CN104544469A - Application of tea tree oil in inhibiting exotoxin secretion of staphylococcus aureus - Google Patents
Application of tea tree oil in inhibiting exotoxin secretion of staphylococcus aureus Download PDFInfo
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- CN104544469A CN104544469A CN201510025162.5A CN201510025162A CN104544469A CN 104544469 A CN104544469 A CN 104544469A CN 201510025162 A CN201510025162 A CN 201510025162A CN 104544469 A CN104544469 A CN 104544469A
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- staphylococcus aureus
- tea tree
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- exotoxin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3481—Organic compounds containing oxygen
Abstract
The invention discloses novel application of tea tree oil in inhibiting exotoxin secretion of staphylococcus aureus. The tea tree oil has broad-spectrum antibacterial, anti-fungal and antioxidant capacities; the minimum inhibitory concentration of the tea tree oil on the staphylococcus aureus is 8-32mL/L, and the taste and mouthfeel of some foods may be changed by the tea tree oil at the concentration. Upon hemolytic tests, the fact that the tea tree oil, at concentration of 0.125-0.5mL/L, can be used for inhibiting the secretion of exotoxin of staphylococcus aureus is discovered. When being combined with other staphylococcus aureus resisting substances as food preservatives, the tea tree oil can be used for effectively inhibiting the secretion of exotoxin of the staphylococcus aureus of the tea tree oil fails to effectively inhibit the growth of a drug-resistant staphylococcus aureus strain. The tea tree oil can be used for preventing poisoning caused by the staphylococcus aureus exotoxin, so that an additional safety guarantee is offered to prevent exotoxin poisoning. Meanwhile, a wider space is also provided in food conditioning by virtue of the tea tree oil.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to tea oil as the purposes suppressing the secretion of staphylococcus aureus exotoxin.
Background technology
Staphylococcus aureus belongs to gram-positive cocci, is distributed widely in nature, is the important pathogen causing purulent disease, is also a kind of significant bacterial causing food pollution and food posioning.After food pollutes by staphylococcus aureus, not only can be putrid and deteriorated, and also part bacterial strain produces golden yellow grape
Coccus enterotoxin and cause food poisoning, the food poisoning caused by Staphylococcus aureus enterotoxin accounts for the first place of whole food posioning.In recent years, in rising trend by the incidence of disease of caused by Staphylococcus aureus hospital infection, its drug resistance also increases year by year.Bacteriological infection has become the one of the main reasons of infectious diseases, and the health of human and animal has been arrived in serious threat, causes the very big concern of basis and clinical research.The exotoxin of staphylococcus aureus secretion mainly comprises and enterotoxin etc.The hemolysin of S. aureus L-forms secretion is one of key factor of its pathogenicity formation, and it is wherein common with alpha hemolysin, toxicity is the strongest, this toxin secretion can be combined by the most cells film such as red blood cell, endothelial cell, immunocyte in host to extracellular, is also extensively by the class virulence factor that people pay close attention to.Enterotoxin mainly refers to the outer strand small protein of born of the same parents that the class formation produced by staphylococcus aureus is under given conditions similar, virulence is similar and antigenicity is different, there is very high heat endurance, the superantigen activity had in addition, belong to superantigen proteins, non-specific T-cell can be stimulated to breed.
Tea oil, have another name called tea tree ethereal oil, it is the volatile oil obtained through steam distillation by the fresh branches and leaves of Myrtaceae melaleuca seeds narrow leaved tea tree, have antibacterial, fungal resistance and the oxidation resistance of wide spectrum, ministry of Health of China has listed tea oil in food flavor list in 2003.
The present invention adopts tea oil as the material suppressing the secretion of staphylococcus aureus exotoxin, lays the foundation for researching and developing novel staphylococcus aureus exotoxin food antiseptic.
Tea oil is the natural plant essential oils of a kind of anti-Staphylococcus aureus exotoxin secretion provided by the invention
summary of the invention:
The object of the invention is for solving staphylococcus aureus variation, drug resistance strengthens gradually, food preservative effectively can not suppress the growth of Staphylococcus aureus in food, and produce toxin, cause the problem of sitotoxismus, and a kind of inhibitor suppressing staphylococcus aureus exotoxin to be secreted is provided.
