© ISO 2017 Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 4 Specific rules for the preparation of miscell[.]
INTERNATIONAL STANDARD ISO 6887-4 Second edition 2017-03 Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 4: Specific rules for the preparation o f miscellaneous products Microbiologie de la chne alimentaire — Préparation des échantillons, de la suspension mère et des dilutions décimales en vue de l’examen microbiologique — Partie 4: Règles spécifiques pour la préparation de produits variés Reference number ISO 6887-4:2017(E) © ISO 2017 ISO 6887-4:2017(E) COPYRIGHT PROTECTED DOCUMENT © ISO 2017, Published in Switzerland All rights reserved Unless otherwise specified, no part o f this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission Permission can be requested from either ISO at the address below or ISO’s member body in the country o f the requester ISO copyright o ffice Ch de Blandonnet • CP 401 CH-1214 Vernier, Geneva, Switzerland Tel +41 22 749 01 11 Fax +41 22 749 09 47 copyright@iso.org www.iso.org ii © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) Contents Page v Scope Normative references Terms and definitions Principle Diluents 5.1 Basic materials 5.2 Diluents for general use 5.2.1 Peptone salt solution 5.2.2 Buffered peptone water 5.3 Diluents for special purposes 5.3.1 Double-strength buffered peptone water 5.3.2 Phosphate buffered diluent 5.4 Distribution and sterilization of the diluent 5.5 Performance testing of diluents 5.6 Enzyme solutions 5.6.1 Alpha-amylase solution 5.6.2 Cellulase solution 5.6.3 Papain solution Apparatus Sampling and sample types Preparation of samples 8.1 General 8.2 Acidic products 8.3 High-fat foods, excluding margarines and spreads (e.g over 20 % of total mass as fat) 8.4 Hard and dry products Specific procedures 9.1 Dehydrated and low a w products 9.1.1 General 9.1.2 Apparatus 9.1.3 Preparation of samples 9.1.4 Preparation of initial suspension 9.1.5 Resuscitation 9.1.6 Water activity 9.2 Flours, cereal grains and by-products and animal feeds 9.3 Gelatine (powdered and leaf) 10 9.3.1 Preparation of samples 10 9.3.2 Preparation of initial suspension 10 9.4 Margarine and spreads 10 9.4.1 Sampling 10 9.4.2 Preparation of test sample 11 9.5 Eggs and egg products 11 9.5.1 Fresh whole eggs 11 9.5.2 Microflora o f whole egg shell 12 9.5.3 Internal microflora 12 9.5.4 Bulk whole liquid egg, egg white and egg yolk 12 9.5.5 Dehydrated whole egg and dried egg white 12 9.5.6 Whole egg microflora (shell plus yolk plus white) 12 9.6 Bakery goods, pastry and cakes 13 9.6.1 General 13 9.6.2 Preparation of samples 13 Foreword © ISO 2017 – All rights reserved iii ISO 6887-4: 01 7(E) 9.7 Fresh fruit and vegetables (pre-packed) 13 9.7.1 Sample preparation of multi-component products 13 13 f ff 9.8 Fermented products or other products containing viable microorganisms 13 9.8.1 General 13 9.8.2 Diluent 14 9.9 Beverages (alcoholic and non-alcoholic drinks and bottled waters, still or carbonated) 14 9.9.1 General 14 14 9.9.3 De-gassing using ultrasound 14 9.10 Alternative protein products (cooked insects, textured vegetable protein 14 9.10.1 General 14 9.10.2 Cooked insects 14 15 9.7.2 Pre- p acked p ro ducts o o ne typ e o 9.9.2 D e- gas s ing by invers io n and mixing ruit o r vegetab le o r myco p ro tein) 9.1 0.3 Textured vegetab le p ro tein and myco p ro tein Bibliography 10 iv Further dilutions © ISO 2017 – All rights reserved ISO 6887-4:2017(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies) The work o f preparing International Standards is normally carried out through 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 collaborates closely with the International Electrotechnical Commission (IEC) on all matters o f electrotechnical standardization The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part In particular the different approval criteria needed for the di fferent types o f ISO documents should be noted This document was dra fted in accordance with the editorial rules of the ISO/IEC Directives, Part (see www.iso org/directives) Attention is drawn to the possibility that some o f the elements o f this document may be the subject o f patent rights ISO shall not be held responsible for identi fying any or all such patent rights Details o f any patent rights identified during the development o f the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso org/patents) Any trade name used in this document is in formation given for the convenience o f users and does not constitute an endorsement For an explanation on the meaning o f ISO specific terms and expressions related to formity assessment, as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso org/iso/foreword.html The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9, Microbiology This second edition cancels and replaces the first edition (ISO 6887-4:2003), which has been technically revised It also incorporates the Amendment ISO 6887-4:2003/Amd.1:2011 and the