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食品安全国家标准 食品微生物学检验 双歧杆菌检验
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GB 4789.34-2016
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标准编号: GB 4789.34-2016 (GB4789.34-2016)
中文名称: 食品安全国家标准 食品微生物学检验 双歧杆菌检验
英文名称: National Food Safety Standard -- Food Microbiological Examination -- Examination of Bifidobacterium
行业: 国家标准
中标分类: X09
字数估计: 12,131
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB 4789.34-2012
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
GB 4789.34-2016: 食品安全国家标准 食品微生物学检验 双歧杆菌检验
GB 4789.34-2016 英文名称: National Food Safety Standard -- Food Microbiological Examination -- Examination of Bifidobacterium
1 范围
本标准规定了双歧杆菌(Bifidobacterium)的鉴定及计数方法。
本标准适用于双歧杆菌纯菌菌种的鉴定及计数。本标准适用于食品中仅含有单一双歧杆菌的菌种
鉴定。本标准适用于食品中仅含有双歧杆菌属的计数,即食品中可包含一个或多个不同的双歧杆菌
菌种。
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:
2.1 恒温培养箱:36℃±1℃。
2.2 冰箱:2℃~5℃。
2.3 天平:感量0.01g。
2.4 无菌试管:
2.5 无菌吸管:1mL(具0.01mL刻度)、10mL(具0.1mL刻度)或微量移液器(200μL~1000μL)及
配套吸头。
2.6 无菌培养皿:直径90mm。
3 培养基和试剂
3.1 双歧杆菌培养基:见A.1。
3.2 PYG培养基:见A.2。
3.3 MRS培养基:见A.3。
3.4 甲醇:分析纯。
3.5 三氯甲烷:分析纯。
3.6 硫酸:分析纯。
3.7 冰乙酸:分析纯。
3.8 乳酸:分析纯。
4 检验程序
双歧杆菌的检验程序见图1。
图1 双歧杆菌的检验程序
5 操作步骤
5.1 无菌要求
全部操作过程均应遵循无菌操作程序。
5.2 双歧杆菌的鉴定
5.2.1 纯菌菌种
5.2.1.1 样品处理:半固体或液体菌种直接接种在双歧杆菌琼脂平板或 MRS琼脂平板。固体菌种或
真空冷冻干燥菌种,可先加适量灭菌生理盐水或其他适宜稀释液,溶解菌粉。
5.2.1.2 接种:接种于双歧杆菌琼脂平板或MRS琼脂平板。36℃±1℃厌氧培养48h±2h,可延长至
72h±2h。
5.2.2 食品样品
5.2.2.1 样品处理:取样25.0g(mL),置于装有225.0mL生理盐水的灭菌锥形瓶或均质袋内,于
8000r/min~10000r/min均质1min~2min,或用拍击式均质器拍打1min~2min,制成1∶10的
样品匀液。冷冻样品可先使其在2℃~5℃条件下解冻,时间不超过18h;也可在温度不超过45℃的
条件解冻,时间不超过15min。
5.2.2.2 接种或涂布:将上述样品匀液接种在双歧杆菌琼脂平板或 MRS琼脂平板,或取0.1mL适当
稀释度的样品匀液均匀涂布在双歧杆菌琼脂平板或 MRS琼脂平板。36℃±1℃厌氧培养48h±2h,
可延长至72h±2h。
5.2.2.3 纯培养:挑取3个或以上的单个菌落接种于双歧杆菌琼脂平板或 MRS琼脂平板。36℃±
1℃厌氧培养48h±2h,可延长至72h±2h。
5.2.3 菌种鉴定
5.2.3.1 涂片镜检:挑取双歧杆菌平板或 MRS平板上生长的双歧杆菌单个菌落进行染色。双歧杆菌
为革兰氏染色阳性,呈短杆状、纤细杆状或球形,可形成各种分支或分叉等多形态,不抗酸,无芽孢,无
动力。
5.2.3.2 生化鉴定:挑取双歧杆菌平板或 MRS平板上生长的双歧杆菌单个菌落,进行生化反应检测。
过氧化氢酶试验为阴性。双歧杆菌的主要生化反应见表1。可选择生化鉴定试剂盒或全自动微生物生