Tea oil suppresses the purposes of staphylococcus aureus exotoxin secretion.
A kind of food preservative, it comprises: tea oil and anti-Staphylococcus aureus element;
A using method for food preservative, tea oil addition is add 0.25-32 mL in every gram of food;
Described tea oil addition is add 0.25-3mL in every gram of food;
0.25-0.5mL is added in described tea oil addition every gram of food.
The invention provides the novelty teabag that tea oil suppresses the secretion of staphylococcus aureus exotoxin, the present inventor utilizes micro-dilution method Antibiotics resistance test, measure tea oil to the minimum inhibitory concentration (MIC) of staphylococcus aureus suspended bacteria and minimum bactericidal concentration (MBC), and then prove that tea oil is to staphylococcus aureus flcating germ inhibition.Measure the inhibitory action of tea oil to alpha hemolysin secretion in staphylococcus aureus by hemolytic experiment, in order to make experimental result have more convincingness, we are except Qualitative observations, can also by carrying out further quantitative assay to the mensuration of absorbance.In addition, protein immunoblot experiment can verify the fungistatic effect of tea oil further.Because enterotoxin can produce tumor necrosis factor TNF-alpha by stimulating expression of macrophage, so take indirect determination enterotoxin by measuring TNF-α.On gene level, utilize real-time RT-PCR technology pair
hla,
sea,
sebthe mechanism of action of tea oil suppression exotoxin secretion is probed in the investigation of three kinds of gene transcription levels, illustrates that tea oil has good effect to the ectotoxic secretion of suppression staphylococcus aureus.This test adopts staphylococcus aureus ATCC 29213 and food separation strain as experimental strain.
Tea oil is to antibacterial, fungal resistance and the oxidation resistance with wide spectrum, and tea oil is to staphylococcus aureus minimum inhibitory concentration at 8-32mL/L, and the existence of this concentration tea oil may change taste and the mouthfeel of some food.We find in hemolytic experiment, the (see figure 1) when 0.125-0.5mL/L, inhibit the ectotoxic secretion of staphylococcus aureus.Tea oil coordinates as food preservative with other anti-Staphylococcus aureus element, when effectively not suppressing resistant Staphylococcus aureus strain growth, effectively can suppress the ectotoxic secretion of staphylococcus aureus.Prevent staphylococcus aureus exotoxin poisoning.For prevention exotoxin is poisoning, many safety guarantee.Also be simultaneously in food modulation, use tea oil to provide more wide space.
Accompanying drawing explanation
Fig. 1 tea oil is on the experimental result of alpha hemolysin haematolysis ability impact in staphylococcus aureus;
Fig. 2 tea oil is to the inhibiting experimental result of staphylococcus aureus China and foreign countries' toxin secretion;
The experimental result of Fig. 3 tea oil Tumor suppression necrosin release;
Fig. 4 tea oil is to the inhibiting experimental result of related gene expression.
Detailed description of the invention
embodiment 1
Micro-dilution method Antibiotics resistance test-tea oil measures the minimum inhibitory concentration (MIC) of staphylococcus aureus suspended bacteria and minimum bactericidal concentration (MBC): picking list bacterium colony from the staphylococcus aureus agar plate bacterial strain preserved, and by colony inoculation in Muller-Hinton meat soup (MHB) culture medium, in 37 DEG C of concussion incubated overnight.Bacterial concentration MHB culture medium is adjusted to OD
600value is equivalent to 5 × 10 for 0.4(
8and be diluted to 1 × 10 further CFU/ml),
