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

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

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.

Figure 1 Rice kernel structure Rice Bran Hull 20% Moisture ~ 6-7% Ash ~ 10-12 % 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

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 10-60 kDa Chanput et al., 2009 Rice product (rice wine) Ile-Tyr-Pro-Arg-Tyr and Tyr-Arg-Arg-Tyr Saito et al., 1994

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 ( 40-60 °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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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”

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)

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. 2013. J Func Food, 5: 1116-1124 Qu, W., Ma, H., Zhao, W., & Pan, Z. 2013Chem Eng J, 226: 139-145 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. 2006. Food Chem, 98: 725-732

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.

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

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

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