ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a dechnical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
ISO（国际标准化组织）是国家标准机构（ISO成员机构）的全球性联合会。制定国际标准的工作通常通过ISO技术委员会进行。每个对已成立十进制委员会的主题感兴趣的成员机构都有权派代表参加该委员会。国际组织，包括政府和非政府组织，与国际标准化组织联络，也参与了这项工作。ISO与国际电工委员会（IEC）在电工标准化的所有问题上密切合作。

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2 .

The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least of the member bodies casting a vote.
技术委员会的主要任务是制定国际标准。技术委员会通过的国际标准草案分发给成员机构进行表决。作为国际标准发布至少需要得到 投票成员机构的批准。

Attention is drawn to the possibility that some of the elements of this/document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all sucl patent rights.
请注意，本文件的某些要素可能成为专利权的主题。ISO 不负责识别任何或所有 sucl 专利权。

ISO 29981|IDF 220 was prepared by Technical Committee ISO/C 34, Food products, Subcommittee SC 5 , Milk and milk products, and the International Dairy Federation (DF). It is being published jointly by ISO and IDF.
国际标准化组织 29981|IDF 220 由 ISO/C 34 技术委员会、食品、SC 5 小组委员会、牛奶和奶制品以及国际乳品联合会 （DF） 编写。它由 ISO 和 IDF 联合发布。

IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with /SO in the development of standard methods of analysis and sampling for milk and milk products.
IDF（国际乳业联合会）是一个全球性的乳品行业联合会，每个成员国都有一个国家委员会。每个国家委员会都有权派代表参加以色列国防军执行技术工作的常设委员会。IDF 与 /SO 合作开发牛奶和奶制品的标准分析和取样方法。

The main task of Standing Committees is to prepare International Standards. Draft International Standards adopted by the Standing Committees are circulated to the National Copmittees for endorsement prior to publication as an International Standard. Publication as an International Slandard requires approval by at least of IDF National Committees casting a vote.
常设委员会的主要任务是制定国际标准。常设委员会通过的国际标准草案在作为国际标准公布之前，先分发给各国委员会批准。作为国际诽谤的出版至少 需要得到以色列国防军国家委员会投票的批准。

Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights.
需要注意的是，本国际标准的某些要素可能成为专利权的主题。IDF 不负责识别任何或所有此类专利权。

ISO 29981|IDF 220 was prepared by Technical Committee ISø/TC 34, Food products, Subcommittee SC 5, Milk and milk products and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF.
国际标准化组织 29981|IDF 220 由技术委员会 ISø/TC 34（食品）、SC 5 小组委员会（牛奶和奶制品）和国际乳品联合会 （IDF） 编制。它由 ISO 和 IDF 联合发布。

All work was carried out by the Joint ISO-IDF Action Team on Lactic acid bacteria and starters of the Standing Committee on Microbiology methods of analysis undep the aegis of its project leaders, Prof. W. Kneifel (AT) and Dr. U. Zitz (AT).
所有工作均由ISO-IDF乳酸菌联合行动小组和微生物学分析方法常设委员会的启动者在其项目负责人W.Kneifel教授（奥地利）和U.Zitz博士（奥地利）的支持下进行。

This International Standard specifies a method for the selective enumeration of presumptive bifidobacteria in milk products by using a colony count technique at under anaerobic conditions.
本国际标准规定了一种 在厌氧条件下使用菌落计数技术选择性计数奶制品中推定双歧杆菌计数的方法。

The method is applicable to milk products such as fermented and non-fermehted milks, milk powders, infant formulae, and starter cultures where these microorganisms are present and viable, and in combination with other lactic acid bacteria. (For proposed quality criteria of dairy produogs, see, for example, Codex Stan 243:2003 [6].)
该方法适用于乳制品，如发酵乳和非含铁剂牛奶、奶粉、婴儿配方奶粉和发酵剂，这些微生物存在并具有活性，并与其他乳酸菌结合使用。（关于乳制品的拟议质量标准，参见，例如，Codex Stan 243：2003 [6]。

Bifidobacteria used in milk products usually belong to the species (e.o. see References [7][8][16]):

a) Bifidobacterium adolescentis;

b) B. animalis subsp. animalis;

c) B. animalis subsp. lactis;

d) B. bifidum;

e) B. breve;

f) B. infantis;

g) B. longum.

