CN110272966A - ATP fluorescence lgCA-lgIAThe method for marking bent method detection antibacterial polyurethane synthetic leather bacteria resistance energy - Google Patents

ATP fluorescence lgCA-lgIAThe method for marking bent method detection antibacterial polyurethane synthetic leather bacteria resistance energy Download PDF

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CN110272966A
CN110272966A CN201910200128.5A CN201910200128A CN110272966A CN 110272966 A CN110272966 A CN 110272966A CN 201910200128 A CN201910200128 A CN 201910200128A CN 110272966 A CN110272966 A CN 110272966A
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sample
atp
group
value
concentration
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李文杰
连素梅
娄巧哲
靳慧达
郝凌云
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/60—Chemiluminescent detection using ATP-luciferin-luciferase system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/25—Shigella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Abstract

The present invention relates to a kind of antibacterial polyurethane synthetic leather antibacterium method for testing performance, and detecting step includes: sample preparation and pretreatment；Lectotype selection and reagent, culture medium are prepared；Culture presevation, activation and bacteria suspension preparation；ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAMark Qu Jianli and inoculation bacterium solution viable bacteria ATP concentration CACalibration；Sample inoculation, culture and elution recycling；Recovered liquid IAMeasurement and its viable bacteria ATP concentration CAAnd TAIt calculates；Antibiotic rate R or antibacterial activity value A is calculated；Evaluation of result；Its special feature is that: accurate quantitative test can be carried out to the synthetic leather bacteria resistance with R or A characterization using ATP fluophotometer.Present invention provide that control sample and antimicrobial sample after elution recycling contact 0h and culture for 24 hours, measure recovered liquid IAAnd with lgIACharacterization and calculating R or A；Evaluation of result foundation is provided.The ATP bioluminescence lgC for the antibacterial polyurethane synthetic leather antibacterium performance detection that the present invention researches and developsA‑lgIABent method is marked, product quality will be supported to be promoted with advanced detection technique.

Description

ATP fluorescence lgCA-lgIAIt marks bent method and detects antibacterial polyurethane synthetic leather bacteria resistance energy Method
Technical field
The present invention relates to the antibacterium performance test methods of antibacterial polyurethane synthetic leather, specifically a kind of application ATP fluorescence Photometer can be carried out precisely quantitative inspection to the antibacterial polyurethane synthetic leather bacteria resistance with antibiotic rate R or antibacterial activity value A characterization The ATP bioluminescence lgC of surveyA-lgIACalibration curve method belongs to leather antibacterial Function detection technical field.
Background technique
Leather is human lives' necessity, due to introducing a large amount of fatting agents and raw materials used skin in leather making process rich in albumen Matter, the nutritional ingredients such as fat cause it easily by microbial infections such as bacterium, fungies.It is improved along with people's living standard Enhance with health perception, leather products anti-microbial property is required constantly soaring；Antibacterial leather comes into being and in daily necessities, clothes The fields such as shoes and hats, communal facility are widely used, application and universal irresistible, promotion correlation of the antimicrobial technology in Leather Industry Quality inspection technology and standardisation requirements are gradually brought into schedule.
Currently, anti-biotic material performance detection technical system is mainly for bacterium and fungi, various countries' bacteria resistance energy both at home and abroad Test method principle is mostly based on the colony counting method being separately cultured to bacterium, first is that the pad pasting training that Japanese Industrial Standards propose Support and impregnate quantitative test method；Two are derived from the antibacterial around-France of U.S. textile industry；Third is that international oscillation flask method；Four It is derived from the dropping method of U.S. textile enterprise；Fifth is that Japanese enterprises are directed to the coverslip method of photocatalyst-type anti-biotic product research and development.It cuts To currently, China has formulateeed and implemented QB/T 4341-2012 " antibacterial polyurethane synthetic leather Anti-microbial Performance Tests method and antibacterial effect Fruit ", but there are following common problems in technology contents and practical application for existing detection method: first is that involved experimental strain mistake It is few, it is difficult to meet the research and development of new product and Quality Control demand that enterprise grows with each passing hour；Second is that experimentation is cumbersome, dependence test is operated by reality The person's of testing professional experiences influence, and test error is big, lack comparativity；Third is that test period between 48 hours~72 hours, the time And economic cost is high.In recent years, increasingly mature in the development of international Bacteria Detection technical field ATP fluorescence analysis, it is flat with tradition Ware method is 98% compared to the correlation of ATP testing result, and accuracy is high and can realize quick detection；Developed country is by the method Apply in HACCP.Domestic ATP correlative study is started late, and is drawn by application demand, during existing ATP fluorescence detector has become The specified health supervision of hygiene department, state and the relevant special inspecting equipment of food safety.Current foreign countries' anti-biotic material performance detection Technical research focuses on testing result accuracy and comparativity to quantification, rapid and summary trend development；ATP is used for reference Fluorescence analysis principle formulates ISO20743:2007-2013 " Textiles-Determination of antibacterial Activity of textile products (the antibacterium performance measurement of textile-antibiotic finish textile) " and ISO 13629-1:2012《Textiles-Determination of antifungal activity of textile Products.Part1 Luminescence (measurement of textile-antibiotic finish textile fungicidal properties) ", it is specified that with sample The fluorimetry of the anti-thin/mould performance of ATP changes of contents characterization after product inoculation, but it is only applicable to water imbibition and control Sample is thin/textile material or poromerics of mould increasing value > 0, it is not suitable for inhaling in terms of the key technologies such as test condition for validity Aqueous poor and control sample bacterium increasing value < 0 synthetic leather material and product；Specific result calculation formula is not provided simultaneously It is assessed with uncertainty of measurement.In addition, the standard method dependent antimicrobial performance characterization parameter is relatively single, antibacterial activity is only related to Value A, and China then usual antibiotic rate R.
Therefore, to resist foreign technology barrier, specification domestic market order pushes industrial transformation upgrading, it would be highly desirable to research section It emulates the advanced, accuracy and reproducibility are high, easy-to-use antibacterial polyurethane synthesising leather performance measuring technology.The art of this patent route The world that integrates with is designed, detection method belongs to the whole world and initiates, and can effectively fill up domestic and international correlative technology field blank.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of application ATP fluophotometers to living with antibiotic rate R or antibacterial Property value A characterization antibacterial polyurethane synthetic leather bacteria resistance can be carried out the ATP bioluminescence lgC of detectionA-lgIACalibration curve method, It is able to solve antibacterial polyurethane synthetic leather or even other product scope anti-biotic materials and product bacteria resistance can accurate quantitative test Problem.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
A kind of detection method of antibacterial polyurethane synthetic leather bacteria resistance energy, comprising: (1) sample preparation and pretreatment；(2) Lectotype selection and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) ATP log concentration value lgCA- Relative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration；(5) sample inoculation, culture And elution recycling；(6) reclaim liquid phase is to fluorescence intensity level IAMeasurement；(7) the viable bacteria ATP concentration C of recovered liquidAAnd TACalculate and Antibiotic rate R and antibacterial activity value A is calculated；(8) evaluation of result；It is characterized in that, using ATP fluophotometer to antibiotic rate R Or the antibacterial polyurethane synthetic leather bacteria resistance of antibacterial activity value A characterization can be carried out the ATP bioluminescence of accurate quantitative test lgCA-lgIACalibration curve method, specific:
In reclaim liquid phase to fluorescence intensity level IAIn measurement:
Quantity, size and the pre-treatment requirement for specifying control sample and antimicrobial sample, testing standard bacterial strain is passed on, After activation, takes continuous switching 2 times Fresh bacterial cultures to prepare bacteria suspension and primary dcreening operation is carried out to its bacterium number.It is trained with test strain Nutrient solution is by 1.0 × 10-3The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10-9mol/L、 7.0×10-8mol/L、7.0×10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard Curve it is linear poor, its ATP concentration series can be changed to 7.0 × 10-10mol/L、7.0×10-9mol/L、7.0×10- 8) and 3.5 × 10 mol/L-11mol/L、3.5×10-10mol/L、3.5×10-9Mol/L, and measure its relative intensity of fluorescence value IA；Draw the lgC of two respective concentrationsA-lgIAStandard curve is derived from fitting equation Y=aA0X+bA0(high concentration), Y =aAX+bA(low concentration) and linearly dependent coefficient RA0 2(high concentration), RA 2(low concentration).It is 6.0 × 10 to bacteria total amount8CFU/ After the bacteria suspension of mL carries out 10 times of gradient dilutions, its relative intensity of fluorescence value I is measuredA, according to high standard fitting equation Y =aA0X+bA0Calculate corresponding viable bacteria ATP concentration CA；It is adjusted to obtain CARange is 5.0 × 10-8Mol/L~9.0 × 10- 8The inoculation bacterium solution of mol/L.Then, 1mL inoculation bacterium solution is added drop-wise to respectively on each group control sample and antimicrobial sample, is adopted immediately (24 ± 1) DEG C, 1min ± 5s, 250r/min~300r/min are carried out to 6 groups of 0h contact samples with 19mL test strain culture solution Oscillation elution, using the relative intensity of fluorescence value I of ATP fluorescent spectrophotometer measuring recovered liquidAC0ij、IAT0ij, according to low concentration mark Directrix curve equation Y=aAX+bACalculate the ATP concentration C of its viable bacteriaAOijAnd TAOij.Meanwhile by other 6 groups of samples in (37 ± 1) DEG C stationary culture after ± 2h, carries out elution recycling using mode identical with 0h contact sample, and apply ATP fluorophotometric for 24 hours Meter measures the relative intensity of fluorescence value I of its recovered liquidACtij、IATtij, calculate corresponding viable bacteria ATP concentration CAtijAnd TAtij；
In antibiotic rate R and antibacterial activity value A is calculated:
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, contacted with 0h and through quiet for 24 hours Set the relative intensity of fluorescence measured value of every control sample and antimicrobial sample recovered liquid after cultivatingWithMake For basic data；In the case where testing condition for validity, its antibiotic rate R is calculatedijWith antibacterial activity value Aij；To the R of every group of sampleijAnd Aij Arithmetic mean of instantaneous value is taken to obtain corresponding RiAnd Ai；The antibiotic rate R and antibacterial activity value A of every batch of antibacterial polyurethane synthetic leather sample be Its three groups of sample RiAnd AiArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement require；
In evaluation of result:
With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that antibacterium grading performance determines mark It is quasi-；As the antibiotic rate R of certain group (part) antibacterial polyurethane synthetic leather samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups When the bacteria resistance of (four) sample can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test； It is calculated through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back (part) antibacterial polyurethane synthetic leather sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining samples anti- Bacterium rate Ri(Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value it is anti-as batch (group) the antibacterial polyurethane synthetic leather sample The evaluation result of bacterium performance.
