FDSC 4763 Ch 9 - Protein Analysis

8/12/2019 FDSC 4763 Ch 9 - Protein Analysis

1/45

FDSC4763AnalysisofFoodProducts


2/45


3/45

Thenature

of

proteins

Proteinsserveanumberoffunctionsinplantsandanimals:

Many

types

of

structural

proteins,

e.g.

collagens,

etc. Manybiochemicallyactiveproteins,e.g.enzymes,cellwall

proteins,scaffoldingproteins,etc.

Proteinsarediverseintheirchemicalproperties.

Proteinsvary

widely

in

their

sizes

and

physical

structures.

Proteinpropertiesaredeterminedbytheiraminoacidsequence(primarystructure),theinteractionamong

aminoacids

in

local

regions

of

apeptide

chain

(secondary

structure),theoverallconformationofapeptidechain(tertiarystructure),andinsomecasestheinteractionsamongpeptidechainsinaproteinmacromolecule

(quaternarystructure).


4/45

Challengesin

analyzing

protein

contentinfoods

Thediversenatureofproteinsinfoodsmakesanalysisdifficult.

Molecular

size

varies,

measured

in

Daltons,

proteins

rangefrom~5000tomorethanamillionDaltons.

Solubilityvaries,someproteinsarereadilysolubleinsaltsolutions,someinacidsolutions,someinalcoholsolutions,andsomearehighlyinsoluble.

Presenceofnonproteinconjugatesvariesconsiderablydependingonproteinfunctionality.


5/45

Waysof

measuring

protein

content

infoods

Determinationof

total

nitrogen.

Proteinsarecomposedofaminoacidsandallaminoacidscontainnitrogen,but

Actualproteincontentvariesbasedonaminoacidprofile.

Othersourcesofnitrogenoccurinmanyfoods.

Detectionandquantificationofpeptidebonds.

Complicatedbythefactthatmolecularsizeofproteinsvaries.

Detection

and

quantification

of

aromatic

amino

acids. Complicatedbythefactthataminoacidprofilesofproteinsvary.


6/45

Waysof

measuring

protein

content

infoods(continued)

Dyebindingcapacity,ultravioletabsorptivity,and/orlightscatteringpropertiesofproteins.

Complicatedbythefactthatallofthesefactorsareinfluencedbyproteinstructure,possiblyincludingprimary,secondary,tertiary,andquaternaryproteinstructures.


7/45

Considerationsin

choosing

aproteinanalysismethod

Whatdegree

of

accuracy

is

required?

Whatdegreeofprecisionisrequired?

What

degree

of

accuracy

and/or

precision

can

we

sacrificeforspeed?

Whatdegreeofaccuracyand/orprecisioncanwe

sacrificefor

cost?

Whatdegreeofaccuracyand/orprecisioncanwesacrificeforsimplicityandeaseofanalysis?


8/45

Usesof

protein

analysis

Nutritionlabeling.

Pricing.

Thevalueofmanycommodities e.g.milk,wheat,etc.

maybe

heavily

influenced

by

protein

content.

Quantificationoffunctionalproperties.

Glutenfunctionalityinwheatflour.

Caseinfunctionalityincheesemaking.

Quantificationofbiologicalactivity.

Enzyme

activity,

e.g.

pectinases,

proteases,

trypsin inhibitors,

etc.


9/45


10/45

Kjeldahl method

Johan Kjeldahl, 1849 - 1900
http://upload.wikimedia.org/wikipedia/commons/9/95/Kjeldahl3.JPG


11/45

Kjeldahl workedfor

Carlsberg

Kjeldahl first developed

his method in 1883.

As of 2009, Carlsberg

was the 4th largest

brewery group in the

world
http://upload.wikimedia.org/wikipedia/commons/4/47/Carlsberg_beer.jpghttp://en.wikipedia.org/wiki/File:Tuborg_Beer_logo.svghttp://en.wikipedia.org/wiki/File:Carlsberg_logo.svg


12/45

Componentsof

the

Kjeldahl

method

TheKjeldahl method

may

be

broken

down

into

three

parts:

Digestion Organicnitrogencompoundsarereducedtoammonium.

Distillation ammoniumisconvertedtoammoniagasanddistilledoff.

Titration ammonia

gas

is

trapped

by

an

acid

solution

andthesolutionistitratedtoquantifytheamountofnitrogenpresentintheoriginalsample.


13/45

Kjeldahl digestion Digestionisthemosttimeconsumingstepintheanalysis.

Thedigestion

reaction

is

made

considerably

faster

by

use

of

catalystandaneutralsalt. Manydifferentcatalystshavebeenusedovertheyears,

includingselenium,

mercury,

and

copper.