Technical Corrigendum ISO 6887-4:2003/Cor.1:2004 A list of all parts in the ISO 6887 series can be found on the ISO website © ISO 2017 – All rights reserved v INTERNATIONAL STANDARD ISO 6887-4:2017(E) Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 4: Specific rules for the preparation o f miscellaneous products WARNING — The use of this document may involve hazardous materials, operations and equipment It is the responsibility of the user of this document to establish appropriate safety and health practices and to determine the applicability of regulatory limitations before use Scope T h i s c u ment s p e ci fie s ru le s exa m i nation o f s p e ci fic fo o d for the prep aration o f s ample s a nd d i lution s for the m icrobiolo gic a l pro duc ts no t covere d i n o ther p ar ts o f I S O 8 7, wh ich de a l with more general categories This document covers a wide range of miscellaneous products, but does not include new products brought on to the market after publication I S O 8 7-1 defi ne s the genera l r u le s for the prep aration o f the i n iti a l s u s p en s ion a nd d i lution s for microbiological examination This document excludes preparation of samples for both enumeration and detection test methods when prep aration de tai l s are s p e c i fie d i n the relevant I nternationa l Standard s This document is applicable to the following products: — acidic (low pH) products; — h ard a nd d r y pro duc ts; — dehyd rate d , — flou rs , whole cere a l gra i n s , cere a l b y-pro duc ts; fre e z e - d rie d a nd o ther low a w pro duc ts (i nclud i ng tho s e with i n h ibitor y prop er tie s) ; — animal feed, cattle cake, kibbles and pet chews; — gelatine (powdered and leaf); — ma rga ri ne s , s pre ad s and non- da i r y pro duc ts with adde d water; — eggs and egg products; — b a ker y go o d s , p a s trie s a nd ca ke s; — — — — fresh fruit and vegetables; fermented products and other products containing viable microorganisms; alcoholic and non-alcoholic beverages; alternative protein products © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) T he Normative references fol lowi ng c u ments are re ferre d to i n the te xt i n s uch a way that s ome or a l l o f thei r content s titute s re qu i rements o f th i s c u ment For date d re ference s , on ly the e d ition c ite d appl ie s For undate d re ference s , the l ate s t e d ition o f the re ference d c u ment (i nclud i ng a ny amend ments) appl ie s ISO 6887-1, Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for microbiological examinations Terms and definitions For the pu rp o s e s o f th i s c ument, the term s a nd defi n ition s gi ven i n I S O 8 7-1 apply ISO and IEC maintain terminological databases for use in standardization at the following addresses: — IEC Electropedia: available at http://www.electropedia org/ — ISO Online browsing platform: available at https://www.iso org/obp/ Principle The general principles for sample preparation and subsequent steps are detailed in ISO 6887-1 This c u ment de s crib e s s p e ci fic me a s u re s for pro duc ts no t covere d i n o ther p a r ts o f I S O 8 An initial suspension is prepared to obtain as uniform a distribution as possible of the microorganisms contained in the test portion For de te c tion me tho d s , the pre - en rich ment or enrich ment s u s p en s ion i s prep are d i n the s a me way, using the medium recommended in the method of examination concerned For enumeration methods f f f Clause us e d or a l l clau s e s o o o d typ e s covere d i n th i s c u ment, genera l d i luents a re c u mente d i n the releva nt I S O 8 7-1 and s p e ci fic d i luents a re i nclude d i n I f ne ce s s ar y, fu r ther d i lution s are prep are d i n order to re duce the nu mb er o f m ic ro orga n i s m s p er un it volu me to a l low, a fter i nc ub ation, ob s er vation o f any grow th (i n the c a s e o f l iqu id me d ia) or colonie s (i n the c a s e o f agar plate s or agar tub e s) as s tate d i n e ach s p e c i fic s tandard Diluents Basic materials Refer to ISO 6887-1 5 Diluents for general use Peptone salt solution Refer to ISO 6887-1 2 Buffered peptone water Refer to ISO 6887-1 © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) Diluents for special purposes Double- strength buffered peptone water 1 Composition and preparation Refer to ISO 6887-1 Application Us e th i s d i luent 7,0 ± 0,5 NOTE for h igh ly ac id ic pro duc ts o f pH ≥ , to pH ˂ 4, to ob ta i n a n i n itia l s u s p en s ion o f pH Buffered peptone water (5.2.2 ) i s s u ffic ient for p ro duc ts o f p H 4, a nd ab ove Phosphate buffered diluent Composition 9,0 g D i s o d iu m hyd ro gen s phate de c a hyd rate (Na2 HPO · 12H O) Po ta s s iu m d i hyd ro gen s ph ate (KH Water 2 PO 4) 1,5 g 000 ml Preparation D i s s olve the comp onents i n the water, b y heati ng i f ne ce s s a r y I f ne ce s s ar y, adj u s t the pH s o that, a fter s teri l i z ation, it i s pH 7, ± , at ° C 3 Application Phosphate buffered solution is used as a diluent for gelatine (9.3) Distribution and sterilization of the diluent Refer to ISO 6887-1 5 Performance testing of diluents Refer to ISO 6887-1 Enzyme solutions 6.1 6.1 Alpha-amylase solution Composition α- amyla s e Water © ISO 2017 – All rights reserved 1,0 g 100 ml ISO 6887-4: 01 7(E) 6.1 Preparation D i s s olve the α- amylas e i n the water and s teri l i ze the s olution b y p a s s i ng th rough a , µm membrane fi lter T he en z yme s olution c a n b e s tore d for up to month at ° C ± ° C or up to month s at ≤ −2 ° C Fi na l comp o s ition o f the α- a myla s e s olution may ne e d to b e adj u s te d dep end i ng on the en z ymatic ac tivity o f the com merc ia l α- amyl as e u s e d and the th icken i ng prop er tie s o f the te s t s ample 6.1 Application T h i s en z yme s olution i s adde d at the rate o f 10 m l to 0 m l o f d i luent (1 % volu me frac tion) 9.1.4.3) to i mprove s olubi l ity o f s wel l i ng s tarch pro duc ts , cere a l s a nd cere a l- contai n i ng pro duc ts ( 6.2 Cellulase solution 6.2 Composition Cellulase Water 6.2 1,0 g 100 ml Preparation D i s s olve the cel lu las e i n the water and s teri l i ze the s olution b y p a s s i ng th rough a , µm membrane fi lter T he en z yme s olution c a n b e s tore d NO TE 6.2 Us e o f fre s h for up to two we eks at ° C ± ° C or up to month at ≤ −2 ° C s olution wi l l en s u re m a xi mu m en z yme ac tivity Application T h i s en z yme s olution i s adde d at the rate o f 10 m l to 0 m l o f d i luent (1 % volu me frac tion) 9.1.4.3) to i mprove s olubi l ity o f c arb ox yme thyl cel lu lo s e, lo c u s t b e a n s , c arob , guar and c a s s i a g um s ( 6.3 6.3 Papain solution Composition Papain Water 6.3 5,0 g 100 ml Preparation D i s s olve the p ap n i n the water a nd s teri l i ze the s olution b y p a s s i ng th rough a , µm membrane fi lter T he en z yme s olution c an b e s tore d NO TE 6.3 Us e o f fre s h for up to month at ° C ± ° C s olution wi l l en s u re m a xi mu m en z yme ac tivity Application T h i s en z yme s olution i s adde d at the rate o f m l to 0 m l o f d i luent (2 % volu me 9.1.4.3 and 9.3) frac tion) to i mprove s olubi l ity o f gelati ne ( Apparatus Us ua l m ic robiolo gic a l l ab orator y e qu ipment particular, the following for genera l u s e (s e e I S O and I S O 8 7-1) a nd , i n © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) 6.1 Homogenizers (blender) Refer to ISO 7218 If a large sample is to be homogenized, the equipment should include a sterile l bowl 6.1 Rotary homogenizer 6.1 Peristaltic homogenizer Re fer to I S O With s teri le pla s tic b ags or fi lter b ags to re tai n p a r tic u late materia l where ne ce s s ar y , sterile 6.2 Domestic grater 6.3 Hammer 6.4 Water baths , cap ab le o f b eing maintained at 44 °C to 47 °C o r as s tated fo r s p ecific p urp o s es 6.5 Sterile scissors knives scalpels 6.6 Sterile spatulas spoons 6.7 Sterile corers 6.8 Stirrer 6.9 Sterile 6.1 Ultrasonic bath , with o p erating frequency o f M H z to 45 M H z or , capable of crushing hard materials other heavy implement , , , or and scoops forceps , for taking samples at depth , capable of operating with a horizontal motion wide-necked flasks or other containers , o f 0 ml cap acity Sampling and sample types C a rr y out s a mpl i ng i n accordance with the s p e c i fic s ta ndard appropri ate to the pro duc t concerne d or see ISO/TS 17728 Some guidance on sampling certain products is included in Clause f parties concerned or cla rity I f a s p e ci fic s tanda rd i s no t avai lable, it i s re com mende d that agre ement b e re ache d on th i s s ubj e c t b y the Preparation of samples 8.1 General All preparations and manipulations shall be carried out using aseptic techniques and sterile equipment (see ISO 7218) General sample preparation procedures are given in ISO 6887-1, but additional detail for some categories is given in 8.2 to 8.4 8.2 Acidic products It is important to consider the end use of the product when testing acidic samples I f the pro duc t i s to b e u s e d as an i ngre d ient i n a fi na l pro duc t o f h igher pH , then the pH o f the i n iti a l s u s p en s ion o f the te s t p or tion s l l b e adj u s te d to pH 7, ± , with the d i luents s p e ci fie d at 5.3.1 or o thers with e qu iva lent bu fferi ng c ap ac ity 5.2.2 or For pH adj u s tment o f mo derately acid ic s a mple s ( pH ≥ , to pH < 4, ) , u s e double - s treng th bu ffere d peptone water (5.3.1) See ISO 6887-1 © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) I f highly acidic (pH < 3,5) samples (e.g low pH fruits and juices or vinegars and pickles) are tested for acid-tolerant and acidophilic spoilage organisms using appropriate media, the pH of such samples shall not be adjusted 8.3 High-fat foods, excluding margarines and spreads (e.g over % of total mass as fat) A diluent with between g/l and 10 g/l o f