- 背景資料
葡聚醣(Glucan)為D-葡萄糖單體藉由糖苷鍵連接的多醣，依據糖苷鍵鍵結方式的差異，可分成α-葡聚醣和β-葡聚醣
常見的α-葡聚醣:
1️⃣直鏈澱粉(amylose)➡️α-1,4糖苷鍵
2️⃣支鏈澱粉(amylopectin)➡️α-1,4糖苷鍵+α-1,6糖苷鍵
3️⃣肝醣(glycogen)➡️α-1,4糖苷鍵+α-1,6糖苷鍵
常見的β-葡聚醣:
1️⃣纖維素(cellulose)➡️β-1,4糖苷鍵➡️植物的細胞壁
2️⃣卡德蘭膠(Curdlan)➡️β-1,3糖苷鍵➡️細菌性胞外多醣體
(1,3)(1,6)-β-D-glucan為真菌和酵母菌細胞壁的成分，在過去的科學研究當中，此種β-葡聚醣可能具有降低發炎反應、降低人體血清中低密度脂蛋白膽固醇含量、增加腸胃道功能或免疫調節作用
- A. MEASUREMENT OF TOTAL GLUCAN (α-glucan + β-glucan) 總葡聚醣測量值
a. Solubilisation and partial hydrolysis of total glucan
1. Mill mushroom or yeast sample to pass a 1.0 mm screen using a centrifugal mill.
2. Add the milled sample (approx. 90 mg, weighed accurately) to a 20 x 12 mm Fisher Brand culture tube. Tap the tube to ensure that all the sample falls to the bottom of the tube.
3. Add 2.0 mL of ice cold 12 M sulphuric acid to each tube, cap the tubes and stir them vigorously on a vortex mixer. Place the tubes in an ice-water bath and NOTE: With each set of determinations, include at least one control yeast or mushroom preparation. Also include reagent blanks and glucose standards of 100 μg (in quadruplicate). Run these through the entire incubation procedure with GOPOD Reagent. The reagent blank consists of 0.2 mL of sodium acetate buffer (200 mM, pH 4.5) + 3.0 mL GOPOD Reagent. The D-glucose standard consists of 0.1 mL of bottle 5 (D-glucose standard, 1 mg/mL) + 0.1 mL of sodium acetate buffer (200 mM, pH 4.5) + 3.0 mL GOPOD Reagent. The absorbances of the samples assayed should not exceed that obtained for the D-glucose control samples. If the sample absorbance exceeds the control values dilute the sample further to achieve a suitable absorbance. 6 incubate for 2 h. Periodically, vigorously stir the tube contents (for 10-15 sec, several times) on a vortex mixer (to ensure complete dissolution of the βglucan).
4. Add 4 mL of water to each tube, cap the tubes and vigorously stir the contents on a vortex mixer for 10 sec. Then add 6 mL of water, cap the tubes and stir the contents for a further 10 sec.
5. Loosen the caps on the tubes and place them in a boiling water bath (~ 100°C). After 5 min, tighten the caps and continue the incubation for 2 h.
6. Cool the tubes to room temperature and carefully loosen the caps.
7. Quantitatively transfer the contents of each tube to a 100 mL volumetric flask using a wash bottle containing 200 mM sodium acetate buffer (pH 4.5).
8. Add 6 mL of 8.0 M NaOH solution to the volumetric flask and adjust to volume with 200 mM sodium acetate buffer (pH 4.5). Mix the contents well by inversion and collect an aliquot of the sample in a microfuge tube.
9. Centrifuge an aliquot of the solution at 13,000 rpm for 5 min.
b. Measurement of total glucan
1. Transfer 0.1 mL aliquots (in duplicate) of filtered or centrifuged extract to the bottom of glass test tubes (16 x 100 mm).
2. Add 0.1 mL of Solution 1 [exo-1,3-β-glucanase plus β-glucosidase] to the bottom of each tube, mix the tube contents on a vortex mixer and incubate at 40°C for 60 min.
3. Add 3.0 mL of GOPOD Reagent to each tube and incubate at 40°C for 20 min.
4. Measure the absorbance of all solutions at 510 nm against the reagent blank. - B. MEASUREMENT OF α-GLUCAN(α-葡聚醣測量值)
a. Solubilisation, hydrolysis and measurement of α-glucan
1. Add the milled sample (approx. 100 mg, weighed accurately) to a 20 x 125 mm Fisher Brand culture tube. Tap the tube to ensure that all the sample falls to the bottom of the tube.
2. Add a magnetic stirrer bar (5 x 15 mm) followed by 2 mL of 1.7 M NaOH to each tube and suspend the pellets by stirring for approx. 20 min in an ice/ water bath over a magnetic stirrer.
3. Add 8 mL of 1.2 M sodium acetate buffer (pH 3.8) to each tube with stirring. Immediately add 0.2 mL of Bottle 2 [amyloglucosidase plus invertase] and 0.05 mL of Bottle 7 [Trehalase], mix well and place the tubes in a water bath at 40°C.
4. Incubate the tubes at 40°C for 60 min with intermittent mixing on a vortex stirrer.
5. For samples containing > 10% α-glucan content; quantitatively transfer the contents of the tube to a 100 mL volumetric flask (using a water wash bottle) and adjust to volume with water. Mix well. Centrifuge an aliquot of the solution at 13,000 rpm for 10 min or filter through Whatman No. 1 filter paper (9 cm).
6. For samples containing < 10% α-glucan content; transfer 2 mL of solution to a microfuge tube and centrifuge at 13,000 rpm for 5 min. For such samples the final volume in the tube is approx. 10.35 mL (however, this volume may vary slightly with the type of sample being analysed). In some cases, an appropriate allowance for volume should be made in the calculations.
7. Transfer 0.1 mL aliquots (in duplicate) of either the diluted or undiluted supernatants into glass test tubes (16 x 100 mm), add 0.1 mL of sodium acetate buffer (200 mM, pH 4.5) plus 3.0 mL of GOPOD reagent and incubate at 40°C for 20 min.
8. Measure the absorbance of all solutions at 510 nm against the reagent blank.
商品特色
商品規格
- 商品規格(100個檢測反應)
Bottle 1: (x2)
exo-1,3-β-Glucanase plus β-Glucosidase ammonium sulphate suspension, 2.0 mL.
Store at 4°C. See individual label for expiry date.
Bottle 2:
Amyloglucosidase plus invertase solution in 50% (v/v) glycerol, 20 mL.
Store below -10 °C. See individual label for expiry date.
NOTE: If stored at 4°C the expiry of this product will be decreased to ≥ 2 years.
Bottle 3:
GOPOD Reagent Buffer.
Buffer (50 mL, pH 7.4). p-hydroxybenzoic acid and sodium azide (0.09%).
Store at 4°C. See individual label for expiry date.
Bottle 4:
GOPOD Reagent Enzymes.
Glucose oxidase plus peroxidase and 4aminoantipyrine.
Freeze-dried powder. Store below -10°C. See individual label for expiry date.
Bottle 5:
D-Glucose standard solution (5 mL, 1.00 mg/mL) in 0.2% (w/v) benzoic acid.
Store sealed at room temperature. See individual label for expiry date.
Bottle 6:
Control yeast β-glucan preparation (~ 2 g, β-glucan content stated on the bottle label).
Store sealed at room temperature. See individual label for expiry date.
Bottle 7:
Trehalase suspension (5 mL).
Store at 4°C. See individual label for expiry date.
