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食品安全国家标准 食品微生物学检验 双歧杆菌的鉴定
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GB 4789.34-2012
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标准编号: GB 4789.34-2012 (GB4789.34-2012)
中文名称: 食品安全国家标准 食品微生物学检验 双歧杆菌的鉴定
英文名称: National food safety standards. Microbiological examination of food. Identification of bifidobacteria
行业: 国家标准
中标分类: C53
国际标准分类: 07.100.30
字数估计: 13,145
旧标准 (被替代): GB/T 4789.34-2008
标准依据: 卫生部公告2012年第9号
发布机构: 中华人民共和国卫生部
范围: 本标准规定了食品中双歧杆菌(Bifidobacterium)的鉴定方法。本标准适用于食品中双歧杆菌的鉴定。
GB 4789.34-2012: 食品安全国家标准 食品微生物学检验 双歧杆菌的鉴定
GB 4789.34-2012 英文名称: National food safety standards -- Microbiological examination of food -- Identification of bifidobacteria
中华人民共和国国家标准
食品安全国家标准
食品微生物学检验 双歧杆菌的鉴定
1 范围
本标准规定了食品中双歧杆菌（Bifidobacterium ）的鉴定方法。
本标准适用于食品中双歧杆菌的鉴定。
2 设备和材料
除微生物实验室常规灭菌及培养设备外，其他设备和材料如下：
a） 恒温培养箱：36 ℃±1 ℃；
b） 气相色谱仪配 FID 检测器；
c） 冰箱：2 ℃～5 ℃；
d） 天平： 感量 0.1 g；
e） 无菌试管：18 mm×180 mm、15 mm×100 mm；
f） 无菌吸管：1 mL（具 0.01 mL 刻度）、10 mL（具 0.1 mL 刻度）
或微量移液器（200 μL～1000 μL）及配套吸头；
g） 无菌锥形瓶：500 mL、250 mL。
3 培养基和试剂
3.1 双歧杆菌培养基：见附录 A 中的 A.1。
3.2 PYG 液体培养基：见附录 A 中的 A.2。
3.3 甲醇：分析纯。
3.4 三氯甲烷：分析纯。
3.5 硫酸：分析纯（体积比）。
3.6 冰乙酸：分析纯（体积比）。
3.7 乳酸：分析纯。
3.8 乙酸标准溶液：吸取分析纯冰乙酸5.7 mL，移入100 mL容量瓶中，加水至刻度，标定，标定方法见附
录B，此溶液浓度约为1 mol/L。
3.9 乙酸标准使用液：将经标定的乙酸标准溶液用水稀释至0.01 mol/L。
3.10 乳酸标准溶液：吸取分析纯乳酸8.4 mL，移入100 mL容量瓶中，加水至刻度，标定，标定方法见附
录B，此溶液浓度约为1 mol/L。
3.11 乳酸标准使用液：将经标定的乳酸标准溶液用水稀释至0.01 mol/L。
4 鉴定程序
双歧杆菌鉴定程序见图 1。
5 操作步骤
5.1 样品制备
5.1.1 样品的全部制备过程均应遵循无菌操作程序。
5.1.2 以无菌操作称取 25 g（mL）样品，置于装有 225 mL 生理盐水的
灭菌锥形瓶内，制成 1:10 的样品匀液。
5.2 稀释及涂布培养步骤
5.2.1 用 1 mL 无菌吸管或微量移液器吸取 1:10 样品匀液 1.0 mL，沿管壁缓慢注于装有 9 mL 生理盐水的
无菌试管中（注意吸管尖端不要触及稀释液），振摇试管或换用 1 支无菌吸管
反复吹打使其混合均匀，制成 1:100 的样品匀液。
5.2.2 另取 1 mL 无菌吸管或微量移液器吸头，按 5.2.1 操作顺序，做 10 倍递增样品匀液，每递增稀释一
次，即换用 1 次 1 mL 灭菌吸管或吸头。
5.2.3 根据待鉴定菌种的活菌数，选择三个连续的适宜稀释度，每个稀释度吸取 0.1 mL 稀释液，用 L 棒
在双歧杆菌琼脂平板进行表面涂布, 每个稀释度作两个平皿。置 36 ℃±1 ℃ 温箱内培养 48 h±2 h，
培养后选取单个菌落进行纯培养。
5.3 纯培养
挑取 3 个或以上的菌落接种于双歧杆菌琼脂平板，厌氧，36 ℃±1 ℃ 培养 48 h。
5.4 镜检及生化鉴定
5.4.1 涂片镜检
双歧杆菌菌体为革兰氏染色阳性，不抗酸、无芽孢，无动力，菌体形态多样，呈短杆状、纤细杆状或
球形，可形成各种分支或分叉形态。
5.4.2 生化鉴定
过氧化氢酶试验为阴性。选取纯培养平板上的三个单个菌落，分别进行生化反应检测，不同双歧杆菌
菌种主要生化反应见附录B。
5.5 有机酸代谢产物测定
气相色谱法测定双歧杆菌的有机酸代谢产物, 见附录 C。
6 报告
根据镜检及生化鉴定的结果（5.4）、双歧杆菌的有机酸代谢产物乙酸与乳酸微摩尔之比大于 1（5.5），
报告双歧杆菌属的种名。
GB 4789.34-2012
National food safety standards. Microbiological examination of food. Identification of bifidobacteria
National Standards of People's Republic of China
National Food Safety Standard
Identification of food microbiology testing bifidobacteria
People's Republic of China Ministry of Health issued
Issued on. 2012-05-17
2012-07-17 implementation
Foreword
This standard replaces GB/T 4789.34-2008 "Microbiological examination of food hygiene inspection Bifidobacterium."
