Solid-state fermentation experiments with amylases
Solid-state fermentation experiments with amylases
Amylase has the ability to catalyze the hydrolysis of starch, starting at the non-reducing end of the starch molecule and breaking down the a-1,4-glucosidic bond to produce glucose. Under alkaline conditions, reducing sugar is co-heated with 3,5-dinitrosalicylic acid, which is reduced to 3-amino-5-nitrosalicylic acid (brownish-red substance), and reducing sugar is oxidized to saccharic acid and other substances.
Operation method
colorimetric method
Principle
Amylase has the ability to catalyze the hydrolysis of starch, starting at the non-reducing end of the starch molecule and breaking down the a-1,4-glucosidic bond to produce glucose. Under alkaline conditions, reducing sugar is co-heated with 3,5-dinitrosalicylic acid, which is reduced to 3-amino-5-nitrosalicylic acid (brownish-red substance), and reducing sugar is oxidized to saccharic acid and other substances. In a certain range, the amount of reducing sugar and the degree of brown-red color shades of the degree of proportionality, can be measured in the 722-type spectrophotometer 540 nm wavelength absorbance value of brown-red substances. Check the standard curve calculation, can find out the content of reducing sugar in the fermentation broth, so as to find out the amylase activity.
Materials and Instruments
Bran Flour Move I. Main instruments, equipment and materials for the experiment Table 2 Determination of reducing sugars For more product details, please visit Aladdin Scientific website.
NaNO3 KH2PO4 (NH4)2SO4 CaCl2 MgSO4-7H2O CoCl2 FeSO4-7H2O ZnSO4
Spectrophotometer Constant temperature water bath Biochemical incubator Magnetic stirrer Triangle bottle
Spectrophotometer, constant temperature water bath, biochemical incubator, magnetic stirrer, triangular flask, bran, flour, NaNO3, KH2PO4, ( NH4)2SO4, CaCl2, MgSO4.7H2O, CoCl2, FeSO4-7H2O, ZnSO4.
II. Experimental methodology, operational steps
1. Determination of reducing sugars
(1) Preparation of standard curvesNine dry test tubes were taken numbered and the glucose standard solution and 3,5-dinitrosalicylic acid reagent at an exact concentration of 1.00 mg/ml was taken in the amounts shown in Table 1. The units in the table are ml.
Shake each tube well, cover with a small funnel, heat in a boiling water bath for 5 min, immediately cool to room temperature with cold water, then add distilled water to each tube to 20.5 ml, plug the mouth of the tube with a rubber stopper and mix well. Do not shake vigorously to avoid the introduction of air bubbles. Under the wavelength of 540 nm, the absorbance values of tubes 1-8 were measured on a spectrophotometer with tube 0 as the blank. The standard curve was plotted using the absorbance value as the vertical coordinate and the mg of glucose as the horizontal coordinate.
Table 1 Reducing sugar standard curve 
(2) Determination of residual reducing sugars in fermentation broths
The fermentation solution was filtered through a single layer of filter paper, and the filtrate was 0.2 ml fixed to 100 ml and diluted 500 times to a sugar content of 2-8 mg/100 ml as a specimen.
Take 4 dry test tubes, number them, and add exactly the amount shown in Table 2 to the solution to be measured and the reagent (ml)
After the reagents were added, the rest of the procedure was the same as that used to make the glucose standard curve, and the absorbance values of the solutions in each tube were determined.
count
The average of the absorbance values of tubes 1, 2 and 3 was used to find the corresponding milligrams of reducing sugar on the standard curve, and the percentage of reducing sugar in the sample was calculated according to the following formula:
(1) Take 5.00 ml of fermentation filtrate and place it in a 100 ml beaker, add 15 ml of water, stir with a magnetic stirrer for 5 min, insert a potentiometer into the beaker and titrate with 0.1 mol/L NaOH standard solution until the acidometer indicates pH=8.20, which is the free acidity and is not measured. Add 10.0 ml of formaldehyde solution, mix immediately and quickly titrate with 0.1 mol/L NaOH standard solution.The solution was rapidly titrated with 0.1 mol/L NaOH standard solution until the pH=9.20 indicated by the acidimeter, and the number of milliliters of NaOH standard solution consumed was counted. At the same time, do the blank experiment with water.
(2) Calculations
Amino nitrogen (%) = (V-V0) x N x 0.014 x 20/5 x 100
Where: V--volume of sodium hydroxide consumed in the test solution after addition of formaldehyde, ml;
V0--volume of sodium hydroxide consumed in the blank solution after addition of formaldehyde, ml;
M - Concentration of NaOH standard solution, mol/L;
0.014 - mass of nitrogen equivalent to 1.00 ml NaOH standard solution, g;
(3) Measurement and calculation of amylase activity
Amylase activity (IU/g dry medium): The amount of enzyme required to release 1 umol of reducing sugar per minute under the conditions of this experiment is referred to as one enzyme activity unit.
(4) Overall experimental procedure:
① Prepare culture medium, sterilize and cool.
② After cooling, inoculate and stir well, and place in 28 ℃ incubator for cultivation.
(iii) Samples were taken every 24 h and leached with 0.2 M disodium hydrogen phosphate and sodium dihydrogen phosphate buffer (pH 6.0) at a certain leaching ratio for 1 h, and centrifuged to obtain the crude enzyme solution. A total of 6 samples were taken.
④ Determine the amylase activity, reducing sugar and protein content of the enzyme solution, respectively.
III. Experimental results
1. Using the concentration of reducing sugar as the vertical coordinate and the incubation time as the horizontal coordinate, plot the change curve of reducing sugar at different times.
2. Using the concentration of amino nitrogen as the vertical coordinate and the incubation time as the horizontal coordinate, plot the change curve of amino nitrogen at different times.
3. Using amylase activity as the vertical coordinate and incubation time as the horizontal coordinate, plot the change curve of amylase activity at different times.