CN110272953A

CN110272953A - ATP bioluminescence lgCA-lgIAThe method of calibration curve method detection antibacterial leather fungicidal properties

Info

Publication number: CN110272953A
Application number: CN201910199725.0A
Authority: CN
Inventors: 李文杰; 连素梅; 娄巧哲; 靳惠达; 郝凌云
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2019-09-24

Abstract

The present invention relates to a kind of detection method of antibacterial leather fungicidal properties, detecting step includes: sample preparation and pretreatment；Lectotype selection and reagent, culture medium are prepared；Culture presevation, activation and spore liquid preparation；OD value-viable bacteria content logarithm lgC_BStandard curve is established；ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria C_ACalibration；Sample inoculation, culture and elution recycling；Recovered liquid I_AMeasurement and its viable bacteria ATP concentration C_AAnd T_AIt calculates；Antibiotic rate R or antibacterial activity value A is calculated；Evaluation of result；Its special feature is that: accurate quantitative test is carried out to the Leather mildew-proof performance with R or A characterization using ATP fluophotometer.Present invention provide that control sample and antimicrobial sample after elution recycling contact 0h and culture 48h, measure recovered liquid I_AAnd with lgI_ACharacterization and calculating R or A.The ATP bioluminescence lgC for the Leather mildew-proof performance detection that the present invention researches and develops_A‑lgI_ACalibration curve method will be promoted with the advanced detection technique support product quality of science.

Description

ATP bioluminescence lgCA-lgIACalibration curve method detects antibacterial leather fungicidal properties Method

Technical field

The present invention relates to the fungicidal properties test method of antibacterial leather, specifically a kind of application ATP fluophotometer to The antibacterial leather fungicidal properties of antibiotic rate R or antibacterial activity value A characterization carries out the ATP bioluminescence lgC of accurate quantitative detection_A- lgI_ACalibration 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, at present daily necessities, The fields such as clothes, shoes and hats, communal facility are widely used, antimicrobial technology Leather Industry application and popularize it is irresistible, therefore, Related 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 both at home and abroad, wherein mould proof detection side Method is originated from American Standard, day mark and Europe superscript substantially, and involved standard mainly includes burying cultivation, agar plate method and moist chamber culture based on soil The ISO 846:1997 " the antibacterial culture of plastic products is assessed " of method, " the antimycotic measurement of antimicrobial macromolecule material of DIN 53793 Method " and GB/T 24128-2009 " plastic mould-proof method for testing performance ", QB/T 4199-2011 " Leather mildew-proof performance test Method " etc..In general, there are following common problems in technology contents and practical application for existing fungicidal properties detection method: First is that due to the mycelium that is formed in mycotic spore growth course can not accurate counting, can only be qualitatively judged, no standard measure Test；Second is that incubation time at least 2 weeks~4 weeks, causing test period longer, time and economy because the fungus growth period is longer It is at high cost；Third is that experimentation is cumbersome, technical difficulty is higher；Relevant operation is affected by human factors greatly, so that test error is big, Lack comparativity.In recent years, increasingly mature in the development of international Bacteria Detection technical field ATP fluorescence analysis, with traditional plate Method is 98% compared to testing result correlation, and accuracy is high and can realize quick detection.By demand pull, external anti-biotic material Energy detection technique research field is directed to current quantification, rapid and summary development trend, has used for reference ATP fluorescence analysis principle Formulate ISO 20743:2007-2013 " 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.Part1Luminescence (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 inoculation, but it is only applicable to water imbibition and control sample Carefully/mould increasing value > 0 weaving or poromerics, in terms of the key technologies such as viable bacteria way of recycling, test condition for validity not Suitable for the leather with unwetted property hard surface and control sample mould increasing value < 0, plastic or other material, while survey is not provided Measure uncertainty evaluation.In addition, the standard method dependent antimicrobial performance characterization parameter is more single, antibacterial activity value A is only related to, 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 leather Performance Testing Technology；The art of this patent highway route design integrates with The world, detection method belong to the whole world and initiate, 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 the ATP bioluminescence lgC that is detected of antibacterial leather fungicidal properties_A-lgI_ACalibration curve method is able to solve anti- Mycoderma leather or even other product scope anti-biotic materials and the accurate quantitative test problem of product fungicidal properties.

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 leather fungicidal properties, comprising: (1) sample preparation and pretreatment；(2) lectotype selection and Reagent, culture medium are prepared；(3) culture presevation, activation and spore suspension preparation；(4) OD value-viable bacteria content logarithm lgC_BStandard Curve is established；(5) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration；(6) sample inoculation, culture and elution recycling；(7) reclaim liquid phase is to fluorescence intensity level I_AMeasurement；(8) it recycles Liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；It is characterized in that, using ATP fluophotometer carries out accurate quantitative test to the antibacterial leather fungicidal properties with antibiotic rate R or antibacterial activity value A characterization ATP bioluminescence lgC_A-lgI_ACalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_AIn measurement:

The cutting of control sample and antimicrobial sample, size, quantity and pre-treatment requirement are specified, by the mark of mould test strain After quasi- bacterial strain is passed on, activated, fresh mycotic spore culture is taken to prepare spore suspension；It is established using MTT colorimetric analysis OD value-viable bacteria content logarithm lgC_BStandard curve is derived from the linear equation Y=a of curve₀X+b₀And coefficient R₀ ²； And viable bacteria content C is carried out to the spore suspension of different dilutions_BCalibration.Then, with ATP fluorescent reagent buffer 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^-8mol/L、7.0×10^-7mol/ L、7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7It is strong to measure its relative fluorescence by mol/L Angle value I_A；Draw the lgC of two respective concentrations_A-lgI_AStandard curve is derived from fitting equation Y=a_A0X+b_A0It is (highly concentrated Degree), Y=a_AX+b_A(low concentration) and linearly dependent coefficient R_A0 ²(high concentration), R_A ²(low concentration).Meanwhile with Cha Shi culture solution pair Viable bacteria content C_BIt is 1.0 × 10⁸CFU/mL~5.0 × 10⁸It is dilute that the mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradients It releases, measures its relative intensity of fluorescence value I_A, and according to high standard fitting equation Y=a_A0X+b_A0Calculate corresponding viable bacteria ATP concentration C_A；It is adjusted to obtain C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7The inoculation bacterium solution of mol/L.Then, to each 0.3mL inoculation bacterium solution is added dropwise in group sample surface to be measured respectively, is eluted immediately with 4.6mL eluent to 6 groups of 0h contact samples Recycling, measures the relative intensity of fluorescence value I of recovered liquid_AC0ij、I_AT0ij, according to low concentration calibration curve equation formula Y=a_AX+b_AIt pushes away Calculate its viable bacteria ATP concentration C_AOijAnd T_AOij.Meanwhile the control sample and antimicrobial sample that 6 groups are sealed in sterilized petri dishes are (28 ± 2) after DEG C, cultivating 48h ± 2h under conditions of (95 ± 2) %RH；Recycling table is eluted using mode identical with 0h contact sample Face residual bacterium and the relative intensity of fluorescence value I for measuring recovered liquid_ACtij、I_ATtij, calculate corresponding viable bacteria ATP concentration C_AtijWith T_Atij；

In antibiotic rate R and antibacterial activity value A is calculated:

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, contacted with 0h and trained through 48h The relative intensity of fluorescence measured value of every control sample and antimicrobial sample recovered liquid after supportingWith As base Plinth data；In the case where testing condition for validity, its mould increasing value F after 48h is cultivated is calculated_ij、G_ijAnd antibiotic rate R_ijAnd antibacterial Activity value A_ij；To the R of every group of sample_ijAnd A_ijArithmetic mean of instantaneous value is taken to obtain corresponding R_iAnd A_i；Every batch of antibacterial leather sample resists Bacterium rate R and antibacterial activity value A is its three groups of sample R_iAnd A_iArithmetic mean of instantaneous value；And the clear related data revision of the convention and measurement be not true Fixed degree requires；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that fungicidal properties is classified criterion； As the antibiotic rate R of certain group (part) antibacterial leather sample_i(R_ij) or antibacterial activity value A_i(A_ij) with other two groups (four) samples When fungicidal properties is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；It is calculated through ATP biology Fluorescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back (part) antibacterial leather sample Fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i (A_ij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial leather sample fungicidal properties.

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, precisely can quickly to survey Determine the viable bacteria amount recycled after antibacterial leather sample culturing specific time, reaches the modernization of Leather mildew-proof performance detection；Can have Effect, which reduces human factor in experimentation, to be influenced, and traditional plate culture qualitative analysis limitation is breached；Realize testing result Quantification, and increase substantially the accuracy of detection 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 lgC_A- relative intensity of fluorescence logarithm lgI_AStandard curve；Construct antibacterial leather fungicidal properties test ATP bioluminescence The mathematical model of real-time quantitative analysis method.Meanwhile country variant consumer perceptions habit is taken into account, take antibiotic rate R and antibacterial Activity value A is correlated performance evaluation index, improves the science and versatility of detection method.

(3) innovative: with existing operation is numerous, the period is long, compared with the conventional method of non-quantitation, 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 leather The precision of fungicidal properties testing result and quantification simultaneously have good reproducibility and comparativity；Test is greatly shortened simultaneously Period reduces testing cost；Presently relevant the field of test technology blank can effectively be filled up.

(4) perspective: to establish with lgC_A、lgI_AStandard curve quantitative analysis method based on linear relationship, innovation is simultaneously Mycotic spore suspension viable bacteria concentration measuring method and leather sample pretreatment mode are enriched, specifies that control sample, standard liquid are dense The technology contents such as degree, determination step, calculation formula, uncertainty；The pioneering recovered liquid viable bacteria directly measured with instrument is relatively glimmering Luminous intensity logarithm lgI_AEvaluation form calculates and determines antibiotic rate R or antibacterial activity value A as a result, and by group and Between-group variation coefficient CV investigates uncertainty of measurement, technically has certain perspective.