This standard compared with GB/T 4789.34-2008, main changes are as follows.
- Modify the standard Chinese name;
- Modify the media and reagents;
- Remove the fructose-6-phosphate phosphatase enzyme ketone (F6PPK) assay and bifidobacteria counting method.
National Food Safety Standard
Identification of food microbiology testing bifidobacteria
1 Scope
This standard specifies the food of bifidobacteria (Bifidobacterium) authentication method.
This standard applies to identify food bifidobacteria.
2 Equipment and Materials
In addition to the microbiological laboratory conventional sterilization and cultivation equipment, other equipment and materials as follows.
a) incubator. 36 ℃ ± 1 ℃;
b) gas chromatograph with FID detector;
c) Refrigerator. 2 ℃ ~ 5 ℃;
d) balance. a sense of the amount of 0.1 g;
e) a sterile tube. 18 mm × 180 mm, 15 mm × 100 mm;
f) sterile pipette. 1 mL (0.01 mL with scale), 10 mL (0.1 mL with scale) or micropipette (200 μL ~ 1000 μL)
And supporting tip;
g) sterile flasks. 500 mL, 250 mL.
3 media and reagents
3.1 Bifidobacterium Medium. See Appendix A for A.1.
3.2 PYG broth. See Appendix A A.2.
3.3 methanol. AR.
3.4 chloroform. AR.
3.5 sulfuric acid. analytically (by volume).
3.6 glacial acetic acid. analytically (by volume).
3.7 lactic acid. AR.
3.8 acetic acid standard solution. lessons AR glacial acetic acid 5.7 mL, transferred to 100 mL volumetric flask, add water to the mark, calibration, calibration method Mitsuke
Record B, the solution concentration of about 1 mol/L.
3.9 acetic acid standard solution. A standard solution of acetic acid diluted with water to 0.01 mol/L.
3.10 acid standard solution. lessons AR lactic acid 8.4 mL, transferred to 100 mL volumetric flask, add water to the mark, calibration, calibration method Mitsuke
Record B, the solution concentration of about 1 mol/L.
3.11 acid standard solution. A standard solution of lactic acid diluted with water calibrated to 0.01 mol/L.
4 identification procedures
Bifidobacterium identification procedure shown in Figure 1.
Figure 1 Bifidobacterium identification procedures
5 steps
5.1 Sample Preparation
All procedures 5.1.1 Samples shall follow aseptic procedures.
5.1.2 Aseptically weigh 25 g (mL) sample, placed in sterile saline containing 225 mL conical flask, made uniform sample 1.10
liquid.
5.2 Dilution and coating step of culturing
Anaerobic, 48 h
36 ℃ ± 1 ℃
Samples 25 g (mL) 225 mL sterile saline
10 fold serial dilutions
Gram stain, catalase test
report
Select two or three suitable dilutions from each 0.1 mL
Bifidobacteria were added to the agar plate coated
Colonies were picked bifidobacteria inoculated agar plate
PYG broth inoculated biochemical reactions
Determination of acetic acid, lactic acid
Anaerobic, 48 h
36 ℃ ± 1 ℃
Anaerobic, 48 h
36 ℃ ± 1 ℃
5.2.1 with 1 mL sterile pipette or micro pipette 1.10 absorbed liquid sample 1.0 mL, slowly along the wall containing 9 mL of saline injection in the
Sterile tube (note not to touch the tip of the pipette dilution), tubes were shaken or replaced with a sterile pipette repeatedly pipetting to mix evenly system
To 1. 100 in sample liquid uniform.
Times, that is replaced with 1 or 1 mL sterile pipette tip.
5.2.3 According to the number of viable bacteria to be identified, a suitable choice of three successive dilutions, each dilution draw 0.1 mL diluent, stick with L
Bifidobacterium agar surface coating, two plates for each dilution. Set 36 ℃ ± 1 ℃ temperature inside cultured 48 h ± 2 h, cultured
Single colonies were selected pure culture.
5.3 pure culture
Picked three or more colonies were inoculated on agar plates bifidobacteria, anaerobic, 36 ℃ ± 1 ℃ cultured 48 h.