The present invention by adopting the above technical scheme, compared with prior art, beneficial effect is:
(1) advanced: using modern precision instrument-ATP fluophotometer as test equipment, it is dense to have reached viable bacteria ATP The modernization of degree measurement and Synthetic Leather antibacterium performance detection；Can effectively reduce human factor in experimentation influences, Avoid the generally existing large error of traditional plate culture；It can ensure the quantification of testing result, while significantly mention The accuracy of the Supreme People's Procuratorate's measured data；Detection technique has certain advance.
(2) scientific: according to ATP fluorescence analysis test philosophy, to establish the viable bacteria ATP concentration pair suitable for multi-cultur es Numerical value lgCA- relative intensity of fluorescence logarithm lgIAStandard curve；It is raw to construct antibacterial polyurethane synthetic leather bacteria resistance energy ATP The mathematical model of object fluorescence real-time quantitative analysis method；And take into account country variant consumer perceptions habit, take antibiotic rate and/it is anti- Bacterium activity value A is correlated performance evaluation index, improves the science and versatility of detection method.
(3) innovative: compared with the conventional method that existing operation is numerous, error is big, the period is long, this patent method was being tested Automation and the higher ATP fluophotometer of intelligent level are introduced in journey, are greatly simplified experimental procedure, are realized synthesis The precision of leather antibacterium performance test results and quantification simultaneously have good reproducibility and comparativity；It is greatly shortened simultaneously Test period reduces testing cost；Presently relevant the field of test technology blank can effectively be filled up.
(4) perspective: to establish with lgCA、lgIAStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously Enrich bacteria suspension viable bacteria concentration characterization and measuring method, specify control sample, standard liquid concentration, determination step, calculation formula, The technology contents such as uncertainty；The pioneering recovered liquid viable bacteria relative intensity of fluorescence value I directly measured with instrumentAIt evaluates as a result Form calculates and determines antibiotic rate R or antibacterial activity value A, and by group and a between-group variation coefficient CV to investigate measurement not true Fixed degree, technically has centainly perspective.
(5) operability: ATP fluophotometer is cheap, it is easy to operate, be widely used, this patent establish with sample It is easy easily that product are inoculated with the synthetic leather bacteria resistance energy ATP fluorometric investigation method that the variation of viable bacteria ATP concentration is attached most importance to after cultivating for 24 hours Row, description of Related Art is clear and specific, should be readily appreciated that and grasps；Have stronger operability in implementation process, is applicable in In the horizontal Experiment on Microbiology personnel of different majors, achievements conversion and popularization and application may advantageously facilitate.
(6) universality: because ATP is prevalent in, life entity is intracellular, and patented method can expand for experimental strain and provide tool The detection technique support for thering is wide spectrum to be worth；The introducing of pertinent instruments can greatly simplify experimental procedure, reduce testing cost；Be conducive to Expand in inspection, learn, grind, produce all circles and promote and apply, leather bacteria resistance energy detection technique realization generalization can be supported, while it can Reference is provided to the product scopes bacteria resistance energy detection technique research such as rubber, plastics.
Further, preferred embodiment of the invention is:
The sample preparation and pretreatment carries out in the steps below:
(1) control sample: being 50g/m from density2Plain polypropylene non-woven cloth on direct clip circle print, use Amount is advisable with sucking 1mL bacterium solution and not spilling over；Reference diameter d=(50 ± 2) mm.Each strain test uses 6 groups of samples；Wherein 0h Contact and for 24 hours culture experiment respectively use 3 groups, every group of 5 samples；
(2) antimicrobial sample: the direct clip circle print directly from antibacterial polyurethane synthetic leather material to be measured or product resists Shape, size, the quantity of bacterium sample are identical as control sample, every group of sample to be tested select one group of control sample as object of reference simultaneously Effectively mark；
(3) sample pre-treatments: selecting high pressure steam sterilization or other suitable sterilization methods according to sample to be tested material, will Each group control sample and antimicrobial sample after sterilizing are respectively put into spare in 50mL sterile conical flask；
The lectotype selection and reagent, culture medium are prepared, and are carried out in the steps below:
(1) General Requirement: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or Deionized water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience；
(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP The ATP bioluminescence rapid detection system of meter, matched reagent box, Special test tube etc. is spent, wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-13Mol/L~10-7mol/L；(37 ± 1) DEG C incubator；(0~50) DEG C ± 1 DEG C, the constant temperature oscillator of (50~300) r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~- 70 DEG C of low temperature refrigerator；2 DEG C~8 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range (30~50) kHz's is super Sound wave washer；The vortex oscillator of the range of speeds (500~3000) r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；
(3) material utensil: the sterile measuring pipette of 1mL, 10mL；The pipette of precision ± 0.01mL；0.05mL,0.1mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)；Capacity The sterile conical flask of 50mL, 150mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；Maxwell bacterium standard opacity tube and Mating test tube；Sterile test tube；Rack for test tube；Diameter is not more than the oese of 4mm；Disposable L stick；Alcolhol burner；Sterilize scissors and tweezer Son；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm；Thermometer；The stopwatch of precision 0.01s； A6 white copy paper；0.7mm core black gel ink pen；
(4) medium/liquid (commercially available medium/liquid can be used): through 121 DEG C of high pressure sterilizations after matched medium/liquid packing 15min, 2 DEG C~8 DEG C storage 30d；
Nutrient agar (NA): 5g beef extract, 10g peptone, 5g sodium chloride, 15g agar are dissolved by heating in 1000mL water In, adjust pH to 7.0~7.2；
Nutrient broth (NB): 3g beef extract, 10g peptone, 5g sodium chloride are dissolved by heating in 1000mL water, pH is adjusted To 7.0~7.2；
Salmonella preservation is with brain heart oxoid meat soup (BHI): by 10g peptone, 5g sodium chloride, 2.5g phosphoric acid hydrogen two Sodium, 2g glucose dissolve by heating in 500mL cattle heart leachate, adjust pH to 7.4 ± 0.2；
The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by 1:500/1:100 Nutrient broth (NB) mixed with 85% sterile saline solution, adjust pH to 7.0~7.2；
(5) ATP fluorescence reaction reagent (or with commercial reagent): in addition to physiological saline and phosphate buffer solution, matched ATP - 20 DEG C~-70 DEG C of fluorescence reaction reagent preservations, use in 6 months；
85% physiological saline；0.005mol/L and the disodium hydrogen phosphate dilution buffer for containing 0.037% sucrose are adjusted PH to 7.2 ± 0.2；121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；
ATP standard reagent stoste: by 60.5mg trinosin (C10H14O13P3Na2·3H2O) it is dissolved in 100mL In water, being configured to concentration is 1 × 10-3The solution of mol/L, -20 DEG C of freezings are sealed 6 months；
ATP fluorescent reagent buffer solution: by 1117mg trishydroxymethylaminomethane, 183mg disodium ethylene diamine tetraacetate, 808mg magnesium acetate, 6.7mg dithiothreitol, the beta-cyclodextrin of 25000mg and 925mg glucose are dissolved by heating in 250mL water In, adjust pH to 7.5 ± 0.2；It is used in 8h；
ATP lysate: 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 is international single Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, the 20mg bovine serum albumin of position/ml is dissolved in 10mL concentration is to adjust in pH to 6.0 ± 0.5,8h in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L and use (1mL cracking ATP concentration in nutrient broth can be down to 10 in 15min by liquid-13Mol/L or less)；
ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, is 10% with 0.2ml concentration Benza mix after, adjust pH to 12.0 ± 0.5；
ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg cow's serum Albumen is dissolved in the ATP fluorescent reagent buffer solution of 30mL, and 15min is stored at room temperature after mixing, is used in 3h；
Culture presevation, activation and the bacteria suspension preparation, carries out in the steps below:
(1) test strain: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or provided by national corresponding culture presevation administrative center and can be traced to the source other Strain)；
(2) culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (sramana is added with capillary syring Salmonella BHI broth), pressure-vaccum makes strain melt dispersion for several times.Then, a little test strain suspension is instilled and 5mL is housed In the test tube of~10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (does not surpass Spend 1 month), inoculation times were no more than for 14 generations；
(3) actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours； Switching 1 time daily, continuous switching are no more than 15d.Test is trained using the 3rd generation~the 14th generation and in the interior Fresh bacterial transferred for 24 hours Support object；
(4) prepared by bacteria suspension: scraping a small amount of Fresh bacterial from strain activation and culture base with oese, test strain is added Bacteria suspension is made in culture solution；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that bacterial suspension is equal It is even.Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, it is white in A6 with 0.7mm core black gel ink pen The parallel lines that 3 length are 10cm are drawn on color copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 108CFU/ ML culture solution is added dropwise until the two turbidity is identical in differences in turbidity)；
The ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution is living Bacterium ATP concentration CACalibration carries out in the steps below:
(1) standard serial solution is determining and prepared by ATP bioluminescence test specimens: using test strain culture solution by 1.0 × 10- 3The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10-9mol/L、7.0×10-8mol/L、 7.0×10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard curve it is linear compared with Its ATP concentration series can be changed to 7.0 × 10 by difference-10mol/L、7.0×10-9mol/L、7.0×10-8) and 3.5 × 10 mol/L-11mol/L、3.5×10-10mol/L、3.5×10-9Mol/L is simultaneously mixed.Then, with sterilizing liquid-transfering gun by the ATP standard of 0.1mL Serial solution is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose is successively added dropwise and mixes It is even；The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes again, as ATP bioluminescence Test Duplicate Samples；
(2) relative intensity of fluorescence value IAMeasurement: according to relative intensity of fluorescence value I in this patentAMeasuring method, with test organisms After kind culture solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, to various concentration The ATP that 0.1mL is added dropwise in three Duplicate Samples of ATP standard solution respectively extracts reagent, and the ATP fluorescence examination of 0.1mL is instilled after mixing Agent；It mixes again, with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and records immediately and (ensure each link operating time Unanimously, cross contamination is avoided).Each Duplicate Samples minute is no more than 15s, with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of relative intensity of fluorescence value is its IAMeasured value；
(3) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with ATP standard series The relative intensity of fluorescence logarithm lgI of solutionAAs abscissa, with its corresponding ATP log concentration value lgCAFor ordinate work Figure；Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrationsA-lgIAStandard curve；And it applies Least square fitting method is derived from the linear equation Y=a of curveA0X+bA0(high concentration), Y=aAX+bA(low concentration) and phase Close coefficients RA0 2(high concentration), RA 2(low concentration).Work as RA0 2And RA 2>=0.98, it when confidence level >=0.95, is done according to this patent Measurement it is effective；
