204 / 311
© 2015 AOAC INTERNATIONAL
(
t
)
+DUGHQHG ¿OWHU SDSHU
²:LWK P UHWHQWLRQ
.
C. Reagents
Note
8VH KLJK TXDOLW\ GLVWLOOHG RU GHLRQL]HG ZDWHU IRU DOO ZDWHU
additions.
(
a
)
Acetate buffer (100 mM, pH 5.0)
²:HLJK J RU SLSHW
P/ JODFLDO DFHWLF DFLG DQG WUDQVIHU LPPHGLDWHO\ WR D ÀDVN
TXDQWLWDWLYHO\ WUDQVIHU ZHLJKHG DFLG ZLWK +
2
2 ULQVHV %ULQJ YROXPH
WR FD P/ :KLOH VWLUULQJ VROXWLRQ RQ D PDJQHWLF VWLU SODWH DGMXVW
S+ WR ZLWK 0 1D2+ VROXWLRQ 'LOXWH WR / ZLWK +
2
O.
7KLV FDQ EH GRQH LQ DQ (UOHQPH\HU ÀDVN RU EHDNHU WKDW KDV EHHQ
PDGH YROXPHWULF E\ ZHLJKLQJ RU WUDQVIHUULQJ / ZDWHU LQWR WKH
YHVVHO DQG WKHQ HWFKLQJ WKH PHQLVFXV OLQH IRU WKH NQRZQ YROXPH
(
b
)
+HDW VWDEOH Į DP\ODVH VROXWLRQ
²/LTXLG KHDW VWDEOH
Į DP\ODVH H[DPSOHV 3URGXFW 7HUPDP\O
/ 1RYR]\PHV
1RUWK $PHULFD )UDQNOLQWRQ 1& 86$ 3URGXFW 0XOWLIHFW $$
/ *HQHQFRU ,QWHUQDWLRQDO 5RFKHVWHU 1< 86$ RULJLQ
Bacillus
licheniformis
RU HTXLYDOHQW 6KRXOG QRW FRQWDLQ JUHDWHU WKDQ
JOXFRVH S+ RSWLPD PXVW LQFOXGH ±
Based on Bacterial Amylase Unit (BAU) method.
—
$SSUR[LPDWHO\
%$8 P/ RI FRQFHQWUDWHG HQ]\PH %$8
LV GH¿QHG DV WKH DPRXQW RI HQ]\PH WKDW ZLOO GH[WULQL]H VWDUFK DW
WKH UDWH RI PJ PLQ DW S+ DQG & ,I PRGL¿FDWLRQV
RI YROXPH GHOLYHUHG DUH QHFHVVDU\ GXH WR HQ]\PDWLF DFWLYLW\ RI
WKH HQ]\PH XVHG WKH YROXPH XVHG SHU WHVW SRUWLRQ VKRXOG GHOLYHU
DSSUR[LPDWHO\
%$8
7KH HQ]\PHV VKRXOG EH RI D SXULW\ PHHWLQJ WKH VSHFL¿FDWLRQV
listed in
991.43
(
see
EXW DV PRGL¿HG EHORZ IRU DSSOLFDWLRQ
LQ WKH DVVD\ IRU GLHWDU\ VWDUFK 7KH HQ]\PH SUHSDUDWLRQ XVHG PXVW
EH YDOLGDWHG ZLWKLQ ODERUDWRU\ WR YHULI\ HI¿FDF\ DV ZHOO DV ODFN
RI LQWHUIHUHQFH 5HFRPPHQGHG YDOLGDWLRQ DQDO\]H
J WHVW
SRUWLRQV RI SXUL¿HG JOXFRVH VXFURVH DQG SXUL¿HG FRUQ VWDUFK ZLWK
WKH HQ]\PDWLF SRUWLRQ RI WKH GLHWDU\ VWDUFK DVVD\ DQG XVLQJ D IUHH
JOXFRVH YDOXH RI ]HUR LQ FDOFXODWLRQV $QDO\VHV ZLWK FDQGLGDWH
HQ]\PH VKRXOG JLYH YDOXHV RI >PHDQ VWDQGDUG GHYLDWLRQ 6' @
JOXFRVH VWDUFK DQG VXFURVH RQ D
dry matter basis. To test for interference from release of glucose
IURP ¿EHU FDUERK\GUDWHV DQDO\]H J WHVW SRUWLRQV RI Į FHOOXORVH
DQG EDUOH\ ȕ JOXFDQ WKDW DUH QRW FRQWDPLQDWHG ZLWK IUHH JOXFRVH
ZLWK WKH HQ]\PDWLF SRUWLRQ RI WKH GLHWDU\ VWDUFK DVVD\ 5HFRYHU\
of these substrates should be less than 0.5% on a dry matter basis
>
see
991.43
(
see
@ 8VH $2$& DSSURYHG PHWKRGV IRU
GHWHUPLQDWLRQ RI GU\ PDWWHUV RI WKH VDPSOHV (Q]\PH SUHSDUDWLRQV
must not contain appreciable concentrations of glucose (<0.5%),
RU EDFNJURXQG DEVRUEDQFH UHDGLQJV ZLOO LQWHUIHUH ZLWK WHVW VDPSOH
measurements.
