CARB

OHYD

RATE

ANALY

SIS(

Ch. 11

+12)

I. A

naly

sis

of S

ugar

sA

-Qua

litat

ive

Det

ecti

on M

etho

dsB-

Qua

ntit

ativ

e A

naly

ses

II-

Ana

lysi

s of

Non

suga

rs(p

olys

acch

arid

es)

A-

Nut

rien

t Po

lisac

char

ides

(sta

rch,

de

xtri

ns,

glyc

ogen

etc

.) -Q

ualit

ativ

e an

alys

es-Q

uant

itat

ive

anal

yses

B-D

ieta

ryFi

ber

orSt

ruct

ural

Po

lysa

ccha

ride

s (c

ellu

lose

, hem

icel

lulo

seet

c.)

a. G

ravi

met

ric

met

hods

: 1.C

rude

fib

er2.

Det

erge

nt m

etho

ds3.

AO

AC

met

hod

b. C

hem

ical

Met

hods

Com

posi

tion

and

Occ

urre

nce

Carb

ohyd

rate

s ar

e th

e m

ost

abun

dant

and

mos

t wi

dely

con

sum

ed(>

%70

)m

acro

fo

od

com

pone

nt.

They

prov

ide

sati

ety,

an

dm

odif

yhu

man

phys

iolo

gica

lpro

cess

es.T

hey

are

a ve

ry h

eter

ogen

eous

gro

up in

str

uctu

re-

all

cont

ain

carb

on,

hydr

ogen

and

oxyg

en,

and

are

alm

ost

excl

usiv

ely

of

plan

tor

igin

(exc

epti

ons:

lact

ose

of m

ilkan

dgl

ycog

enin

mea

t).

CLA

SSIF

ICA

TIO

N:

I. M

ono

and

Olig

osa

chha

ride

s(S

ugar

s):

Suga

rs h

ave

nutr

itio

nal

and

met

abol

ic f

unct

ions

;the

ygo

vern

rhe

olog

ical

prop

erti

es,

caus

e br

owni

ng

reac

tion

s,

have

ro

le

in

food

st

abili

ty,

ar

e ra

w m

ater

ials

fo

r fe

rmen

tati

ons.

a. M

onos

acha

ride

s: P

olyh

ydro

xyal

dose

san

d ke

tose

s

Pent

oses

: Xyl

ose,

arab

inos

e(C 5

)

Hex

oses

: G

luco

se, f

ruct

ose(

C 6 )

OH

OH

CHH

CO

H

CH

H

HOH

C CH 2O

H

O

C

OH

O OH

OH

HO

CH 2O

H

1

23

456

b. O

ligos

acch

arid

es:

Som

esu

gar u

nits

(>1,

<10)

are

cond

ense

d to

geth

er

form

ing

di-,

tri-,

tetra

-sa

ccha

rides

: S

acha

rose

, lac

tose

, ra

ffino

se, s

tach

yose

O OH

OH

HO

CH

2O

HC

H2O

H

OC

H2O

H

O

HO

OH

H1

23

4

5 6

II. Non

suga

rs: P

olys

achh

arid

es

a.

Nut

rien

t po

lysa

char

ides

: Th

ey

are

met

abol

ic

rese

rves

of

plan

ts a

nd a

nim

als

,and

are

bas

ical

ly a

ll po

lym

ers

of g

luco

se (d

extr

ins,

sta

rch,

gly

coge

n)

b. S

truc

tura

l po

lysa

char

ides

: Th

ey c

onst

itut

e th

e ri

gid

mec

hani

cal

cell

wall

stru

ctur

e in

pl

ants

. Ce

llulo

se( p

olym

er o

f b-

D-G

luco

se b

(1, 4

) lin

kage

.

Repe

atin

g ce

llobi

ose

moi

ety)

O OH

OHCH

2O

H

OH

OO

O

O OH

OHCH

2O

H

O OH

OHCH

2O

H

O OH

OHCH

2O

H

O OH

OHCH

2O

H

O OH

OHCH

2O

H

O OH

OHCH

2O

H

O OH

OHCH

2O

H

n

O OH

OHCH

2O

H

OH

OO

O

O OH

OHCH

2O

H

O OH

OHCH

2O

H

O OH

OHCH

2O

H

O OH

OHCH

2O

H

O OH

OHCH

2O

H

Pect

in(g

alac

turo

nic

acid

and

met

hylg

alac

turo

nate

poly

mer

)

Hom

oglic

ans

are

poly

mer

s of

sam

e su

gar

(i.e.

xyla

n:xy

lose

poly

mer

) H

eter

oglic

ans

are

poly

mer

s of

mor

e th

an o

ne s

ugar

gro

up(i.

e.ar

abin

ogal

acta

ns)

O OH

OH

OH

OO

O

O OH

OH

O OH

OH

O OH

OH

O OH

OH

O OH

OH

CO

OH

CO

OC

H3

CO

OC

H3

CO

OH

FUN

CTI

ON

AL

PRO

PER

TIES

OF

CH

O’s

-Sto

rage

mat

eria

lfor

carb

onan

den

ergy

in

live

syst

ems

-Com

pone

ntof

Stru

ctur

e:

1.V

isco

sity

con

trol

2.Te

xtur

e co

ntro

l

3.Em

ulsi

fyin

g ag

ent

4.St

abili

zer d

ueto

wat

erbi

ndin

gca

paci

ty

Carb

ohyd

rate

con

tent

s of

som

e fo

ods

Hon

ey: 7

5-82

% f

ruct

ose

+ gl

ucos

e +

sach

aros

e

App

les:

