WO2018018784A1 - 一种益生菌抗菌家用纺织品抗菌性能的测试方法 - Google Patents

一种益生菌抗菌家用纺织品抗菌性能的测试方法 Download PDF

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WO2018018784A1
WO2018018784A1 PCT/CN2016/105675 CN2016105675W WO2018018784A1 WO 2018018784 A1 WO2018018784 A1 WO 2018018784A1 CN 2016105675 W CN2016105675 W CN 2016105675W WO 2018018784 A1 WO2018018784 A1 WO 2018018784A1
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sample
pipette
antibacterial
culture
cfu
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PCT/CN2016/105675
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French (fr)
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汪明星
刘金抗
陈永兵
陈凤
葛玲
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紫罗兰家纺科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

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  • the invention particularly relates to a test method for antibacterial properties of probiotic antibacterial household textiles.
  • the present invention provides a test method for antibacterial properties of probiotic antibacterial household textiles.
  • a test method for antibacterial properties of probiotic antibacterial household textiles comprising the following steps:
  • a loop of bacteria was taken from the slant of the 3rd to 10th generation of the bacteria preservation strain tube, and streaked on the nutrient agar plate for culture, the culture temperature was 36 ° C -38 ° C, the culture time was 18 h - 24 h; A typical colony was picked from the plate and inoculated in 20 mL of nutrient broth at a temperature of 36 ° C to 38 ° C. The rotation speed was 130 r / min. After shaking for 18 h to 20 h, the inoculum suspension was prepared. Determination by spectrophotometer, the number of viable bacteria should reach 1*10 9 CFU/ml—5*10 9 CFU/ml. This fresh bacterial solution should be used as soon as possible within 4 hours to ensure the activity of the inoculated bacteria;
  • Pipette 2mL - 3mL from the bacterial suspension that is, take the upper limit of Staphylococcus aureus, take the lower limit of E. coli, transfer to a test tube containing 9mL nutrient broth, mix well; pipette 1ml into another branch with 9mL In the test tube of the nutrient broth, mix well; pipette 1 mL into a test tube containing 9 mL of 0.03 mol/L PBS buffer and mix well; pipette 5 mL into a conical flask containing 45 mL of 0.03 mol/L PBS buffer.
  • the inoculum contains a trace amount of nutrient broth for The sample is inoculated, and the inoculum should be used as soon as possible within 4 hours to maintain the activity of the inoculum;
  • the probiotic antibacterial fabric and its untreated control sample were respectively cut into a size of about 5 mm * 5 mm, and 0.75 g ⁇ 0.05 g was weighed as a sample, wrapped in a small piece of paper, and a multi-sample was weighed according to the experiment. Each sample is wrapped in small pieces of paper;
  • the small paper bag containing the sample is placed in an autoclave and sterilized at 121 ° C and 103 kPa for 15 minutes; (4) Sample bottling
  • the inhibition rate is calculated according to formula (A.1):
  • A The number of Staphylococcus aureus on the probiotic-prepared sample cloth after 6 days, the unit is (CFU/mL);
  • composition and content of the nutrient broth are as follows: beef extract 3g; peptone 5g; deionized water 1000ml; after sterilization, the pH value is 6.8 ⁇ 0.2.
  • the components and contents of the nutrient agar medium are as follows: beef extract 3 g; peptone 5 g; agar powder 15 g; deionized water 1000 ml; after sterilization, the pH value is 6.8 ⁇ 0.2.
  • the composition and content of the PBS buffer are as follows: 2.84 g of disodium hydrogen phosphate; 1.36 g of potassium dihydrogen phosphate; 1000 ml of deionized water; after sterilization, the pH is 7.2-7.4, at 5 ° C - 10 ° C Save the reserve at the temperature.
  • test sample and the control sample are placed in a buffer containing the inoculum, and the mechanism of cutting the bacterial food chain by the culture sample is starved to reduce the number of colonies.
  • the antibacterial properties of the test samples were examined under the conditions of the specified temperature and humidity under the bacteriostatic rate after 3 days (E. coli) and 6 days (S. aureus).
  • a test method for antibacterial properties of probiotic antibacterial household textiles comprising the following steps:
  • a loop of bacteria was taken from the slanted surface of the 3rd to 10th generation of the bacterial preservation strain, and streaked on the nutrient agar plate, cultured at a temperature of 36 ° C, and cultured for 18 hours; the inoculation ring was used to pick out the plate.
  • a typical colony was inoculated into 20mL nutrient broth at a temperature of 36 ° C and a rotation speed of 130 r / min. After shaking for 18 h, the inoculum suspension was prepared. The content of the bacterial liquid was determined by spectrophotometry. Up to 1*10 9 CFU/ml, this fresh bacterial solution should be used as soon as possible within 4 hours to ensure the activity of the inoculated bacteria;
  • Pipette 2mL from the bacterial suspension which is the upper limit of Staphylococcus aureus, take the lower limit of E. coli, transfer to a test tube containing 9mL nutrient broth, mix well; pipette 1ml into another with 9mL of nutrient meat In the test tube, mix thoroughly; pipette 1 mL into a test tube containing 9 mL of 0.03 mol/L PBS buffer and mix well; pipette 5 mL into a conical flask containing 45 mL of 0.03 mol/L PBS buffer and mix thoroughly.