(
c
)
Diluted amyloglucosidase solution.
—Dilute concentrated
amyloglucosidase with 100 mM sodium acetate buffer,
C
(
a
), to
JLYH P/ RI VROXWLRQ SHU WHVW SRUWLRQ ZLWK WR P/ H[FHVV $GG
RI QHHGHG EXIIHU WR DQ DSSURSULDWHO\ VL]HG JUDGXDWHG F\OLQGHU
Pipet concentrated amyloglucosidase into buffer, rinsing tip by
WDNLQJ XS DQG H[SHOOLQJ EXIIHU LQ WKH JUDGXDWHG F\OLQGHU %ULQJ WR
GHVLUHG YROXPH ZLWK DGGLWLRQDO EXIIHU &DS F\OLQGHU ZLWK SODVWLF
¿OP DQG LQYHUW F\OLQGHU UHSHDWHGO\ WR PL[ 7KH FRQFHQWUDWHG
amyloglucosidase used should not contain greater than 0.5%
JOXFRVH DQG VKRXOG KDYH D S+ RSWLPXP RI
DQG S+ VWDELOLW\
EHWZHHQ ± H[DPSOH RI FRQFHQWUDWHG DP\ORJOXFRVLGDVH
3URGXFW ( $0*') 0HJD]\PH ,QWHUQDWLRQDO ,UHODQG /WG %UD\
&R :LFNORZ ,UHODQG RULJLQ
Aspergillus niger
RU HTXLYDOHQW
(
1
)
Based on release of glucose from soluble starch or
glycogen.
² 8 P/ XQLW RI HQ]\PH DFWLYLW\ LV GH¿QHG DV
WKH DPRXQW RI HQ]\PH UHTXLUHG WR UHOHDVH PROH JOXFRVH PLQ DW
S+ DQG &
(
2
)
Based on p-nitrophenyl-
ȕ
-maltoside method.
² XQLWV P/
XQLW LV GH¿QHG DV WKH DPRXQW RI HQ]\PH UHTXLUHG WR UHOHDVH PROH
p
-nitrophenol from
p
QLWURSKHQ\O ȕ PDOWRVLGH PLQ DW S+ DQG
& )ROORZ WKH SURWRFRO GHVFULEHG LQ
C
(
b
) for standards and
SURFHGXUH IRU WHVWLQJ DGHTXDF\ RI HQ]\PH DFWLYLW\ DQG ODFN RI VLGH
DFWLYLW\
7KH HQ]\PH XVHG PXVW EH YDOLGDWHG ZLWKLQ ODERUDWRU\ WR YHULI\
HI¿FDF\ DV ZHOO DV ODFN RI LQWHUIHUHQFH 8VH WKH VDPH YDOLGDWLRQ
SURFHGXUH DV GHVFULEHG IRU KHDW VWDEOH Į DP\ODVH
C
(
b
).
(
d
)
Benzoic acid solution (0.2%)
²:HLJK J EHQ]RLF DFLG
VROLG $&6 UHDJHQW !
SXULW\ DQG DGG WR D ÀDVN %ULQJ ÀDVN
WR / YROXPH ZLWK +
2
2 $GG PDJQHWLF VWLU EDU VWRSSHU ÀDVN DQG
DOORZ WR VWLU RYHUQLJKW WR GLVVROYH EHQ]RLF DFLG 7KLV FDQ EH GRQH
LQ DQ (UOHQPH\HU ÀDVN RU EHDNHU WKDW KDV EHHQ PDGH YROXPHWULF E\
ZHLJKLQJ RU WUDQVIHUULQJ / ZDWHU LQWR WKH YHVVHO DQG WKHQ HWFKLQJ
WKH PHQLVFXV OLQH IRU WKH NQRZQ YROXPH
(
e
)
GOPOD reagent.
