!

! 1!

!!!!!!!!

MILK!QUALITY!AND!SAFETY!TESTING!MANUAL!!!!

AUTHORS:!!

PAUL!EBNER,!PhD!Associate!Professor!Purdue!University!!

College!of!Agriculture!Animal!Sciences!

!MOHAMMAD!ALAM!GHORYAR,!PhD!

Associate!Professor!Herat!University!

Faculty!of!Agriculture!!

SHOAIB!AHMAD!SHAKHES,!DVM,!MVSc.,!Associate!Professor!Herat!University!

Faculty!of!Veterinary!Science!!

AHMAD!ZIA!GHANIZADAH,!MSc.,!Associate!Professor!Herat!University!Faculty!of!Science!!

!!!!

Prepared'for'the'USWDP'Food'Technology'Bridge'Program'at'Herat'University' '

!

! 2!

TABLE!OF!CONTENTS!!

! ! ! ! ! ! ! ! ! ! !!!!!!!!!Page!!INTRODUCTION! ! ! ! ! ! ! ! ! X!!MILK!QUALITY!TESTS! ! ! ! ! ! ! X!!! 1.!!General!Physical!Tests! !

2.!!Clot!of!Boiling!(COB)!Test! ! ! ! ! ! X!! 3.!!Alcohol!Test! ! ! ! ! ! ! ! X!! 4.!!Alcohol/Alizarin!(AA)!Test! ! ! ! ! ! X!! 5.!!Acidity!Test! ! ! ! ! ! ! ! X!! 6.!!Resazurin!Test! ! ! ! ! ! ! ! X!! 7.!!Protein!Content! ! ! ! ! ! ! ! X!! 8.!!Fat!Content! ! ! ! ! ! ! ! X!! 9.!!Milk!Analysis!Equipment! ! ! ! ! ! X!!MICROBIAL!TESTS! ! ! ! ! ! ! ! X!!

1.!!Total!Plate!Count!(TPC)! ! ! ! ! ! X!! 2.!!Total!Coliform!Test! ! ! ! ! ! ! X!!TESTS!FOR!ADULTERATION! ! ! ! ! ! X!!! 1.!!Using!a!Lactometer! ! ! ! ! ! ! X!

2.!!General!Test!for!Inhibitors! ! ! ! ! ! X!3.!!Soap!Test! ! ! ! ! ! ! ! ! X!4.!!Starch!Test! ! ! ! ! ! ! ! X!5.!!Sugar!Test! ! ! ! ! ! ! ! X!6.!!Acid`based!Preservatives! ! ! ! ! ! X!7.!!Sodium!(bi`)!Carbonate! ! ! ! ! ! X!

! 8.!!Formaldehyde!Test! ! ! ! ! ! ! X!! 9.!!Antibiotic!Residues!! ! ! ! ! !POWDERED!MILK! ! ! ! ! ! ! ! X!!! Processing!Technology! ! ! ! ! ! ! X!! Sampling!Considerations! ! ! ! ! ! ! X!! General!Physical!Tests! ! ! ! ! ! ! X!! Sediment!Test! ! ! ! ! ! ! ! X!! Solubility!Test! ! ! ! ! ! ! ! X!!REFERENCES!! ! ! ! ! ! ! ! X!! !

!

! 3!

INTRODUCTION!!

!There!are!many!factors!that!affect!milk!quality!and!safety.!!At!the!same!time,!there!are!numerous!quality!and!safety!tests!that!can!easily!be!done!at!the!farm,!in!the!market,!or!in!the!lab!based!on!both!microbiology!and!chemistry.!!!!Many!milk!quality!and!safety!issues!are!associated!with!microbial!growth!in!the!milking!environment!and!the!milk!itself.!!Most!of!these!microbes!are!bacteria,!but!milk!can!also!become!contaminated!with!molds!or!yeast.!!These!microbes!can!contaminate!milk!at!almost!any!point!of!the!production.!!The!types!and!concentrations!of!microbes!found!in!milk!are!significantly!influenced!by:!

!1)!!animal!husbandry!and!care;!!2)!!animal!health;!!3)!!milk!handling!and!storage;!and!4)!!processing.!!!!

Therefore,!assuring!milk!quality!is!a!team!effort!requiring!animal!scientists,!veterinarians,!chemists,!microbiologists,!and!food!technicians.!!!!

Collection!!Microbes!are!regularly!introduced!to!milk!during!milk!collection.!!Therefore,!it!is!important!to!recognize!that!milk!quality!and!safety!starts!at!the!farm.!!The!biggest!contributors!to!contamination!of!milk!during!collection!are!animal!health!and!unsanitary!milking!conditions.!!1.!!Animal!Health.!!Milking!animals!are!often!affected!by!mastitis.!!Mastitis!is!inflammation!of!the!udder.!!This!inflammation!is!most!often!caused!by!bacterial!infections.!!When!bacterial!concentrations!are!high,!the!bacteria!can!easily!be!transferred!to!milk!during!the!milking!process.!!Mastitis!can!be!prevented!or!controlled!by!proper!sanitation!and,!in!severe!cases,!the!use!of!antibiotics.!!It!is!important!to!note!that!milk!collected!from!cows!that!are!being!treated!with!antibiotics!should!be!discarded!as!the!antibiotics!and!antibiotic!residues!can!easily!be!transferred!to!milk.!!Milk!containing!antibiotics!or!antibiotic!residues!is!considered!adulterated.!!!!2.!!Sanitation.!!Unsanitary!milking!conditions!regularly!lead!to!contamination!of!milk.!!The!milking!area!in!general!should!be!kept!as!clean!as!possible.!!Accumulation!of!feces/urine!

“Microbes”'is'a'general'term'for'microorganisms'including'viruses,'bacteria,'yeasts,'molds,'and'protozoa.'''

Mastitis:''Inflammation'of'the'udder'and'udder'tissue.''In'some'cases,'the'udder'and'tissue'can'be'visibly'damaged.''Other'cases'are'“subclinical”'in'that'the'udder'is'infected,'but'may'not'show'any'symptoms.''Mastitis'is'often'transferred'from'cow'to'cow'through'milk'equipment,'milkers'(unwashed'hands),'housing,'and'any'bedding.'''

!

! 4!

greatly!increases!the!likelihood!of!microbial!contamination!of!milk.!!Similarly,!milk!collectors!can!easily!introduce!bacteria!to!both!the!milking!animal!and!the!collected!milk!through!unwashed!hands!and!clothing.!!!!!Improperly!or!insufficiently!cleaned!milk!collection!containers!often!lead!to!contamination!of!milk.!!Milk!is!high!in!nutrients!and!is!actually!used!often!to!grow!bacteria!in!the!lab.!!As!such,!milk!left!in!a!collection!container!(even!trace!amounts!of!milk)!will!support!the!growth!of!many!different!types!of!bacteria.!!If!new!milk!is!added!to!the!contaminated!container,!it!too!becomes!contaminated.!!Therefore,!it!is!important!to!use!containers!that!can!be!easily!cleaned.!!!!At!the!same!time,!if!incorrect!chemicals!are!used!to!clean!milk!collection!containers!or!the!containers!are!not!properly!rinsed!after!cleaning,!new!milk!can!become!contaminated!with!harmful!chemicals.!!These!chemicals!can!be!harmful!to!milk!consumers!and!can!also!make!milk!unusable!for!cheese,!yoghurt,!or!other!processed!dairy!products.!!

Holding!or!Transportation!!It!should!always!be!assumed!that!fresh!milk!contains!some!level!of!bacterial!contamination.!!Therefore,!controlling!or!limiting!microbial!growth!in!milk!is!key!to!ensuring!milk!quality!and!safety.!!Milk!is!considered!a!“TCS”!food!or!a!food!where!safety!and!quality!is!dependent!on!controlling!time!and!temperature.!!Lower!temperatures!(e.g.,!refrigeration)!limit!microbial!growth.!!Keeping!fresh!milk!under!unrefrigerated!conditions!(e.g.,!room!temperature!or!higher)!for!long!periods!allows!bacteria!to!rapidly!grow!to!very!high!concentrations.!In!many!countries,!fresh!milk!must!be!refrigerated!within!45!minutes!of!collection.!!In!other!countries!this!is!not!feasible!and!alternative!measures!to!limit!microbial!growth!must!be!used.!!!!

Impact!of!Contamination!!

Milk!with!high!levels!of!bacterial!contamination!is!problematic!for!several!reasons.!!Foremost!is!safety.!!Dairy!environments!(e.g.,!animals,!feces,!barns,!milking!equipment,!etc.)!are!often!contaminated!with!common!bacterial!pathogens!such!as!Salmonella,!E.*coli,!and!Campylobacter.!!These!bacteria!can!cause!diarrhea,!renal!failure,!and,!in!severe!cases,!death.!!They!are!especially!serious!for!infants!and!children!and!the!elderly.!!The!second!issue!is!quality.!!Milk!with!higher!levels!of!contamination!spoils!very!quickly.!!When!bacteria!are!allowed!to!grow!to!high!levels,!milk!can!become!very!acidic!producing!sour!flavors.!!With!some!time,!sour!milk!will!coagulate!and,!in!general,!is!not!usable.!!This!can!significantly!reduce!“shelfTlife”!and!profitability.!!Finally,!high!levels!of!contamination!can!impact!further!processing!of!milk!into!different!products!such!as!cheese,!cream,!ice!cream,!and!yoghurt!as!we!will!see!later.!!

ShelfMlife:''how'long'a'product'can'remain'on'a'store'shelf'before'it'becomes'spoiled,'rotten,'or'otherwise'unusable.''The'shelfMlife'of'high'quality,'pasteurized,'and'refrigerated'milk'is'generally'around'7M10'days.'