15%

(fru

ctos

e+gl

ucos

e+sa

ccha

rose

+st

arch

)

Dri

ed f

igs:

30%

fruc

tose

+ 4

2%gl

ucos

e

Dat

es: 5

0% s

acch

aros

e

Seed

less

rai

sins

: 70%

fruc

tose

+glu

cose

; Gra

pes:

16-

17%

Mel

ons,

pea

ches

, tan

geri

nes

: <10

% s

acch

aros

e

Carr

ots:

10%

CHO

Ches

tnut

s: 3

3% s

tarc

h

Whi

te f

lour

: 70

% s

tarc

h , W

hite

Brea

d:50

% C

HO

Corn

-pat

atoe

s: %

15 s

tarc

h; C

orn

Flak

es: 8

6% C

HO

Chic

ken

brea

st: 0

%

Shor

t-cu

t to

CHO Q

uant

ificat

ion:

In g

ener

al(in

mos

t fo

od c

ompo

siti

on t

able

san

dfo

rnu

trit

iona

lla

belli

ng),

it is

ass

umed

tha

t:

Tot

alCH

O(w

ithf

iber

)=10

0-[M

oist

ure%

+Pro

tein%+F

at%+A

sh%]

or

CHO w

itho

ut c

rude

fiber

=N-f

ree

extr

act

In

calc

ulat

ing

the

calo

rifi

c va

lues

of

fo

ods,

su

gars

an

d nu

trie

nt p

olys

acch

arid

es o

f CH

O’s

are

gene

rally

ass

umed

as

havi

ng 4

cal/

g an

d st

ruct

ural

pol

ysac

char

ides

(i.e

.cru

de f

iber

) as

ha

ving

0

cal/

g.

H

owev

er,

nove

l “D

iete

tic

food

in

gred

ient

s”no

w in

clud

e co

mpo

nent

s wh

ich

are

part

ially

m

etab

oliz

ed

and

ther

efor

e do

prov

ide

som

eca

lori

es

(i.e.

olig

osac

char

ides

:1.2

kca

l/g;

sug

ar a

lcoh

ols:

2.4

kcal

/g)

thus

co

ntri

buti

ng t

o a

rath

er n

ew c

once

pt, “

diet

ary

fibr

e”.

The

alte

rnat

ive

mea

ns o

f qu

anti

fica

tion

of c

alor

ies

is b

y us

e of

a B

OM

B Ca

lorimet

er,

whic

hm

easu

res

hea

t pr

oduc

edwh

ensa

mpl

eis

bur

nt.

Tota

lCar

bohy

drat

es

CH

O’s

are

dest

roye

dby

heat

and

acid

. C

ontin

ued

heat

ing

in p

rese

nce

of a

cid

lead

sto

prod

ucts

that

cond

ense

with

phen

olic

com

poun

dsto

prod

uce

colo

red

com

poun

ds,th

enqu

antif

ied

byU

V-V

issp

ectro

phpo

tom

etric

read

ings

of a

bsor

banc

eat

490

nm

.

Ex: P

heno

l-Sul

furic

Aci

dM

etho

d(p.

172-

3)

.A

naly

ses

of S

ugar

s(W

ater

-sol

uble

CHO

’s)SA

MPL

E PR

EPA

RATI

ON

:Sa

mpl

e ha

s to

be

hom

ogen

ized

bef

ore

anal

yses

. -R

emov

al o

f “L

ipid

s an

d Pr

otei

ns"

is r

equi

red

befo

re s

tart

ing

anal

yses

, sin

ce t

hey

caus

e tu

rbid

ity

duri

ng a

naly

ses

whic

h in

terf

eres

wit

h po

lari

zati

on a

nd r

efra

ctio

n:Li

pids

are

extr

acte

d wi

th p

etro

leum

eth

er, b

ut c

are

shou

ld b

e ta

ken

not

to e

xcee

d 50

°C, s

ince

hig

her

tem

pera

ture

s m

ight

so

lubi

lize

star

ch.

Prot

eins

are

prec

ipit

ated

with

hea

vy m

etal

s us

ing

[Pb

acet

ate]

or

[ZnO

Hor

Zn

ferr

ocya

nide

]. Pr

otei

ns a

re c

opre

cipi

tate

din

co

lloid

al f

orm

. Ca

rrez

solu

tion

(1+1

) [%

15 Z

nSO

4 +

%30

K fe

rric

yani

de) i

s an

oth

er a

lter

nati

ve.

-Neu

tral

izin

gan

y ac

idit

y (b

y Ca

CO3)

, sin

ce a

cids

may

cau

se

hydr

olys

is o

f su

gars

. -R

emov

al o

f pi

gmen

ts b

y ad

sorp

tion

an

char

coal

-Ina

ctiv

atin

gen

zym

es [H

gCl 2

inac

tiva

tes

enzy

mes

]

Sam

ple

prep

arat

ion

and

extra

ctio

nsc

hem

e(fi

g.11

.1)

-Dry

sam

ple

-Fro

mde

hydr

ated

mat

eria

l, e

xtra

ctlip

idsw

ith95

:5

Chl

orof

orm

-met

hano

l

-Ext

ract

resi

due

with

80%

etha

nol

-Rem

ove

any

inte

rfer

ance

(i.e.

by

ion-

exch

ange

, Se

ppak

cartr

idge

etc)

Mon

o an

ddi

sacc

harid

esar

eob

tain

ed.

