CN109161578A - A kind of environment-friendly function detection method of hemp stalk core fibre film - Google Patents
A kind of environment-friendly function detection method of hemp stalk core fibre film Download PDFInfo
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Abstract
The invention discloses a kind of environment-friendly function detection methods of the hemp stalk core fibre film for the environmental technology field for belonging to space environment purification.The detection of the antibacterial activity in vitro of preparation and hemp stalk core extract including test medicine hemp stalk core fibre film, big waste of flax core viscose fiber has more apparent inhibition and killing effect to staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans, has inhibition to anaerobic bacteria and aerobic bacteria and kills effect.The present invention using hemp stalk core fibre be made natural fiber film in outdoor air because of the foreign flavor indoors of the generations such as haze particulates emission Pm2.5, formaldehyde; the special adsorption filtration effect having; and there is apparent degerming bacteriostasis, to achieve the purpose that environment purification, protection human health, prevention disease.Used a variety of materials are environment-friendly materials in design.It can be widely applied to air and living environment purification and related separation purifying.
Description
Technical field
The invention belongs to the environmental technology field of space environment purification, in particular to a kind of rings of hemp stalk core fibre film
Protect function detecting method.
Background technique
Haze source is industrial discharge pollution, urban construction finishing, dust on the roads, life heating smoke evacuation and car tail
Gas etc..Subtle dust, volatility acid mist such as nitric acid-sulfuric acid mist, organic hydrocarbon compounds, heavy metal etc. are contained in haze, this
It is the main component of heavy industry air pollutants, fugitive dust, vehicle exhaust a bit.Thermal power generation, smelting especially based on fire coal
The woodenware processing of gold, chemical industry and dust concentration, building materials industry, are one of the main sources of air suspension microparticle and sulfide.
And vehicle exhaust majority is that microparticle content is bigger in nitrogen oxides and oxycarbide, especially diesel automobile exhaust.
It is known as the London of the title of " mist is all ", Englishman introspects bitter pill caused by air pollution, this island country
Capital has sea wind to brush, and should not have haze according to reason." smog episode in london " has expedited the emergence of generation as a note weight caused by haze
The appearance of the upper first air pollution control law case " clean air act " in boundary.Pass through a series of measures, by 1975, London
Haze day was reduced to 15 days by annual tens days, was dropped within 1980 5 days.After the eighties, the automobile quantity in London is increased sharply, when
When vehicle guaranteeding organic quantity reached 2,440,000, traffic jam environment is serious.Since 1981, number that London is gone out by bus
Amount increases 20%, accounts for the 43% of all working strokes, traffic pollution replaces industrial pollution to become the primary of London air quality
It threatens.British government has to adopt a series of measures this by automobile bring air pollution to fight.This motor-driven vehicle
Air pollution caused by gas, not only in Britain, Germany, France in European Union member countries, the economy such as Italy and the U.S., Japan
Also all since vehicle guaranteeding organic quantity increases sharply, traffic produced air pollution caused by motor-vehicle tail-gas is more than modern industry for flourishing country
Pollution, people endure the hardship of haze to the fullest extent, and various countries take different countermeasures, such as:
Not so its member state of European Union requirements year air number of days not up to standard will face 4.5 hundred million dollars no more than 35 days
Significant fine；
Germany is forbidden to the implementation of certain class vehicle, or forbids all vehicle drivings in seriously polluted region.2013, moral
State encourages motor vehicle to install exhaust gas cleaning apparatus energetically, and the car owner for installing filter can get public subsidies.
In the puzzlement for also falling into haze in recent years, the buildings such as Eiffel Tower almost disappear in haze for France.Method
Implement automobile odd or even number and many kinds of measures such as travel in turn in state capital Paris；
Milan, ITA city levies taxes to the automobile of pollution most serious, and working day 7, seriously polluted automobile must when 19
2 to 10 Euros of taxes, which must be paid, just can enter urban district.
Japan also once endured the hardship of air pollution to the fullest extent at industrialization initial stage.Respiratory tract along 600 multidigit national highways of Tokyo in 1999
Disease is just by indicting local government and eight Automobile Enterprises, through expert appraisal, car tail with vehicle exhaust injury body
PM2.5 caused by gas truly has strong carcinogenesis.Exactly this lawsuit pushed Japanese government to the attention of PM2.5 and
The resolution of traffic is set about managing by local government.Tokyo legislation demands automobile installs filter additional, and diesel-engined vehicle is forbidden to sail
The city Ru Gai.What all taxis in Tokyo used is all natural gas.Urban afforestation is the important measures of Japanese pollution administration.Tokyo
Regulation, newly-built building must have greenery patches, it is necessary to do roof greening.
Statistical analysis, interior decoration bring organic solvent, toluene, formaldehyde etc. and luxurious finishing, it is a large amount of using big
Fibrous gypsum finishing, such interior can contain higher radioactive radon, it has been proved that other than smoking, this is second for Canada
The reason of position causes lung cancer.Indoor pollution influences the morbidity of respiratory disease very big., it is not only to cause indoor child
The inducement of the leukaemia of son, the stimulation to human respiratory are also bigger.In addition there are indoor a large amount of blanket, ground
Blanket, furniture etc., these are exactly anaphylactogen, especially mite class, easily cause asthma；According to statistics, 15% tracheitis, bronchus
Scorching, lung cancer is caused by air pollution, 22% chronic lung disease is caused by air pollution；35.75% respiratory disease be by
Caused by air pollution；Formaldehyde in 72.2% children's house；This is one group of reflection compatriots because of outdoor or room air pollution
Seriously affect the data of health.Thus for, control atmosphere pollution, purify the air of a room it is imperative！！
For the filtration processing of the machine property guarded against of haze molecule (being commonly called as Pm2.5), up to the present, still lack one kind
Pm2.5 molecule can be not only filtered out made of natural material, but also can be to companion possible in the floating dust by carbon dioxide atmosphere
With disinfect pathogen have inhibit and kill effect device guard against and the relevant technologies.The technology of the present invention and device commandment development aiming at
Solve this practical challenges.