6cFU/ml.With MHB culture medium dilution tea oil liquid, liquid final concentration is made to be 256 mg/ml.Add often ranked first in hole of aseptic 96 hole microtiter plates the liquid that 200 μ l final concentrations are 256 mg/ml, in the 2nd hole to the 10th hole, add 100 μ l MHB culture mediums.Performing in the operation of often arranging, from first hole of often arranging, shifting out 100 μ l concentration with pipettor is that the liquid of 256 mg/ml joins in the 2nd hole of row separately and carries out dilution and mix, and then join the 3rd hole from the solution that the 2nd hole sucking-off 100 μ l concentration is 128 mg/ml, by that analogy, until the 10th hole of often arranging, often dilute mixing one hole and change a rifle head.Add 200 μ l MHB culture mediums as negative control often arranging in the 11st hole, add bacterium liquid that 100 μ l MHB culture mediums and 100 μ l have diluted as normal growth control wells often arranging in the 12nd hole.Add the dilution bacterium liquid that 100 μ l mix up often ranked first the 10th Zhong Mei hole, hole again.So often ranked first hole to be respectively to the 10th hole herb liquid concentration: 128 mg/ml, 64 mg/ml, 32 mg/ml, 16 mg/ml, 8 mg/ml, 4 mg/ml, 2 mg/ml, 1 mg/ml, 0.5mg/ml, 0.25 mg/ml, in each hole, DMSO content is all less than 1 %.96 hole microtiter plates are placed in 37 DEG C of incubators and hatch 24 h.Its OD value is measured, the growing state of bacterial detection by ELIASA (λ=660 nm).MIC is the lowest concentration of drug of energy bacteria growing inhibiting.Experiment in triplicate, calculates MIC.Draw 10 μ l liquid inoculations from the first hole to MIC hole on MHA flat board, in 37 DEG C of incubators, hatch 24 h.Observed result, the lowest concentration of drug having no thalli growth in inoculation flat board is
mBC.Tea oil is to staphylococcus aureus suspended bacteria (be shown in table 1)
embodiment 2
tea oil is on the mensuration of alpha hemolysin haematolysis ability impact in staphylococcus aureus:picking list bacterium colony from the staphylococcus aureus agar plate bacterial strain preserved, and by colony inoculation in Muller-Hinton meat soup (MHB) culture medium, in 37 DEG C of concussion incubated overnight.Bacterial concentration pancreas peptone soybean broth culture medium (TSB) is adjusted to OD
600value is 0.05, then continues to be cultured to OD
600value reaches 0.3, and start sub-bottle dosing, the bacterium liquid by same volume is divided in the triangular flask of five 100 ml, and adds the tea oil medicine storage liquid of variable concentrations, makes its final concentration be 1/128MIC ~ 1/16MIC(0.0625 ~ 0.5 mg/ml).Continue the bacterium liquid cultivating various different pharmaceutical concentration gradient (comprising blank group of not dosing), make it all to reach logarithmic growth later stage (OD
600nm=2.5), namely in blank group and other each dosing groups, the sum of bacterium is identical, and this in period bacterium secreted exotoxin in supernatant.By centrifugal for bacterium liquid 4 DEG C, with centrifugal 1 min of the rotating speed of 5500 r/min.After taking out supernatant, add benzene mesyl fluoride wherein, its final concentration is 0.025mM.Then the supernatant adding benzene mesyl fluoride is placed in-20 DEG C, refrigerator preserve and for subsequent use.Thoroughly wash the rabbit blood of the fresh defiber of taking-up with hemolysin buffer solution, then centrifugally become clarification, with 2500 r/min rotating speeds, till centrifugal 2 ~ 5 min to supernatant.The hemolysin buffer solution of 875 μ l is joined in the supernatant samples of 100 μ l different pharmaceutical concentration process and also mix gently; 25 μ l defiber rabbit blood are joined in 100 μ l culture medium negative controls, mixes gently.Then both will be placed in and be placed on incubator hatch half an hour at 37 DEG C.After half an hour, take out, at room temperature centrifugal about 1 min of the centrifugal rotating speed with 6000 r/min, finally measures the light absorption value of supernatant under 450 nm conditions.Can obviously find out from qualitative results, along with the increase of tea oil drug concentration, in each centrifuge tube, the color of liquid reduces gradually, and the generation of Drug inhibition haemolysis is described, namely inhibits the secretion of alpha hemolysin.From quantitative experimental result, when tea oil drug concentration increases, under the condition that total number of bacteria is identical, its absorbance reduces gradually, illustrate that haemolysis situation obviously weakens, further demonstrate the suppression (see figure 1) that the secretion of alpha hemolysin in bacterium receives medicine.