The following referenced documents are indispensable for the application of this document. For dated references, only the edition cites applies. For undated references, the latest edition of the referenced documents (including any amendments) applies.
以下引用文件对于本文档的应用是必不可少的。对于注明日期的引用文件，仅适用引用的版本。对于未注明日期的引用文件，适用引用文件的最新版本（包括任何修订）。

ISO 6887-1, Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microblological examination - Part 1: General rules for the preparation of the initial suspension and decimal dilutiops
ISO 6887-1，食品和动物饲料的微生物学 - 用于微生物学检查的测试样品的制备、初始悬浮液和小数稀释液 - 第 1 部分：初始悬浮液和小数稀释液制备的一般规则

ISO 6887-5, Microbiology offood and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 5: Specific rules for the preparation of milk and milk products
ISO 6887-5，食品和动物饲料的微生物学 - 用于微生物检查的测试样品的制备、初始悬浮液和小数稀释度 - 第 5 部分：牛奶和奶制品制备的具体规则

ISO 7218, Microbiology of food and animal feeding stuffs - General requirements and guidance for microbiological examinations
ISO 7218，食品和动物饲料的微生物学 - 微生物学检查的一般要求和指南

ISO 29981:2010(E)

IDF 220:2010(E)

ISO/TS 11133-1, Microbiology of food and animal feeding stuffs - Guidelines on preparation and production of culture media - Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory
ISO/TS 11133-1，食品和动物饲料的微生物学-培养基的制备和生产指南-第1部分：实验室培养基制备质量保证的一般准则

ISO 14461-1 IIDF 169-1, Milk and milk products - Quality control in microbiological laboratdries - Part 1: Analyst performance assessment for colony counts
ISO 14461-1 IIDF 169-1，牛奶和奶制品 - 微生物实验室的质量控制 - 第 1 部分：菌落计数的分析员绩效评估

ISO 14461-2|IDF 169-2, Milk and milk products - Quality control in microbiological laporatories - Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps
国际标准化组织 14461-2|IDF 169-2，牛奶和奶制品 - 微生物实验室的质量控制 - 第 2 部分：平行板菌落计数可靠性的测定和后续稀释步骤

For the purposes of this document, the following terms and definitions apply.

anaerobic microorganisms that form lenticular or round whitish colonjes, partially star shaped or trilobate of diameter 1 mm to 4 mm on transgalactosylated oligosaccharides-mupirocin lithium salt (TOS-MUP) medium under the conditions specified in this International Standard
在本国际标准规定的条件下，在跨半乳糖基化低聚糖-莫匹罗星锂盐 （TOS-MUP） 培养基上形成直径为 1 毫米至 4 毫米的透镜状或圆形白色结肠、部分星形或三叶状结肠的厌氧微生物

4.1 The antibiotic, mupirocin lithium salt (MUP), inhibis the growth of most lactic acid bacteria commonly used in fermented and non-fermented dairy products.
4.1 抗生素莫匹罗星锂盐（MUP）可抑制发酵和非发酵乳制品中常用的大多数乳酸菌的生长。

Owing to the proven selectivity of the MUP antibiotic when added to the medium, usually there is no growth of typical yogurt bacteria (Streptococcus thermophills, Lactobacillus delbrueckii subsp. bulgaricus), mesophilic cultures (e.g. Lactococcus lactis), Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus rhamnosus on the medium specified.
由于 MUP 抗生素在加入培养基时已被证明具有选择性，因此在指定的培养基上，通常不会生长典型的酸奶菌（嗜热链球菌、保加利亚德氏乳杆菌亚种）、嗜温培养物（例如乳酸乳球菌）、嗜酸乳杆菌、干酪乳杆菌和鼠李糖乳杆菌。

This property has been tested with a representative number of reference strains and isolates.