(4) it is inoculated with the viable bacteria ATP concentration C of bacterium solutionACalibration: according to relative intensity of fluorescence value I in this patentAMeasuring method is right Bacteria total amount is 6.0 × 10 after the turbidimetry primary dcreening operation of Maxwell8The bacteria suspension of CFU/mL carries out 10 times of gradient dilutions, and it is opposite to measure it Fluorescence intensity level IA；According to high standard fitting equation Y=aA0X+bA0Calculate and demarcate the viable bacteria ATP concentration of dilution bacterium solution CA.It is adjusted through test strain culture solution, obtains CARange is 5.0 × 10-8Mol/L~9.0 × 10-8The inoculation bacterium solution of mol/L is surveyed Determine and records relative intensity of fluorescence value I of the 1mL inoculation bacterium solution after the dilution of 19mL eluent in 1minA0；
Sample inoculation, culture and the elution recycling, carries out in the steps below:
1mL inoculation bacterium solution is drawn (with lgC with sterilizing pipetteB-lgIBCalibration curve is derived from same branch test organisms with bacterium solution Kind stoste test tube, 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used), it is added drop-wise to each group control sample and antimicrobial sample surface to be measured respectively On, and smeared bacterium solution uniformly with disposable L stick (attachment is inoculated with bacterium solution but does not hang drop), so that its is covered sample whole surface, together When ensure that bacterium solution is not contacted with bottle wall.19mL examination is injected separately into the conical flask for filling 6 groups of 0h contact samples immediately after inoculation Bacteria culture fluid is tested, is sealed against being placed on constant temperature oscillator；(24 ± 1) DEG C are shaken with the revolving speed of 250r/min~300r/min 1min ± 5s is swung, and using mixing liquid in bottle as the recovered liquid of each group sample to be tested.Meanwhile the cone that other 6 groups of samples will be filled The sealing of shape bottle, (37 ± 1) DEG C stationary culture after ± 2h, 19mL test strain culture solution are injected into bottle and is stood again for 24 hours Then conical flask is placed on constant temperature oscillator by 5min, (24 ± 1) are DEG C with the speed oscillation 1min of 250r/min~300r/min ± 5s, and using mixing liquid in bottle as each group sample to be tested recovered liquid；
The reclaim liquid phase is to fluorescence intensity level IAMeasurement carries out in the steps below:
(1) instrument and reagent set relative intensity of fluorescence background value calibration: use sterilizing liquid-transfering gun by 0.1mL test strain culture The ATP lysate of liquid, 0.8mL phosphate buffer solution and 0.1mL containing 0.037% sucrose is separately added into same branch sterile test tube In and mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, stand 5min~ 30min, as skip test Duplicate Samples.The ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records (ensuring that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values)；
(2) reclaim liquid phase is to fluorescence intensity level IAMeasurement: it if blank reagent group background level meets instrument requirement, uses The ATP lysate for phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose that sterilize Be separately added into same branch sterile test tube, after mixing well by the above-mentioned mixed solution of 0.1mL successively move to three instruments without In bacterium test tube, 5min~30min is stood, as ATP fluorometric investigation Duplicate Samples.It is added dropwise 0.1mL's respectively into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, uses its phase of ATP fluorescent spectrophotometer measuring immediately To fluorescence intensity level IAAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute No more than 15s, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquidAMeasurement Value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the opposite of three Duplicate Samples of recovered liquid Fluorescence intensity level IA)；
The recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and antibacterial activity value A is calculated, in the steps below It carries out:
(1) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate control sample and antibacterial After sample is contacted through 0h and is cultivated for 24 hours, the viable bacteria ATP concentration C of recovered liquidAAnd TA；Relevant calculation (uses instrument matched reagent box When, the viable bacteria ATP concentration C of 1mL recovered liquid is calculated according to its practical sample volumeAAnd TA) see formula (1)~(12):
In formula:
CA0And CAtThe average ATP concentration that -3 groups of 0h are contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；
CA0iAnd CAtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
WithThe measured value of-every 0h contact control sample recovered liquid relative intensity of fluorescence after cultivating for 24 hours, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAtThe average ATP concentration that -3 groups of 0h are contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；
TA0iAnd TAtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
(2) condition for validity is tested
If the recovered liquid and 1mL inoculation bacterium solution of every 0h contact control sample are after the dilution of 19mL eluent in 1min Relative intensity of fluorescence measured value is close, i.e.,Every control sample recovered liquid relative intensity of fluorescence survey after cultivating for 24 hours The logarithm of definite valueThat is CAtij≥0.1×CA0ij；Then when 3 groups of 0h contact control sample reclaim liquid phase pair Fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent correlative measurement Measure uncertainty requirement), the measurement carried out according to this patent method is effective.
(3) bacterium increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample after cultivating for 24 hours, bacterium increasing value Fij、GijRespectively according to formula (13), (14) it calculates:
In formula:
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
TA0ijAnd TAtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(4) antibiotic rate R is calculated
It tests under condition for validity, the antibiotic rate R of every antibacterial polyurethane synthetic leather sampleij, every group and every batch of sample it is anti- Bacterium rate RiIt is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every antibacterial polyurethane synthetic leather sample, %；Sample group i=1,2,3；Every group of sample number into spectrum J=1,2,3,4,5；
RiThe antibiotic rate of-every group antibacterial polyurethane synthetic leather sample, %；Sample group i=1,2,3；
R-every batch of antibacterial polyurethane synthetic leather sample antibiotic rate, %；
CAtijAnd TAtij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after cultivating for 24 hours, single Position is mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
WithAfter cultivating for 24 hours, the relative intensity of fluorescence of recovered liquid is measured for-every antimicrobial sample and control sample Value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(5) antibacterial activity value A is calculated
It tests under condition for validity, the antibacterial activity value A of every antibacterial polyurethane synthetic leather sampleij, every group and every batch of sample Antibacterial activity value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every antibacterial polyurethane synthetic leather sample；Sample group i=1,2,3；Every group of sample is compiled Number j=1,2,3,4,5；
AiThe antibacterial activity value of-every group antibacterial polyurethane synthetic leather sample, sample group i=1,2,3；
A-every batch of antibacterial polyurethane synthetic leather sample antibacterial activity value；
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(6) data revision of the convention requirement: in the concentration and inoculation bacterium solution, sample recovered liquid of calibration ATP standard serial solution Viable bacteria ATP concentration CAWhen, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as CALess than 100mol/L When, " rounding up " round numbers；Work as CAWhen not less than 100mol/L, preceding 2 bit digital is taken after the 3rd bit digital " rounding up ", after Face replaces digit with 0；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals afterwards.Control sample and After antimicrobial sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate Rij、Ri, R calculate As a result three effective digitals are taken；Bacterium increasing value Fij、GijWith antibacterial activity value Aij、Ai, A calculated result take two effective digitals；
(7) uncertainty of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample and contacts through 0h and for 24 hours After culture, reclaim liquid phase is in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and C V calculated result remains into 2 significant digits), judge ATP fluorimetric assay for biological materials method being applied to Synthetic Leather antibacterium The reproducibility of performance test；In regulation group and between-group variation coefficient CV≤10%.
5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours of every group of 0h contact, 5 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after cultivating for 24 hours that 3 groups of 0h are contacted Part antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and for 24 hours After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours of every group of 0h contact, 5 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after cultivating for 24 hours that 3 groups of 0h are contacted Part antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and for 24 hours After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
The evaluation of result of the antibacterial polyurethane synthetic leather sample bacteria resistance energy, carries out in the steps below:
(1) with reference to health industry common practice and the requirement of dependent antimicrobial product standard, following classification criterion is taken:
If using antibiotic rate R as correlated performance evaluation index: working as Rij/Ri(staphylococcus aureus when/R < 90% With salmonella Rij/Ri/ R < 80%), sample is without antibacterial actions；As 90%≤Rij/Ri(golden yellow grape when/R < 99% 80%≤R of coccus and salmonellaij/Ri/ R < 90%), sample has antibacterial actions；As 99%≤Rij/Ri/ R < 99.9% When (90%≤R of staphylococcus aureus and salmonellaij/Ri/ R < 99%), sample antibacterial actions are stronger；Work as Rij/Ri/R (staphylococcus aureus and salmonella R when >=99.9%ij/Ri/ R >=99%), sample antibacterial actions are extremely strong；
If using antibacterial activity value A as correlated performance evaluation index: working as Aij/Ai(Staphylococcus aureus when/A < 1.0 Bacterium and salmonella Aij/Ai/ A < 0.5), sample is without antibacterial actions；As 1.0≤Aij/Ai(Staphylococcus aureus when/A < 2.0 0.5≤A of bacterium and salmonellaij/Ai/ A < 1.0), sample has antibacterial actions；As 2.0≤Aij/AiIt is (golden yellow when/A < 3.0 1.0≤A of color staphylococcus and salmonellaij/Ai/ A < 2.0), sample antibacterial actions are stronger；Work as Aij/Ai(gold when/A >=3.0 Staphylococcus aureus and salmonella Aij/Ai/ A >=2.0), sample antibacterial actions are extremely strong；
(2) as the antibiotic rate R of certain group (part) antibacterial polyurethane synthetic leather samplei(Rij) or antibacterial activity value Ai(Aij) and its When the bacteria resistance of his two groups of (four) samples can be compared to a levels be at least differed, one group of (part) sample is extracted again and is repeated Experiment；It is calculated through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If front and back Two groups of antibacterial polyurethane synthetic leather sample antibacterium performance levels are identical, then abandon it；Take other two groups (four) remaining samples anti- Bacterium rate Ri(Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value it is anti-as batch (group) the antibacterial polyurethane synthetic leather sample The evaluation result of bacterium performance；
Specific embodiment
Below with reference to preferred embodiment, the present invention will be described in detail, so that advantages and features of the invention can be easier to It is readily appreciated by one skilled in the art, so as to make a clearer definition of the protection scope of the present invention.
The present embodiment is with the antibacterial polyurethane synthetic leather sample bacteria resistance through nano silver system antimicrobial finish working process It can be illustrated for detection.
