Experiment 1: production of microbial enzyme

1. Materials:
Rice bran (g)

Pectin powder (g)

Inoculum : Aspergillus ozyae

Citrate buffer (0.01M; pH 5.8)

Pectin substrate solution

Equipments:

Erlenmeyer flask, beaker, spectrophotometer, water bath, sucrose concentration tester, glass rod,
centrifuge, filter membrane, pH meter, refrigerator, pipette,…

2. Medium preparation:
Prepare the medium for microorganism in erlenmeyer flasks.

a. Effect of medium moisture content:

Medium 50 60 66.7 75 80
moisture content
(%W)
Rice bran (g) 50 50 50 50 50

b. Effect of medium composition:

Medium number 1 2 3 4 5
Rice bran (g) 50 50 50 50 50
Pectin powder 1 2 3 4 5
(%)
Moisture content 66.7 66.7 66.7 66.7 66.7
(%W)

c. Effect of fermentation duration

Duration (hrs) 24 36 48 60 72
Rice bran (g) 50 50 50 50 50
Pectin powder 1 1 1 1 1
(%)
Moisture content 66.7 66.7 66.7 66.7 66.7
(%W)

d. Inoculation :
The inoculum has been prepared in test tubes by Mr Hoa. We use Aspergillus ozyae
microorganism in this experiment.

- Add 10ml distilled water into a tube, try to resuspend the fungal spores on the surface of
agar without break it.
- Add this suspension into the erlent that has been prepared.
- Mix well and keep them in exact duration at room temperature.
- When the duration finish, we keep the erlent at 4oC for the next class to test enzyme
activity.

e. Enzyme recovery and pectinase activity:

- Mix well 1g of culture with 5ml citrate buffer for 10mins and then go to filtrate.
- Centrifuge the extraction with 10,000xg in 15mins at 4oC.
- Mix 0.1ml of supernatant with 3ml of pectin solution.
- Put it into water bath at 30oC for enzyme activity
- And then go to evaluate the OD value by spectrophotometer at 235nm
3. Result and disscusion:
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
H1 H2 H3 H4 H5
Each of microorganism have survival curve of moisture content to growth. The moisture
content also effect the growth of microorganism. If content of moisture too high or too
low, the microorganism is inhibited. At 75%W, the largest numbers of microorganism
survive and grow and most of them are inhibited at 60%W
0.18

0.16

0.14

0.12

0.1

0.08

0.06

0.04

0.02

0
MC1 MC2 MC3 MC4 MC5
The medium composition in this experiment also effect production of pectinase enzyme.
Based on this graph we can see if the medium composition too low , of course it means
the substrate is not enough for microorganism, therefore, the production of pectinase
enzyme is low . If the medium composition too high, the microorganism can’t use
substrate completely and it may become toxic for microorganism. This is the reason for
the low number of production of pectinase enzyme. This graph show us at 1.5% medium
composition ( pectin powder ) , the production of pectinase enzyme is highest

0.12

0.1

0.08

0.06

0.04

0.02

0
D1 D2 D3 D4 D5
According to this graph, the more fermentation duration, the more production of
pectinase enzyme is. Because if we have more time, the number of microorganism who
 used the substrate for production of pectinase enzyme, is increase with time. Therefore, at
72h, the production of pectinase enzyme is highest

Experiment 2: production of microbial biomass

1. Materials:
Inoculum : saccharomyces cerevisiae

Malt (g)

DNS reagent

Sugar standard: glucose 0.1%

Equipment:

Water-bath, Spectrophotometer, blender, beaker, glucose concentration tester, shaker, pipette,

erlenmeyer flask, botlle,microsope, haemocytometry,…

2. Malt solution preparation for microorganism culture:

- Grind 200g of germinated malt with 1 liter of water; at first, add water not more than 1
liter to blender for grinding easier and then add more water until 1 liter enough.
- Get all malt solution and transfer it to becher.
- Put the becher in the water bath to hydrolyse the starch at 520 C duration: 20 mins, then
630 C duration: 30 mins, then 730 C duration: 30 mins, and at last 1000 C duration 15
mins
- After cooling down the becher, going to filtrate to get the liquid solution.
- Test the sucrose concentration, if this value is more than 10 0 Brix, the solution must be
diluted to 100, if this value is less than 100 Brix, add more glucose to 100 Brix.
Test the value pH, this value must be higher 4,5, if less than this value, the malt solution
have to be adjusted by NaOH.
- Prepare 2 erlent and 1 bottle then fill each of them 200ml liquid solution. Cap 2 erlent by
wood cotton, cover the cap by plastic. Keep the rest of liquid solution for beer
fermentation.
- Put to autoclave for sterilization : 121oC, 15 mins.
3. Inoculation :
After sterilization, add inoculum and label 2 erlent and 1 bottle, keep it in 3 diferent
conditions.
The inoculum is saccharomyces cerevisiae

Sample 1: The fermentation is carried out in a thermostat:

  Temperature: 30oC
 Agitation: 125rpm
 Time: 24h

Sample 2: The fermentation is carried out in room temperature (with oxygen)

 Temperature: 22-30oC
 Agitation: 0 rpm
 Time: 24h
Sample 3: The fermentation is carried out in room temperature (without oxygen)

 Temperature: 22-30oC
 Agitation: 0 rpm
 Time: 24h
The sample will be tested whenever we have class
4. Reducing sugars:
Label and set test tube as table below:

Tube number 1 2 3 4 5 6 7 8 9
Standard glucose 0 0 1 2 3 4 5
0.1% (ml)
Water (ml) 5 5 4 3 2 1 0
The concentration 0 0 0.2 0.4 0.6 0.8 1
in each tube

Determination of reducing sugars

- Pipette 1mL diluted sample into a tube

- Add 1mL DNS solution
- Put the tube in a water-bath at 100oC for 5min
- Cool the tube to ambient temperature
- Add 10mL distilled water and mix
- And then go to check OD value by the spectrophotometric absorbance at 540nm
5. Yeast cell number:

- Yeast cell concentration is quantified by haemocytometry, using a Thoma counting chamber

- Dead cells are determined by the methylen blue staining.
- We mix the cell culture with the methylen blue staining at 1:1 ratio
- Inject ~15 µl of solution per side, using the loading notch. Make sure that the chamber is
fully loaded with liquid, plus some excess in the channels beside it.
- The number of yeast cell is calculated by:
Number of cells/ml = (a * 4000 * 103 * 10n)/b
Where:
a : number of cell in 5 large squares
b: number of smallest square in 5 large squares
103: 1000mm3 = 1ml
10n: diluted factor