化鉴定系统。
5.2.3.3 有机酸测定:测定双歧杆菌的有机酸代谢产物(可选项),见附录B。
5.3 双歧杆菌的计数
5.3.1 纯菌菌种
5.3.1.1 固体和半固体样品的制备:以无菌操作称取2.0g样品,置于盛有198.0mL稀释液的无菌均质
杯内,8000r/min~10000r/min均质1min~2min,或置于盛有198.0mL稀释液的无菌均质袋中,用
拍击式均质器拍打1min~2min,制成1∶100的样品匀液。
5.3.1.2 液体样品的制备:以无菌操作量取1.0mL样品,置于9.0mL稀释液中,混匀,制成1∶10的样
品匀液。
5.3.2 食品样品
5.3.2.1 样品处理:取样25.0g(mL),置于装有225.0mL生理盐水的灭菌锥形瓶或均质袋内,于
8000r/min~10000r/min均质1min~2min,或用拍击式均质器拍打1min~2min,制成1∶10的
样品匀液。冷冻样品可先使其在2℃~5℃条件下解冻,时间不超过18h;也可在温度不超过45℃的
条件解冻,时间不超过15min。
5.3.3 系列稀释及培养
用1mL无菌吸管或微量移液器,制备10倍系列稀释样品匀液,于8000r/min~10000r/min均
质1min~2min,或用拍击式均质器拍打1min~2min。每递增稀释一次,即换用1次1mL灭菌吸管
或吸头。根据对样品浓度的估计,选择2个~3个适宜稀释度的样品匀液,在进行10倍递增稀释时,吸
取1.0mL样品匀液于无菌平皿内,每个稀释度做两个平皿。同时,分别吸取1.0mL空白稀释液加入
两个无菌平皿内作空白对照。及时将15mL~20mL冷却至46℃的双歧杆菌琼脂培养基或 MRS琼
脂培养基(可放置于46℃±1℃恒温水浴箱中保温)倾注平皿,并转动平皿使其混合均匀。从样品稀释
到平板倾注要求在15min内完成。待琼脂凝固后,将平板翻转,36℃±1℃厌氧培养48h±2h,可延
长至72h±2h。培养后计数平板上的所有菌落数。
5.3.4 菌落计数
5.3.4.1 可用肉眼观察,必要时用放大镜或菌落计数器,记录稀释倍数和相应的菌落数量。菌落计数以
菌落形成单位(colony-formingunits,CFU)表示。
5.3.4.2 选取菌落数在30CFU~300CFU之间、无蔓延菌落生长的平板计数菌落总数。低于30CFU
平均数。
5.3.4.3 其中一个平板有较大片状菌落生长时,则不宜采用,而应以无片状菌落生长的平板作为该稀释
度的菌落数;若片状菌落不到平板的一半,而其余一半中菌落分布又很均匀,即可计算半个平板后乘以
2,代表一个平板菌落数。
5.3.4.4 当平板上出现菌落间无明显界线的链状生长时,则将每条单链作为一个菌落计数。
5.3.5 结果的表述
5.3.5.1 若只有一个稀释度平板上的菌落数在适宜计数范围内,计算两个平板菌落数的平均值,再将平
均值乘以相应稀释倍数,作为每克或每毫升中菌落总数结果。
GB 4789.34-2016
GB
NATIONAL STANDARD OF
THE PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Food Microbiological
Examination - Examination of Bifidobacterium
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
People 's Republic of China;
China Food and Drug Administration.
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Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Medium and reagent ... 4
4 Inspection procedures ... 5
5 Operation steps ... 6
Annex A Medium and reagent ... 12
Annex B Detection of organic acid metabolites of Bifidobacterium ... 14
Foreword
This Standard replaces GB 4789.34-2012 National Food Safety Standard -
Food Microbiological Examination - Identification of Bifidobacterium.