Copper

is

usually

usednowbecauseitisrelativelynontoxic.

Inorganicsalts,suchaspotassiumsulfate(K2SO4),raisetheboilingpointofthedigestingacid.andthusthetemperature

ofthe

reaction.

The

boiling

point

of

aconcentrated

sulfuric

acidsolutionisabout330 C. TheadditionofK2SO4elevatestheboilingpointtoaround400 C.Thissignificantlydecreasesthelengthoftimerequiredfordigestion.


14/45

Kjeldahl digestion the

chemistry

The

basic

chemical

reaction

is

as

follows: OrganicN+H2SO4 (NH4)2SO4+H2O+CO2+

othersamplematrixbyproducts.


15/45

Kjeldahl distillation Thepurposeof

distillation,is

to

separatetheammoniafromthedigestionmixture.

Thisis

accomplished

by

raisingthepHofthemixtureusingsodiumhydroxide(45%NaOH

solution).This

converts

theammonium(NH4+)

ionsinthedigestionmixturetogaseous

ammonia(NH3).


16/45

Kjeldahl distillation the

chemistry

ofcapturingtheammonia

Thebasicchemicalreactionisasfollows:

NH

3+H

3B

O

3

NH

4+ +

H

2B

O

3

Notethattheabovereactionusesboricacidasthereceivingsolution. Otheracidshavebeenusedaswell.


17/45

Kjeldahl titration

Acidisaddedtotitratetheborate: H2BO3 +H+ H3BO3

Notethatthenumberofhydrogenionsconsumedisequaltothe

numberof

ammonia

gas

moleculescaptured.


18/45

Kjeldahl titrationDifferentmethodsofdetectingtheendpointareused:

Colorimetric differentindicatorsareused,includingiodine,methylred/methylblue,andindophenol.

pH pHismeasuredafterdistillationintoaknownvolumeofboricacid(musthaveboricacidinexcess).

Directmeasurementofammoniausingionchromatography.


19/45

Kjeldahl calculations

Formulafor

calculating

%

nitrogen

in

asample:

Supposeweuse0.163mlof0.01NHCl (correctedvolume)totitrateoursample,whichoriginallyweighed

2.0368

g.

What

is

our

%

N

(wet

basis)?

Ifourdrymatterinthesampleis15.39%,whatisourdry

basis

%

N?

1.12%

% N = N HCl xcorrected acid volume (ml)

sample weight (g)x 100x

14 g N

mol

7.28%


20/45

Kjeldahl calculations(cont)

NotethatKjeldahl measurestotalnitrogen.

Mustcovertnitrogentoprotein.

StandardConversionfactoris6.25

Based

on

meat,

in

which

protein

is

fairly

uniformly

16%

N

byweight.

Notethatotherconversionfactorsmaybeappropriateforotherfoods(seeTable92intext).

Usingthestandardconversionfactor,whatisour%protein(wetbasis)fromourpreviousexample?

7%


21/45

Moresample

calculations

Ifourdrybasis%proteinis27.5%andourdrybasis%

moistureis

566%,

what

is

our

wet

basis

%

nitrogen

(assumestandardconversionfactor)?

First, calculate the % dry matter in the original sample.

Recall that % moisture (dry basis) = (weight of water / weightof dry matter) x 100. Therefore, if the dry basis % moisture is

566%, then:

x / (100-x) = 5.66, where x = the portion of water in 100grams of the original sample.

Solving for x, we get x = 0.85 = 85% moisture (wet basis).

Thus, dry matter is 15% (100% - 85%) by weight in theoriginal wet sample.


22/45

Moresample

calculations

(continued)Second, convert dry basis % protein to wet basis % protein:

Because the % dry matter in the original sample was

15%, our conversion factor is (1 / 0.15) = 6.67. Toconvert, we divide the dry basis % protein by our

conversion factor.

27.5% / 6.67 = 4.1% protein (wet basis).

Third, convert wet basis % protein to wet basis % nitrogen

using the standard conversion factor:

4.1% / 6.25 = 0.7% nitrogen (wet basis).


23/45

Advantagesand

disadvantages

of

Kjeldahl method

Advantages: Verylittlesamplepreparationisneeded. Sizereductionis

typicallyallthatmaybeneeded.

Workswell

for

almost

all

food

types.

Mayberelativelyinexpensive.

Canbeveryaccurate(isofficialmethodforcrudeprotein).

Disadvantages: Measurestotalnitrogen,includingnonproteinsources.

Relativelytimeconsuming.

Reagentscan

be

corrosive

and/or

toxic.

