名稱:直鏈淀粉/支鏈淀粉（膠淀粉）檢測(cè)試劑盒
規(guī)格: 100次
產(chǎn)地:愛(ài)爾蘭
用途:用于測(cè)量谷物和面粉中直鏈淀粉/支鏈淀粉的比例和淀粉含量。
標(biāo)配:6個(gè)玻璃瓶
在水中的溶解度:易溶
pH值:中性
氣味:無(wú)
狀態(tài):粉末和液體
保持期:10年以上（冰箱內(nèi)）
成分:
名稱 比例
Concanavelin A 1瓶（玻璃）
淀粉葡萄糖苷酶/α-淀粉酶 1瓶（玻璃）
葡萄糖緩沖液 1瓶（聚丙烯
直鏈淀粉/支鏈淀粉(膠淀粉)檢測(cè)試劑盒 100次 愛(ài)爾蘭 型號(hào):K-AMYL
庫(kù)號(hào)：M248897 查看hh
midwest-group
midwest-group
midwest-group
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midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
名稱:直鏈淀粉/支鏈淀粉(膠淀粉)檢測(cè)試劑盒
規(guī)格: 100次
產(chǎn)地:愛(ài)爾蘭
用途:用于測(cè)量谷物和面粉中直鏈淀粉/支鏈淀粉的比例和淀粉含量。
標(biāo)配:6個(gè)玻璃瓶
在水中的溶解度:易溶
pH值:中性
氣味:無(wú)
狀態(tài):粉末和液體
保持期:10年以上(冰箱內(nèi))
成分:
名稱 比例
Concanavelin A 1瓶（玻璃）
淀粉葡萄糖苷酶/α-淀粉酶 1瓶（玻璃）
葡萄糖緩沖液 1瓶（聚丙烯）
葡萄糖檢測(cè)試劑 1瓶（玻璃）
葡萄糖標(biāo)準(zhǔn)品和淀粉參考品 各1瓶（玻璃和塑料）
詳細(xì)信息
前言:
谷類淀粉的許多特性決定其終用途，這些取決于直鏈淀粉/支鏈淀粉的比率。這些特性包括糊化和凝膠化，溶解度，抗性淀粉的形成以及整顆大米的烹飪和構(gòu)造特性。因此，淀粉中直鏈淀粉含量的測(cè)定是淀粉加工的一個(gè)重要的質(zhì)量參數(shù)。
常用的測(cè)定谷物淀粉中直鏈淀粉含量的方法是利用電勢(shì)，電流測(cè)定或直鏈淀粉的碘結(jié)合能力比色測(cè)定直鏈淀粉-碘色合配合物。然而，這些方法具有不確定性。支 鏈淀粉-碘復(fù)合物也可以形成，這樣降低了利用非比色法測(cè)定的游離碘離子的濃度，并且用比色法測(cè)量時(shí)，該復(fù)合物可能和直鏈淀粉-碘復(fù)合物吸收相同波長(zhǎng)的光。 這種復(fù)合物致使直鏈淀粉的測(cè)定含量超過(guò)實(shí)際含量，需要進(jìn)行校正。Gibson等詳細(xì)列舉了使用這些方法所遇到的許多其他問(wèn)題。
支鏈淀粉結(jié)合ConA的特殊復(fù)合物為淀粉中直鏈淀粉的測(cè)定提供了一種替代方法，而且不存在不確定性問(wèn)題。在pH值，溫度和離子強(qiáng)度的條件下，ConA 特異性結(jié)合分支多糖并形成沉淀，這種結(jié)合以多個(gè)非還原性末端基團(tuán)上的α-D-吡喃葡萄糖基或α-D-吡喃甘露糖基單位為基礎(chǔ)。因此，ConA可以有效結(jié)合 淀粉中的支鏈淀粉成分，但是不能結(jié)合線性為主的直鏈淀粉成分。
此方法是Yun和Matheson改進(jìn)的ConA方法。分析之前用乙醇預(yù)處理去除脂質(zhì)。
原理：
淀粉樣品通過(guò)加熱*地溶解在二甲基亞砜（DMSO）里。用乙醇沉淀淀粉去除其中的脂質(zhì)，回收沉淀的淀粉。用醋酸/鹽溶液溶解沉淀的樣品，加入ConA， 特異性沉淀支鏈淀粉，離心去除沉淀。單位體積上清液中的直鏈淀粉用酶水解為D-葡萄糖，然后用葡萄糖氧化酶/過(guò)氧化物酶試劑進(jìn)行測(cè)定。另外一份單位體積醋 酸/鹽溶液中的總淀粉同樣用酶水解為D-葡萄糖，然后加入葡萄糖氧化酶/過(guò)氧化物酶，用比色法測(cè)定。
根據(jù)ConA沉淀樣品的上清液和與總淀粉樣品中的GOPOD在510 nm處的吸光光度值之比判斷直鏈淀粉在總淀粉中的含量。
該方法適用于所有的純淀粉和谷物粉。
精確性
樣品如為純淀粉，相對(duì)標(biāo)準(zhǔn)偏差為<5%。
樣品如為谷物面粉，相對(duì)標(biāo)準(zhǔn)偏差為～10%。
試劑盒：
瓶子1：凍干的Con A （伴刀豆球蛋白 A concanavalin A) ，200 mg，-20℃下穩(wěn)定性>5年。
瓶子2：淀粉葡糖苷酶【200U，條件為消化對(duì)硝基苯基β-麥芽糖苷（也就是3300U，條件為pH4.5，40℃下消化淀粉）】加上真菌α-淀粉酶（500U ，條件為pH5.0，40℃下消化 Ceralpha 試劑），2mL，4℃下穩(wěn)定性>5年。
瓶子3：GOPOD 試劑緩沖液。磷酸鉀緩沖液（1M，pH7.4），對(duì)羥苯甲酸（0.22M）和*（0.02% W/W）。4℃下穩(wěn)定性> 3年。
瓶子4：GOPOD試劑酶。葡糖氧化酶（>12,000U）加上過(guò)氧化物酶（>650U）和4-氨基安替比林（80mg）。凍干粉，-20℃下穩(wěn)定性>5年。
瓶子5：D-葡萄糖標(biāo)準(zhǔn)溶液（5 mL,1.0mg/mL）溶于苯甲酸0.2%（w/v）。室溫下穩(wěn)定性>5年。
瓶子6：淀粉參考樣品（含有特定含量的直鏈淀粉），室溫下穩(wěn)定性>5年。
Amylose/Amylopectin Kits
For the measurement of amylose/amylopectin ratio and content in cereal starches and flours. Based on a Con A precipitation procedure.
