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OBJECTIVES:

1. To learn the how to use the Fibertec filtering aid machine.


2. To determine the amount of dietary fiber in food sample.

EQUIPMENT:
1. Flask (FOSS)
2. Shaking water bath
3. Micropipette
4. Aluminium foil
5. Measuring cylinder
6. pH meter
7. glass rod/ spatula
8. Thermometer
9. Crucible (FOSS glass crucible for dietary fiber)
10.Foss FIBERTEC 1023 for filtering aid

CHEMICAL REAGENTS:
1. Phosphate buffer solution, 0.08M with pH 6.0 (0.2)
- Prepared by dissolving 1.4g of sodium phosphate anhydrate (Na 2HPO4)
(or 1.753g dehydrate) and 9.68g sodium phosphate monobasic
monohydrate (NaH2PO4) (or 10.94g dehydrate) in approximately 700ml
distilled water. Dilute to 1L with water. pH was checked.
2. Sodium hydroxide solution 0.275N
- Prepared by dissolving 11.00g NaOH in approx. 700mL of distilled water
and dilute to 1L water.
3. Hydrochloric acid solution 0.325N
- Dilute 325mL of 1.0N HCl to 1L distilled water.
4. Megazyme enzymatic Kit
- -amylase (thermostable), 50 L
- protease, 100L
- amyloglucosidase, 200L
5. chemicals:
- Each flask will needed 280mL of 95% ethanol.
- 10mL of 78% ethanol, 95% ethanol, and acetone for washing purpose.
- Celite 545 (Diatomaceous Earth)

Sample preparation:
-

Samples with high fat were defatted with petroleum ether.

Samples with 50% of carbohydrate were sugar extracted with ethanol

78%.
Weight of samples needed: 1.000g

PROSEDURE:
1. Duplicate 1g samples were weighed, accurate to 0.1mg into the flask.
Sample weights should be differ by less than 20 mg from each other. 50
mL phosphate buffer was added to each beaker and pH was checked (pH
must be 6.00.1). The pH was adjusted when required.
2. 50 L -amylase solution was added to the flask.
3. The beaker was covered with aluminium foil and was placed in boiling
water both for 30 minutes.
4. The solution was cooled to room temperature.
5. By adding 10 mL 0.275N NaOH solution the pH was adjusted to pH
7.50.1. The pH was checked with pH meter.
6. 100L of protease was added.
7. The beaker was covered with aluminium foil and incubated at 60C with
continuous agitation for 30 min.
8. By cooling and adding 10 mL of 0.325N HCl solution the pH was adjusted
to pH 4.50.2. The pH was checked with pH meter.
9. 200L amyloglucosidase was added, covered and incubated 60C for 30
min.
10.280 mL of 95% ethanol was added and preheated to 60C and at room
temperature precipitate was formed.
11.Crucible (blank) was weighed and Celite 545 1g was added then wet with
78% ethanol using Fibertec filtering aid machine. Suction was applied
when using Fibertec machine to achieve even mat of celite.
12.Precipitation of fiber was filtered by using Fibertec filtering aid.
13.Residue from the filtering was washed with three 20mL portions of 78%
ethanol, two 10 mL portions of 95% ethanol and two 10 mL portions of
acetone.
14.Crucible was dried overnight in 105C air oven.
15.Cooled in the dessicator and weighed. Crucible and celite weights were
substract to determine weight of residue.
16. The residue was analysed from one samples of duplicate for protein
(using kjedahl, N 6.25 conversion factor), and one for ashing (5 hours at
525C furnace).