!

! 5!

Pasteurization!significantly!increases!the!quality!and!safety!of!milk.!!Pasteurization!is!simply!the!use!of!heat!to!kill!microbes.!!For!milk,!the!minimum!pasteurization!standards!requite!heating!milk!to!63C!(143F)!for!30!minutes.!!It!is!important!to!note!that!pasteurization!is!designed!to!kill!pathogenic!bacteria,!but!not!all!bacteria.!!As!such,!milk!pasteurized!under!standard!conditions!should!not!be!considered!sterile!and!milk!that!has!high!amounts!of!microbial!growth!before!pasteurization!will!still!spoil!much!more!quickly!after!pasteurization!than!milk!with!low!amounts!of!microbial!growth.!!!!!!!!

Assessing!Milk!Quality!!There!are!numerous!easy!and!rapid!tests!to!determine!milk!quality.!!These!tests!rely!on!some!very!well!defined!properties!of!milk!including!its!basic!composition!and!the!presence!or!absence!of!different!compounds.!!Milk!Composition.!!Milk!is!approximately!87%!water.!!The!remaining!constituents!are!referred!to!as!“solids”!and!are!what!separates!many!of!the!physical!properties!of!milk!from!other!liquids.!!“Total!solids”!(TS)!refers!to!all!nonTwater!components!of!milk!including!fats,!proteins,!sugars,!vitamins,!and!minerals.!!Fats!make!up!about!4%!of!fresh!milk,!but!can!vary!slightly!depending!on!animal!species!and!breed.!!The!remaining!solids!are!often!called!“solidsTnon!fat”!(SNF).!!Of!the!SNF!portion,!the!majority!is!protein,!which!makes!up!around!5%!of!milk,!and!sugars,!which!make!up!around!3.5%!of!milk.!!The!remainder!is!vitamins!and!minerals!(0.5%).!!!!The!total!solids!give!milk!distinct!qualities!from!other!liquids.!!One!of!the!first!milk!quality!tests!is!to!measure!its!density.!!The!proteins,!sugars,!vitamins,!and!minerals!essentially!add!weight!to!milk.!!Because!the!composition!of!milk!varies!only!slightly!from!animal!to!animal,!the!density!of!fresh!milk!should!always!be!within!an!established!range!(see!table!below).!!When!water!is!added!to!milk,!or!fat!is!removed,!or!adulterants!are!added,!the!density!of!the!milk!often!changes.!Fortunately,!the!density!of!milk!is!easily!measured!with!a!lactometer!and!that!process!is!described!later!in!the!section!entitled!“Using!a!Lactometer”.!!!!Microbial!Contamination.!!There!are!numerous!ways!to!measure!microbial!growth!and!concentrations!in!milk.!!!Tests!like!the!Total!Plate!Count!Test!or!Coliform!Test!(each!are!described!later)!are!standard!microbiology!tests!aimed!at!measuring!the!amount!or!type!of!bacteria!present!in!milk.!!!!!When!bacteria!grow,!however,!they!often!produce!high!amounts!of!acid.!!In!milk,!high!amounts!of!acid!produce!very!sour!flavors!and!will!significantly!reduce!the!shelfTlife!and!usability!of!the!milk.!!Many!milk!quality!tests!are!designed!to!measure!contamination!based!on!the!pH!or!acidity!of!milk.!!There!are!several!tests!described!below!that!can!give!a!preliminary!indication!that!the!acidity!of!the!milk!is!too!high!(qualitative!test)!and!other!test!that!can!more!closely!measure!the!percentage!or!amount!of!acidity!(quantitative!tests).!!!Abnormal!milk/Adulteration.!!Milk!is!often!adulterated!for!different!reasons.!!Sometimes,!colostrum!can!be!combined!with!collected!milk.!!Colostrum!is!the!milk!first!produced!by!the!mother!immediately!after!giving!birth.!!It!is!generally!high!in!antibodies!and!other!

!

! 6!

proteins!that!can!impact!taste,!color,!usability,!and!quality!of!milk.!!More!often,!however,!milk!may!be!intentionally!adulterated.!!“Adulterants”!are!additives!that!may!mask!or!hide!problems!with!the!milk,!make!milk!appear!of!higher!quality!than!it!really!is,!change!its!color,!or!increase!its!volume.!!For!example,!buffers!such!as!sodium!carbonate!can!be!added!to!old!milk!or!milk!with!high!bacterial!growth!to!prevent!acidity!and!give!the!appearance!that!the!milk!fresh.!!Water!is!sometimes!added!to!milk!to!increase!volume!and,!in!turn,!payment.!!Detecting!water!in!milk!is!rather!easy,!however,!as!diluted!milk!is!less!dense.!!Therefore,!additional!adulterants!such!as!sugar,!salt,!other!solids!are!sometimes!added!to!milk!diluted!with!water!to!increase!its!density.!!!In!other!cases,!chemicals!high!in!nitrogen!may!be!added!to!milk!to!give!the!appearance!that!the!milk!is!higher!in!protein!or!other!qualities.!!This!is!seen!in!systems!where!milk!producers!are!given!higher!prices!for!their!milk!based!on!quality.!!Other!chemicals!may!be!added!to!milk!to!prevent!growth!of!microbes.!!Some!of!the!most!common!chemical!preservatives,!such!as!formaldehyde,!are!extremely!toxic.!These!additions!are!always!violations!and!there!are!numerous!cases!where!people!were!poisoned!when!the!adulterant!was!a!toxic!substance.!!!!Table!1.!!General!Properties!of!Milk!!Property! ! ! !!!!!!!!!!!!!Whole!Milk,!Non`homogenized!!pH! ! ! ! ! 6.5!–!6.7!Acidity! ! ! ! 0.16!–!0.18%!Density! ! ! ! 1.030!–!1.035!Kg/L!Acidity! ! ! ! 0.16!–!0.18%!Freezing!Point! ! ! T0.512!to!T0.550!C!Water! ! ! ! ! 87%!(whole!milk)!Total!Solids! ! ! ! 13%!! Fat! ! ! ! 4%!(whole!milk,!nonThomogenized)!! NonTfat!solids!! ! 9%!! ! Protein! ! 3.5%!! ! Sugars!! ! 5.0%!! ! Vitamins/Mineral! <0.5%!TPC! ! ! ! ! <100,000!CFU/mL!(raw!milk)*!! ! ! ! ! <200!CFU/mL!(pasteurized!milk)*!Coliforms! ! ! ! <100!CFU/mL!(raw!milk)*!! ! ! ! ! Undetectable!(pasteurized!milk)*!!*these*standards*differ*from*country*to*country.**' *

Adulteration:''The'addition'of'any'substance'or'compound'(adulterant)'to'milk,'intentionally'or'accidentally,'that'reduces'the'quality'and'safety'of'milk.'''

!

! 7!

GENERAL!MILK!QUALITY!TESTS!!

1.!!Physical!Appearance!!

To!conduct!a!preliminary!examination!of!a!milk!sample,!first!look!at!the!container!and!assess!its!overall!cleanliness.!!Examine!seals!or!lids!to!see!if!they!are!working!properly.!!!Next,!open!the!container!and!immediately!smell!the!milk.!!The!milk!should!not!contain!any!off!odors.!!Off!odors!can!be!produced!by!the!animal’s!diet,!chemical!adulteration,!natural!hormones!in!the!milk,!the!milk!collection!environment,!and,!most!commonly,!bacterial!growth.!!Milk!with!high!bacterial!counts!will!have!a!characteristic!sour!odor.!!!Fresh!milk!should!appear!white.!Chemical!adulteration!and!colostrum!can!produce!off!colors.!!If!a!small!amount!of!milk!is!allowed!to!run!down!a!polished!surface,!it!should!leave!a!streak!of!milk.!!Finally,!the!milk!should!be!free!of!clumping.!!Milk!that!is!spoiled!or!otherwise!unusable!due!to!microbial!growth!is!often!easy!to!identify!based!on!simply!on!appearance!and!odor.!!The!majority!of!milk!quality!parameters,!however,!require!some!more!advanced!testing!for!assessment,!many!of!which!are!described!below.!!!!

!! !

!

! 8!

2.!!Clot!on!Boiling!Test!(COB)!!The!COB!test!is!an!old,!but!very!easy!test.!The!test!is!designed!to!determine!the!overall!quality!of!the!milk.!!If!used,!it!should!be!considered!a!preliminary!test,!but!it!can!also!be!easily!done!in!the!field!or!on!the!farm.!!The!COB!test!simply!boils!milk!and!looks!at!physical!characteristics!once!the!milk!cools.!!When!high!quality!milk!is!boiled,!it!will!remain!a!fluid!with!a!general!milk!appearance!once!it!is!cooled.!!Milk!that!is!acidic!(an!indication!of!bacterial!growth)!or!milk!that!contains!colostrum!or!other!adulterations!often!cannot!withstand!boiling!and!will!coagulate!and!form!lumps!or!flakes!once!it!is!cooled.!!Materials:!Milk!sample!Spoon!Flame!!Procedure:!1.!!Start!flame!away!from!all!flammable!objects.!2.!!Place!milk!in!spoon!using!a!pipette!or!similar!device.!3.!!Hold!milk/spoon!over!flame!until!milk!boils.!4.!!Allow!milk!to!cool.!5.!!Check!for!coagulation.!!

!!!!!!!!!!!!

1! 2! 3!

