Tiêu chuẩn iso 11816 2 2016

Milk and milk products — Determination of alkaline phosphatase activity — Part 2 Fluorimetric method for cheese Lait et produits laitiers — Détermination de l’activité de la phosphatase alcaline — Par[.]

Milk and milk products —

Part 2:

L it et produits laitiers — Déte mination de l’activité de la

p osph tase alcaline —

Partie 2: Méth de flu rimétrique p ur le froma e

IDF 155-2:2016(E)

© ISO an IDF 2016, P blshed in Switzer lan

A ll rig hts r eserved Unles otherw ise spe ified, nopar of this p blc tion ma y be r eprod c d or utilz d otherwise in an form

or b an me ns, ele tr onic or me hanic l, inclu in p oto opying , or posin on the internet or an intr anet , without prior

written permis ion Permis ion c n be r eq esed fr om either ISO at the ad r es below or ISO’s member bod y in the c u ty of

the r eq eser

ISO c pyrig ht ofic Int ernational Dairy F ederation

C de Blan on et 8 • C 4 1 Siver B idin • B Aug st e Reyer 70/B • B-1030 Brus els

CH-1 14 Vernier, Geneva, Sw itzerlan

IDF 155-2:2016(E)

F r ewords i v

1 Sc ope 1

2 Nor mati ve r eferenc e 1

3 Terms an definitions 1

4 Principle 1

5 Re g ents 2

6 A pparatus 3

7 S mpl ng 4

8 Preparatio of test sample 4

9 Proc ed re .4

9.1 Ve ification of ins rument pe formanc 4

9.1.1 General 4

9.1.2 Daiy instrument es s 5

9.1.3 C ntr ols 5

9.2 Reag ent contr ols to tes the suitabi ty of r ead y to use wor king subs rate (5.3) 5

9.3 Calbration

6 9.4 Dete mination 6

9.5 Tes sample r elated contr ols 7

9.5.1 Re ommended negative an p sitive contr ol tes s 7

9.5.2 Inte fe ing subs anc tes 8

9.5.3 Heat-s a le micr obial alkalne phosphatase control tes 8

10 Calculation and ex pr es io of r esults 8

1 1 Calbration ratio 8

1 2 Calculation 9

1 2.1 Supe natant 9

1 2.2 Che se 9

1 3 Ex pr es ion of r esult 1

11 Precision 10

1 1 Inter la or atory tes 1

1 2 Repeatabi ty 1

1 3 Reprod cibi ty 1

12 Test rep rt 10

A nne x A (informative)Inter laborator y tr ial 11

A nne x B (informative)Examples of preparatio of a test sample 13

Biblog raphy 15

ISO (he Int ernational Org nization for Stan ardization) is a worldwide fede ation of national s an ards

b dies (ISO membe b dies) The work of pr paring Int ernational Stan ards is normal y car ied out

through ISO t ech ical committ ees Each membe b dy int er st ed in a subje t for w hich a t ech ical

committ ee has be n es a lshed has the right t o be r pr sent ed on that committ ee Int ernational

org nizations, g ove nmental an non-g ove nmental, in laison with ISO, also take p rt in the work

ISO cola orat es closely with the Int ernational Ele trot ech ical C mmis ion (IEC) on al matt ers of

ele trot ech ical s an ardization

The proc d r s used t o develo this document an those int en ed for it furthe maint enanc ar

desc ibed in the ISO/IEC Dir ctives, Part 1 In p rticular the dife ent a pro al c it eria ne ded for the

dife ent ty es of ISO document should be not ed This document was draft ed in ac ordanc with the

edit orial rules of the ISO/IEC Dir ctives, Part 2 ( e www.iso.org dir ctives)

A tt ention is drawn t o the p s ibi ity that some of the element of this document ma be the subje t of

p t ent right ISO shal not be held r sponsible for identifying any or al such p t ent right Detai s of

any p t ent right identif ied d ring the develo ment of the document wi be in the Introduction an / r

on the ISO ls of p t ent de larations r c ived ( e www.iso.org p t ent )

Any trade name used in this document is information given for the convenienc of use s an does not

cons itut e an en orsement

F or an ex lanation on the meaning of ISO spe if ic t erms and ex r s ions r lat ed t o conformity

as es ment, as wel as information a out ISO’ s adhe enc t o the WTO principles in the Te h ical

Bar ie s t o Trade (TBT) se the folowing URL: F or word - Sup lementary information

