CN113969300A - Eucalyptus leaf composite active extract process and antibacterial application thereof - Google Patents

Eucalyptus leaf composite active extract process and antibacterial application thereof Download PDF

Info

Publication number
CN113969300A
CN113969300A CN202010718542.8A CN202010718542A CN113969300A CN 113969300 A CN113969300 A CN 113969300A CN 202010718542 A CN202010718542 A CN 202010718542A CN 113969300 A CN113969300 A CN 113969300A
Authority
CN
China
Prior art keywords
fresh
eucalyptus
protein polypeptide
leaves
hydrolysate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010718542.8A
Other languages
Chinese (zh)
Inventor
李钧翔
陈成
陆益
康思宁
周碧柳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Enlightenment Hemei Biotechnology Jiaxing Co ltd
Original Assignee
Enlightenment Hemei Biotechnology Jiaxing Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Enlightenment Hemei Biotechnology Jiaxing Co ltd filed Critical Enlightenment Hemei Biotechnology Jiaxing Co ltd
Priority to CN202010718542.8A priority Critical patent/CN113969300A/en
Publication of CN113969300A publication Critical patent/CN113969300A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/28—Myrtaceae [Myrtle family], e.g. teatree or clove
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

Abstract

The application relates to a method for extracting eucalyptus leaf with compound activity and antibacterial application thereof, which takes widely planted eucalyptus globulus leaves in China as raw materials, develops a method for compounding aqueous phase extract micromolecule peptide, proves good safety and antibacterial effect through experimental evidence, and has wide application prospect in natural and safe antibacterial products.

