THE UNIQUE NUTRITIONAL/

NUTRACEUTICAL
PROPERTIES OF RICE
PROTEIN

RICE UTILIZATION WORKSHOP

Chateau Bourbon, New Orleans, LA
March 12-13, 2009

Navam Hettiarachchy, PhD

University Professor
Ronny Horax
Arvind Kannan
Food Science Department
University of Arkansas, Fayetteville
 OVER VIEW
‰INTRODUCTION
‰RICE ENDOSPERM PROTEIN
‰RICE BRAN PROTEIN
‰UNIQUE PROPERTIES OF RICE PROTEIN
) Gluten Free
) Hypoallergenic
) Hypocholesterolemic
) Antioxidant
) Antihypertensive
) Anti-cancer
Native Unfolding
INTRODUCTION
Introduction: Structure and composition of Rough Rice

Rice Bran
(8%)
Hull Subaleurone layer Endosperm
(21%) (70%)

Embryo
(1%)
Radicle

Rachilla

Source: Juliano, 1985

 Rough Rice Brown Rice

Rice Flour Milled Rice Rice Bran

Proximate Composition of Rice, Bran, and
Hull
Nutrient (g/100 g)* Rough rice Brown rice Milled rice Rice bran Rice hull
Crude protein (N x
5.95 ) 5.8-7.7 7.1-8.3 6.3-7.1 11.3-14.9 2.0-2.8
Crude fat 1.5-2.3 1.6-2.8 0.3-0.5 15.0-19.7 0.3-0.8
Crude fiber 7.2-10.4 0.6-1.0 0.2-0.5 7.0-11.4 34.5-45.9
Crude ash 2.9-5.2 1.0-1.5 0.3-0.8 6.6-9.9 13.2-21.0
Carbohydrates 64-73 73-87 77-89 34-62 22-34
Neutral detergent
fiber 16.4-19.2 2.9-3.9 0.7-2.3 24-29 66-74

*at 14% moisture

Sources: Juliano, 1985; Eggum et al., 1982; Pedersen and Eggum, 1983.
 Vitamins and Minerals in Rice, Bran,
and Hull

Nutrient (per g) Rough rice Brown rice Milled rice Rice bran Rice hull
Thiamine (µg) 2.6-3.3 2.9-6.1 0.2-1.1 12.0-24.0 0.9-2.1
Riboflavin (µg) 0.6-1.1 0.4-1.4 0.2-0.6 1.8-4.3 0.5-0.7
Niacin (µg) 29-56 35-53 13-24 267-499 16-42
α-Tocopherol (µg) 9.0-20 9.0-25 trace-3.0 26-130 60-130
Calcium (mg) 0.1-0.8 0.1-0.5 0.1-0.3 0.3-1.2 0.6-1.3
Phosphorus (mg) 1.7-3.9 1.7-4.3 0.8-1.5 11.0-25.0 0.3-0.7
Phytin P (mg) 1.8-2.1 1.3-2.7 0.2-0.7 9.0-22 0
Iron (µg) 14-60 2.0-52 2.0-28 86-43 43-258
Zinc (µg) 1.7-3.1 0.6-2.8 0.6-2.3 4.3-25.8 0.9-4.0

Sources: Juliano, 1985; Pedersen and Eggum, 1983

Preparation of Protein Rice Fractions from Rice
Endosperm Flour
Rice Flour (100g)

Defatted (hexane 400 ml)

Air-dried (hood 24 h)

Extracted (water 400 ml, 20 oC, 4 h)

Supernatant-1
(Albumin)
Residue
Salt extraction
Supernatant-2 (400 ml 0.86M, 20 oC, 4 h)
(Globulin)
Residue
Alkali extraction
Supernatant-3 (400 ml 0.02M NaOH, 30 min)
(Glutelin)
Residue
Alcohol extraction
Supernatant-4 (300 ml 70% , 20 oC,4 h)
(Prolamin)
Residue
(Crude carbohydrate)
Preparation of Protein from Heat-Stabilized
Defatted Rice Bran (HDRB)