Summary of the invention
The object of the present invention is to provide a kind of environment-friendly function detection methods of hemp stalk core fibre film, which is characterized in that tool
Steps are as follows for body:
Step 1, the preparation of test medicine hemp stalk core fibre powder: by the hemp stalk core powder by dry, super pulverization process
2000 grams, 80% alcohol reflux is added to extract 3 times；Ethyl alcohol dosage is 80% second for being for the first time 5 times of hemp stalk core powder respectively
Alcohol；Second 4 times 80% ethyl alcohol, 3 times of third time 80% ethyl alcohol；It extracting solution will be concentrated three times, recycling design to no ethyl alcohol
Taste stands more than for 24 hours, removes the chlorophyll for being sunken to lower layer, retains supernatant；Solution is diluted 5 times, it is big to be transferred to D101 respectively
Hole resin, AB-8 macroreticular resin, SP-70 macroporous resin column, and washed respectively with water, 30% ethyl alcohol, 70% ethyl alcohol, 80% ethyl alcohol
It is de-, variant concentration ethanol eluent and pure water eluent are collected, is concentrated under reduced pressure removes solvent respectively, remain solid fraction after recycling
Object is hemp stalk core fibre powder given the test agent；
Step 2, the detection of the antibacterial activity in vitro of hemp stalk core extract, big waste of flax core viscose fiber is to golden yellow Portugal
Grape coccus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans have it is obvious inhibit and killing effect, all to anaerobic bacteria and aerobic bacteria
There is inhibition and kills effect；It specifically includes:
(1) material:
1.1, strain subject includes: escherichia coli Escherichia coli [CMCC (B) 44103], golden yellow grape
Coccus Staphyloccocus aureus (ATCC29213), monokaryon hyperplasia Li Shi spy bacterium Listeria monocytogenes
[LM CMCC (B) 54002], bacillus subtilis Bacillus subtilis [CMCC (B) 63501], bacillus cereus
Bacillus cereus [CMCC (B) 63301], proteus vulgaris Proteus vulgaris [CMCC (B) 49027], wound
Cold salmonella Salmonella typhi (ATCC 14028) is provided by Capital Normal University's department of microbiology；Trichophyton rubrum
Trichophyton rubrum (BMU01672), alpha fungus Trichophyton mentagrophytes (ATCC
28185), microsporum canis Microsporum canis (ATCC 22019), Candida albicans Candida albicans
[CMCC (B) 98001] is provided by Peking University fungi and nosomycosis research center and is contained bacterial strain.
1.2 test drugs, equipment D101 resin (Tianjin sea light resin Co., Ltd)；AB-8, SP-70 resin (Japan
Mitsubishi Chemical, Japan), dimethyl sulfoxide (amresco company, the U.S.), 0.22 μm of miillpore filter, beef extract-peptone culture
The phenol red beef extract-peptone culture solution of base, glucose (contains glucose 1%, phenol red 0.002%)；Potato dextrose medium
(PDA), oat medium, Sabouraud dextrose peptone agar (SDA) the above material are limited from the damp biotechnology of Beijing section sky
Company；
(2), detection process
The preparation of 2.1 indicator bacteria bacteria suspensions, weighs 9 each 8.0mg of sample and is dissolved in 2mL dimethyl sulfoxide, mass concentration
For 4 000 μ g/mL；It is stored in after 0.22 μm of filter degerming spare in 4 DEG C of refrigerator；By prepared for reagent object
The beef extract-peptone body culture medium of stoste carries out 1:2 from the 1st pipe to the 9th pipenTimes dilute, wherein n be 1,2 ... .., 9；It sets
In 35 DEG C, 120r/min shaking table culture 18~take out afterwards for 24 hours；Obtain indicator bacteria bacteria suspension；
2.2, it is respectively 1 × 10 by concentration6Each 100 μ L of the indicator bacteria bacteria suspension of CFU/mL is coated in the beef extract prepared
On peptone plate, 4 Oxford cups of internal diameter 6mm, outer diameter 8mm, high 10mm are put on the plate coated, gently pressurization makes
Itself and culture medium contact tight, be added in Oxford cup 1.1 in above-mentioned steps 2 described in measuring samples, wherein at first 3
Add same concentration, each 200 μ L of the same sample in Oxford cup, the dimethyl sulfoxide of 200 μ L is added in the 4th Oxford cup, then will
There is the plate of Oxford cup coated to be put into 35 DEG C of incubators culture 18~take out afterwards for 24 hours, observe in plate is around Oxford cup
It is no to have inhibition zone, and to the size for thering is the Oxford cup of inhibition zone vernier caliper to measure, record inhibition zone, the size of inhibition zone
Show drug to the power of the inhibiting effect of bacterium；Sterile test tube 12 of φ 13mm × 100mm are taken, are arranged in a row, every pipe
The phenol red beef extract-peptone culture solution 1mL of glucose is added, the drug stoste that concentration is 4000 μ g/mL is added in the 1st pipe
1.0mL is mixed, and is then drawn 1mL and is put into the 2nd pipe, is drawn in 1mL to the 3rd pipe again after mixing, so continuous proportion dilution,