Content: 100 assays per kit
Appearance Four glass vials plus two plastic vials
Specific Gravity Not applicable
Solubility in Water Most components readily soluble.
pH Value neutral
Odour none
Form Powders and liquid.
Stability stable in a refrigerator for ten or more years
Ingredients
Name Proportion
Concanavelin A 1 vial (glass)
Amyloglucosidase/a-Amylase 1 vial (glass)
Glucose Buffer 1 vial (polypropylene)
Glucose Assay reagent 1 vial (glass)
Glucose standard and Starch reference 1 vial each (glass and plastic)
INTRODUCTION:
Many of the properties of cereal starches that determine their
suitability for particular end-uses are dependent upon their
amylose/amylopectin ratios. These properties include gelatinisation
and gelation characteristics, solubility, the formation of resistant
starch, and for rice, the cooking and textural characteristics of whole
grains1-5. Thus, the measurement of the amylose content of starches is
an important quality parameter for starch processing.
Amylose is most commonly determined in cereal starches by the
potentiometric, ampometric or colourimetric measurement of the
iodine binding capacity of the amylose with the resultant formation of
amylose-iodine inclusion complexes6-10. However, these methods are
subject to uncertainties. Amylopectin-iodine complexes also form, and
these reduce the concentration of free iodine measured by the noncolourimetric
methods and may absorb at similar wavelengths to
amylose-iodine complexes in colourimetric methods. These
complexes lead to an overestimation of the amylose, requiring
corrections to be applied. Many of the other problems experienced
in the use of these methods are detailed by Gibson et al.11.
The specific formation of amylopectin complexes with the lectin
concanavalin A (Con A) offers an alternative approach to amylose
measurement in starches, that is not subject to these uncertainties12,13.
Under defined conditions of pH, temperature and ionic strength,
Con A specifically complexes branched polysaccharides based on
a-D-glucopyranosyl or a-D-mannopyranosyl units at multiple nonreducing
end-groups with the formation of a precipitate. Thus, Con A
effectively complexes the amylopectin component of starch but not
the primarily linear amylose component.
The procedure described in this booklet13 is a modification of a Con A
method developed by Yun and Matheson (1990)13. It uses an ethanol
pretreatment step to remove lipids prior to analysis [modified from
Morrison and Laignelet (1983)7].
PRINCIPLE:
Starch samples are compley dispersed by heating in dimethyl
sulphoxide (DMSO). Lipids are removed by precipitating the starch in
ethanol and recovering the precipitated starch. After dissolution of
the precipitated sample in an acetate/salt solution, amylopectin is
specifically precipitated by the addition of Con A and removed by
centrifugation. The amylose in an aliquot of the supernatant is
enzymically hydrolysed to D-glucose, which is analysed using glucose
oxidase/peroxidase reagent. The total starch in a separate aliquot of
the acetate/salt solution is similarly hydrolysed to D-glucose and
1
measured colourimetrically by glucose oxidase/peroxidase. The
concentration of amylose in the starch sample is estimated as the
ratio of GOPOD absorbance at 510 nm of the supernatant of the
Con A precipitated sample, to that of the total starch sample.
This procedure is applicable to all pure starch samples and to cereal
flours.
ACCURACY:
Repeated analyses of a set of samples yielded repeatability (within
laboratory) relative standard deviations of < 5 % for pure starches
and ~ 10 % for cereal flours.
KITS:
Kits suitable for performing 100 assays are available from Megazyme.
The kits contain the full assay method plus:
Bottle 1: Freeze dried Con A (200 mg). Stable for > 5 years
at -20°C.
Bottle 2: Amyloglucosidase [200 U on p-nitrophenyl ßmaltoside
(i.e.: 3300 U on starch at pH 4.5 at 40°C)]
plus fungal a-amylase (500 U on Ceralpha Reagent
at pH 5.0 and 40°C), 2 mL. Stable for > 5 years
at 4°C.
Bottle 3: GOPOD Reagent Buffer. Potassium phosphate
buffer (1 M, pH 7.4), p-hydroxybenzoic acid (0.22 M)
and sodium azide (0.02 % w/w). Stable for > 3 years
at 4°C.
Bottle 4: GOPOD Reagent Enzymes. Glucose oxidase
(> 12,000 U) plus peroxidase (> 650 U) and
4-aminoantipyrine (80 mg). Freeze-dried powder.
Stable for > 5 years at -20°C.
Bottle 5: D-Glucose standard solution (5 mL, 1.0 mg/mL) in
0.2 % (w/v) benzoic acid. Stable for > 5 years at
room temperature.
Bottle 6: Starch reference sample (with specified content of
amylose). Stable for > 5 years at room temperature.
2
3
PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS:
1. Dissolve the contents of bottle 1 in 50 mL of Con A solvent
(Buffer 3, page 4). Divide into aliquots of appropriate size
and store in polypropylene tubes at -20°C between use and
keep cool during use if possible. Stable for > 2 years at -20°C.
2. Dissolve the contents of bottle 2 in 20 mL of sodium acetate
buffer (100 mM, pH 4.5). Divide into appropriay sized
aliquots and store in polypropylene tubes at -20°C between
use and keep cool during use if possible. Stable for > 2 years
at -20°C.