1.**Add*a*small*amount*of*milk*sample*to*the*spoon*using*a*pipette*or*similar*device.*

2.**Be*careful*not*to*overfill*the*spoon,*as*the*milk*will*easily*boil*over*the*sides*once*it*is*heated.*

3.**Hold*the*spoon/milk*sample*over*a*flame*until*milk*thoroughly*boils.*

!

! 9!

Reading!Results:!!

!!

!!Notes!on!the!COB!test:!

!!!!

!! !

1! 2! 3!

1.**Milk*with*little*to*no*microbial*contamination/*growth.**This*milk*has*a*neutral*pH*and*withstands*boiling*and*has*a*normal*milk*appearance*once*it*cools.*'

2.**Milk*with*lower*pH*due*to*bacterial*growth.**Such*milk*cannot*withstand*boiling*and*shows*coagulation*around*the*edges.***

3.**Milk*with*very*low*pH*due*to*bacterial*growth.**Milk*with*a*very*low*pH*will*show*heavy*coagulation*and*flakes*when*boiled.*

!

! 10!

3.!!Ethanol/Alcohol!Test!!The!ethanol/alcohol!test!is!a!very!rapid!test!to!determine!overall!quality!of!milk.!!It!relies!on!how!milk!reacts!with!ethanol.!!High!quality!or!otherwise!normal!milk!will!not!react!with!ethanol.!!Milk!that!is!high!in!acidity!(low!pH),!contains!colostrum,!or!contains!salt!concentrates!(indication!of!mastitis)!will!often!coagulate!or!form!flakes!when!exposed!to!ethanol.!!!Materials:!Milk!Sample!68%!Ethanol!(often!abbreviated!“EtOH”)!Mixing!Container!!Procedure:!1.!!Combine!equal!parts!68%!ethanol!and!milk!sample!(example:!!10!mL!of!milk!sample!+!10!mL!of!68%!ethanol).!

2.!!Mix!thoroughly.!3.!!Incubate!at!room!temperature!for!10!minutes.!4.!!Check!samples!for!coagulation.!!Reading!Results:!

!!

Notes!on!Alcohol!Test:!!!!!!!!!! !

1.**The*samples*on*the*right*are*milk*samples*after*the*addition*of*ethanol*and*a*10*minute*incubation*at*room*temperature.**Sample*A*is*fresh*milk*with*little*to*no*microbial*growth.**It*does*not*react*with*alcohol*and*retains*a*normal*milk*appearance.**Sample*B*has*a*low*pH*(high*acid)*due*to*microbial*growth.**This*milk*reacts*with*alcohol*and*heavy*coagulation*and*flaking*can*be*seen*on*the*sides*of*the*flask.*

*

A! B!1!

!

! 11!

4.!!Alcohol/Alizarin!(AA)!Test!!The!AA!test!is!very!similar!to!the!basic!alcohol/ethanol!test.!!The!only!difference!is!the!addition!of!alizarin!to!the!alcohol.!!Alizarin!is!a!color!indicator!that!changes!based!on!the!pH!of!the!milk.!!The!AA!test!is!an!example!of!a!semiTquantitative!test!as!it!can!give!you!an!estimate!of!the!level!of!acidity!of!the!milk.!!The!test!is!conducted!similarly!to!the!alcohol/ethanol!test,!but!the!analysis!takes!into!account!color!changes.!!!!Materials:!Milk!Sample!Alizarin!Solution!(4.0g!Alizarin!dissolved!in!1L!61%!ethanol)!Mixing!Container!!Procedure:!1.!!Combine!equal!parts!alizarin!solution!and!milk!sample!(example:!!10!mL!of!milk!sample!+!10!mL!of!alizarin!solution).!

2.!!Mix!thoroughly.!3.!!Incubate!at!room!temperature!for!10!minutes.!4.!!Check!samples!for!coagulation!and!record!color.!!Reading!Results:!The!normal!pH!of!fresh!milk!is!around!6.6!–!6.7.!!High!levels!of!microbial!growth!will!produce!acid,!which!lowers!the!pH!of!the!milk.!!An!estimation!of!the!acidity!of!the!milk!samples!can!be!made!using!the!table!below:!!Measurement!!!Normal! ! Slightly!Acidic! Acidic!! Alkaline!!pH! ! !!!!6.6!–!6.7! ! 6.4!–!6.6! ! 6.3!or!lower! 6.8!or!higher!Color! ! !!!!Red/Brown!! Yellow/Brown! Yellow!! Pink/Purple!Physical! !!!!No!coagulation! No!coagulation! Coagulation!! No!coagulaT!Characteristics! ! ! ! ! ! ! ! tion!*!!*may!have!flakes.!! !

!

! 12!

!!!!!!!!!!!!!

!Notes!on!the!Alcohol/Alizarin!test:!

!!!!!!!!!

1.**The*picture*above*shows*several*different*milk*samples*combined*with*alizarin*solution*after*a*10*minute*incubation.**Sample*A*is*fresh*milk*with*little*to*no*bacterial*growth.**Once*alizarin*solution*is*added,*there*is*no*color*change*and*it*remains*a*red/brown*color.**There*is*also*no*coagulation.**The*samples*increase*in*acidity*from*B*N*E*due*to*higher*levels*of*bacterial*growth.**With*higher*acidity,*the*samples*increase*in*coagulation*and*become*more*yellow.***

A! B! C! D! E!

!

! 13!

5.!!Acidity!Test!!The!acidity!test!is!only!a!little!more!involved!than!the!previous!alcoholTbased!tests,!but!will!give!you!more!quantitative!information.!!The!test!is!used!to!determine!the!percentage!of!acidity.!!Like!the!Alizarin!test,!the!acidity!test!uses!a!color!indicator!(phenophtalein).!!The!percentage!acidity!for!normal!quality!milk!is!around!0.16%!to!0.18%.!!!!Materials:!Milk!Sample!Flask!for!Mixing!10!mL!Pipette!Burette!(0.1!mL!graduations)!Mixing!Utensil!(e.g.,!glass!rod)!Phenophtalein!Solution!(0.5%!phenophtalein!in!50%!ethanol)!Sodium!Hydroxide!(NaOH;!base,!0.1M)!!Procedure:!1.!!Add!9!mL!of!the!milk!sample!to!a!flask.!2.!!Add!1!mL!of!the!phenophtalein!solution!to!the!milk!sample.!3.!!Slowly!and!gradually,!add!sodium!hydroxide!from!the!burette!to!the!milk!sample.!4.!!Stop!the!assay!when!the!milk!sample!reaches!a!pink!color.!!Record!the!amount!of!sodium!hydroxide!needed!to!obtain!the!pink!color.!!!

!Using!a!Burette:!

!!!!!!!!!!!!

!!!!

1.**Fill*the*bottom*burette*container*with*0.1M*NaOH.**Squeeze*the*container*to*push*liquid*into*the*burette.***

2.*Fill*the*burette*until*the*bottom*of*the*meniscus*is*at*zero.***

3.**Open*the*valve*slowly*to*adjust*the*flow*rate*of*0.1M*NaOH.***

Closed!

Open!

1! 2! 3!

!

! 14!

!Recording!Results:!!Add!0.1M!NaOH!to!the!milk!sample!until!it!reaches!the!color!of!the!picture!to!the!right.!!Record!the!volume!(mL)!of!sodium!hydroxide!needed!to!develop!the!pink!color.!!Divide!this!number!by!10!to!get!the!percentage!acid!of!the!milk!sample.!!Example:'!1.8!mL!of!NaOH!were!required!to!change!the!milk!sample!from!normal!milk!color!to!pink.!!Therefore,!the!percentage!acidity!is!1.8!/!10!=!0.18%.!!This!would!be!a!normal!acidity!percentage!for!fresh!milk!without!much!contamination!or!microbial!growth.!!Milk!that!is!acidic!will!require!higher!volumes!of!NaOH!to!become!pink!and!will!therefore!have!a!higher!acidity!percentage.!!!!

!!!!Notes!on!the!Acidity!Test:! !

1.**The*addition*of*sodium*hydroxide*is*stopped*when*milk*reaches*a*pink*color*similar*to*the*sample*here.**

1!

!

! 15!

6.!!Resazurin!Test!!The!Resazurin!Test!is!a!common!test!to!determine!overall!bacterial!growth!in!a!milk!sample.!!Like!the!alizarin!and!acidity!tests,!it!uses!a!color!indicator,!which!allows!a!qualitative!assessment!of!milk!quality.!!The!test!requires!an!incubator!where!milk!samples!can!be!incubated!at!37C.!!The!test!is!somewhat!rapid!(1!hr),!but!can!be!extended!for!up!to!three!hours.!!!!Materials:!Milk!Sample!Sterile!Test!Tubes!with!Stoppers!Resazurin!Solution!(~10!g!resazurin!dissolved!in!200!mL!distilled/sterile!water)!!Procedure:!!1.!!Add!1!mL!of!resazurin!solution!to!the!test!tube.!2.!!Add!9!mL!of!milk!sample!to!the!test!tube!containing!resazurin!solution.!3.!!Place!stopper!on!test!tube.!4.!!Place!tube!in!37C!incubator.!5.!!Once!sample!reaches!approximately!37C,!invert!tube!to!mix!sample.!6.!!Incubate!1!hr!at!37C.!7.!!Observe!and!record!results!after!1!hr.!!Recording!Results:!Color! ! ! ! Quality!Blue!(no!color!change)! Excellent!Blue!to!purple! ! Good!Purple!to!pink! ! Fair!Pink!to!whitish!pink! ! Poor!White! ! ! ! Bad!!

!!!!