Compared with GB 4789.34-2012, the main changes in this Standard are as
follows.
- added the counting method for Bifidobacterium;
- added MRS medium;
- modified the application scope of this Standard;
- modified Annex B as optional.
National Food Safety Standard - Food Microbiological
Examination - Examination of Bifidobacterium
1 Scope
This Standard specifies the identification and counting method for
Bifidobacterium.
This Standard applies to the identification and counting of pure bacteria strain
of Bifidobacterium. This Standard applies to the identification of bacteria
when the food only contains single Bifidobacterium. This Standard applies to
the counting when the food only contains Bifidobacterium, i.e., the food may
contain one or more different Bifidobacterium species.
2 Equipment and materials
In addition to microbial laboratory routine sterilization and training equipment,
other equipment and materials are as follows.
2.1 Constant temperature incubator. 36°C ± 1°C.
2.2 Refrigerator. 2°C ~ 5°C.
2.3 Balance. resolution of 0.01 g.
2.4 Sterile test tube. 18mm × 180mm, 15mm × 100mm.
2.5 Sterile pipettes. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale) or
micro-pipettes (200μL ~ 1000μL) and matching tips.
2.6 Sterile petri dish. 90 mm in diameter.
3 Medium and reagent
3.1 Bifidobacterium culture medium. see A.1.
3.2 PYG medium. see A.2.
3.3 MRS medium. see A.3.
3.4 Methanol. analytically pure.
5 Operation steps
5.1 Sterile requirements
All operating procedures shall follow the sterile procedures.
5.2 Identification of Bifidobacterium
5.2.1 Pure bacteria species
5.2.1.1 Sample processing. semi-solid or liquid species are directly
inoculated on Bifidobacterium agar plates or MRS agar plates. For solid
bacteria or vacuum freeze-dried bacteria, it shall add an appropriate amount
of sterilized saline or other suitable diluent to dissolve the bacteria powder.
5.2.1.2 Vaccination. inoculate on Bifidobacterium agar plates or MRS agar
plates. Perform anaerobic culture at 36°C ± 1°C for 48h ± 2h that can be
extended to 72h ± 2h.
5.2.2 Food sample
5.2.2.1 Sample processing. take 25.0g (mL) of sample into a sterile conical
homogenizing at 8000r/min ~ 10000r/min for 1min ~ 2min or using slapping
homogenizer to beat 1min ~ 2min to make 1.10 sample homogenizing
solution. The frozen sample may be thawed at 2°C ~ 5°C for not more than
18h or at a temperature not greater than 45°C for not more than 15min.
5.2.2.2 Inoculation or coating. inoculate the above sample homogenizing
solution on a Bifidobacterium agar plate or MRS agar plate OR take 0.1mL of
sample homogenizing solution of appropriate dilution to evenly coat on a
Bifidobacterium agar plate or MRS agar plate. Perform anaerobic culture at
36°C ± 1°C for 48h ± 2h that can be extended to 72h ± 2h.
on Bifidobacterium agar plates or MRS agar plates. Perform anaerobic
culture at 36°C ± 1°C for 48h ± 2h that can be extended to 72h ± 2h.
5.2.3 Identification of bacteria
5.2.3.1 Smear microscopy. pick up Bifidobacterium single colony growing in
Bifidobacterium plate or MRS plate for dyeing. Bifidobacterium is
Gram-positive, short rod-shaped, slender rod-shaped or spherical, can form a
variety of branches or bifurcation and other forms, non-acid, no spores, no
power.
5.2.3.2 Biochemical identification. pick up Bifidobacterium single colony
place in 9.0mL of diluent; well mix to make 1.10 sample homogenizing
solution.
5.3.2 Food sample
5.3.2.1 Sample processing. take 25.0g (mL) of sample; place in a sterile
conical flask or homogeneous bag containing 225.0mL of physiological saline,
homogenizing at 8000r/min ~ 10000r/min for 1min ~ 2min, or using slapping
homogenizer to beat 1min ~ 2min to make 1.10 sample homogenizing
solution. The frozen sample may be thawed at 2°C ~ 5°C for not more than
18h or at 45°C for not more than 15min.