Description

Eucalyptus leaf composite active extract process and antibacterial application thereof
Technical Field
The invention relates to the field of plant extract antibiosis, and particularly relates to an extraction method of a eucalyptus leaf composite active extract and related application thereof.
Background
The epidemic situation of the new coronary pneumonia is mainly spread through droplets, and in order to avoid spreading of the epidemic situation and protect medical staff and the public, environmental disinfection is very important for controlling the spread of pathogenic bacteria, so that the selection of a proper disinfectant is very necessary.
The environmental disinfectants which are mainly used at present are chemical disinfectants represented by chlorine-containing disinfectants. The chemical environmental disinfectants have certain safety problems in the using process, such as bad smell of most environmental disinfectants; the chlorine-containing disinfectant can release toxic gas chlorine when being stored, and the high-concentration chlorine can be mixed with combustible gases such as acetylene, hydrogen and the like to cause explosion or fire; meanwhile, the chlorine-containing disinfectant is easy to cause corrosive damage to the skin of a person due to the problem of excessive concentration in use and potential food safety risks caused by the residual of partial food surfaces; in addition, the excessive use of the disinfectant containing chlorine can acidify the air, induce various respiratory diseases and reduce the immunity of the human body.
Therefore, the natural extract components of the environmental plant source with strong disinfection and sterilization effects, high safety and pleasant smell are screened and optimized, and the natural and safe antiviral and antibacterial spray product is developed, so that the natural and safe antiviral and antibacterial spray product is necessary for improving the working biological environments of medical workers and people in epidemic situations and relieving the anxiety mood of patients.
Eucalyptus (Eucalyptus) is a plant of the Myrtaceae family (Mytaceae) and contains the genera Eucalyptus (Eucalyptus), California (Angophora) and Umbellium (Corymbra). It is one of the highest angiosperm plants in the earth, and has various varieties, known varieties of which are 945 varieties; the planting area is wide, and more than 50 countries in the world are planted; eucalyptus has a very high economic value, is an excellent tree species with the functions of appreciation, material utilization and medicine, and people use the artificially planted eucalyptus in the aspects of furniture manufacture, building engineering, paper pulp raw materials and the like. The eucalyptus trunks can be used as materials for bridges, buildings, pulp, artificial boards and the like; the eucalyptus bark can be used as a boiler descaling retarder and a green new energy; the eucalyptus leaves can be used for producing essential oil, citral, eucalyptol, etc. In addition, eucalyptus bark and leaves can be used as medicinal materials for treating diarrhea, fever, toothache, etc.
The eucalyptus species include Eucalyptus globulus Labill, Eucalyptus robusta, Eucalyptus smithii, Eucalyptus robusta, Eucalyptus lobutanus, and Eucalyptus globulus Labill. The Eucalyptus globulus Labill and Eucalyptus robusta are main varieties for extracting eucalyptus oil. The eucalyptus oil contains eucalyptol, is colorless or yellowish liquid, has pungent and refreshing fragrance, and is mainly used in toothpaste, mouthwash, food and medicine. The eucalyptus globulus and the straight-rod eucalyptus globulus are excellent in species, and the eucalyptus oil is extracted from branches and leaves of the eucalyptus globulus, so that the quality is optimal.