(5) operability: ATP fluophotometer is cheap, it is easy to operate, be widely used, the OD value-that this patent is established Viable bacteria content logarithm lgC_BMethod is simple for calibration curve method and Leather mildew-proof performance ATP fluorometric investigation, and the relevant technologies are said Ming and Qing is clear and specific, should be readily appreciated that and grasps；Have stronger operability in implementation process, it is horizontal to be suitable for different majors Experiment on Microbiology personnel may advantageously facilitate achievement transfer conversion and promote and apply.

(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 mildew-proof performance detection technology realization generalization can be supported, while it can be right The product scopes fungicidal properties detection technique research such as plastics, rubber provides reference.

Further, preferred embodiment of the invention is:

The sample preparation and pretreatment carries out in the steps below:

(1) leather samples: by leather to be measured be placed in (20 ± 2) DEG C, (65 ± 5) %RH normal air environment in be adjusted to Few 48h, it is ensured that free air circulation simultaneously touches each surface of leather.Then, according to Fig. 1, Fig. 2 in QB/T2707-2005, Fig. 3, Shown in Fig. 4, using each related shadow stripe region as sampling point；The obvious shortcomings such as scratch, knife wound can not occur in leather sampling region, If sampling point is unable to satisfy the requirement of sample size, can be sampled from its ridge line other side corresponding site or adjacent regions.It is fixed Behind position, sample is cut from the grain (or imitative grain) that whole Zhang Ge, half stern back leather, shoulder leather or abdomen side are removed from office using mould knife, if without grain (or imitative grain), then cut sample from leather using face；Keep mould knife inner surface perpendicular to cut material surface in sampling process, it is interior Outer surface in knife-edge part at (20 ± 1) ° angle, and the depth on the angle wedge shape side be greater than cut leather thicknesses (mould knife cut legend sees Fig. 1 in QB/T 2707-2005)；

(2) control sample: raw cattle hide is selected to be processed into leather (chemical industry material used in process according to conventional leather-making technology Material is without fungicide and mould inhibitor ingredient) after, it is sampled with the circular mode knife that interior diameter is 50mm ± 1mm, thickness of sample is not more than 10mm；Then, sample surface to be measured is placed in below the ultraviolet disinfecting of 254nm, 40W upwards and irradiates 30min at 1.0m, and is close It is encapsulated in sterilized petri dishes, (- 10~-20) DEG C freezen protective is spare.Each strain test uses 6 groups of control samples；Wherein 0h connects Touching and 48h culture experiment respectively use 3 groups, every group of 5 samples；

(3) according to the preparation method of control sample, it is identical that raw material, technique, size, quantity antimicrobial sample: are obtained Leather sample, putting it into mass concentration to impregnate ± 1h for 24 hours in 0.5% benzene thiophene cyanogen solution (ensures that benzene thiophene cyanogen contains in sample Amount is 250mg/kg~350mg/kg)；After taking-up by sample surface to be measured upwards, be placed horizontally in Biohazard Safety Equipment, it is natural It dries and is sealed in sterilized petri dishes, (- 10~-20) DEG C freezen protective is spare.Every group of antimicrobial sample selects one group of control sample As object of reference and effectively identify；

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 degree meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-11Mol/L~10^-6mol/L；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~ The microplate reader of 4.0Abs；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) % The constant temperature and humidity incubator of RH ± 2%RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating Centrifuge tube；The vortex oscillator of the range of speeds (500~3000) r/min；The steam of (121 ± 2) DEG C, (103 ± 5) kPa Sterilizer；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range The ultrasonic cleaner of (30~50) kHz；The ultraviolet disinfecting of wavelength 254nm, power 40W；The pH of precision ± 0.1 (25 DEG C) Meter；Electric furnace；

(3) material utensil: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.1mL, 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)； Filter sizes are not more than 0.45 μm of syringe-driven filter；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；96 Hole flat-bottomed plates；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the inoculation of 4mm Ring；L stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；It is interior straight The circular mode knife of diameter 50mm；DEG C ± 0.2 DEG C of thermometer；The hygrometer of %RH ± 1%RH；Precision The stopwatch of 0.01s；Aseptic filter paper；

(4) reagent: the benzene thiophene cyanogen solution that mass concentration is 0.5%；MTT (the tetramethyl azo azoles of 5mg/mL, pH value 7.4 Salt) solution (0.45 μm of membrane filtration degerming, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilizations after following reagent packing 30min, the physiological saline of 5 DEG C~10 DEG C storage 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are appointed One kind is selected, 0.05% wetting agent solution is configured to；

(5) medium/liquid (commercially available medium/liquid can be used): 121 DEG C of high pressure sterilizations after matched medium/liquid packing 30min, 2 DEG C~8 DEG C storage 30d；

Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen Dipotassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose are dissolved by heating in 1000mL containing 0.05% wetting In the aqueous solution of agent, adjust pH to 6.0~6.5 (25 DEG C)；

One dextrose culture-medium of potato (mycotic spore actication of culture and its total plate count plate count use): 300g is new Fresh potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Filter to take juice, into filtrate be added 20g glucose, 1000mL is settled to after 0.1g chloramphenicol, 20g agar；

(6) ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction examination - 20 DEG C~-70 DEG C of agent preservations, use in 6 months；

Dilution buffer: 0.005mol/L and the disodium phosphate soln for containing 0.037% sucrose, adjusting pH to 7.2 ± 0.2；121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；

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 Sharpe culture solution can be down to 10 in 15min by liquid^-11Mol/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 (fungal cell ATP extraction efficiency be not less than 80%)；

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL ATP fluorescent reagent buffer solution, be stored at room temperature 15min after mixing, used in 3h；

Culture presevation, activation and the spore suspension preparation, carries out in the steps below:

(1) test strain: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by Other strains that national Culture Collection is provided and can be traced to the source)；

(2) mould test strain culture presevation: is inoculated in simultaneously by potato-dextrose culture-medium inclined-plane with sterile working Indicate the date, 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3 DEG C~10 DEG C preservation 4 months；

(3) actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C ~30 DEG C of culture 7d~14d are until generate a large amount of spores.Mold species test tube plug must not be extracted before spore suspension by preparing, and every Only for spore liquid of preparation after test tube opening, suspension is prepared using the mycotic spore newly cultivated every time；

(4) prepared by spore suspension: the sterile water of 10mL being added into strain test tube, gently scrapes media surface with oese The spore stoste injection of wash-off is equipped with the sterile taper of jumping a queue of 15 beades and 45mL Cha Shi culture solution by fresh mycotic spore In bottle, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, sterile absorbent cotton or eight layers of yarn will be covered with The glass funnel of cloth is placed on conical flask, and filtering spore suspension removes mycelia and culture medium fragment；By filtrate move to sterilizing from In heart pipe, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；It is heavy that spore is cleaned with 50mL Cha Shi culture solution again Starch is simultaneously centrifuged, after repeated washing 3 times, with the spore sediment after the dilution centrifugation of Cha Shi culture solution.Every kind of test mould is pressed Spore suspension is prepared according to the above method, the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used；

The OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration, in the steps below It carries out:

(1) bacterium solution OD value measures: carrying out primary dcreening operation using spore total amount of the blood counting chamber to mixing spore suspension, uses Cha Shi Culture solution is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and with culture solution As blank control sample liquid, zeroing correction is carried out to microplate reader.Then, the spore liquid of the above-mentioned different dilutions of 100 μ L is distinguished 96 hole flat-bottomed plates are injected, each dilution does 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength； The viable bacteria OD value of each hole mixed solution is measured, the viable bacteria OD value of different dilution spore suspensions is its 3 multiple holes OD measured values Arithmetic mean of instantaneous value.Meanwhile the method referring to as defined in national standard GB 4789.15-2016, it is appropriate to carry out to above-mentioned mixing spore suspension After dilution, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B；

(2) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilutions The viable bacteria OD value of spore suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard Curve；The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ².Work as R₀ ²≥ 0.98, when confidence level >=0.95, the spore liquid viable bacteria content C that is measured according to this patent method_BEffectively；

The ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution is living Bacterium ATP concentration C_ACalibration carries out in the steps below:

(1) determination of ATP standard serial solution and its test specimens preparation: ATP fluorescent reagent buffer 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^-8mol/L、7.0×10^-7mol/L、 7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7mol/L.It then, will with sterilizing liquid-transfering gun The above-mentioned ATP standard solution of 0.1mL is moved to respectively in three sterile test tubes, and 0.05mL sterile water and 0.35mL physiology salt is successively added dropwise Aqueous solution simultaneously mixes, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution test specimens are moved to respectively again It is each that 0.4mL physiological saline is added dropwise and mixes in three sterile test tubes, as second level ATP standard solution test specimens；And it is moved with sterilizing Liquid rifle moves to the second level ATP standard solution test specimens of 0.1mL various concentration in three instrument sterile test tubes respectively, as ATP standard solution tests Duplicate Samples；

(2) blank background value calibration: the aqueous solution, 0.35mL with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent are raw The ATP lysate of reason salt water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；It stands Level-one blank sample is used as after 10min~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, is added dropwise 0.4mL physiological saline simultaneously mixes, as second level blank sample.Then, 0.1mL second level blank sample is moved to respectively with sterilizing liquid-transfering gun In three instrument sterile test tubes, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples is mentioned Reagent is taken, instills the ATP fluorescent reagent of 0.1mL after mixing again.3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and examination Agent group background values (or calibrating background according to instrument operation instruction)；