Until the 10th pipe, and draw 1mL from the 10th pipe and discard；Only add 1mL solvent as solvent control group, the 12nd pipe in 11st pipe
For the growth control that drug is not added；
2.3, above-mentioned each 1mL of the inoculum prepared is added in every pipe, keeps the final bacterium solution of every pipe dense for antibacterial detection
Degree is 1 × 106CFU/mL；1st pipe drug quality concentration into the 10th pipe is respectively 2 000,1 000,500,250,125,
62.5,31.2,15.6,7.8,3.9μg/mL.Inoculated dilution tube is stoppered plug, is placed in 35 DEG C of normal air incubator
It is middle be incubated for 18~for 24 hours, bacterial growth make glucose fermentation produce acid, with phenolic red indicator reflect turn yellow, the positive pipe be yellow；Nothing
Bacterial growth, liquid is still clear red person's negative tube in test tube；To visually observe the highest drug less than bacterium colony growth
Diluted concentration is minimal inhibitory concentration MIC of the sample to this kind of bacterium；Observation is taken still to measure pipe for clear red person
Culture solution is coated on beef-protein medium plate in MIC negative tube, and 35 DEG C of constant temperature incubations 18~for 24 hours；Observe and record bacterium
Fall number, the minimum concentration of clump count < 3 or asepsis growth is the minimum bactericidal concentration MBC of Chinese fiber crops sample；
2.4, drug measures the minimal inhibitory concentration of fungi, weighs each 6.4mg of above 9 samples and is dissolved in 2 mL dimethyl
In sulfoxide, mass concentration is 3200 μ g/mL；It is spare that 4 DEG C of refrigerators are stored in after 0.22 μm of filter degerming；Take 3200 μ
Each sample of g/mL obtains the sample of 64 μ g/mL after first diluting 50 times with RPMI-1640 culture medium, then carry out again twice it is dilute
It releases, obtains diluting whole mass concentration μ g/mL being respectively 32,16,8,4,2,1,0.5,0.25,0.125 and 0.06, totally 10 matter
Measure concentration；The drug of each mass concentration is added in 96 porocyte culture plates respectively, every 100 μ L of hole；1~10 is classified as drug
Each mass concentration, 11 are classified as growth control, and 12 are classified as blank control and Vehicle controls；
2.5, for the detection of specific strain, dermatophyte is cultivated into 7~10d on PDA；Trichophyton rubrum is in oat
7~10d is cultivated on culture medium；Candida albicans cultivates 24~48h on SDA；With 0.9% normal saline flushing skin tinea
Bacterium colony surface, takes bacteria suspension in test tube, settles 10min, supernatant is taken to be counted as 0.5 Maxwell turbidity than turbid instrument, it is equivalent to 1 ×
105~5 × 105CFU/mL；It is 1 × 10 with the final bacteria suspension concentration that RPMI-1640 culture medium dilutes 100 times3~5 ×
103CFU/mL；Candida albicans is counted as 0. Maxwell turbidity than turbid instrument and is equivalent to 1 × 103~5 × 103CFU/mL；Use RPMI-
The final bacteria suspension concentration that 1640 culture mediums dilute 2000 times is 0.5 × 103~2.5 × 103CFU/mL takes 100 μ L bacteria suspensions
It is added in the prepared column of drug sensitive plate 1~11；35 DEG C of incubator cultures, wherein dermatophyte 4~6d of culture is observed as a result, white
Color candida albicans cultivates 24~48h, observes result；
(3) result
3.1, the beef extract-peptone plate for being placed with Oxford cup is taken out the inhibition zone of bacterium by drug, observes Oxford cup
Whether surrounding has inhibition zone, then uses the diameter of vernier calliper dipstick metering inhibition zone；Find out from inhibition zone result, in 70% ethyl alcohol
The antibacterial activity that de- object on 3 kinds of resins of elution has had, wherein with eluate has a broad antifungal spectrum under AB-8 resin；
3.2, in minimum-bacteriostat mass concentration MIC result it can be seen that the effect that the inhibitions fungi of C1 grows be it is best,
In addition to A1, A2, A3 are insensitive to microsporum canis, and remaining bacteriostasis is obvious, most strong with the inhibiting effect of C1；
3.3, minimal bactericidal concentration (MBC)；A1 is best to Escherichia coli bactericidal effect, and C1 is to wax bacillus
With bactericidal effect, but effect is unobvious, and B1 is more apparent to proteus and salmonella bactericidal effect.
1:2 is carried out in (the 2) -2,1 of the step 2nAgain diluted n be 1,2 ... .., 9；I.e. extension rate is respectively 1:
2、1∶4、1∶8、1∶16、1∶32、1∶64、1∶128、1∶256、1∶512。
The invention has the advantages that using hemp stalk core fibre be made natural fiber film in outdoor air because of haze
The special adsorption filtration effect that particulates emission Pm2.5, foreign flavor indoors (formaldehyde etc.) have, and there is the antibacterial work of apparent degerming
With to achieve the purpose that environment purification, protection human health, prevention disease.Used a variety of materials are ring in design
Protect material.It can be widely applied to air and living environment purification and related separation purifying.