Wherein, the origin of the Eucalyptus globulus Labill is imported in Australia, and then cultivated in Guangxi, Yunnan, Sichuan and other places in large quantities, so that China has rich Eucalyptus globulus Labill resources. The eucalyptus globulus is an ideal plant raw material with rapid growth and low cost, and long-term practice shows that the eucalyptus globulus leaf water-phase extract has obvious insecticidal, disinfection and sterilization effects. The Takahashi team reports that eucalyptus varieties of e.globulus, e.macular a and e.viminalis have good inhibition effects on staphylococcus aureus, bacillus cereus, fecal escherichia coli, acnes acetonide, escherichia coli, trilobate, and the like; the Aya Abu-Jafar group uses eucalyptus varieties of E camaldulensis, E camaldulensis (Dehn) and E torelliana (R.Muell) as materials, and proves that the related eucalyptus varieties have obvious inhibition effects on herpes simplex virus, varicella-zosterit virus and human enterovirus (poliovirus I, coxsackie virus, echovirus and the like).
In the research process, a large amount of bioactive components are found in the eucalyptus leaves, and the eucalyptus leaves are mainly identified to contain eucalyptus oil, flavonoids, organic acids, tannins, phenols, amino acids and protein compounds. The components have good effects in resisting bacteria, killing parasite, resisting inflammation, regulating plant, etc. For example: the inclusion jade team tests the nutrient content, the amino acid content, the trace elements and the like of 25 varieties of eucalyptus leaves in China, and researches show that each eucalyptus leaf contains 9% of crude protein. However, according to the situation of the currently published literature, the antibacterial activity of proteins, particularly small molecular peptides, in eucalyptus leaves has not been reported, and the antibacterial activity of hydrolyzed polypeptides in eucalyptus leaves has not been studied systematically in the literature.
In addition, methods for extracting eucalyptus active substances are various, and mainly include solvent extraction, ultrasonic extraction, microwave extraction, Supercritical Fluid Extraction (SFE) techniques, and the like. However, these methods have problems, or the method has strong limitations (the extract is easily decomposed and oxidized to thermally unstable effective components), or the cost is high (supercritical fluid extraction), and when the active ingredients of eucalyptus leaves are extracted, the extraction method should be adjusted sufficiently according to the property and structure of the extracted ingredients in order to obtain the best effect.
In the patent, the inventor takes the eucalyptus globulus leaves widely planted in China as a raw material, develops a technical route of a composite aqueous phase extract small molecular peptide part, adopts a comprehensive extraction method of filtration, alcohol extraction and enzymolysis, particularly selects a hydrolysis peptide mixture with a certain molecular weight, and proves that the hydrolysis peptide mixture has good safety and antibacterial effect through experiments, and has wide application prospect in natural and safe antibacterial products.
Disclosure of Invention
The invention relates to a method for extracting a composite active extract of eucalyptus leaves and related eucalyptus leaf polypeptide hydrolysate, in some embodiments, a fresh eucalyptus leaf protein polypeptide hydrolysate, which is obtained by the steps of:
1) washing fresh picked leaves of Eucalyptus globulus Labill, removing impurities, sieving and crushing to 40-100 meshes, placing in 0.4-1% sodium chloride solution according to the material-liquid ratio of 1: 6-12, and extracting in 80 ℃ water bath for 4-6 h;
2) concentrating the obtained crude extract of fresh folium Eucalypti Globueli to original concentration of 0.5-1.2g/ml after filtering under reduced pressure;
3) extracting a crude extract of fresh eucalyptus leaves by using 80% ethanol at room temperature, and then freeze-drying residues obtained after ethanol extraction to obtain a crude protein raw material of the fresh eucalyptus leaves;
4) carrying out enzymolysis on a fresh eucalyptus leaf protein raw material to obtain a fresh eucalyptus leaf protein polypeptide hydrolysate;