(3) ATP standard solution relative intensity of fluorescence value I_AMeasurement: from low to high according to concentration, to various concentration ATP The ATP that 0.1mL is added dropwise in three test Duplicate Samples of standard solution respectively extracts reagent, and the ATP for instilling 0.1mL after mixing again is glimmering Light reagent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records (really It is consistent to protect each link operating time, avoids cross contamination).Each Duplicate Samples minute is no more than 15s, with each concentration ATP mark The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of quasi- solution is its I_AMeasured value；

(4) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with various concentration ATP The relative intensity of fluorescence logarithm lgI of standard solution_AAs abscissa, with its ATP log concentration value lgC_AFor ordinate mapping； Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And application is most Small two multiply the linear equation Y=a that fitting process is derived from curve_A0X+b_A0(high concentration), Y=a_AX+b_AIt is (low concentration) and related Coefficients R_A0 ²(high concentration), R_A ²(low concentration).Work as R_A0 ²And R_A ²>=0.98, it when confidence level >=0.95, is done according to this patent Measurement is effective；

(5) it is inoculated with bacterium solution viable bacteria ATP concentration C_ACalibration: with Cha Shi culture solution to viable bacteria content C_BIt is 1.0 × 10⁸CFU/mL ~5.0 × 10⁸The mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradient dilutions, measures its relative intensity of fluorescence value I_A；Root According to high standard fitting equation Y=a_A0X+b_A0It calculates and demarcates corresponding viable bacteria ATP concentration C_A, through Cha Shi culture solution tune It is whole, obtain C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1min_A0；

Sample inoculation, culture and the elution recycling, carries out in the steps below:

(1) inoculated and cultured: taking out each group control sample and antimicrobial sample from refrigerator, thaws to room temperature；With sterilizing liquid relief 0.3mL inoculation bacterium solution is added dropwise (with lgC to its surface to be measured in rifle respectively_A-lgI_ACalibration curve is derived from same branch with spore liquid and mixes Spore liquid test tube, 0 DEG C ± 1 DEG C saves, and 2h is interior to be used), it is with L stick (attachment is inoculated with bacterium solution but does not hang drop) that bacterium solution smearing is uniform, It is set to cover sample whole surface.Ware lid is covered, is sealed the plate equipped with 6 groups of 48h contact samples with medical adhesive tape；(28± 2) DEG C, (95 ± 2) %RH cultivates 48h ± 2h；

(2) elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution are simultaneously anti-with liquid transfer gun head Mould washing lotion in multiple pressure-vaccum plate, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL) less than 4.9mL after mixing.Each group Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould；

The reclaim liquid phase is to fluorescence intensity level I_AMeasurement carries out in the steps below:

(1) instrument and reagent set relative intensity of fluorescence background value calibration: according to lgC_A-lgI_ABlank when standard curve is established Background calibration methods, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values；

(2) reclaim liquid phase is to fluorescence intensity level I_AMeasurement: if background level meets instrument requirement, with sterilizing liquid-transfering gun The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube, 3000r/min shakes test tube 30s； After being stored at room temperature 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min.It will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence.To The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquid_AMeasured value；

The recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated, by following steps It is rapid to carry out:

(1) recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, calculate that each group sample connects through 0h Touch the viable bacteria ATP concentration C of recovered liquid after 48h is cultivated_AAnd T_A.Relevant calculation is shown in formula (1)~(12):

In formula:

C_A0And C_AtThe average ATP concentration that -3 groups of 0h are contacted and control sample recycles viable bacteria after 48h is cultivated, unit are to rub You are every liter (mol/L)；

C_A0iAnd C_AtiThe average ATP concentration that-every group 0h is contacted and control sample recycles viable bacteria after 48h is cultivated, unit are Mole every liter (mol/L)；Sample group i=1,2,3；

C_A0ijAnd C_AtijThe ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after 48h is cultivated, 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 48h is cultivated 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；

a_A- low concentration standard curve lgC_A-lgI_ASlope；b_A- low concentration standard curve lgC_A-lgI_AIn vertical axis intercept；

T_A0And T_AtThe average ATP concentration that -3 groups of 0h are contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, unit are to rub You are every liter (mol/L)；

T_A0iAnd T_AtiThe average ATP concentration that-every group 0h is contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, unit are Mole every liter (mol/L)；Sample group i=1,2,3；

T_A0ijAnd T_AtijThe ATP concentration that-every 0h is contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, 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 48h is cultivated 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 recovered liquid and 0.3mL inoculation the bacterium solution 1min after the dilution of 4.6mL eluent of every 0h contact control sample Interior relative intensity of fluorescence measured value is close, i.e.,Every after 48h is cultivated control sample recovered liquid relative fluorescence it is strong Spend the logarithm of measured valueThat is C_Atij≥0.1×C_A0ij.Then when 3 groups of 0h contact control sample reclaim liquid phase To fluorescent strength determining value I_AC0ijGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent correlation Uncertainty of measurement requirement), the measurement carried out according to this patent method is effective；

(3) mould increasing value F_ij、G_ijIt calculates

Every control sample and antimicrobial sample are after 48h is cultivated, mould increasing value F_ij、G_ijRespectively according to formula (13), (14) it calculates:

In formula:

F_ijAnd G_ijThe mould increasing value of-every control sample and antimicrobial sample after 48h is cultivated, sample group i=1, 2,3；Every group of sample number into spectrum j=1,2,3,4,5；

C_A0ijAnd C_AtijThe average ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after 48h is cultivated, unit It 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 0h contact and after 48h is cultivated control sample recovered liquid relative intensity of fluorescence measured value, RLU；Group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

T_A0ijAnd T_AtijThe average ATP concentration values that-every 0h is contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, 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；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(4) antibiotic rate R is calculated

It tests under condition for validity, the antibiotic rate R of every antibacterial leather sample_ij, every group and every batch of sample antibiotic rate R_iAnd R It is calculated respectively according to formula (15), (16), (17):

In formula:

R_ijThe antibiotic rate of-every antibacterial leather sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2, 3,4,5；

R_iThe antibiotic rate of-every group antibacterial leather sample, %；Sample group i=1,2,3；

R-every batch of antibacterial leather sample antibiotic rate, %；

C_AtijAnd T_Atij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after 48h is cultivated, 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 48h is cultivated, 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；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(5) antibacterial activity value A is calculated

It tests under condition for validity, the antibacterial activity value A of every antibacterial leather sample_ij, the antibacterial of every group and every batch of sample it is living Property value A_iIt is calculated respectively according to formula (18), (19), (20) with A:

In formula:

A_ijThe antibacterial activity value of-every antibacterial leather sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2, 3,4,5；

A_iThe antibacterial activity value of-every group antibacterial leather sample, sample group i=1,2,3；

A-every batch of antibacterial leather sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(6) data revision of the convention requirement: calibration mycotic spore liquid viable bacteria content C_BAnd ATP concentration of standard solution and inoculation bacterium Liquid, sample recovered liquid viable bacteria ATP concentration C_AWhen, it is provided with reference to the data revision of the convention in GB 4789.2-2016 in relation to clump count, when C_BLess than 100CFU/mL or C_AWhen less than 100mol/L, " rounding up " round numbers；Work as C_BNot less than 100CFU/mL or C_AIt is not small When 100mol/L, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；Can also with 10 index Form indicates that " rounding up " uses two effective digitals afterwards.Control sample and antimicrobial sample through 0h contact and 48h culture after, The relative intensity of fluorescence measured value round numbers of recovered liquid, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increases Value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals；

(7) uncertainty of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h 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 Leather mildew-proof performance test Reproducibility；In regulation group and between-group variation coefficient CV≤10%.

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated 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 48h is trained 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 48h is cultivated 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 48h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

5 control samples, 5 antimicrobial samples and 5 control samples after 48h is cultivated of every group of 0h contact, 5 it is anti- Bacterium sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively according to public affairs 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 through 0h contact and 48h After culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples, 15 after 48h is cultivated that 3 groups of 0h are contacted Part antimicrobial sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively 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 and compiles Number k=1,2,3；In formulaWithRespectively every group of control sample and antimicrobial sample contacted through 0h and After 48h culture, the relative intensity of fluorescence measured value of each Duplicate Samples):

The evaluation of result of the antibacterial leather sample fungicidal properties 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 R_ij/R_iWhen/R < 80%, sample is without mildew-proof function； As 80%≤R_ij/R_iWhen/R < 90%, sample has mildew-proof function；As 90%≤R_ij/R_iWhen/R < 99%, sample mildew-proof function It is moderate；As 99%≤R_ij/R_iWhen/R < 99.9%, sample mildew-proof function is stronger；Work as R_ij/R_iWhen/R >=99.9%, sample is mould proof It acts on extremely strong；

If using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mould proof work With；As 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function It is moderate；As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function pole By force；

(2) as the antibiotic rate R of certain group (part) antibacterial leather sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups When the fungicidal properties of (four) sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Meter It is calculated through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back antibacterial Leather sample fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial work Property value A_i(A_ij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial leather sample fungicidal properties；

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 said by taking the antibacterial leather sample fungicidal properties detection impregnated through 0.5% benzene thiophene cyanogen solution as an example It is bright.