polysorbate 80 [polyoxyethylene (20) sorbitan monooleate] according to the estimated fat content shall be used to improve emulsification during suspension (e.g for a fat content of 40 %, add g/l) Alternative sur factants and emulsifiers are available under various trade names, but the proportions to use should be determined by the laboratory 8.4 Hard and dry products Do not homogenize hard or dry products in a rotary homogenizer (6.1.1) for more than 2,5 at a time to avoid excessive heating that may damage the microorganisms present Homogenize dry and hard heterogeneous products by mincing or grinding the laboratory sample Avoid excessive heating during this process by homogenizing for periods o f no more than at a time, with suitable rest intervals applied, depending on the product being processed Mince or grind until the sample is visibly homogeneous Resuscitation at laboratory ambient temperature (18 °C to 27 °C) for up to h is recommended to assist in the recovery o f stressed organisms from all hard and dry products Specific procedures 9.1 9.1 Dehydrated and low a w products General The following are regarded as dehydrated products: — dehydrated meats and vegetables; — dried soups, bouillon cubes and gravy mixes; — powdered beverages (tea, cocoa and cocoa-based products, co ffee, dehydrated fruit juice); — raw cellulose, soluble cellulose, dextrin, sorbitol, sugars, glucose, glutamate; — herbs, spices, flavourings and colourings; — polysaccharide gelling agents, alginates, gums, etc.; — coconut, partially dehydrated vegetable/yeast/meat/fish extracts; — chocolate and fectionery (bars or sweets); — dehydrated whole egg and dried egg white; — cereals, flours, animal feeds; — powdered or pelletized viable microorganisms (e.g yeasts for bakery) 9.1 Apparatus Plastic bags with filter inserts (6.1.2) are recommended to assist in the pipetting of products with substantial insoluble matter in suspension © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) 9.1 Preparation of samples 6.6) and then weigh out 5.2.2) to minimize M i x p owdere d pro duc ts thorough ly i n thei r conta i ner u s i ng a s teri le i mplement ( u s i ng as ep tic te ch n ique s Weigh acc urately i nto a pre - d i s p en s e d volume o f d i luent ( o s mo tic s ho ck to the m ic ro flora O ther pro duc ts may re qui re bre a ki ng or c utti ng up with s teri le to ol s i nto s ma l l pie ce s b e fore ta ki ng the test portion 9.1 Preparation of initial suspension 9.1 4.1 Powdered products, completely soluble I t i s no t ne ce s s a r y to homo gen i z e fu l ly s oluble pro duc ts me ch an ic a l ly a s m i xi ng b y hand i s ade quate Prepare the initial suspension in accordance with ISO 6887-1 9.1 4.2 Other less soluble or non-powdered products 6.1.1) or peristaltic homogenizer (6.1.2) P rep a re the i n iti a l s u s p en s ion u s i ng a ro tar y b lender ( 9.1 4.3 Products which swell in water For a l l pro duc ts that s wel l i n water (e g p olys accha ride s and gu m gel l i ng agents , dehyd rate d p a rsley or chives), make further dilutions (1 in 20, in 50 or in 100, as appropriate) until a suitable suspension is obtained Record the use of additional diluent to ensure the correct calculation of enumeration test results Where greater dilutions are made, the number of inoculated plates for enumeration tests shall be increased to ensure a minimum of 0,1 g of the test portion is distributed between all plates when low counts are expected T he s olubi l ity o f s ome s ub s tance s i s i mprove d b y the add ition o f a s p e ci fic en z yme s olution to the i n itia l suspension in buffered peptone water (5.2.2 ) S ome exa mp le s o f s uitable en z yme s are the fol lowi ng: f 5.6.1) for swelling starch products, cereals and cerealcontaining products; — % (volume fraction) cellulase (5.6.2 f cassia gums; — % (volume fraction) papain (5.6.3) for gelatine — % (volu me rac tion) a lph a- amyl as e ) 9.1 4.4 For fo o d ( or c a rb ox yme thyl cel lu lo s e, lo c u s t b e an s , c arob , guar a nd Inhibitory food materials materia l s that contai n i n h ibitor y s ub s ta nce s (e g onion p owder, garl ic, orega no , p epp ers , cer tai n te as and co ffe e s , vitam i n prem i xe s and h igh ly s a lte d pro duc ts) , it i s ne ce s s ar y to de cre a s e the anti m icrobi a l ac tivity b e fore te s ti ng b y u s i ng s p e c ia l prep aration pro ce dure s s uch as the fol lowi ng: — use of greater dilutions (e.g in 100 for cinnamon and oregano and in 000 for cloves); f SO ) to the buffered peptone water (5.2.2 concentration of 0,5 % (w/v); — use of diluent (5.2.2 f premixes; — use of higher dilutions for products containing more than 10 % (mass fraction) salt (sodium chloride) f diluent or enrichment broth) does not exceed % (w/v) — add ition o p o ta s s iu m s u lph ite (K ) to ach ieve a fi na l ) at ° C ± ° C , to aid d i s s olution, a nd h igher d i lution s (e g i n 0) or vita m i n to en s u re the to ta l