The committ ee r sp nsible for this document is ISO/TC 34, Fo d produc ts , Subcommitt ee SC 5, Mik a d

mik produc ts an the Int ernational Dairy F ede ation (IDF) It is being publshed jointly b ISO an IDF

This se on edition of ISO 1 8 6- 2|IDF 1 5- 2 canc ls an r plac s the firs edition

(ISO 1 8 6- 2|IDF 1 5- 2:2 0 ), w hich has be n t ech ical y r vised

ISO 1 8 6|IDF 1 5 consis s of the folowing part , u de the g ene al title Mik a d mik produc ts —

Determin tio  o alkalne p os ph tase ac tiv it y:

— Part 1: Flu rimetric meth d fr mik a d mik-b s ed drink s

— Part 2 : Flu rimetric meth d fr che s e

IDF 155-2:2016(E)

IDF (he Int ernational Dairy F ede ation) is a non-prof it priv t e se t or org nization r pr senting the

int er s s of v rious s akeholde s in dairying at he glo al level IDF membe s ar org niz d in National

Committ ees, w hich ar national as ociations comp sed of r pr sentatives of dairy-r lat ed national

int er s groups inclu ing dairy farme s, dairy proc s ing in us ry, dairy sup le s, academics an

g ove nments / fo d control authorities

ISO and IDF col a orat e closely on al matt ers of s an ardization r lating t o methods of analy sis

an sampl ng for mi k and mi k prod ct Sinc 2 0 , ISO an IDF jointly publ sh their Int ernational

Stan ards using the log os an r fe enc n mbe s of b th org nizations

A tt ention is drawn t o the p s ibi ity that some of the element of this document ma be the subje t of

p t ent right IDF shal not be held r sp nsible for identifying any or al such p t ent right Detais of

any p t ent right identified d ring the develo ment of the document wi be in the Introd ction an / r

on the ISO ls of p t ent de larations r c ived ( e www.iso.org p t ent )

Any trade name used in this document is information given for the convenienc of use s an does not

cons itut e an en orsement

ISO 1 8 6|IDF 1 5 was pr p r d b the IDF Stan ing C mmitt ee on An ly tic al Meth ds  fr Proc es s in  

Aids a d Indic ators an ISO Te h ical C mmitt eeISO/TC 34, Fo d produc ts , Subcommitt ee SC 5, Mik a d

mik produc ts

The work was car ied out b the IDF ISO Proje t Groupon Alkalne p os ph tase ac ti vit y in che se (P0 6),

of the Stan ing C mmitt ee on An ly tic al Meth ds fr Proc es s in  Aids a d Indic ators , un e the aegis of

it proje t leade Mrs M Nicolas (FR)

This ISO/IDF Int ernational Stan ard canc ls an r plac sISO 1 8 6- 2|IDF 1 5- 2:2 0 , w hich has be n

t ech icaly r vised

ISO 1 8 6|IDF 1 5 consis s of the folowing part , u de the g ene al title Mik a d mik produc ts —

Dete min tio  o alkalne p os ph tas e ac ti vit y:

— Part 1: Flu rimetric meth d fr mik a d mik-b s ed drink s

— Part 2 : Flu rimetric meth d fr che s e

IDF 155-2:2016(E)

Milk and milk pr oducts — Determination ofalkaline

Part 2:

This p rt of ISO 1 8 6|IDF 1 5 spe ifies a fluorimetric method for the det ermination of alkalne

phosphatase (ALP, EC 3.1.3.1) activity in che se

This method is a plca le t o sof che ses, semi-hard an hard che ses pro ided that the mould is only

on the surfac of the che se an not also in the in e part (e.g blue veined che ses) F or larg e hard

che ses, spe if ic con itions of samplng a ply ( e Clause 7)

The ins rument can r ad activities in thesupe natant up t o 7 0 0mi lu it pe ltr (mU/l)

2 Normati ve referenc e

The folowing document , in w hole or in p rt, ar normatively r fe enc d in this document an ar

in ispensa le for it a pl cation F or dat ed r fe enc s, only the edition cit ed a ples F or un at ed

r fe enc s, the lat es edition of the r fe enc d document ( inclu ing any amen ment )a ples

ISO 5 2 -1, Ac c uracy (truenes s  a d  pr c i s ion )  o  meas ur ment  meth ds  a d  r s ults  —  Part  1:  G ene al 

princ iples a d de initio s

ISO 5 2 - 2, Ac c uracy (truenes s a d pr c i s ion ) o meas ur ment meth ds a d r s ults — Part 2 : B as ic meth d 

fr the determin tion o r peata i t y a d r produc ibi t y o a s ta dard meas ur ment meth d