3. Dilute the contents of bottle 3 (GOPOD Reagent Buffer) to
1 L with distilled water. Use immediay.
SAFETY CONSIDERATIONS:
1. Dimethyl sulphoxide (DMSO) is listed in the Merck Index (No.
3255) as a skin irritant and thus it should be used with caution.
It is absorbed through the skin and can cause irritation to both
skin and eyes. Wear protective clothing and gloves and avoid
splashing the solvent. Use in a fume cupboard where possible.
2. Concanavalin A is harmful by inhalation, skin contact and
ingestion. Effects may be irreversible and may involve
teratogenesis. Wear protective clothing, gloves and a
particulate mask when handling crystalline Con A and gloves
when handling solutions containing Con A.
3. Sodium azide is a toxic chemical and should be treated
accordingly. It is added to buffers solely as a preservative. It
can be deleted from buffer recipes, but buffers should then be
stored at 4°C.
NOTE: If this buffer is accidentally stored at -20°C, some salt will
crystallise. Ensure all crystalline material is dissolved in the 1 L of
distilled water.
4. Dissolve the contents of bottle 4 with 20 mL of solution 3
and quantitatively transfer this to the bottle containing
solution 3. Cover this bottle with aluminium foil to protect
the enclosed reagent from light. This is Glucose
Determination Reagent (GOPOD Reagent). Stable for
approx. 3 months when stored in the dark at 2-5°C or
> 12 months at -20°C.
5 & 6. Use the contents of bottles 5 and 6 as supplied.
Stable for > 5 years at room temperature.
BUFFERS AND SOLVENTS (not enclosed):
1. Sodium Acetate Buffer (100 mM, pH 4.5)
Add 5.9 mL of glacial acetic acid (1.05 g/mL) to 900 mL of
distilled water. Adjust the pH to pH 4.5 by the addition of 1 M
(4 g/100 mL) sodium hydroxide solution (approx. 30 mL is
required). Add 0.2 g of sodium azide and adjust the volume to
1 L. Stable for > 2 years at room temperature.
2. Concentrated Con A Solvent (600 mM, pH 6.4 sodium
acetate buffer)
Dissolve 49.2 g of anhydrous sodium acetate (Sigma cat. no.
71183), 175.5 g of sodium chloride (Sigma cat. no. S 7652),
0.5 g of CaCl2.2H2O (Sigma cat. no. C 5080),
0.7 g of MgCl2.6H2O (Sigma cat. no. M 2670)
and 0.7 g of MnCl2.4H2O (Sigma cat. no. M 3634) in 900 mL of
distilled water. Adjust the pH to 6.4 by dropwise addition of
glacial acetic acid and then adjust the volume to 1 L with
distilled water. Stable for 2 weeks at 4°C.
3. Con A Solvent (working concentration)
Dilute 30 mL of Concentrated Con A Solvent to 100 mL with
distilled water. Use on the day of preparation.
4. Dimethyl sulphoxide (DMSO)
Analytical reagent grade (BDH Analar cat. no. 10323). Stable for
5 years at room temperature.
EQUIPMENT:
1. Glassware:
- Volumetric flask (25 mL);
- glass test tubes (16 x 120 mm, 15 mL);
- screw capped sample tubes (Kimax®) (10 mL).
2. Micro-pipettors, to dispense 50-1000 μL (e.g. Gilson Pipetman).
3. Positive displacement pipettor e.g. Eppendorf Multipette®.
4. Eppendorf® microfuge tubes (2.0 mL capacity).
5. Boiling water bath.
4
NOTE: When preparing this buffer mixture, it is essential that the
pH is adjusted very carefully. If the pH drops significantly below
6.4, a precipitate forms, and this will not redissolve on pH
adjustment. Consequently, this buffer must be discarded and a
fresh batch prepared.
6. Bench centrifuge (capable of 2,000 g).
7. Vortex mixer (e.g. IKA®Yellowline Test Tube Shaker TTS2).
8. Spectrophotometer (set at 510 nm).
9. Stop clock.
10. Analytical balance.
11. Microfuge (capable of 14,000 g).
12. Thermostated water bath set at 40°C.
PRECAUTIONS:
1. Starch samples must be pretreated with ethanol as described to
remove lipids. If samples are not treated with ethanol, the
amylose contents in some samples may be underestimated by as
much as 50 %.
ASSAY PROCEDURE:
A. Starch Pretreatment
1. Accuray weigh starch or flour sample (20-25 mg to the
nearest 0.1 mg) into a 10 mL screw capped Kimax® sample tube.
Record the sample weight to the nearest 0.1 mg.
2. Add 1 mL of DMSO to the tube while gently stirring it at low
speed on a vortex mixer. Cap the tube and heat the tube
contents in a boiling water bath until the sample is compley
dispersed (approx. 1 min). Ensure that no gelatinous lumps of
starch are remaining.
3. Vigorously mix the contents of the sealed tube at high speed on
a vortex mixer, place the tube in a boiling water bath and heat it
for 15 min, with intermittent high-speed stirring on a vortex
mixer.
4. Store the tube at room temperature for approx. 5 min and add
2 mL of 95 % (v/v) ethanol with continuous stirring on a vortex
mixer. Add a further 4 mL of ethanol, cap the tube and invert to
mix. A starch precipitate will form. Allow the tube to stand for
15 min (or overnight if desired).
5. Centrifuge the tubes at 2,000 g, 5 min, discard the supernatant
and drain the tubes on tissue paper for 10 min. Ensure that all
of the ethanol has drained. Use the pellet in the subsequent
amylose and starch determinations.
5
NOTE: Include a reference sample with each batch.
Duplicate every fifth test sample.
6. Add 2 mL of DMSO (with gentle vortex mixing) to the starch
pellet. Place the tube in a boiling water bath for 15 min and mix
occasionally. Ensure that there are no gelatinous lumps.