Specific detection method carries out in the steps below:

(1) sample preparation and pretreatment

The sampling of 1.1 leathers: by leather to be measured be placed in (20 ± 2) DEG C, (65 ± 5) %RH normal air environment in be adjusted to Few 48h, it is ensured that free air circulation simultaneously touches each surface of leather.Then, according to Fig. 1, Fig. 2, figure in QB/T 2707-2005 3, shown in Fig. 4, using each related shadow stripe region as sampling point；Leather sampling region scratch, knife wound etc. can not occurs and obviously lack It falls into, if sampling point is unable to satisfy the requirement of sample size, can be sampled from its ridge line other side corresponding site or adjacent regions. After positioning, sample is cut from the grain (or imitative grain) that whole Zhang Ge, half stern back leather, shoulder leather or abdomen side are removed from office using mould knife, if without grain Face (or imitative grain), then cut sample from leather using face；Keep mould knife inner surface perpendicular to cut material surface in sampling process, Surfaces externally and internally in knife-edge part at (20 ± 1) ° angle, and the depth on the angle wedge shape side be greater than cut leather thicknesses (mould knife cutting legend See Fig. 1 in QB/T 2707-2005).

1.2 control samples: raw cattle hide is selected to be processed into leather (chemical industry material used in process according to conventional leather-making technology Material is without fungicide and mould inhibitor ingredient) after, it is sampled with the circular mode knife that interior diameter is 50mm ± 1mm, thickness of sample is not more than 10mm；Then, sample surface to be measured is placed in below the ultraviolet disinfecting of 254nm, 40W upwards and irradiates 30min at 1.0m, and is close It is encapsulated in sterilized petri dishes, (- 10~-20) DEG C freezen protective is spare.Each strain test uses 6 groups of control samples；Wherein 0h connects Touching and 48h culture experiment respectively use 3 groups, every group of 5 samples.

1.3 antimicrobial samples: according to the preparation method of control sample, it is identical that raw material, technique, size, quantity are obtained Leather sample, putting it into mass concentration to impregnate ± 1h for 24 hours in 0.5% benzene thiophene cyanogen solution (ensures that benzene thiophene cyanogen contains in sample Amount is 250mg/kg~350mg/kg)；After taking-up by sample surface to be measured upwards, be placed horizontally in Biohazard Safety Equipment, it is natural It dries and is sealed in sterilized petri dishes, (- 10~-20) DEG C freezen protective is spare.Every group of antimicrobial sample selects one group of control sample As object of reference and effectively identify.

(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 degree meter, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-11Mol/L~10^-6mol/L；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~ The microplate reader of 4.0Abs；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) % The constant temperature and humidity incubator of RH ± 2%RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating Centrifuge tube；The vortex oscillator of the range of speeds (500~3000) r/min；The steam of (121 ± 2) DEG C, (103 ± 5) kPa Sterilizer；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range The ultrasonic cleaner of (30~50) kHz；The ultraviolet disinfecting of wavelength 254nm, power 40W；The pH of precision ± 0.1 (25 DEG C) Meter；Electric furnace.

2.3 material utensils: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；0.05mL, The single track changeable fluid liquid-transfering gun and sterile liquid transfer gun head of 0.1mL, 0.2mL, 1mL, 5mL, 10mL (measurement error is less than 1%)； Filter sizes are not more than 0.45 μm of syringe-driven filter；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；96 Hole flat-bottomed plates；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the inoculation of 4mm Ring；L stick；The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；It is interior straight The circular mode knife of diameter 50mm；DEG C ± 0.2 DEG C of thermometer；The hygrometer of %RH ± 1%RH；Precision The stopwatch of 0.01s；Aseptic filter paper.

2.4 reagents: the benzene thiophene cyanogen solution that mass concentration is 0.5%；MTT (the tetramethyl azo azoles of 5mg/mL, pH value 7.4 Salt) solution (0.45 μm of membrane filtration degerming, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilizations after following reagent packing 30min, the physiological saline of 5 DEG C~10 DEG C storage 30d:85%；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are appointed One kind is selected, 0.05% wetting agent solution is configured to.

2.5 medium/liquids (can use commercially available medium/liquid): 121 DEG C of high pressure sterilizations after matched medium/liquid packing 30min, 2 DEG C~8 DEG C storage 30d；

One dextrose culture-medium of potato (mycotic spore actication of culture and its total plate count plate count use): 300g is new Fresh potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Filter to take juice, into filtrate be added 20g glucose, 1000mL is settled to after 0.1g chloramphenicol, 20g agar.

2.6ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction examination - 20 DEG C~-70 DEG C of agent preservations, use in 6 months；

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL ATP fluorescent reagent buffer solution, be stored at room temperature 15min after mixing, used in 3h.

(3) culture presevation, activation and spore suspension preparation

3.1 test strains: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by Other strains that national Culture Collection is provided and can be traced to the source).

3.2 culture presevation: mould test strain is inoculated in simultaneously by potato-dextrose culture-medium inclined-plane with sterile working Indicate the date, 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3 DEG C~10 DEG C preservation 4 months.

3.3 actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C ~30 DEG C of culture 7d~14d are until generate a large amount of spores.Mold species test tube plug must not be extracted before spore suspension by preparing, and every Only for spore liquid of preparation after test tube opening, suspension is prepared using the mycotic spore newly cultivated every time.

The preparation of 3.4 spore suspensions: the sterile water of 10mL is added into strain test tube, gently scrapes media surface with oese The spore stoste injection of wash-off is equipped with the sterile taper of jumping a queue of 15 beades and 45mL Cha Shi culture solution by fresh mycotic spore In bottle, 3000r/min shakes test tube 2min, breaks up spore ball, mixes spore liquid.Then, sterile absorbent cotton or eight layers of yarn will be covered with The glass funnel of cloth is placed on conical flask, and filtering spore suspension removes mycelia and culture medium fragment；By filtrate move to sterilizing from In heart pipe, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；It is heavy that spore is cleaned with 50mL Cha Shi culture solution again Starch is simultaneously centrifuged, after repeated washing 3 times, with the spore sediment after the dilution centrifugation of Cha Shi culture solution.Every kind of test mould is pressed Spore suspension is prepared according to the above method, the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used.

(4) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration

The measurement of 4.1 bacterium solution OD values: primary dcreening operation is carried out using spore total amount of the blood counting chamber to mixing spore suspension, uses Cha Shi Culture solution is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and with culture solution As blank control sample liquid, zeroing correction is carried out to microplate reader.Then, the spore liquid of the above-mentioned different dilutions of 100 μ L is distinguished 96 hole flat-bottomed plates are injected, each dilution does 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength； The viable bacteria OD value of each hole mixed solution is measured, the viable bacteria OD value of different dilution spore suspensions is its 3 multiple holes OD measured values Arithmetic mean of instantaneous value.Meanwhile the method referring to as defined in national standard GB 4789.15-2016, it is appropriate to carry out to above-mentioned mixing spore suspension After dilution, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B。

4.2OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilutions The viable bacteria OD value of spore suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard Curve；The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ².Work as R₀ ²≥ 0.98, when confidence level >=0.95, the spore liquid viable bacteria content C that is measured according to this patent method_BEffectively.

(5) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration

The determination of 5.1ATP standard serial solution and its test specimens preparation: ATP fluorescent reagent buffer 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^-8mol/L、7.0×10^-7mol/L、 7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7mol/L.It then, will with sterilizing liquid-transfering gun The above-mentioned ATP standard solution of 0.1mL is moved to respectively in three sterile test tubes, and 0.05mL sterile water and 0.35mL physiology salt is successively added dropwise Aqueous solution simultaneously mixes, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution test specimens are moved to respectively again It is each that 0.4mL physiological saline is added dropwise and mixes in three sterile test tubes, as second level ATP standard solution test specimens；And it is moved with sterilizing Liquid rifle moves to the second level ATP standard solution test specimens of 0.1mL various concentration in three instrument sterile test tubes respectively, as ATP standard solution tests Duplicate Samples.

5.2 blank background value calibrations: the aqueous solution, 0.35mL with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent are raw The ATP lysate of reason salt water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；It stands Level-one blank sample is used as after 10min~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, is added dropwise 0.4mL physiological saline simultaneously mixes, as second level blank sample.Then, 0.1mL second level blank sample is moved to respectively with sterilizing liquid-transfering gun In three instrument sterile test tubes, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples is mentioned Reagent is taken, instills the ATP fluorescent reagent of 0.1mL after mixing again.3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and examination Agent group background values (or calibrating background according to instrument operation instruction).

5.3ATP standard solution relative intensity of fluorescence value I_AMeasurement: from low to high according to concentration, to various concentration ATP The ATP that 0.1mL is added dropwise in three test Duplicate Samples of standard solution respectively extracts reagent, and the ATP for instilling 0.1mL after mixing again is glimmering Light reagent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records (really It is consistent to protect each link operating time, avoids cross contamination).Each Duplicate Samples minute is no more than 15s, with each concentration ATP mark The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of quasi- solution is its I_AMeasured value.

5.4ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with various concentration ATP The relative intensity of fluorescence logarithm lgI of standard solution_AAs abscissa, with its ATP log concentration value lgC_AFor ordinate mapping； Calibration curve is carried out to mathematical relationship between the two, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And application is most Small two multiply the linear equation Y=a that fitting process is derived from curve_A0X+b_A0(high concentration), Y=a_AX+b_AIt is (low concentration) and related Coefficients R_A0 ²(high concentration), R_A ²(low concentration).Work as R_A0 ²And R_A ²>=0.98, it when confidence level >=0.95, is done according to this patent Measurement is effective.