Additionally, TOS-agar enhances the growth of bifidobacteria used in dairy products (see Reference [17]).

NOTE 1 Examination under a microscope at a magnification of 100 times and oil immersion in contrast phase illumination shows rods of very varied sprapes, usually curved and clubbed, often branched, arranged singly, in pairs, in V shaped arrangements, in chains, in pa/sades of parallel cells, or in rosettes occasionally exhibiting swollen coccoid forms.
注1 在显微镜下以100倍的放大倍数和油浸在对比相照下检查，可以看到非常多样化的柱状杆，通常是弯曲的和棒状的，通常是分枝的，单独排列的，成对排列的，呈V形排列的，呈链状排列的，平行细胞的pa/sades中，或偶尔表现出肿胀的球状形状的莲座状。

NOTE 2 Bifidobacteria are ngh-acid-fast, non-spore-forming, gram-positive, non-motile and catalase-negative chemoorganotrophs, which produce acetic acid and lactic acid. Glucose is degraded exclusively and characteristically by the fructose-6-phosphate shunt ji which fructose-6-phosphate phosphoketolase (F6PPK, EC 4.1.2.22) cleaves fructose-6phosphate into acetyl phosphaye and erythrose-4-phosphate.
注2 双歧杆菌是抗酸、不形成孢子、革兰氏阳性、非运动和过氧化氢酶阴性的化学有机营养菌，产生乙酸和乳酸。葡萄糖仅由果糖-6-磷酸分流剂降解，果糖-6-磷酸磷酸酮醇酶（F6PPK，EC 4.1.2.22）将果糖-6磷酸裂解成乙酰基磷和赤藓糖-4-磷酸。

NOTE 3 The optimum growhth temperature is between and . For further details, see Reference [9].

4.2 Inoculation of appropriate decimal dilutions of the homogenized sample into TOS-agar containing MUP using the pour plate technique, is followed by anaerobic incubation at for 72 h .
4.2 使用倾板技术将均一化样品的适当十进制稀释液接种到含有 MUP 的 TOS 琼脂中，然后在 72 小时下厌氧孵育 。

4.3 The colonies are counted.

NOTE Optionally, selected isolates from the plates can be confirmed by means of appropriate tests (e.g. F6PPK assay, see Reffrences [14][15]).
注：可选地，从板中选择的分离物可以通过适当的测试来确认（例如F6PPK测定，参见参考文献[14][15]）。

4.4 The humber of bifidobacteria per gram of sample is calculated from the number of colonies obtained on plates at dhution levels so as to give a significant result.
4.4 每克样品中双歧杆菌的蜂数是根据在平板上获得的菌落数计算得出的，以得出显著的结果。

Use only reagents of recognized analytical grade, unless otherwise specified, and distilled gr demineralized water or water of equivalent purity.
除非另有说明，否则仅使用公认的分析级试剂，以及蒸馏 gr 软化水或同等纯度的水。

See ISO 6887-5 and ISO/TS 11133-1 for bașic materials.

See ISO 6887-5 for the preparation of diluents.

To ensure comparability of the specified colony-forming unit (CFU) results, observe the following requirements.

a) Use quarter-strength Ringer's solution, or any other suitable diluent which is specified in ISO 6887-5 and proven to be equivalent.
a） 使用四分之一强度的林格氏溶液，或 ISO 6887-5 中规定并证明等效的任何其他合适稀释剂。

b) Sterilize in bulk and use an adequate sterile dispenser unit.

c) Adjust the diluent to room temperature. Transfer the diluept by dripping, without incorporating air.

d) The uncertainty of measurement of volumes used shall be in accordance with the requirements of ISO 6887-1.