7. On removing the tubes from the boiling water bath, immediay
add 4 mL of Con A solvent (Buffer 3; page 4), mix thoroughly
and then quantitatively transfer the tube contents (by repeated
washing with Con A solvent) to a 25 mL volumetric flask. Dilute
to volume with Con A solvent (this is Solution A). If
necessary, filter this solution through Whatman No. 1 filter
paper (this step will be necessary for whole flour samples).
B. Con A Precipitation of Amylopectin and Determination
of Amylose
1. Transfer 1.0 mL of Solution A to a 2.0 mL Eppendorf®
microfuge tube. Add 0.50 mL of Con A solution (bottle 1), cap
the tube and gently mix by repeated inversion. Avoid frothing
of the sample.
2. Allow the tube to stand for 1 h at room temperature.
Centrifuge at 14,000 g for 10 min in a microfuge at room
temperature.
3. Transfer 1 mL of the supernatant to a 15 mL centrifuge tube.
Add 3 mL of 100 mM sodium acetate buffer, pH 4.5. This
reduces the pH to ~ 5. Mix the contents, lightly stopper (with a
marble) and heat in a boiling water bath for 5 min to denature
the Con A.
6
NOTE: This solution should be analysed within 2 h.
4. Place the tube in a water bath at 40°C and allow to equilibrate
for 5 min. Add 0.1 mL of amyloglucosidase/a-amylase enzyme
mixture (page 3; solution 2) and incubate at 40°C for 30 min.
Centrifuge the tube at 2,000 g for 5 min.
5. To 1.0 mL aliquots of the supernatant add 4 mL of GOPOD
Reagent (Reagent B). Incubate at 40°C for 20 min. Incubate the
Reagent Blank and the D-Glucose Controls concurrently.
6. Read the absorbance of each sample and the D-glucose controls
at 510 nm against the reagent blank.
C. Determination of Total Starch
1. Mix 0.5 mL of Solution A with 4 mL of 100 mM sodium acetate
buffer, pH 4.5.
2. Add 0.1 mL of amyloglucosidase/a-amylase solution and incubate
the mixture at 40°C for 10 min.
3. Transfer 1.0 mL aliquots (in duplicate) of this solution to glass
test tubes, add 4 mL of GOPOD Reagent (solution 4) and mix
well. Incubate at 40°C for 20 min. This incubation should be
performed concurrently with the samples and standards from
Section B above.
CALCULATION OF AMYLOSE CONTENT (%):
Amylose, % (w/w)
= Absorbance (Con A Supernatant) x 6.15 x 100
Absorbance (Total Starch Aliquot) 9.2 1
= Absorbance (Con A Supernatant) x 66.8
Absorbance (Total Starch Aliquot)
Where 6.15 and 9.2 are dilution factors for the Con A and Total
Starch extracts respectively.
7
NOTE:
The Reagent Blank is prepared by adding 1.0 mL of 100 m M
sodium acetate buffer (Buffer 1; page 4) to 4.0 mL of GOPOD
Reagent and incubating at 40°C for 20 min.
D-Glucose Controls (duplicate)comprise 0.1 mL of D-glucose
standard solution (1 mg/mL), 0.9 mL of sodium acetate buffer and
4.0 mL of GOPOD Reagent. This value is not used in the
calculation, however, we suggest that it is performed to ensure that
there are no problems with this part of the assay.
REFERENCES:
1. Juliano, B.O. (1971) Cereal Sci.Today 16, 334-338, 340, 360.
2. Berry, C. S., l’Anson, K., Miles, M. J.,Morris,V. J. and Russel, P. L. J.
(1988) Cereal Sci. 8, 203-206.
3. Sievert, D. and Pomeranz,Y. (1989) Cereal Chem. 66, 342-347.
4. Tester, R. F. and Morrison,W. R. (1990) Cereal Chem. 67, 551-557.
5. Leloup,V. M., Colonna, P. and Buleon,A. (1991) J. Cereal Sci. 13,
1-13.
6. Matheson, N. K. (1971) Phytochem. 10, 3213-3219.
7. Morrison,W. R. and Laignet, B. (1983) J. Cereal Sci. 1, 9-20.
8. Knutson, C.A. (1986) Cereal Chem. 63, 89-92.
9. Chrastil, J. (1987) Carbohydr. Res. 159, 154-158.
10. International Organisation for Standardisation (1987)
ISO 6647:1987E. Rice: determination of amylose content.
11. Gibson,T. S., Solah,V.A. and McCleary, B.V. (1996) “A procedure to
measure amylose in cereal starches and flours with Con A.” J.
Cereal Science 25, 111-119.
12. Matheson, N. K. and Welsh, L.A. (1988) Carbohydr. Res. 180,
301-313.
13. Yun, S. H. and Matheson, N. K. (1990) Starch/Starke 42, 302-305.
8
ACKNOWLEDGEMENTS:
The procedure described in this booklet was developed in
association with the Biological and Chemical Research Institute,
NSW Agriculture, Rydalmere, NSW, Australia. We also
acknowledge many valuable discussions with Professor N. K.
Matheson during the development of this procedure.
9
Figure 1: Effect of Con A concentration on the level of amylose determined
in starch samples by the modified Con A procedure. n = high
amylose maize starch (74.4 % w/w amylose), l = rice starch
(16.9 % w/w amylose), o = maize starch (1.9 % w/w amylose).
The vertical dashed line represents the conditions used in the final
assay format.
Figure 2: Effect of precipitation reaction time on the amylose determined in
starch samples by the modified Con A procedure. n = high
amylose maize starch (74.4 % w/w amylose), l = rice starch
(16.9 % w/w amylose), o = maize starch (1.9 % w/w amylose).
The vertical dashed line represents the conditions used in the final
assay format.