HDRB (100 g)+DI water (1 L)

Adjusted to pH 10.0 using 3M NaOH

Stirred for 3 h at 20 oC

Centrifuged (15,000 x g for 10 min)

Residue
Re-extracted Supernatant

Supernatant Combined Supernatant

Isoelectrically precipitated at pH 4.5

Centrifuged (15,000 x g for 10 min)

Freeze-dried

Protein
 Rice Endosperm Protein Protein

) DSM, Switzerland – Functional Protein

Rice Bran Protein

) BENE0 – Group, Belgium (Functional-in
progress)
 Essential Amino Acid Composition (g/100 g protein) of Rice, Bran,
Soybean, Milk, and Egg Albumin

Amino acid Rough rice Brown rice Milled rice Rice bran Soybean Milk (Casein) Egg Albumin

Histidine 1.6-2.9 2.4-2.6 2.3-2.7 2.8-3.5 2.7-5.9 2.8 2.7

Isoleucine 3.2-5.0 3.6-4.6 3.7-4.8 2.8-4.3 1.5-4.0 4.7 6.1
Leucine 7.2-9.2 8.3-8.9 8.4-8.6 7.2-8.0 4.4-7.3 9.1 9.7
Lysine 3.4-4.9 3.9-4.3 3.4-4.2 5.0-5.7 4.7-13.8 7.5 8.1
Methionine 1.6-3.6 2.3-2.5 2.3-3.0 1.8-2.4 1.2-5.4 1.9 4.3
Phenylalanine 3.3-6.1 5.0-5.3 4.6-5.4 4.7-5.0 2.5-5.0 5 6.9
Threonine 3.2-4.7 3.9-4.0 4.4-5.3 4.0-4.4 1.5-4.5 4.9 5
Tryptophan 1.3-2.1 1.3-1.5 0.6 0.6-1.3 1.3 1.5 1.5
Valine 4.8-7.4 5.0-6.6 5.8-7.9 5.1-6.3 1.7-4.2 7.4 7.7

Rerefences 1 1 1 1 2,3 4 5,6

1. Juliano, 1985; 2.Hettiarachchy, 2008 (unpublished); 3. Rackis et al., 1961; 4.

Daenzer et al., 2001; 5. Matoba et al., 1982; 6. Lang et al., 1998
Unique Properties of Rice
Proteins

) Gluten Free
) Hypoallergenic
) Hypocholesterolemic
) Antioxidant
) Antihypertensive
) Anti-cancer
 Gluten-free
) Gluten can cause celiac disease that damage the
mucose of the small intestine leading to
malabsorption of nutrients to gluten intolerant
people (Thompson T, 1997; Collin et al. 2004)

) Rice protein is gluten free

Hypo-allergenic
) Some proteins can cause allergic reactions such
as Baker’s asthma, severe dermatitis, and ezcema
to sensitive people (Usui et al. 2001)

) Rice protein is hypo-allergenic

 Hypocholesterolemic
)Very little information

Our Study-Hypocholesterolemic

¾Rice bran protein concentrate

9 Hamsters
9 Soy protein vs. rice bran protein
9 Rice bran protein four times more effective
 IMMUNOMODULATORY AND ANTIOXIDANT
ACTIVITIES OF RICE ENDOSPERM AND BRAN
PEPTIDES AND PROTEIN HYDROLYSATES

¾ Immunomodulatory peptides  derived  from  tryptic

hydrolysates of  rice  proteins  stimulated  superoxide anions 
(reactive  oxygen  species‐(ROS)  that  triggers  non‐specific 
immune defense systems (Hartmann and Meisel, 2007).

¾ Antioxidant  activity  of  phytate‐free  crude  rice  bran  protein 

hydrolysate was found to be increased compared to crude rice 
bran protein hydrolysate (Adebiyi et al,2006).

www.afrihealth.com/.../antioxidant_diag.gif
 The Angiotensin Cascade

Angiotensin converting
enzyme (ACE) converts the
decapeptide angiotensin I into
the octapeptide angiotensin II
through cleavage of the
carboxyl terminal dipeptide
histidyl-leucine.