Specific detection method carries out in the steps below:
(1) sample preparation and pretreatment
1.1 control samples: being 50g/m from density2Plain polypropylene non-woven cloth on direct clip circle print, use Amount is advisable with sucking 1mL bacterium solution and not spilling over；Reference diameter d=(50 ± 2) mm.Each strain test uses 6 groups of samples；Wherein 0h Contact and for 24 hours culture experiment respectively use 3 groups, every group of 5 samples.
1.2 antimicrobial samples: the direct clip circle print directly from antibacterial polyurethane synthetic leather material to be measured or product resists Shape, size, the quantity of bacterium sample are identical as control sample, every group of sample to be tested select one group of control sample as object of reference simultaneously Effectively mark.
1.3 sample pre-treatments: selecting high pressure steam sterilization or other suitable sterilization methods according to sample to be tested material, will Each group control sample and antimicrobial sample after sterilizing are respectively put into spare in 50mL sterile conical flask.
(2) lectotype selection and reagent, culture medium are prepared
2.1 General Requirements: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or Deionized water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience.
2.2 instrument and equipments: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；The light of fluorescence containing ATP The ATP bioluminescence rapid detection system of meter, matched reagent box, Special test tube etc. is spent, wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-13Mol/L~10-7mol/L；(37 ± 1) DEG C incubator；(0~50) DEG C ± 1 DEG C, the constant temperature oscillator of (50~300) r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~- 70 DEG C of low temperature refrigerator；2 DEG C~8 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range (30~50) kHz's is super Sound wave washer；The vortex oscillator of the range of speeds (500~3000) r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace.
2.3 material utensils: the sterile measuring pipette of 1mL, 10mL；The pipette of precision ± 0.01mL；0.05mL,0.1mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)；Capacity The sterile conical flask of 50mL, 150mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；Maxwell bacterium standard opacity tube and Mating test tube；Sterile test tube；Rack for test tube；Diameter is not more than the oese of 4mm；Disposable L stick；Alcolhol burner；Sterilize scissors and tweezer Son；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm；Thermometer；The stopwatch of precision 0.01s； A6 white copy paper；0.7mm core black gel ink pen.
2.4 medium/liquids (can use commercially available medium/liquid): through 121 DEG C of high pressure sterilizations after matched medium/liquid packing 15min, 2 DEG C~8 DEG C storage 30d；
Nutrient agar (NA): 5g beef extract, 10g peptone, 5g sodium chloride, 15g agar are dissolved by heating in 1000mL water In, adjust pH to 7.0~7.2；
Nutrient broth (NB): 3g beef extract, 10g peptone, 5g sodium chloride are dissolved by heating in 1000mL water, pH is adjusted To 7.0~7.2；
Salmonella preservation is with brain heart oxoid meat soup (BHI): by 10g peptone, 5g sodium chloride, 2.5g phosphoric acid hydrogen two Sodium, 2g glucose dissolve by heating in 500mL cattle heart leachate, adjust pH to 7.4 ± 0.2；
The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by 1:500/1:100 Nutrient broth (NB) mixed with 85% sterile saline solution, adjust pH to 7.0~7.2.
2.5 ATP fluorescence reaction reagents (or with commercial reagent): in addition to physiological saline and phosphate buffer solution, matched - 20 DEG C~-70 DEG C of ATP fluorescence reaction reagent preservations, use in 6 months；
85% physiological saline；0.005mol/L and the disodium hydrogen phosphate dilution buffer for containing 0.037% sucrose are adjusted PH to 7.2 ± 0.2；121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；
ATP standard reagent stoste: by 60.5mg trinosin (C10H14O13P3Na2·3H2O) it is dissolved in 100mL In water, being configured to concentration is 1 × 10-3The solution of mol/L, -20 DEG C of freezings are sealed 6 months；
ATP fluorescent reagent buffer solution: by 1117mg trishydroxymethylaminomethane, 183mg disodium ethylene diamine tetraacetate, 808mg magnesium acetate, 6.7mg dithiothreitol, the beta-cyclodextrin of 25000mg and 925mg glucose are dissolved by heating in 250mL water In, adjust pH to 7.5 ± 0.2；It is used in 8h；
ATP lysate: 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 is international single Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, the 20mg bovine serum albumin of position/ml is dissolved in 10mL concentration is to adjust in pH to 6.0 ± 0.5,8h in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L and use (1mL cracking ATP concentration in nutrient broth can be down to 10 in 15min by liquid-13Mol/L or less)；
ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, is 10% with 0.2ml concentration Benza mix after, adjust pH to 12.0 ± 0.5；
ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg cow's serum Albumen is dissolved in the ATP fluorescent reagent buffer solution of 30mL, and 15min is stored at room temperature after mixing, is used in 3h.
(3) culture presevation, activation and bacteria suspension preparation
3.1 test strains: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or provided by national corresponding culture presevation administrative center and can be traced to the source other Strain).
3.2 culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (sramana is added with capillary syring Salmonella BHI broth), pressure-vaccum makes strain melt dispersion for several times.Then, a little test strain suspension is instilled and 5mL is housed In the test tube of~10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (does not surpass Spend 1 month), inoculation times were no more than for 14 generations.
3.3 actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours； Switching 1 time daily, continuous switching are no more than 15d.Test is trained using the 3rd generation~the 14th generation and in the interior Fresh bacterial transferred for 24 hours Support object.
The preparation of 3.4 bacteria suspensions: a small amount of Fresh bacterial is scraped from strain activation and culture base with oese, test strain is added Bacteria suspension is made in culture solution；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that bacterial suspension is equal It is even.Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, it is white in A6 with 0.7mm core black gel ink pen The parallel lines that 3 length are 10cm are drawn on color copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 108CFU/ ML culture solution is added dropwise until the two turbidity is identical in differences in turbidity).
(4) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration
4.1 standard serial solutions are determining and prepared by ATP bioluminescence test specimens: using test strain culture solution by 1.0 × 10- 3The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10-9mol/L、7.0×10-8mol/L、 7.0×10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard curve it is linear compared with Its ATP concentration series can be changed to 7.0 × 10 by difference-10mol/L、7.0×10-9mol/L、7.0×10-8) and 3.5 × 10 mol/L-11mol/L、3.5×10-10mol/L、3.5×10-9Mol/L is simultaneously mixed.Then, with sterilizing liquid-transfering gun by the ATP standard of 0.1mL Serial solution is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose is successively added dropwise and mixes It is even；The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes again, as ATP bioluminescence Test Duplicate Samples.
4.2 relative intensity of fluorescence value IAMeasurement: according to relative intensity of fluorescence value I in this patentAMeasuring method, with test organisms After kind culture solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, to various concentration The ATP that 0.1mL is added dropwise in three Duplicate Samples of ATP standard solution respectively extracts reagent, and the ATP fluorescence examination of 0.1mL is instilled after mixing Agent；It mixes again, with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and records immediately and (ensure each link operating time Unanimously, cross contamination is avoided).Each Duplicate Samples minute is no more than 15s, with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of relative intensity of fluorescence value is its IAMeasured value.
4.3ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with ATP standard series The relative intensity of fluorescence logarithm lgI of solutionAAs abscissa, with its corresponding ATP log concentration value lgCAFor ordinate work Figure；Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrationsA-lgIAStandard curve；And it applies Least square fitting method is derived from the linear equation Y=a of curveA0X+bA0(high concentration), Y=aAX+bA(low concentration) and phase Close coefficients RA0 2(high concentration), RA 2(low concentration).Work as RA0 2And RA 2>=0.98, it when confidence level >=0.95, is done according to this patent Measurement it is effective.
The viable bacteria ATP concentration C of 4.4 inoculation bacterium solutionsACalibration: according to relative intensity of fluorescence value I in this patentAMeasuring method is right Bacteria total amount is 6.0 × 10 after the turbidimetry primary dcreening operation of Maxwell8The bacteria suspension of CFU/mL carries out 10 times of gradient dilutions, and it is opposite to measure it Fluorescence intensity level IA；According to high standard fitting equation Y=aA0X+bA0Calculate and demarcate the viable bacteria ATP concentration of dilution bacterium solution CA.It is adjusted through test strain culture solution, obtains CARange is 5.0 × 10-8Mol/L~9.0 × 10-8The inoculation bacterium solution of mol/L is surveyed Determine and records relative intensity of fluorescence value I of the 1mL inoculation bacterium solution after the dilution of 19mL eluent in 1minA0。
(5) sample inoculation, culture and elution recycling
1mL inoculation bacterium solution is drawn (with lgC with sterilizing pipetteB-lgIBCalibration curve is derived from same branch test organisms with bacterium solution Kind stoste test tube, 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used), it is added drop-wise to each group control sample and antimicrobial sample surface to be measured respectively On, and smeared bacterium solution uniformly with disposable L stick (attachment is inoculated with bacterium solution but does not hang drop), so that its is covered sample whole surface, together When ensure that bacterium solution is not contacted with bottle wall.19mL examination is injected separately into the conical flask for filling 6 groups of 0h contact samples immediately after inoculation Bacteria culture fluid is tested, is sealed against being placed on constant temperature oscillator；(24 ± 1) DEG C are shaken with the revolving speed of 250r/min~300r/min 1min ± 5s is swung, and using mixing liquid in bottle as the recovered liquid of each group sample to be tested.Meanwhile the cone that other 6 groups of samples will be filled The sealing of shape bottle, (37 ± 1) DEG C stationary culture after ± 2h, 19mL test strain culture solution are injected into bottle and is stood again for 24 hours Then conical flask is placed on constant temperature oscillator by 5min, (24 ± 1) are DEG C with the speed oscillation 1min of 250r/min~300r/min ± 5s, and using mixing liquid in bottle as the recovered liquid of each group sample to be tested.
(6) reclaim liquid phase is to fluorescence intensity level IAMeasurement
6.1 instruments and reagent set relative intensity of fluorescence background value calibration: use sterilizing liquid-transfering gun by 0.1mL test strain culture The ATP lysate of liquid, 0.8mL phosphate buffer solution and 0.1mL containing 0.037% sucrose is separately added into same branch sterile test tube In and mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, stand 5min~ 30min, as skip test Duplicate Samples.The ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records (ensuring that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values).