  • the inoculum contains a trace amount of nutrient broth, used to inoculate the sample, the inoculum should be in Use as soon as possible within 4 hours to maintain the activity of the inoculum;
  • the probiotic antibacterial fabric and its untreated control sample were respectively cut into a size of about 5 mm * 5 mm, and 0.70 g was weighed as a sample, wrapped in a small piece of paper, and a multi-sample was weighed according to the experimental needs. All samples are wrapped in small pieces of paper;
  • the small paper bag containing the sample is placed in an autoclave, and sterilized at 121 ° C, 103 kPa for 15 minutes;
  • test tube draw 0.9mL into the sterilized plate, pour the nutrient agar medium into 15mL, and dilute each tube to make two plates for counting, that is, the number of colonies should be within 15%, otherwise
  • the data were ineffective, solidified at room temperature, inverted plate, Escherichia coli was cultured on probiotic-treated fabric at 36 ° C for 72 h, staphylococcus 144 h, and the number of colonies per plate was recorded.
  • the untreated control was inoculated with "0" contact time, diluted 100 In the double plate, the number of colonies is controlled between 200 CFU, otherwise the test accuracy is affected;
  • the inhibition rate is calculated according to formula (A.1):
  • A The number of Staphylococcus aureus on the probiotic-prepared sample cloth after 6 days, the unit is (CFU/mL);
  • the components and contents of the nutrient broth are as follows: beef extract 3 g; peptone 5 g; deionized water 1000 ml; after sterilization, pH 6.6.
  • the components and contents of the nutrient agar medium are as follows: beef extract 3 g; peptone 5 g; agar powder 15 g; Deionized water 1000ml; after sterilization, the pH is 6.6.
  • the components and contents of the PBS buffer were as follows: 2.84 g of disodium hydrogen phosphate; 1.36 g of potassium dihydrogen phosphate; 1000 ml of deionized water; after sterilization, the pH was 7.2, and stored at 5 ° C for use.
  • a test method for antibacterial properties of probiotic antibacterial household textiles comprising the following steps:
  • a loop-inoculation bacteria was taken from the slant of the 3rd to 10th generation of the bacterial preservation strain test tube, and the cultured agar plate was streaked and cultured at a culture temperature of 38 ° C for 24 hours; the inoculation loop was used to pick out the plate.
  • a typical colony was inoculated into 20mL nutrient broth at a temperature of 38 ° C and a rotational speed of 130 r / min. After shaking for 20 h, the inoculum suspension was prepared. The content of the bacterial liquid was determined by spectrophotometry. Up to 5*10 9 CFU/ml, this fresh liquid should be used as soon as possible within 4 hours to ensure the activity of the inoculated bacteria;
  • Pipette 3mL from the bacterial suspension that is, take the upper limit of Staphylococcus aureus, take the lower limit of E. coli, transfer it to the test tube containing 9mL nutrient broth, mix well; take 1ml and transfer to another with 9mL of nutrient meat In the test tube, mix thoroughly; pipette 1 mL into a test tube containing 9 mL of 0.03 mol/L PBS buffer and mix well; pipette 5 mL into a conical flask containing 45 mL of 0.03 mol/L PBS buffer and mix thoroughly.
  • the inoculum contains a trace amount of nutrient broth, used to inoculate the sample, the inoculum should be in Use as soon as possible within 4 hours to maintain the activity of the inoculum;
  • the probiotic antibacterial fabric and its untreated control sample were respectively cut into a size of about 5 mm * 5 mm, and 0.80 g was weighed as a sample, wrapped in a small piece of paper, and a multi-sample was weighed according to the experimental needs. All samples are wrapped in small pieces of paper;
  • the small paper bag containing the sample is placed in an autoclave, and sterilized at 121 ° C, 103 kPa for 15 minutes;
  • the data is invalid, solidified at room temperature, inverted plate, Escherichia coli cultured on probiotic treatment fabric at 38 ° C for 72 h, Staphylococcus 144 h, the number of colonies per plate was recorded; wherein, the untreated control was inoculated with "0" contact time, In the 100-fold dilution plate, the number of colonies is controlled between 250 CFU, otherwise the test accuracy is affected;
  • the inhibition rate is calculated according to formula (A.1):
  • A The number of Staphylococcus aureus on the probiotic-prepared sample cloth after 6 days, the unit is (CFU/mL);
  • composition and content of the nutrient broth are as follows: beef extract 3g; peptone 5g; deionized water 1000ml; After sterilization, the pH was 7.0.
  • the components and contents of the nutrient agar medium were as follows: beef extract 3 g; peptone 5 g; agar powder 15 g; deionized water 1000 ml; after sterilization, pH was 7.0.
  • the components and contents of the PBS buffer were as follows: 2.84 g of disodium hydrogen phosphate; 1.36 g of potassium dihydrogen phosphate; 1000 ml of deionized water; after sterilization, the pH was 7.4, and stored at a temperature of 10 ° C for use.