Use freshly prepared transgalactosylated oligosag-harides-mupirocin lithium salt (TOS-MUP) culture medium, which has not been exposed to direct sunlight
使用新鲜制备的跨半乳糖基化寡糖基化寡糖苷 - 哈里德斯 - 莫匹罗星锂盐 （TOS-MUP） 培养基，该培养基未暴露在阳光直射下

5.3.1 Basic medium (TOS-propionate agar medium, see Reference [10])

A TOS mixture is obtained by enzymatic hydrolysis of lactose using Aspergillus oryceae -galactosidase. The TOS mixture contains galactose (Gal) and glucose (Glc) units in accordance with the formula
TOS混合物是通过使用曲霉菌- 半乳糖苷酶对乳糖进行酶水解而获得的。根据公式，TOS混合物含有半乳糖（Gal）和葡萄糖（Glc）单位

where

The TOS mixture is purified by chromatography under defined conditions (see References [18][19]). The total sugar content ( mass fraction) includes a certain proportion of tri-, tetra-, penta- and hexasaccharides. (Modification of the ratio of oligosaccharides has no significant effect on the potential of the medium.)
TOS混合物在规定的条件下通过色谱法纯化（参见参考文献[18][19]）。总糖含量（ 质量分数）包括一定比例的三糖、四糖、五糖和六糖。（低聚糖比例的改变对培养基的潜力没有显着影响。

Suspend the ingredients in 950 ml water while heating carefully (e.g. using a hotplate or a water bath) with frequent agitation until completely dissolved.
将成分悬浮在 950 毫升水中，同时小心加热（例如使用加热板或水浴），并经常搅拌直至完全溶解。

Distribute in portions of 190 ml into bottles of 250 ml capacity. Adjust the pH (6.6), if necessary, so that after autoclaving a final pH of -units is obtained at .
将 190 毫升的部分分配到 250 毫升容量的瓶子中。如有必要，调节pH值（6.6），以便在高压灭菌后获得-单位的 最终pH 值。

Autoclave the basic medium at for 15 min .

If not used immediately, cool the prepared basic medium, unless otherwise specified. Store the medium between and for a maximum of 1 week under conditions not producing any change in its composition.
如果不立即使用，除非另有说明，否则请冷却准备好的碱性培养基。将培养基储存在不超过其成分变化的 条件下，最多 储存 1 周。

TOS-medium is sensitive to heat, thus excessive heat treatment can negatively influence the properties of the medium. Complete TOS-propionate media are commercially available and have a composition in accordance with this International Standard. However, if (he medium is made up in the laboratory, the results can differ significantly from one preparation to another. Therefore media should be validated to ensure that growth performance of bifidobacteria, indicated by CFU results, are on a comparable level (see also ISO/TS 11133-1).
TOS介质对热敏感，因此过度的热处理会对介质的性能产生负面影响。完整的 TOS-丙酸盐培养基是市售的，其成分符合本国际标准。但是，如果（培养基是在实验室中配制的，则结果可能因一种制剂与另一种制剂而有很大差异。因此，应对培养基进行验证，以确保双歧杆菌的生长性能（由 CFU 结果指示）处于可比水平（另见 ISO/TS 11133-1）。

Immediately before use, dissolve, for example, 50 mg MUP in 50 ml of water, or other amounts in the same proportion. Sterilize the solution obfained by filtration through a membrane (pore size ) as specified in 5.3.3.
使用前，将50mg MUP溶解在50ml水中，或以相同比例溶解其他量。按照 5.3.3 中的规定，对通过膜（孔径 ）过滤而褪色的溶液进行灭菌。

Immediately before use, mel 190 ml portions of the prepared basic medium (5.3.1) under steam or equivalent. Cool it in a water bath ( 6.5 ) to . Add 10 ml of MUP supplement solution (5.3.2) to each portion by using a syringe equipped with a sterile filter unit of pore size (6.11) shortly before pouring. Mix carefully while avoiding formation of air bubbles.
使用前，将 190 ml 制备的碱性培养基 （5.3.1） 部分置于蒸汽或等效物中。在水浴（6.5）中冷却至 。在倾倒前不久，使用配备有孔径 （6.11）的无菌过滤单元的注射器向每部分加入10ml MUP补充溶液（5.3.2）。小心混合，同时避免形成气泡。

Put the completed medium back in the water bath (6.5) at until it is ready to be poured.

The complete TOS-MUP medium shall have a final MUP concentration of .