5) further optimizing the antibacterial polypeptide components of the eucalyptus by ultrafiltering the protein polypeptide hydrolysate of the fresh eucalyptus leaves; filtering fresh folium Eucalypti Globueli protein polypeptide hydrolysate with membrane, and separating with cellulose membrane of 4kDa (Mw) to obtain active extracts with different components with molecular weight of Mw less than 4kDa and Mw greater than 4kDa, wherein the component with Mw less than 4kDa is the fresh folium Eucalypti Globueli protein polypeptide hydrolysate.
In one aspect, the enzymatic hydrolysis step of step 4 is as follows:
hydrolyzing fresh folium Eucalypti Globueli protein material at 37 deg.C with pepsin (pH 2.0) and trypsin (pH8.0) for 3 hr; the concentration of pepsin and trypsin is 5% (w/w), the enzyme is inactivated in boiling water for 10 min, then centrifuged at 9000g for 10 min at 4 ℃, and the soluble part of the supernatant is subjected to secondary enzymolysis by pepsin and trypsin and freeze-dried to obtain the fresh eucalyptus leaf protein polypeptide hydrolysate.
In another aspect, the membrane pore size in the membrane filtration of step 5 is 0.22 μm.
In other embodiments, the present application relates to a method for extracting a composite active extract of eucalyptus leaves, comprising the steps of:
1) washing fresh picked leaves of Eucalyptus globulus Labill, removing impurities, sieving, pulverizing into 40-100 mesh, placing into 0.4% -1% sodium chloride solution according to the material-liquid ratio of 1: 6-12, and extracting in 80 deg.C water bath for 4-6 h;
2) concentrating the obtained crude extract of fresh folium Eucalypti Globueli to original concentration of 0.5-1.2g/ml after filtering under reduced pressure;
3) extracting a crude extract of fresh eucalyptus leaves by using 80% ethanol at room temperature, and then freeze-drying residues obtained after ethanol extraction to obtain a crude protein raw material of the fresh eucalyptus leaves;
4) carrying out enzymolysis on a fresh eucalyptus leaf protein raw material to obtain a fresh eucalyptus leaf protein polypeptide hydrolysate;
5) further optimizing the antibacterial polypeptide components of the eucalyptus by ultrafiltering the protein polypeptide hydrolysate of the fresh eucalyptus leaves; fresh eucalyptus leaf protein polypeptide hydrolysate is subjected to membrane filtration and then separated by a cellulose membrane of 4kDa (Mw) to obtain active extracts of different fractions having molecular weights Mw < 4kDa and Mw > 4kDa, and the fractions having Mw < 4kDa are selected for further activity verification tests.
In one aspect, the enzymatic hydrolysis step of step 4 is as follows:
hydrolyzing fresh folium Eucalypti Globueli protein material at 37 deg.C with pepsin (pH 2.0) and trypsin (pH8.0) for 3 hr; the concentration of pepsin and trypsin is 5% (w/w), the enzyme is inactivated in boiling water for 10 min, then centrifuged at 9000g for 10 min at 4 ℃, and the soluble part of the supernatant is subjected to secondary enzymolysis by pepsin and trypsin and freeze-dried to obtain the fresh eucalyptus leaf protein polypeptide hydrolysate.
In another aspect, the membrane pore size in the membrane filtration of step 5 is 0.22 μm.
In other embodiments, the application relates to the use of the fresh eucalyptus leaf protein polypeptide hydrolysate in the preparation of an antibacterial agent.
In other embodiments, the application relates to an antibacterial agent comprising said fresh eucalyptus leaf protein polypeptide hydrolysate.
Drawings
FIG. 1: directly inhibiting bacteria according to the experimental result; FIG. 2: results of drug susceptibility test
FIG. 3: punching test results; FIG. 4: antibacterial experimental results on mask
Detailed Description
Example 1 separation and extraction Process
1. Washing fresh picked Eucalyptus globulus Labill leaves, removing impurities, sieving, pulverizing into 40-100 mesh, placing into 0.4% -1% sodium chloride solution at a material-liquid ratio of 1: 6-12(g/ml), and extracting in 80 deg.C water bath for 4-6 hr;