concentration i n the i n itia l s u s p en s ion (no t i nclud i ng a ny s a lt content o © ISO 2017 – All rights reserved the ISO 6887-4: 01 7(E) I f any o f the s e te ch n ique s i s u s e d , s pi ke d s a mp le pro ce s s control s s l l b e i nclude d at fi rs t u s e to veri fy the effectiveness of the neutralization process chosen 9.1 4.5 Us e Cocoa and cocoa-containing products either UH T milk or re s titute d non- fat for re s titution) a s the pre - en rich ment bro th and S T E C B P W may b e u s e d a s a genera l d i luent NOTE dry milk p owder (10 g/ l water; s teri l i z e d de te c tion o f the s ign i fic ant p atho gen s for a fter Salmonella spp o ther te s ts Milk is used to neutralize the bactericidal effect of cocoa or cocoa-containing products The probable i n h ib ito r y fac tor 11 13 i n co co a i s a ntho c ya n i n [ ] , [ ] ,[ ] P rehe at the d i luent to ° C to ° C Weigh the test portion (e.g 25 g) into a plastic bag (6.1.2), add the warmed diluent (e.g 225 ml) to ach ieve a i n 10 i n itia l s u s p en s ion and m i x by hand i m me d i ately L e ave the s u s p en s ion at lab orator y ambient temp eratu re (1 ° C to ° C ) T hen, m i x comple tely i n a p eri s ta ltic homo gen i zer For co co a p owder a nd any o ther s a mple s for wh ich for m i n to m i n to melt 60 s ± s may b e h igh ly contam i nate d with Gram-p o s itive bacteria as a result of inadequate thermal processing, addition of 0,45 ml % (w/v) aqueous brilliant green solution (1 g/100 ml water) to the initial suspension of 250 ml can reduce inhibition of low levels of Gram-negative target organisms during non-selective pre-enrichment 12 Whether this is used [ ] ,[ ] dep end s on the typ e o f s a mple and lab orator y e xp erience with s uch s ample s When large portions of solid chocolate or other cocoa-containing materials which cannot be broken up e a s i ly a re te s te d, it may b e ne ce s s ar y to melt the cho colate at a temp erature b e twe en 42 ° C and 47 ° C , for no longer than ne ce s s ar y, b e fore ta ki ng the te s t p or tion For cho colate pro duc ts conta i n i ng >2 % fat, u n le s s the pro duc ts a l re ady contai n s u ffic ient emu l s i fier, add s u ffic ient p olys orb ate [ p olyox ye thylene (2 0) s orbita n mono ole ate] or o ther emu l s i fier to the diluent (see 8.3 and ISO 6887-1) 9.1 4.6 Confectionery (bars or sweets) P re -he at the d i luent to ° C to ° C Weigh out the test portion in a plastic bag (6.1.2 6.3), to aid dispersion ) and add the warme d d i luent M i x i m me d iately b y hand to d i s tribute the te s t p or tion Ver y h ard s we e ts or c a nd ie s may a l s o b e p ar tia l ly cr u she d with a he av y obj e c t, s uch as m mer ( L e ave the s u s p en s ion at l ab orator y ambient temp erature (1 ° C to ° C ) 6.1.2) for m i n to m i n to d i s s olve T hen, m i x comple tely u s i ng the p eri s ta ltic homo geni z er ( 9.1 Resuscitation In general, leave the initial suspension of low-moisture products requiring resuscitation for up to h at f cases are detailed in the relevant International Standards lab orator y ambient temp eratu re (1 ° C to ° C ) b e fore prep a ri ng a ny 9.1 u r ther d i lution s S ome s p e c i fic Water activity Table ISO 21527-2) give s e xample s of fo o d typ e s and typic a l nge s o f water ac tivity for guidance when te s t me tho d s s p e c i fy d i fferent pro ce du re s to b e u s e d dep end i ng on the water ac tivity (e g I S O 7-1 and © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) Table — Water activity of different products (adapted from Reference [10] ) Water activity[ ] ≥0,95 aw Highly perishable foods, fresh and canned fruits, vegetables, meats, fish, milk, cooked sausages, breads ≥0,91 Cheeses (e.g cheddar, Swiss, muenster, provolone), cured meats, some fruit juice concentrates, foods containing 55 % (mass fraction) sucrose or 12 % (mass fraction) salt ≥0,87 Fermented sausages, sponge cakes, dry cheeses, margarine, foods containing 65 % (mass fraction) sucrose or 15 % (mass fraction) salt ≥0,80 Most fruit juice concentrates, sweetened condensed milk, maple and fruit syrups, flour, rice, pulses (15 % to 17 % mass fraction moisture), fruit cake, country-style ham, fondants, high-sugar cakes Jam, marmalade, marzipan, glace fruits, some marshmallows ≥0,75 ≥0,65 Rolled oats (≅10 % mass fraction moisture), jelly, fudge, molasses, some dried fruits, nuts ≥0,60 Dried fruits (15 % to 20 % mass fraction moisture), to ffees, caramels, honey, cereal bars, pet chews, granulated foods, cereals, cereal products and grains Noodles (12 % mass fraction moisture), spices (10 % mass fraction moisture) Nougat, whole egg powder (5 % mass fraction moisture), chocolate ≥0,50 ≥0,40 ≥0,30 ≥0,03 9.2 E xamples of product Biscuits and cookies, crackers, dehydrated sauce powders Whole milk powders, instant co ffee, dehydrated soups Flours, cereal grains and by-products and animal feeds Mix dry powders well in the sample