and γ-terpinene in the Alpinia oxyphylla essential oils. Efficient, pure and stable extraction methods can enable the Alpinia oxyphylla essential oils to retain more antibacterial components, which can continue to play a role in the antibacterial process, thereby improving the antibacterial properties of the Alpinia oxyphylla essential oils.
[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. An application of an essential oil for preparing an antibacterial agent capable of preventing and controlling foodborne pathogenic bacteria contamination, characterized in that: The Alpinia oxyphylla essential oil is a volatile oil component extracted from the traditional Chinese medicine Alpinia oxyphylla, and the foodborne pathogenic bacteria are Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas fragrans.
2. The use according to claim 1, characterized in that: The preparation method of the described Qiyi essential oil comprises the following steps: (1) Freezing treatment: taking ripe fruits of Alpinia oxyphylla and freezing them at a freezing temperature of -10°C to -2°C for 2 to 5 hours to obtain frozen Alpinia oxyphylla; (2) thawing treatment: adding water at a temperature of 70° C. to 90° C. to the frozen Alpinia oxyphylla obtained in step (1), soaking for 1 to 3 hours, draining the water, and obtaining thawing Alpinia oxyphylla; (3) Final treatment: the thawed Alpinia oxyphylla obtained in step (2) is crushed and sieved to obtain a powder; the powder is mixed with water and allowed to stand and soak for 2.2 to 2.5 hours, and then placed in water vapor for distillation for 5 to 7 hours, the upper oily liquid is collected, and anhydrous sodium sulfate is added to dry to obtain Alpinia oxyphylla essential oil.
3. The use according to claim 2, characterized in that: The freezing temperature in step (1) is -5°C.
4. The use according to claim 2, characterized in that: The freezing treatment time in step (1) is 2.5 hours.
5. The use according to claim 2, characterized in that: The mass ratio of the powder to water in step (3) is 1:(10.5-11.5).
6. The use according to claim 2, characterized in that: The soaking time in step (3) is 2.4 hours.
7. The use according to claim 2, characterized in that: The distillation temperature in step (3) is 215-240°C.
8. The use according to claim 7, characterized in that: The distillation temperature in step (3) is 230°C.
9. The use according to claim 2, characterized in that: The distillation time in step (3) is 5.5 h.
10. The use according to any one of claims 1 to 9, characterized in that: The AI fu essential oil can be used directly or prepared into a bacterial agent for use.