3 Terms and definitions

F or the purposes of this document, the fol owing t erms and def initionsa ply

3.1

alkal ne ph sp atase activity

ALP activity

activity of the alkal ne phosphatase pr sent in the prod ct, det ermined b the spe ified proc d r

Not e 1 to entry: T e alkaline p osp atase activity is e pres ed as mil iu its of enzyme activity per gram of

sample (mU/ )

3.2

unit of alkal ne phosphatase activity

amount of alkal ne phosphatase enzyme that catalyses the trans ormation of 1 µmol of subs rate

pe min te

The alkalne phosphatase activity of the sample is measur d b a contin ous fluorimetric dir ct

kinetic as a A non-fluor sc nt aromatic mono hosphoric est er subs rat e, 2′- 2-benzothiazoly l]-6

hydro y enzothiazole phosphat e, in thepr senc of any alkalne phosphatase de ived from the sample,

un e g oes hydroly sis of it phosphat e radical, prod cing a highly fluor sc nt prod ct Fluorimetric

IDF 155-2:2016(E)

measur ment of alkalne phosphatase (ALP) activity is measur d at 3 °C over a 3 min pe iod w hen

using the Fluoro hos

®1)

This inclu es pr -incub tion of subs rat e an sample, folowed b multiple

kinetic r adings of the r action rat e

NOTE Although this is a 3 min test, the f ir t min te is an eq ilibration period to ensure that the sample is

at 3 °C Me surements of activity are actualy made from the b gin ing of the secon min t e t o the en of the

third min t e (i.e.over a 2 min period)

Use only r ag ent of r co niz d analytical grade, unles othe wise spe ified, an dis i ed or

demine al z d wat er or wat er of eq iv lent purity

subs rate p wde ,

molar mas 58 g rams pe mole

This is a non-fluor sc nt aromatic mono hosphoric est er subs rat e, 2′- 2-benzothiazoly

l]-6′-hydro y benzothiazole phosphat e

This subs rat e r mains s a le for 2 years from the dat e of man factur , pro ided it is st or d in

uno ened b t les at a t empe atur betwe n 2 °Can 8 °C Prot ect a ains lght

5.2 Substrate bufer solution, diethanolamine (DEA ) bufe solution, c(DEA ) = 2,4 mol/l, w ith

pH10,0

The subs rat e bufe solution r mains s a le for 2 years from the dat e of man factur , pro ided it is

st or d in u o ened b t les at a t empe atur betwe n 2 °C an 8 °C Prot ect a ains l ght

5.3 Wor king substrate

Alow the Fluoro hos

®

subs rat e (5.1) an the subs rat e bufe solution (5.2) t o come t o ro m

t empe atur Ad the cont ent of one b t le subs rat e bufe solution (240 ml) (5.2)t o that of one b t le

Fluoro hos

®

subs rat e (14 mg) (5.1) an mix wel b inve sion for 3 min Use ambe glas t o prot ect

a ains l ght

Alow the o tained solution t o s an at ro m t empe atur for at leas 3 min prior t o use

Use the analog -t o-digital (A/D ) t es given in 9.2 t o t es the suita i ty of the r ady t o use working

subs rat e Do not use the working subs rat e if a r ading a o e 1 2 0 F U (fluor sc nc u it ) is

6′-hydr ox benzothiazole] in substrate bufe solution (5.2)

Theworking cal brat or solutions r main s a le for 1 months from man facturing dat e w hen st or d in

uno ened b t les at a t empe atur betwe n 2 °Can 8 °C

1) Fluor op os is a r eg istered trademark T is information is g iven for the con venience of user of this document

an do s not constitute an en or ement by either ISO or IDF of these pr od cts E uivalent pr od cts ma y b used if

they can b show n to le d to the same r esults

2) T e reag ents specified in 5.1 to 5.6 an the ap aratus specified in 6.1 to 6.4 (e cept 6.3.1) ar e a vaila le fr om

Advanced Instruments, Inc T wo Tech olog y Wa y, Norwo d, Mas ach s etts 0 062, USA T e man factur er

ma y chang e packag ing config urations su plied w ith the Fluor op os Test System T e user should r efer to the

man factur er’s instructions for pr eparing r eag ents if difer ent fr om those specified herein Fluorop os an

Fluor oyelow are reg ister ed tr ademarks of Advanced Instruments Inc.T is information is g iven for the con venience

of user of this document an do s not constitute an en or ement by either ISO or IDF of these pr od cts