embodiment 3
Western blot analysis (western blotting)-tea oil is to the inhibiting mensuration of staphylococcus aureus China and foreign countries' toxin secretion: the preparation first carrying out sample.From the staphylococcus aureus agar plate bacterial strain preserved, be inoculated in TSB culture medium, 37 DEG C of concussions are cultivated.Bacterial concentration is regulated to make its OD
600reach 0.05, then continue at 37 DEG C of concussion cultivations.As its OD
600when reaching 0.3, start sub-bottle dosing, the bacterium liquid by same volume is divided in the triangular flask of 5 100 ml, and adds the tea oil medicine storage liquid of variable concentrations, makes its final concentration be 1/128MIC ~ 1/16MIC(0.0625 ~ 0.5 mg/ml).Cultivate the bacterium liquid of various different pharmaceutical concentration gradient (comprising blank group of not dosing), make it all to reach logarithmic growth later stage (OD
600nm=2.5), namely in blank group and other each dosing groups, the sum of bacterium is identical, and this in period bacterium secreted exotoxin in supernatant.After collecting bacterium supernatant, add appropriate trichloroacetic acid, overnight precipitation.Next day, with centrifugal 30 min of the rotating speed of 8500 r/min, abandons supernatant.Add appropriate ice ethanol washing again, again centrifugal, 8500 r/min, 30 min.Discard ice ethanol, after oven dry, with Tris buffer solution, precipitation is dissolved.Sample is made for subsequent use after mixing with sample-loading buffer loading buffer.The concrete test method of protein immunoblot: the preparation of a. SDS-PAGE gel: by formulated separation gel.After abundant mixing, the separation gel of ot-yet-hardened is poured in the glass plate clipped in advance, fill with 4/5 to glass plate, after having added separation gel, then use deionized water fluid-tight.After gelling to be separated is solid, start the concentrated glue preparing 5%.Glue is concentrated according to formulated.The concentrated glue prepared is poured into separation gel upper strata, to full.After inserting the comb of 1.5mm, after leaving standstill about 20min, concentrated gelling is solid.B. electrophoresis: get 24 μ l samples, add sample in the sample well that concentrated glue is formed, after being connected by supply unit, starts to carry out electrophoresis, constant voltage 120V.After about 2h, complete electrophoresis.C. transferring film: shear out two pieces of threeply filter paper, one piece of pvdf membrane, size needs consistent with separation gel.During migration: adopt half dry type transferring film instrument to carry out transferring film.According to the order of filter paper → pvdf membrane → glue → filter paper, filter paper, glue, pvdf membrane are put into transferring film instrument, after alignment, fully drive bubble away, after covering transferring film instrument upper cover, start transferring film.Constant voltage 15V, transferring film 40min.D. close: be put in confining liquid after being taken out by pvdf membrane, room temperature closes 2h, and horizontal shaker slowly rocks.E. primary antibodie is hatched: antibody dilutes by instruction confining liquid to specifications.According to 1:10000 dilution proportion alpha hemolysin; According to 1:8000 dilution proportion enterotoxin A; According to 1:5000 dilution proportion enterotoxin B.4 DEG C are spent the night.F. wash film: next day, wash film twice with TBST solution, each 10min; Use TBS solution again instead and wash film once, 10min.G. immune response (hatching two to resist): according to the ratio of 1:5000, HRPO is marked goat anti-rabbit igg antibody (namely two resist) and dilute with confining liquid.Incubated at room 1h.H. film is washed: TBST solution washes film twice, each 10min; Use TBS solution again instead and wash film once, 10min.I. ECL luminescent solution carries out chemical development.Experimental result shows, and when tea oil drug concentration increases, when total number of bacteria is identical, in bacterium, ectotoxic secretion is suppressed, and its secretory volume reduces gradually.Illustrate tea oil for enterotoxin SEA, SEB in staphylococcus aureus and and the secretion of alpha hemolysin have good inhibitory action (see figure 2).
