11th week

Hello BMS students!!! Its da 11th week already, 9 more weeks to go, 2 more campus discussions and we are done for MP/SIP...

This week, I'm going to blog about a technique which I learn during my SIP. Although it is not related to most of our biomedical science subjects, I find it very interesting. The technique is to determine the total dietary fiber in food using the Enzymatic-Gravimetric method.

The principle of this technique is as follows:

Duplicate samples of dried foods, fat extracted if containing >10% fat, are gelatinized with Termamyl. Termamyl is a heat stable alpha-amylase. They are then enzymatically digested with protease and amyloglucosidase to remove protein and starch. It is noted that whenever analyzing mixed diets, always extract fat prior to determining total dietary fiber. Four volumes of ethyl alcohol are added to precipitate soluble dietary fiber. The total residue is filtered, washed with 78% ethyl alcohol, 95% ethyl alcohol and acetone. After drying, the residue is weighed. One duplicate is analyzed for protein whereas the other is incinerated at 525 degrees celsius and ash is determined.

The calculation for total dietary fiber = weight residue - weight (protein + ash)

The apparatus required for this technique are:
1. Fritted crucible
2. vacuum source
3. vacuum oven
4. desicator
5. muffle furnace
6. water baths
7. beakers
8. analytical balance
9. pH meter

The reagents for the technique are as follows:
1. 98% ethanol
2. 78% ethanol
3. acetone
4. phosphate buffer
5. termamyl
6. protease
7. amyloglucosidase
8. sodium hydroxide
9. hydrochloric acid solution
10. celite

Procedure
Note: Run blank through entire procedure along with samples to measure any contributions from reagents to reside

1. Weigh duplicate 1g samples accurate to 0.1mg into beakers. Sample weight must not differ by >20mg
2. 50ml of pH 6.0 phosphate buffer is added to each beaker
3. check pH and adjust pH to 5.8-6.2pH if necessary
4. Add 0.1 termamyl solution
5. cover beaker with aluminium foil and place in boiling water bath for 15 minutes. Shakre gently at 5 minutes interval
6. cool solutions to room temperature
7. adjust to pH 7.3-7.7pH by adding 10ml of 0.275M sodium hydroxide solution
8. add 5mg of protease
9. Cover beaker with aluminium foil and incubate for 30 mins at 60 degrees celsius with continous agitation
10. cool to room temperature
11. add 10ml of 0.325M hydrochloric acid solution
12. measure pH and add acid dropwise if neccessary to attain final pH of 4.0-4.6
13. add 0.3ml of amyloglucosidase and cover with aluminium foil
14. incubate for 30 minutes at 60 degrees celsius with continous agitation
15. add 280ml of 95% ethyl alcohol preheated to 60 degrees celsius
16. let precipitate form at room temperature for 1 hour
17. weigh crucible containing celite to nearest 0.1mg then wet and redistribute bed of celite in cricible using stream of 78% ethyl alcohol
18. apply suction to draw celite onto fritted glass as even mat
19. wash residue excessively with 3 portions of 20ml 78% ethyl alcohol, 2 portions of 10ml 95% ethyl alcohol and 2 portions of 10ml acetone
20. If gums are form, break surface film with spatula to improve filtration
21. time for filtration and washing varies from 0.1 to 6 hours
22. dry crucible containing residue overnight in 70 degrees celsius vacuum oven or 105 degrees celsius air oven
23. cool in desicator and weigh to nearest 0.1mg
24. subtract crucible and celite weight to determine weight of residie
25. incinerate second residue sample of duplicate for 5 hours at 525 degrees celsius
26. cool in desicator and weigh to nearest 0.1mg
27. subtract crucible and celite weight to determine ash

Andika Putra Anda Indera
TG01