Specific embodiment
The present invention provides a kind of environment-friendly function detection method of hemp stalk core fibre film, is said below with reference to embodiment
It is bright.
Specific step is as follows for the detection method of the environment-friendly function of the hemp stalk core fibre film:
Step 1, the preparation of test medicine hemp stalk core fibre powder: will be by the dry hemp stalk core through super pulverization process
2000 grams of powder, 80% alcohol reflux is added to extract 3 times；Ethyl alcohol dosage is respectively 80% second for being for the first time 5 times of hemp stalk core powder
Alcohol；Second 4 times 80% ethyl alcohol, 3 times of third time 80% ethyl alcohol；It extracting solution will be concentrated three times, recycling design to no ethyl alcohol
Taste stands more than for 24 hours, removes the chlorophyll for being sunken to lower layer, retains supernatant；Solution is diluted 5 times, it is big to be transferred to D101 respectively
Hole resin, AB-8 macroreticular resin, SP-70 macroporous resin column, and washed respectively with water, 30% ethyl alcohol, 70% ethyl alcohol, 80% ethyl alcohol
It is de-, variant concentration ethanol eluent and pure water eluent are collected, is concentrated under reduced pressure removes solvent respectively, remain solid fraction after recycling
The hemp stalk core fibre powder given the test agent that object is；
Step 2, the detection of the antibacterial activity in vitro of hemp stalk core extract, big waste of flax core viscose fiber is to golden yellow Portugal
Grape coccus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans have more apparent inhibition and killing effect, to anaerobic bacteria and aerobic bacteria
There is inhibition and kills effect；It specifically includes:
(1) material:
1.1 strain subjects include: escherichia coli Escherichia coli [CMCC (B) 44103], golden yellow grape
Coccus Staphyloccocus aureus (ATCC29213), monokaryon hyperplasia Li Shi spy bacterium Listeria monocytogenes
[LM CMCC (B) 54002], bacillus subtilis Bacillus subtilis [CMCC (B) 63501], bacillus cereus
Bacillus cereus [CMCC (B) 63301], proteus vulgaris Proteus vulgaris [CMCC (B) 49027], wound
Cold salmonella Salmonella typhi (ATCC 14028) is provided by Capital Normal University's department of microbiology；Trichophyton rubrum
Trichophyton rubrum (BMU01672), alpha fungus Trichophyton mentagrophytes (ATCC
28185), microsporum canis Microsporum canis (ATCC 22019), Candida albicans Candida albicans
[CMCC (B) 98001] is provided by Peking University fungi and nosomycosis research center and is contained bacterial strain.
1.2 test drugs, equipment D101 resin (Tianjin sea light resin Co., Ltd)；AB-8, SP-70 resin (Japan
Mitsubishi Chemical, Japan), dimethyl sulfoxide (amresco company, the U.S.), 0.22 μm of miillpore filter, beef extract-peptone culture
The phenol red beef extract-peptone culture solution of base, glucose (contains glucose 1%, phenol red 0.002%)；Potato dextrose medium
(PDA), oat medium, Sabouraud dextrose peptone agar (SDA) the above material are limited from the damp biotechnology of Beijing section sky
Company；
(2), detection process
The preparation of 2.1 indicator bacteria bacteria suspensions, weighs 9 each 8.0mg of sample and is dissolved in 2mL dimethyl sulfoxide, mass concentration
For 4 000 μ g/mL；It is stored in after 0.22 μm of filter degerming spare in 4 DEG C of refrigerator；By prepared for reagent object
The beef extract-peptone body culture medium of stoste carries out 1:2 from the 1st pipe to the 9th pipenTimes dilute, wherein n be 1,2 ... .., 9；
(i.e. extension rate is respectively 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64,1: 128,1: 256,1: 512).Be placed in 35 DEG C,
120r/min shaking table culture 18~take out afterwards for 24 hours；Obtain indicator bacteria bacteria suspension；
2.2, it is respectively 1 × 10 by concentration6Each 100 μ L of the indicator bacteria bacteria suspension of CFU/mL is coated in the beef extract prepared
On peptone plate, 4 Oxford cups of internal diameter 6mm, outer diameter 8mm, high 10mm are put on the plate coated, gently pressurization makes
Itself and culture medium contact tight, be added in Oxford cup 1.1 in above-mentioned steps 2 described in measuring samples, wherein at first 3
Add same concentration, each 200 μ L of the same sample in Oxford cup, the dimethyl sulfoxide of 200 μ L is added in the 4th Oxford cup, then will
There is the plate of Oxford cup coated to be put into 35 DEG C of incubators culture 18~take out afterwards for 24 hours, observe in plate is around Oxford cup
It is no to have inhibition zone, and to the size for thering is the Oxford cup of inhibition zone vernier caliper to measure, record inhibition zone, the size of inhibition zone
Show drug to the power of the inhibiting effect of bacterium；Sterile test tube 12 of φ 13mm × 100mm are taken, are arranged in a row, every pipe
The phenol red beef extract-peptone culture solution 1mL of glucose is added, the drug stoste that concentration is 4 000 μ g/mL is added in the 1st pipe
1.0mL is mixed, and is then drawn 1mL and is put into the 2nd pipe, is drawn in 1mL to the 3rd pipe again after mixing, so continuous proportion dilution,
