May 05, 2019
A simple, accurate, low cost method for starch quantification in green microalgae
- Tze Ching Yong1,
- Chia-Sheng Chiu1,
- Ching-Nen Nathan Chen1
- 1National Sun Yat-Sen University
External link: https://doi.org/10.1186/s40529-019-0273-y
Protocol Citation: Tze Ching Yong, Chia-Sheng Chiu, Ching-Nen Nathan Chen 2019. A simple, accurate, low cost method for starch quantification in green microalgae. protocols.io https://dx.doi.org/10.17504/protocols.io.2mhgc36
Manuscript citation:
Yong TC, Chiu C, Chen CN, Optimization of a simple, accurate and low cost method for starch quantification in green microalgae. Botanical Studies doi: 10.1186/s40529-019-0273-y
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 05, 2019
Last Modified: May 05, 2019
Protocol Integer ID: 22921
Harvest microalgal cells from 10 mL culture using swing bucket centrifugation (2600 g for 3 min).
Transfer the cells to a 2-mL screw cap microtube. Spin briefly and get rid of the medium using a pippet. Freeze the cells immediately at -15 °C in an ice-crude sea salt mix.
Add 1 mL methanol/tetrahydrofuran mix (v/v =1/3) to the cell pellet to extract pigments. Shake the microtube occasionally. Extract the pigments for at least 30 min.
Centrifuge at 16,000 g at 4 °C for 10 min. Discard the supernatant. Repeat the pigment extraction until the pellet becomes white, then dry the pellet at 65 °C in an oven for one hour.
Wash the pellet out of the microtube repeatedly using 50 mM, pH 5 sodium phosphate buffer, 5 mL in total, and transfer to a 15-mL tube.
Autoclave the cell suspension at 134 °C for one hour to disintegrate the starch granules.
Mix well and transfer 1 mL of the autoclaved sample to a 2-mL microtube, add 0.5 g acid-washed glass beads and then use a mini-beadbeater (BioSpec Products, USA) to smash the cells and release the starch (three cycles at the highest speed).
Transfer 0.5 mL of the beated sample to a microtube, add 0.5 mL sodium phosphate buffer (50 mM, pH 5) and 2 units (in 20 μL) of glucoamylase(1) for overnight digestion at 50 °C. Add the same amount of glucoamylase again the next morning for the second digestion in the same conditions for 7 hours.
After the double digestions, centrifuge the sample at 16,000 g at room temperature for 10 min and measure the glucose in the sample using the glucose assay protocol (the DNS method, see below).
Glucose assay using the DNS method
Glucose assay using the DNS method
Build the standard curve for glucose assay: Prepare 10 mM glucose solution in 50 mM sodium phosphate buffer, pH 5.0, and do 2-fold serial dilutions until 312 μM using the buffer.
Add 0.5 mL the glucose solution (or a sample) to 2 mL of the DNS reagent(2) in a test tube (need to include 2 blanks to set zero). Mix well then heat the mixture in boiling water for 5 min. Cool down in tap water and measure the OD540 of the glucose standards and the samples.
Build the standard curve of OD540 against glucose quantities. Use the regression equation to calculate the glucose quantities in the samples.
Recipes and notes
Recipes and notes
(1) Glucoamylase (TCI Chemicals, Tokyo, Japan; Cat. # M0035) prep. : Take 100 units enzyme and dissolve it in 1 mL of 50 mM sodium phosphate buffer, pH 5 (17 mg of the enzyme powder contains about 100 units enzyme; the enzyme powder contains about 6000 units/g). This enzyme prep is good for 2 weeks if stored at 4 °C. Unit definition: amount of protein to cause degadation of 10 mg starch to glucose in 30 min (in specified conditions).
(2) DNS reagent prep: Dissolve 2 g NaOH in 70 mL H2O, add 1 g of 3,5-dinitrosalicylic acid (DNS), then add 30 g potassium sodium tartrate. When the all chemicals are dissolved (takes about 2 - 3 days using sonication occasionally), bring the final volume to 100 mL. The final concentrations of the chemicals: 0.5 M NaOH, 44 mM dinitrosalicylicacid, 1 M potassium sodium tartrate.
(3) This protocol is based on the manuscript “Optimization of starch quantificationin green microalgae: a simple, accurate, low cost method using glucoamylase and dinitrosalicylic acid” submitted to PLOS ONE.