6.2 reclaim liquid phases are to fluorescence intensity level IAMeasurement: it if blank reagent group background level meets instrument requirement, uses The ATP lysate for phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose that sterilize Be separately added into same branch sterile test tube, after mixing well by the above-mentioned mixed solution of 0.1mL successively move to three instruments without In bacterium test tube, 5min~30min is stood, as ATP fluorometric investigation Duplicate Samples.It is added dropwise 0.1mL's respectively into three Duplicate Samples ATP extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, uses its phase of ATP fluorescent spectrophotometer measuring immediately To fluorescence intensity level IAAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples minute No more than 15s, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquidAMeasurement Value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the opposite of three Duplicate Samples of recovered liquid Fluorescence intensity level IA)。
(7) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and antibacterial activity value A is calculated
7.1 recovered liquid viable bacteria ATP concentration CsAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate control sample and antibacterial After sample is contacted through 0h and is cultivated for 24 hours, the viable bacteria ATP concentration C of recovered liquidAAnd TA；Relevant calculation (uses instrument matched reagent box When, the viable bacteria ATP concentration C of 1mL recovered liquid is calculated according to its practical sample volumeAAnd TA) see formula (1)~(12):
In formula:
CA0And CAtThe average ATP concentration that -3 groups of 0h are contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；
CA0iAnd CAtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
WithThe measured value of-every 0h contact control sample recovered liquid relative intensity of fluorescence after cultivating for 24 hours, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAtThe average ATP concentration that -3 groups of 0h are contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；
TA0iAnd TAtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are Mole every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5.
7.2 test conditions for validity
If the recovered liquid and 1mL inoculation bacterium solution of every 0h contact control sample are after the dilution of 19mL eluent in 1min Relative intensity of fluorescence measured value is close, i.e.,Every control sample recovered liquid relative intensity of fluorescence survey after cultivating for 24 hours The logarithm of definite valueThat is CAtij≥0.1×CA0ij；Then when 3 groups of 0h contact control sample reclaim liquid phase pair Fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent correlative measurement Measure uncertainty requirement), the measurement carried out according to this patent method is effective.
7.3 bacterium increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample after cultivating for 24 hours, bacterium increasing value Fij、GijRespectively according to formula (13), (14) it calculates:
In formula:
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
TA0ijAnd TAtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are to rub You are every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope.
7.4 antibiotic rate R are calculated
It tests under condition for validity, the antibiotic rate R of every antibacterial polyurethane synthetic leather sampleij, every group and every batch of sample it is anti- Bacterium rate RiIt is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every antibacterial polyurethane synthetic leather sample, %；Sample group i=1,2,3；Every group of sample number into spectrum J=1,2,3,4,5；
RiThe antibiotic rate of-every group antibacterial polyurethane synthetic leather sample, %；Sample group i=1,2,3；
R-every batch of antibacterial polyurethane synthetic leather sample antibiotic rate, %；
CAtijAnd TAtij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after cultivating for 24 hours, single Position is mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
WithAfter cultivating for 24 hours, the relative intensity of fluorescence of recovered liquid is measured for-every antimicrobial sample and control sample Value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope.
7.5 antibacterial activity value A are calculated
It tests under condition for validity, the antibacterial activity value A of every antibacterial polyurethane synthetic leather sampleij, every group and every batch of sample Antibacterial activity value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every antibacterial polyurethane synthetic leather sample；Sample group i=1,2,3；Every group of sample is compiled Number j=1,2,3,4,5；
AiThe antibacterial activity value of-every group antibacterial polyurethane synthetic leather sample, sample group i=1,2,3；
A-every batch of antibacterial polyurethane synthetic leather sample antibacterial activity value；
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours control sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope.
7.6 data revisions of the convention requirement: in the concentration and inoculation bacterium solution, sample recovered liquid of calibration ATP standard serial solution Viable bacteria ATP concentration CAWhen, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as CALess than 100mol/L When, " rounding up " round numbers；Work as CAWhen not less than 100mol/L, preceding 2 bit digital is taken after the 3rd bit digital " rounding up ", after Face replaces digit with 0；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals afterwards.Control sample and After antimicrobial sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate Rij、Ri, R calculate As a result three effective digitals are taken；Bacterium increasing value Fij、GijWith antibacterial activity value Aij、Ai, A calculated result take two effective digitals.
7.7 uncertainties of measurement: this patent method, which mainly passes through, to be calculated control sample and antimicrobial sample and contacts through 0h and for 24 hours After culture, reclaim liquid phase is in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and C V calculated result remains into 2 significant digits), judge ATP fluorimetric assay for biological materials method being applied to Synthetic Leather antibacterium The reproducibility of performance test；In regulation group and between-group variation coefficient CV≤10%.
5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours of every group of 0h contact, 5 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after cultivating for 24 hours that 3 groups of 0h are contacted Part antimicrobial sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and for 24 hours After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
5 control samples, 5 antimicrobial samples and 5 control samples after cultivating for 24 hours of every group of 0h contact, 5 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples, 15 after cultivating for 24 hours that 3 groups of 0h are contacted Part antimicrobial sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to public affairs Formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k =1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are through 0h contact and for 24 hours After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):
(8) evaluation of result
8.1, with reference to health industry common practice and the requirement of dependent antimicrobial product standard, take following classification criterion:
If using antibiotic rate R as correlated performance evaluation index: working as Rij/Ri(staphylococcus aureus when/R < 90% With salmonella Rij/Ri/ R < 80%), sample is without antibacterial actions；As 90%≤Rij/Ri(golden yellow grape when/R < 99% 80%≤R of coccus and salmonellaij/Ri/ R < 90%), sample has antibacterial actions；As 99%≤Rij/Ri/ R < 99.9% When (90%≤R of staphylococcus aureus and salmonellaij/Ri/ R < 99%), sample antibacterial actions are stronger；Work as Rij/Ri/R (staphylococcus aureus and salmonella R when >=99.9%ij/Ri/ R >=99%), sample antibacterial actions are extremely strong；
If using antibacterial activity value A as correlated performance evaluation index: working as Aij/Ai(Staphylococcus aureus when/A < 1.0 Bacterium and salmonella Aij/Ai/ A < 0.5), sample is without antibacterial actions；As 1.0≤Aij/Ai(Staphylococcus aureus when/A < 2.0 0.5≤A of bacterium and salmonellaij/Ai/ A < 1.0), sample has antibacterial actions；As 2.0≤Aij/AiIt is (golden yellow when/A < 3.0 1.0≤A of color staphylococcus and salmonellaij/Ai/ A < 2.0), sample antibacterial actions are stronger；Work as Aij/Ai(gold when/A >=3.0 Staphylococcus aureus and salmonella Aij/Ai/ A >=2.0), sample antibacterial actions are extremely strong.
8.2 work as the antibiotic rate R of certain group (part) antibacterial polyurethane synthetic leather samplei(Rij) or antibacterial activity value Ai(Aij) and its When the bacteria resistance of his two groups of (four) samples can be compared to a levels be at least differed, one group of (part) sample is extracted again and is repeated Experiment；It is calculated through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures.If front and back Two groups of antibacterial polyurethane synthetic leather sample antibacterium performance levels are identical, then abandon it；Take other two groups (four) remaining samples anti- Bacterium rate Ri(Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value it is anti-as batch (group) the antibacterial polyurethane synthetic leather sample The evaluation result of bacterium performance.
Laboratory biosafety qualification, instrument and equipment, medium/liquid, chemical reagent, reference culture used in the present embodiment:
(1) Laboratory biosafety qualification
In the two stage biological safety experiment room that institute registration number is CNAS BL0059, by having secondary advanced techniques academic title Microorganism detection professional complete related experiment.
(2) instrument and equipment
2.1 two stage biological safety cabinets: 1300 series of secondary B2 type Biohazard Safety Equipment of Thermo Scientific, workbench Surface area 0.55m2, capacity 1130m3/ h, filter efficiency 99.99% (0.3 μm).
2.2 superclean benches: American blend Thermo ScientificTM HeraguardTM, model ECO ultra-clean work Make platform, inside width × depth × height=920mm × 585mm × 645mm；Air velocity is 0.15m/s~0.25m/s and 0.36m/s ~0.45m/s.
2.3 ATP bioluminescence rapid detection systems: the portable system SUREATP fluorescence of Hygiena company, the U.S. Detector, box containing matched reagent, plastics Special test tube etc.；ATP content detection lower limit 4 × 10-18Mol/ml, the inspection of microorganism total amount Rising limit 1.0CFU/ml, RLU range of readings 0~9999, detection time 10s, 1000 times/s of sampling rate, measurement error ± 5%.
2.4 constant temperature and humidity incubators: Guan Sen biotechnology (Shanghai) Co., Ltd. model WS-380H, volume 380L, temperature control The constant temperature and humidity incubator of ± 0.8 DEG C of range (0~50) DEG C, wet range (30~95) %RH ± 2%RH of control.
2.5 constant-temperature shaking incubators: the permanent model WS-380H in Shanghai one, ± 0.1 DEG C of temperature control range (4~65) DEG C, oscillation frequency Rate (40~300) r/min, timing range (1~99) h.
2.6 pressure steam sterilizers: the autoclave of Japanese Sanyo company model MLS-3780, volume 75L, sterilizing ± 2 DEG C of temperature (105~135) DEG C, maximum pressure 0.235MPa, timing range (1~250) min.
2.7 low temperature refrigerators: the superfreeze storage of Zhong Kemeiling low temperature Science and Technology Co., Ltd. model DW-HL398 Case, dischargeable capacity 398L, -10 DEG C~-86 DEG C of storage temperature.
2.8 refrigerating boxes: the Medical refrigerator of Zhong Kemeiling low temperature Science and Technology Co., Ltd. model YC-968L effectively holds Long-pending 968L, ± 0.1 DEG C of storage temperature (2~8) DEG C.
2.9 electronic balances: the electronic balance of Japanese Shimadzu model AUX220, range 220g, precision ± 0.1mg.
2.10 ultrasonic cleaners: the supersonic wave cleaning machine of Shanghai Yi Jing ultrasonic instrument Co., Ltd model YQ-120C, Capacity 3.2L, supersonic frequency 40KHz/28KHz/25KHz, timing range 1min~30min/99min.
2.11 vortex oscillators: the vortex oscillator of American blend Coleparmer Votex-Genie2, the range of speeds (500~3000) r/min ± 3r/min, timing range 1s~60s.
2.12 pH meters: the accurate pH meter of Shanghai precision instrumentation Co., Ltd model MP512-03, range ability (- 2.000~19.999) pH ± 0.002pH, ± 0.4 DEG C of temperature range (- 10~110) DEG C.