  • a test method for antibacterial properties of probiotic antibacterial household textiles comprising the following steps:
  • a loop of bacteria was taken from the slant of the 3rd to 10th generation of the bacterial preservation strain, and streaked on the nutrient agar plate for culture, the culture temperature was 37 ° C, and the culture time was 21 h; the inoculation ring was used to pick out the plate.
  • a typical colony was inoculated into 20mL nutrient broth at a temperature of 37 ° C and a rotation speed of 130 r / min. After shaking for 19 h, the inoculum suspension was prepared.
  • the bacterial liquid content was determined by spectrophotometry. Up to 3*10 9 CFU/ml, the fresh bacterial solution should be used as soon as possible within 4 hours to ensure the activity of the inoculated bacteria;
  • Pipette 2.5mL from the bacterial suspension that is, take the upper limit of Staphylococcus aureus, take the lower limit of E. coli, transfer it to the test tube containing 9mL nutrient broth, mix well; pipette 1ml into another with 9mL nutrition Mix well in the test tube of the broth; pipette 1 mL into a test tube containing 9 mL of 0.03 mol/L PBS buffer and mix well; pipette 5 mL into a conical flask containing 45 mL of 0.03 mol/L PBS buffer, fully Mix and dilute to the number of live bacteria 3.5*10 5 CFU/mL, thus the fixed 4 dilution procedures.
  • the inoculum contains a trace amount of nutrient broth for inoculating the sample. Use as soon as possible within 4 hours to maintain the activity of the inoculum;
  • the probiotic antibacterial fabric and its untreated control sample were respectively cut into a size of about 5 mm * 5 mm, and 0.75 g was weighed as a sample, wrapped in a small piece of paper, and a multi-sample was weighed according to the experimental needs. All samples are wrapped in small pieces of paper;
  • the small paper bag containing the sample is placed in an autoclave, and sterilized at 121 ° C, 103 kPa for 15 minutes;
  • the difference between the number of colonies should be within 15%. Otherwise, The data were ineffective, solidified at room temperature, inverted plate, E. coli was cultured on probiotic-treated fabric at 37 ° C for 72 h, staphylococcus 144 h, and the number of colonies per plate was recorded. Among them, the untreated control was inoculated with "0" contact time, diluted 100 In the double plate, the number of colonies is controlled between 230 CFU, otherwise the test accuracy is affected;
  • the inhibition rate is calculated according to formula (A.1):
  • A The number of Staphylococcus aureus on the probiotic-prepared sample cloth after 6 days, the unit is (CFU/mL);
  • the components and contents of the nutrient broth are as follows: beef extract 3 g; peptone 5 g; deionized water 1000 ml; after sterilization, pH 6.8.
  • the components and contents of the nutrient agar medium were as follows: beef extract 3 g; peptone 5 g; agar powder 15 g; deionized water 1000 ml; after sterilization, pH was 6.8.
  • the components and contents of the PBS buffer were as follows: 2.84 g of disodium hydrogen phosphate; 1.36 g of potassium dihydrogen phosphate; 1000 ml of deionized water; after sterilization, the pH was 7.3, and stored at a temperature of 8 ° C for use.
  • test sample and the control sample are placed in a buffer containing the inoculum, and the mechanism of cutting the bacterial food chain by the culture sample is starved to reduce the number of colonies.
  • the antibacterial properties of the test samples were examined under the conditions of the specified temperature and humidity under the bacteriostatic rate after 3 days (E. coli) and 6 days (S. aureus).
  • Staphylococcus aureus (ATCC 6538) and Escherichia coli (8099) used in this test may be harmful to people or the environment, so the test should be carried out by experienced inspectors trained by relevant testing techniques. This appendix does not identify all possible security issues. It is the responsibility of the user to take appropriate safety and health measures as well as specific measures to ensure compliance with the relevant national regulations.
  • Probiotic antibacterial household textiles are classified into three antibacterial grades: A grade, AA grade and AAA grade according to the number of washable times. See Table 1 below for the inhibition rate of probiotic antibacterial household textiles.