I.a.

Qua

litat

ive

Det

ecti

on M

etho

ds f

or s

ugar

s:1-

Tota

lsug

ars

::Met

hods

bas

ed o

n co

lour

reac

tion

s of

sug

ars.

-Wit

h st

rong

m

iner

al

acid

s (H

2SO

4, H

Cl,

H3P

O4

etc.

) EX

:CH

O+P

heno

l-H

2SO

4ye

llow-

oran

geco

lour

due

to c

onde

nsat

ion

of d

egra

ding

sug

ar p

rodu

cts

: 490

nmac

ids+

hex

oses

HM

F in

dica

tor

acid

s+pe

ntos

esfu

rfur

ol-W

ith

ant

hron

ein

con

c. H

2SO

4,ch

arac

teri

stic

blu

e-gr

een

colo

ur: 6

20nm

2-Spe

cific

Ana

lyse

sof

Ind

ividua

lSug

ars:

M

etho

dsba

sed

on r

educ

ing

prop

erti

es o

f m

onos

acch

arid

esas

wel

l as

red

ucin

g di

sacc

hari

des

like

lact

ose

and

mal

tose

(Cu+

+ is

red

uced

toCu

+):

A-F

EHLI

NG

Reac

tion

( Ben

edic

t’s T

est,

Mun

son

Wal

ker-

Grav

imet

ric;

La

neEy

non-

Volu

met

ric,

Som

ogyi

-Nel

son:

spe

ctro

phot

omet

ric)

:Fe

hlin

gA

:35g

CuS

O4

/500

ml;

Fehl

ing

B:[1

73g

NaK

C 4H

406(

Roch

elle

sal

t)+5

0g N

aOH

]/50

0 m

l

OC

uC

uSO

2su

gar

redu

cing

4

→

+

++→Cu

Cu

B. –

Rea

ctio

n wi

th T

olle

n's

reag

ent:

[]

oA

g]

Ag[

NH

suga

rsre

duci

ngio

n2

3

→

+

Silv

er i

ons

is

redu

ced

to t

o m

etal

lic s

ilver

, wh

ich

is

depo

site

d on

a g

lass

sur

face

, for

min

g a

“mir

ror”

.

C. F

orm

atio

n of

“osa

zone

s”:

Oxi

dati

ve c

leav

age

of h

ydro

xyl

grou

ps o

f su

gars

and

phen

ylhi

draz

ine

fo

rm

beau

tifu

l cr

ysta

lline

ye

llow

com

poun

ds, c

alle

d os

azon

es(“

diph

enyl

hydr

azon

e”)

3. P

aper

Chr

amot

ogra

phy:

I

s by

far

t

he b

est

and

sim

ples

t qu

alit

ativ

em

etho

d to

dis

ting

uish

qua

litat

ivel

y be

twee

n va

riou

s su

gars

in f

oods

.Ex

: Su

gar

solu

tion

is

spot

ted

on F

ilter

pap

er;f

or d

evel

opin

g so

lven

t sy

stem

:

[Phe

nol-H

2O,

80:2

0]

or

[H2O

:but

anol

:ace

tic

acid

, 5:

1:4]

or

man

y ot

her

alte

rnat

ive

solv

ent

syst

ems

can

be

used

. Pe

culia

riti

es o

f di

ffer

ent

suga

rs l

ead

to d

iffe

renc

es i

n M

igra

tion

rat

es, r

esul

ting

in

diff

eren

t bu

t ty

pica

l R f

valu

e fo

r di

ffer

ent

suga

rs in

a g

iven

sol

vent

sys

tem

. Gen

eral

ly:

Hig

her

Rfva

lues

indi

cate

hig

her

mob

ility

, sm

alle

r M

W :

Pent

ose>

hexo

se>d

isac

char

ides

>tri

sach

arid

es>

And

ket

ohex

ose>

aldo

hexo

se

Then

the

chr

omat

ogra

m i

s sp

raye

d w

ith

a vi

sual

isat

ion

spra

y-re

agen

t (E

x. p

-ani

nisi

nein

n-b

utan

ol) o

r ph

loro

gluc

inol

in H

Cl, a

nd

afte

rdr

ying

for

5-10

min

, is

put

in

an o

ven

at

105°

C fo

r vi

sual

isat

ion.

ABC

AB

S

Solv

ent

Rf =

A S

For S

pot A

Thin

Lay

er c

hrom

atog

raph

ySi

lica

gel a

s sta

tiona

ry p

hase

(250

µm

)Q

uant

itativ

e by

den

sito

mer

(tim

e va

ries w

ith so

lven

t, ge

nera

lly

over

nigh

t):

ABC

AB

S

Solv

ent

Rf =

A S

For S

pot A

Gas

and

Liqu

id C

hrom

atog

raph

yGC

and

HPL

C: F

or q

uant

ific

atio

n of

indi

vidu

al s

ugar

com

pone

nts,

HPL

C yi

elds

mos

t sa

tisf

acto

ry r

esul

ts.A

lso,

GC

follo

wing

for

mat

ion

ofvo

lati

le

sily

lder

ivat

ives

of

suga

rs w

ith

TMS

(tri

met

hyls

ilyle

ther

) can

be

used

fo

r qu

anti

tati

ve a

naly

ses

of in

divi

dual

sug

ars.

This

is a

req

uire

men

t, s

ince

com

poun

ds m

ust

be v

olat

ile.

