1、BS ISO21527-2:2008ICS 07.100.30NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDMicrobiology of foodand animal feedingstuffs Horizontalmethod for theenumeration of yeastsand mouldsPart 2: Colony count technique inproducts with water activity less thanor equal to
2、0,95This British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31 August2008 BSI 2008ISBN 978 0 580 57109 1Amendments/corrigenda issued since publicationDate CommentsBS ISO 21527-2:2008National forewordThis British Standard is the UK implementation of ISO 2
3、1527-2:2008.The UK participation in its preparation was entrusted to TechnicalCommittee AW/9, Microbiology.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users ar
4、e responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS ISO 21527-2:2008Reference numberISO 21527-2:2008(E)ISO 2008INTERNATIONAL STANDARD ISO21527-2First edition2008-07-01Microbiology of food and animal feeding stuffs Horizontal me
5、thod for the enumeration of yeasts and moulds Part 2: Colony count technique in products with water activity less than or equal to 0,95 Microbiologie des aliments Mthode horizontale pour le dnombrement des levures et moisissures Partie 2: Technique par comptage des colonies dans les produits activit
6、 deau infrieure ou gale 0,95 BS ISO 21527-2:2008ISO 21527-2:2008(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installe
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10、without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2008 All
11、 rights reservedBS ISO 21527-2:2008ISO 21527-2:2008(E) ISO 2008 All rights reserved iiiForeword 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 t
12、hrough ISO technical committees. Each member body interested in a subject for which a technical 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 colla
13、borates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. 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 Sta
14、ndards. 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 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements o
15、f this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 21527-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. ISO 21527 consists of the following parts, under the genera
16、l title Microbiology of food and animal feedings stuffs Horizontal method for the enumeration of yeasts and moulds: Part 1: Colony count technique in products with water activity greater than 0,95 Part 2: Colony count technique in products with water activity less than or equal to 0,95 This part of
17、ISO 21527, together with ISO 21527-1, cancel and replace ISO 7698:1990, ISO 7954:1987 and ISO 13681:1995. BS ISO 21527-2:2008ISO 21527-2:2008(E) iv ISO 2008 All rights reservedIntroduction Because of the large variety of food and feed products, the applications of the horizontal method specified in
18、ISO 21527 (all parts) may not be appropriate for certain products. In this case, different methods, which are specific to these products, may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt shall be made to apply the horizontal method as specified in ISO
19、21527 (all parts) as far as possible. When ISO 21527 (all parts) is next reviewed, account will be taken of all information then available regarding the extent to which the horizontal method has been followed and the reasons for deviations from this method in the case of particular products. The har
20、monization of test methods cannot be immediate, and for certain groups of products International Standards and/or national standards may already exist that do not comply with the horizontal method as specified in ISO 21527 (all parts). It is hoped that when such standards are reviewed they will be c
21、hanged to comply with ISO 21527 (all parts) so that eventually the only remaining departures from this horizontal method will be those necessary for well-established technical reasons. BS ISO 21527-2:2008INTERNATIONAL STANDARD ISO 21527-2:2008(E) ISO 2008 All rights reserved 1Microbiology of food an
22、d animal feeding stuffs Horizontal method for the enumeration of yeasts and moulds Part 2: Colony count technique in products with water activity less than or equal to 0,95 WARNING It is essential that enumeration of moulds is carried out with the greatest care to protect the operator and to prevent
23、 contamination of the atmosphere with mould spores. 1 Scope This part of ISO 21527 specifies a horizontal method for the enumeration of viable osmophilic yeasts and xerophilic moulds in products intended for human consumption or feeding of animals that have a water activity less than or equal to 0,9
24、5 (dry fruits, cakes, jams, dried meat, salted fish, grains, cereals and cereal products, flours, nuts, spices and condiments, etc. Annex A), by means of the colony count technique at 25 C 1 C (Reference 3). This part of ISO 21527 does not apply to dehydrated products with water activity less than o
25、r equal to 0,60 (dehydrated cereals, oleaginous products, spices, leguminous plants, seeds, powders for instant drinks, dry products for domestic animals, etc.) and does not allow the enumeration of mould spores (Reference 3). Neither the identification of fungal flora nor the examination of foods f
26、or mycotoxins lie within the scope of this part of ISO 21527. The method specified in this part of ISO 21527 is not suitable for enumeration of halophilic xerophilic fungi (i.e. Polypaecilum pisce, Basipetospora halophila) such as may be found in dried fish. 2 Normative references The following refe
27、renced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 6887 (all parts), Microbiology of food and animal feeding stuff
28、s Preparation of test samples, initial suspension and decimal dilutions for microbiological examination ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations ISO 8261, Milk and milk products General guidance for the preparation of
29、 test samples, initial suspensions and decimal dilutions for microbiological examination ISO/TS 11133 (all parts), Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media ISO 21527-1, Microbiology of food and animal feedings stuffs Horizontal method f
30、or the enumeration of yeasts and moulds Part 1: Colony count technique in products with water activity greater than 0,95 BS ISO 21527-2:2008ISO 21527-2:2008(E) 2 ISO 2008 All rights reserved3 Terms and definitions For the purposes of this document the terms and definitions given in ISO 21527-1 and t
31、he following apply. 3.1 osmophilic yeast xerophilic mould fungus which is capable of growth at a water activity less than or equal to 0,95 4 Principle 4.1 Surface-inoculated plates are prepared using a specified selective culture medium. Depending on the expected number of colonies, a specified quan
32、tity of the sample (if the product is liquid), or of an initial suspension (in the case of other products), or decimal dilutions of the sample/suspension are used. Additional plates can be prepared under the same conditions, using decimal dilutions of the test sample or of the initial suspension. 4.