5.4 microscopy and biochemical identification
5.4.1 smear
Bifidobacterium bacteria to Gram-positive, not acid, no spores, no power, cell morphology varied, were short rod-shaped, slender rod-like or
5.4.2 biochemical identification
Catalase test was negative. Select three pure cultures of single colonies on the plate were detected biochemical reactions, different Bifidobacterium
The main strains of biochemical reactions in Appendix B.
5.5 Determination of Organic Acid Metabolites
Gas Chromatography organic metabolites of bifidobacteria, see Appendix C.
6 Report
According to the results of microscopic examination and biochemical identification of (5.4), bifidobacteria and lactic acid organic acid metabolite ratio greater than 1 micromolar (5.5),
Species of the genus Bifidobacterium report.
Appendix A
A.1 Bifidobacterium agar
A.1.1 ingredients
Peptone 15.0 g
Yeast extract 2.0 g
Glucose 20.0 g
0.5 g of soluble starch
Sodium chloride 5.0 g
Tomatoes leachate 400.0 mL
Tween 80 1.0 mL
Agar 15.0 g ~ 20.0 g
Add distilled water to 1 000.0 mL
A.1.2 Method
A.1.2.1 cysteine salt solution
Cysteine weighed 0.5 g, 1.0 mL of hydrochloric acid was added, to dissolve all cysteine, cysteine formulated as a salt solution.
A.1.2.2 tomatoes leachate
Wash fresh tomatoes weighed chopped, add the same amount of distilled water was heated in a water bath at 100 ℃, stirred 90 min, and then use gauze
Filtering, correction pH7.0, after the leaching solution aliquots, 121 ℃ autoclave 15 min ~ 20 min.
A.1.2.3 medium
℃ autoclaving 15 min ~ 20 min. When you use heat to melt the agar, cooling to 50 ℃ use.
A.2 PYG broth
A.2.1 ingredients
Peptone 10.0 g
Glucose 2.5 g
Yeast extract 5.0 g
Cysteine -HCl 0.25 g
Salt solution 20.0 mL
Vitamin K1 solution 0.5 mL
Add distilled water to 500.0 mL
A.2.2 Method
A.2.2.1 salt solution
Weigh anhydrous calcium chloride 0.2 g, magnesium sulfate 0.2 g, dipotassium phosphate 1.0 g, potassium dihydrogen phosphate 1.0 g, sodium bicarbonate, 10.0 g, chloride
Sodium 2.0 g, add distilled water to 1000 mL.
A.2.2.2 Hemin solution (5mg/mL)
Hemin weighed 0.5 g was dissolved in 1 mol/L sodium hydroxide, 1.0 mL, add distilled water to 1000 mL, 121 ℃ autoclaving 15
min ~ 20 min.
A.2.2.3 vitamin K1 solution
A.2.2.4 medium
In addition to a solution of hemin and vitamin K1 solution outside, A.2.1 remaining ingredients was added distilled water, heated to dissolve, correcting pH 6.0, plus
The neutral red solution. After dispensing 121 ℃ autoclaving 15 min ~ 20 min. When you use heat to melt the agar, and added to a solution of Hemin
Vitamin K1 solution, cooled to 50 ℃ use.
Appendix B
Bifidobacterium strain major biochemical reactions
Bifidobacterium strain major biochemical reactions in Table B.1.
Table B.1 Bifidobacterium strain major biochemical reactions
No. Item
(B.bifidum)
Bifidobacterium infantis
(B.infantis)
Longum
(B.longum)
Bifidobacterium
(B.adolescentis)
Bifidobacterium animalis
(B.animalis)
(B.breve)
1 Glycerol - - - - - -
2 erythritol - - - - - -
3 D- arabinose - - - - - -
4 L- arabinose - - -
5 D- ribose - -
6 D- xylose - d
7 L- xylose - - - - - -
8 Adon alcohol - - - - - -
10 D- galactose dd
11 D- glucose
12 D- fructose ddd
13 D- mannose - - - -
14 L- sorbose - - - - - -
15 L- rhamnose - - - - - -
16 dulcitol - - - - - -
Inositol 17 - - - - -
Mannitol 18 - - - - - -
20 α- mannose-methyl -D- glucoside - - - - - -
21 α- methyl -D- glucoside - - - - -
22 N- acetyl - glucosamine - - - - -
23 amygdalin (almond glycosides) - - - -
24 Arbutin - - - - - -
25 esculin - - -
26 Salicin (Liu alcohol) - - -
27 D- cellobiose - - d - -
28 D- maltose -
30 D- melibiose -
31 D- sucrose -
32 D- trehalose (Tan sugar) - - - - - -
33 inulin (purpurea root powder) - - - - - -
34 D- melezitose - - - -
35 D- Raffinose -
Table B.1 (continued) Bifidobacterium strain major biochemical reactions
project
Bifidobacterium bifidum
Bifidobacterium infantis
(B.infantis)
Longum
(B.longum)
Bifidobacterium
(B.adolescentis)
Bifidobacterium animalis
(B.animalis)
Breve
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