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Abstract

本发明公开了一种益生菌抗菌家用纺织品抗菌性能的测试方法，包括如下步骤：金黄色葡萄球菌(ATCC 6538)、大肠杆菌(8099)菌液培养和准备；试样准备；试样灭菌；试样装瓶；"0"接触时间振荡接触、稀释培养及菌落数测定；结果计算。本发明将试验样品及对照样品上放置于含有接种菌液的缓冲液中，通过培养试样将细菌食物链切断的作用机理，将其饿死，从而减少菌落数。在规定的温湿度条件下，以培养3天(大肠杆菌)和6天(金黄色葡萄球菌)后的抑菌率考察测试样品的抗菌性能。

Description

一种益生菌抗菌家用纺织品抗菌性能的测试方法 技术领域
本发明具体涉及一种益生菌抗菌家用纺织品抗菌性能的测试方法。
背景技术
由于传统家居用品的原料、生产、运输、销售中受到一定程度的环境和人工污染，从而导致产品携带污染物和病菌；给广大的消费者带来使用中的健康隐患；传统家居用品是在普通环境下进行生产，自然界的温湿度、空气中有害物质、粉尘，以及家居用品原料本身就携带病毒、细菌、化学有害物等；少数产品甚至为携带病毒和病菌的载体。
发明内容
发明目的：本发明提供一种益生菌抗菌家用纺织品抗菌性能的测试方法。
技术方案：一种益生菌抗菌家用纺织品抗菌性能的测试方法，包括如下步骤：
(1)金黄色葡萄球菌(ATCC 6538)、大肠杆菌(8099)菌液培养和准备
从3代—10代的细菌保存菌种试管斜面中取一接种环细菌，在营养琼脂平板上划线，进行培养，培养温度为36℃-38℃，培养时间为18h—24h；用接种环从平板中挑出一个典型菌落，接种于20mL营养肉汤中，温度为36℃-38℃，转速为130r/min，振荡培养18h—20h，即制成了接种菌悬液；菌液含量采用分光光度计法测定，活菌数应达到1*109CFU/ml—5*109CFU/ml,此新鲜菌液应在4h内尽快使用，以保证接种菌的活性；
用吸管从细菌悬液中吸取2mL—3mL,即为金黄色葡萄球菌取上限，大肠杆菌取下限，移入装有9mL营养肉汤的试管中，充分混匀；吸取1ml移入另一支装有 9mL营养肉汤的试管中，充分混匀；吸取1mL移入装有9mL0.03mol/L PBS缓冲液的试管中，充分混匀；吸取5mL移入装有45mL0.03mol/L PBS缓冲液的三角烧瓶中，充分混匀，稀释至含活菌数目3*105CFU/mL—4*105CFU/mL，由此固定的4次稀释程序，此接种菌液中含有微量的营养肉汤，用来对试样接种，此接种菌液应在4h内尽快使用，以保持接种菌的活性；
(2)试样准备
将益生菌抗菌织物及其未处理对照试样分别剪成约5mm*5mm大小，称取0.75g±0.05g作为一份试样，用小纸片包好，根据实验需要称取多分试样，每份试样均用小纸片包好；
(3)试样灭菌
将装有试样的小纸包放入高压灭菌锅，于121℃、103kPa灭菌15min备用；(4)试样装瓶
准备8个三角烧瓶，在其中4个烧瓶中各加入一份益生菌抗菌织物，4个烧瓶中各加入未处理对照样一份，然后在每个烧瓶中个加入70m L±0.1m L 0.03mol/L PBS缓冲液；
(5)“0”接触时间振荡接触、稀释培养及菌落数测定
用吸管往8个试样烧瓶中各加入5ml接种菌液，盖好瓶塞，再将此8个烧瓶置于恒温振荡器上，在24℃±1℃，以100r/min—150r/min，振荡1min±5s后，从每个烧瓶中吸取1mL±0.1mL试液，移入装有9mL±0.1mL 0.03mol/L PBS缓冲液的试管中，充分混匀；用10倍稀释法再进行1次稀释，充分混匀；用吸管从每个稀释倍数的试管中分别吸取1mL±0.1mL移入灭菌的平皿，倾注营养琼脂培养基15mL，每个稀释倍数的试管分别吸液制作两个平板进行计数，即二者的菌落数相差应在15％以内，否则数据无效，室温凝固，倒置平板，以37℃±1℃在益生 菌处理面料上培养大肠杆菌72h，葡萄球菌144h，记录每个平板的菌落数；其中，未处理对照样接种“0”接触时间，稀释100倍平板中，菌落数控制在200CFU—250CFU之间，否则影响试验精确度；
(6)结果计算
以分离益生菌提取纯计数葡萄球菌为例，抑菌率按式(A.1)计算：
Figure PCTCN2016105675-appb-000001
式中：
Y——抑菌率，单位为％；
A——6天后经益生菌整理样布上的金黄色葡萄球菌数，单位为(CFU/mL)；
B——6天后未整理对照样布上的金黄色葡萄球菌数，单位为(CFU/mL)。
作为优化：所述营养肉汤的成分及含量如下：牛肉膏3g；蛋白胨5g；去离子水1000ml；灭菌后，pH值为6.8±0.2。
作为优化：所述营养琼脂培养基的成分及含量如下：牛肉膏3g；蛋白胨5g；琼脂粉15g；去离子水1000ml；灭菌后，PH值为6.8±0.2。
作为优化：所述PBS缓冲液的成分及含量如下：磷酸氢二钠2.84g；磷酸二氢钾1.36g；去离子水1000ml；灭菌后，pH值为7.2—7.4,在5℃—10℃的温度下保存备用。