container, using a sterile implement (6.6), before weighing out the test portion Weigh the test portion accurately and add it to the required volume o f peptone salt solution (5.2.1) to minimize osmotic shock to the microflora This is the initial suspension at a in 10 dilution For flours, take a proportion o f the required volume o f diluent and add the test portion Mix well by hand and then add the remainder of the diluent to obtain a in 10 dilution Be fore homogenization, leave to stand for 20 to 30 at laboratory ambient temperature (18 °C to 27 °C) to assist resuscitation o f damaged organisms I f the viscosity o f the suspension increases so that it becomes too thick or viscous to mix well or to pipette, add a further equal volume of peptone salt solution to produce a in 20 initial suspension and record this to ensure correct calculation of enumeration test results Mix for 60 s ± s using a peristaltic homogenizer (6.1.2 ) for flours or a rotary blender (6.1.1) for cereal grains or animal feeds A test portion of 50 g is required when testing cereals and other heterogeneous products to improve the reliability o f test results In this case, use a in suspension, homogenize and then make a further in dilution to obtain the initial in 10 suspension NOTE Hard materials (e.g grains and seeds) can puncture plastic bags (6.1.2); double- or triple-bagging can help to prevent leakage and subsequent contamination Test portions can also be partially crushed with a heavy object, such as a hammer (6.3), before homogenizing Animal feeds are presented for testing in a variety o f forms, but the general rules for cereal products above apply to many For pet chews, kibble (extruded product) and similar hard products, immerse in diluent at a in 10 dilution, then leave to soak for approximately be fore massaging by hand for 30 s ± s Mix © ISO 2017 – All rights reserved ISO 6887-4:2017(E) when the products have softened using a peristaltic homogenizer (6.1.2 ) or rotary blender (6.1.1) for 60 s ± s, i f necessary 9.3 Gelatine (powdered and leaf) 9.3.1 Preparation of samples Take a test portion o f 20 g o f the laboratory sample using aseptic techniques 9.3.2 Preparation of initial suspension Trans fer this test portion to a 500 ml sterile flask (6.9) Add 180 ml of phosphate buffered diluent (5.3.2) and mix to disperse the granules in the liquid Leave the gelatine to adsorb the diluent for 60 at laboratory ambient temperature (18 °C to 27 °C) Place the flask in a water bath (6.4) at 44 °C to 47 °C for a maximum o f 30 min; mix frequently to dissolve the gelatine to obtain the initial in 10 suspension Alternatively, papain may be used to dissolve the gelatine (see 9.1.4.3) 9.4 Margarine and spreads 9.4.1 Sampling 9.4.1.1 General Samples may be taken from within the bulk product or from within and/or on the sur face o f packaged items ready for sale, using aseptic techniques throughout 9.4.1.2 Bulk or pre-wrapped products o f ≥1 kg To determine the microbiological quality o f bulk product, examine only core samples First, remove a slice o f mm to mm thickness from the outer layer with a sterilized spatula (6.6) or knife (6.5) Push a sterile metal corer (6.7 ) into the product diagonally without going all the way through Turn the corer in a full circle, then remove it with the cylindrical sample Transfer a portion of this core sample to a sterile container or plastic bag (6.1.2), using a spatula (6.6) or knife (6.5 ), but retain the upper 25 mm to plug the hole made by the corer Take one or more core samples to obtain an adequate laboratory sample NOTE Any other sampling method (such as taking one mass o f at least 500 g) is permitted i f the product is 9.4.1.3 Pre-wrapped product o f ≤1 kg regarded as homogeneous The laboratory sample shall be made up o f one or more pre-wrapped, intact items Remove the wrapping and take a representative test sample from one or more packs using aseptic techniques Remove the outer mm section be fore sampling i f the packs are ≥500 g Take the test sample using a sterile instrument or use a sterile corer (6.7 ) to take a cylindrical portion through the laboratory sample I f the customer requests testing o f the product sur face only, remove the test sample by scraping su fficient material from the sur face with a sterile implement 10 © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) 9.4.2 Preparation of test sample 9.4.2 General Weigh 50 g from the laboratory sample containing a volume-to-mass ratio o f water o f sterilized flask or other container (6.9) 9.4.2 W % into a Preparation of the aqueous phase (primary dilution) Pre-warm a volume o f [50 − (50 · W/100)] ml o f diluent (5.2) in a water bath (6.4) at 44 °C to 47 °C and add it to the test sample in the container In these circumstances, ml of the aqueous phase is equivalent to g of the margarine or spread in the test sample (see ISO 6887-1) EXAMPLE For a 50 g test sample of margarine with an 84 % fat content and therefore a volume-to-mass