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This standard is developed in accordance with the rules given in GB/T 1.1-2009.
This standard was proposed by and is under the jurisdiction of China National Institute of Standardization.
Method for determination of functional microorganism in biologic products
1 Scope
This standard specifies the basic principles, general requirements and examination methods for determination of functional microorganism in biologic products.
This standard is applicable to examination of lactobacillus plantarum, lactobacillus acidophilus, bacillus subtilis, bacillus licheniformis, enterococcus faecalis, enterococcus faecium, candida utilis and saccharomyces cerevisiae.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB 4789.1 National food safety standard - Food microbiological examination - General guidelines
GB 4789.28 National food safety standard - Food microbiological examination - Quality requirements for medium and reagents
GB 19489 Laboratories - General requirements for biosafety
GB/T 27405 Criterion on quality control of laboratories - Microbiological testing of food
RB/T 151 Guidelines for the estimation of measurement uncertainty of food microbiological quantitative detection
SN/T 0330 General rules for microbiological examinations of export food
SN/T 2632 Technical codes for routine microbial culture collection
SN/T 2660 Preservation methods for culture collection in foodstuff microbiology laboratory
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
biologic products
industrial products related to biology
Note: the biologic products in this standard refer in particular to the viable bacteria preparations produced by the functional microorganisms after industrial production and propagation, and the products with such preparations.
3.2
functional microorganism
viable microbial strains with precise regulation and improvement functions under certain conditions
3.3
colony-forming units; CFU
colony formed by growth and propagation of single thalli or gathered thallus on solid medium during culture and counting of viable bacteria
4 General
4.1 The examination of functional microorganism implemented in accordance with this standard shall comply with the requirements of SN/T 0330.
4.2 Biosafety requirements for laboratories shall meet the requirements of GB 19489.
4.3 The laboratory personnel shall meet the requirements of GB 4789.1.
4.4 Strain preservation shall meet the requirements of SN/T 2632 and SN/T 2660.
4.5 The quality requirements for culture media and reagents shall meet the requirements of GB 4789.28.
4.6 The laboratory quality control shall meet the requirements of GB/T 27405.
4.7 The quantitative determination value is rounded off and reported in unit of CFU/g/(CFU/mL). The estimation of measurement uncertainty of quantitative determinations is performed in accordance with the requirements of RB/T 151.
4.8 The examined samples may be treated only after the reporting of examination result. The sample shall be subjected to innocent treatment.
5 Lactobacillus plantarum
5.1 Equipment and materials
In addition to the conventional sterilization and culture equipment in microbiological laboratory, other equipment and materials are as follows.
5.1.1 Thermostatic anaerobic incubator: 37±1℃.
5.1.2 Refrigerator: 2~5℃.
5.1.3 Balance: with a sensibility of 0.1g.
5.1.4 Homogenizer and aseptic homogenizing bag or homogenizing cup.
5.1.5 Oscillator.
5.1.6 Aseptic pipette: 1mL (with a scale division of 0.01mL), 10mL (with a scale division of 0.1mL) or micropipettor and sucker.
5.1.7 Aseptic conical flask: with a volume of 250mL or 500mL.
5.1.8 Aseptic culture dish: with a diameter of 90mm.
5.1.9 Microscope: 10×~100×.
5.1.10 pH meter or pH colorimetric tube or precision pH test paper.
5.2 Culture media and reagents
5.2.1 MRS (Man Rogosa Sharpe) agar medium: see A.1 in Annex A.
5.2.2 MRS broth medium: see A.2.
5.2.3 Sterilized saline water: see A.10.
5.2.4 Gram stain solution: see A.11.
5.2.5 Biochemical identification kit for bacteria.
5.3 Examination procedure
See Figure 1 for the examination procedure of lactobacillus plantarum.
Figure 1 Examination procedure of lactobacillus plantarum
5.4 Operation steps
5.4.1 Sample preparation
5.4.1.1 Solid and semi-solid samples
Weigh 25g sample, place it in an aseptic homogenizing bag containing 225mL normal saline, and slap for 1~2min with a slap-type homogenizer to prepare 1:10 homogeneous sample solution; or place it in an aseptic homogenizing cup containing 225mL sterilized saline water, and homogenize for 1~2min at a speed of 8000~10000r/min to prepare 1:10 homogeneous sample solution.
5.4.1.2 Liquid sample
Firstly, shake the liquid sample sufficiently, pipette 25mL sample with an aseptic pipette and put it into an aseptic conical flask (with appropriate quantity of aseptic glass beads placed in advance) containing 225mL normal saline, oscillate the flask well to prepare 1:10 homogeneous sample solution.
5.4.2 Sample dilution
5.4.2.1 Pipet 1mL 1:10 homogeneous sample solution with a 1mL aseptic pipette or a micropipettor, slowly inject it into an aseptic test tube (take care that the pipette tip shall not contact with the diluent) containing 9mL normal saline along the tube wall, oscillate the test tube or slap it with another aseptic pipette repetitively for uniform mixing, thus preparing 1:100 homogeneous sample solution.
5.4.2.2 Take another 1mL aseptic pipette or micropipettor sucker, operate in the above sequence to prepare 10-fold increasing homogeneous sample solutions, replace the 1mL aseptic pipette or sucker once for each serial dilution.
5.4.3 Culture
Based on the estimated bacteria content in the to-be-examined sample, select 2~3 consecutive appropriate dilutions, and pipet 0.1mL homogeneous sample solution at each dilution to inoculate on MRS agar plates, coat the inoculum on agar surface carefully and quickly with a coating rod as possible and the coating rod shall not contact with the plate edge. Two plates are inoculated at each dilution. After coating, keep the plates still for 10min to allow the inoculum to be completely absorbed by the culture medium. Invert the plates and place them in an anaerobic incubator at 37±1℃ for 48±2h.
Foreword i
1 Scope
2 Normative references
3 Terms and definitions
4 General
5 Lactobacillus plantarum
6 Lactobacillus acidophilus
7 Bacillus subtilis
8 Bacillus licheniformis
9 Enterococcus faecalis
10 Enterococcus faecium
11 Candida utilis
12 Saccharomyces cerevisiae
Annex A (Normative) Culture media and reagents
Annex B (Normative) Biochemical identification characteristics of functional microorganism
Annex C (Informative) Main identification characteristics of common bacteria of lactobacillus and enterococcus