2.13 electric furnaces: the 1KW closed temp.-adjustable electric furnace of Changzhou Deco Instrument Ltd. model DLD-1KW.
(3) medium/liquid
The medium/liquid of Beijing overpass technical concern Co., Ltd production.
(4) chemical reagent
Trinosin standard items and prepare ATP fluorescent reagent buffer solution, ATP lysate, ATP extracting solution and A series of biochemical reagents needed for ATP fluorescent reagent are purchased from U.S.'s Amresco product of Shanghai Jin Pan Biotechnology Co., Ltd agency Board.
(5) reference culture
Escherichia coli ATCC 8739 and staphylococcus aureus ATCC 6538 is purchased from Chinese industrial microorganism fungus kind Preservation administrative center.
The detection data and result of the present embodiment calculate:
Using ATP fluophotometer through lgCA-lgIABacteria resistance of the calibration curve method to antibacterial Synthetic Leather sample It can be carried out detection, coherent detection data and Calculation of Measuring Uncertainty result are shown in Table 1~table 4 (Shigella and Salmonella respectively The testing result of bacterium is close with escherichia coli and the testing result of staphylococcus aureus respectively).
1 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result (escherichia coli)
2 relative intensity of fluorescence value Calculation of Measuring Uncertainty result (escherichia coli) of table
3 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result (staphylococcus aureus)
4 relative intensity of fluorescence Calculation of Measuring Uncertainty result (staphylococcus aureus) of table
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair Equivalent transformation made by bright description, is applied directly or indirectly in other relevant technical fields, and is included in this hair In bright scope of patent protection.

Claims (9)

1. a kind of detection method of antibacterial polyurethane synthetic leather bacteria resistance energy, comprising: (1) sample preparation and pretreatment；(2) it sets Alternative type and reagent, culture medium are prepared；(3) culture presevation, activation and bacteria suspension preparation；(4) ATP log concentration value lgCAPhase To fluorescence intensity logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CACalibration；(5) sample inoculation, culture and Elution recycling；(6) reclaim liquid phase is to fluorescence intensity level IAMeasurement；(7) the viable bacteria ATP concentration C of recovered liquidAAnd TAIt calculates and anti- Bacterium rate R and antibacterial activity value A is calculated；(8) evaluation of result；It is characterized in that, using ATP fluophotometer to antibiotic rate R or The antibacterial polyurethane synthetic leather bacteria resistance of antibacterial activity value A characterization can be carried out the ATP bioluminescence lgC of accurate quantitative testA- lgIACalibration curve method, specific:
In reclaim liquid phase to fluorescence intensity level IAIn measurement:
Quantity, size and the pre-treatment requirement for specifying control sample and antimicrobial sample, testing standard bacterial strain is passed on, is activated Afterwards, it takes continuous switching 2 times Fresh bacterial cultures to prepare bacteria suspension and primary dcreening operation is carried out to its bacterium number, with test strain culture solution By 1.0 × 10-3The ATP standard stock solution of mol/L is diluted to high and low concentration standard serial solution: 7.0 × 10-9mol/L、7.0× 10-8mol/L、7.0×10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard curve It is linear poor, its ATP concentration series can be changed to 7.0 × 10-10mol/L、7.0×10-9mol/L、7.0×10-8mol/L) With 3.5 × 10-11mol/L、3.5×10-10mol/L、3.5×10-9Mol/L, and measure its relative intensity of fluorescence value IA；Draw two The lgC of respective concentrationA-lgIAStandard curve is derived from fitting equation Y=aA0X+bA0(high concentration), Y=aAX+bAIt is (low Concentration) and linearly dependent coefficient RA0 2(high concentration), RA 2(low concentration) is 6.0 × 10 to bacteria total amount8The bacteria suspension of CFU/mL After carrying out 10 times of gradient dilutions, its relative intensity of fluorescence value I is measuredA, according to high standard fitting equation Y=aA0X+bA0It pushes away Calculate corresponding viable bacteria ATP concentration CA；It is adjusted to obtain CARange is 5.0 × 10-8Mol/L~9.0 × 10-8The inoculation bacterium of mol/L Then 1mL inoculation bacterium solution is added drop-wise on each group control sample and antimicrobial sample respectively, uses 19mL test strain immediately by liquid Culture solution elutes the oscillation of 6 groups of 0h contact samples progress (24 ± 1) DEG C, 1min ± 5s, 250r/min~300r/min, application The relative intensity of fluorescence value I of ATP fluorescent spectrophotometer measuring recovered liquidAC0ij、IAT0ij, according to low concentration calibration curve equation formula Y= aAX+bACalculate the ATP concentration C of its viable bacteriaAOijAnd TAOij, meanwhile, by other 6 groups of samples in (37 ± 1) DEG C stationary culture for 24 hours ± After 2h, elution recycling is carried out using mode identical with 0h contact sample, and using its recovered liquid of ATP fluorescent spectrophotometer measuring Relative intensity of fluorescence value IACtij、IATtij, calculate corresponding viable bacteria ATP concentration CAtijAnd TAtij；
In antibiotic rate R and antibacterial activity value A is calculated:
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, contacted with 0h and through stationary culture for 24 hours The relative intensity of fluorescence measured value of every control sample and antimicrobial sample recovered liquid afterwardsWithBased on Data；In the case where testing condition for validity, its antibiotic rate R is calculatedijWith antibacterial activity value Aij；To the R of every group of sampleijAnd AijTake arithmetic Average value obtains corresponding RiAnd Ai；The antibiotic rate R and antibacterial activity value A of every batch of antibacterial polyurethane synthetic leather sample are its three groups Sample RiAnd AiArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement require；
In evaluation of result:
With reference to health industry common practice and the requirement of dependent antimicrobial product standard, antibacterium grading performance criterion is determined；When The antibiotic rate R of certain group (part) antibacterial polyurethane synthetic leather samplei(Rij) or antibacterial activity value Ai(Aij) and other two groups (four) When the bacteria resistance of sample can be compared to a levels be at least differed, one group of (part) sample is extracted again and repeats to test；Calculate it Through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back (part) antibacterial Synthetic Leather sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate Ri (Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value as batch (group) antibacterial polyurethane synthetic leather sample bacteria resistance The evaluation result of energy.
2. the detection method of antibacterial polyurethane synthetic leather bacteria resistance energy according to claim 1, which is characterized in that described Sample preparation and pretreatment, in the steps below carry out:
(1) control sample: being 50g/m from density2Plain polypropylene non-woven cloth on direct clip circle print, dosage with Sucking 1mL bacterium solution, which is not spilt over, to be advisable；Reference diameter d=(50 ± 2) mm, each strain test use 6 groups of samples；Wherein 0h is contacted Culture experiment respectively uses 3 groups for 24 hours, every group of 5 samples；
(2) antimicrobial sample: direct clip circle print, antibacterial sample directly from antibacterial polyurethane synthetic leather material to be measured or product Shape, size, the quantity of product are identical as control sample, and every group of sample to be tested selects one group of control sample as object of reference and effective Mark；
(3) sample pre-treatments: high pressure steam sterilization or other suitable sterilization methods are selected according to sample to be tested material, will be sterilized Each group control sample and antimicrobial sample afterwards is respectively put into spare in 50mL sterile conical flask.
3. the detection method of antibacterial polyurethane synthetic leather bacteria resistance energy according to claim 1, which is characterized in that described Lectotype selection and reagent, culture medium prepare, in the steps below carry out:
(1) General Requirement: test analytical reagents and meet tertiary effluent as defined in GB/T 6682-2008 (distilled water or go from Sub- water), laboratory has the safe qualification of two stage biological, and personnel have regular Microbiological Lab's working experience；
(2) instrument and equipment: the superclean bench of two stage biological safety cabinet or lustration class not less than 100；Fluorophotometric containing ATP The ATP bioluminescence rapid detection system of meter, matched reagent box, Special test tube etc., wherein ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10-13Mol/L~10-7mol/L；(37 ± 1) DEG C incubator；(0~50) DEG C ± 1 DEG C, the constant temperature oscillator of (50~300) r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~- 70 DEG C of low temperature refrigerator；2 DEG C~8 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range (30~50) kHz's is super Sound wave washer；The vortex oscillator of the range of speeds (500~3000) r/min；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；
(3) material utensil: the sterile measuring pipette of 1mL, 10mL；The pipette of precision ± 0.01mL；0.05mL,0.1mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)；Capacity The sterile conical flask of 50mL, 150mL, 250mL, 500mL；The sterile petri dish of diameter 90mm；Maxwell bacterium standard opacity tube and Mating test tube；Sterile test tube；Rack for test tube；Diameter is not more than the oese of 4mm；Disposable L stick；Alcolhol burner；Sterilize scissors and tweezer Son；Cotton ball soaked in alcohol (75%)；The ruler of scale division value 1mm；Thermometer；The stopwatch of precision 0.01s； A6 white copy paper；0.7mm core black gel ink pen；
(4) medium/liquid (commercially available medium/liquid can be used): through 121 DEG C of high pressure sterilization 15min after the packing of matched medium/liquid, 2 DEG C~8 DEG C of storage 30d；
Nutrient agar (NA): 5g beef extract, 10g peptone, 5g sodium chloride, 15g agar are dissolved by heating in 1000mL water, adjusted Save pH to 7.0~7.2；
Nutrient broth (NB): by 3g beef extract, 10g peptone, 5g sodium chloride dissolve by heating in 1000mL water, adjust pH to 7.0~7.2；
Salmonella preservation is with brain heart oxoid meat soup (BHI): by 10g peptone, 5g sodium chloride, 2.5g disodium hydrogen phosphate, 2g Glucose dissolves by heating in 500mL cattle heart leachate, adjusts pH to 7.4 ± 0.2；
The culture solution of escherichia coli and Shigella/staphylococcus aureus and salmonella: by the battalion of 1:500/1:100 It supports meat soup (NB) to mix with 85% sterile saline solution, adjusts pH to 7.0~7.2；
(5) ATP fluorescence reaction reagent (or with commercial reagent): in addition to physiological saline and phosphate buffer solution, matched ATP fluorescence - 20 DEG C~-70 DEG C of reaction reagent preservations, use in 6 months；
85% physiological saline；0.005mol/L and contain 0.037% sucrose disodium hydrogen phosphate dilution buffer, adjust pH to 7.2±0.2；121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；
ATP standard reagent stoste: by 60.5mg trinosin (C10H14O13P3Na2·3H2O it) is dissolved in 100mL water, Being configured to concentration is 1 × 10-3The solution of mol/L, -20 DEG C of freezings are sealed 6 months；
ATP fluorescent reagent buffer solution: by 1117mg trishydroxymethylaminomethane, 183mg disodium ethylene diamine tetraacetate, 808mg Magnesium acetate, 6.7mg dithiothreitol, the beta-cyclodextrin of 25000mg and 925mg glucose dissolve by heating in 250mL water, adjust Save pH to 7.5 ± 0.2；It is used in 8h；
ATP lysate: by 4.6 international units/ml apyrase (EC:3.6.1.5) and 46 international units/ml Adenosine phosphate deaminase (EC:3.5.4.6 or EC:3.5.4.17), 37mg sucrose, that 20mg bovine serum albumin is dissolved in 10mL is dense Degree is in the 2-morpholine ethane sulfonic acid buffer solution of 0.05mol/L, and adjusting use in pH to 6.0 ± 0.5,8h, (1mL lysate exists The ATP concentration in nutrient broth can be down to 10 in 15min-13Mol/L or less)；
ATP extracting solution: 45mg trishydroxymethylaminomethane is dissolved by heating in 9.8ml water, the benzene for being 10% with 0.2ml concentration After pricking the mixing of oronain solution, pH to 12.0 ± 0.5 is adjusted；
ATP fluorescent reagent: by 16mg luciferase (EC:1.13.12.7), the D- fluorescein of 12.6mg, 56mg bovine serum albumin It is dissolved in the ATP fluorescent reagent buffer solution of 30mL, 15min is stored at room temperature after mixing, is used in 3h.