有益效果：本发明将试验样品及对照样品上放置于含有接种菌液的缓冲液中，通过培养试样将细菌食物链切断的作用机理，将其饿死，从而减少菌落数。在规定的温湿度条件下，以培养3天(大肠杆菌)和6天(金黄色葡萄球菌)后的抑菌率考察测试样品的抗菌性能。
具体实施方式
下面结合具体实施例对本发明进行详细阐述。
具体实施例一：
一种益生菌抗菌家用纺织品抗菌性能的测试方法，包括如下步骤：
(1)金黄色葡萄球菌(ATCC 6538)、大肠杆菌(8099)菌液培养和准备
从3代—10代的细菌保存菌种试管斜面中取一接种环细菌，在营养琼脂平板上划线，进行培养，培养温度为36℃，培养时间为18h；用接种环从平板中挑出一个典型菌落，接种于20mL营养肉汤中，温度为36℃，转速为130r/min，振荡培养18h，即制成了接种菌悬液；菌液含量采用分光光度计法测定，活菌数应达到1*109CFU/ml,此新鲜菌液应在4h内尽快使用，以保证接种菌的活性；
用吸管从细菌悬液中吸取2mL,即为金黄色葡萄球菌取上限，大肠杆菌取下限，移入装有9mL营养肉汤的试管中，充分混匀；吸取1ml移入另一支装有9mL营养肉汤的试管中，充分混匀；吸取1mL移入装有9mL0.03mol/L PBS缓冲液的试管中，充分混匀；吸取5mL移入装有45mL0.03mol/L PBS缓冲液的三角烧瓶中，充分混匀，稀释至含活菌数目3*105CFU/mL，由此固定的4次稀释程序，此接种菌液中含有微量的营养肉汤，用来对试样接种，此接种菌液应在4h内尽快使用，以保持接种菌的活性；
(2)试样准备
将益生菌抗菌织物及其未处理对照试样分别剪成约5mm*5mm大小，称取0.70g作为一份试样，用小纸片包好，根据实验需要称取多分试样，每份试样均用小纸片包好；
(3)试样灭菌
将装有试样的小纸包放入高压灭菌锅，于121℃、103kPa灭菌15min备用；
(4)试样装瓶
准备8个三角烧瓶，在其中4个烧瓶中各加入一份益生菌抗菌织物，4个烧瓶中各加入未处理对照样一份，然后在每个烧瓶中个加入69.9m L 0.03mol/L PBS缓冲液；
(5)“0”接触时间振荡接触、稀释培养及菌落数测定
用吸管往8个试样烧瓶中各加入5ml接种菌液，盖好瓶塞，再将此8个烧瓶置于恒温振荡器上，在23℃，以100r/min，振荡55s后，从每个烧瓶中吸取0.9mL试液，移入装有8.9mL 0.03mol/L PBS缓冲液的试管中，充分混匀；用10倍稀释法再进行1次稀释，充分混匀；用吸管从每个稀释倍数的试管中分别吸取0.9mL移入灭菌的平皿，倾注营养琼脂培养基15mL，每个稀释倍数的试管分别吸液制作两个平板进行计数，即二者的菌落数相差应在15％以内，否则数据无效，室温凝固，倒置平板，以36℃在益生菌处理面料上培养大肠杆菌72h，葡萄球菌144h，记录每个平板的菌落数；其中，未处理对照样接种“0”接触时间，稀释100倍平板中，菌落数控制在200CFU之间，否则影响试验精确度；
(6)结果计算
以分离益生菌提取纯计数葡萄球菌为例，抑菌率按式(A.1)计算：
Figure PCTCN2016105675-appb-000002
式中：
Y——抑菌率，单位为％；
A——6天后经益生菌整理样布上的金黄色葡萄球菌数，单位为(CFU/mL)；
B——6天后未整理对照样布上的金黄色葡萄球菌数，单位为(CFU/mL)。
所述营养肉汤的成分及含量如下：牛肉膏3g；蛋白胨5g；去离子水1000ml；灭菌后，pH值为6.6。
所述营养琼脂培养基的成分及含量如下：牛肉膏3g；蛋白胨5g；琼脂粉15g； 去离子水1000ml；灭菌后，PH值为6.6。
所述PBS缓冲液的成分及含量如下：磷酸氢二钠2.84g；磷酸二氢钾1.36g；去离子水1000ml；灭菌后，pH值为7.2,在5℃的温度下保存备用。
具体实施例二：
一种益生菌抗菌家用纺织品抗菌性能的测试方法，包括如下步骤：
(1)金黄色葡萄球菌(ATCC 6538)、大肠杆菌(8099)菌液培养和准备
从3代—10代的细菌保存菌种试管斜面中取一接种环细菌，在营养琼脂平板上划线，进行培养，培养温度为38℃，培养时间为24h；用接种环从平板中挑出一个典型菌落，接种于20mL营养肉汤中，温度为38℃，转速为130r/min，振荡培养20h，即制成了接种菌悬液；菌液含量采用分光光度计法测定，活菌数应达到5*109CFU/ml,此新鲜菌液应在4h内尽快使用，以保证接种菌的活性；
用吸管从细菌悬液中吸取3mL,即为金黄色葡萄球菌取上限，大肠杆菌取下限，移入装有9mL营养肉汤的试管中，充分混匀；吸取1ml移入另一支装有9mL营养肉汤的试管中，充分混匀；吸取1mL移入装有9mL0.03mol/L PBS缓冲液的试管中，充分混匀；吸取5mL移入装有45mL0.03mol/L PBS缓冲液的三角烧瓶中，充分混匀，稀释至含活菌数目4*105CFU/mL，由此固定的4次稀释程序，此接种菌液中含有微量的营养肉汤，用来对试样接种，此接种菌液应在4h内尽快使用，以保持接种菌的活性；
(2)试样准备