Waste

disposal

can

be

aproblem.


24/45


25/45

Dumas(Nitrogen

Combustion)

method

Principle: Samplesareburnedinpure

oxygenat700to1000C.

Nitrogencontaining

compoundsareconvertedtoN2andnitrogenoxides.

Nitrogen

oxides

are

reduced

to

N2inacoppercolumnat~600C

Nitrogengasisassayedusing

gaschromatography

(GC).


26/45

DumasMethod

Equipment


27/45


28/45

Infraredspectroscopy

Principle:

Usesnearinfrared(NIR)ormidinfrared(MIR)radiationtodetectandquantifypeptidebondsinasample.


29/45

Advantagesand

disadvantages

of

Infraredspectroscopy

Advantages: Requiresnohazardouschemicals.

Veryrapid,typicallyunder2minutestoanalyzeasample.

Verylittle

training

required

to

use

equipment.

Specifictoprotein,notaffectedbynonproteinnitrogen.

Suitableforawiderangeofproducts.

Disadvantages: Equipmentisrelativelyexpensivetopurchaseandmaintain.

Equipmentmustbecalibratedtospecificproductsinorderto

giveaccurate

results.


30/45

Biuret MethodPrinciple:

Cupric

ions

(Cu++

)

will

complex

with

peptide

bonds

in

a

proteinmoleculeunderalkalineconditons.

Theproteincoppercomplexabsorbslightwithpeakabsorbanceoccursat540nm. (Thisgivesthesampleapurplishcolor.) Thedegreeofabsorbanceisproportionaltotheamountofproteininthesample.


31/45

Biuret Method:Procedure

Biuret reagent(solutionofcoppersulfate[CuSO4],NaOH,

and

potassium

sodium

tatrate [NaKC4H4O6])is

mixedatabouta5:1ratiowithaproteinsolution.

Mixtureisallowedtostandforapproximately15to30minutesatroomtemperature.

Absorbanceisreadat540nmusinga

spectrophotometer

and

a

Biuret reagent

blank.Proteinconcentrationiscalculatedusingastandard

curve,typicallyconstructedusingbovineserum

albumin(BSA).


32/45

Biuret methodapplications

The

Biuret Method

is

commonly

used

to

determine

proteinconcentrationin:

Cerealproducts.

Meats. Soybeanproducts.

Animalfeeds.

Proteinconcentrates

and

isolates.


33/45

Advantagesand

disadvantages

of

Biuret Method

Advantages: Simplemethod,requiresnospecializedequipment.

Relativelyrapid,typically~30minutestoanalyzeasample.

Specific

to

proteins,

not

affected

by

non

protein

nitrogen. Rarelyencounterinterferencefromothercomponents.

Disadvantages: Notparticularlysensitive.

Colormayshiftdependingonproteinaminoacidprofileandstructure.

Samplesmustberelativelyclearandnonturbid.

Mustbe

standardized

against

aknown

protein,

e.g.

results

reportedasBSAequivalents. Thisusuallyreducesaccuracy.


34/45

LowryMethod

Principle:

Combines

Biuret Methodwithadditionalreagentsandreactionstoboostsensitivity.

Thecomplexofcupricions(Cu++)withpeptidebonds

underalkaline

conditons reduces

cupric

ions

to

cuprous

ions(Cu+).

Alongwithtyrosineandtyptophan sidechains,cuprous

ions

react

to

reduce

Folin

Ciocalteu Reagent

(phosphomolybdic/phosphotungstic acid).

Thisproducesabluecolorthatcanbereadat750nm(highsensitivity)or500nm(lowsensitivity). The

amountof

absorbance

is

proportional

to

the

number

of

peptidebonds.


35/45

LowryMethod

applications

The

Lowry

Method

is

commonly

used

to

determine

proteinconcentrationin:

Isolatedcellfractions.

Chromatographyfractions.

Enzymepreparations.

Otherproductsofproteinbiochemistry.

Notgenerallyusedforcomplexfoodsystems;maybeusedonisolated,purifiedfoodproteins. Somewhat

supersededby

the

BCA

method.


36/45

Advantages

and

disadvantages

of

LowryMethod Advantages:

Simplemethod,

requires

no

specialized

equipment.

ProceduresaresimilartoBiuret Method,typically~11.5hourstoanalyzeasample.

Specific

to

proteins,

not

affected

by

non

protein

nitrogen. MethodismuchmoresensitivethanBiuret method. MethodislesssensitivetoturbiditythanBiuret method.

Disadvantages: Color

may

shift

depending

on

protein

amino

acid

profile

and

structure. MoreaffectedbyothersamplecomponentsthanBiuret

method,especiallyoxidizing/reducingagents.