10
Figure 3: Effect of sample size on the amylose determined in starch samples
by the
直鏈淀粉/支鏈淀粉(膠淀粉)檢測(cè)試劑盒 100次 愛(ài)爾蘭 型號(hào):K-AMYL
庫(kù)號(hào)：M248897 查看hh
midwest-group
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midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
midwest-group
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名稱:直鏈淀粉/支鏈淀粉(膠淀粉)檢測(cè)試劑盒
規(guī)格: 100次
產(chǎn)地:愛(ài)爾蘭
用途:用于測(cè)量谷物和面粉中直鏈淀粉/支鏈淀粉的比例和淀粉含量。
標(biāo)配:6個(gè)玻璃瓶
在水中的溶解度:易溶
pH值:中性
氣味:無(wú)
狀態(tài):粉末和液體
保持期:10年以上(冰箱內(nèi))
成分:
名稱 比例
Concanavelin A 1瓶（玻璃）
淀粉葡萄糖苷酶/α-淀粉酶 1瓶（玻璃）
葡萄糖緩沖液 1瓶（聚丙烯）
葡萄糖檢測(cè)試劑 1瓶（玻璃）
葡萄糖標(biāo)準(zhǔn)品和淀粉參考品 各1瓶（玻璃和塑料）
詳細(xì)信息
前言:
谷類淀粉的許多特性決定其終用途，這些取決于直鏈淀粉/支鏈淀粉的比率。這些特性包括糊化和凝膠化，溶解度，抗性淀粉的形成以及整顆大米的烹飪和構(gòu)造特性。因此，淀粉中直鏈淀粉含量的測(cè)定是淀粉加工的一個(gè)重要的質(zhì)量參數(shù)。
常用的測(cè)定谷物淀粉中直鏈淀粉含量的方法是利用電勢(shì)，電流測(cè)定或直鏈淀粉的碘結(jié)合能力比色測(cè)定直鏈淀粉-碘色合配合物。然而，這些方法具有不確定性。支 鏈淀粉-碘復(fù)合物也可以形成，這樣降低了利用非比色法測(cè)定的游離碘離子的濃度，并且用比色法測(cè)量時(shí)，該復(fù)合物可能和直鏈淀粉-碘復(fù)合物吸收相同波長(zhǎng)的光。 這種復(fù)合物致使直鏈淀粉的測(cè)定含量超過(guò)實(shí)際含量，需要進(jìn)行校正。Gibson等詳細(xì)列舉了使用這些方法所遇到的許多其他問(wèn)題。
支鏈淀粉結(jié)合ConA的特殊復(fù)合物為淀粉中直鏈淀粉的測(cè)定提供了一種替代方法，而且不存在不確定性問(wèn)題。在pH值，溫度和離子強(qiáng)度的條件下，ConA 特異性結(jié)合分支多糖并形成沉淀，這種結(jié)合以多個(gè)非還原性末端基團(tuán)上的α-D-吡喃葡萄糖基或α-D-吡喃甘露糖基單位為基礎(chǔ)。因此，ConA可以有效結(jié)合 淀粉中的支鏈淀粉成分，但是不能結(jié)合線性為主的直鏈淀粉成分。
此方法是Yun和Matheson改進(jìn)的ConA方法。分析之前用乙醇預(yù)處理去除脂質(zhì)。
原理：
淀粉樣品通過(guò)加熱*地溶解在二甲基亞砜（DMSO）里。用乙醇沉淀淀粉去除其中的脂質(zhì)，回收沉淀的淀粉。用醋酸/鹽溶液溶解沉淀的樣品，加入ConA， 特異性沉淀支鏈淀粉，離心去除沉淀。單位體積上清液中的直鏈淀粉用酶水解為D-葡萄糖，然后用葡萄糖氧化酶/過(guò)氧化物酶試劑進(jìn)行測(cè)定。另外一份單位體積醋 酸/鹽溶液中的總淀粉同樣用酶水解為D-葡萄糖，然后加入葡萄糖氧化酶/過(guò)氧化物酶，用比色法測(cè)定。
根據(jù)ConA沉淀樣品的上清液和與總淀粉樣品中的GOPOD在510 nm處的吸光光度值之比判斷直鏈淀粉在總淀粉中的含量。
該方法適用于所有的純淀粉和谷物粉。
精確性
樣品如為純淀粉，相對(duì)標(biāo)準(zhǔn)偏差為<5%。
樣品如為谷物面粉，相對(duì)標(biāo)準(zhǔn)偏差為～10%。
試劑盒：
瓶子1：凍干的Con A （伴刀豆球蛋白 A concanavalin A) ，200 mg，-20℃下穩(wěn)定性>5年。
瓶子2：淀粉葡糖苷酶【200U，條件為消化對(duì)硝基苯基β-麥芽糖苷（也就是3300U，條件為pH4.5，40℃下消化淀粉）】加上真菌α-淀粉酶（500U ，條件為pH5.0，40℃下消化 Ceralpha 試劑），2mL，4℃下穩(wěn)定性>5年。
瓶子3：GOPOD 試劑緩沖液。磷酸鉀緩沖液（1M，pH7.4），對(duì)羥苯甲酸（0.22M）和*（0.02% W/W）。4℃下穩(wěn)定性> 3年。
瓶子4：GOPOD試劑酶。葡糖氧化酶（>12,000U）加上過(guò)氧化物酶（>650U）和4-氨基安替比林（80mg）。凍干粉，-20℃下穩(wěn)定性>5年。
瓶子5：D-葡萄糖標(biāo)準(zhǔn)溶液（5 mL,1.0mg/mL）溶于苯甲酸0.2%（w/v）。室溫下穩(wěn)定性>5年。
瓶子6：淀粉參考樣品（含有特定含量的直鏈淀粉），室溫下穩(wěn)定性>5年。
Amylose/Amylopectin Kits
For the measurement of amylose/amylopectin ratio and content in cereal starches and flours. Based on a Con A precipitation procedure.
Content: 100 assays per kit
Appearance Four glass vials plus two plastic vials
Specific Gravity Not applicable
Solubility in Water Most components readily soluble.
pH Value neutral
Odour none
Form Powders and liquid.