将益生菌抗菌织物及其未处理对照试样分别剪成约5mm*5mm大小，称取0.80g作为一份试样，用小纸片包好，根据实验需要称取多分试样，每份试样均用小纸片包好；
(3)试样灭菌
将装有试样的小纸包放入高压灭菌锅，于121℃、103kPa灭菌15min备用；
(4)试样装瓶
准备8个三角烧瓶，在其中4个烧瓶中各加入一份益生菌抗菌织物，4个烧瓶中各加入未处理对照样一份，然后在每个烧瓶中个加入70.1mL 0.03mol/L PBS缓冲液；
(5)“0”接触时间振荡接触、稀释培养及菌落数测定
用吸管往8个试样烧瓶中各加入5ml接种菌液，盖好瓶塞，再将此8个烧瓶置于恒温振荡器上，在25℃，以150r/min，振荡1min±5s后，从每个烧瓶中吸取1.1mL试液，移入装有9.1mL 0.03mol/L PBS缓冲液的试管中，充分混匀；用10倍稀释法再进行1次稀释，充分混匀；用吸管从每个稀释倍数的试管中分别吸取1.1mL移入灭菌的平皿，倾注营养琼脂培养基15mL，每个稀释倍数的试管分别吸液制作两个平板进行计数，即二者的菌落数相差应在15％以内，否则数据无效，室温凝固，倒置平板，以38℃在益生菌处理面料上培养大肠杆菌72h，葡萄球菌144h，记录每个平板的菌落数；其中，未处理对照样接种“0”接触时间，稀释100倍平板中，菌落数控制在250CFU之间，否则影响试验精确度；
(6)结果计算
以分离益生菌提取纯计数葡萄球菌为例，抑菌率按式(A.1)计算：
Figure PCTCN2016105675-appb-000003
式中：
Y——抑菌率，单位为％；
A——6天后经益生菌整理样布上的金黄色葡萄球菌数，单位为(CFU/mL)；
B——6天后未整理对照样布上的金黄色葡萄球菌数，单位为(CFU/mL)。
所述营养肉汤的成分及含量如下：牛肉膏3g；蛋白胨5g；去离子水1000ml； 灭菌后，pH值为7.0。
所述营养琼脂培养基的成分及含量如下：牛肉膏3g；蛋白胨5g；琼脂粉15g；去离子水1000ml；灭菌后，PH值为7.0。
所述PBS缓冲液的成分及含量如下：磷酸氢二钠2.84g；磷酸二氢钾1.36g；去离子水1000ml；灭菌后，pH值为7.4,在10℃的温度下保存备用。
具体实施例三：
一种益生菌抗菌家用纺织品抗菌性能的测试方法，包括如下步骤：
(1)金黄色葡萄球菌(ATCC 6538)、大肠杆菌(8099)菌液培养和准备
从3代—10代的细菌保存菌种试管斜面中取一接种环细菌，在营养琼脂平板上划线，进行培养，培养温度为37℃，培养时间为21h；用接种环从平板中挑出一个典型菌落，接种于20mL营养肉汤中，温度为37℃，转速为130r/min，振荡培养19h，即制成了接种菌悬液；菌液含量采用分光光度计法测定，活菌数应达到3*109CFU/ml,此新鲜菌液应在4h内尽快使用，以保证接种菌的活性；
用吸管从细菌悬液中吸取2.5mL,即为金黄色葡萄球菌取上限，大肠杆菌取下限，移入装有9mL营养肉汤的试管中，充分混匀；吸取1ml移入另一支装有9mL营养肉汤的试管中，充分混匀；吸取1mL移入装有9mL0.03mol/L PBS缓冲液的试管中，充分混匀；吸取5mL移入装有45mL0.03mol/L PBS缓冲液的三角烧瓶中，充分混匀，稀释至含活菌数目3.5*105CFU/mL，由此固定的4次稀释程序，此接种菌液中含有微量的营养肉汤，用来对试样接种，此接种菌液应在4h内尽快使用，以保持接种菌的活性；
(2)试样准备
将益生菌抗菌织物及其未处理对照试样分别剪成约5mm*5mm大小，称取0.75g作为一份试样，用小纸片包好，根据实验需要称取多分试样，每份试样均用小纸片包好；
(3)试样灭菌
将装有试样的小纸包放入高压灭菌锅，于121℃、103kPa灭菌15min备用；
(4)试样装瓶
准备8个三角烧瓶，在其中4个烧瓶中各加入一份益生菌抗菌织物，4个烧瓶中各加入未处理对照样一份，然后在每个烧瓶中个加入70mL 0.03mol/L PBS缓冲液；
(5)“0”接触时间振荡接触、稀释培养及菌落数测定
用吸管往8个试样烧瓶中各加入5ml接种菌液，盖好瓶塞，再将此8个烧瓶置于恒温振荡器上，在24℃，以100r/min，振荡1min后，从每个烧瓶中吸取1mL±0.1mL试液，移入装有9mL 0.03mol/L PBS缓冲液的试管中，充分混匀；用10倍稀释法再进行1次稀释，充分混匀；用吸管从每个稀释倍数的试管中分别吸取1mL移入灭菌的平皿，倾注营养琼脂培养基15mL，每个稀释倍数的试管分别吸液制作两个平板进行计数，即二者的菌落数相差应在15％以内，否则数据无效，室温凝固，倒置平板，以37℃在益生菌处理面料上培养大肠杆菌72h，葡萄球菌144h，记录每个平板的菌落数；其中，未处理对照样接种“0”接触时间，稀释100倍平板中，菌落数控制在230CFU之间，否则影响试验精确度；
(6)结果计算
以分离益生菌提取纯计数葡萄球菌为例，抑菌率按式(A.1)计算：
Figure PCTCN2016105675-appb-000004
式中：
Y——抑菌率，单位为％；
