MANIPAL INTERNATIONAL UNIVERSITY

ANALYZING THE COMPONENT OF RICE BRAN & RICE

STRAW TO PRODUCE THE ANIMAL FEEDS

Present by : AMIN RAHMAN BIN SABEER MOHAMED

Matrix no : 1000113

Industrial Supervisor : EN. MOHD FIRKHRY FADHLY BIN ABDUL GHANI

Academic Supervisor : PROF. DR. AROKIARAJ PAPPUSAMY

Department : INDUSTRIAL BIOTECHNOLOGY RESEARCH CENTRE (IBRC)

COMPANY PROFILE

Sirim Berhad

Standards and Industrial Research Institute of Malaysia

(SIRIM).

A corporate organization wholly owned by the Malaysian

Government.

Main headquarters – Shah Alam, Selangor

OBJECTIVES

The objectives of SIRIM are:

1) To innovate and develop processes, product and technologies for industry.

2) To promote standardization and quality.

3) To provide technical services for industry and the public.

INTRODUCTION Rice Bran

A by product of rice milling industry.

Research by Mohammed et al. (2014) revealed that rice bran is an incredible source of the:

Vitamins

Minerals

Amino acids

Essential fatty acids

Dietary fibre and

more than 100 antioxidant nutrients

that helps to fight against disease and promote good health

CONT’ Rice Straw

The vegetative part of the rice plant.

Cut at grain harvest or after.

It may be burned and left on the field before the next

ploughing.

Good source of energy, but is poor in protein (Heuze & Tran,

2013).

It has low digestibility.

Low in energy with a crude protein content of only 3–

5 percent.

OBJECTIVE

-To analyze the content of rice bran

and rice straw.

-To gain the working experience.

TASKS

Moisture Content test

Carbohydrate test

Lignin test

Cellulose test

Enzyme Activity

Enzyme Kinetic

METHODOLOGY

Moisture Content test- Oven- Dry method.

Carbohydrate test- Phenol Sulphuric Acid method.

Lignin test- Tappi 222 test method.

Cellulose test- Tappi 203 test method.

Enzyme activity- FP Assay, CMC Assay, Glucose

Enzyme Kinetic- Method of CMC assay (different concentration of CMC solution)

Moisture Content test

1. The Petri dish was weighed before place the sample.

2. 1 gram of sample was added and incubated at 90°C for 16 hours.

3. The sample was weighed and minus the weight of original Petri dish.

PHENOL SULPHURIC ACID METHOD (TOTAL

CARBOHYDRATE) 100 mg of sample weighed into boiling tube.

The boiling tube was kept in boiling water bath for 3 hours with 5 mL of 2.5 NHCL and cooled to room temperature.

Solid sodium carbonate was added to neutralized the mixture.

The volume was made up to 100 mL and centrifuged.

0.2, 0.4, 0.6, 0.8 and 1 mL of working standard were pipette out into a series of test tube.

0.1 and 0.2 mL of the sample solution were pipette out in two separate test tubes. Made up the volume in each tube to 1 mL with distilled water.

CONT’

The blank was set with 1 mL of distilled water.

1 mL of phenol solution was added to each tube.

5 mL of 96% sulphuric acid was added to each tube

and shake well.

After 10 minutes the content in the test tubes were

shake and placed in a water bath at 25- 30 ̊C for 20

min.

The color was read at 490 nm.

LIGNIN TEST

1 gram of sample was

weighed

Placed in a beaker and 15 ml of 72% of sulfuric acid was added

The sample was stirred in

acid until fully

dissolved.

Transfered into a schott bottle and distilled water was

added until the total volume

reached 575 ml.

The solution was autoclaved and

allowed it to cool

The solution was filtered

The filtrate washed using hot water (3

times)

And dried in an oven for 2 days at

90°C

The sample placed in vesicator for 1 day

The dried sample weighed.

ALPHA- CELLULOSE

25 mL of the filtrate and 10.0 mL of 0.5N potassium dichromate solution were pipette into a 250-mL flask.

Add cautiously, while swirling the flask, 50 mL of concentrated H₂SO₄.

Allowed the solution to remain hot for 15 min, then add 50 mL of water and cool to room temperature.

2 to 4 drops of Ferroin indicator were added and titrated with 0.1N ferrous ammonium sulfate solution to a purple color.

Make a blank titration substituting the pulp filtrate with 12.5 mL of 17.5% NaOH and 12.5 mL of water.

BETA- AND GAMMA- CELLULOSE 50 mL of the pulp filtrate was pipette into a 100-mL graduated

cylinder having a ground glass stopper.

50 mL of 3N H₂SO₄ was added and mix thoroughly by inverting.

The cylinder was heat submerged in a hot water bath at about

70-90C for a few minutes to coagulate the beta-cellulose.

Allowed the precipitate to settle for several hours, preferably

overnight, then decant or filter, if necessary, to obtain a clear

solution.

50 mL of the clear solution and 10.0 mL of 0.5N K₂Cr₂O₇ was

pipette into a 300-mL flask.

90 mL of concentrated H2SO4 was cautiously added.

The solution was allowed to remain hot for 15 min, then proceed

with titration as outlined in alpha cellulose method.

Made a blank titration substituting the solution with 12.5 mL of

17.5% NaOH, 12.5 mL of water and 25 mL of 3N H₂SO₄.