Stability stable in a refrigerator for ten or more years
Ingredients
Name Proportion
Concanavelin A 1 vial (glass)
Amyloglucosidase/a-Amylase 1 vial (glass)
Glucose Buffer 1 vial (polypropylene)
Glucose Assay reagent 1 vial (glass)
Glucose standard and Starch reference 1 vial each (glass and plastic)
INTRODUCTION:
Many of the properties of cereal starches that determine their
suitability for particular end-uses are dependent upon their
amylose/amylopectin ratios. These properties include gelatinisation
and gelation characteristics, solubility, the formation of resistant
starch, and for rice, the cooking and textural characteristics of whole
grains1-5. Thus, the measurement of the amylose content of starches is
an important quality parameter for starch processing.
Amylose is most commonly determined in cereal starches by the
potentiometric, ampometric or colourimetric measurement of the
iodine binding capacity of the amylose with the resultant formation of
amylose-iodine inclusion complexes6-10. However, these methods are
subject to uncertainties. Amylopectin-iodine complexes also form, and
these reduce the concentration of free iodine measured by the noncolourimetric
methods and may absorb at similar wavelengths to
amylose-iodine complexes in colourimetric methods. These
complexes lead to an overestimation of the amylose, requiring
corrections to be applied. Many of the other problems experienced
in the use of these methods are detailed by Gibson et al.11.
The specific formation of amylopectin complexes with the lectin
concanavalin A (Con A) offers an alternative approach to amylose
measurement in starches, that is not subject to these uncertainties12,13.
Under defined conditions of pH, temperature and ionic strength,
Con A specifically complexes branched polysaccharides based on
a-D-glucopyranosyl or a-D-mannopyranosyl units at multiple nonreducing
end-groups with the formation of a precipitate. Thus, Con A
effectively complexes the amylopectin component of starch but not
the primarily linear amylose component.
The procedure described in this booklet13 is a modification of a Con A
method developed by Yun and Matheson (1990)13. It uses an ethanol
pretreatment step to remove lipids prior to analysis [modified from
Morrison and Laignelet (1983)7].
PRINCIPLE:
Starch samples are compley dispersed by heating in dimethyl
sulphoxide (DMSO). Lipids are removed by precipitating the starch in
ethanol and recovering the precipitated starch. After dissolution of
the precipitated sample in an acetate/salt solution, amylopectin is
specifically precipitated by the addition of Con A and removed by
centrifugation. The amylose in an aliquot of the supernatant is
enzymically hydrolysed to D-glucose, which is analysed using glucose
oxidase/peroxidase reagent. The total starch in a separate aliquot of
the acetate/salt solution is similarly hydrolysed to D-glucose and
1
measured colourimetrically by glucose oxidase/peroxidase. The
concentration of amylose in the starch sample is estimated as the
ratio of GOPOD absorbance at 510 nm of the supernatant of the
Con A precipitated sample, to that of the total starch sample.
This procedure is applicable to all pure starch samples and to cereal
flours.
ACCURACY:
Repeated analyses of a set of samples yielded repeatability (within
laboratory) relative standard deviations of < 5 % for pure starches
and ~ 10 % for cereal flours.
KITS:
Kits suitable for performing 100 assays are available from Megazyme.
The kits contain the full assay method plus:
Bottle 1: Freeze dried Con A (200 mg). Stable for > 5 years
at -20°C.
Bottle 2: Amyloglucosidase [200 U on p-nitrophenyl ßmaltoside
(i.e.: 3300 U on starch at pH 4.5 at 40°C)]
plus fungal a-amylase (500 U on Ceralpha Reagent
at pH 5.0 and 40°C), 2 mL. Stable for > 5 years
at 4°C.
Bottle 3: GOPOD Reagent Buffer. Potassium phosphate
buffer (1 M, pH 7.4), p-hydroxybenzoic acid (0.22 M)
and sodium azide (0.02 % w/w). Stable for > 3 years
at 4°C.
Bottle 4: GOPOD Reagent Enzymes. Glucose oxidase
(> 12,000 U) plus peroxidase (> 650 U) and
4-aminoantipyrine (80 mg). Freeze-dried powder.
Stable for > 5 years at -20°C.
Bottle 5: D-Glucose standard solution (5 mL, 1.0 mg/mL) in
0.2 % (w/v) benzoic acid. Stable for > 5 years at
room temperature.
Bottle 6: Starch reference sample (with specified content of
amylose). Stable for > 5 years at room temperature.
2
3
PREPARATION OF REAGENT SOLUTIONS/SUSPENSIONS:
1. Dissolve the contents of bottle 1 in 50 mL of Con A solvent
(Buffer 3, page 4). Divide into aliquots of appropriate size
and store in polypropylene tubes at -20°C between use and
keep cool during use if possible. Stable for > 2 years at -20°C.
2. Dissolve the contents of bottle 2 in 20 mL of sodium acetate
buffer (100 mM, pH 4.5). Divide into appropriay sized
aliquots and store in polypropylene tubes at -20°C between
use and keep cool during use if possible. Stable for > 2 years
at -20°C.
3. Dilute the contents of bottle 3 (GOPOD Reagent Buffer) to
1 L with distilled water. Use immediay.
SAFETY CONSIDERATIONS:
1. Dimethyl sulphoxide (DMSO) is listed in the Merck Index (No.
3255) as a skin irritant and thus it should be used with caution.
It is absorbed through the skin and can cause irritation to both
skin and eyes. Wear protective clothing and gloves and avoid
splashing the solvent. Use in a fume cupboard where possible.
2. Concanavalin A is harmful by inhalation, skin contact and
ingestion. Effects may be irreversible and may involve
teratogenesis. Wear protective clothing, gloves and a
particulate mask when handling crystalline Con A and gloves
when handling solutions containing Con A.
3. Sodium azide is a toxic chemical and should be treated
accordingly. It is added to buffers solely as a preservative. It
can be deleted from buffer recipes, but buffers should then be
stored at 4°C.
NOTE: If this buffer is accidentally stored at -20°C, some salt will
crystallise. Ensure all crystalline material is dissolved in the 1 L of
distilled water.