Until the 10th pipe, and draw 1mL from the 10th pipe and discard；Only add 1mL solvent as solvent control group, the 12nd pipe in 11st pipe
For the growth control that drug is not added；
2.3, above-mentioned each 1mL of the inoculum prepared is added in every pipe, makes the final bacterial concentration of every pipe for antibacterial detection
About 1 × 106CFU/mL；1st pipe drug quality concentration into the 10th pipe is respectively 2 000,1 000,500,250,125,
62.5,31.2,15.6,7.8,3.9μg/mL.Inoculated dilution tube is stoppered plug, is placed in 35 DEG C of normal air incubator
It is middle be incubated for 18~for 24 hours, bacterial growth make glucose fermentation produce acid, with phenolic red indicator reflect turn yellow, the positive pipe be yellow；Nothing
Bacterial growth, liquid is still clear red person's negative tube in test tube；To visually observe the highest drug less than bacterium colony growth
Diluted concentration is minimal inhibitory concentration MIC of the sample to this kind of bacterium；Observation is taken still to measure pipe for clear red person
Culture solution is coated on beef-protein medium plate in MIC negative tube, and 35 DEG C of constant temperature incubations 18~for 24 hours；Observe and record bacterium
Fall number, the minimum concentration of clump count < 3 or asepsis growth is the minimum bactericidal concentration MBC of Chinese fiber crops sample；
2.4, drug measures the minimal inhibitory concentration of fungi, weighs each 6.4mg of above 9 samples and is dissolved in 2 mL dimethyl
In sulfoxide dimethyl sulfoxide, mass concentration is 3200 μ g/mL；It is standby that 4 DEG C of refrigerators are stored in after 0.22 μm of filter degerming
With.Each sample for taking 3 200 μ g/mL obtains the sample of 64 μ g/mL, then after first diluting 50 times with RPMI-1640 culture medium
Twice of dilution is carried out again, obtains diluting whole mass concentration μ g/mL being respectively 32,16,8,4,2,1,0.5,0.25,0.125 and
0.06, totally 10 mass concentrations；The drug of each mass concentration is added in 96 porocyte culture plates respectively, every 100 μ L of hole；
1~10 is classified as each mass concentration of drug, and 11 are classified as growth control, and 12 are classified as blank control and Vehicle controls；
2.5, for the detection of specific strain, dermatophyte is cultivated into 7~10d on PDA；Trichophyton rubrum is in oat
7~10d is cultivated on culture medium；Candida albicans cultivates 24~48h on SDA；With 0.9% normal saline flushing dermatophyte
Surface is fallen, takes bacteria suspension in test tube, 10min is settled, takes supernatant to be counted as 0.5 Maxwell turbidity than turbid instrument, be equivalent to 1 × 105
~5 × 105CFU/mL；It is 1 × 10 with the final bacteria suspension concentration that RPMI-1640 culture medium dilutes 100 times3~5 × 103CFU/
mL；Candida albicans is counted as 0. Maxwell turbidity than turbid instrument and is equivalent to 1 × 103~5 × 103CFU/mL；It is cultivated with RPMI-1640
The final bacteria suspension concentration that base dilutes 2000 times is 0.5 × 103~2.5 × 103CFU/mL；Candida albicans is than turbid instrument meter number
For 0.5 Maxwell turbidity, it is equivalent to 1 × 103~5 × 103CFU/mL；2000 times of final bacterium is diluted with RPMI-1640 culture medium
Suspension concentration is 0.5 × 103~2.5 × 103CFU/mL takes 100 μ L bacteria suspensions to be added in the prepared column of drug sensitive plate 1~11；
35 DEG C of incubator cultures, wherein 4~6d of dermatophyte culture observation is as a result, 24~48h of Candida albicans culture observes result；
(3) result
3.1, the beef extract-peptone plate for being placed with Oxford cup is taken out the inhibition zone of bacterium by drug, observes Oxford cup
Whether surrounding has inhibition zone, then uses the diameter of vernier calliper dipstick metering inhibition zone；Find out from inhibition zone result, in 70% ethyl alcohol
De- object on 3 kinds of resins of elution has preferable antibacterial activity, different resins different volumes score ethyl alcohol as shown in Table 1
Sample shown in substance markers, table 2 is eluted to the size of each bacterium antibacterial circle diameter, wherein with eluate antibacterial under AB-8 resin
Spectrum is wide.
1 different resins different volumes score ethanol elution substance markers of table
2 sample of table to the size of each bacterium antibacterial circle diameter (N=3)
Note: "-" is represented without inhibition zone.Compared with negative control,**p<0.001
* it is found in experimental result, there is no apparent inhibition zones by sample A2, A3, C2C3, therefore omit in the table.
3.2, minimum-bacteriostat mass concentration (MIC) the results are shown in Table shown in 3, table 4, it can be seen that the suppression of C1 from table 3, table 4
The effect of fungi processed growth be it is best, in addition to A1, A2, A3 are insensitive to microsporum canis, remaining bacteriostasis is bright
It is aobvious, it is most strong with the inhibiting effect of C1.
Minimal inhibitory concentration (MIC) of 3 sample of table to G- and G+
Note: "-" indicates that liquid when maximum concentration in test tube is still red, i.e. aseptic result.