2. filtering under reduced pressure, and concentrating the crude extract of fresh folium Eucalypti Globueli to give crude drug concentration of 0.5-1.2 (g/ml);
3. 1 kg of fresh eucalyptus leaf crude extract was extracted with 10L of 80% ethanol (v/v) at room temperature for 48 hours. And freeze-drying the residue after ethanol extraction to obtain a crude protein raw material of fresh eucalyptus leaves.
4. Fresh eucalyptus leaf protein material was hydrolyzed using pepsin (pH 2.0) and trypsin (pH8.0) at 37 ℃ for 3 hours (pH was adjusted prior to use of pepsin and trypsin in succession). The concentration of pepsin and trypsin is 5% (w/w), the enzyme is inactivated in boiling water for 10 minutes, then centrifuged at 9000g for 10 minutes at 4 ℃, and the soluble part of the supernatant is subjected to enzymolysis again by pepsin and trypsin and then freeze-dried to obtain the fresh eucalyptus leaf protein polypeptide hydrolysate.
5. The eucalyptus antibacterial polypeptide components (AMPs) are further optimized by ultrafiltration of the fresh eucalyptus leaf protein polypeptide hydrolysate. Fresh eucalyptus leaf protein polypeptide hydrolysate was filtered through a 0.22 μm membrane and separated using a 4kDa (Mw) cellulose membrane. Obtaining different fractions with molecular weight Mw < 4kDa and Mw > 4kDa, selecting the fraction with Mw < 4kDa, and freeze-drying. Before use, sterile PBS is used for redissolving according to the mass ratio of 1: 10, and the components are used for detecting the antibacterial activity of the components on escherichia coli.
Example 2: efficacy detection method
First, direct inhibition experiment of bacteria
The main reagents are as follows: sterile water, fresh eucalyptus leaf protein polypeptide hydrolysate (Mw < 4kDa), double antibody (penicillin 100ug/ml, streptomycin 50ug/ml), MH culture medium (beef powder 2.0g/L, soluble starch 1.5g/L, acid hydrolyzed casein 17.5g/L, pH 7.5), Escherichia Coli E-Coli MG1655 (Qinghua Bonus gift) (2 Tsuan 10 Boyu university)8one/mL);
2. diluting Escherichia coli by 1000 times, and mixing 100uL of bacteria solution with 100uL of sterile water/fresh folium Eucalypti Globueli protein polypeptide hydrolysate/double-antibody mixture;
3. uniformly coating 200 mu L of bacterial liquid on an MH culture medium, and waiting for drying;
4. the mixture was placed upside down in an incubator at 37 ℃ for 24 hours, and the results were observed.
Results of the bacterial direct inhibition experiments show that (see fig. 1): fresh eucalyptus leaf protein polypeptide hydrolysate and double-antibody-free (E-Coli MG1655) bacterial colony, and sterile water content is more than 100 cfu/mL; the fresh eucalyptus leaf protein polypeptide hydrolysate obtained by the application has a good growth inhibition effect on bacteria.
Second, drug sensitivity test
1. Preparing blank drug sensitive tablets (Hangzhou microbial agent Co., Ltd., product number: S1099), fresh folium Eucalypti Globueli crude protein material, fresh folium Eucalypti Globueli protein polypeptide hydrolysate (Mw less than 4kDa), double antibody (penicillin 100ug/ml, streptomycin 50ug/ml), 75% alcohol, MH culture medium (beef powder 2.0g/L, soluble starch 1.5g/L, acid hydrolyzed casein 17.5g/L, pH 7.5), Escherichia Coli E-Coli MG1655 (present in Qinghua university) 2 T108Per mL;
2. diluting Escherichia coli by 100 times, uniformly coating 300 μ L of bacterial liquid on MH culture medium, and air drying;
3. soaking the blank drug sensitive tablet in fresh eucalyptus leaf protein polypeptide hydrolysate, and drying in a 50 ℃ oven for later use;
4. repeating the step 3 to prepare the crude protein raw material of fresh eucalyptus leaves and the drug sensitive tablets of double-resistant alcohol with 75 percent;
5. sticking the blank drug sensitive tablet on the culture medium, slightly pressing, and sticking the double-resistant drug sensitive tablet, the fresh eucalyptus leaf protein polypeptide hydrolysate drug sensitive tablet and the 75% alcohol drug sensitive tablet by the same method;
5. the mixture was placed upside down in an incubator at 37 ℃ for 24 hours, and the results were observed.
