Rice Bran white / brown (Before stabilization )

Chemical
Test Method
required
Nutritional
01. Moisture Oven dry method – AOAC method
02. Fat Soxhlet extraction
03. Fiber
04. Protein Kjeldahl nitrogen method
05. Reducing sugar
06. Total Ash Accurately weigh 5 g of sample in a weighing silica dish and ignite at 550°C for 4-6 hrs.

 Water-Soluble and -
Insoluble Ash -
 Acid-Insoluble ash
 Alkalinity of the Ash
1. Boil the ash in a one of the silica dish with 25 ml water.
2. Filter through an ash less filter paper and thoroughly wash with hot water
(The combined filtrates and washing should not exceed 60 ml)
3. Transfer the filter paper to the original silica dish.
4. Ignite, cool and note the weight.
5. The tare weight deducted from this weight gives water- insoluble ash content.
6. Calculate the water-soluble ash content as follows.
Water soluble ash= Total ash -Water insoluble ash
1 add ash silica dish, add 25 ml of dil. HCI (10% wt/wt)
2. Cover with a watch glass and, then boil gently over a low flame for 5 minutes.
3. Filter using an ash less filter paper.
4. Wash the filter paper thoroughly with hot water.
5. Return the paper to the original silica dish, ignite, cool and weigh the Insoluble ash.
1. add ash to silica dish, add 10 ml of 0.1 N HCl
2. Dissolve by warming on a water bath, cool, and titrate the excess acid with 0.1 N NaOH
using Methyl
Orange indicator. (Let the alkali consumed be B ml)
3. Carry out a blank titration using 10 ml of 0.1 N HCl. (Let the blank value be A ml)
4. Calculate the Alkalinity of ash express as number of ml of 0.1 N acid required to
neutralize the ash from
100 g of the sample.
07. Mineral P, Ca, Fe , Mg ,Na AOAC METHOD.
08. Carbohydrate Carbohydrate was calculated by subtracting the values of moisture, protein, crude fiber,
fat and ash, from 100.
09. Antioxidant DHHP assay method:  DPPH
 methanol

10. Flavonoid

11. Polyphenolic content Folin–Ciocalteu method  Folin–

 One millilitre of 0.01 g/ml rice bran extract was mixed with 5 ml Folin– Ciocalteu Ciocalteu
reagent (diluted tenfold with distilled water) and 4 ml (7.5 g/100 ml) sodium reagent
carbonate.  sodium
 After 1 h at room temperature, the absorption of clear solutions was read at 765 carbonate.
nm.  ethanolic
gallic acid
 For the preparation of calibration curve, different concentrations of ethanolic
solutions
gallic acid solutions were mixed with the same reagents as described above, and
after 30 min, the absorption of clear solutions was measured.
 The amount of total phenolic compounds was expressed as gallic acid equivalents
(GAE) in milligrams per rice bran extract.
 The experiment was repeated thrice and the mean value was reported
Physical
12. Color Colorimeter

13. Bulk density bulk density of rice bran was determined by pouring the sample into a 25 ml volumetric
flask by shaking and lightly tapping the bottom of flask on a plastic pad (Chastril 1990).
The flask was filled up to the mark with rice bran, weighed and bulk density of rice bran
was calculated. bulk density was carried out in triplicate
Pouring Method:
Materials Needed:
Rice bran sample
Graduated cylinder or container of known volume
Procedure:

14. Water absorption
15. Swelling power
16. Relative density
pH
acid value
saponification value
17. saturated potassium iodide
solution
Ensure that the graduated cylinder or container is clean and dry.
Pour the rice bran into the graduated cylinder carefully, avoiding excessive compaction or
air entrapment.
Gently tap the cylinder to settle the rice bran and remove any air voids.
Record the initial volume of the rice bran in the graduated cylinder.
Weigh the cylinder with the rice bran sample (total weight, including the container).
Calculate the bulk density using the formula:
Bulk Density = (Weight of Rice Bran) / (Volume of Rice Bran)
Five grams of RB was suspended in 30 mL of water; the mixture was stirred 7 times with a No chemical
10 min interval, centrifuged at 2300 rpm for 25 min, and the residue was dried. WAC was required
calculated as grams of water absorbed per 100 g of dried pellet
Cite - (Rice Bran Stabilization and Oil Extraction Using the Microwave-Assisted Method and
Its Effects on GABA and Gamma-Oryzanol Compounds)
No chemical
required
Chemical Properties
In bran oil – pH meter
Ethyl alcohol
Ether
Titrate with NaOH
Phenolphthalein
Ethanol
KOH
Titrate with Sulfuric
acid
18. Iodine value

(Nielsen, 2017)
19. Smoke point

To a liquid fat sample, neutralized 95% ethanol and phenolphthalein indicator are added. ethanol
20. Free Fatty Acids (FFAs) The sample then is titrated with NaOH and the percent FFA calculated (AOAC) phenolphthalein