5.5 inoculation bacterium solution viable bacteria ATP concentration Cs_ACalibration: with Cha Shi culture solution to viable bacteria content C_BIt is 1.0 × 10⁸CFU/mL ~5.0 × 10⁸The mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradient dilutions, measures its relative intensity of fluorescence value I_A；Root According to high standard fitting equation Y=a_A0X+b_A0It calculates and demarcates corresponding viable bacteria ATP concentration C_A, through Cha Shi culture solution tune It is whole, obtain C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1min_A0。

(6) sample inoculation, culture and elution recycling

6.1 inoculated and cultureds: taking out each group control sample and antimicrobial sample from refrigerator, thaws to room temperature；With sterilizing liquid relief 0.3mL inoculation bacterium solution is added dropwise (with lgC to its surface to be measured in rifle respectively_A-lgI_ACalibration curve is derived from same branch with spore liquid and mixes Spore liquid test tube, 0 DEG C ± 1 DEG C saves, and 2h is interior to be used), it is with L stick (attachment is inoculated with bacterium solution but does not hang drop) that bacterium solution smearing is uniform, It is set to cover sample whole surface.Ware lid is covered, is sealed the plate equipped with 6 groups of 48h contact samples with medical adhesive tape；(28± 2) DEG C, (95 ± 2) %RH cultivates 48h ± 2h.

6.2 elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately (i.e. Cha Shi culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate, sufficiently elution are simultaneously anti-with liquid transfer gun head Mould washing lotion in multiple pressure-vaccum plate, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL) less than 4.9mL after mixing.Each group Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould.

(7) reclaim liquid phase is to fluorescence intensity level I_AMeasurement

7.1 instruments and reagent set relative intensity of fluorescence background value calibration: according to lgC_A-lgI_ABlank when standard curve is established Background calibration methods, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values.

7.2 reclaim liquid phase is to fluorescence intensity level I_AMeasurement: if background level meets instrument requirement, with sterilizing liquid-transfering gun The ATP lysate of 4.9mL recovered liquid and 0.1mL is separately added into same branch sterile test tube, 3000r/min shakes test tube 30s； After being stored at room temperature 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min.It will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence.To The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd it records and (ensures that each link operating time is consistent, avoid cross contamination).Each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquid_AMeasured value.

(8) recovered liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated

8.1 recovered liquid viable bacteria ATP concentration Cs_AAnd T_AIt calculates

In formula:

With- every 0h contact and after 48h is cultivated 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.

8.2 test conditions for validity

If recovered liquid and 0.3mL inoculation the bacterium solution 1min after the dilution of 4.6mL eluent of every 0h contact control sample Interior relative intensity of fluorescence measured value is close, i.e.,Every after 48h is cultivated control sample recovered liquid relative fluorescence it is strong Spend the logarithm of measured valueThat is C_Atij≥0.1×C_A0ij.Then when 3 groups of 0h contact control sample reclaim liquid phase To fluorescent strength determining valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent correlation Uncertainty of measurement requirement), the measurement carried out according to this patent method is effective.

8.3 mould increasing value F_ij、G_ijIt calculates

In formula:

a_A- low concentration standard curve lgC_A-lgI_ASlope.

8.4 antibiotic rate R are calculated

In formula:

R-every batch of antibacterial leather sample antibiotic rate, %；

a_A- low concentration standard curve lgC_A-lgI_ASlope.

8.5 antibacterial activity value A are calculated

In formula:

A-every batch of antibacterial leather sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope.

8.6 data revisions of the convention requirement: calibration mycotic spore liquid viable bacteria content C_BAnd ATP concentration of standard solution and inoculation bacterium Liquid, sample recovered liquid viable bacteria ATP concentration C_AWhen, it is provided with reference to the data revision of the convention in GB 4789.2-2016 in relation to clump count, when C_BLess than 100CFU/mL or C_AWhen less than 100mol/L, " rounding up " round numbers；Work as C_BNot less than 100CFU/mL or C_AIt is not small When 100mol/L, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；Can also with 10 index Form indicates that " rounding up " uses two effective digitals afterwards.Control sample and antimicrobial sample through 0h contact and 48h culture after, The relative intensity of fluorescence measured value round numbers of recovered liquid, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increases Value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals.

8.7 uncertainties of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h 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 Leather mildew-proof performance test Reproducibility；In regulation group and between-group variation coefficient CV≤10%.

(9) evaluation of result

9.1, with reference to health industry common practice and the requirement of dependent antimicrobial product standard, take following classification criterion:

If using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mould proof work With；As 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function It is moderate；As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function pole By force.

(2) as the antibiotic rate R of certain group (part) antibacterial leather sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups When the fungicidal properties of (four) sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Meter It is calculated through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures.If two groups of front and back antibacterial Leather sample fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial work Property value A_i(A_ij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial leather sample fungicidal properties.

Detection qu alification, instrument and equipment used in the present embodiment and test equipment, medium/liquid, chemical reagent, reference culture:

(1) bio-safety 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 and test 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 Scientific^TMHeraguard^TM, model ECO ultra-clean work Platform, inside width × depth × height=920mm × 585mm × 645mm；Air velocity be 0.15m/s~0.25m/s and 0.36m/s~ 0.45m/s。

2.3ATP bioluminescence rapid detection system: the portable system SURE ATP 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 microplate reader: the full-automatic microplate reader of American blend Thermo Scientific, model Multiskan FC, wave Long range (340~850) nm ± 1nm, range of readings (0.0~6.0) Abs ± 0.001Abs；The corresponding measurement accuracy of 405nm is ± 1% (0.0Abs~3.0Abs) or ± 2% (3.0Abs~4.0Abs).

2.5 Photobiology microscopes: have the adjustable eyepiece of 10 times of wide visual fields and 4 times, 10 times, 20 times, 40 times and 100 times The tri- mesh research grade biomicroscope of Olympus BX43 of flat-field achromatic objective lens.

2.6 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.

2.7 constant water bath box: the electric heating constant temperature sink of upper sea base Wei test apparatus equipment Co., Ltd model DK-S420, Volume 15L, ± 0.5 DEG C of temperature control range (5~99.9) DEG C, timing range 1min~999min.

2.8 constant temperature oscillators: the permanent model WS-380H in Shanghai one, ± 0.1 DEG C of temperature control range (4~65) DEG C, frequency of oscillation (40~300) r/min, timing range (1~99) h.

2.9 refrigerated centrifuges: the vertical refrigerated centrifuge of Beijing Xin Nuolihua Instrument Ltd. model DL7M-12L turns Fast 8000r/min ± 20r/min, 12300 × g of relative centrifugal force；Capacity 14400ml, timing range 1s~99h59min99s, ± 1 DEG C of temperature control range (- 20~40) DEG C, centrifuge tube specification Ф 74mm × 168mm.

2.10 pressure steam sterilizers: Japanese Sanyo company model MLS-3780 autoclave, volume 75L, sterilizing temperature ± 2 DEG C, maximum pressure 0.235MPa of degree (105~135) DEG C, timer (1~250) min.

2.11 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.

2.12 refrigerators: the cryogenic box ultralow temperature ice of the glad experimental instruments and equipment limited model HXL-25-250AD of Dongguan City sky Case, ± 0.1 DEG C of refrigerating box (2~10) DEG C, ± 0.1 DEG C of household freezer (- 30~20) DEG C.

2.13 electronic balances: the electronic balance of Japanese Shimadzu model AUX220, range 220g, precision ± 0.1mg.

2.14 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.

2.15 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.

2.16 ultraviolet lamp tubes: the ultraviolet disinfection lamp of LONGPRO brand UVC series, power 40W, wavelength 254nm.

2.17pH meter: 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.

2.18 electric furnaces: the 1KW closed temp.-adjustable electric furnace of Changzhou Deco Instrument Ltd. model DLD-1KW.

2.19 blood counting chambers: the blood counting chamber that refinement specification in Shanghai is 25 × 16.

(3) medium/liquid

The medium/liquid of Beijing overpass technical concern Co., Ltd production.

(4) chemical reagent

Benzene thiophene cyanogen solution, tetramethyl azo azoles salt, trinosin standard items and preparation ATP fluorescent reagent buffering are molten A series of biochemical reagents needed for liquid, ATP lysate, ATP extracting solution and ATP fluorescent reagent are limited purchased from Shanghai gold side biotechnology U.S.'s Amresco brand of corporate agent.

(5) reference culture

Aspergillus niger ATCC 16404, Chaetomium globosum ATCC 6205, penicillium chrysogenum ATCC 9179 are purchased from the common micro- life of China Object culture presevation administrative center.

The detection data and result of the present embodiment calculate:

Using ATP fluophotometer through lgC_A-lgI_ACalibration curve method examines the fungicidal properties of antibacterial leather sample It surveys, coherent detection data and Calculation of Measuring Uncertainty the results are shown in Table 1~table 2.