The results of the drug sensitivity test (drug sensitivity patch method) (see fig. 2) are as follows:
the double-antibody is 25.9mm, the protein polypeptide hydrolysate of fresh eucalyptus leaves is 10.9mm, the crude protein raw material of fresh eucalyptus leaves is 8mm, the 75% alcohol is 9.4mm, and the sterile water is N/A;
the experimental principle is as follows: the antimicrobial agent diffuses around within the agar and its concentration decreases in a gradient, so that bacterial growth is inhibited within a certain distance around the paper sheet. After overnight culture, a bacteriostasis zone is formed, the larger the bacteriostasis zone is, the larger the sensitivity of the bacterium to the medicine is, otherwise, the smaller the sensitivity of the bacterium is, and if the bacteriostasis zone is not formed, the drug resistance of the bacterium to the medicine is determined.
Therefore, the experimental result shows that: the bacteria circle of the polypeptide hydrolysate is larger than that of alcohol bacteria, and the sensitivity of the bacteria to the related polypeptide hydrolysate is larger than that to the alcohol; therefore, the fresh eucalyptus leaf protein polypeptide hydrolysate obtained by the method has a good growth inhibition effect on bacteria.
Thirdly, punching experiment:
1. preparing a plurality of blank drug sensitive tablets, a crude protein raw material of fresh eucalyptus leaves, a protein polypeptide hydrolysate of fresh eucalyptus leaves, a double antibody (penicillin 100ug/ml and streptomycin 50ug/ml), 75% alcohol, an MH culture medium (beef powder 2.0g/L, soluble starch 1.5g/L, acid hydrolyzed casein 17.5g/L, pH 7.5), and escherichia Coli E-Coli MG1655 (present in Qinghua university, 2 Twai 10-8one/mL);
2. diluting Escherichia coli by 100 times, uniformly coating 300 μ L of bacterial liquid on MH culture medium, and air drying;
3. punching 5 holes in the culture medium respectively;
4. adding sterile water, fresh folium Eucalypti Globueli crude protein material, fresh folium Eucalypti Globueli protein polypeptide hydrolysate, double antibody, 75% alcohol, and 20 μ L;
5. the mixture was cultured in an incubator at 37 ℃ for 24 hours, and the results were observed.
The inhibition zone quantification results (see fig. 3) were as follows: 27mm of double-antibody, 8.2mm of fresh eucalyptus leaf crude protein raw material, 11.6mm of fresh eucalyptus leaf protein polypeptide hydrolysate, 9.7mm of 75% alcohol and sterile water N/A; the results show that: the antibacterial effect of the fresh eucalyptus leaf protein polypeptide hydrolysate is superior to that of alcohol.
Fourth, antibacterial experiment on mask
1. The mouth-nose contact position of a mask (product of Mediciom of Madikang: 8445872) used for the past day is 10cm2The left and the right are cut into two halves;
2. the two pieces of gauze mask are respectively put into sterile water containing 20mL of 5% fresh eucalyptus leaf protein polypeptide hydrolysate and fully mixed for 1 hour, then the gauze mask is removed, and 500uL of TSA culture medium is respectively smeared.
3. The mixture was cultured in an incubator at 37 ℃ for 24 hours, and the results were observed.
The results show (see fig. 4): the fresh eucalyptus leaf protein polypeptide hydrolysate treated group is sterile, the untreated group is more than 100cfu/mL, and the fresh eucalyptus leaf protein polypeptide hydrolysate can play a good antibacterial role.
The eucalyptus leaf protein polypeptide hydrolysate can effectively inhibit the growth of bacteria, has better antibacterial and bactericidal effects than alcohol, has good safety, is easier to accept and use for a long time than antibiotics, and belongs to a natural and safe antibacterial product.
The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its central concept. It should be noted that it would be apparent to those skilled in the art that various changes and modifications can be made in the invention without departing from the principles of the invention, and such changes and modifications are intended to be covered by the appended claims.