IDF 155-2:2016(E)

Mix g ently prior t o use t o ensur o timal r sult

5.4.1 Calbr ator solutio A , containing 0 μmol/l of Fluor oyelow

The daiy ins rument control solution r mains s a le for 1 months from man facturing dat e w hen

st or d in u o ened b t les at a t empe atur between 2 °C an 8 °C

Mix g ently prior t o use t o ensur o timal r sult

5.6 Fluor ophos

®

che se e x tractio bufer , diethanolamine (DEA ) bufe , pH 8,0 w ith mag nesium

and Triton X-1 0

The che se extraction bufe r mains s a le for 3 years from man facturing dat e w hen st or d in

uno ened b t lesat a t empe atur betwe n 2 °Can 8°C

5.7 P ositi ve, negati ve and Ph sphacheck-N™ c ontrols

6 A pparatus

Usual la orat ory eq ipment and, in p rticular, the fol owing

6.1 Fiter fluor imeter, with the mos aticaly contr oled cuvette holde , ca a le of o erating at

3 °C ± 1°C an right-ang le o tics, alowing excitation at a wa veleng th of 4 0 nm an emis ion betwe n

5 0 nm an 5 0 nm [e.g Fluorophos

6.3.2 P ositi ve-displac ement or air -displac ement pipet e, of ca acity 0,07 ml

6.4 He tin block, ca a le of maintaining a temperatur e of 3 °C± 1 °C, suita le for holding cuvetes

6.5 Plastic parafin fim (e.g Parafim

®3)

) or othe suitable la or atory-g rade fim

6.6 Vor te x mixer

6.7 Gr inding devic e

6.8 Glas be ker, of ca acity 5 ml (of a prox imately diamete 2 mm an leng th 3 mm) an 1 ml

(of a pr ox imately diamete 2 mm an leng th 3 mm)

3) Par afilm

®

an Ultra turrax

®

ar e examples of a suita le prod ct a vaila le commer cialy T is information is

g iven for the con venience of user of this document an do s not constitute an en or ement by either ISO or IDF of

these pr od cts

IDF 155-2:2016(E)

6.9 High-spe d h mog enizer (e.g Ultra turrax

®3)

) pr ovided with the s em of diamete of

a prox imately 6 mm to 8mm

6.10 One-mar k v lumetric flasks, of ca acity 2 ml

6.11 Centr ifug e, ca a le of c ntrifug ing at 1 0 0 at 4 °C

6.12 Glas test tube, of a pr ox imately diamete 1 mm an leng th 1 cm

6.13 Glas Pasteur pipete, an air-displac ment pipete can also be used

6.14 Wate bath, ca a le of maintaining a temperatur e of 6 °C ± 1°C

A r pr sentative sample should be sent t o the la orat ory It should not ha e be n damag ed or alt er d

d ring transp rt or st orag e

Samplng is not p rt of the method spe ified in this p rt of ISO 1 8 6|IDF 1 5 A r commen ed samplng

method isgiven in ISO 7 7|IDF 5

[1]

Howeve , ISO 7 07|IDF 5 is not suita le for large hard che ses w he e the whey curd mixtur has be n

scalded at t empe atur s a o e 5 °C If the che se is made from raw mik, the ALP a ctivity is not

homogenously dis ributed within these che ses The a ctivity is high in the outer la e ofthe che se w he l,

betwe n 0 cm to 4 cm below, the rin of the rou d side, but ve y low or even u det ecta le in the cor

Samples of larg e hard che ses, the efor , shal be sampled b taking a p rtion of 1 cm, taken at 0,5 cm

below the rind of the rou d side ( e Figur B.1)

In case of doubt r g rding the ty e of che se, betwe n a hard an a semi-hard che se, proc ed t o the

samplng as desc ibed for larg e hard che ses

8 Preparation oftest sample

R emo e the rin or the surfac from the t es sample with a clean k ife E sur that the t es sample

is not contaminat ed with surfac mic oflora d ring it pr p ration Espe ialy for sof che se with

moulded surfac , r mo e al the rin but in a la e as thin as p s ible, so as t o a oid el minating the

fat layer u de the mould surfac ( e Figur B.2) F or larg e hard che ses, proc ed as desc ibed un e