Suga

rs f

orm

vol

atile

Trim

ethy

lsily

lEth

ers:

Ethe

r lin

kage

Trim

ethy

lchl

oros

ilane

SiSi

Cl

CH

3

CH

3

CH

3

O

CH

3

CH

3

CH

3

Liqu

id C

hrom

atog

raph

y

Car

bohy

drat

e A

naly

sis (

Wat

ers A

ssoc

s. H

PLC

840

380

Stat

iona

ry

Phas

e)

Solv

ents

yste

m:

H2O

/CH

3CN

Fruc

tose

Glu

cose

Mal

totri

ose

Mal

tose

•Th

e m

ost c

omm

only

use

d de

tect

or fo

r ca

rboh

ydra

te a

naly

sis

is th

e di

ffere

ntia

l re

fract

ive

inde

x (R

I) de

tect

or. B

ecau

se it

re

spon

ds to

all

anal

ytes

who

se re

fract

ive

inde

x di

ffers

from

that

of t

he m

obile

pha

se,

it fu

nctio

ns a

s a

univ

ersa

l det

ecto

r. It

is

pref

erre

d ov

er t

radi

tiona

l UV

det

ecto

r for

no

n-ch

rom

opho

ricco

mpo

unds

like

ca

rboh

ydra

tes.

En

han

ced

Se

nsit

ivit

y a

nd

Red

uce

d R

un

Tim

es U

sin

g G

rad

ien

ts

42

DE

Co

rn S

yru

p M

alt

ose

Olig

om

ers

•Col

umn:

Pre

vail™

Car

bohy

drat

e E

S,

•25

0 x

4.6m

m•M

obile

Pha

se:

Wat

er:A

ceto

nitri

le(3

8:62

)

•F

low

rate

:1.

0mL/

min

•Col

umn

Tem

p:A

mbi

ent

•Inje

ctio

n Vo

lum

e:5µ

L

Co

lum

n:

Prev

ail™

Car

boh

ydra

te E

S,

250 x

4.6

mm

Mo

bile P

hase

:A

:Ace

tonitrile

B:

Wat

er

Gra

die

nt:

Tim

e:

0 1

5

25

%B

:35 50 50

Flo

wra

te:

1.0

mL/

min

Co

lum

n T

em

p:

Am

bie

nt

Inje

ctio

n V

olu

me:

5µL

1.

Glu

cose

2.

Mal

tose

3.

Mal

totr

iose

4.

Mal

tote

trao

se5

.M

alto

pen

taos

e6

.M

alto

hex

aose

7.

Mal

tohep

taos

e8

.M

alto

octa

ose

9.

Mal

tonon

aose

10

.M

alto

dec

aose

11

.M

alto

undec

aose

12

.M

alto

dod

ecao

se1

3.

Mal

totr

idec

aose

9353

9354

Iso

crati

cG

rad

ien

t

•Col

umn:

Pre

vail™

Car

bohy

drat

e E

S, 2

50 x

4.

6mm

•Mob

ile P

hase

:W

ater

:Ace

toni

trile

(30:

70)

•Flo

wra

te:

1.0m

L/m

in•C

olum

n Te

mp:

Am

bien

t•In

ject

ion

Volu

me:

5µL

Su

gar

Alc

oh

ols

1.

Iso-

eryt

hrito

l2

.Xyl

itol

3.

Man

nitol

4.

Mal

tito

l

9352

•Col

umn:

Pre

vail™

Car

bohy

drat

e E

S,

250

x 4.

6mm

•Mob

ile P

hase

:W

ater

:Ace

toni

trile

(25:

75)

•Flo

wra

te:

1.0m

L/m

in•C

olum

n Te

mp:

Am

bien

t•In

ject

ion

Volu

me:

20µL

1.

Fruct

ose

2.

Glu

cose

3

.Sucr

ose •C

olum

n:P

reva

il™C

arbo

hydr

ate

ES

, 25

0 x

4.6m

m•M

obile

Pha

se:

A: A

ceto

nitri

leB

: Wat

er•G

radi

ent:

Tim

e:0

30

•%

B:

25 5

0•F

low

rate

:1.

0mL/

min

•Col

umn

Tem

p:A

mbi

ent

•Inje

ctio

n Vo

lum

e:20

µL

Ora

ng

e J

uic

eV

eg

eta

ble

Ju

ice

9331

9317

1.

Fruct

ose

2.

Glu

cose

3.

Sucr

ose

Co

lum

n:

Prev

ail™

Car

boh

ydra

te E

S, 250 x

4.6

mm

Mo

bile P

hase

:A

:Ace

tonitrile

B:

Wat

er

Gra

die

nt:

Tim

e:

0 1

5%

B:

25 40

Flo

wra

te:

1.0

mL/

min

Co

lum

n T

em

p:

Am

bie

nt

Inje

ctio

n V

olu

me:

5µL

Dete

cto

r:ELS

D 2

000

Clo

ver

Ho

ney

9365

I.b.

Qua

ntit

ativ

e Su

gar

Ana

lyse

s M

etho

dsTh

e fo

llowi

ng

met

hods

giv

e to

tal

suga

r co

nten

t an

d al

ways

req

uire

cal

ibra

tion

wit

h st

anda

rd s

ugar

sol

utio

ns.

b.1-

Chem

ical M

etho

ds:

Chem

ical

met

hods

are

bas

ed

on

redu

cing

pr

oper

ties

of

m

onos

acch

arid

es.