Claims (8)

1. A fresh eucalyptus leaf protein polypeptide hydrolysate obtained by the following steps:
1) washing fresh picked leaves of Eucalyptus globulus Labill, removing impurities, sieving and crushing to 40-100 meshes, placing in 0.4-1% sodium chloride solution according to the material-liquid ratio of 1: 6-12, and extracting in 80 ℃ water bath for 4-6 h;
2) concentrating the obtained crude extract of fresh folium Eucalypti Globueli to original concentration of 0.5-1.2g/ml after filtering under reduced pressure;
3) extracting a crude extract of fresh eucalyptus leaves by using 80% ethanol at room temperature, and then freeze-drying residues obtained after ethanol extraction to obtain a crude protein raw material of the fresh eucalyptus leaves;
4) carrying out enzymolysis on a fresh eucalyptus leaf protein raw material to obtain a fresh eucalyptus leaf protein polypeptide hydrolysate;
5) further optimizing the antibacterial polypeptide components of the eucalyptus by ultrafiltering the protein polypeptide hydrolysate of the fresh eucalyptus leaves; filtering fresh folium Eucalypti Globueli protein polypeptide hydrolysate with membrane, and separating with cellulose membrane of 4kDa (Mw) to obtain active extracts with different components with molecular weight of Mw less than 4kDa and Mw greater than 4kDa, wherein the component with Mw less than 4kDa is the fresh folium Eucalypti Globueli protein polypeptide hydrolysate.
2. The fresh eucalyptus leaf protein polypeptide hydrolysate of claim 1, wherein the enzymolysis step of step 4 is as follows:
hydrolyzing fresh folium Eucalypti Globueli protein material at 37 deg.C with pepsin (pH 2.0) and trypsin (pH8.0) for 3 hr; the concentration of pepsin and trypsin is 5% (w/w), the enzyme is inactivated in boiling water for 10 min, then centrifuged at 9000g for 10 min at 4 ℃, and the soluble part of the supernatant is subjected to secondary enzymolysis by pepsin and trypsin and freeze-dried to obtain the fresh eucalyptus leaf protein polypeptide hydrolysate.
3. The fresh eucalyptus leaf protein polypeptide hydrolysate as claimed in any one of claims 1 or 2, wherein the membrane pore size in the membrane filtration of step 5 is 0.22 μm.
4. A method for extracting a compound active extract of eucalyptus leaves comprises the following steps:
1) washing fresh picked leaves of Eucalyptus globulus Labill, removing impurities, sieving and crushing by a 40-100 mesh sieve, and mixing according to a material-liquid ratio of 1: 6 to 12, putting the mixture into a sodium chloride solution with the concentration of 0.4 to 1 percent, and extracting the mixture for 4 to 6 hours in a water bath at the temperature of 80 ℃;
2) concentrating the obtained crude extract of fresh folium Eucalypti Globueli to original concentration of 0.5-1.2g/ml after filtering under reduced pressure;
3) extracting a crude extract of fresh eucalyptus leaves by using 80% ethanol at room temperature, and then freeze-drying residues obtained after ethanol extraction to obtain a crude protein raw material of the fresh eucalyptus leaves;
4) carrying out enzymolysis on a fresh eucalyptus leaf protein raw material to obtain a fresh eucalyptus leaf protein polypeptide hydrolysate;
5) further optimizing the antibacterial polypeptide components of the eucalyptus by ultrafiltering the protein polypeptide hydrolysate of the fresh eucalyptus leaves; fresh eucalyptus leaf protein polypeptide hydrolysate is subjected to membrane filtration and then separated by a cellulose membrane of 4kDa (Mw) to obtain active extracts of different fractions with molecular weight Mw < 4kDa and Mw > 4kDa, and the fractions with Mw < 4kDa are selected for further activity verification.
5. The method of claim 4, wherein the enzymolysis step of the step 4 is as follows:
hydrolyzing fresh folium Eucalypti Globueli protein material at 37 deg.C with pepsin (pH 2.0) and trypsin (pH8.0) for 3 hr; the concentration of pepsin and trypsin is 5% (w/w), the enzyme is inactivated in boiling water for 10 min, then centrifuged at 9000g for 10 min at 4 ℃, and the soluble part of the supernatant is subjected to secondary enzymolysis by pepsin and trypsin and freeze-dried to obtain the fresh eucalyptus leaf protein polypeptide hydrolysate.
6. The method according to any one of claims 4 or 5, wherein the membrane pore size in the membrane filtration of step 5 is 0.22 μm.
7. Use of the fresh eucalyptus leaf protein polypeptide hydrolysate of claims 1-3 in the preparation of an antibacterial agent.
8. An antibacterial agent comprising the fresh eucalyptus leaf protein polypeptide hydrolysate of any one of claims 1 to 3.
CN202010718542.8A 2020-07-23 2020-07-23 Eucalyptus leaf composite active extract process and antibacterial application thereof Pending CN113969300A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010718542.8A CN113969300A (en) 2020-07-23 2020-07-23 Eucalyptus leaf composite active extract process and antibacterial application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010718542.8A CN113969300A (en) 2020-07-23 2020-07-23 Eucalyptus leaf composite active extract process and antibacterial application thereof

