1. Food Science and Technology
Bioactive properties of rice bran protein hydrolysates
School of Chemical Engineering
Chatchaporn (Gib) Uraipong
Food Science and Technology

2. Presentation outline Background Problem statement
Current solution and objectives
Research Approach
Experiments progress results
Degree of hydrolysis
Ultrafiltrations and their biological activities
Ion exchange chromatography and their biological activities
Conclusion
Future Direction

3. World and Australian Rice Production
- Annual Australian production = 1.2 million tonnes from approx. 150,000ha
- Australian consumption = 10 kg/person/year.
- Up to 85% of the rice produced in Australia is exported.

4. Figure 1 Rice kernel structure
Rice Bran
Hull 20%
Moisture ~ 6-7%
Ash ~ %
Fat ~ 15-20%
Fiber ~ 11%
Protein ~ 10-15%
Carbohydrate ~ 50%
Pericarp
Seed coat
Rice bran
8-10%
Nucellus
Alleurone layer
Endosperm 69.5%
Embryo 2.5%
Figure 1 Rice kernel structure

5. Bioactive properties on rice protein/peptides
Source
Active protein and peptides
Bioactivity
References
Rice endosperm
Rice albumin
Immunomodulatory activity
Takahashi et al., 1996
Rice endosperm hydrolysate
Lys-
His-Asn-Arg-Gly-Asp-Glu-Phe
Antioxidative activity
Zhang et al., 2009
Phe-Arg-Asp-Glu-His-Lys-Lys
Zhang et al., 2010
Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg
Opioid activity
Takahashi et al., 1994
Thr-Gln-Val-Tyr
ACE inhibitory activity
Li et al., 2007
Rice bran protein hydrolysate
Tyr-Leu-
Ala-Gly-Met-Asn
Adebiyi et al., 2009
Protein fraction with molecular weight ranging from kDa
Chanput et al., 2009
Rice product
(rice wine)
Ile-Tyr-Pro-Arg-Tyr and Tyr-Arg-Arg-Tyr
Saito et al., 1994

6. What we know about enzymatic hydrolysis on rice/rice bran protein
Enzymatic rice bran protein hydrolysates
Safe and mild treatment
More bioactive properties found in protein hydrolysates
Rice bran protein is hypoallergenic
No evidence on forming toxic substances
(lysino-alanine)
Easier process to control and give higher yields
It is cheaper than acid/alkaline hydrolysis
It is a easier process to control and give higher yield products compared to acid/alkaline hydrolysis
It is developed under mild condition of pH ( 6-8) and temperature ( °C)
It does not reduce nutritional qualities
No evidence about forming toxic substances like lysino-alanine (chemical hydrolysis can destroy L-form amino acid and produce D-form amino acid)

7. Problem statement Rice bran is a low value product
Large amount of by-product (from rice milling and rice bran oil industry)
Limited utilization of rice bran protein/peptides, as human food
Limited knowledge on bioactive properties of rice bran protein/peptides
Limited understanding on enzymatic process

8. Project Goal
Enhancing the health promoting potential of rice bran protein and increase its commercial value by producing bioactive peptides through enzymatic hydrolysis

9. Objective
1. To study the relationship between degree of hydrolysis and the biological activities on four main rice bran proteins
2. To study the effect of various enzyme preparations on the enzymatic hydrolysis of defatted rice bran protein
3. To purify the bioactive peptides using ultrafiltration and ion exchange chromatography
4. To isolate and identify active peptides

10. Research approach Protein extraction Enzymatic hydrolysis
Modified Osborne protein fractionation to remove some phytochemicals
Enzymatic hydrolysis
To understand the type of protease for production of rice bran bioactive peptides and their properties
Peptide purification
Ultrafiltration and determined their biological functions
Ion Exchange Chromatography (IEX) and determined their biological functions
Identification
LC-MS/MS
Mode of inhibition

11. Degree of Hydrolysis
%DH= 27±0.2%
%DH= 15±0.4%
%DH= 28±0.1%
%DH= 16±0.8%
Figure 2 Degree of hydrolysis of RBPH ( rice bran protein hydrolysates) prepared using
four different proteases

12. DPPH radical scavenging capacity assay
Figure 3 DPPH scavenging activity of RBPH prepared using four different proteases

13. ABTS cation radical scavenging capacity assay
Figure 4 ABTS scavenging activity of RBPH prepared using four different proteases

14. α-Glucosidase inhibitory activity of crude hydrolysates
% inhibition= 43±2.1%
% inhibition= 41±0.2%
Figure 6 α-Glucosidase inhibitory activity of RBPH prepared using four different proteases