33、2 The plates are then aerobically incubated at 25 C 1 C for 5 d to 7 d. If necessary, the agar plates are left to stand in diffuse daylight for 1 d to 2 d. 4.3 Colonies/propagules are then counted and, if required (to distinguish yeast colonies from bacterial colonies), the identity of any doubtful
34、colonies is confirmed by examination with a binocular magnifier or microscope. 4.4 The number of yeasts and moulds per gram or per millilitre of sample is calculated from the number of colonies/propagules/germs obtained on plates chosen at dilution levels producing countable colonies. Moulds and yea
35、sts are counted separately, if necessary. 5 Diluent and culture medium For current laboratory practice, see ISO/TS 11133 (all parts). 5.1 Diluent 5.1.1 General See ISO 6887 (all parts), ISO 8261 and the specific International Standard dealing with the product concerned. The use of a diluent containi
36、ng a sufficient amount of solute e.g. a 20 % to 35 % (mass concentration) solution of glycerol or D-glucose is recommended to minimize osmotic shock to xerophilic mould and osmophilic yeast cells when serial dilutions are made prior to plating (References 1, 3). NOTE It is possible to add surface-ac
37、tive agents such as sodium poly(oxyethylene)sorbitanmonooleate 1)0,05 % (mass concentration) to diluents to reduce clumping of mould spores and conidia (Reference 3). Except for specific preparation of the test sample, the use of 0,1 % (mass concentration) peptone water broth as diluent is recommend
38、ed . 1) Tween 80 is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. BS ISO 21527-2:2008ISO 21527-2:2008(E) ISO 2008 All rights reserved 35.1.2
39、 Composition of 0,1 % (mass concentration) peptone water broth Enzymatic digest of animal or vegetal tissues 1,0 g Water 1 000 ml 5.1.3 Preparation of 0,1 % (mass concentration) peptone water broth Dissolve the components in the water, by heating if necessary. If necessary, adjust the pH so that, af
40、ter sterilization, it is 7,0 0,2 at 25 C. 5.2 Culture medium 5.2.1 Dichloran 18 % (mass concentration) glycerol agar (DG18) (References 4, 5, 6) 5.2.1.1 Composition Casein enzymatic digest 5,0 g D-Glucose (C6H12O6) 10,0 g Potassium dihydrogenphosphate (KH2PO4) 1,0 g Magnesium sulfate (MgSO4 H2O) 0,5
41、 g Dichloran (2,6-dichloro-4-nitroaniline) 0,002 g Glycerol anhydrous 220 g Agar 12 g to 15 g aChloramphenicol 0,1 g Water, distilled or deionized 1000 ml aDepending on the gel strength of the agar. 5.2.1.2 Preparation 5.2.1.2.1 General Suspend all the ingredients except chloramphenicol in the water
42、 and bring to the boil to dissolve completely. If necessary, adjust the pH (6.4) so that after sterilization it is 5,6 0,2 at 25 C. Add 10 ml of a 1 % (mass concentration) solution of chloramphenicol in ethanol and mix. Dispense the medium in quantities into suitable containers (6.5) of suitable cap
43、acity. Sterilize by autoclaving at 121 C for 15 min. Immediately cool the medium in a water bath (6.3) maintained at a temperature of 44 C to 47 C. Cool to below 50 C and dispense 15 ml amounts into sterile Petri dishes (6.6). Allow the medium to solidify, and dry, if necessary, the surface of the p
44、lates as described in ISO 7218 and ISO/TS 11133 (all parts). Use immediately, or store in the dark, according to ISO/TS 11133 (all parts) until required. CAUTION Avoid exposure of the medium to light, since cytotoxic breakdown products can result in underestimation of mycoflora in samples. BS ISO 21
45、527-2:2008ISO 21527-2:2008(E) 4 ISO 2008 All rights reserved5.2.1.2.2 Optional addition of chlortetracycline hydrochloride Where bacterial overgrowth may be a problem, chloramphenicol (50 mg/l) and chlortetracycline (50 mg/l) are recommended. In this case, prepare the basic medium as described above
46、, with only chloramphenicol 50 mg, dispense it in quantities of 100 ml and sterilize. Since it is relatively unstable, freshly prepare also a 0,1 % (mass concentration) solution of chlortetracycline hydrochloride in water and sterilize by filtration. Just prior to use, add 5 ml of this solution asep
47、tically to 100 ml of the basic medium, and pour plates. Gentamicin is not recommended, as it has been reported to cause inhibition of some yeast species (Reference 3). 5.2.1.2.3 Optional addition of trace elements In order for moulds to exhibit their full morphology, particularly any pigments they n
48、ormally produce, they need trace elements that may not be present in DG18. To identify moulds on this medium, add the following trace element solution at 1 ml per litre of the medium, prior to autoclaving: ZnSO4 7H2O 1g; CuSO4 5H2O 0,5 g; water, distilled or deionized 100 ml (Reference 2). 5.2.1.3 P
49、erformance testing for the quality assurance of the culture medium 5.2.1.3.1 General DG18 medium is a solid medium. Productivity and selectivity shall be tested according to ISO/TS 11133 (all parts) according to the following specifications: 5.2.1.3.2 Productivity Incubation: 5 d at 25 C 1 C Strains: Saccharomyces cerevisiae ATCC 9763 Wallemia sebi ATCC 42694 Aspergillus restrictus ATCC 42693 Eurotium rubrum ATCC 42690 or strains recorded as equivalent in other