ratio o f about 16 %, the aqueous phase represents ml o f water Add [50 − (50 × 16/100)] = 42 ml o f diluent This can also be calculated by taking the sample weight o f 50 g and multiplying by the fat content o f 84 % (i.e 50 × 84 / 100 = 42 ml) Place the container in the water bath (6.4) at 44 °C to 47 °C until the product has completely melted The time taken shall not exceed 20 Mix in a peristaltic homogenizer (6.1.2) for 60 s ± s and then for further 30 s increments until an emulsion is produced Leave the container at laboratory ambient temperature (18 °C to 27 °C) so that the fatty (upper) layer and the aqueous (lower) layer are separated fully Use the aqueous layer to take the test portion (1 ml corresponds to g o f the test portion) and prepare the initial suspension in accordance with ISO 6887-1 9.4.2 Preparation of an enrichment or pre-enrichment suspension See the relevant International Standard for the microorganism to be detected or enumerated I f the method requires enrichment or pre-enrichment, the sample may be an entire portion o f the product rather than the aqueous layer (9.4.2.2), but this shall be recorded 9.5 Eggs and egg products 9.5 9.5 1 Fresh whole eggs General Eggs used for routine microbiological examination shall not have any visible cracks in the shells Eggs may be examined singly or in batches according to the purpose o f the testing Where specified by the customer, carry out pooling o f the contents o f several eggs according to ISO 6887-1 Examination o f whole eggs may be carried out with or without cleaning/disin fecting o f the eggshell as required by the customer To examine only the contents, always disin fect the eggs be fore opening For detection o f pathogens (which may also be found on the outside o f the egg), disin fection o f the shell may not be required, but agreement on the procedure to be used shall be reached between the parties 9.5 Disinfecting the shell Remove any dirt or faeces with a damp tissue and blot dry Wearing sterile gloves and using a clean gauze or wipe soaked in a solution of either 70 % (volume fraction) ethanol or isopropanol to water, wipe the entire shell surface This reduces the risk of contamination o f the egg yolk and albumen when the egg is broken open to remove the contents © ISO 2017 – All rights reserved 11 ISO 6887-4:2017(E) Allow to dry completely without re-contaminating the shells be fore breaking the egg to take the test portion 9.5.2 Microflora o f whole egg shell 9.5.2.1 Method by rinsing the whole egg shell Place the whole intact egg in a peristaltic homogenizer bag (6.1.2) or other sterile container and add a known volume of the diluent or culture medium required in the test method Then, massage or rotate the egg care fully in the liquid Remove the egg and use the liquid as the initial suspension to continue with the test method 9.5.2.2 Friction method Use sterile gauze (or other equivalent fabric/paper) soaked in diluent or the required culture medium Hold the gauze with sterile forceps and rub over the entire eggshell Place the pieces o f gauze in the volume o f diluent or culture medium required by the test method 9.5.2.3 Soaking method Break the egg aseptically and discard the contents into a bowl or beaker Retain the shell and place it in a peristaltic homogenizer bag (6.1.2) with the required volume of diluent or culture medium Massage and crush the shell in the bag by hand and use this as the initial suspension 9.5.3 Internal microflora Using fresh sterile gloves for each egg, break the egg open aseptically into a sterile container I f the yolk and white are to be examined separately, separate them and place each in a di fferent sterile container Add peptone salt solution (5.2.1) to give a in 10 dilution for the yolk and in 40 dilution for the white to overcome inhibition by the naturally occurring lysozyme To examine the whole egg contents, place all o f the yolk and white (o f approximately 20 ml) in a sterile container with 180 ml of buffered peptone water (5.2.2) or into the appropriate diluent or enrichment broth required by the specific International Standard and use this as the initial in 10 suspension 9.5.4 Bulk whole liquid egg, egg white and egg yolk These bulk products may or may not be pasteurized For bulk whole liquid egg or egg yolks, dilute in 10 with bu ffered peptone water (5.2.2) For bulk liquid egg whites, use a in 40 suspension in buffered peptone water (5.2.2) to overcome inhibition by the naturally occurring lysozyme 9.5.5 Dehydrated whole egg and dried egg white Treat as dehydrated products (see 9.1) 9.5.6 Whole egg microflora (shell plus yolk plus white) Using aseptic techniques, break the egg and place the shell and contents in a sterile plastic bag (6.1.2) or other container Crush and shake the mixture to homogenize it by hand 12 © ISO 2017 – All rights reserved ISO 6887-4: 01 7(E) Take the required test portion to make the initial suspension 9.6 9.6.1 Bakery goods, pastry and cakes