embodiment 4
The mensuration of the experiment-cytokine TNF-α of tea oil Tumor suppression necrosin release: due to the generation of tumor necrosis factor TNF-alpha in the secretion meeting stimulating expression of macrophage of enterotoxin in staphylococcus aureus, so carried out the secretion of indirect proof enterotoxin by the mensuration of TNF-α.ATCC 29213 and food separation strain JL-20110 two strain bacterium is adopted to test in this experiment.Concussion of spending the night in 37 ° of C constant-temperature tables is cultivated and is increased to exponential phase to bacterium, i.e. OD
600nm=2.5.By at the bacterium liquid 37 DEG C that diluted, 1h cultivated by shaking table.Bacterium liquid is placed in 5 conical flasks with every bottle of 15ml respectively, according to the dosing of different pharmaceutical concentration gradient, i.e. 1/128MIC ~ 1/16MIC(0.0625 ~ 0.5 mg/ml), cultivate in 37 DEG C of constant-temperature tables, after 4h, with filter, filtrate is filtered, collect filtrate.-20 DEG C save backup.After cell Raw 264.7 is cultured to some, plating cells.In 96 orifice plates, add 200 μ L containing cell culture medium, make every porocyte number be no less than 10
5individual.After cell attachment, in the cell of 96 orifice plates, add bacterium liquid according to variable concentrations gradient, after 24h, dull and stereotyped centrifugal, 1000 r/min, 10 min.After centrifugal, get supernatant ,-20 DEG C save backup.ELISA method measures TNF-alpha content.Determine the number in hole used.1 × wash buffer washes plate twice, and after patting dry, gauge orifice and blank well add 100 μ L sample diluting liquids, and sample sky adds 50 μ L sample diluting liquids and 50 μ L samples.Add 100 μ L standard items in gauge orifice, doubling dilution is to last hole.Again porose in add 50 μ L 1 × Assay buffer dilute Biotin-Conjugate.Post sealer, at 18 ~ 25 DEG C, leave standstill 2h.1 × wash buffer washes plate three times, after patting dry, adds the HRP that 100 μ L 1 × Assay buffer have diluted.Stick sealed membrane, at 18 ~ 25 DEG C, leave standstill 1h.1 × wash buffer washes plate three times, after patting dry, adds 100 μ L Substrate Solution.37 DEG C of lucifuges place 10min.100 μ L stop buffers are added rapidly after taking-up.Absorbance is surveyed under 450nm.Experimentally result is known, and along with the increase of tea oil drug concentration, TNF-α burst size reduces (see figure 3).
embodiment 5
The enterotoxin SEA that real time fluorescent quantitative RT-PCR (real-time RT-PCR tests)-tea oil is secreted staphylococcus aureus, the impact of SEB and alpha hemolysin related gene expression: from the staphylococcus aureus agar plate bacterial strain preserved, be inoculated in TSB culture medium, 37 DEG C of concussions are cultivated.Bacterial concentration is regulated to make its OD
600reach 0.05, then continue at 37 DEG C of concussion cultivations.As its OD
600when reaching 0.3, start sub-bottle dosing, the bacterium liquid by same volume is divided in the triangular flask of 5 100 ml, and adds the tea oil medicine storage liquid of variable concentrations, makes its final concentration be 1/8MIC ~ 1/2MIC(0.25 ~ 1 μ g/ml).The bacterium liquid (comprising blank group of not dosing) through the process of different pharmaceutical concentration is cultivated in concussion of spending the night in 37 DEG C of constant-temperature tables, until bacterium increases to exponential phase, i.e. and OD
600nm=2.0, make each group of total number of bacteria identical.Collected after centrifugation bacterium mud, saves backup.In bacterium mud, add 0.5 ml sample dissociation liquid, fully add 0.5 ml sample dissociation liquid again after mixing, after vortex, leave standstill 10 min.Add 200 μ l trichloroacetic acids, after placing 15 min, centrifugal, 12000 r/min, 15 min, 4 DEG C.Get supernatant, add 500 μ l isopropyl alcohols, rock after leaving standstill again centrifugal gently, 12000 r/min, 15 min, 4 DEG C.1 ml 75% ethanol is added in precipitation, centrifugal.RNA concentration is surveyed after finally carrying out sample dissolution with 60 μ l DEPC water.Reactant ligand system to specifications, makes RNA template, carries out reversing.Pcr amplification is carried out to specifications after reversion.By the display of real-time RT-PCR result, increase with tea oil drug-treated concentration, under the condition that total number of bacteria is identical, tea oil can suppress the expression (Fig. 4) of exotoxin related gene.