1.**The*picture*to*the*left*shows*several*milk*samples*after*a*resazurin*test.**Sample*A*is*fresh*milk*with*little*to*no*bacterial*growth.**After*the*resazurin*solution*is*combined*with*the*milk*sample*and*incubated,*good*quality*milk*will*remain*blue*with*no*color*change.**Samples*B*N*E*increase*in*acidity*from*left*to*right*due*to*higher*amounts*of*bacterial*growth.**The*color*of*the*samples*changes*as*well,*becoming*light*purple*to*pink.**Sample*E*is*extremely*acidic*and*shows*heavy*coagulation*along*with*a*color*change.*

A! B! C! D! E!1!

!

! 16!

!Notes!on!the!Reszurin!Test:!

! !

!

! 17!

7.!!Protein!Content!Test!(Formaldehyde!Titration)!!This!test!will!give!an!estimate!of!the!percentage!protein!in!a!milk!sample.!!The!assay!uses!formaldehyde,!which!reacts!with!amino!acids!of!proteins.!!When!formaldehyde!is!mixed!with!protein,!the!solution!becomes!acidic.!!The!protein!percentage!is!estimated!by!measuring!the!amount!of!acid!produced!upon!the!addition!of!formaldehyde.!!This!assay!is!conducted!in!the!same!way!as!the!acidity!test!and!uses!the!same!equipment!as!well!as!the!same!color!indicator.!!The!pictures!included!in!the!section!on!the!Acidity!Test!can!be!used!as!instructionals.!!!!Materials:!Flask!(>50mL)!Burette!(0.1!mL!graduations)!10!mL!Pipette!Stirring!Rod!Potassium!Oxalate!(saturated!aqueous)!

“saturated”*means*the*highest*amount*of*the*compound*that*will*dissolve*in*water.**For*potassium*oxalate,*it*is*approximately*35g*in*1L*of*water.***

Phenophtalein!Solution!(0.5%!phenophtalein!in!50%!ethanol)!40%!Formalin!Solution!0.1!M!Sodium!Hydroxide!(NaOH)!!Procedure:!1.!!Add!10!mL!of!milk!sample!to!flask.!2.!!Add!0.4!mL!of!potassium!oxalate!and!0.5!mL!of!phenophtalein!solution!to!the!milk!sample.!

3.!!Allow!sample!to!stand!for!2!min!and!add!sodium!hydroxide!(with!burette)!until!the!sample!becomes!pink!(see!“Acidity!Test”!for!pink!color!sample).!

4.!!Add!2!mL!of!formalin!to!return!the!sample!to!normal!color.!5.!!Continue!to!add!sodium!hydroxide!until!sample!becomes!pink!again.!6.!!Record!the!amount!of!0.1M!NaOH!needed!to!turn!the!milk!sample!pink!AFTER!the!addition!of!formaldehyde.!!!

!Recording!Results:!The!percentage!protein!is!the!number!of!mL!of!sodium!hydroxide!added!after!the!addition!of!formaldehyde!multiplied!by!1.74.!!!!Example:'2.1!mL!of!NaOH!were!required!to!make!the!milk!sample!turn!pink!AFTER!formaldehyde!was!added.!!Therefore,!the!percentage!protein!of!the!milk!sample!is!2.1!X!1.74!=!3.65%.!!The!protein!of!milk!should!be!approximately!3.6T4.0%.!!!Pasteurized!milk!often!has!a!lower!protein!percentage!(~3.6%).!!Sheep!milk!is!in!general!is!often!higher!in!protein!(~4.0%).!!!While!the!percentage!of!protein!can!differ!based!on!animal!breed!and!diet,!milk!samples!that!have!protein!percentages!much!lower!or!much!higher!than!these!numbers!may!be!adulterated.!!!!

!

! 18!

Notes!on!the!Protein!Percentage!Assay:! !

!

! 19!

8.!!Milkfat!Test!!There!are!several!ways!to!measure!the!percentage!of!fat!in!a!milk!sample.!!Most!of!these!tests!use!sulfuric!acid!in!some!manner.!!This!is!due!to!all!components!of!milk!being!soluble!in!sulfuric!acid!except!fat.!!Therefore,!adding!sulfuric!acid!will!separate!the!fat!content!from!rest!of!the!milk.!!The!test!described!below!likely!requires!the!least!amount!of!reagents!and!equipment.!!Materials:!Milk!Sample!90%!Sulfuric!Acid!Graduated!container!that!can!be!centrifuged!at!low!speeds!!Procedure:!1.!!Combine!equal!parts!sulfuric!acid!and!milk!sample.!!E.g.,!20mL!sulfuric!acid!+!20mL!milk!sample.!

2.!!Thoroughly!mix!sample.!3.!!Centrifuge!at!low!speed!(e.g.,!1000rpm)!for!1!min.!!Fat!has!less!mass!than!other!milk!constituents!(including!water),!so!it!will!rise!to!the!top.!

4.!!Measure!fat!fraction.!!Recording!Results:!The!percentage!of!fat!is!calculated!by!dividing!the!fat!fraction!by!the!amount!of!milk!in!the!sample.!!For!example,!if!the!fat!fraction!is!0.7mL!then!the!fat!percentage!would!be!0.7mL!/!20mL!=!0.035!or!3.5%.!!!!Notes!on!the!Milkfat!Test:!! !

!

! 20!

9.!!Milk!Analysis!Machines!!Different!types!of!equipment!and!machines!are!available!for!milk!analysis.!!Such!machines!can!estimate!fat%,!protein%,!and!lactose%.!!Most!will!also!estimate!freezing!point!or!density!as!an!indication!of!whether!water!has!been!added!to!the!milk.!!Milk!analyzers!usually!use!one!of!two!types!of!technology:!!ultrasound!or!infrared.!!Ultrasound!analyzers!have!the!benefit!of!often!being!less!expensive,!while!infrared!analyzers!are!generally!more!accurate.!!!

!!!!!!!!!!!!!!!!!!!!! !

1.**Example*of*a*lowNcost,*ultrasound*milk*analyzer.***

2.**Milk*is*drawn*into*the*analyzer*from*one*sample.**The*analyzer*also*contains*a*pH*probe*to*simultaneously*measure*acidity*from*a*second*sample*(in*researcher’s*hand).*

!

! 21!

MICROBIAL!TESTS!!

1.!!Total!Plate!Count!(TPC)!!All!fresh!milk!will!contain!bacteria.!!TPC!tests!measure!overall!bacterial!levels.!!They!measure!total!bacteria,!but!do!not!distinguish!between!nonTharmful!bacteria!and!harmful!or!pathogenic!bacteria.!!There!are!other!tests!that!are!used!to!detect!specific!pathogens!such!as!Salmonella,!E.*coli,!or!Campylobacter.!!!Higher!bacterial!levels!can!significantly!reduce!the!shelfTlife!of!milk!and!usability.!!Pasteurization!will!destroy!many!of!these!bacteria,!but!if!levels!are!too!high!or!milk!is!not!refrigerated!for!long!periods!prior!to!pasteurization,!high!bacterial!levels!will!produce!acid!and!quickly!spoil!the!milk.!!!There!are!different!ways!to!conduct!a!TPC!assay.!!Described!below!are!the!pourTplate!method!and!the!spreadTplate!method.!!They!are!both!very!similar!with!the!pourTplate!generally!being!more!accepted,!but!the!spreadTplate!method!being!sufficient.!!Additionally,!the!terms!APC!(aerobic!plate!count),!SPC!(standard!plate!count),!and!TPC!are!often!used!interchangeably,!but!TPC!is!probably!used!most!often.'!!!Materials:!Medium!(most*simple*media*such*as*LB,*nutrient,*etc.*are*acceptable)!! Enzymatic!digestion!of!casein!5.0!g!

Yeast!extract!2.5!g!Glucose,!anhydrous!1.0!g!Agar!17g!Bring!up!to!1!L!with!distilled!water!Autoclave!or!otherwise!sterilize!

Ringer’s!Solution!(other*isotonic*solutions*such*as*PBS*can*also*be*used.**Ringer’s*solution*is*isotonic*relative*to*most*animal*fluids)!

! NaCl!6.5g!!KCl!0.42g!!!CaCl2!0.25g!Calcium!bicarbonate!0.2g!!Bring!up!to!1!L!

Petri!Dishes!Test!Tubes!Pipettes!Cell!Spreader!Ethanol!Incubator!(37C)!!

! !

!

! 22!

Pour`plate!TPC!!Sample'Preparation:'**if*possible,*it’s*best*to*do*all*of*these*tests*in*duplicate.**Taking*an*average*of*your*results*will*give*you*a*more*accurate*estimation*of*the*bacterial*concentration*1.!!Add!9mL!of!Ringer’s!solution!to!7!individual!test!tubes.!!Label!the!test!tubes!T1,!T2,!T3….!2.!!Add!1mL!of!milk!sample!to!the!tube!labeled!“T1”!and!mix!thoroughly.!3.!!Take!1mL!of!the!“T1”!mix!sample!and!add!it!to!the!tube!labeled!“T2”.!4.!!Repeat!for!the!remaining!tubes.!

!'''''''''''

'Plating:'1.!!Add!1mL!of!the!undiluted!milk!sample!to!an!empty,!sterile!petri!dish.!2.!!Add!9mL!of!medium!(cooled!to!below!45C)!to!the!petri!dish!and!mix!gently,!but!thoroughly.!!Label!this!plate!“1”!

3.!!Repeat!for!all!test!tubes!labeling!each!plate!with!the!corresponding!test!tube!number!(T1,!T2,!T3,!etc.).!

4.!!Incubate!at!37C!for!24hr.!!!!!!