Minimal inhibitory concentration (MIC) of 4 sample of table to fungi
Note: general dose-dependant drug resistance is 16-32 μ g/ml in * clinical commonly used drug Fluconazole (FCZ) experiment, but 6 μ g/
Ml is drug resistance.According to 1992, U.S. clinical and laboratory standards institute draft " liquid dilution method of saccharomycete is anti-true
Bacterium drug sensitivity test reference scheme " file name is M=27P, i.e., >=32 μ g/ml is believed that insensitive.
3.3, minimal bactericidal concentration (MBC) the results are shown in Table shown in 5.As can be seen from Table 5, A1 kills Escherichia coli
Bacterium effect is best, and C1 has bactericidal effect to wax bacillus, but effect is unobvious, and B1 is to proteus and sramana
Salmonella bactericidal effect is more apparent.
Minimum bactericidal concentration (MBC) of 5 sample of table to G- and G+
Note: "-" representative can not measure its MBC value, clump count > 3
4 kinds of gram-positive bacterias are used in this experiment and 3 kinds of Gram-negative bacterias do antibacterium screening experiment, are resisted true
The screening at bacterium position is using the hungmao mentagrophyte and Trichophyton mentagrophytes of superficial portion dermatophytid infection pathogenic bacteria and dog microspore
Bacterium, this 3 kinds of fungies occupy significant proportion in dermatophytid infection, and deep infection is used as screening using Candida albicans
The representative of bacterial strain, these common bacterial strains can preferably evaluate the antibacterial activity of the extract as screening active ingredients carrier.Chinese fiber crops
In bar core treatment process, for enriched composition, the substances such as tannin chlorophyll are removed, adsorbed using alcohol precipitation, macroporous absorbent resin etc.
Method.It is eluted during screening ethanol elution concentration using chemical means detection 30% ethanol elution of discovery and 70% obvious
Difference, flavones or saponin(e amount also have difference；Flavones amount is higher than 50% ethanol elution object, but two in 70% ethanol elution object
Flavone compound or phenylpropanoids classification contained by person are close；In 80% or 80% or more ethanol elution
When, saponin(e amount is higher than 70% ethanol elution in eluate, and the chemical component in eluate has significant difference.Therefore this experiment
The middle active material that opposed polarity position is studied using 30%, 70%, 80% ethanol elution.30% ethyl alcohol of this studies have shown that
Eluate has stronger inhibiting effect to Escherichia coli, thus it is speculated that may be related with phenols contained therein, organic acidic material.Before
The amount of phase chemical experiment research 70% ethanol elution its general flavone as the result is shown is up to 50%, and implying may be with contained therein
Flavonoids, the ingredient of Phenylpropanoid Glycosides class, amount it is related.Equally, in 80% ethanol elution object its total saposins amount of chemical assay compared with
Height is stronger to the inhibiting effect of superficial portion fungal infection bacterial strain.3 kinds of resins are under 70% ethanol elution, and eluent is to being surveyed
Most of bacterium have inhibiting effect, show the has a broad antifungal spectrum at the position, also demonstrate that 70% ethanol elution is anti-thin
The effective elution volume score of bacterium active site.The antifungal activity position of dermatophyte is concentrated in 80% eluent, this is
Research different parts active material provides an important reference in next step.
In fact, the present invention using hemp stalk core fibre be made natural fiber film in outdoor air because haze nocuousness is micro-
The foreign flavor indoors of the generations such as grain Pm2.5, formaldehyde, the special adsorption filtration effect having, the preparation of hemp stalk core fibre film are
Be relatively easy to: first preparation material: pulverized < 80-200 mesh hemp stalk core fibre powder, carboxymethyl cellulose
(CMC) and medical non-woven fabrics；Specific preparation method: (heating) is mixed in 0.2-1.0%CMC aqueous solution and hemp stalk core fibre powder
Uniformly, grainless object is answered in solution, by thickness < 0.2-0.8Cm be uniformly applied to non-woven fabrics (wide 50-80cm, long 100cm~,
Naturally dry or heated-air drying to obtain the final product, then use ultraviolet lamp radiation sterilization, spare.
By above-mentioned test, big waste of flax core viscose fiber is to staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and white
Color candida albicans has more apparent inhibition and killing effect, as shown in minimal inhibitory concentration (MIC) of 3 sample of table to G- and G+；To detesting
Oxygen bacterium and aerobic bacteria have inhibition and kill effect, have excellent antibacterial characteristics.Hemp viscose fiber have antibiotic property be because
Cannabinoids and its derivative be present in the lignin for remaining in viscose fiber or in conjunction with fiber caused by.Different from other
Lignin structure in fiber, lignin structure is a kind of reticular structure in big waste of flax core, is radially formed " fibre bundle " group,
Its cross section is irregular triangle, polygon and oblateness etc., rough surface, there are many crackle and hole, during fiber has
Chamber, middle cavity volume often account for mono- l/2 of l/3 of fibrocyte total volume, and hemp large specific surface area, hole is big, more gaps, empty
Gap rate is high, and is connected with many gaps of fiber surface genesis analysis and hole, " fibre bundle " inside and " fibre bundle " group
Between many gaps for being similarly dispersed with and hole.This special structure makes it rich in oxygen, has brilliant moisture absorption
Property and gas permeability.This just makes under moisture conditions, and the anaerobism der Pilz metabolism and physiological activity of survival and reproduction are pressed down
System, it is difficult to survive, effectively inhibit the oxidative phosphorylation of microorganism, influence mitosis, hinder microbial respiratory.In hemp
In bar core viscose fiber, there are lignin cellulose carbohydrate complex.Cellulose glucose ring and lignin equal part
Son is in three-dimensional structure, it is envisaged that in the ingredient based on cellulose, is dispersed with denier lignin molecule, changes
The original structure for having become cellulose keeps the amorphous region of big waste of flax core viscose fiber loosely organized, and accessibility is larger, same rich
It is oxygenous, there is brilliant hygroscopicity and gas permeability, anaerobism der Pilz metabolism and physiological activity are suppressed.