4. the detection method of antibacterial polyurethane synthetic leather bacteria resistance energy according to claim 1, which is characterized in that described Culture presevation, activation and bacteria suspension preparation, in the steps below carry out:
(1) test strain: escherichia coli ATCC 8739；Staphylococcus aureus ATCC 6538；Salmonella ATCC 14028；Shigella CMCC 51105 (or other strains that is provided and can be traced to the source by national corresponding culture presevation administrative center)；
(2) culture presevation: opening freeze-drying lactobacillus pipe with sterile working, and adequate nutrition meat soup (salmonella is added with capillary syring With BHI broth), pressure-vaccum make for several times strain melt dispersion, then, by a little test strain suspension instill equipped with 5mL~ In the test tube of 10mL nutrient broth, (37 ± 1) DEG C culture for 24 hours~48h；As slant preservation bacterium, (5 ± 1) DEG C storage (is no more than 1 month), inoculation times were no more than for 14 generations；
(3) actication of culture: slant preservation bacterium is transferred nutrient agar slant medium, and (37 ± 1) DEG C culture 18h~for 24 hours；Daily Switching 1 time, continuously switching is no more than 15d, and test is using the 3rd generation~the 14th generation and in the interior Fresh bacterial cultures transferred for 24 hours；
(4) prepared by bacteria suspension: scraping a small amount of Fresh bacterial from strain activation and culture base with oese, test strain culture is added Bacteria suspension is made in liquid；1000r/min shakes test tube 1min or with 30cm amplitude of oscillation shaking test tube 80 times, it is ensured that and bacterial suspension is uniform, Appropriate bacteria suspension is moved in sterile test tube matched with Maxwell standard opacity tube, with 0.7mm core black gel ink pen in A6 white The parallel lines that 3 length are 10cm are drawn on copy paper；Estimate test tube and standard opacity tube (nominal concentration 6.0 × 108CFU/mL) Differences in turbidity, be added dropwise culture solution until the two turbidity it is identical until.
5. the detection method of antibacterial polyurethane synthetic leather bacteria resistance energy according to claim 1, which is characterized in that described ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration CA Calibration carries out in the steps below:
(1) standard serial solution is determining and prepared by ATP bioluminescence test specimens: using test strain culture solution by 1.0 × 10-3mol/ The ATP standard stock solution of L is diluted to high and low concentration standard serial solution: 7.0 × 10-9mol/L、7.0×10-8mol/L、7.0× 10-7Mol/L is (if instrument upper limit of detection is unable to reach 10-7Mol/L magnitude or high standard curve it is linear poor, can will Its ATP concentration series is changed to 7.0 × 10-10mol/L、7.0×10-9mol/L、7.0×10-8) and 3.5 × 10 mol/L-11mol/ L、3.5×10-10mol/L、3.5×10-9Mol/L is simultaneously mixed, then, with sterilizing liquid-transfering gun that the ATP standard series of 0.1mL is molten Liquid is moved to respectively in three sterile test tubes, and phosphate buffer of the 0.9mL containing 0.037% sucrose and mixing is successively added dropwise；Again will The mixed solution of 0.1mL various concentration is moved to respectively in three instrument sterile test tubes, parallel as the test of ATP bioluminescence Sample；
(2) relative intensity of fluorescence value IAMeasurement: according to relative intensity of fluorescence value I in this patentAMeasuring method is trained with test strain After nutrient solution is as blank sample liquid validation instrument and reagent set background；According to the sequence of concentration from low to high, marked to various concentration ATP The ATP that 0.1mL is added dropwise in three Duplicate Samples of quasi- solution respectively extracts reagent, and the ATP fluorescent reagent of 0.1mL is instilled after mixing；Again Secondary mixing, immediately with its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring and record (ensure that each link operating time is consistent, Avoid cross contamination), each Duplicate Samples minute is no more than 15s, relatively glimmering with each three Duplicate Samples of concentration ATP standard solution The arithmetic mean of instantaneous value of light intensity value is its IAMeasured value；
(3) ATP log concentration value lgCARelative intensity of fluorescence logarithm lgIAStandard curve is established: with ATP standard serial solution Relative intensity of fluorescence logarithm lgIAAs abscissa, with its corresponding ATP log concentration value lgCAFor ordinate mapping；It is right Mathematical relationship between the two carries out calibration curve, draws the lgC of two respective concentrationsA-lgIAStandard curve；And application is minimum Two multiply the linear equation Y=a that fitting process is derived from curveA0X+bA0(high concentration), Y=aAX+bA(low concentration) and phase relation Number RA0 2(high concentration), RA 2(low concentration), works as RA0 2And RA 2>=0.98, when confidence level >=0.95, the survey done according to this patent It is fixed effective；
(4) it is inoculated with the viable bacteria ATP concentration C of bacterium solutionACalibration: according to relative intensity of fluorescence value I in this patentAMeasuring method, to through wheat Bacteria total amount is 6.0 × 10 after family name's turbidimetry primary dcreening operation8The bacteria suspension of CFU/mL carries out 10 times of gradient dilutions, measures its relative fluorescence Intensity value IA；According to high standard fitting equation Y=aA0X+bA0Calculate and demarcate the viable bacteria ATP concentration C of dilution bacterium solutionA, It is adjusted through test strain culture solution, obtains CARange is 5.0 × 10-8Mol/L~9.0 × 10-8The inoculation bacterium solution of mol/L, measurement And record relative intensity of fluorescence value I of the 1mL inoculation bacterium solution after the dilution of 19mL eluent in 1minA0。
6. the detection method of antibacterial polyurethane synthetic leather bacteria resistance energy according to claim 1, which is characterized in that described Sample inoculation, culture and elution recycling, in the steps below carry out:
1mL inoculation bacterium solution is drawn (with lgC with sterilizing pipetteB-lgIBCalibration curve is derived from same branch test strain original with bacterium solution Liquid test tube, 2 DEG C of ± 0.2 DEG C of preservations, 2h is interior to be used), it is added drop-wise on each group control sample and antimicrobial sample surface to be measured respectively, and Bacterium solution is smeared uniformly with disposable L stick (attachment is inoculated with bacterium solution but does not hang drop), so that its is covered sample whole surface, ensures simultaneously Bacterium solution is not contacted with bottle wall, is injected separately into 19mL test strain into the conical flask for filling 6 groups of 0h contact samples immediately after inoculation Culture solution is sealed against being placed on constant temperature oscillator；(24 ± 1) are DEG C with the speed oscillation 1min of 250r/min~300r/min ± 5s, and using mixing liquid in bottle as the recovered liquid of each group sample to be tested, meanwhile, the conical flask for filling other 6 groups of samples is close Envelope, (37 ± 1) DEG C stationary culture after ± 2h, 19mL test strain culture solution are injected into bottle and stands 5min again, then for 24 hours Conical flask is placed on constant temperature oscillator, (24 ± 1) DEG C, and will with speed oscillation 1min ± 5s of 250r/min~300r/min Recovered liquid of the mixing liquid as each group sample to be tested in bottle.