A——6天后经益生菌整理样布上的金黄色葡萄球菌数，单位为(CFU/mL)；
B——6天后未整理对照样布上的金黄色葡萄球菌数，单位为(CFU/mL)。
所述营养肉汤的成分及含量如下：牛肉膏3g；蛋白胨5g；去离子水1000ml；灭菌后，pH值为6.8。
所述营养琼脂培养基的成分及含量如下：牛肉膏3g；蛋白胨5g；琼脂粉15g；去离子水1000ml；灭菌后，PH值为6.8。
所述PBS缓冲液的成分及含量如下：磷酸氢二钠2.84g；磷酸二氢钾1.36g；去离子水1000ml；灭菌后，pH值为7.3,在8℃的温度下保存备用。
本发明将试验样品及对照样品上放置于含有接种菌液的缓冲液中，通过培养试样将细菌食物链切断的作用机理，将其饿死，从而减少菌落数。在规定的温湿度条件下，以培养3天(大肠杆菌)和6天(金黄色葡萄球菌)后的抑菌率考察测试样品的抗菌性能。
本试验所采用的金黄色葡萄球菌(ATCC 6538)、大肠杆菌(8099)可对人员或环境有危害，因此试验应由经相关检测技术训练且有经验的检测人员从事。本附录并未指出所有可能的安全问题。使用者有责任采取适当的安全、健康措施及具体仿佛措施，并保证符合国家有关法规规定的条件。
益生菌抗菌家用纺织品按耐水洗次数不同分为A级、AA级、AAA级三个抗菌级别。对测试益生菌抗菌家用纺织品的抑菌率指标见下表1。
表1益生菌抗菌家用纺织品的抑菌率指标
Figure PCTCN2016105675-appb-000005
本发明不局限于上述最佳实施方式，任何人在本发明的启示下都可得出其他各种形式的产品，但不论在其形状或结构上作任何变化，凡是具有与本申请相同或相近似的技术方案，均落在本发明的保护范围之内。

Claims (4)

  1. 一种益生菌抗菌家用纺织品抗菌性能的测试方法，其特征在于：包括如下步骤：
    (1)金黄色葡萄球菌(ATCC 6538)、大肠杆菌(8099)菌液培养和准备
    从3代—10代的细菌保存菌种试管斜面中取一接种环细菌，在营养琼脂平板上划线，进行培养，培养温度为36℃-38℃，培养时间为18h—24h；用接种环从平板中挑出一个典型菌落，接种于20mL营养肉汤中，温度为36℃-38℃，转速为130r/min，振荡培养18h—20h，即制成了接种菌悬液；菌液含量采用分光光度计法测定，活菌数应达到1*109CFU/ml—5*109CFU/ml,此新鲜菌液应在4h内尽快使用，以保证接种菌的活性；
    用吸管从细菌悬液中吸取2mL—3mL,即为金黄色葡萄球菌取上限，大肠杆菌取下限，移入装有9mL营养肉汤的试管中，充分混匀；吸取1ml移入另一支装有9mL营养肉汤的试管中，充分混匀；吸取1mL移入装有9mL0.03mol/L PBS缓冲液的试管中，充分混匀；吸取5mL移入装有45mL0.03mol/L PBS缓冲液的三角烧瓶中，充分混匀，稀释至含活菌数目3*105CFU/mL—4*105CFU/mL，由此固定的4次稀释程序，此接种菌液中含有微量的营养肉汤，用来对试样接种，此接种菌液应在4h内尽快使用，以保持接种菌的活性；
    (2)试样准备
    将益生菌抗菌织物及其未处理对照试样分别剪成约5mm*5mm大小，称取0.75g±0.05g作为一份试样，用小纸片包好，根据实验需要称取多分试样，每份试样均用小纸片包好；
    (3)试样灭菌
    将装有试样的小纸包放入高压灭菌锅，于121℃、103kPa灭菌15min备用；
    (4)试样装瓶
    准备8个三角烧瓶，在其中4个烧瓶中各加入一份益生菌抗菌织物，4个烧瓶中各加入未处理对照样一份，然后在每个烧瓶中个加入70m L±0.1m L0.03mol/L PBS缓冲液；
    (5)“0”接触时间振荡接触、稀释培养及菌落数测定
    用吸管往8个试样烧瓶中各加入5ml接种菌液，盖好瓶塞，再将此8个烧瓶置于恒温振荡器上，在24℃±1℃，以100r/min—150r/min，振荡1min±5s后，从每个烧瓶中吸取1mL±0.1mL试液，移入装有9mL±0.1mL0.03mol/L PBS缓冲液的试管中，充分混匀；用10倍稀释法再进行1次稀释，充分混匀；用吸管从每个稀释倍数的试管中分别吸取1mL±0.1mL移入灭菌的平皿，倾注营养琼脂培养基15mL，每个稀释倍数的试管分别吸液制作两个平板进行计数，即二者的菌落数相差应在15％以内，否则数据无效，室温凝固，倒置平板，以37℃±1℃在益生菌处理面料上培养大肠杆菌72h，葡萄球菌144h，记录每个平板的菌落数；其中，未处理对照样接种“0”接触时间，稀释100倍平板中，菌落数控制在200CFU—250CFU之间，否则影响试验精确度；
    (6)结果计算
    以分离益生菌提取纯计数葡萄球菌为例，抑菌率按式(A.1)计算：
    Figure PCTCN2016105675-appb-100001
    式中：
    Y——抑菌率，单位为％；
    A——6天后经益生菌整理样布上的金黄色葡萄球菌数，单位为(CFU/mL)；
    B——6天后未整理对照样布上的金黄色葡萄球菌数，单位为(CFU/mL)。