The fat or oil sample is dissolved in glacial acetic acid isooctane (3:2). Upon addition of glacial acetic acid
excess potassium iodide, which reacts with the peroxides, iodine is produced (Eq. 23.14). potassium iodide
The solution then is titrated with standardized sodium thiosulfate using a starch indicator sodium thiosulfate
21. Peroxide Value
(aocs ) starch indicator

in rice bran oil :-

Chemical
Test Method
required
 Nutritional
22. Moisture Oven dry method – AOAC method
23. Fat Soxhlet extraction
24. Protein Kjeldahl nitrogen method
25. Reducing sugar
26. Total Ash Accurately weigh 5 g of sample in a weighing silica dish and ignite at 550°C for 4-6 hrs.

 Water-Soluble and -
Insoluble Ash -
 Acid-Insoluble ash
1. Boil the ash in a one of the silica dish with 25 ml water.
2. Filter through an ash less filter paper and thoroughly wash with hot water
(The combined filtrates and washing should not exceed 60 ml)
3. Transfer the filter paper to the original silica dish.
4. Ignite, cool and note the weight.
5. The tare weight deducted from this weight gives water- insoluble ash content.
6. Calculate the water-soluble ash content as follows.
Water soluble ash= Total ash -Water insoluble ash
1 add ash silica dish, add 25 ml of dil. HCI (10% wt/wt)
2. Cover with a watch glass and, then boil gently over a low flame for 5 minutes.
3. Filter using an ash less filter paper.
4. Wash the filter paper thoroughly with hot water.
5. Return the paper to the original silica dish, ignite, cool and weigh the Insoluble ash.

1. add ash to silica dish, add 10 ml of 0.1 N HCl

 Alkalinity of the Ash
27. Mineral
2. Dissolve by warming on a water bath, cool, and titrate the excess acid with 0.1 N NaOH
using Methyl
Orange indicator. (Let the alkali consumed be B ml)
3. Carry out a blank titration using 10 ml of 0.1 N HCl. (Let the blank value be A ml)
4. Calculate the Alkalinity of ash express as number of ml of 0.1 N acid required to
neutralize the ash from
100 g of the sample.
P, Ca, Fe , Mg ,Na AOAC METHOD.
28. Carbohydrate Carbohydrate was calculated by subtracting the values of moisture, protein, crude fiber, fat
and ash, from 100.
29. Oryzanol  Exactly 1.0 g dried bran was weight into screw cap test tube and added 4 ml of (Hexane or ethyl
solvent (hexane, ethyl acetate, isopropanol and butanol). acetate or
 Extraction was done by mixing the substances for 30 minutes at room temperature isopropanol or
(27 C) and incubated overnight. butanol)
 Centrifugation was used for separate the bran from miscella for 5 minutes at 3,000
rpm.
 Supernatants was collected and recorded for -oryzanol absorption spectra. The
brand of RBO were dissolved in solvent and the -oryzanol in the oil absorbance
was done by using a U-100 UV-vis spectrophotometer (HITASHI, Japan).
 Quantification of -oryzanol in solvent was determined by standard curve.
30. Antioxidant DHHP assay method:  DPPH
 methanol

31. Flavonoid

32. Polyphenolic content Folin–Ciocalteu method  Folin–

33. Total Anthocyanin Content
 One millilitre of 0.01 g/ml rice bran extract was mixed with 5 ml Folin– Ciocalteu Ciocalteu
reagent (diluted tenfold with distilled water) and 4 ml (7.5 g/100 ml) sodium reagent
carbonate.  sodium
 After 1 h at room temperature, the absorption of clear solutions was read at 765 carbonate.
nm.  ethanolic
gallic acid
 For the preparation of calibration curve, different concentrations of ethanolic gallic
solutions
acid solutions were mixed with the same reagents as described above, and after 30
min, the absorption of clear solutions was measured.
 The amount of total phenolic compounds was expressed as gallic acid equivalents
(GAE) in milligrams per rice bran extract.
 The experiment was repeated thrice and the mean value was reported
Total anthocyanin contents of the rice bran were measured using a spectrophotometric pH potassium chloride
 differential sodium acetate
The rice bran extracts were mixed thoroughly with 0.025 M potassium chloride (pH 1) buffer
buffer.
The absorbance of the mixture was then measured at 515 and 700 nm against distilled
water blank.
The rice bran extracts were then combined similarly with sodium acetate buffer (pH 4.5),
and the absorbance of these solutions was measured at the same wavelengths
The anthocyanin content was calculated as total anthocyanins
total anthocyanins (mg/100 g) of DW of sample;
A x MW x DF x 1000/ =(ε x C)

