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GB 4789.35-2010 (GB4789.35-2010)

GB 4789.35-2010_English: PDF (GB4789.35-2010)
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GB 4789.35-2010English70 Add to Cart 0--9 seconds. Auto-delivery National food safety standard -- Food microbiological examination: Lactic acid bacteria Obsolete GB 4789.35-2010

BASIC DATA
Standard ID GB 4789.35-2010 (GB4789.35-2010)
Description (Translated English) National food safety standard - Food microbiological examination: Lactic acid bacteria
Sector / Industry National Standard
Classification of Chinese Standard C53
Classification of International Standard 07.100.30
Word Count Estimation 10,187
Date of Issue 2010-03-26
Date of Implementation 2010-06-01
Older Standard (superseded by this standard) GB/T 4789.35-2008
Quoted Standard GB/T 4789.34
Regulation (derived from) Circular of the Ministry of Health (2010)7
Issuing agency(ies) Ministry of Health of People's Republic of China
Summary This Chinese standard specifies the lactic acid foods containing lactic acid bacteria (lactic acid bacteria) test methods. This standard applies to foods containing lactic acid bacteria Lactobacillus test.

Standards related to: GB 4789.35-2010

GB 4789.35-2010
GB
National Standard of the People’s Republic of China
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
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative references ... 4 
3 Terms and Definitions ... 4 
4 Apparatuses and materials ... 4 
5 Culture medium and reagents ... 5 
6 Inspection procedure ... 5 
7 Operation steps ... 7 
8 Results and report ... 10 
9 Identification of lactic acid bacteria (Optional) ... 10 
Appendix A ... 12 
Foreword 
This standard will replace GB/T 4789.35-2008 Microbiological examination of food
hygiene-Examination of lactic acid bacteria in foods.
Compared with GB/T 4789.35-2008, 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 4789.35-1996, GB/T 4789.35-2003, GB/T 4789.35-2008.
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.
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.1.
5.2 MC culture medium (modified chalmers culture medium). see appendix A.2.
5.3 0.5% sucrose fermentation tube. see appendix A.3.
5.4 0.5% cellobiose fermentation tube. see appendix A.3.
5.5 0.5% maltose fermentation tube. see appendix A.3.
5.6 0.5% mannitol fermentation tube. see appendix A.3.
5.7 0.5% salicin fermentation tube. see appendix A.3.
5.8 0.5% sorbitol fermentation tube. see appendix A.3.
5.9 0.5% lactose fermentation tube. see appendix A.3.
5.10 Esculin fermentation tube. see appendix A.4.
5.11 Gram dye. see appendix A.5.
5.12 Li-mupirocin. chemically pure.
6 Inspection procedure 
Inspection procedure of lactic acid bacteria is shown in Figure 1.
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 3rd digit. It
may also use 10-exponential form to express. After rounding off according to the
rounding rules, take 2 significant digits.
7.5.3 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
9.2.1 Identify bifidobacterium according to the provisions in GB/T 4789.34.
9.2.2 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.
9.2.3 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 + - - - + +
A.5.2.1 Composition
Iodide 1.0g
Potassium iodide 2.0g
Distilled water 300mL
A.5.2.2 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.5.3.1 Composition
Safranine 0.25g
95% ethanol 10mL
Distilled water 90mL
A.5.3.2 Preparation method
Add safranine into ethanol to dissolve. Use distilled water to dilute.
A.5.4 Staining methods
A.5.4.1 Fix the smear on flame of alcohol lamp. Drop-add crystal violet staining
solution. Dye for 1min. Use water to wash it.
A.5.4.2 Drop-add Gram iodine solution into it. After 1min, use water to wash it.
A.5.4.3 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.5.4.4 Drop-add counterstain into it. After 1min, use water to wash it. When it is dried,
use microscopy to inspect it.
...