!!!!!!!!!!!

!

1.**Take*care*to*thoroughly*mix*each*tube*before*beginning*the*following*dilution.*

2.**The*final*dilution*should*have*1mL*more*than*the*other*dilutions*as*shown*by*the*white*arrow.***

1! 2!

1.**Add*1mL*of*the*diluted*sample*directly*to*the*sterile,*empty*petri*dish.*

2.**Add*9mL*of*cooled*medium/agar*to*the*sample*in*the*petri*dish.***

3.**Gently*mix*the*sample*and*the*agar*together*by*moving*left*to*right*and*up*and*down.***

1! 2! 3!

!

! 23!

Spread`plate!TPC!!

With!the!spreadTplate!method,!the!diluted!milk!samples!are!added!directly!to!petri!dishes!that!already!contain!agar!medium.!!!!Sample'Preparation:'**if*possible,*it’s*best*to*do*these*tests*in*duplicate.**Taking*an*average*of*your*results*will*give*you*a*more*accurate*estimation*of*the*bacterial*concentration*1.!Add!900uL!Ringer’s!solution!to!7!individual!microcentrifuge!tubes.!!Label!the!tubes!starting!with!“T2”,!and!continue!!

2.!!Add!100uL!of!milk!sample!to!the!tube!labeled!“T2”!and!mix!thoroughly.!3.!!Take!100uL!of!the!“T2”!mix!sample!and!add!it!to!the!tube!labeled!“T3”.!4.!!Repeat!for!the!remaining!tubes.!!Plating:'1.!!Add!100uL!from!the!undiluted!milk!sample!to!the!first!plate!and!spread!sample!with!a!sterile!spreader.!Label!this!plate!“T1”.!!!

2.!!Repeat!for!samples!from!each!test!tube!labeling!each!plate!with!the!corresponding!test!tube!number!(T2,!T3,!T4,!etc.).!!

3.!!Once!plates!have!dried,!turn!them!upside!down!before!placing!them!in!the!incubator.!!This!is!prevent!condensation!from!dripping!onto!the!agar.!!!

3.!!Incubate!at!37C!for!24hr.!!!!!!!!!!!!!!!!!!!!!!!!!!!

1.**Dip*the*spreader*in*ethanol.* 2.**Carefully*hold*spreader*over*flame*to*burn*ethanol*and*sterilize*spreader.*

1! 2!

3.**Apply*100*uL*of*the*sample*to*the*center*of*the*plate.*

4.**Spin*the*plate*and*uniformly*spread*bacteria*from*the*inside*of*the*plate*to*the*outside*being*careful*not*to*touch*the*edges*of*the*plate.*

3! 4!

!

! 24!

!Recording!Results:!Plates!with!25T250!colonies,!like!the!example!to!the!right,!should!be!used!to!most!accurately!count!colonies.!!When!there!are!>250!colonies!on!a!plate,!do!not!try!and!count!them.!!Just!record!that!plate!as!TNTC!(too!numerous!to!count)!and!go!to!the!next!readable!plate.!!!!Bacterial!concentrations!are!reported!in!colony!forming!units!(CFU)!per!mL.!!As!such,!some!calculations!have!to!be!done!to!determine!the!true!concentration.!!!With!the!pourTplate!method,!1mL!of!the!milk!sample!was!added!to!the!first!plate.!!If!that!plate!had!100!colonies,!the!bacterial!concentration!of!the!milk!sample!would!be!100!CFU/mL.!!This!is!an!unlikely!scenario!with!raw!milk.!!The!following!example!is!more!realistic.!!!!!Plate! !! !!!!!1! !!!!!!T1! !!!!!!T2! !!!!!!!T3! !!!!!!!!T4! !!!!!!!T5!! !!!!T6!!!!!!!!!T7!Colonies! TNTC! !!TNTC!!!!!TNTC!!!!205!!!!!!!!!20!!!!!!!!!2!!!!!!!!0!!!!!!!!!!0!! ! TNTC! !!TNTC!!!!!TNTC!!!!190!!!!!!!!!22!!!!!!!!1!!!!!!!!!0!!!!!!!!!!0!!In!this!scenario,!taking!into!account!the!dilution!and!choosing!the!T3!plates!for!calculation!(they!have!between!25!and!250!colonies),!the!estimated!bacterial!concentration!would!be:!!((205!X!103)!+!(190!X!103))!/!2!=!!197.5!X!103!CFU/mL!!Bacterial!concentration!are!normally!recorded!in!scientific!notation,!so!the!concentration!would!be!1.98!X!105!CFU/mL.!!Note!that!you!will!often!see!bacterial!concentrations!recorded!in!log10!as!well.!!So,!here!the!concentration!could!also!be!recorded!as!log10!(1.98!X!105)!=!5.29!log10!CFU/mL.!!Calculating!the!bacterial!concentration!with!the!spread!plate!method!is!similar,!but!there!is!an!extra!dilution!as!only!100!uL!was!added!to!the!plate!instead!of!1!mL!like!the!pourTplate!method.!!Therefore,!there!is!no!“1”!plate.!!Directly!plating!only!100!uL!of!the!milk!sample!is!already!diluted!1/10,!so!that!is!the!“T1”!plate.!!!!Plate! !! !!!!!T1! !!!!!!T2! !!!!!!T3! !!!!!!!T4! !!!!!!!!T5! !!!!!!!T6!!!!!!!T7!!!!!!!!T8!Colonies! !!TNTC!!!TNTC!!!!!185!!!!!!!!!19! !!!!!!!!2!!!!!!!!!!!0!!!!!!!!!0!!!!!!!!!0!! ! TNTC! !!TNTC!!!!!196!!!!!!!!!18!!!!!!!!!!1!!!!!!!!!!0!!!!!!!!!!0!!!!!!!!!0!!As!in!the!scenario!above,!you!would!choose!the!T3!plates!as!they!have!25T250!colonies.!!The!estimated!bacterial!concentration!would!be:!!

1.**Example*of*the*type*of*plate*that*is*ideal*for*counting*colonies.**There*are*between*25*and*250*colonies*that*are*well*isolated*from*each*other.***

1!

!

! 25!

((185!X!103)!+!(196!X!103))!/!2!=!!190.5!X!103!or!1.90!X!105!CFU/mL.!!In!log10!it!would!be!5.27!log10!CFU/mL.!!!!It!normally!takes!some!practice!and!experience!to!produce!accurate!plate!counts.!!Going!slowly,!thoroughly!mixing!samples,!changing!pipette!tips!whenever!possible,!and!always!plating!the!highest!dilutions!first!can!help!in!reducing!error.!!!!Notes!on!the!TPC!Test:! !

!

! 26!

2.!!Total!Coliform!Test!!TPC!tests!measure!total!bacteria!in!a!milk!sample!(good!and!bad).!!A!coliform!test!is!more!specific.!!Coliforms!are!bacteria!that!are!generally!found!in!the!intestinal!systems!of!animals!and,!in!turn,!animal!feces.!!So,!the!presence!of!coliforms!in!a!milk!sample!(or!water!sample,!etc.)!is!an!indication!of!fecal!contamination.!!!!The!coliform!test!is!done!just!like!a!TPC!test,!but!uses!a!“selective”!medium.!!This!medium!allows!coliforms!to!grow!while!inhibiting!other!types!of!bacteria,!thereby!“selecting”!coliforms.!!In!the!protocol!here,!violet!red!bile!agar!(VRBA)!is!used,!which!is!regularly!recommended!for!testing!milk!and!dairy!products!for!coliforms.!!It!is!a!standard!medium!to!detect!lactose!fermenting!coliforms.!!The!medium!contains!crystal!violent!and!bile!salts,!which!inhibit!the!growth!of!most!gramTpositive!bacteria.!!!!Neutral!Red!is!a!dye!and!is!including!as!a!pH!indicator.!!Lactose!fermenting!colonies!(likely!coliforms)!appear!pink/red!and!may!be!surrounded!by!pink/red!zones!indicating!precipitation!of!bile!salts.!!Note!that!VRBA!does!not!get!autoclaved.!!It!is!only!boiled!prior!to!pouring!plates.!!!!The!procedure!is!exactly!the!same!as!the!TPC—either!the!pour!plate!or!spread!plate!method.!!Only!the!growth!medium!is!different.!!

!!!!!!!!!!!!!!!!!!!!!!

Notes!on!the!Coliform!Test:!! !

1.**E.!coli*grown*on*VRBA*agar.**Typical*colonies*are*pink.*

2.**Closer*detail*of*E.!coli*on*VRBA*agar.**Lighter*pink*zones*can*be*seen*surrounding*the*bacterial*colonies*indicating*precipitation*of*bile*salts.***

1! 2!

!

! 27!

TESTS!FOR!ADULTERATION!!

1.!!Using!a!Lactometer!!A!lactometer!can!be!used!for!several!purposes.!!It!is!basically!a!tool!to!measure!the!specific!gravity!or!mass!of!milk.!!For!example,!milk!has!a!higher!specific!gravity!than!water,!so!a!lactometer!can!be!used!to!test!for!added!water.!!It!can!also!be!used!to!estimate!total!solids!of!milk.!!The!total!solids!are!basically!the!nonTwater!portions!of!milk!and!make!up!about!13%!of!all!milk!(~4%!fat!and!~9%!protein,!lactate!and!other!nonTfat!solids).!!!The!quality!of!milk!for!further!processing,!such!as!cheese!or!yogurt,!is!dependent!in!many!ways!upon!the!total!solids!concentration!of!the!starting!milk.!!Measuring!Specific!Gravity!of!Milk!!Materials:!Cylinder!(<1L)!Lactometer!!Procedure:!1.!!Heat!fresh!milk!sample!to!40C!and!cool!to!room!temperature.!(Heating*reduces*the*size*of*fat*globules,*which*can*impact*the*lactometer)!