Mustbe

standardized

against

aknown

protein,

e.g.

results

reportedasBSAequivalents. Thisusuallyreducesaccuracy.


37/45

DyeBinding

Methods

Principle: Dye

molecules

bind

to

proteins,

oftentoNterminalaminoacids.

Bounddyemoleculeseffecta

changein

the

solutions

absorbance

orfluorescence.

Degreeofchangeisproportional

to

the

amount

of

protein

present. Absorbanceorfluorescencecomparedtoastandard

curvetocalculateproteinconcentration.

Manydifferentspecifictestsexist,manyareintendedfortesting

specific

types

of

foods.


38/45

Anionicdye

binding

Applications: Milk;wheatflour;soyproducts;meats.

Advantages: Rapid(lessthan15minutes,typically). Relativelyaccurateandprecise.

Does

not

measure

non

nitrogen

proteins. Maybeusedspecificallytoquantitate changesinlysine

contentincerealproducts.

Disadvantages:

Notsensitive

to

less

than

milligram

quantities

of

protein.

Calibrationcurverequiredforeachtypeofsamplebeinganalyzed.

Willnotdetecthydrolyzedproteins. Non

protein

components

(e.g.

starch)

may

bind

dye

and

introduceinaccuracies.


39/45

Bradforddye

binding

UsesCoomassie BrilliantBluedye. Applications:

Worts (beerproducts).

Potatoes. Enzymeconcentrations.

Advantages: Veryrapid(~2minutes). Verysensitive. Lesssusceptibletointerferencethanmanyotherchemical

methods. Disadvantages:

Calibrationcurverequiredforeachtypeofsamplebeinganalyzed maybeveryspecifictoagivenproduct.

Proteindye

complex

can

be

sticky.

Must

use

glass

or

plastic

cuvettes foranalysis.


40/45

Bicinchoninic Acid(BCA)

Method

Principle:

Cupricionsarereducedtocuprousionsunderalkalineconditions(similar

to

Biuret reaction).

Cuprousionsreactwithbicinchoninic Acid

(BCA)

to

formacomplexthatabsorbsat562nm(purplishcolor).

BCA Molecule


41/45

BCAMethod

Uses

Applications: Oftenusedinproteinpurificationandisolation,similarto

Lowrymethod.

Not

used

for

measuring

protein

in

complex

foodsystems.

Advantages: AssensitiveasLowrymethod,butprocedureissimpler.

ReagentismorestablethanLowrymethod.

Relativelylesssusceptibletointerferencefromnonproteincomponents.

Disadvantages: Colorofreactionproductsisnotstablewithtime.

Reducingcompoundswillinterferewiththetest.

Colorof

reaction

products

will

vary

according

to

protein

structure,similartoLowryMethod.


42/45


43/45

UVAbsorption

applications

TheUV

Absorption

Method

is

commonly

used

to

determineproteinconcentrationin:

Milk.

Meat. Purifiedproteinpreparations.

Notoften

used

for

complex

food

systems

containing

proteinswithdifferingsolubilitycharacteristics.


44/45

UVAbsorption

advantages

and

disadvantages

Advantages: Veryrapid(onceproteinissolubilized). Nointerferencefromothercommonnitrogencontaining

components,e.g.ammoniumsulfateandotherbuffering

agents. Nondestructive preparedproteinsolutionscanbeusedforotherpurposesafterUVabsorbanceismeasured.

Disadvantages:

Nucleicacids

interfere

with

the

test

as

they

also

absorb

at

280

nm. Proportionsoftyrosineandtryptophancanvaryamong

differentfoodproteins,calibrationmayberequired.

Proteinsolutions

must

be

clear,

colorless,

and

fairly

pure

for

themethodtobeaccurate.


45/45

Proteinanalysis

final

thoughts

Manydifferentmethodsofproteinanalysisexist

mustmatch

method

to

foodstuff

and

be

aware

of

the

limitationofanygivenmethod.

Methodsthatmeasuretotalnitrogen(e.g.Kjeldahl andDumas)

will

usually

overestimate

crude

protein

content.

Methodsthatmeasureproteindirectlywillusuallygivedifferentresultswithdifferenttypesofproteins proper

calibrationis

amust.

Measurementsofcrudeproteinwillusuallynotbesufficienttodetermineactualproteinnutritivevalue,e.g.

Protein

Efficiency

Ratio

(PER).

Date post:	03-Jun-2018
Category:	Documents
Upload:	rita-farida
View:	221 times
Download:	0 times

FDSC 4763 Ch 9 - Protein Analysis

Documents