Use a 1mL sterile pipet or micro pipette to prepare 10 times serial sample
homogenizing solution, homogenizing at 8000r/min ~ 10000r/min for 1min ~
2min, or using slapping homogenizer to beat 1min ~ 2min. Dilute once for
every increment, i.e., switch a 1mL sterile pipet or pipet head once. According
to the estimation of the sample concentration, select two to three sample
homogenizing solution of appropriate dilution. When performing 10 times
increment dilution, pipette 1.0mL of sample in the sterile plate. Do two plates
for each dilution. Meanwhile, respectively pipette 1.0mL of blank diluent into
two sterile plates for blank control. Timely pour 15mL ~ 20mL of
placed in 46°C ± 1°C constant temperature water bath for insulation) into the
plate, rotate the plate to make it evenly mixed. From sample dilution to plate
pouring, it requires 15 min. After agar solidification, flip the plate, perform
anaerobic culture at 36°C ± 1°C for 48h ± 2h that can be extended to 72h ± 2h.
Count the number of colonies on the plate after incubation.
5.3.4 Colony counting
5.3.4.1 It can be observed with the naked eye, if necessary, with a
magnifying glass or colony counter. Record the dilution factor and the
corresponding number of colonies. The colony counting is expressed in
5.3.4.2 Select the total number of colony colonies counted between 30CFU
and 300CFU, and the total number of colonies without flake colonies. Record
the number of specific colonies on plates below 30CFU. When it exceeds
300CFU, it can be recorded as countless. The number of colonies per dilution
shall be the average of two plates.
5.3.4.3 If one of the plates is growing with large flake colonies, it shall not be
used. It shall use the plate without flake colonies as the number of colonies of
this dilution. If the number of flake colonies is less than half of the plate, and
Annex A
A.1 Bifidobacterium agar medium
A.1.1 Ingredients
Peptone 15.0 g
Yeast extract 20.0 g
Glucose 20.0 g
Soluble starch 0.5 g
Sodium chloride 5.0 g
Tomato extract 400.0 mL
Tween 80 1.0 mL
Agar powder 20.0 g
Adding distilled water to 1000.0 mL
A.1.2 Preparation
A.1.2.1 Preparation of cysteine solution. weigh 0.5 g of cysteine solution,
add into 1.0 mL of hydrochloric acid to make cysteine all dissolved and
prepare cysteine salt solution.
A.1.2.2 Preparation of tomato extract. wash the fresh tomatoes and scrape
them; add the same amount of distilled water and heat in a 100°C water bath,
stir for 90 min, then filter with gauze, calibrate to pH 7.0 ± 0.1; after
A.1.2.3 Preparation. add all components of A.1.1 into distilled water,
dissolve by heating, and then add into the cysteine solution, calibrate to pH
6.8 ± 0.1; after sub-packaging the leaching solution, autoclave at 121°C for
15 min ~ 20min.
A.2 PYG liquid medium
A.2.1 Composition
Peptone 10.0 g
Glucose 2.5 g
Yeast 5.0 g
Salt solution 20.0 mL
Vitamin K1 solution 0.5 mL
Hemoglobin solution 5 mg/mL 2.5 mL
Adding distilled water to 500.0 mL
Annex B
Detection of organic acid metabolites of Bifidobacterium
B.1 Preparation of Bifidobacterium culture medium
Inoculate the Bifidobacterium cultured on Bifidobacterium agar plate or MRS
agar plate into PYG liquid medium. Then use non-inoculated PYG liquid
B.2 Preparation of standard solution
B.2.1 Acetic acid standard solution. accurately pipette 5.7 mL of pure acetic
acid, add water and dilute to 100.0 mL, shake and calibrate, prepare about
1.0 mol/L acetic acid standard solution. Calibration method. accurately weigh
3.0 g of acetic acid, add 15.0 mL of water, 2 drops of phenolphthalein
indicator solution, use 1.0 mol/mL sod...
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