Free

al

dehy

deor

ket

ogr

oup

redu

ces

alka

line

solu

tion

s of

m

etal

lic s

alts

to

met

al o

xide

s(Cu

O) o

r fr

ee m

etal

(Ag)

.

-Gra

vim

etri

c m

etho

ds(C

u-M

unso

n-W

alke

r):

Wei

ghin

g pr

ecip

itat

ed m

etal

oxi

de-T

itri

met

ric

met

hods

a) L

ane-

Eyno

n:Su

gar

solu

tion

is a

dded

fro

m a

bur

etto

a

vigo

rous

ly

boili

ng

mix

ture

of

Fe

hlin

gA

+B

solu

tion

m

ixtu

re.

Nea

r th

e en

d po

int,

met

hyle

nebl

ue i

ndic

ator

wi

ll ch

ange

fro

m b

lue

to c

olor

less

in e

xces

s of

red

ucin

g su

gars

.b)

Alk

alin

e fe

rric

yani

dem

etho

d:2K

3Fe(

CN) 6

+2KI

2K 4

Fe(C

N) 6

+I2

Libe

rate

d io

dine

is t

itra

ted

with

Na 2

S 20 3

b.2-

Phys

ical

met

hods

-Ins

trum

enta

l met

hods

(p.18

4)

Mic

rosc

opy:

esp

ecia

llyfo

rst

arch

orig

inde

term

inat

ion

anal

ysis

Spec

ific

grav

ity:

hyd

rom

eter

sca

libra

ted

in B

rix

degr

ees

(suc

rose

% b

ywe

ight

)

Spec

trop

hoto

met

ric

met

hods

: Sin

ce in

the

ran

ge (0

-80µ

g )

the

abso

rban

ce v

alue

at

490n

m f

or r

educ

ed c

oppe

r is

in li

near

ra

nge,

spe

ctro

phot

omet

ric

read

ings

can

be

used

for

qu

anti

fica

tion

of

tota

l sug

ar c

onte

nt.

-Ref

ract

omet

ry:

Incr

easi

ng s

ugar

con

cent

rati

on i

ncre

ases

the

an

gle

of r

efra

ctio

n of

a s

olut

ion,

whi

ch i

s al

so a

ffec

ted

by

conc

entr

atio

n te

mpe

ratu

re.

Po

cket

an

d A

bbé

type

s of

re

frac

tom

eter

syi

eld

rati

o of

sol

uble

sol

ids.

Res

ults

are

pre

cise

on

ly f

or p

ure

suga

r so

luti

ons.

-Pol

arim

etry

(see

11.6

.5):

This

is

a

nond

estr

ucti

ve

and

rapi

d m

etho

d de

pend

ing

on t

he f

act

that

sug

ar s

olut

ions

, lik

eal

lco

mpo

unds

cont

aini

ngch

iral

carb

on,

have

the

abi

lity

to r

otat

e a

plan

e of

pol

ariz

ed li

ght

thro

ugh

an a

xis

para

llel t

o it

s di

rect

ion

of

prop

ogat

ion,

an

d th

e sp

ecif

ican

gle

of

rota

tion

be

ing

pr

opor

tion

al w

ith

suga

r m

olec

ular

stru

ctur

ean

dco

ncen

trat

ion.

D

extr

orot

ator

y -p

lane

pol

ariz

ed li

ght r

otat

ed c

lock

wise

(or

to th

e ri

ght)

Levo

rata

tory

-pla

ne p

olar

ized

ligh

t rot

ated

cou

nter

cloc

kwise

.

Inve

rt S

ugar

---w

hen

sucr

ose

has b

een

hydr

olyz

ed in

to g

luco

se a

nd fr

ucto

se,

the

rota

tion

chan

ges f

rom

dex

tror

otat

ory

(+66

.5) t

o le

voro

tato

ry (-

19.8

). S

o,

sucr

ose

solu

tion

is ca

lled

“Inv

ert S

ugar

”.IC

UM

SA-I

nter

nati

onal

Co

mm

issi

on

for

Uni

fied

M

etho

ds

of

Suga

r A

naly

sis

cite

s po

lari

met

ryin

sta

ndar

d m

etho

ds f

or

suga

r ın

dust

ry.

For

sach

aros

e=+6

6.5°

For

inve

rt s

ugar

sol

utio

n(gl

ucos

e+fr

ucto

se)=

-20°

-

b.3.

Enzy

mat

icM

etho

ds:G

luco

se O

xida

seSy

stem

Glu

cose

Oxi

dase

D-G

luco

se

+ O

2G

luco

nic

Aci

d +

H

2O2

Pero

xida

seH

2O2+

0

-Dia

nisi

dine

2 H

2O +

Oxi

dize

d 0-

Dia

nisi

dine

(Col

orle

ss)

(Bro

wn)

OC

H3

H3C

O

H2N

NH

2

H3C

OO

CH

3

HN

NH

II. A

. Ana

lysi

s of

Nut

rien

t Po

lisac

char

ides

STARC

H(1

1.4.