4. Dissolve the contents of bottle 4 with 20 mL of solution 3
and quantitatively transfer this to the bottle containing
solution 3. Cover this bottle with aluminium foil to protect
the enclosed reagent from light. This is Glucose
Determination Reagent (GOPOD Reagent). Stable for
approx. 3 months when stored in the dark at 2-5°C or
> 12 months at -20°C.
5 & 6. Use the contents of bottles 5 and 6 as supplied.
Stable for > 5 years at room temperature.
BUFFERS AND SOLVENTS (not enclosed):
1. Sodium Acetate Buffer (100 mM, pH 4.5)
Add 5.9 mL of glacial acetic acid (1.05 g/mL) to 900 mL of
distilled water. Adjust the pH to pH 4.5 by the addition of 1 M
(4 g/100 mL) sodium hydroxide solution (approx. 30 mL is
required). Add 0.2 g of sodium azide and adjust the volume to
1 L. Stable for > 2 years at room temperature.
2. Concentrated Con A Solvent (600 mM, pH 6.4 sodium
acetate buffer)
Dissolve 49.2 g of anhydrous sodium acetate (Sigma cat. no.
71183), 175.5 g of sodium chloride (Sigma cat. no. S 7652),
0.5 g of CaCl2.2H2O (Sigma cat. no. C 5080),
0.7 g of MgCl2.6H2O (Sigma cat. no. M 2670)
and 0.7 g of MnCl2.4H2O (Sigma cat. no. M 3634) in 900 mL of
distilled water. Adjust the pH to 6.4 by dropwise addition of
glacial acetic acid and then adjust the volume to 1 L with
distilled water. Stable for 2 weeks at 4°C.
3. Con A Solvent (working concentration)
Dilute 30 mL of Concentrated Con A Solvent to 100 mL with
distilled water. Use on the day of preparation.
4. Dimethyl sulphoxide (DMSO)
Analytical reagent grade (BDH Analar cat. no. 10323). Stable for
5 years at room temperature.
EQUIPMENT:
1. Glassware:
- Volumetric flask (25 mL);
- glass test tubes (16 x 120 mm, 15 mL);
- screw capped sample tubes (Kimax®) (10 mL).
2. Micro-pipettors, to dispense 50-1000 μL (e.g. Gilson Pipetman).
3. Positive displacement pipettor e.g. Eppendorf Multipette®.
4. Eppendorf® microfuge tubes (2.0 mL capacity).
5. Boiling water bath.
4
NOTE: When preparing this buffer mixture, it is essential that the
pH is adjusted very carefully. If the pH drops significantly below
6.4, a precipitate forms, and this will not redissolve on pH
adjustment. Consequently, this buffer must be discarded and a
fresh batch prepared.
6. Bench centrifuge (capable of 2,000 g).
7. Vortex mixer (e.g. IKA®Yellowline Test Tube Shaker TTS2).
8. Spectrophotometer (set at 510 nm).
9. Stop clock.
10. Analytical balance.
11. Microfuge (capable of 14,000 g).
12. Thermostated water bath set at 40°C.
PRECAUTIONS:
1. Starch samples must be pretreated with ethanol as described to
remove lipids. If samples are not treated with ethanol, the
amylose contents in some samples may be underestimated by as
much as 50 %.
ASSAY PROCEDURE:
A. Starch Pretreatment
1. Accuray weigh starch or flour sample (20-25 mg to the
nearest 0.1 mg) into a 10 mL screw capped Kimax® sample tube.
Record the sample weight to the nearest 0.1 mg.
2. Add 1 mL of DMSO to the tube while gently stirring it at low
speed on a vortex mixer. Cap the tube and heat the tube
contents in a boiling water bath until the sample is compley
dispersed (approx. 1 min). Ensure that no gelatinous lumps of
starch are remaining.
3. Vigorously mix the contents of the sealed tube at high speed on
a vortex mixer, place the tube in a boiling water bath and heat it
for 15 min, with intermittent high-speed stirring on a vortex
mixer.
4. Store the tube at room temperature for approx. 5 min and add
2 mL of 95 % (v/v) ethanol with continuous stirring on a vortex
mixer. Add a further 4 mL of ethanol, cap the tube and invert to
mix. A starch precipitate will form. Allow the tube to stand for
15 min (or overnight if desired).
5. Centrifuge the tubes at 2,000 g, 5 min, discard the supernatant
and drain the tubes on tissue paper for 10 min. Ensure that all
of the ethanol has drained. Use the pellet in the subsequent
amylose and starch determinations.
5
NOTE: Include a reference sample with each batch.
Duplicate every fifth test sample.
6. Add 2 mL of DMSO (with gentle vortex mixing) to the starch
pellet. Place the tube in a boiling water bath for 15 min and mix
occasionally. Ensure that there are no gelatinous lumps.
7. On removing the tubes from the boiling water bath, immediay
add 4 mL of Con A solvent (Buffer 3; page 4), mix thoroughly
and then quantitatively transfer the tube contents (by repeated
washing with Con A solvent) to a 25 mL volumetric flask. Dilute
to volume with Con A solvent (this is Solution A). If
necessary, filter this solution through Whatman No. 1 filter
paper (this step will be necessary for whole flour samples).
B. Con A Precipitation of Amylopectin and Determination
of Amylose
1. Transfer 1.0 mL of Solution A to a 2.0 mL Eppendorf®
microfuge tube. Add 0.50 mL of Con A solution (bottle 1), cap
the tube and gently mix by repeated inversion. Avoid frothing
of the sample.
2. Allow the tube to stand for 1 h at room temperature.
Centrifuge at 14,000 g for 10 min in a microfuge at room
temperature.
3. Transfer 1 mL of the supernatant to a 15 mL centrifuge tube.
Add 3 mL of 100 mM sodium acetate buffer, pH 4.5. This
reduces the pH to ~ 5. Mix the contents, lightly stopper (with a
marble) and heat in a boiling water bath for 5 min to denature
the Con A.