2.!!Mix!cooled!milk!sample!and!add!to!cylinder.!!Add!enough!milk!so!that!the!lactometer!will!not!touch!the!bottom!of!the!cylinder.!

3.!!Lower!the!lactometer!into!the!milk!sample!and!allow!it!to!settle!(float).!!This!can!take!up!to!1!min.!

4.!!Record!the!lactometer!reading!at!the!bottom!of!the!meniscus.!5.!!Record!the!temperature.!!!

!!!!!!!!!!!!!!!!!

!!

1.**Gently*insert*the*lactometer*into*the*milk*sample.*

2.**Once*the*lactometer*settles,*read*the*density*reading*at*the*bottom*of*the*meniscus.*

3.**Read*the*temperature*on*the*thermometer*portion*of*the*lactometer.*

1! 2! 3!

!

! 28!

!Recording!Results:!!In!the!example!above,!the!lactometer!reads!120!and!the!temperature!is!65F.!!If!the!milk!is!too!warm!or!too!cold,!the!lactometer!reading!has!to!be!corrected.!!The!acceptable!range!with!the!lactometer!pictured!above!is!60F!+/10!degrees.!!The!lactometer!reading!must!be!adjusted!for!the!temperature!if!it!is!outside!of!this!range!(+1!for!every!10!degrees!above!60F!and!T1!for!every!10!degrees!below!60F).!!!!The!lactometer!pictured!above!is!“New!York”!style!lactometer.!!The!“quevenne”!style!is!also!common!throughout!the!world.!!The!only!difference!is!the!scale.!!With!both!lactometers,!a!reading!of!0!translates!to!a!specific!gravity!of!1!(water).!!To!determine!the!specific!gravity!with!the!New!York!scale:!!

!!!!!!lactometer!reading!!X!!0.29!Specific!gravity!=! ! ! ! ! ! ! +!1!

1000!From!the!sample!above:!!

!!!!!!!120!X!0.29!Specific!gravity!=! ! ! ! ! ! ! +!1!=!1.0348!Kg/mL!! ! ! ! ! 1000!!This!is!the!normal!specific!gravity!for!fresh,!unadulterated!milk.!!The!examples!below!illustrate!how!a!lactometer!can!used!as!a!first!step!in!determining!if!milk!is!adulterated.!!

!!!!!!!!!!!!

!! !1.**Here*the*lactometer*is*

placed*in*water.**Water*is*much*less*dense*than*milk*and*the*lactometer*sinks*to*a*much*lower*reading*(0).***

2.**This*milk*sample*is*diluted*with*water*(about*50mL*water*was*added*to*450mL*of*milk).**The*milk*appears*normal,*but*its*density*is*much*lower*as*indicated*by*the*lactometer*reading*(80).*

1! 2!

!

! 29!

2.!!Using!a!pH!Meter!!A!pH!meter,!if!used!properly,!is!a!valuable!tool!for!testing!milk!quality.!!Most!pH!meters!require!regular!calibration!for!accurate!measurement.!!A!proper!calibration!prior!to!starting!any!experiments!can!save!a!lot!of!time!and!produce!much!more!accurate!results.!!Different!pH!meters!have!different!protocols!for!calibration!and!calibration!should!follow!the!manufacturer’s!instructions.!!Below!is!a!standard!protocol!typical!of!most!pH!meters.!!!!

!!!!!!!!!!!!!!!!!!!!!

!

1! 2! 3!

4.**Press*the*“CAL”*button*and*insert*the*probe*into*the*pH*7.0*solution.*

5.**Once*the*screen*stops**blinking,*remove*the*probe.**The*meter*will*adjust*to*pH*7.0.***

6.**After*washing*the*probe,*press*the*“CAL”*button*and*insert*the*probe*into*the*pH*4.0*solution.***

4! 5! 6!

1.**Start*by*cleaning*the*probe.**This*is*done*by*rinsing*the*probe*with*clean,*distilled*water.***

2.**Dry*the*probe*gently*with*soft,*clean*tissue*paper*or*a*towel.***

3.**Be*especially*careful*with*the*electrode*end*of*the*probe.**It*is*easily*damaged.**Dry*it*simply*by*blotting*the*end*with*the*paper.*

1! 2! 3!

!

! 30!

!!!!!!!!!!!!!!

!!!!!

!!

Notes!on!Using!a!Ph!Meter:!! !

6.**Once*the*screen*stops*blinking,*remove*the*probe.**The*meter*will*adjust*to*pH*7.0.***

7.***Repeat*the*procedure*one*last*time*with*the*pH*10.0*solution.**The*pH*meter*is*now*calibrated.*

6! 7!

!

8.***After*calibration,*insert*the*probe*into*the*test*sample.**Record*final*pH*reading.*

!

! 31!

2.!!General!Test!for!Inhibitors!!Many!adulterants!are!added!to!milk!to!slow!or!stop!bacterial!growth.!!In!doing!so,!these!adulterants!extend!the!shelfTlife!of!milk!or!can!give!the!appearance!that!the!milk!is!fresh.!!Some!of!these!adulterants!are!health!hazards!(e.g.,!high!concentrations!of!antibiotics,!formaldehyde,!some!acids,!etc.).!!All!adulterants!that!inhibit!bacterial!growth!can!significantly!impact!the!further!processing!of!milk!as!many!of!these!processes!rely!on!bacterial!fermentation,!such!as!in!cheese!and!yoghurt!production.!!The!General!Test!for!Inhibitors!can!be!used!as!a!preliminary!test!to!determine!if!milk!contains!any!bacterial!inhibiting!adulterants.!!Materials:!Milk!Samples!Bacterial!Starter!Culture!(pure*cultures*can*be*used,*but*fresh*yoghurt*or*buttermilk*can*also*be*used)!

Test!Tubes!!Incubator!!Procedure:!1.!!Heat!fresh!milk!to!90C!for!5!min.!2.!!Cool!milk!to!37C!or!below.!!!3.!!Add!1mL!of!starter!culture!to!each!milk!sample.!4.!!Incubate!milk!samples!at!37C!for!3!hr.!5.!!Measure!acid!production!using!the!Acidity!Test!or!by!pH!meter.!!Recording!Results:!When!bacteria!grow!in!milk!they!produce!acid.!!Many!inhibitors!(e.g.,!antibiotics,!formaldehyde,!preservatives,!etc.)!will!reduce!bacterial!growth.!!Therefore,!if!inhibitors!are!present!in!the!milk!sample,!there!will!be!reduced!bacterial!growth!of!the!starter!culture!compared!to!milk!without!any!inhibitors!and!reduced!acid!production.!!The!percentage!acidity!in!milk!with!inhibitors!will!then!be!lower!than!that!of!normal!fresh!milk.!!If!using!a!pH!meter,!after!incubation!with!the!starter!culture,!milk!with!inhibitors!will!have!a!higher!pH!than!that!of!milk!without!inhibitors.!!!!!!!!!! !

!

! 32!

Recording!Results:!!

!!!!!!!!!!!!

!!!!!!

!!!

!!

! !

1.**Two*milk*samples*that*appear*identical,*but*one*contains*an*inhibitor.*

3.**After*the*same*treatment,*the*pH*of*the*milk*sample*that*contains*the*inhibitor*is*only*5.63,*indicating*that*the*milk*contains*some*substance*that*is*inhibiting*the*growth*of*bacteria*the*production*of*acid.*

2.**After*adding*the*starting*culture*and*incubating*for*3*hours,*the*pH*of*the*milk*without*the*inhibitor*is*4.82*indicating*the*growth*of*bacteria*and*acid*production.*

1! 3!

5.63!

2!

4.82!

1!

!

! 33!

3.!!Soap!Test!!When!soap!is!added!to!milk,!the!milk!often!becomes!thicker.!!Therefore,!milk!with!soap!as!an!adulterant!can!appear!to!have!more!cream.!!As!soaps!are!bases,!testing!for!the!presence!of!soap!is!a!basic!test!of!pH.!!This!can!be!done!using!the!same!phenophtalein!solution!that!is!used!for!the!Acidity!test.!!!!Materials:!Milk!Sample!Phenophtalein!solution!(0.5%!phenophtalein!in!50%!ethanol)!!Procedure:!1.!!Add!10mL!of!milk!sample!to!test!tube.!2.!!Add!phenophtalein!solution!in!drops!to!the!milk!sample.!3.!!Check!for!color!changes.!!Recording!Results:!

!!!!!

!Notes!on!the!Soap!Test:!!!!!!! !

1.**Two*milk*samples*appear*identical,*but*one*has*been*adulterated*with*soap.*

2.**When*phenophtalein*solution*is*added*to*both*samples,*the*adulterated*sample*becomes*pink*indicating*the*presence*of*a*basic*chemical,*soap*in*this*case.***1! 2!

!

! 34!