Clause 7 Grin the t es sample b means of a grin ing mi l or othe a pro riat e devic (6.7) an mix

thoroughly K eep the pr p r d sample in an airtight containe

9.1 Verification of instrument perfor mance

9.1.1 General

It is important t o che k ins rument pe formanc for drif , s ra l ght an s a i ity prior t o analysing

t es samples F olow g ood la orat ory practic s an ards w hen o e ating the filt er fluorimet er (6.1)

Q ualty control t es s inclu esthe folowing:

a) The daiy A /D (analog -t o-digital)t es , used t o che k the pro e functioning of the eq ipment

b) The dai y ins rument control t es , using the dai y ins rument control solution (5.5) t o monit or any

ele tronic or o tical drif in the fluorimet er

IDF 155-2:2016(E)

c) The use of ext ernal p sitive, neg tive an normal controls, desc ibed in 9.1.3, is r commen ed for

monit oring daiy ins rument pr cision paramet ers

9.1.2 Dail y instr ument tests

9.1.2.1 When using the Fluor ophos

®

ins rument , pe form the A /D tes daiy befor e testing commenc s

A cc s the A/D t es through the “ SETUP men ” Pr s “ SETUP” key, then sele t men it em “A/D Tes ”

b pr s ing < or > W ith nothing in the cuvett e holde , pr s START Alow the f igur s a pearing on the

displa sc e n t o s a i z The displa should r ad 3 2 ± 4 If the r ading is out ide that rang e, clean

the ex citation an emis ion f ilt ers an r peat he A /D t es

9.1.2.2 Dispense 2,0 ml of the daiy ins rument control solution (5.5) into a la eled cuvete, using the

pipete (6.3.1) Plac the cuvete in the heating block (6.4)set at 3 °C for 2 min Inse t he pr e-warmed

cuvete into the cuvete holde Close the ld When the displa y is s a le, r ecor d the displa yed v lue,

which should be 60 ± 1 If out ide that rang e, use the smal scr ewdrive suppled to slowly turn the

p tentiomete screw on the left-han side of the ins rument clockw ise or anticlockwise, as ne es ary,

u ti the displa y reads 6 2 A llow the n mbe s to eq i bratefor 1 min

9.1.3 C ntrols

Pe form p sitive, neg tive an Phosphache k-N™ controls

)

using a powde ed mik b se, with

phosphatase and pr se v tive (5.7)

The P ospha he k

®

p s eurization controls r main s a le for 1 months from man facturing date

when s or d in uno ened an unr cons ituted bot les at a tempe atur between 2 °C and 8 °C Onc

r cons ituted, the controls ar s a le for 3 da s (7 h) at a tempe atur betw een 2 °C an 8 °C Do

not fr e e

Alow the controls t o come t o ro m t empe atur R econs itut e the Phospha he k

®

p st eurization

controls befor use R emo e the metal an rub e st op e Ad 3,0 ml of deionised wat er at ro m

t empe atur , using the pipett e (6.3.1) R eplac the st op e an mix g ently b inversion for 1 min an

then let tan for 1 min Do not hake the controls or al ow them t o fo m Mix g ently befor each uset o

ensur o timal r sult

Aft er calbrating an u used channel with the neg tive control, analy se the thr e control solutions ( i.e

positive, neg tive an P ospha he k-N™) b ad ing 0,0 5 ml of each control solution t o 2 ml of pr

-warmed subs rat e Pe form the ALP t es

The r ading for the neg tive control shal be < 0 mU/l, the P ospha he k-N™ control shal be betwe n

1 mU/l an 40 mU/l an the p sitive control shal be 5 0 mU/l ± 1 0 mU/l

9.2 Reag ent c ontrols to test the suitabi ity of r ead y to use wor king substrate (5.3)

Dispense 2,0 ml of the wor king subs rat e (5.3) int o a la eled cuvett e, using the pipett e (6.3.1) Plac the

cuvett e in the heating block (6.4) set at 3 °C for 2 min Inse t the pr -w armed cuvett e with the wor king

subs rat e into the cuvette holde Close the ld When the displa is s a le, r cord the displa ed v alue

Fr shly made subs rat e alone in the A /D mode usual y gives a displa r ading of a out 6 0 F U w hich

inc eases over time

Do not use the working subs rat e w hen a displa r ading of a o e 1 2 0 F U is o tained

4) T e contr ols specified an instrument performance check instructions ar e a vaila le from Advanced Instruments

Inc, Two Tech olog y Wa y, Norwo d, MA 0 062, USA T is information is g iven for the con enience of user of this

document an do s not constitute an en or ement by either ISO or IDF of these pr od cts

IDF 155-2:2016(E)