6
NOTE: This solution should be analysed within 2 h.
4. Place the tube in a water bath at 40°C and allow to equilibrate
for 5 min. Add 0.1 mL of amyloglucosidase/a-amylase enzyme
mixture (page 3; solution 2) and incubate at 40°C for 30 min.
Centrifuge the tube at 2,000 g for 5 min.
5. To 1.0 mL aliquots of the supernatant add 4 mL of GOPOD
Reagent (Reagent B). Incubate at 40°C for 20 min. Incubate the
Reagent Blank and the D-Glucose Controls concurrently.
6. Read the absorbance of each sample and the D-glucose controls
at 510 nm against the reagent blank.
C. Determination of Total Starch
1. Mix 0.5 mL of Solution A with 4 mL of 100 mM sodium acetate
buffer, pH 4.5.
2. Add 0.1 mL of amyloglucosidase/a-amylase solution and incubate
the mixture at 40°C for 10 min.
3. Transfer 1.0 mL aliquots (in duplicate) of this solution to glass
test tubes, add 4 mL of GOPOD Reagent (solution 4) and mix
well. Incubate at 40°C for 20 min. This incubation should be
performed concurrently with the samples and standards from
Section B above.
CALCULATION OF AMYLOSE CONTENT (%):
Amylose, % (w/w)
= Absorbance (Con A Supernatant) x 6.15 x 100
Absorbance (Total Starch Aliquot) 9.2 1
= Absorbance (Con A Supernatant) x 66.8
Absorbance (Total Starch Aliquot)
Where 6.15 and 9.2 are dilution factors for the Con A and Total
Starch extracts respectively.
7
NOTE:
The Reagent Blank is prepared by adding 1.0 mL of 100 m M
sodium acetate buffer (Buffer 1; page 4) to 4.0 mL of GOPOD
Reagent and incubating at 40°C for 20 min.
D-Glucose Controls (duplicate)comprise 0.1 mL of D-glucose
standard solution (1 mg/mL), 0.9 mL of sodium acetate buffer and
4.0 mL of GOPOD Reagent. This value is not used in the
calculation, however, we suggest that it is performed to ensure that
there are no problems with this part of the assay.
REFERENCES:
1. Juliano, B.O. (1971) Cereal Sci.Today 16, 334-338, 340, 360.
2. Berry, C. S., l’Anson, K., Miles, M. J.,Morris,V. J. and Russel, P. L. J.
(1988) Cereal Sci. 8, 203-206.
3. Sievert, D. and Pomeranz,Y. (1989) Cereal Chem. 66, 342-347.
4. Tester, R. F. and Morrison,W. R. (1990) Cereal Chem. 67, 551-557.
5. Leloup,V. M., Colonna, P. and Buleon,A. (1991) J. Cereal Sci. 13,
1-13.
6. Matheson, N. K. (1971) Phytochem. 10, 3213-3219.
7. Morrison,W. R. and Laignet, B. (1983) J. Cereal Sci. 1, 9-20.
8. Knutson, C.A. (1986) Cereal Chem. 63, 89-92.
9. Chrastil, J. (1987) Carbohydr. Res. 159, 154-158.
10. International Organisation for Standardisation (1987)
ISO 6647:1987E. Rice: determination of amylose content.
11. Gibson,T. S., Solah,V.A. and McCleary, B.V. (1996) “A procedure to
measure amylose in cereal starches and flours with Con A.” J.
Cereal Science 25, 111-119.
12. Matheson, N. K. and Welsh, L.A. (1988) Carbohydr. Res. 180,
301-313.
13. Yun, S. H. and Matheson, N. K. (1990) Starch/Starke 42, 302-305.
8
ACKNOWLEDGEMENTS:
The procedure described in this booklet was developed in
association with the Biological and Chemical Research Institute,
NSW Agriculture, Rydalmere, NSW, Australia. We also
acknowledge many valuable discussions with Professor N. K.
Matheson during the development of this procedure.
9
Figure 1: Effect of Con A concentration on the level of amylose determined
in starch samples by the modified Con A procedure. n = high
amylose maize starch (74.4 % w/w amylose), l = rice starch
(16.9 % w/w amylose), o = maize starch (1.9 % w/w amylose).
The vertical dashed line represents the conditions used in the final
assay format.
Figure 2: Effect of precipitation reaction time on the amylose determined in
starch samples by the modified Con A procedure. n = high
amylose maize starch (74.4 % w/w amylose), l = rice starch
(16.9 % w/w amylose), o = maize starch (1.9 % w/w amylose).
The vertical dashed line represents the conditions used in the final
assay format.
10
Figure 3: Effect of sample size on the amylose determined in starch samples
by the modified Con A procedure. n = high amylose maize starch
(74.4 % w/w amylose), l = rice starch (16.9 % w/w amylose),
o = maize starch (1.9 % w/w amylose). The vertical dashed lines
represent the conditions used in the final assay format.
Figure 4: Mixed amylose/amylopectin standard curve for the modified Con A
procedure. The regression equation is y = 0.956 x + 3.259 and
the correlation coefficient 0.999. Nominal amylose contents are
predicted values based on the proportion of ICN potato amylose
(~ 100 % w/w amylose) and waxy maize starch (~ 0 % w/w
amylose based on potentiometric iodine titration) in the mixed
modified Con A procedure. n = high amylose maize starch
(74.4 % w/w amylose), l = rice starch (16.9 % w/w amylose),
o = maize starch (1.9 % w/w amylose). The vertical dashed lines
represent the conditions used in the final assay format.
Figure 4: Mixed amylose/amylopectin standard curve for the modified Con A
procedure. The regression equation is y = 0.956 x + 3.259 and
the correlation coefficient 0.999. Nominal amylose contents are
predicted values based on the proportion of ICN potato amylose
(~ 100 % w/w amylose) and waxy maize starch (~ 0 % w/w
amylose based on potentiometric iodine titration) in the mixed