Publications (1)

Publication Number Publication Date
CN113969300A true CN113969300A (en) 2022-01-25

Family

ID=79585341

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010718542.8A Pending CN113969300A (en) 2020-07-23 2020-07-23 Eucalyptus leaf composite active extract process and antibacterial application thereof

Country Status (1)

Country Link
CN (1) CN113969300A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2181507A1 (en) * 1972-04-26 1973-12-07 Boutroux Andre Animal and vegetable proteolysates prepn - for treatment of undernutrition
CN102341105A (en) * 2009-02-03 2012-02-01 微生物公司 Bismuth-thiols as antiseptics for epithelial tissues, acute and chronic wounds, bacterial biofilms and other indications
CN103096720A (en) * 2010-08-12 2013-05-08 微生物公司 Bismuth-thiols as antiseptics for agricultural, industrial and other uses
US20190160117A1 (en) * 2017-11-29 2019-05-30 Access Business Group International Llc Method and topical composition for modification of a skin microbiome

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2181507A1 (en) * 1972-04-26 1973-12-07 Boutroux Andre Animal and vegetable proteolysates prepn - for treatment of undernutrition
CN102341105A (en) * 2009-02-03 2012-02-01 微生物公司 Bismuth-thiols as antiseptics for epithelial tissues, acute and chronic wounds, bacterial biofilms and other indications
CN103096720A (en) * 2010-08-12 2013-05-08 微生物公司 Bismuth-thiols as antiseptics for agricultural, industrial and other uses
US20190160117A1 (en) * 2017-11-29 2019-05-30 Access Business Group International Llc Method and topical composition for modification of a skin microbiome

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
周汉军;王挥;龚吉军;唐静;唐雅珂;毛雪琳;: "蓝桉叶精油对砂糖橘的保鲜效果", 食品工业科技, vol. 37, no. 10, pages 347 - 350 *
唐云 等: "蓝桉的化学成分及其药理活性研究进展", 中草药, vol. 46, no. 6, pages 923 - 931 *
曹素芳;陈运娇;陈洪璋;欧阳文;叶锡光;曹庸;赵力超;: "桉叶月见草素B的单体制备及其体内外稳定性研究", 林产化学与工业, no. 01, pages 112 - 118 *

Similar Documents

Publication Publication Date Title
Anandan et al. Effect of different physical and chemical treatments on detoxification of ricin in castor cake
CN106538638B (en) A kind of Chinese medicine antiseptic and preparation method thereof
CN103689010B (en) Medicine for hog house disinfection
KR101670953B1 (en) Manufacturing method for sterilization deodorant and sterilization deodorant thereof
CN1739358A (en) Mosquito killing disinfectant
CN109105398A (en) A kind of plant source extraction bactericidal deodorant and preparation method thereof for environmental improvement
CN102428972A (en) Liquidd mosquito-repellent incense made of folium artemisiae argyi and preparation method thereof
CN105284942A (en) Novel botanical insecticide, preparation method and application of novel botanical insecticide
CN105379780A (en) Pesticide, as well as preparation method and application thereof
CN102038014A (en) Compound plant-source acaricide and preparation method thereof
CN104762136A (en) Compound tea tree essential oil with sterilization and disinfection effects and preparation method of compound tea tree essential oil
CN111265598A (en) Composite broad-spectrum antibacterial agent and preparation method thereof
CN112021340A (en) Preparation and application of nano composite bactericidal antiviral material
CN104189162B (en) For the thimerosal and the preparation method that clean a wound
CN101647475B (en) Incense for eliminating plague
CN113969300A (en) Eucalyptus leaf composite active extract process and antibacterial application thereof
CN104491121B (en) It is a kind of to be used to treat antibacterial Essence of breathing problem and preparation method thereof
KR101227091B1 (en) Food additive agents using worm-fermented liquid or worm wood-carbonized liquid
CN109907080A (en) A kind of organic vegetable pest control chemicals and its preparation and application
CN109601561A (en) A kind of Chinese medical extract for air sanitizer and preparation method thereof, environment-friendly composite air sanitizer and preparation method thereof
CN108935536A (en) A kind of natural drug and preparation method thereof inhibiting wawter bloom
KR100775395B1 (en) Compounds for red tide prevention comprising natural plant extracts
CN108273106A (en) A kind of anti-sense medicine is fragrant
KR102206192B1 (en) Method for manufacturing eco-friendly pest repellent for horticulture and eco-friendly pest repellent for horticulture manufactured thereby
KR102221700B1 (en) Method for manufacturing natural pest repellent and natural pest repellent manufactured thereby

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB02 Change of applicant information
CB02 Change of applicant information

Address after: 314000 room 14f1401-b, No. 705, Asia Pacific Road, Nanhu District, Jiaxing City, Zhejiang Province

Applicant after: Hemei Biotechnology (Zhejiang) Co.,Ltd.

Address before: 314000 room 14f1401-b, No. 705, Asia Pacific Road, Nanhu District, Jiaxing City, Zhejiang Province

Applicant before: Enlightenment Hemei Biotechnology (Jiaxing) Co.,Ltd.