4.!!Starch!Test!!Like!soap,!starch!can!be!added!to!milk!as!a!thickener!to!give!the!appearance!that!the!milk!is!high!in!cream.!!Starch!can!also!be!added!to!adulterated!milk!to!increase!its!density!(sugars!are!part!of!the!total!solids!of!milk!and!part!of!what!makes!milk!heavier!than!water).!!In!doing!so!it!can!mask!the!effects!of!diluting!milk!with!water!by!giving!the!appearance!that!the!adulterated!milk!has!the!same!density!as!unadulterated!milk.!Starch!is!a!complex!carbohydrate!(polyTsaccharide)!that!reacts!with!iodine,!unlike!the!primary!milk!sugars!lactose!and!galactose,!which!are!single!and!diTsaccharides.!!Thus,!the!presence!of!starch!can!easily!be!detected!by!adding!a!small!amount!of!iodine.!!Materials:!Milk!Sample!Test!Tubes!Water!bath!(or!other!device!that!can!hold!boiling!water)!Iodine!Solution!(1%)!!Procedure:!1.!!Add!5mL!milk!sample!to!a!test!tube!that!can!be!boiled.!2.!!Boil!milk!samples!for!5!min.!3.!!Allow!samples!to!cool!to!room!temperature.!4.!!Add!2T3!drops!of!iodine!solution!to!each!sample.!5.!!Observe!color!changes.!!Recording!Results:!

!!

!!Notes!on!the!Starch!Test:!! !

1.**Two*milk*samples*that*appear*identical.**One*sample*has*been*adulterated*with*starch.***

2.**After*boiling*and*the*addition*of*iodine,*the*sample*adulterated*with*starch*becomes*dark*blue*to*black.**

1! 2!

!

! 35!

5.!!Table!Sugar!Test!!Like!starch,!table!sugar!can!be!added!to!milk!to!increase!the!total!solids.!!For!instance,!if!milk!is!diluted!with!water!to!increase!its!volume,!the!milk!density!will!decrease.!!Adding!table!sugar!increases!the!concentration!of!solids!and,!in!doing!so,!increases!its!density!to!appear!like!normal!milk.!!Sucrose!is!also!a!component!of!most!kinds!of!synthetic!milks.!There!is!a!fundamental!different!between!table!sugar!(sucrose)!and!the!sugars!present!in!normal!milk.!!Sucrose!is!ketose!sugar!that!will!react!with!resorcinol!when!it!is!reduced.!!To!test!for!sucrose,!an!acid!is!added!to!reduce!sucrose.!!Resorcinol!is!then!added.!!The!reduced!sucrose!will!then!react!with!resorcinol!to!form!a!red!color.!!!!! !

!

! 36!

6.!!Acid`based!Preservatives!Test!!Acids!are!often!used!in!food!preservation,!but!they!should!not!be!added!to!milk!that!is!sold!as!fresh!milk.!!Preservatives!are!inhibitors!and!will!reduce!bacterial!growth,!which!will!extend!the!milk!shelfTlife,!but!preservatives!also!interfere!with!any!fermentation!processes!needed!to!make!yoghurt,!cheese,!or!similar!products.!!The!test!below!is!designed!to!identify!acidTbased!preservatives!such!as!benzoic!acid,!but!the!General!Test!for!Inhibitors!can!also!be!used!to!detect!inhibitors!in!general.!!Materials:!Milk!Samples!Sulfuric!Acid!Ferric!Chloride!(5%)!!Procedure:!1.!!Place!10mL!of!milk!sample!in!an!appropriate!test!tube.!2.!!Add!approximately!500uL!of!sulfuric!acid!to!sample!and!shake!thoroughly.!3.!!Add!approximately!500uL!of!ferric!chloride!solution!and!shake!well.!4.!!Observe!any!color!changes.!!Recording!Results:!The!presence!of!acidTbased!preservatives!will!result!in!a!color!change!in!the!adulterated!milk!sample.!!These!color!changes!can!range!from!red!to!purple.!!Milk!that!has!not!been!adulterated!will!remain!white!to!slightly!yellow!due!to!sulfuric!acid.!

!! !

1.**Two*milk*samples*that*appear*identical,*but*one*sample*has*been*adulterated*with*an*acidNbased*preservative.***

2.**The*color*change*in*the*right*sample*indicates*the*presence*of*an*acidNbased*preservative.***

1! 2!

A! B!

!

! 37!

7.!!Test!for!Sodium!(bi`)!Carbonate!!Sodium!carbonate!and!sodium!bicarbonate!can!be!added!to!milk!to!prevent!acidity.!!As!milk!ages,!bacteria!will!grow!and!produce!acid.!!Sodium!carbonate!is!a!buffer!and!is!used!to!neutralize!that!acid.!!Therefore,!sodium!carbonate!is!added!to!milk!with!high!amounts!of!bacteria!to!raise!the!pH!and!give!the!appearance!that!the!milk!is!fresh.!!Below!are!two!basic!tests!for!testing!for!the!presence!of!sodium!carbonate.!!!!Sodium!Carbonate!Test!1!Materials:!Milk!Samples!Rosalic!Acid!(1%;!also!called!Aurin!or!Corralin)!Test!Tubes!!Procedure:!1.!!Add!10mL!of!milk!sample!to!a!test!tube.!2.!!Add!100uL!of!rosalic!acid!solution.!3.!!Observe!any!color!changes.!!Recording!Results:!Sodium!Carbonate!Test!2!Materials:!Milk!Samples!Beaker,!Test!Tube,!or!Similar!Container!Water!Bath,!Heat!Block,!or!Similar!Heating!Device!!Procedure:!1.!!Add!2T3mL!of!milk!sample!to!a!test!tube.!2.!!Heat!milk!samples!at!90C!until!milk!has!evaporated.!!If!other!heat!sources!are!used,!be!careful!not!to!burn!milk.!

3.!!Observe!color!changes.!!Recording!Results:!

!!!!

1.**Three*milk*samples*that*appear*identical,*but*two*samples*have*different*concentrations*of*sodium*carbonate.*

2.**The*A*sample*is*unadulterated*milk.**Samples*B*and*C*contain*increasing*amounts*of*sodium*carbonate.**Upon*heating,*the*milk*begins*to*evaporate*and*the*sodium*carbonate*leaves*a*yellow*to*orange*residue.***

1! 2!A! C!B!

!

! 38!

Notes!on!the!Acid`based!Preservatives!Test:!!!!!!!!!!!!!!!!!!! !

!

! 39!

8.!!Formaldehyde!Test!!As!mentioned!previously,!formaldehyde!can!be!added!to!milk!to!coverup!or!mask!contamination!problems.!!This!poses!a!clear!human!health!hazard,!as!formaldehyde!is!highly!toxic.!!Like!in!the!general!test!for!inhibitors,!it!is!always!a!good!idea!to!include!a!sample!of!high!quality,!fresh,!unadulterated!milk!when!conducting!the!formaldehyde!test!for!comparison.!!!!Materials:!Milk!Sample!Test!tubes!!10%!Ferric!Chloride!Solution!90%!Sulfuric!Acid!!Procedure:!1.!!Add!5mL!of!milk!sample!and!5mL!of!sterile!water!to!a!test!tube.!2.!!Add!1!drop!of!10%!ferric!chloride!solution!to!10mL!of!sulfuric!acid.!3.!!Add!the!ferric!chloride/sulfuric!acid!solution!to!milk!sample!by!pouring!gently!along!the!side!of!the!tube.!!!

4.!!Observe!color!changes.!!Recording!Results:!Milk!that!contains!formaldehyde!will!have!a!purple/red!color!when!the!ferric!chloride/sulfuric!acid!is!added!to!the!milk.!!Normal!milk!will!be!green!and!will!change!to!brown!if!left!to!stand.!!This!test!will!detect!formaldehye!at!10ppm!or!about!1mL!of!formaldehyde!in!100!liters!of!milk.!!!

!!!!!!!!!!!!!!!!!!!!

1.**Add*the*ferric*chloride/sulfuric*acid*solution*to*the*milk*sample*by*applying*the*liquid*to*the*side*of*the*tube*in*drops.*

2.**The*sample*labeled*“A”*is*fresh,*unadulterated*milk.**After*sitting,*the*bottom*becomes*dark*brown.**The*sample*labeled*“B”*is*milk*adulterated*with*formaldehyde.**The*bottom*is*a*red/purple.****

1! 2!

A! B!

!

! 40!

!!Notes!on!the!Formaldehyde!Test:!!! !

!

! 41!

9.!!Antibiotic!Residue!Tests!!When!animals!are!treated!with!antibiotics,!antibiotic!residues!can!easily!pass!to!the!milk.!!This!can!cause!problems!for!further!processing!that!relies!on!fermentation!(e.g.,!yogurt,!cheese,!etc.).!Therefore,!it!is!important!to!separate!milk!from!animals!being!treated!with!antibiotics!from!milk!from!untreated!animals.!!!!Numerous!tests!are!available!to!test!for!the!presence!of!general!and!specific!antibiotics!in!the!milk.!!Basic!tests!simply!combine!milk!samples!with!bacterial!cultures!and!test!for!bacterial!growth.!!If!antibiotics!are!present,!bacterial!growth!will!be!inhibited.!!In!these!tests,!a!color!indicator!is!usually!included!to!measure!pH.!!If!bacteria!grow!and!produce!acid,!the!sample!will!change!colors!(negative).!!Samples!that!do!not!change!color!indicate!that!bacterial!growth!is!inhibited!(positive).!!In!this!way,!the!tests!are!identical!in!principle!to!the!General!Test!for!Inhibitors.!!Tests!for!specific!antibiotics!usually!rely!on!immunoTchemistry.!!Milk!samples!are!added!to!a!paper!strip!that!contains!compounds!that!will!bind!to!any!antibiotics!residues!present.!!If!bound,!the!strip!changes!colors!indicating!a!positive!sample.!!!!

!! !1.**Examples*of*antibiotic*residue*test*strips*used*in*dairy*industries*around*the*globe.**A*small*milk*sample*is*applied*to*the*strip*and*the*strip*is*incubated.**If*antibiotics*are*present,*bands*(red)*appear*in*distinct*patterns.******

!