1):M

ost

abun

dant

CHO c

ompo

nent

of f

oods

.-Q

ualit

ativ

e m

etho

dsfo

rst

arch

dete

rmin

atio

n:

-Pol

ariz

ing

light

Mic

rosc

opy:

inta

ct s

tarc

h gr

anul

es c

an b

e id

enti

fied

as t

oth

eir

sour

ce:l

ike

pota

toe,

cor

n, w

heat

sinc

e gr

anul

esi

ze ,

shap

e, io

dine

stai

ning

char

acte

rist

ics

of e

ach

diff

er(s

ee11

.6.1,

p.18

4)

-Iod

ine

test

: lin

ear

stra

igh-

chai

n am

ylos

ere

acts

wit

h io

dine

tfo

rm

a bl

ue-b

lack

st

arch

-iodi

de

com

plex

, wh

erea

s th

bran

ched

ch

ains

of

amyl

opec

tins

form

pa

le

re

d vi

ole

com

plex

es.

Thou

gh c

olor

int

ensi

ty i

s no

t sp

ectr

opho

tom

etri

cre

acti

on

is

very

se

nsit

ive(

0.00

2g/m

l) an

d ap

plic

able

t

star

ch,d

extr

ins

and

glyc

ogen

).

-Qua

ntit

ativ

e M

etho

dsfo

rto

talS

tarc

h:

Star

chHyd

rolysis:

Follo

wing

Carr

ezTre

atmen

t*fo

rre

mov

ing

inte

rfer

ing

subs

tanc

es,

star

chis h

ydro

lized

with

acids

(0.4

NH

2SO

4)

and/

or en

zymes

[

firs

twi

th

αan

d β-

amylas

e en

zymes

to br

eak

amylos

ech

ains

to

malto

se a

nd g

luco

se;t

hen

with

amylog

luco

sida

se(o

rgluc

oamylos

e) e

nzym

eto

bre

ak a

mylop

ectins

]to

gluco

se,

which

can

then

be

dete

rmined

co

lorimet

rica

llywi

th

anth

rone

reag

ent

or

quan

tified

by

any

othe

r met

hod

(See

Fig.

11-

7,p.

180.

)

*CARR

EZ T

REATM

ENT:F

irst

addi

nga

solution

of p

otas

ium

hexa

cyan

ofer

rate

K 4[F

e(CN

) 6] th

enfo

llowe

dby

firs

tZn

SO

4 an

dth

enNaO

Hso

lution

s, t

hus

brea

king

any

emulsion

s, p

recipita

ting

prot

eins

, th

enge

tting

rid

of p

igmen

tsaf

ter

which

the

clea

rfiltra

teis u

sed

dire

ctly

in s

pecific

enzy

me

cata

lyse

dre

action

sfo

rea

chclas

sof

CH

O..

Hyd

roco

lloid

sot

hert

han

Sta

rch:

Food

Gum

s•

Stru

ctur

alhe

tero

geni

ty:li

near

orbr

anch

ed,

neut

ralo

rani

onic

,diff

erin

gso

lubi

lity

degr

ees;

ther

efor

eve

ryco

mpl

exan

alyt

ical

sche

mes

have

been

deve

lope

d(F

ig.1

1-8,

p.1

82-3

)

•M

etho

dsde

pend

on e

xtra

ctio

nfo

llow

edby

fract

iona

tion

and

hydr

olys

isfo

llow

edby

spec

ific

iden

tific

atio

nte

sts

Pec

tin•

~80%

is D

-gal

actu

roni

cac

id.

•S

apon

ifyw

ithN

aOH

, the

nac

idify

and

add

Ca+

2

topr

ecip

itate

pect

in.

•C

olle

ctC

ape

ctat

e, w

ash,

dry

and

wei

gh.

•D

egre

eof

est

erifi

catio

n(D

E):

The

isol

ated

pect

inis

was

hed

with

acid

ified

alco

holt

oco

nver

tto

acid

. Afte

rwas

hing

, titr

atio

nw

ithN

aOH

solu

tion

give

sno

nest

erifi

edgr

oups

. Afte

rsap

onifi

catio

nof

pec

tins,

bac

ktit

ratio

nw

ithst

anda

rdis

edac

idso

lutio

nto

dete

rmin

eex

cess

base

give

sD

E.

“Die

tary

Fibe

r”Fo

odco

mpo

nent

sno

t di

gest

ible

bym

amm

als

(Ch.

12)

•H

elp

prot

ect

agai

nst

colo

nca

ncer

•H

elp

norm

aliz

ebl

ood

lipid

s•

Redu

ceca

rdio

vasc

ular

dise

ases

•Sl

owgl

ucos

eab

sorb

tion

and

insu

linse

cret

ion

•H

elps

prev

ent

cons

tipa

tion

•D

RV(D

ieta

ryRe

fere

nce

Valu

e): 2

5g/2

000k

cal

Stru

ctur

al P

olis

acch

arid

es:

Stru

ctur

al P

olys

acch

arid

es a

re c

ompo

nent

s of

cel

l wal

ls o

fpl

ants

Plan

t ce

ll wa

ll co

ntai

nsLi

gnin

(non

CHO

)Ce

lulo

seCr

ude

fibe

r**

Hem

icel

lulo

seD

ieta

ry f

iber

*Pe

ctin

Gu

ms

Muc

ilage

Prot

eins

&Lip

ids

Die

tary

Fibe

r*:

Sum

of

the

nond

iges

tibl

eco

mpo

nent

sof

foo

ds

a. I

nsol

uble

fibe

r: C

rude

Fibe

r**:

b.So

lubl

efib

er:P

ecti

n+Gu

ms+

Muc

ilage

(Hyd

roco

lloid

s)

Solubl

efib

er

Tota

l, s

olub

le, i

nsol

uble

diet

ary

fiber

con

tent

sof

sel

ecte

dfo

ods

g/10

0 g

of F

ood

16.9

29.