  2. 根据权利要求1所述的益生菌抗菌家用纺织品抗菌性能的测试方法，其特征在于：所述营养肉汤的成分及含量如下：牛肉膏3g；蛋白胨5g；去离子水1000ml； 灭菌后，pH值为6.8±0.2。
  3. 根据权利要求1所述的益生菌抗菌家用纺织品抗菌性能的测试方法，其特征在于：所述营养琼脂培养基的成分及含量如下：牛肉膏3g；蛋白胨5g；琼脂粉15g；去离子水1000ml；灭菌后，PH值为6.8±0.2。
  4. 根据权利要求1所述的益生菌抗菌家用纺织品抗菌性能的测试方法，其特征在于：所述PBS缓冲液的成分及含量如下：磷酸氢二钠2.84g；磷酸二氢钾1.36g；去离子水1000ml；灭菌后，pH值为7.2—7.4,在5℃—10℃的温度下保存备用。
PCT/CN2016/105675 2016-07-26 2016-11-14 一种益生菌抗菌家用纺织品抗菌性能的测试方法 WO2018018784A1 (zh)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108485953A (zh) * 2018-04-23 2018-09-04 莱茵技术（上海）有限公司 一种细菌培养装置及用于口含烟抑菌性能测试的方法
CN109207554A (zh) * 2018-09-06 2019-01-15 西安开米股份有限公司 用ttc琼脂培养基快速检测日化产品抑菌效果的方法
CN111321196A (zh) * 2020-02-21 2020-06-23 浙江方圆检测集团股份有限公司 一种贴膜法检测抗菌性能用大肠埃希氏菌悬浮培养基及其制备方法
CN113430245A (zh) * 2021-08-03 2021-09-24 广州百仕肽生物科技有限公司 一种伊枯草菌素抑菌活性的定量测定方法

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402216A (zh) * 2017-08-17 2019-03-01 宝洁公司 用于演示牙刷刷毛的抗细菌功效的方法
CN109609590A (zh) * 2018-12-21 2019-04-12 扬州倍加洁日化有限公司 一种湿巾产品的抗菌性能测试方法
CN110272968A (zh) * 2019-03-15 2019-09-24 李文杰 ATP荧光lgCB-lgIB标曲法评价一次性卫生用品真菌杀灭效果的方法
CN110272969A (zh) * 2019-03-15 2019-09-24 李文杰 ATP生物荧光lgCA-lgIA标准曲线法评价日化品细菌杀灭效果的方法
CN109762873A (zh) * 2019-03-27 2019-05-17 苏州纳工坊健康科技有限公司 一种杀菌剂抗菌效果检测方法和杀菌剂存放装置
CN110819691A (zh) * 2019-12-06 2020-02-21 石家庄学院 一种消毒剂灭菌效果的鉴定和评价方法
CN111206067B (zh) * 2020-02-18 2021-03-09 威莱(广州)日用品有限公司 湿纸巾防腐效果的评价方法
CN112611740B (zh) * 2020-11-27 2022-10-28 中纺协（北京）检验技术服务有限公司 一种纺织品抗病毒性能检测方法
CN113933459A (zh) * 2021-09-18 2022-01-14 中国日用化学研究院有限公司 一种织物洗涤剂除菌性能的测试评价方法
CN113984752A (zh) * 2021-11-02 2022-01-28 福建省产品质量检验研究院 一种快速检测食品接触材料生物抗菌性能试验方法
CN114459981A (zh) * 2021-12-22 2022-05-10 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) 一种用于检测口罩细菌过滤效率的方法
CN114703652A (zh) * 2022-03-23 2022-07-05 青岛理工大学 一种纳米抗菌纺织品的整理工艺及其抗菌活性评价方法
CN115980321A (zh) * 2022-12-06 2023-04-18 安徽锦昕医疗用品有限公司 隔离服抗菌性能检测装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHENZHEN BEYOND HAIWEI CHEMICAL CO. LTD: "Textiles-Evaluation for Antibacterial Activity-Part 3: Shake Flask Method", NATIONAL STANDARDS OF THE PEOPLE'S REPUBLIC OF CHINA, 2008 *

Cited By (6)

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