Anthocyanin content was expressed as milligrams of Cy-3-G equivalents per 100 g of DW of

sample for triplicate extracts

where A is absorbance= (A515 - A700) pH 1.0 - (A515 - A700) pH 4.5

MW is molecular weight for Cy-3-G=449.2;
ε is the molar absorptivity of Cy-3-G= 26900;
C is the concentration of the buffer in mg/mL;
DF is the dilution factor (0.1 mL of free phenolic extract sample was diluted to 10 mL, DF=
100; 2 mL of bound phenolic extract was diluted to 10 mL, DF = 5)

Physical
34. Color Colorimeter

35. Bulk density bulk density of rice bran was determined by pouring the sample into a 25 ml volumetric
flask by shaking and lightly tapping the bottom of flask on a plastic pad (Chastril 1990).
The flask was filled up to the mark with rice bran, weighed and bulk density of rice bran
was calculated. bulk density was carried out in triplicate
Pouring Method:

Materials Needed:
Rice bran sample
Graduated cylinder or container of known volume
Procedure:
 Ensure that the graduated cylinder or container is clean and dry.
Pour the rice bran into the graduated cylinder carefully, avoiding excessive compaction or
air entrapment.
Gently tap the cylinder to settle the rice bran and remove any air voids.
Record the initial volume of the rice bran in the graduated cylinder.
Weigh the cylinder with the rice bran sample (total weight, including the container).
Calculate the bulk density using the formula:
Bulk Density = (Weight of Rice Bran) / (Volume of Rice Bran)
36. Water absorption Five grams of RB was suspended in 30 mL of water; the mixture was stirred 7 times with a No chemical
10 min interval, centrifuged at 2300 rpm for 25 min, and the residue was dried. WAC was required
calculated as grams of water absorbed per 100 g of dried pellet
Cite - (Rice Bran Stabilization and Oil Extraction Using the Microwave-Assisted Method and
Its Effects on GABA and Gamma-Oryzanol Compounds)
(Stabilisation, Method and Compounds, 2022)
37. Swelling power No chemical
required
38. Relative density

Chemical Properties
pH In bran oil – pH meter
Ethyl alcohol
Ether
acid value
Titrate with NaOH
Phenolphthalein
Ethanol
KOH
saponification value
Titrate with Sulfuric
acid
39. saturated potassium iodide
solution

40. Iodine value

(Nielsen, 2017)
41. Smoke point

To a liquid fat sample, neutralized 95% ethanol and phenolphthalein indicator are added. ethanol
42. Free Fatty Acids (FFAs) The sample then is titrated with NaOH and the percent FFA calculated (AOAC) phenolphthalein

The fat or oil sample is dissolved in glacial acetic acid isooctane (3:2). Upon addition of glacial acetic acid
excess potassium iodide, which reacts with the peroxides, iodine is produced (Eq. 23.14). potassium iodide
The solution then is titrated with standardized sodium thiosulfate using a starch indicator sodium thiosulfate
43. Peroxide Value
(aocs ) starch indicator
Extraction method
RBO was extracted from rice bran (50 g) by Soxhlet extraction for 3 h using hexane as the solvent. The AOCS official methods (Official AOCS 1997,
Method Ca 5a-40) ( Isolation of oryzanol from crude rice bran oil pdf)

(Rice Bran Stabilization and Oil Extraction Using the) - oil yield is higher when the sample is heated to 120 ◦C. Additionally, the 9:1 ratio has a
better extraction performance, and ethanol proved to be the most suitable solvent for the MAE compared to isopropanol, as the highest yields
were obtained with ethanol

Bran Oil

Melting Point -
The capillary tube melting point, also known as the complete melting point or clear point, is the temperature at which fat heated at a
given rate becomes completely clear and liquid in a one-end closed capillary.

Cloud Point - is the temperature at which a cloud is formed in a liquid fat due to the beginning of crystallization.
The sample is heated to 130 °C and then cooled with agitation. The temperature of first crystallization is taken to be the point at which
a thermometer in the fat is no longer visible.
Sample Preparation:
Obtain a representative sample of the fat or lipid you want to test. Ensure that the sample is homogeneous and free from any visible
particles or impurities.
Filling the Test Tubes:
Carefully pour a small amount of the fat sample into a clean and dry test tube. Avoid introducing air bubbles into the sample.
Cap the Test Tubes:
Seal the test tubes tightly with caps to prevent any evaporation during the experiment.
Heating and Observation:
Set up a water bath or temperature-controlled heating device with a thermometer or temperature sensor to monitor the temperature
accurately.
Place the test tubes containing the fat sample into the water bath or heating device.
Gradually increase the temperature of the water bath while gently swirling or stirring the test tubes to ensure uniform heating.
Observe the test tubes for the appearance of cloudiness or haze inside the fat sample as the temperature rises. The cloud point is the
temperature at which the cloudiness or haze becomes visible in the sample.
Iodine Value – is defined as the grams of iodine absorbed per 100 g of sample. AOAC
Saponification Value
Acid value