737.

71O

atbr

an

2.89

6.66

2.01

5.25

1.02

1.41

Gre

enB

eans

Dry

bean

s

3.93

2.81

1.10

Car

rots

9.29

4.17

5.07

Pru

nes

1.12

0.59

0.53

Apr

icot

s

Tota

lDF

Inso

lubl

eD

FS

olub

leD

FFo

od

Bot

anis

tscl

assi

fyfib

er a

s:

1.C

ellw

allP

olys

acch

arid

es2.

Non

-cel

lwal

lpol

ysac

char

ides

1.C

ellw

allP

:S.:

Cel

lulo

se(lo

ngch

ain

mol

ecul

esm

ade

upof

glu

cose

arra

nged

in

mic

rofib

rils,

with

par

alle

l gro

ups

form

ing

stro

ngbu

ndle

s)H

emic

ellu

lose

(het

erog

eneo

usm

olec

ules

cont

aini

nga

num

bero

f su

gars

,and

tyin

gce

llulo

sem

icro

fibril

s; s

olub

lein

dilu

teal

kali

but n

ot in

w

ater

, Pe

ctin

(uro

nic

acid

poly

mer

solu

ble

in h

otw

ater

and

form

sge

ls)

2. N

on-c

ellw

allP

.S.:P

olys

acch

arid

esno

t inv

olve

d in

cel

lwal

lstru

ctur

e an

d ar

e no

t nut

ritio

nal.

They

incl

ude

hydr

ocol

loid

ssu

chas

muc

ilage

s(gu

aran

dlo

cust

bean

sgu

ms

) pla

ntex

udat

egu

ms(

arab

ic, t

raga

cant

hgu

ms)

and

alga

lpol

ysac

char

ides

(alg

inat

es, a

gar.

Car

rage

enan

). G

ener

ally

they

for

m

visc

ous

solu

tions

in w

ater

, but

ins

olub

le in

alc

ohol

s.

Lign

inis

a n

on-C

HO

thre

edi

men

sion

alpo

lym

erw

ith~4

0 ph

enol

units

cova

lent

lylin

ked

tohe

mic

ellu

lose

.

Die

tary

Fibe

r ME

THO

DS

•1.

Gra

vim

etric

:Dig

estib

leC

HO

’s, l

ipid

s, p

rote

ins

are

sele

ctiv

ely

solu

biliz

edby

chem

ical

san

den

zym

es; u

ndig

estib

les

are

colle

cted

byfil

tratio

nan

dqu

antit

ated

byw

eigh

ing.

•2.

Che

mic

al:D

iges

tible

CH

O’s

are

rem

oved

byen

zym

atic

dige

stio

n, fi

ber c

ompo

nent

sar

ehy

drol

ysed

byac

id, a

ndm

onos

acch

arid

esin

the

acid

hydr

olys

ate

repr

esen

tfib

er.

Extr

actio

n Pr

oced

ures

for S

elec

ted

fiber

com

pone

nts:

Cellu

lose

: In

solu

ble

resi

due

from

ext

ract

ion

with

17

.5%

NaO

H; c

an b

e so

lubi

lized

by a

ddin

g 72

% H

2SO

4

Pect

in: E

xtra

ct w

ith h

ot w

ater

or a

queo

us a

mm

oniu

m o

xala

te

(0.5

%).

whi

ch c

hela

tes

calc

ium

and

sol

ubiliz

esot

herw

ise

inso

lubl

e C

a pe

ctat

em

olec

ules

. The

n pr

ecip

itate

with

95%

et

hano

l.

Hem

icel

lulo

ses:

Ext

ract

with

25%

KO

H o

r 10%

NaO

H; m

ake

extra

ct a

cidi

c by

add

ing

acet

ic a

cid.

The

n pr

ecip

itate

by

addi

ng 4

vol

umes

of e

than

ol

Lign

in:.

Del

igni

ficat

ion:

Tre

at w

ith C

lor s

ulfit

e to

bre

ak c

hain

a.Gr

avim

etric

Met

hods

:a.

1.

Crud

e Fi

ber

::Si

nce

1850

s, i

t is

bein

gde

term

ined

as

mat

eria

l ins

olub

le in

dilu

te a

cid

and

dilu

te a

lkal

i und

er s

peci

fied

co

ndit

ions

(Wee

nde

met

hod

). Se

quen

tial

extr

acti

onof

sam

ple

with

1.25

N H

2SO

4, 1.2

5NN

aOH

, the

inso

lubl

ere

sidu

ebe

ing

drie

dwe

ighe

d,

ashe

dto

corr

ect

for

min

eral

s. T

ecat

orIn

c. h

as a

utom

ated

the

W

eend

em

etho

d(F

iber

Tec)

.Th

isan

alys

isdo

esno

t de

tect

hem

icel

lulo

se,

pect

ins

and

hydr

ocol

loid

s,

but

only

cellu

lose

and

ligni

n.

Sout

hgat

eha

s de

velo

ped

the

met

hod.

a.2.