7. the detection method of antibacterial polyurethane synthetic leather bacteria resistance energy according to claim 1, which is characterized in that described Reclaim liquid phase to fluorescence intensity level IAMeasurement carries out in the steps below:
(1) instrument and reagent set relative intensity of fluorescence background value calibration: with sterilizing liquid-transfering gun by 0.1mL test strain culture solution, The ATP lysate of phosphate buffer solution and 0.1mL of the 0.8mL containing 0.037% sucrose is separately added into same branch sterile test tube And mix well, then the above-mentioned mixed solution of 0.1mL is successively moved in three instrument sterile test tubes, standing 5min~ 30min, as skip test Duplicate Samples, the ATP that 0.1mL is added dropwise respectively into three Duplicate Samples extracts reagent, instills after mixing The ATP fluorescent reagent of 0.1mL；It mixes again, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediatelyAAnd it records (ensuring that each link operating time is consistent, avoid cross contamination), each Duplicate Samples minute is no more than 15s, with three blank The arithmetic mean of instantaneous value of Duplicate Samples relative intensity of fluorescence value is tested (or to use according to pertinent instruments as instrument and reagent set background values Illustrate to calibrate background values)；
(2) reclaim liquid phase is to fluorescence intensity level IAMeasurement: if blank reagent group background level meets instrument requirement, with sterilizing The ATP lysate of phosphate buffer solution and 0.1mL of the liquid-transfering gun by 0.1mL recovered liquid, 0.8mL containing 0.037% sucrose is distinguished It is added in same branch sterile test tube, the above-mentioned mixed solution of 0.1mL is successively moved into three sterile examinations of instrument after mixing well Guan Zhong stands 5min~30min, and as ATP fluorometric investigation Duplicate Samples, the ATP that 0.1mL is added dropwise respectively into three Duplicate Samples is mentioned Reagent is taken, the ATP fluorescent reagent of 0.1mL is instilled after mixing；It mixes again, with ATP fluorescent spectrophotometer measuring, it is relatively glimmering immediately Light intensity value IAAnd record and (ensure that each link operating time is consistent, avoid cross contamination), each Duplicate Samples minute does not surpass 15s is crossed, using the arithmetic mean of instantaneous value of three ATP fluorometric investigation Duplicate Samples relative intensity of fluorescence values as the I of recovered liquidAMeasured value (or instrument matched reagent box is used, it is required according to operation instructions, directly upper machine measures the relatively glimmering of three Duplicate Samples of recovered liquid Light intensity value IA)。
8. the detection method of antibacterial polyurethane synthetic leather bacteria resistance energy according to claim 1, which is characterized in that described Recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates and antibiotic rate R and antibacterial activity value A is calculated, carry out in the steps below:
(1) recovered liquid viable bacteria ATP concentration CAAnd TAIt calculates
According to ATP low concentration standard curve lgCA-lgIALinear equation Y=aAX+bA, calculate control sample and antimicrobial sample After contacting through 0h and cultivating for 24 hours, the viable bacteria ATP concentration C of recovered liquidAAnd TA；Relevant calculation (when using instrument matched reagent box, root The viable bacteria ATP concentration C of 1mL recovered liquid is calculated according to its practical sample volumeAAnd TA) see formula (1)~(12):
In formula:
CA0And CAt- 3 groups of 0h contact and after cultivating for 24 hours control sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；
CA0iAnd CAtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after cultivating for 24 hours, unit are mole Every liter (mol/L)；Sample group i=1,2,3；
CA0ijAnd CAtij- every 0h contact and after cultivating for 24 hours control sample recycling viable bacteria ATP concentration, unit is mole every It rises (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
WithThe measured value of-every 0h contact control sample recovered liquid relative intensity of fluorescence after cultivating for 24 hours, RLU；Sample Product group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；bA- low concentration standard curve lgCA-lgIAIn vertical axis intercept；
TA0And TAt- 3 groups of 0h contact and after cultivating for 24 hours antimicrobial sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；
TA0iAnd TAtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after cultivating for 24 hours, unit are mole Every liter (mol/L)；Sample group i=1,2,3；
TA0ijAnd TAtij- every 0h contact and after cultivating for 24 hours antimicrobial sample recycling viable bacteria ATP concentration, unit is mole every It rises (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
(2) condition for validity is tested
If the recovered liquid of every 0h contact control sample and 1mL inoculation bacterium solution are opposite in 1min after the dilution of 19mL eluent Fluorescent strength determining value is close, i.e.,Every after cultivating for 24 hours control sample reclaim liquid phase to fluorescent strength determining value LogarithmThat is CAtij≥0.1×CA0ij；Then when 3 groups of 0h contact control sample recovered liquid relative fluorescence Strength detection valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent measurement of correlation not Degree of certainty requirement), the measurement carried out according to this patent method is effective；
(3) bacterium increasing value Fij、GijIt calculates
Every control sample and antimicrobial sample after cultivating for 24 hours, bacterium increasing value Fij、GijIt is counted respectively according to formula (13), (14) It calculates:
In formula:
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1,2,3；Often Group sample number into spectrum j=1,2,3,4,5；
CA0ijAnd CAtij- every 0h contact and after cultivating for 24 hours control sample recycling viable bacteria ATP concentration, unit is mole every It rises (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours control sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
TA0ijAnd TAtij- every 0h contact and after cultivating for 24 hours antimicrobial sample recycling viable bacteria ATP concentration, unit is mole every It rises (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(4) antibiotic rate R is calculated
It tests under condition for validity, the antibiotic rate R of every antibacterial polyurethane synthetic leather sampleij, every group and every batch of sample antibiotic rate RiIt is calculated respectively according to formula (15), (16), (17) with R:
In formula:
RijThe antibiotic rate of-every antibacterial polyurethane synthetic leather sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j= 1,2,3,4,5；
RiThe antibiotic rate of-every group antibacterial polyurethane synthetic leather sample, %；Sample group i=1,2,3；
R-every batch of antibacterial polyurethane synthetic leather sample antibiotic rate, %；
CAtijAnd TAtij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after cultivating for 24 hours, and unit is Mole every liter (mol/L)；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every antimicrobial sample and control sample after cultivating for 24 hours, the relative intensity of fluorescence measured value of recovered liquid, RLU；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(5) antibacterial activity value A is calculated
It tests under condition for validity, the antibacterial activity value A of every antibacterial polyurethane synthetic leather sampleij, every group and every batch of sample it is anti- Bacterium activity value AiIt is calculated respectively according to formula (18), (19), (20) with A:
In formula:
AijThe antibacterial activity value of-every antibacterial polyurethane synthetic leather sample；Sample group i=1,2,3；Every group of sample number into spectrum j= 1,2,3,4,5；
AiThe antibacterial activity value of-every group antibacterial polyurethane synthetic leather sample, sample group i=1,2,3；
A-every batch of antibacterial polyurethane synthetic leather sample antibacterial activity value；
FijAnd GijThe bacterium increasing value of-every control sample and antimicrobial sample after cultivating for 24 hours, sample group i=1,2,3；Often Group sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours control sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
With- every 0h contact and after cultivating for 24 hours antimicrobial sample recovered liquid relative intensity of fluorescence measured value, RLU； Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；
aA- low concentration standard curve lgCA-lgIASlope；
(6) data revision of the convention requirement: in the concentration of calibration ATP standard serial solution and the viable bacteria of inoculation bacterium solution, sample recovered liquid ATP concentration CAWhen, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count；Work as CAWhen less than 100mol/L, " rounding up " round numbers；Work as CAWhen not less than 100mol/L, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind use 0 replaces digit；It can also be indicated with 10 exponential form, " rounding up " uses two effective digitals, control sample and antibacterial afterwards After sample is contacted through 0h and cultivated for 24 hours, reclaim liquid phase is to fluorescent strength determining value round numbers, antibiotic rate Rij、Ri, R calculated result Take three effective digitals；Bacterium increasing value Fij、GijWith antibacterial activity value Aij、Ai, A calculated result take two effective digitals；
(7) uncertainty of measurement: this patent method mainly passes through calculating control sample and antimicrobial sample and contacts through 0h and cultivate for 24 hours Afterwards, reclaim liquid phase in the group of fluorescent strength determining value and between-group variation coefficient CV=σ ÷ μ × 100% (μ, σ and CV count Calculate result and remain into 2 significant digits), judge ATP fluorimetric assay for biological materials method being applied to Synthetic Leather bacteria resistance energy The reproducibility of test；In regulation group and between-group variation coefficient CV≤10%；
5 control samples, 5 antimicrobial samples and 5 control samples, the 5 antibacterial samples after cultivating for 24 hours that every group of 0h is contacted Product, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (21), (22) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2,3； In formulaWithAfter respectively every group of control sample and antimicrobial sample are contacted through 0h and cultivated for 24 hours, respectively The relative intensity of fluorescence measured value of Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples after cultivating for 24 hours of 3 groups of 0h contact, 15 it is anti- Bacterium sample, arithmetic mean of instantaneous value of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (23), (24) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
5 control samples, 5 antimicrobial samples and 5 control samples, the 5 antibacterial samples after cultivating for 24 hours that every group of 0h is contacted Product, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAnd Respectively according to formula (25) and (26) (sample group i=1,2,3 is calculated；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k=1,2,3； In formulaWithAfter respectively every group of control sample and antimicrobial sample are contacted through 0h and cultivated for 24 hours, respectively The relative intensity of fluorescence measured value of Duplicate Samples):
15 control samples, 15 antimicrobial samples and 15 control samples after cultivating for 24 hours of 3 groups of 0h contact, 15 it is anti- Bacterium sample, standard deviation of the reclaim liquid phase to fluorescent strength determining valueAndRespectively according to formula (27) and (28) calculate (sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；ATP tests Duplicate Samples number k= 1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample are contacted through 0h and are trained for 24 hours After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):
9. the detection method of antibacterial polyurethane synthetic leather bacteria resistance energy according to claim 1, which is characterized in that described Evaluation of result, in the steps below carry out:
(1) with reference to health industry common practice and the requirement of dependent antimicrobial product standard, following classification criterion is taken:
If using antibiotic rate R as correlated performance evaluation index: working as Rij/Ri(staphylococcus aureus and sand when/R < 90% Door Salmonella Rij/Ri/ R < 80%), sample is without antibacterial actions；As 90%≤Rij/Ri(staphylococcus aureus when/R < 99% With 80%≤R of salmonellaij/Ri/ R < 90%), sample has antibacterial actions；As 99%≤Rij/RiWhen/R < 99.9% (90%≤R of staphylococcus aureus and salmonellaij/Ri/ R < 99%), sample antibacterial actions are stronger；Work as Rij/Ri/R≥ (staphylococcus aureus and salmonella R when 99.9%ij/Ri/ R >=99%), sample antibacterial actions are extremely strong；
If using antibacterial activity value A as correlated performance evaluation index: working as Aij/AiWhen/A < 1.0 (staphylococcus aureus and Salmonella Aij/Ai/ A < 0.5), sample is without antibacterial actions；As 1.0≤Aij/AiWhen/A < 2.0 (staphylococcus aureus and 0.5≤A of salmonellaij/Ai/ A < 1.0), sample has antibacterial actions；As 2.0≤Aij/Ai(golden yellow Portugal when/A < 3.0 1.0≤A of grape coccus and salmonellaij/Ai/ A < 2.0), sample antibacterial actions are stronger；Work as Aij/AiIt is (golden yellow when/A >=3.0 Staphylococcus and salmonella Aij/Ai/ A >=2.0), sample antibacterial actions are extremely strong；
(2) as the antibiotic rate R of certain group (part) antibacterial polyurethane synthetic leather samplei(Rij) or antibacterial activity value Ai(Aij) with other two When the bacteria resistance of group (four) sample can be compared to a levels be at least differed, one group of (part) sample is extracted again and is repeated in fact It tests；It is calculated through ATP bioluminescence lgCA─lgIAThe antibiotic rate or antibacterial activity value that calibration curve method measures, if front and back two Group antibacterial polyurethane synthetic leather sample antibacterium performance level is identical, then abandons it；Take other two groups (four) remaining sample antibacterials Rate Ri(Rij) or antibacterial activity value Ai(Aij) arithmetic mean of instantaneous value it is anti-thin as batch (group) the antibacterial polyurethane synthetic leather sample The evaluation result of bacterium performance.
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