Claims (2)
1. a kind of environment-friendly function detection method of hemp stalk core fibre film, which is characterized in that specific step is as follows:
Step 1, the preparation of test medicine hemp stalk core fibre powder: by the hemp stalk core powder 2000 by dry, super pulverization process
Gram, add 80% alcohol reflux to extract 3 times；Ethyl alcohol dosage is 80% ethyl alcohol for being for the first time 5 times of hemp stalk core powder respectively；Second
80% ethyl alcohol of secondary 4 times of 80% ethyl alcohol, 3 times of third time；It extracting solution will be concentrated three times, recycling design to no ethanol flavor is stood
More than for 24 hours, the chlorophyll for being sunken to lower layer is removed, retains supernatant；By solution dilute 5 times, be transferred to respectively D101 macroreticular resin,
AB-8 macroreticular resin, SP-70 macroporous resin column, and collected each with water, 30% ethyl alcohol, 70% ethyl alcohol, 80% ethanol elution respectively
Different concentration ethanol eluent and pure water eluent are concentrated under reduced pressure remove solvent respectively, and it is Chinese fiber crops that decorating film is remained after recycling
Bar core fibre powder given the test agent；
Step 2, the detection of the antibacterial activity in vitro of hemp stalk core extract, big waste of flax core viscose fiber is to Staphylococcus aureus
Bacterium, Escherichia coli, Pseudomonas aeruginosa and Candida albicans have obvious inhibition and killing effect, have inhibition to anaerobic bacteria and aerobic bacteria
It is acted on killing；It specifically includes:
(1) material:
1.1, strain subject includes: escherichia coli Escherichia coli [CMCC (B) 44103], staphylococcus aureus
Staphyloccocus aureus (ATCC29213), monokaryon hyperplasia Li Shi spy bacterium Listeria monocytogenes [LM
CMCC (B) 54002], bacillus subtilis Bacillus subtilis [CMCC (B) 63501], bacillus cereus
Bacillus cereus [CMCC (B) 63301], proteus vulgaris Proteus vulgaris [CMCC (B) 49027], typhoid fever
Salmonella Salmonella typhi (ATCC 14028) is provided by Capital Normal University's department of microbiology；Trichophyton rubrum
Trichophyton rubrum (BMU01672), alpha fungus Trichophyton mentagrophytes (ATCC
28185), microsporum canis Microsporum canis (ATCC 22019), Candida albicans Candida albicans
[CMCC (B) 98001] is provided by Peking University fungi and nosomycosis research center and is contained bacterial strain；
1.2 test drugs, equipment D101 resin (Tianjin sea light resin Co., Ltd)；AB-8, SP-70 resin (Mitsubishi
Chemistry, Japan), dimethyl sulfoxide (amresco company, the U.S.), 0.22 μm of miillpore filter, beef-protein medium, Portugal
The phenol red beef extract-peptone culture solution of grape sugar (contains glucose 1%, phenol red 0.002%)；Potato dextrose medium (PDA),
Oat medium, Sabouraud dextrose peptone agar (SDA) the above material come from Beijing Ke Haoze Bioisystech Co., Ltd；
(2), detection process
The preparation of 2.1 indicator bacteria bacteria suspensions, weighs 9 each 8.0mg of sample and is dissolved in 2mL dimethyl sulfoxide, mass concentration 4
000μg/mL；It is stored in after 0.22 μm of filter degerming spare in 4 DEG C of refrigerator；By prepared for reagent object stoste
Beef extract-peptone body culture medium from the 1st pipe to the 9th pipe carry out 1:2n times dilute, wherein n be 1,2 ... .., 9；Be placed in 35 DEG C,
120r/min shaking table culture 18~take out afterwards for 24 hours；Obtain indicator bacteria bacteria suspension；
2.2, it is respectively 1 × 10 by concentration6Each 100 μ L of the indicator bacteria bacteria suspension of CFU/mL is coated in the beef extract-peptone prepared
On plate, 4 Oxford cups of internal diameter 6mm, outer diameter 8mm, high 10mm are put on the plate coated, gently pressurization makes itself and culture
Base contacts tight, measuring samples described in 1.1 in above-mentioned steps 2 is added in Oxford cup, wherein in preceding 3 Oxford cups
Add each 200 μ L of same concentration, the same sample, the dimethyl sulfoxide of 200 μ L is added in the 4th Oxford cup, then there will be Oxford cup
The plate coated be put into 35 DEG C of incubators culture 18~take out afterwards for 24 hours, observe in plate whether have around Oxford cup it is antibacterial
Circle, and to the size for thering is the Oxford cup of inhibition zone vernier caliper to measure, record inhibition zone, the size of inhibition zone shows drug pair
The power of the inhibiting effect of bacterium；Sterile test tube 12 of φ 13mm × 100mm are taken, are arranged in a row, glucose phenol is added in every pipe
The drug stoste 1.0mL that concentration is 4 000 μ g/mL is added in the 1st pipe, mixes, so by red beef extract-peptone culture solution 1mL
1mL is drawn afterwards to be put into the 2nd pipe, is drawn in 1mL to the 3rd pipe again after mixing, so continuous proportion dilution, until the 10th pipe, and