15. Angiotensin I converting enzyme (ACE) inhibitory activity of crude hydrolysates
% inhibition=73±3.2%
% inhibition=81±1.0%
Figure 5 ACE inhibitory activity of RBPH prepared using four different proteases

16. Summary I
Protamax catalysed albumin (AP) and glutelin (GP) at 120 min exhibited strongest ACE inhibitory activity
Alcalase assisted albumin (AA) and glutelin (GA) at 240 min presented highest free radical scavenging and anti α-glucosidase activity
Bioactive properties of crude hydrolysates appeared to relate the degree of hydrolysis
Type of protease effect the biological functions of crude hydrolysates

17. Biological activities of Ultrafiltration Fractions
Figure 6 Biological functions of ultrafiltration fractions

18. Summary II
Peptides MW <3KDa exhibited strongest bioactive activities
The ultrafiltration fractions on hydrolysates showed potent synergism on antioxidant activities (including ABTS radical scavengind and reducing power)
Alcalase catalysed glutelin (MW <3KDa) exhibited strongest α-glucosidase inhibitory activity
Protamax catalysed albumin (MW <3KDa) showed highest antihypertensive activity

19. Antioxidant activity for IEX fraction
Alcalase-assisted glutelin was selected to study on antioxidant activity
Mini Macro-Prep® High Q Cartridge- Strong anion exhange
Figure 7 IEX fractionation and reducing power activity of Alcalase-assisted glutenin <3KDa

20. Antidiabetic activity of IEX fraction
Alcalase-assisted glutelin was selected to study on anti α-glucosidase activity
Figure 8 IEX fractionation and α-glucosidase inhibitory of Alcalase-assisted glutenin <3KDa

21. Antihypertensive function of activity of IEX fraction
Protamax-assisted albumin was selected to study on antihypertensive activity
Figure 9 IEX fractionation and ACE inhibitory of Protamax-assisted albumin <3KDa

22. Kinetic mode of inhibition
Kinetic of angiotensin inhibition peptide
No inhibitor
AP 5
(mg/mL)
AP 10
Vmax
0.051±0.07
0.034±0.01
0.025±0.01 Km
1.83±0.5
1.85±0.2
1.85±0.1 n 8 R2
0.913
0.989
0.996
Kinetic of α-glucosidase inhibition peptide
No inhibitor
GA 0.5 (0.5mg/mL)
GA 1 (mg/mL)
Vmax
0.42±0.03
0.26±0.01
0.19±0.01 Km
2.2±0.4
1.9±0.2
1.8±0.2 n 8 R2
0.968
0.980
0.989
“A non-competitive inhibition rice bran peptide”

23. A non-competitive inhibition of ACE inhibition rice bran peptide
Enzyme
E + I I-E (inactive)
E-S + I I-E-S (inactive)
Inhibitor
Substrate
E- I + S I-E-S(inactive)

24. The ACE inhibitory potential of cereal and legume protein hydrolysates
Sample
IC50 value
(mg mL-1)
Reference
Rice bran
0.92
Our Study
Red bean
0.07
Rui et al, 2013
Lentil
1.11
Jakubczyk and Baraniak, 2013
Soy
66.4
Chiang et al, 2006
Wheat germ
0.45
Qu et al, 2013
References
Rui, X., Boye, J. I., Simpson, B. K., & Prasher, S. O J Func Food, 5:
Qu, W., Ma, H., Zhao, W., & Pan, Z. 2013Chem Eng J, 226:
Jakubczyk, A., & Baraniak, B. 2013, Int J Food Sci tech., 48: 2363–2369
Chiang, W.D., Tsou, M,J., Tsai, Z.Y., & Tsai, T.C Food Chem, 98:

25. Conclusion
The type of enzymes and fractionation had a significant effect on the bioactive peptide of hydrolysates
The biological activities of rice bran protein hydrolysates related to degree of hydrolysis
Rice bran protein hydrolysates had strong antioxidant and anti α-glucosidase activities, and moderately high on angiotensin I converting enzyme inhibitory
Rice bran protein hydrolysates have potential to be developed to bioactive ingredient as nutraceutical/ functional food.

26. Simulation Human Digestion Isolation and Identification
Future works
Determination biological functions of rice bran protein on human digestion
Stability test of the produced bioactive peptides
Simulation Human Digestion
Identification molecular mass and peptide sequencing (LC/MS-MS)
Isolation and Identification

27. Acknowledgements Supervisor : Dr Jian Zhao , University of NSW
Rural Industries Research and Development Corporation (RIRDC)
SunRice® Australia
Novozyme® Australia

28. Thank you