ENZYME ACTIVITY TEST FOR CMC-ASE

Stock dilute solution

1 ml dilute sample was added in test

tube

Add 1 ml of citrate buffer pH 4.8 and add 1 ml

of CMC

Incubate at 50°C for 1 hour

Add 3 ml of DNS

Incubate at boiling

temperature for 15 minutes

Add 1 ml of 40% potassium

tartarate

Add 20 ml of Distilled Water

Cool down the sample in room

temperature

Absorbance at 540 nm using spectrophotometer.

Enzyme activity test for FP- ase method 1. 0.5 mL of diluted enzyme solution was taken into the

test tube.

2. 1 mL 0.1 mM citrate buffer pH 4.8 and 50 mg (1x6

cm strip) of Whatman No. 1 filter paper that has

been curled round a glass rod was added.

3. Test tube was incubated for 1 hour at 50 ̊C.

4. After incubation, 3 mL DNS reagent was added.

5. Then boiled for 15 minutes and 1 mL of 40% sodium

potassium tartarate was added.

6. 20 mL of distilled water was added.

7. After cooling to room temperature, absorbance was

measured at 550 nm.

ENZYME ACTIVITY TEST FOR GLUCOSE

METHOD

10 mM glucose solution were taken and diluted

to final volume of 1 mL.

In 1 mL standard solution, 2 mL 0.l M citrate

buffer, pH 4.8 was added.

3 mL DNS reagent was added and boiled for 15

minutes.

After boiled, 1 mL 40% sodium tartarate was

added in hot tubes.

20 mL of distilled water was added.

The tubes were cooled at room temperature and

the absorbance was measured at 550 nm.

ENZYME KINETIC

Stock dilute solution

1 ml dilute sample was added in test

tube

1 ml of citrate buffer pH 4.8 and 1 ml of

different concentration of CMC was added.

Incubated at 50°C for 1 hour

3 ml of DNS was added.

Incubated at boiling

temperature for 15 minutes

1 ml of 40% potassium

tartarate was added.

20 ml of distilled water was added.

The sample was cooled at room

temperature

Absorbance at 540 nm using spectrophotometer.

RESULTS

MOISTURE CONTENT TEST

Rice Bran Rice Straw

Weight of Petri Dish 94.39 gram 93.62 gram

Sample 1 gram 1 gram

Weight after incubation 95.25 gram 94.52 gram

Difference of weight 0.84 gram 0.90 gram

Moisture % 14% 10%

Moisture% = Weight of original sample – weight of dried sample x 100

weight of original sample

RESULT OF LIGNIN TEST

Sample Weigh of

sample

Weight of

Petri Dish

before

incubation

Weight of

Petri Dish

after

incubation

Differences

in Weight

(%)

Rice Bran 1 gram 2.69 3.78 0.09 gram

(9%)

Rice Straw 1 gram 2.74 3.70 0.08 gram

(8%)

ALPHA, BETA AND GAMMA CELLULOSE

α- cellulose β- cellulose γ- cellulose

Rice Bran 62.34877666 26.26777657 11.38344693

Rice Straw 59.28866667 25.21842677 9.49290666

GLUCOSE IN ENZYME ACTIVITY TEST

0.0909 0.1828

0.9263

1.2463

1.9699

2.4862

y = 0.5045x - 0.6154

R² = 0.9738

-0.5

0

0.5

1

1.5

2

2.5

3

0 1 2 3 4 5 6 7

Ab

sorb

an

ce

[Glucose], mg/ml

Abs (550nm)

Abs (550nm)

Linear (Abs (550nm))

[CMC HTec],

mg/ml Abs (540nm)

Standard Factor CMC-ase Activity

Blank 0.4262 - -

1 0.8128 1.23 1.641856

2 0.9263 2.02 1.871126

3 1.2463 2.41 2.517526

4 1.9699 2.02 3.979198

5 2.48862 2.02 5.0270124

[CMC CTec],

mg/ml Abs (540nm)

Standard Factor CMC-ase Activity

Blank 0.3574 - -

1 1.5441 0.65 2.177181

2 1.8277 1.04 2.577057

3 2.1311 1.41 3.004851

4 2.7854 1.41 3.927414

5 3.7484 1.33 5.285244

[Filter Paper Assay HTec], mg/ml Abs (550nm)

Blank 0.07

1 0.9722

2 1.7059

3 2.0465

4 2.438

5 3.5793

[Filter Paper Assay CTec], mg/ml Abs (550nm)

Blank 0.0747

1 0.8794

2 1.4484

3 1.5541

4 1.9328

5 2.673

[Enzyme Kinetics HTec], mg/ml Abs (540nm)

Blank 0.0875

0.8 0.4899

1 0.6062

1.2 0.8879

1.4 2.1804

1.6 2.6088

1.8 3.1877

2 4.461

[Enzyme Kinetics CTec], mg/ml Abs (540nm)

Blank 0.0904

0.8 0.3191

1 0.9931

1.2 1.5585

1.4 1.7958

1.6 2.3257

1.8 2.9981

2 4.3732

CONCLUSION Diet of rice straw alone is not sufficient even to

maintain the animals weight unless supplementary

protein is provided. (Food and Agriculture

Organization)

All of the nutrients in rice bran were significantly

effective and can be useful in food application as

edible oil extraction and food supplement

(Mohammed et al. 2014).

Learn about the process and method of analysis the

chemical content in sample.

Know how to use equipment in proper way.