Dete

rgen

tM

etho

ds:

Two

met

hods

have

been

deve

lope

d:

1. A

cid

dete

rgen

tfi

ber

met

hod

: det

erm

ines

ligni

n+

cellu

lose

2.N

eutr

al-d

eter

gent

fibe

r= d

eter

min

esac

idde

terg

entf

iber

+ he

mic

ellu

lose

ND

F =

Neu

tral

-det

erge

nt f

iber

(hem

icel

lulo

se+c

ellu

lose

+lig

nin)

ND

F-A

DF

= he

mic

ellu

lose

(Nei

ther

incl

ude

pect

ins

orhy

droc

ollo

ids)

Grou

nd d

ry s

ampl

e(1

mm

)

Refl

ux w

ith

H2S

O4

and

CTA

B so

l’n

Cellu

lose

, lig

nin

Sepa

rate

all

solu

ble

com

pone

nts

Dry

wei

gh

Filt

er, w

ash

with

hot

wate

r an

d ac

eton

e

ACI

D D

ETER

GENT F

IBER

Fig

1: T

he a

cid

dete

rgen

t fibe

r met

hod

Det

erge

nt M

etho

d fo

r Dieta

ry F

iber

Grou

nd d

ry s

ampl

e(1

mm

)

Refl

ux w

ith

SLS*

+ED

TA s

ol’n

Filt

er, w

ash

with

hot

wate

r an

d ac

eton

e

Dry

,wei

ghin

solu

bles

(W1)

Det

erm

ine

ash

(W2)

by

Burn

ing

Sepa

rate

all

solu

ble

com

pone

nts

Cellu

lose

, lig

nin,

min

eral

sH

emic

ellu

lose

(W1)

+ash

NDF

= W

1 –

W2

Fig

2: T

he n

eutr

al d

eter

gent

(*SL

S:So

dium

Laur

ylSu

lfat

e)fibe

r met

hod

Enzy

mat

ic M

etho

d fo

r Dieta

ry F

iber

Grou

nd, d

efat

ted

sam

ple

Aut

ocla

ve, E

xtra

ctio

n,

Cent

rifu

gati

on

Cent

rifu

gate

Extr

act

Am

ylog

luco

sida

sean

d pa

ncre

atin

for

hydr

olys

ing

star

ch a

nd p

rote

ins

Filt

rati

on

Was

h, d

ry, w

eigh

Inso

lubl

e di

etar

y fi

ber

Mem

bran

e Fi

ltra

tion

Was

h, d

ry, w

eigh

Solu

ble

diet

ary

fibe

r

Fig

3:Det

erminat

ion

of d

ieta

ry f

iber

using

enz

ymat

ic(M

euse

r)met

hod

a.3.

The

AO

AC

Met

hod

•Th

ism

etho

dco

mbi

nes

crud

efi

ber,

det

erge

ntfi

ber

and

Sout

hgat

em

etod

olog

ies

(see

fig.

12.1,

and

Tabl

e12

-1, p

.192

-5

for

tota

ldie

tary

fibe

r an

alys

is).

•Pr

inci

ple:

D

ryfa

t-ex

trac

ted

grou

ndfo

odsa

mpl

esar

een

zym

atic

ally

dige

sted

with

α-am

ylas

e, a

myl

oglu

cosi

dase

, and

prot

ease

tore

mov

epr

otei

nsan

dst

arch

. Ins

olub

lefi

ber

is c

olle

cted

byfi

ltra

tion

. Sol

uble

fibe

r is

pre

cipi

tate

dby

brin

ging

filt

rate

to78

%

eth

anol

and

colle

cted

byfi

ltra

tion

. The

filt

ered

fibe

r re

sidu

esar

ewa

shed

with

etha

nola

ndac

eton

e, o

ven

–dri

edan

dwe

ighe

d.

The

fibe

r re

sidu

eis

ana

lyse

dfo

rpr

otei

n an

das

h.

Tota

lfib

er=

resi

due

fibe

r we

ight

-(w

tof

pro

tein

+ w

tof

ash

)

b. C

hem

ical

Die

tary

Fib

er A

naly

ses

ENGL

YST-

CUM

MIN

GS P

roce

dure

Def

init

ion:

Fi

ber=

Sum

of a

llno

n-st

arch

mon

osac

char

ides

+ lig

nin

Prin

cipl

eof

EN

GLYS

T-CU

MM

INGS

Pro

cedu

re:

Star

chis

fir

stge

lati

nise

dan

dth

enen

zym

atic

ally

(pul

lula

nase

and

amyl

oglu

cosi

dase

)dig

este

d; r

emai

ning

non-

star

chpo

lysa

ccha

ride

sar

ehy

drol

ysed

byH

2SO

4 to

liber

ate

free

mon

osac

char

ides

, whi

char

ede

term

ined

eith

erby

GC o

rby

colo

rim

etri

cm

etho

ds. T

here

sult

is t

otal

diet

ary

fibe

r.

Tom

easu

rein

solu

ble

fibe

r, e

xtra

ctth

ewa

ter-

solu

ble

fibe

r by

heat

ing

the

dige

sted

mix

ture

for

30 m

inut

esin

a b

oilin

gwa

ter-

bath

.The

prec

ipit

ate

afte

rce

ntri

fugi

ngis

inso

lubl

efi

ber.

So

cont

inue

the

sam

ewa

ywi

thth

ispr

ecip

itat

ewi

thth

ehy

drol

ysis

step

. So

lubl

efi

ber=

Tot

alfi

ber-

Inso

lubl

efi

ber

Dra

wbac

ks:li

gnin

and

resi

stan

tst

arch

are

not

mea

sure

dby

this

met

hod.

Any

suga

rin

itia

llypr

esen

tsh

ould

be e

xtra

cted

prev

ious

ly, o

rwi

llyi

eld

high

resu

lts.

. Se

eFi

g. 1

2-2,

p. 1

96-7

.