—(
1
)
Mixture of glucose oxidase,
7000 U/L, free from catalase activity; peroxidase, 7000 U/L;
and 4-aminoantipyrine, 0.74 mM
²3UHSDUH E\ GLVVROYLQJ J
1D
2
+32 GLEDVLF DQK\GURXV DQG J .+
2
PO LQ FD P/ +
2
O
LQ D / YROXPHWULF ÀDVN 8VH +
2
O to rinse chemicals into bulb of
ÀDVN 6ZLUO WR GLVVROYH FRPSOHWHO\ $GG J SKHQRO $&6 JUDGH
DQG
J DPLQRDQWLS\ULQH 8VH +
2
O to rinse chemicals into
EXOE RI ÀDVN 6ZLUO WR GLVVROYH FRPSOHWHO\ $GG JOXFRVH R[LGDVH
8 DQG SHUR[LGDVH
8 ULQVH HQ]\PHV LQWR ÀDVN
ZLWK +
2
2 DQG VZLUO JHQWO\ WR GLVVROYH ZLWKRXW FDXVLQJ H[FHVVLYH
IRDPLQJ %ULQJ WR / YROXPH ZLWK +
2
2 6HDO DQG LQYHUW UHSHDWHGO\
WR PL[ )LOWHU VROXWLRQ WKURXJK D JODVV ¿EHU ¿OWHU ZLWK P
retention,
B
(
s
6WRUH LQ D VHDOHG DPEHU ERWWOH DW FD & 5HDJHQW
life: 1 month. Before use in test sample determinations, determine a
VWDQGDUG FXUYH IRU WKH UHDJHQW XVLQJ D SRLQW VWDQGDUG FXUYH XVLQJ
C
(
e
) and
C
(
f
) according to
D
(
b
).
(
2
$OWHUQDWLYHO\ XVH DQRWKHU $2$& DSSURYHG JOXFRVH
VSHFL¿F DVVD\ WKDW KDV SDVVHG LQ ODERUDWRU\ YDOLGDWLRQ WR DFFXUDWHO\
determine glucose concentrations of glucose standard solutions
DQG JLYH YDOXHV HTXLYDOHQW WR WKH YDOXHV OLVWHG IRU GHWHUPLQDWLRQ
RI HI¿FDF\ RI HQ]\PHV 5HFRPPHQGHG YDOLGDWLRQ DQDO\]H DOO ¿YH
JOXFRVH ZRUNLQJ VWDQGDUG VROXWLRQV DQG PJ WHVW SRUWLRQV RI
SXUL¿HG JOXFRVH SXUL¿HG VXFURVH DQG SXUL¿HG FRUQ VWDUFK WKDW KDYH
EHHQ SURFHVVHG WKURXJK WKH HQ]\PDWLF K\GURO\VLV SRUWLRQ RI WKH
GLHWDU\ VWDUFK SURFHGXUH DQG XVLQJ D IUHH JOXFRVH YDOXH RI ]HUR LQ
FDOFXODWLRQV 7KH JOXFRVH YDOXHV RI WKH ZRUNLQJ VWDQGDUG VROXWLRQV
VKRXOG EH SUHGLFWHG J JOXFRVH P/ 2Q D GU\ PDWWHU EDVLV WKH
FRQWURO VDPSOH JOXFRVH VKRXOG JLYH D GLHWDU\ VWDUFK YDOXH PHDQ
6' RI FRUQ VWDUFK DW
DQG VXFURVH
(
f
)
Glucose working standard solutions.
—0, 250, 500, 750, and
J P/ 'HWHUPLQH WKH GU\ PDWWHU RI SRZGHUHG FU\VWDOOLQH
glucose (purity !
E\ DQ $2$& DSSURYHG PHWKRG :HLJK
approximately 62.5, 125, 187.5, and 250 mg portions of glucose and
UHFRUG ZHLJKW WR
J 5LQVH HDFK SRUWLRQ RI JOXFRVH IURPZHLJK
SDSHU LQWR D VHSDUDWH P/ YROXPHWULF ÀDVN ZLWK
EHQ]RLF
acid solution,
C
(
d
DQG VZLUO WR GLVVROYH %ULQJ HDFK VWDQGDUG WR
P/ YROXPH ZLWK
EHQ]RLF DFLG VROXWLRQ
C
(
d
WR JLYH IRXU
LQGHSHQGHQW JOXFRVH VWDQGDUG VROXWLRQV 7KH
EHQ]RLF DFLG
solution,
C
(
d
VHUYHV DV WKH J P/ VWDQGDUG VROXWLRQ 0XOWLSO\
weight of glucose by dry matter percentage and percentage purity
DV SURYLGHG E\ WKH PDQXIDFWXUHU LQ WKH FHUWL¿FDWH RI DQDO\VLV DQG
GLYLGH E\ P/ WR FDOFXODWH DFWXDO JOXFRVH FRQFHQWUDWLRQV RI WKH
solutions. Prepare solutions at least one day before use to allow