Can obviously find out from (Fig. 1) qualitative results, along with the increase of tea oil drug concentration, in each centrifuge tube, the color of liquid reduces gradually, and the generation of Drug inhibition haemolysis is described, namely inhibits the secretion of alpha hemolysin.From quantitative experimental result, when tea oil drug concentration increases, under the condition that total number of bacteria is identical, its absorbance reduces gradually, illustrates that haemolysis situation obviously weakens, and further demonstrates the secretion of alpha hemolysin in bacterium and receives the suppression of medicine.Experimental result (Fig. 2) shows, and when tea oil drug concentration increases, when total number of bacteria is identical, in bacterium, ectotoxic secretion is suppressed, and its secretory volume reduces gradually.Illustrate tea oil for enterotoxin SEA, SEB in staphylococcus aureus and and the secretion of alpha hemolysin have good inhibitory action.Experimentally result (Fig. 3) is known, and along with the increase of tea oil drug concentration, in ATCC 29213 and food separation strain JL-20110 two groups experiment, TNF-α burst size reduces.By real-time RT-PCR result (Fig. 4) display, increase with tea oil drug-treated concentration, under the condition that total number of bacteria is identical, tea oil can suppress the expression of exotoxin related gene.
Claims (5)
1. tea oil suppresses the purposes of staphylococcus aureus exotoxin secretion.
2. a food preservative, it comprises: tea oil and anti-Staphylococcus aureus element.
3. a using method for food preservative, is characterized in that: tea oil addition is addition is add 0.25-32 mL in every gram of food.
4. the using method of a kind of food preservative according to claim 3, is characterized in that: described tea oil addition is add 0.25-3mL in every gram of food.
5. the using method of a kind of food preservative according to claim 4, is characterized in that: the addition of described tea tree tea oil is addition is add 0.25-0.5mL in every gram of food.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999013892A1 (en) * | 1997-09-12 | 1999-03-25 | Laboratorios Pisa, S.A. De C.V. | Antimastitic pharmaceutical composition and preparation process |
| US6534548B1 (en) * | 2001-10-02 | 2003-03-18 | Kimberly-Clark Worldwide, Inc. | Isoprenoid compositions for the inhibition of exoprotein production from gram positive bacteria |
| CN101437413A (en) * | 2006-05-12 | 2009-05-20 | 可口可乐公司 | Beverage preservatives |
| CN102469830A (en) * | 2009-09-08 | 2012-05-23 | 马斯公司 | Composition for prevention against spoilage by moulds and yeasts and uses and products related thereto |
| CN102845512A (en) * | 2012-07-28 | 2013-01-02 | 海南大学 | Water-soluble tea tree oil/chitosan antibacterial compound |
-
2015
- 2015-01-19 CN CN201510025162.5A patent/CN104544469A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999013892A1 (en) * | 1997-09-12 | 1999-03-25 | Laboratorios Pisa, S.A. De C.V. | Antimastitic pharmaceutical composition and preparation process |
| US6534548B1 (en) * | 2001-10-02 | 2003-03-18 | Kimberly-Clark Worldwide, Inc. | Isoprenoid compositions for the inhibition of exoprotein production from gram positive bacteria |
| CN101437413A (en) * | 2006-05-12 | 2009-05-20 | 可口可乐公司 | Beverage preservatives |
| CN102469830A (en) * | 2009-09-08 | 2012-05-23 | 马斯公司 | Composition for prevention against spoilage by moulds and yeasts and uses and products related thereto |
| CN102845512A (en) * | 2012-07-28 | 2013-01-02 | 海南大学 | Water-soluble tea tree oil/chitosan antibacterial compound |
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| 于丹丹: "茶树精油在果实采后保鲜中的作用", 《果树学报》 * |
| 王懿: "茶树油抗菌活性的实验研究`", 《医药导报》 * |
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Inventor after: Guo Na Inventor after: Shi Ce Inventor after: Zhao Ziwen Inventor after: Yu Lu Inventor after: Meng Rizeng Inventor after: Zhao Xingchen Inventor after: Liu Zonghui Inventor after: Li Xiaoxu Inventor after: Zhu Xuanzhi Inventor before: Guo Na Inventor before: Shi Ce Inventor before: Zhao Ziwen Inventor before: Yu Lu Inventor before: Meng Rizeng Inventor before: Zhao Xingchen Inventor before: Liu Zonghui Inventor before: Li Xiaoxu Inventor before: Zhu Xuanzhi |
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