1mL is drawn from the 10th pipe to discard；Only add 1mL solvent as solvent control group in 11st pipe, the 12nd pipe is the life that drug is not added
Long control；
2.3, above-mentioned each 1mL of the inoculum prepared is added in antibacterial detection in every pipe, makes the final bacterial concentration 1 of every pipe
×106CFU/mL；1st pipe drug quality concentration into the 10th pipe is respectively 2 000,1 000,500,250,125,62.5,
31.2, inoculated dilution tube is stoppered plug by 15.6,7.8,3.9 μ g/mL, is placed in 35 DEG C of normal air incubator and is incubated for
18~for 24 hours, bacterial growth makes glucose fermentation produce acid, reflects with phenolic red indicator and turns yellow, and positive pipe is yellow；No bacterium is raw
Long, liquid is still clear red person's negative tube in test tube；It is dense less than the highest drug dilution of bacterium colony growth to visually observe
Degree is minimal inhibitory concentration MIC of the sample to this kind of bacterium；The MIC for taking observation still to measure pipe for clear red person is negative
Culture solution is coated on beef-protein medium plate in pipe, and 35 DEG C of constant temperature incubations 18~for 24 hours；Observe and record clump count, bacterium
The minimum concentration for falling several < 3 or asepsis growth is the minimum bactericidal concentration MBC of Chinese fiber crops sample；
2.4, drug measures the minimal inhibitory concentration of fungi, weighs each 6.4mg of above 9 samples and is dissolved in 2mL dimethyl sulfoxide
In, mass concentration is 3200 μ g/mL；It is spare that 4 DEG C of refrigerators are stored in after 0.22 μm of filter degerming；Take 3200 μ g/mL
Each sample, the sample of 64 μ g/mL is obtained after first diluting 50 times with RPMI-1640 culture medium, twice of dilution is then carried out again, obtains
It is respectively 32,16,8,4,2,1,0.5,0.25,0.125 and 0.06 to dilution end mass concentration μ g/mL, totally 10 mass concentrations；
The drug of each mass concentration is added in 96 porocyte culture plates respectively, every 100 μ L of hole；1~10 to be classified as each quality of drug dense
Degree, 11 are classified as growth control, and 12 are classified as blank control and Vehicle controls；
2.5, for the detection of specific strain, dermatophyte is cultivated into 7~10d on PDA；Trichophyton rubrum is in oat medium
7~10d of upper culture；Candida albicans cultivates 24~48h on SDA；With 0.9% normal saline flushing skin tinea bacterium colony table
Face takes bacteria suspension in test tube, settles 10min, takes supernatant to be counted as 0.5 Maxwell turbidity than turbid instrument, be equivalent to 1 × 105~5 ×
105CFU/mL；It is 1 × 10 with the final bacteria suspension concentration that RPMI-1640 culture medium dilutes 100 times3~5 × 103CFU/mL；It is white
Color candida albicans is counted as 0. Maxwell turbidity than turbid instrument and is equivalent to 1 × 103~5 × 103CFU/mL；It is diluted with RPMI-1640 culture medium
2000 times of final bacteria suspension concentration is 0.5 × 103~2.5 × 103CFU/mL takes 100 μ L bacteria suspensions that prepared susceptibility is added
In the column of plate 1~11；35 DEG C of incubator cultures, wherein 4~6d of dermatophyte culture is observed as a result, 24~48h of Candida albicans culture
Observe result；
(3) result
3.1, the beef extract-peptone plate for being placed with Oxford cup is taken out the inhibition zone of bacterium by drug, and observing around Oxford cup is
It is no to have inhibition zone, then use the diameter of vernier calliper dipstick metering inhibition zone；Find out from inhibition zone result, the 3 of 70% ethanol elution
The antibacterial activity that de- object on kind resin has had, wherein with eluate has a broad antifungal spectrum under AB-8 resin；
3.2, in minimum-bacteriostat mass concentration MIC result it can be seen that the effect of the inhibitions fungi growth of C1 be it is best, remove A1,
A2, A3 are insensitive to microsporum canis outer, and remaining bacteriostasis is obvious, most strong with the inhibiting effect of C1；
3.3, minimal bactericidal concentration (MBC)；A1 is best to Escherichia coli bactericidal effect, and C1, which has wax bacillus, to be killed
Bacterium effect, but effect is unobvious, and B1 is more apparent to proteus and salmonella bactericidal effect.
2. a kind of environment-friendly function detection method of hemp stalk core fibre film according to claim 1, which is characterized in that the step
Rapid 2 (2) -2, it is 1,2 that 1:2n times of diluted n is carried out in 1 ... .., 9；I.e. extension rate is respectively 1: 2,1: 4,1: 8,1: 16,1
∶32、1∶64、1∶128、1∶256、1∶512。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110438198A (en) * | 2019-09-05 | 2019-11-12 | 湖南农业大学 | The ramee variety and organ screening technique of antibiotic are prepared based on grass meal extract |
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