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1 Translated English of Chinese Standard: Translated by: National Standard of the People s Republic of China GB National food safety standard Food microbiological examination: Lactic acid bacteria Issued on: March 26, 2010 Implemented on: June 01, 2010 Issued by: Ministry of Health of the People s Republic of China Page 1 of 15

2 Table of Contents Foreword Scope Normative references Terms and Definitions Apparatuses and materials Culture medium and reagents Inspection procedure Operation steps Results and report Identification of lactic acid bacteria (Optional) Appendix A Page 2 of 15

3 Foreword This standard will replace GB/T Microbiological examination of food hygiene-examination of lactic acid bacteria in foods. Compared with GB/T , the main changes of this standard are as follows: - Modify the counting methods of the total number of lactic acid bacteria, lactobacillus, bifidobacterium and streptococcus thermophilus. This standard s appendix A is normative. The previous versions replaced by this standard are: - GB , GB/T , GB/T Page 3 of 15

4 National food safety standard Food microbiological examination: Lactic acid bacteria 1 Scope This standard specifies test methods of lactic acid bacteria in foods containing lactic acid bacteria. This standard applies to the test of lactic acid bacteria in foods containing lactic acid bacteria. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited apply. For undated references, the latest edition of the referenced document (including any amendments) apply. 3 Terms and Definitions 3.1 Lactic acid bacteria It is known as a kind of bacteria which can ferment sugar and mainly produce large amounts of lactic acid. The lactic acid bacteria in this standard mainly refers to lactobacillus, bifidobacterium and streptococcus. 4 Apparatuses and materials In addition to conventional sterilization and cultivation apparatuses, other apparatuses and materials are as follows: 4.1 Incubator: 36 C±1 C. 4.2 Refrigerator: 2 C~5 C. 4.3 Homogenizer and sterile homogeneous bag, homogeneous cup or sterile mortar. Page 4 of 15

5 4.4 Balance: Sensitivity is 0.1g. 4.5 Sterile tubes: 18mm 180mm, 15mm 100mm. 4.6 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale), or micropipette and tips. 4.7 Sterile Erlenmeyer flasks: 500mL, 250mL. 5 Culture medium and reagents 5.1 MRS (man rogosa sharpe) culture medium and improved Li-mupirocin MRS culture medium: see appendix A MC culture medium (modified chalmers culture medium): see appendix A % sucrose fermentation tube: see appendix A % cellobiose fermentation tube: see appendix A % maltose fermentation tube: see appendix A % mannitol fermentation tube: see appendix A % salicin fermentation tube: see appendix A % sorbitol fermentation tube: see appendix A % lactose fermentation tube: see appendix A Esculin fermentation tube: see appendix A Gram dye: see appendix A Li-mupirocin: chemically pure. 6 Inspection procedure Inspection procedure of lactic acid bacteria is shown in Figure 1. Page 5 of 15

6 7.5.2 When the colony number is more than or equal to 100CFU, after the 3rd digit rounded off by the rounding rules, take the first 2 digits. Use 0 to replace the 3 rd digit. It may also use 10-exponential form to express. After rounding off according to the rounding rules, take 2 significant digits Weighing sampling shall use CFU/g as the unit to report. Volume sampling shall use CFU/mL as the unit to report. 8 Results and report Issue the report according to the counting results of colonies. Use CFU/g (ml) as the unit to report. 9 Identification of lactic acid bacteria (Optional) 9.1 Pure culture Select 3 or more single colonies. Inoculate streptococcus thermophilus on MC agar plates. Inoculate lactobacillus on MRS agar plates. Use anaerobic method to cultivate it for 48h at 36 C±1 C. 9.2 Identification Identify bifidobacterium according to the provisions in GB/T Microscopy of smear: Lactobacillus is in multiple form long-rod shaped, curved-rod shaped or short-rod shaped. Without bacillus, gram dye is positive. Streptococcus thermophilus can be in the ball-shaped or cue-shaped. The diameter is 0.5µm~2.0µm. They are arranged in pair or in chain. Without bacillus, gram dye is positive Major biochemical reactions of lactic acid bacteria are shown in Table 1 and Table 2. Table 1 Carbohydrates reactions of common lactic acid bacteria genus Strain Esculin Cellobiose Maltose Mannitol Salicin Sorbitol Sucrose Raffinose L. casei subsp. casei L. Delbrueckii subsp. bulgaricus L. acidophilus d L. reuteri ND Page 10 of 15

7 A Composition Iodide 1.0g Potassium iodide 2.0g Distilled water 300mL A Preparation method Mix iodide with potassium iodide. Add a little of distilled water. Shake it fully. After it is completely dissolved, add distilled water to 300mL. A.5.3 Safranine counterstain A Composition Safranine 0.25g 95% ethanol 10mL Distilled water 90mL A Preparation method Add safranine into ethanol to dissolve. Use distilled water to dilute. A.5.4 Staining methods A Fix the smear on flame of alcohol lamp. Drop-add crystal violet staining solution. Dye for 1min. Use water to wash it. A Drop-add Gram iodine solution into it. After 1min, use water to wash it. A Drop-add 95% ethanol to de-stain for 15s~30s, until the staining solution is washed out. Do not de-stain excessively. Use water to wash it. A Drop-add counterstain into it. After 1min, use water to wash it. When it is dried, use microscopy to inspect it. END Page 15 of 15