General Bakery goods, including sweet pastries and cakes, are made from flour, butter, eggs and other ingredients and some also include dairy or fruit products As such, they should be treated in the same way as other multi-component products (see ISO 6887-1) The test portion o f relatively homogeneous products, such as loaves o f bread, rolls and other finished items made from plain dough, should be taken according to the purpose of the testing For example, sur face samples may be required for investigations o f mould spoilage 9.6.2 Preparation of samples For packaged pre-cooked products, open the packaging aseptically Take pieces of each component in proportion to the amounts in the whole product Alternatively, homogenize the entire laboratory sample to reflect the microflora o f the whole item and take a representative test portion Treat biscuits or cookies in the same way as dehydrated products i f they are hard and low in moisture (9.1) 9.7 9.7.1 Fresh fruit and vegetables (pre-packed) Sample preparation of multi-component products For multi-component products (those containing pieces of different fruit or vegetables), take pieces of each component in proportion to the amounts in the whole product to provide the test portion Alternatively, homogenize the entire laboratory sample to reflect the microflora o f the whole item and take a representative test portion Dilute the test portion in 10 with buffered peptone water (5.2.2) Homogenize using a peristaltic homogenizer (6.1.2) until a suitable initial suspension is obtained 9.7.2 Pre-packed products of one type of fruit or vegetable Weigh the test portion and dilute in 10 with buffered peptone water (5.2.2) Homogenize using a peristaltic homogenizer (6.1.2) until a suitable initial suspension is obtained 9.8 9.8.1 Fermented products or other products containing viable microorganisms General Such products are examined for contamination by microorganisms other than those used as starter cultures for fermentations or as the active constituent microflora o f probiotic products To test these products for contaminants, use suitable inhibitors to suppress growth of the starter culture or probiotic organisms © ISO 2017 – All rights reserved 13 ISO 6887-4: 01 7(E) 9.8.2 Diluent Use buffered peptone water (5.2.2 ) routi nely or at double - s treng th ( 5.3.1 ) i f the s ample i s h igh ly ac id ic ( pH < 4, ) I n the c a s e o f ye a s t c u ltu re s or fermentation s , add an anti- fu nga l agent (e g c yclohe xi m ide or nys tati n at a concentration of 50 mg/kg or amphotericin at 10 mg/kg) to the counting medium to reduce overgrow th b y u nwa nte d ye a s ts and mou ld s For o ther pro duc ts , us e an antibio tic ac tive aga i n s t the m ic ro flora o f the pro duc t b ei ng e xam i ne d (s e e ISO 27205 for probiotics ) Record the use and concentration of the antibiotic and add these details to the test report [ 9.9 ] Beverages (alcoholic and non-alcoholic drinks and bottled waters, still or carbonated) 9.9.1 To General de te c t contam i nation o f the s e pro duc ts , use membrane fi ltration o f s p e c i fie d volu me s th rough ,45 µm s teri le membrane s (s e e I S O 819 9) For c a rb onate d b everage s , prel i m i na r y de - ga s s i ng i s re qui re d to en s u re acc urate volu me s a re fi ltere d or pipetted 9.9.2 De-gassing by inversion and mixing I nver t the lab orator y s ample b y hand (th rough an arc o f cm five ti me s) a nd then lo o s en the c ap ca re fu l ly to rele a s e evolve d c a rb on d ioxide Tighten the c ap aga i n a nd rep e at the pro ce s s unti l no more ga s i s evolve d Ta ke the te s t p or tion b y pip e tti ng or fi lteri ng acc u rately Us e a s ep tic te ch n ique throughout 9.9.3 De-gassing using ultrasound U ltra s ou nd may a l s o b e u s e d to de - ga s c arb onate d b everage s I nver t the conta i ner (th rough a n arc o f c m five ti me s) to m i x a nd as ep tica l ly de c ant 10 % o f the contents into a sterile container 6.10) for 120 s ± s Check more than twice to minimize potential damage to microorganisms in the sample Replace the l id o f the contai ner lo o s ely and place it i n a n u ltra s onic b ath ( for a ny rema i n i ng ga s and, i f ne ce s s ar y, rep e at the u ltras ou nd tre atment D o no t rep e at th i s pro ce du re Ta ke the te s t p or tion by pip e tti ng or fi lteri ng acc u rately Us e as ep tic te ch n ique th roughout 9.1 Alternative protein products (cooked insects, textured vegetable protein or mycoprotein) 9.1 0.1 General M any o f the s e pro duc ts may b e h and le d u s i ng the genera l prep aration pro ce du re s given i n I S O 8 7-1 Some additional details are given in 9.10.2 and 9.10.3 for particular products 9.1 0.2 Cooked insects Weigh the test portion into a plastic bag (6.1.2) and dilute in 10 with buffered peptone water (5.2.2) Homogenize in a peristaltic homogenizer until a suitable initial suspension is obtained 14 © ISO 2017 – All rights reserved