! 42!

POWDERED!MILK!!

1.!!Processing!and!Technology!!Quality!and!safety!testing!of!powdered!milk!is,!in!most!cases,!similar!to!tests!used!for!fluid!milk.!!Once!the!milk!is!reconstituted,!all!tests!that!are!used!for!fluid!milk!can!be!used!for!powdered!milk.!!It!is!important!to!note!that!powdered!milk,!once!reconstituted,!should!retain!all!of!the!qualities!and!characteristics!of!fluid!milk.!!!Below!is!a!table!that!describes!some!acceptable!properties!of!powdered!milk.!!!!! ! ! ! ! Milkfat!(%)! ! Maximum!! Total!Solids!(%)!! ! ! ! ! ! ! ! Water!(%)!!Whole!milk!powder! ! ! !!26!–!42! ! !!!!!!5! ! ! 34!Partially!skimmed!milk!powder! !1.5!–!26! ! !!!!!!5! ! ! 34!Skim!milk!powder!! ! ! !!!!!!1.5! ! ! !!!!!!5! ! ! 34!!!Two!basic!methods!are!used!to!remove!the!water!from!milk!to!produce!powdered!milk:!!rollerTdrying!and!sprayTdrying.!!The!method!used!to!produce!the!powdered!milk!can!influence!the!results!of!some!tests.!!In!most!cases,!this!is!due!to!the!process!of!rollerTdrying,!which!uses!higher!heat!than!spray!drying.!!The!result!is!that!rollerTdrying!often!damages!milk!proteins.!!In!doing!so,!rollerTdried!powdered!milk!is!often!less!soluble,!more!often!has!offTflavors,!and!is,!in!general,!lower!quality!than!sprayTdried!powdered!milk.!!!

! !

!

! 43!

2.!!Preliminary!Physical!Test!of!Powdered!Milk!!Since!powdered!milk!is!simply!milk!with!the!water!removed,!it!should!retain!all!of!the!qualities!of!fluid!milk.!!Upon!initial!inspection,!the!powder!should!be!a!uniform!white!to!slightly!offTwhite!(cream!color).!!!It!should!be!free!of!any!brown!or!yellow!colors.!!The!powder!itself!should!taste!slightly!sweet!and!have!no!off!tastes!or!odors!that!are!also!found!fluid!milk.!!Powdered!milk!should!also!be!free!of!any!burnt!flavors!or!odors.!!Finally,!the!powder!should!appear!dry,!with!uniform!granules,!and!free!of!clumps!or!lumps!that!indicate!improper!moisture!content!or!storage.!!! !

!

! 44!

3.!!Measurement!of!Scorched!Particles!!As!mentioned!above,!rollerTdrying!uses!high!heat!which!can!damage!powdered!milk.!!Often,!high!heat!will!produce!scorched!or!burnt!milk.!!This!can!significantly!lower!the!solubility!and!nutritive!value!of!the!powdered!milk.!!Testing!powdered!milk!to!determine!the!level!of!scorched!particles!is!easy,!quick,!and!involves!very!few!materials.!!Materials:!Milk!sample!Filter!Pad!Flask!Scorched!Particle!Standard!!Procedure:!1.!!Combine!powdered!milk!(25.0g!skim!milk!or!32.5g!whole!milk)!with!250mL!sterile!water.!!!

2.!!Mix!at!high!speed!for!1!min.!3.!!Let!milk!sit!for!10!min.!3.!!Pass!milk!sample!through!filter.!4.!!Collect!filter!and!compare!visually!to!the!scorched!particle!standard.!!

!!!!!!!!!!!!!!!!!!!!

!!!!!

1.**Place*the*filter*in*the*top*of*a*funnel*or*similar*device.*

2.**Slowly*pour*the*sample*over*the*filter*and*collect*waste*milk*in*the*container*below.***

1! 2!

!

! 45!

Recording!results:!Comparing!the!filters!to!the!standard!below!will!give!the!milk!a!grade!(A!–!D)!with!A!being!highest!quality!and!D!being!lowest!quality.!!!!

!!!!!!!!!!!!!!!!!!

!Notes!on!the!Scorched!Particles!Test:! !

2.**This*sample*is*from*high*quality*powdered*milk*with*little*to*no*scorched*particles.**It*would*receive*an*“A”*grade.*

3.**This*sample*is*from*lower*quality*powdered*with*a*high*concentration*of*scorched*particles.**It*would*receive*a*“D”*grade.***

1.**These*are*the*standards*developed*by*the*American*Dry*Milk*Institute.**Other*standards*are*available*as*well.***

1! 2! 3!

!

! 46!

4.!!Solubility!Test!!If!powdered!milk!is!not!soluble,!it!loses!its!nutritive!value.!!The!acceptable!solubility!standards,!however,!differ!for!rollerTdried!and!sprayTdried!powdered!milk.!!In!general,!rollerTdried!powdered!milk!is!less!soluble!than!sprayTdried!powdered!milk!due!to!the!process.!!Here!again,!the!high!heat!tends!to!damage!more!milk!proteins!during!roller!drying!than!in!sprayTdrying!making!it!more!difficult!to!completely!dissolve!rollerTdried!powdered!milk.!!The!solubility!test!is!easy,!but!requires!a!centrifuge.!!The!protocol!described!here!can!be!scaled!up!or!down!to!match!the!type!and!size!of!centrifuge!available.!!Materials:!Milk!Sample!Flask!Centrifuge!Centrifuge!tubes!!Procedure:!1.!!Combine!powdered!milk!(10g!for!skim!milk,!13g!for!whole!milk)!with!100mL!sterile!water!in!a!flask.!

2.!!Shake!vigorously!for!1!min.!3.!!Let!milk!sit!for!10!min.!4.!!Shake!again!briefly.!5.!!Transfer!reconstituted!milk!to!a!50mL!centrifuge!tube.!6.!!Centrifuge!samples!at!5000rpm!for!5!min!to!sediment!solids.!7.!!Remove!supernatant!(liquid)!being!careful!not!to!disturb!the!solid!pellet!at!the!bottom!of!the!tube.!

8.!!Resuspend!pellet!in!50mL!sterile!water!and!repeat!steps!6!and!7.!9.!!Measure!the!pellet!in!the!bottom!of!the!tube!in!mL.!!Recording!Results:!The!size!of!the!pellet!left!in!the!bottom!of!the!centrifuge!tube!is!the!solubility!index.!!Lower!quality!milk!powders!will!contain!larger!pellets!as!large!portions!of!the!powdered!milk!do!not!completely!dissolve.!!Below!is!a!table!showing!solubility!standards!for!different!types!of!powdered!milk.!!!!! ! Maximum!Acceptable!Solubility!Index!!! ! ! ! Spray`dried! ! Roller`dried!!Whole!milk!powder! ! !!!!!!!!1%! ! !!!!15%!Skim!milk!powder! ! !!!!!!!!1%!!!!!!!!!!! !!!!! !!!!15%!!!!!!!

!

! 47!

In!our!example,!we!have!scaled!the!experiment!down!to!accompany!of!centrifuge.!!Our!starting!volume!was!25mL!(2.5g!powdered!milk!in!25mL!water).!

!!!!!!!!!!!!!

!!

!!!!!!!!!!!!

!!!!!!

!!!

1.**A*pellet*will*form*in*the*bottom*of*the*tube*after*centrifugation.**The*above*example*is*a*pellet*from*a*high*quality*(sprayNdried)*powdered*milk*sample.***

2.**This*pellet*is*from*a*lower*quality*powdered*milk*sample.**The*dark*area*is*scorched*milk.***

3.**Resuspend*the*pellet*with*1mL*of*water*until*it*is*completely*dissolved.*****

4.**The*resuspended*pellet*of*the*high*quality*milk*sample*measures*approximately*1.2mL.***

5.**The*resuspended*pellet*of*the*lower*quality*milk*measures*approximately*2.3mL.***

1! 2! 3!

4! 5!

!

! 48!

!!!The!percentage!solubility!is!calculated!as!follows:!!%!solubility!!!!! Final!Volume!–!1!mL!!!! ! !!=! ! ! ! ! ! ! ! !! ! !!!!!!!!!Starting!volume!in!step!1! ! ! ! ! !!!!!!!!In!our!example:!!High!Quality!Sample! ! ! 1.2mL!–!1mL!%!Solubility! ! ! =! ! ! !!!=!0.8%!! ! ! ! ! !!!!!!!!!25!!Low!Quality!Sample! ! ! 2.3mL!–!1mL!%!Solubility! ! ! =! ! ! !!!=!5.2%!! ! ! ! ! !!!!!!!!!25!!! ! ! ! ! ! !Notes!on!the!Solubility!Test:!!! !

!

! 49!

REFERENCES!and!RESOURCES!!

Food!and!Agriculture!Organization!(FAO).!!Milk!Processing!Guide.!!Available!at:!http://www.fao.org/ag/AGAinfo/resources/documents/MPGuide/mpguide2.htm!!O’Connor,!CB.!!1995.!!Rural!Dairy!Technology.!!International!Livestock!Research!Institute.!!Available!at:!!https://cgspace.cgiar.org/handle/10568/570!!AMRITA!Vishwa!Vidyapeetham!University.!!2014.!!Detection!of!Adulteration!in!Milk.!!Available!at:!!http://vlab.amrita.edu/?sub=3&brch=63&sim=1091&cnt=1.!!US!Food!and!Drug!Administration.!!2012.!!Import!Reinspection:!!Milk.!!Available!at:!!http://www.fda.gov.!!!!