1 relative intensity of fluorescence measured value of table and antibiotic rate R, antibacterial activity value A calculated result

2 relative intensity of fluorescence Calculation of Measuring Uncertainty result 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

1. a kind of detection method of antibacterial leather fungicidal properties, comprising: (1) sample preparation and pretreatment；(2) lectotype selection and examination Agent, culture medium are prepared；(3) culture presevation, activation and spore suspension preparation；(4) OD value-viable bacteria content logarithm lgC_BStandard is bent Line is established；(5) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration；(6) sample inoculation, culture and elution recycling；(7) reclaim liquid phase is to fluorescence intensity level I_AMeasurement；(8) it recycles Liquid viable bacteria ATP concentration C_AAnd T_AIt calculates and antibiotic rate R and antibacterial activity value A is calculated；(9) evaluation of result；It is characterized in that, using ATP fluophotometer carries out accurate quantitative test to the antibacterial leather fungicidal properties with antibiotic rate R or antibacterial activity value A characterization ATP bioluminescence lgC_A-lgI_ACalibration curve method, specific:

In reclaim liquid phase to fluorescence intensity level I_AIn measurement:

The cutting of control sample and antimicrobial sample, size, quantity and pre-treatment requirement are specified, by the standard bacteria of mould test strain After strain is passed on, activated, fresh mycotic spore culture is taken to prepare spore suspension；OD value-is established using MTT colorimetric analysis Viable bacteria content logarithm lgC_BStandard curve is derived from the linear equation Y=a of curve₀X+b₀And coefficient R₀ ²；And it is right The spore suspension of different dilutions carries out viable bacteria content C_BCalibration, then, with ATP fluorescent reagent buffer 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^-8mol/L、7.0×10^-7mol/L、 7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7Mol/L measures its relative intensity of fluorescence Value I_A；Draw the lgC of two respective concentrations_A-lgI_AStandard curve is derived from fitting equation Y=a_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and linearly dependent coefficient R_A0 ²(high concentration), R_A ²(low concentration), meanwhile, with Cha Shi culture solution to viable bacteria Content C_BIt is 1.0 × 10⁸CFU/mL~5.0 × 10⁸The mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradient dilutions, surveys Fixed its relative intensity of fluorescence value I_A, and according to high standard fitting equation Y=a_A0X+b_A0Calculate corresponding viable bacteria ATP concentration C_A；It is adjusted to obtain C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7Then the inoculation bacterium solution of mol/L is waited for each group sample It surveys surface and 0.3mL inoculation bacterium solution is added dropwise respectively, elution recycling is carried out to 6 groups of 0h contact samples with 4.6mL eluent immediately, is measured The relative intensity of fluorescence value I of recovered liquid_AC0ij、I_AT0ij, according to low concentration calibration curve equation formula Y=a_AX+b_ACalculate its viable bacteria ATP concentration C_AOijAnd T_AOij, meanwhile, the control sample and antimicrobial sample that 6 groups are sealed in sterilized petri dishes (28 ± 2) DEG C, After cultivating 48h ± 2h under conditions of (95 ± 2) %RH；Recycling remained on surface bacterium is eluted using mode identical with 0h contact sample And measure the relative intensity of fluorescence value I of recovered liquid_ACtij、I_ATtij, calculate corresponding viable bacteria ATP concentration C_AtijAnd T_Atij；

In antibiotic rate R and antibacterial activity value A is calculated:

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, contacted with 0h and after 48h is cultivated it is every The relative intensity of fluorescence measured value of part control sample and antimicrobial sample recovered liquidWith Based on number According to；In the case where testing condition for validity, its mould increasing value F after 48h is cultivated is calculated_ij、G_ijAnd antibiotic rate R_ijAnd antibacterial activity Value A_ij；To the R of every group of sample_ijAnd A_ijArithmetic mean of instantaneous value is taken to obtain corresponding R_iAnd A_i；The antibiotic rate R of every batch of antibacterial leather sample It is its three groups of sample R with antibacterial activity value A_iAnd A_iArithmetic mean of instantaneous value；And the clear related data revision of the convention and uncertainty of measurement are wanted It asks；

In evaluation of result:

With reference to health industry common practice and the requirement of dependent antimicrobial product standard, determine that fungicidal properties is classified criterion；When certain The antibiotic rate R of group (part) antibacterial leather sample_i(R_ij) or antibacterial activity value A_i(A_ij) mould proof with other two groups (four) samples When performance is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；It is calculated through ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back (part) antibacterial leather sample is mould proof Performance level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i(A_ij) Evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial leather sample fungicidal properties.

2. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that the sample preparation And pre-treatment, it carries out in the steps below:

(1) leather samples: by leather to be measured be placed in (20 ± 2) DEG C, (65 ± 5) %RH normal air environment in adjust at least 48h, it is ensured that free air circulation simultaneously touches each surface of leather, then, according to Fig. 1, Fig. 2, Fig. 3, figure in QB/T2707-2005 Shown in 4, using each related shadow stripe region as sampling point；The obvious shortcomings such as scratch, knife wound can not occur in leather sampling region, such as Fruit sampling point is unable to satisfy the requirement of sample size, can sample from its ridge line other side corresponding site or adjacent regions, positioning Afterwards, using mould knife from whole Zhang Ge, half stern back leather, shoulder leather or abdomen side remove from office grain (or imitative grain) cut sample, if without grain (or Imitative grain), then sample is cut from leather using face；Keep mould knife inner surface perpendicular to cut material surface in sampling process, it is inside and outside Surface in knife-edge part at (20 ± 1) ° angle, and the depth on the angle wedge shape side be greater than cut leather thicknesses (mould knife cut legend see QB/ Fig. 1 in T 2707-2005)；

(2) control sample: selecting raw cattle hide according to conventional leather-making technology to be processed into leather, (chemical materials used in process is not Containing fungicide and mould inhibitor ingredient) after, it is sampled with the circular mode knife that interior diameter is 50mm ± 1mm, thickness of sample is not more than 10mm； Then, sample surface to be measured is placed in below the ultraviolet disinfecting of 254nm, 40W upwards and irradiates 30min at 1.0m, and be sealed in In sterilized petri dishes, (- 10~-20) DEG C freezen protective is spare, and each strain test uses 6 groups of control samples；Wherein 0h contact and 48h culture experiment respectively uses 3 groups, every group of 5 samples；

(3) antimicrobial sample: according to the preparation method of control sample, the identical leather of raw material, technique, size, quantity is obtained Sample, putting it into mass concentration to impregnate ± 1h for 24 hours in 0.5% benzene thiophene cyanogen solution (ensures that benzene thiophene cyanogen content is in sample 250mg/kg~350mg/kg)；After taking-up by sample surface to be measured upwards, be placed horizontally in Biohazard Safety Equipment, spontaneously dry And be sealed in sterilized petri dishes, (- 10~-20) DEG C freezen protective is spare, every group of antimicrobial sample select one group of control sample as Object of reference simultaneously effectively identifies.

3. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that 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 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, Special test tube etc., ATP fluophotometer wavelength range 300nm~650nm, ATP Concentration Testing range 10^-11Mol/L~10^-6mol/L；Wave-length coverage 400nm~760nm, range of readings 0.0Abs~4.0Abs's Microplate reader；Amplification factor 40 ×~400 × Photobiology microscope；(25~30) DEG C ± 1 DEG C, (20~95) %RH ± 2% The constant temperature and humidity incubator of RH；46 DEG C ± 1 DEG C of constant water bath box；Revolving speed >=8000r/min centrifuge and mating centrifuge tube； The vortex oscillator of the range of speeds (500~3000) r/min；The pressure steam sterilizer of (121 ± 2) DEG C, (103 ± 5) kPa；- 20 DEG C~-80 DEG C of low temperature refrigerator；0 DEG C~10 DEG C of refrigerating box；The electronic balance of sensibility reciprocal 0.001g；Frequency range (30~50) The ultrasonic cleaner of kHz；The ultraviolet disinfecting of wavelength 254nm, power 40W；The pH meter of precision ± 0.1 (25 DEG C)；Electric furnace；

(3) material utensil: blood counting chamber and dedicated coverslip；The sterile measuring pipette of 1mL, 10mL；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%)；Filter hole Diameter is not more than 0.45 μm of syringe-driven filter；The sterile conical flask and bottle stopper of capacity 100mL, 250mL, 500mL；96 holes are flat Culture plate；The sterile petri dish of diameter 90mm；Glass funnel；Sterile cock test tube；Diameter is not more than the oese of 4mm；L stick； The bead of diameter 5mm；Alcolhol burner；Sterilize tweezers；Medical adhesive tape；Absorbent cotton and gauze for biochemistry detection；Interior diameter 50mm Circular mode knife； Thermometer；Hygrometer；The second of precision 0.01s Table；Aseptic filter paper；

(4) reagent: the benzene thiophene cyanogen solution that mass concentration is 0.5%；5mg/mL, the MTT (tetramethyl azo azoles salt) of pH value 7.4 are molten Liquid (0.45 μm of membrane filtration degerming, 4 DEG C~6 DEG C are kept in dark place 15d)；121 DEG C of high pressure sterilization 30min after the packing of following reagent, 5 DEG C~10 DEG C storage 30d:85% physiological saline；N monomethyl ethanesulfonic acid, Tween 80, two pungent sulfonation sodium succinates are chosen any one kind of them, It is configured to 0.05% wetting agent solution；

(5) medium/liquid (commercially available medium/liquid can be used): 121 DEG C of high pressure sterilization 30min after the packing of matched medium/liquid, 2 DEG C ~8 DEG C of storage 30d；

Cha Shi culture solution (preparation of mycotic spore liquid, bacteria suspension dilution and sample elution use): by 2g sodium nitrate, 1g phosphoric acid hydrogen two Potassium, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g ferrous sulfate, 30g sucrose, which are dissolved by heating, contains 0.05% wetting agent in 1000mL Aqueous solution in, adjust pH to 6.0~6.5 (25 DEG C)；

One dextrose culture-medium of potato (mycotic spore actication of culture and its total plate count plate count use): by the fresh horse of 300g Bell potato removes the peel stripping and slicing, boils 20min~30min in 1000mL water；Juice is filtered to take, 20g glucose, 0.1g are added into filtrate 1000mL is settled to after chloramphenicol, 20g agar；

(6) ATP fluorescence reaction reagent (or with commercial reagent): in addition to phosphate buffer solution, matched ATP fluorescence reaction reagent- 20 DEG C~-70 DEG C preservations, use in 6 months；

Dilution buffer: 0.005mol/L and the disodium phosphate soln for containing 0.037% sucrose adjust pH to 7.2 ± 0.2； 121 DEG C of high pressure sterilizations 15min, 2 DEG C~8 DEG C storage 30d；

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 Sharpe culture solution can be down to 10 in 15min^-11Mol/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, adjust pH to 12.0 ± 0.5 (fungal cell ATP extraction efficiency is not less than 80%)；

ATP fluorescent reagent: 0.7mg luciferase, the D- fluorescein of 12.6mg, 56mg bovine serum albumin are dissolved in 30mL's ATP fluorescent reagent buffer solution is stored at room temperature 15min, uses in 3h after mixing.

4. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that the strain is protected Hiding, activation and spore suspension preparation, carry out in the steps below:

(1) test strain: aspergillus niger ATCC 16404；Chaetomium globosum ATCC 6205；Penicillium chrysogenum ATCC 9179 (or by country Other strains that Culture Collection is provided and can be traced to the source)；

(2) culture presevation: mould test strain is inoculated in by potato-dextrose culture-medium inclined-plane with sterile working and is indicated Date, 28 DEG C~30 DEG C culture to inclined-planes cover with mycotic spore (7d~14d)；3 DEG C~10 DEG C preservation 4 months；

(3) actication of culture: with oese scrape preservation bacterium spore, be inoculated with potato-dextrose culture-medium inclined-plane, 28 DEG C~30 DEG C culture 7d~14d must not extract mold species test tube plug, every test tube before preparing spore suspension until generate a large amount of spores Only for spore liquid of preparation after opening, suspension is prepared using the mycotic spore newly cultivated every time；

(4) prepared by spore suspension: the sterile water of 10mL being added into strain test tube, the fresh of media surface is gently scraped with oese The spore stoste injection of wash-off is equipped with the sterile conical flask of jumping a queue of 15 beades and 45mL Cha Shi culture solution by mycotic spore In, 3000r/min shakes test tube 2min, breaks up spore ball, then sterile absorbent cotton or eight layers of gauze will be covered with by mixing spore liquid Glass funnel be placed on conical flask, filtering spore suspension removes mycelia and culture medium fragment；Filtrate is moved into sterilizing centrifugation Guan Zhong, 8000r/min separating treatment at least 10min, removes supernatant at room temperature；Spore precipitating is cleaned with 50mL Cha Shi culture solution again Object is simultaneously centrifuged, after repeated washing 3 times, with Cha Shi culture solution dilution centrifugation after spore sediment, every kind of test mould according to The above method prepares spore suspension, and the spore liquid of each test strain is mixed in equal volume；0 DEG C~7 DEG C storages, 4d is interior to be used.

5. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that the OD value-work Bacterial content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration carries out in the steps below:

(1) bacterium solution OD value measures: carrying out primary dcreening operation using spore total amount of the blood counting chamber to mixing spore suspension, is cultivated with Cha Shi Liquid is 6.0 × 10 to concentration⁸The mixing spore liquid of CFU/mL carries out 1:10,1:15,1:20 and is serially diluted, and using culture solution as Blank control sample liquid carries out zeroing correction to microplate reader, and then, the spore liquid of the above-mentioned different dilutions of 100 μ L is injected separately into 96 hole flat-bottomed plates, each dilution do 3 multiple holes；It is added dropwise the MTT solution of 20 μ L to every hole, (28 ± 1) DEG C, (95 ± 2) after %RH cultivates 30min；Maximum absorption band is scanned in 560nm~610nm wave-length coverage, determines maximum absorption wavelength；It surveys The viable bacteria OD value of fixed each hole mixed solution, the viable bacteria OD value of different dilution spore suspensions are the calculation of its 3 multiple holes OD measured values Art average value, meanwhile, the method referring to as defined in national standard GB 4789.15-2016 carries out above-mentioned mixing spore suspension appropriate dilute After releasing, in (28 ± 1) DEG C culture 3d, and bacterium colony counting is carried out to plate, demarcate its viable bacteria content C_B；

(2) OD value-viable bacteria content logarithm lgC_BStandard curve is established and inoculating spores liquid C_BCalibration: with different dilution spores The viable bacteria OD value of suspension is as ordinate, with its viable bacteria content logarithm lgC_BFor abscissa, OD value-lgC is drawn_BStandard curve； The linear equation Y=a of curve is derived from using least square fitting method₀X+b₀And coefficient R₀ ², work as R₀ ²>=0.98, it sets Letter is horizontal >=0.95 when, the spore liquid viable bacteria content C that is measured according to this patent method_BEffectively.

6. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that the ATP concentration Logarithm lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established and inoculation bacterium solution viable bacteria ATP concentration C_ACalibration, is pressed State step progress:

(1) determination of ATP standard serial solution and its test specimens preparation: ATP fluorescent reagent buffer solution is by 1.0 × 10^-3mol/L ATP standard stock solution be diluted to high and low concentration standard serial solution: 7.0 × 10^-8mol/L、7.0×10^-7mol/L、7.0×10^-6Mol/L and 2.1 × 10^-9mol/L、2.1×10^-8mol/L、2.1×10^-7Mol/L then will be on 0.1mL with sterilizing liquid-transfering gun It states ATP standard solution to move to respectively in three sterile test tubes, 0.05mL sterile water and 0.35mL normal saline solution is successively added dropwise And mix, as level-one ATP standard solution test specimens；0.1mL level-one ATP standard solution test specimens are moved into three nothings respectively again It is each that 0.4mL physiological saline is added dropwise and mixes in bacterium test tube, as second level ATP standard solution test specimens；And it will with sterilizing liquid-transfering gun The second level ATP standard solution test specimens of 0.1mL various concentration are moved to respectively in three instrument sterile test tubes, are marked as ATP Quasi- solution testing Duplicate Samples；

(2) blank background value calibration: aqueous solution, 0.35mL physiology salt with sterilizing liquid-transfering gun by 0.1mL containing 0.05% wetting agent The ATP lysate of water and 0.05mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；Stand 10min Level-one blank sample is used as after~20min；0.1mL level-one blank sample is moved in an other sterile test tube again, it is raw that 0.4mL is added dropwise Reason salt water simultaneously mixes, and as second level blank sample, then, 0.1mL second level blank sample is moved to three Zhi Yi respectively with sterilizing liquid-transfering gun In device special sterile test tube, as skip test Duplicate Samples；The ATP that 0.1mL is successively added dropwise into three Duplicate Samples extracts reagent, Instill the ATP fluorescent reagent of 0.1mL after mixing again, 3000r/min shakes test tube 5s, immediately with ATP fluorescent spectrophotometer measuring its Relative intensity of fluorescence value I_AAnd record and (ensure that each link operating time is consistent, avoid cross contamination), when each Duplicate Samples measure Between be no more than 15s, using the arithmetic mean of instantaneous value of three skip test Duplicate Samples relative intensity of fluorescence values as instrument and reagent set sheet Floors (or calibrating background according to instrument operation instruction)；

(3) ATP standard solution relative intensity of fluorescence value I_AMeasurement: from low to high according to concentration, to various concentration ATP standard The ATP that 0.1mL is added dropwise in three test Duplicate Samples of solution respectively extracts reagent, instills the ATP fluorescence examination of 0.1mL after mixing again Agent；3000r/min shakes test tube 5s, uses its relative intensity of fluorescence value of ATP fluorescent spectrophotometer measuring I immediately_AAnd it records and (ensures each The link operating time is consistent, avoids cross contamination), each Duplicate Samples minute is no more than 15s, molten with each concentration ATP standard The arithmetic mean of instantaneous value of three Duplicate Samples relative intensity of fluorescence values of liquid is its I_AMeasured value；

(4) ATP log concentration value lgC_ARelative intensity of fluorescence logarithm lgI_AStandard curve is established: with various concentration ATP standard The relative intensity of fluorescence logarithm lgI of solution_AAs abscissa, with its ATP log concentration value lgC_AFor ordinate mapping；To two Mathematical relationship between person carries out calibration curve, draws the lgC of two respective concentrations_A-lgI_AStandard curve；And application minimum two Multiply the linear equation Y=a that fitting process is derived from curve_A0X+b_A0(high concentration), Y=a_AX+b_A(low concentration) and related coefficient R_A0 ²(high concentration), R_A ²(low concentration), works as R_A0 ²And R_A ²>=0.98, when confidence level >=0.95, the measurement done according to this patent Effectively；

(5) it is inoculated with bacterium solution viable bacteria ATP concentration C_ACalibration: with Cha Shi culture solution to viable bacteria content C_BIt is 1.0 × 10⁸CFU/mL~5.0 ×10⁸The mould mixing spore liquid of CFU/mL carries out continuous 10 times of gradient dilutions, measures its relative intensity of fluorescence value I_A；According to height Concentration standard curve equation Y=a_A0X+b_A0It calculates and demarcates corresponding viable bacteria ATP concentration C_A, adjust, obtain through Cha Shi culture solution To C_ARange is 5.0 × 10^-7Mol/L~9.0 × 10^-7The inoculation bacterium solution of mol/L measures and records 0.3mL inoculation bacterium solution warp Relative intensity of fluorescence value I after the dilution of 4.6mL eluent in 1min_A0。

7. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that the sample connects Kind, culture and elution recycling, carry out in the steps below:

(1) inoculated and cultured: taking out each group control sample and antimicrobial sample from refrigerator, thaws to room temperature；With sterilizing liquid-transfering gun to 0.3mL inoculation bacterium solution is added dropwise (with lgC in its surface to be measured respectively_A-lgI_ACalibration curve is derived from same branch with spore liquid and mixes spore Liquid test tube, 0 DEG C ± 1 DEG C saves, and 2h is interior to be used), it is with L stick (attachment is inoculated with bacterium solution but does not hang drop) that bacterium solution smearing is uniform, make it Sample whole surface is covered, ware lid is covered, is sealed the plate equipped with 6 groups of 48h contact samples with medical adhesive tape；(28±2)℃, (95 ± 2) %RH cultivates 48h ± 2h；

(2) elution recycling: after 6 groups of 0h contact sample inoculation moulds, 4.6mL eluent is drawn with sterilizing liquid-transfering gun immediately and (is examined Family name's culture solution), each sample inoculation surface of repeated flushing at least 4 times in plate are sufficiently eluted and are blown repeatedly with liquid transfer gun head Mould washing lotion in plate is inhaled, until lawn again moves into washing lotion in sterile test tube after being broken up；3000r/min shakes test tube 2min, as the recovered liquid of sample to be tested (if recovered liquid adds eluent to 4.9mL), each group less than 4.9mL after mixing Sample after 48h is cultivated uses type of elution identical with 0h contact sample to recycle mould.

8. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that the reclaim liquid phase To fluorescence intensity level I_AMeasurement carries out in the steps below:

(1) instrument and reagent set relative intensity of fluorescence background value calibration: according to lgC_A-lgI_ABlank background when standard curve is established Calibration method, with the wetting agent solution of Cha Shi culture solution substitution 0.05%, determining instrument and reagent set background values；

(2) reclaim liquid phase is to fluorescence intensity level I_AMeasurement:, will with sterilizing liquid-transfering gun if background level meets instrument requirement The ATP lysate of 4.9mL recovered liquid and 0.1mL are separately added into same branch sterile test tube, and 3000r/min shakes test tube 30s；Room After temperature stands 20min, then the ATP extraction reagent of 5.0mL is added dropwise and mixes；It is stored at room temperature 10min, it will with sterilizing liquid-transfering gun The above-mentioned mixed solution of 0.1mL is moved to respectively in three instrument sterile test tubes, tests Duplicate Samples as ATP bioluminescence, to The ATP fluorescent reagent of 0.1mL is respectively added dropwise in three Duplicate Samples, 3000r/min shakes test tube 5s, uses ATP fluophotometer immediately Measure its relative intensity of fluorescence value I_AAnd record and (ensure that each link operating time is consistent, avoid cross contamination), each Duplicate Samples Minute is no more than 15s, using the arithmetic mean of instantaneous value of three ATP bioluminescences test Duplicate Samples relative intensity of fluorescence value as to The I of sample recovered liquid_AMeasured value.

9. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that the recovered liquid is living Bacterium ATP concentration C_AAnd T_AIt 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 C_AAnd T_AIt calculates

According to ATP low concentration standard curve lgC_A-lgI_ALinear equation Y=a_AX+b_A, calculate each group sample through 0h contact and The viable bacteria ATP concentration C of recovered liquid after 48h culture_AAnd T_A, relevant calculation is shown in formula (1)~(12):

In formula:

C_A0And C_At- 3 groups of 0h contact and after 48h is cultivated control sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；

C_A0ijAnd C_Atij- every 0h contact and after 48h is cultivated 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 48h is cultivated 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；

T_A0And T_At- 3 groups of 0h contact and after 48h is cultivated antimicrobial sample recycling viable bacteria average ATP concentration, unit is mole every It rises (mol/L)；

T_A0ijAnd T_Atij- every 0h contact and after 48h is cultivated 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 48h is cultivated 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 0.3mL inoculation bacterium solution of every 0h contact control sample are after the dilution of 4.6mL eluent in 1min Relative intensity of fluorescence measured value is close, i.e.,Every control sample recovered liquid relative intensity of fluorescence survey after 48h is cultivated The logarithm of definite valueThat is C_Atij≥0.1×C_A0ij, then when 3 groups of 0h contact control sample reclaim liquid phases are to glimmering Luminous intensity measured valueGroup in and when between-group variation coefficient CV≤10% (involved calculation formula is shown in this patent measurement of correlation Uncertainty requirement), the measurement carried out according to this patent method is effective；

(3) mould increasing value F_ij、G_ijIt calculates

Every control sample and antimicrobial sample are after 48h is cultivated, mould increasing value F_ij、G_ijIt is counted respectively according to formula (13), (14) It calculates:

In formula:

F_ijAnd G_ijThe mould increasing value of-every control sample and antimicrobial sample after 48h is cultivated, sample group i=1,2,3；Often Group sample number into spectrum j=1,2,3,4,5；

C_A0ijAnd C_AtijThe average ATP concentration that-every 0h is contacted and control sample recycles viable bacteria after 48h is cultivated, 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 48h is cultivated control sample recovered liquid relative intensity of fluorescence measured value, RLU； Group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4,5；

T_A0ijAnd T_AtijThe average ATP concentration values that-every 0h is contacted and antimicrobial sample recycles viable bacteria after 48h is cultivated, unit It 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；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(4) antibiotic rate R is calculated

It tests under condition for validity, the antibiotic rate R of every antibacterial leather sample_ij, every group and every batch of sample antibiotic rate R_iDistinguish with R It is calculated according to formula (15), (16), (17):

In formula:

R_ijThe antibiotic rate of-every antibacterial leather sample, %；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4, 5；

R-every batch of antibacterial leather sample antibiotic rate, %；

C_AtijAnd T_Atij- every antimicrobial sample and control sample recycle the average ATP concentration of viable bacteria after 48h is cultivated, 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 are after 48h is cultivated, 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；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(5) antibacterial activity value A is calculated

It tests under condition for validity, the antibacterial activity value A of every antibacterial leather sample_ij, every group and every batch of sample antibacterial activity value A_iIt is calculated respectively according to formula (18), (19), (20) with A:

In formula:

A_ijThe antibacterial activity value of-every antibacterial leather sample；Sample group i=1,2,3；Every group of sample number into spectrum j=1,2,3,4, 5；

A-every batch of antibacterial leather sample antibacterial activity value；

a_A- low concentration standard curve lgC_A-lgI_ASlope；

(6) data revision of the convention requirement: calibration mycotic spore liquid viable bacteria content C_BAnd ATP concentration of standard solution and inoculation bacterium solution, sample Recovered liquid viable bacteria ATP concentration C_AWhen, with reference to the data revision of the convention regulation in GB 4789.2-2016 in relation to clump count, work as C_BIt is less than 100CFU/mL or C_AWhen less than 100mol/L, " rounding up " round numbers；Work as C_BNot less than 100CFU/mL or C_AIt is not less than When 100mol/L, take preceding 2 bit digital after the 3rd bit digital " rounding up ", behind with 0 replace digit；Can also with 10 index shape Formula indicates that " rounding up " is returned afterwards using two effective digitals, control sample and antimicrobial sample after 0h is contacted and 48h is cultivated Receive the relative intensity of fluorescence measured value round numbers of liquid, antibiotic rate R_ij、R_i, R calculated result take three effective digitals；Mould increasing value F_ij、G_ijWith antibacterial activity value A_ij、A_i, A calculated result take two effective digitals；

(7) uncertainty of measurement: this patent method, which mainly passes through, calculates control sample and antimicrobial sample through 0h contact and 48h culture 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 the reproduction that ATP fluorimetric assay for biological materials method is applied to Leather mildew-proof performance test Property；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 48h is cultivated 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 formulaWithRespectively every group of control sample and antimicrobial sample are after 0h is contacted and 48h is cultivated, respectively The relative intensity of fluorescence measured value of Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples after 48h is cultivated 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 48h is trained 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 48h is cultivated that every group of 0h is contacted Product, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively 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 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

15 control samples, 15 antimicrobial samples and 15 control samples after 48h is cultivated of 3 groups of 0h contact, 15 it is anti- Bacterium sample, the standard deviation of the relative intensity of fluorescence measured value of recovered liquidAndRespectively 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 48h is trained After supporting, the relative intensity of fluorescence measured value of each Duplicate Samples):

10. the detection method of antibacterial leather fungicidal properties according to claim 1, which is characterized in that the result is commented Valence carries out in the steps below:

If using antibiotic rate R as correlated performance evaluation index: working as R_ij/R_iWhen/R < 80%, sample is without mildew-proof function；When 80%≤R_ij/R_iWhen/R < 90%, sample has mildew-proof function；As 90%≤R_ij/R_iWhen/R < 99%, sample mildew-proof function is suitable In；As 99%≤R_ij/R_iWhen/R < 99.9%, sample mildew-proof function is stronger；Work as R_ij/R_iWhen/R >=99.9%, the mould proof work of sample With extremely strong；

If using antibacterial activity value A as correlated performance evaluation index: working as A_ij/A_iWhen/A < 0.5, sample is without mildew-proof function； As 0.5≤A_ij/A_iWhen/A < 1.0, sample has mildew-proof function；As 1.0≤A_ij/A_iWhen/A < 2.0, sample mildew-proof function is suitable In；As 2.0≤A_ij/A_iWhen/A < 3.0, sample mildew-proof function is stronger；Work as A_ij/A_iWhen/A >=3.0, sample mildew-proof function is extremely strong；

(2) as the antibiotic rate R of certain group (part) antibacterial leather sample_i(R_ij) or antibacterial activity value A_i(A_ij) and other two groups (four) When the fungicidal properties of sample is compared to a levels are at least differed, one group of (part) sample is extracted again and repeats to test；Calculate its warp ATP bioluminescence lgC_A─lgI_AThe antibiotic rate or antibacterial activity value that calibration curve method measures, if two groups of front and back antibacterial leather sample Product fungicidal properties level is identical, then abandons it；Take other two groups (four) remaining sample antibiotic rate R_i(R_ij) or antibacterial activity value A_i (A_ij) evaluation result of the arithmetic mean of instantaneous value as batch (group) the antibacterial leather sample fungicidal properties.