Angiotensinogen
Renin
Angiotensin-I Angiotensin-II

Angiotensin-I Converting Enzyme

Ref: Jack M. Ginsburg. 1998. Renal Physiology, regulation of sodium and extracellular fluid volume
 Antihypertensive
[Angiotensin-1 converting enzyme (ACE) inhibition activity]

Principle of the ACE Inhibition Assay

The ACE mediates the cleavage of a synthetic substrate, N-hippuryl-l-histidyl-l-
leucine into hippuric acid and the dipeptide, histidyl-leucine. This reaction is
stopped by addition of hydrochloric acid. Hippuric acid is then complexed with
cyanuric chloride and the absorbance of the hippuric acid-cyanuric acid complex
is measured at 228 nm. One unit of ACE activity is defined as the amount of
enzyme required to release one μmol of hippuric acid per minute and per liter of
serum at 37°C (Hurst and Lovell-Smith, 1981).
Angiotensin-I Angiotensin-II

Angiotensin-I Converting Enzyme

N-hippuryl-
histidyl-leucine Hippuric acid + Histidyl-leucine

(synthetic + cyanuric acid

substrate) Hippuric acid-cyanuric acid complex

Absorbance at 228 nm
PREPARATION OF PEPTIDES FROM RICE BRAN
‐ ENZYMATIC APPROACH

Protein
Rice bran

S P

E ES E
Enzyme substrate complex
Protein
Peptide
 FRACTIONATION OF PEPTIDES BY ULTRAFILTRATION 

Rice bran peptide hydrolysate

UF (50kDa) Nominal cut‐offs

< 50kDa             > 50kDa
UF (10kDa)
Evaluated for 
Nominal cut‐offs
functional properties

10‐50kDa           < 10kDa
Nominal cut‐offs       UF (5kDa)
Fractionating peptides by UF columns

< 5kDa              5‐10kDa
 ACE Inhibition of Rice Endosperm Protein Peptide
Fractions

A R P I+P e ps in
100 A R P I+A lc a la s e
A R P I+Liquipa no l
90 T R P I+P e ps in
T R P I+A lc a la s e
80 T R P I+Liquipa no l

70
ACE inhibition (%)

60

50

40

30

20

10

0
< 5 kDa 5-10 kDa 10-50 kDa > 50 kDa

Peptide fractions

*ARPI: rice protein isolate extracted under alkali condition (pH 11); TRPI: rice protein
isolate extracted using heat stable alpha-amylase (Termamyl).
(Hettiarachchy & Horax, unpublished data)
 Gastrointestinal Juices Treatment of Bran 
Peptides

Method Used ‐ Protein Research Laboratory

S P

E ES E
Enzyme substrate complex

Protein
GI tract

Source: www.drugdevelopment-technology.com/projects/
ACE Inhibition of Rice Bran Peptide Fractions

90

80

70
ACE Inhibition (%)

60

50
RBH
40 GJRH
30 GIJRH

20

10

0
w/ o UF <1 k D a 1- 5 k D a 5 - 10 k D a 10 - 5 0 k D a >5 0 k D a

Fractions

*RBH-Rice bran hydrolysate; GJRH-Gastric (pepsin) juice resistant hydrolysate; GIJRH-

Gastrointestinal (pepsin + pancreatin) juice resistant hydrolysate; Values with different
lower case letters are significantly different within fractions
(Hettiarachchy & Henderson, unpublished data)
 ANTICANCER ACTIVITY TESTING OF PEPTIDES

Human cancer cells

Cultured in 25/75 cm2 flasks with ATCC recommended

respective growth media at 37°C

Cells trypsinized and plated on 24/96 well plates – allowed to

form monolayer at 37°C

Peptide fractions incubated on cells on 24 and 96-well plates

with fresh growth media – incubated for 24-48h at 37°C

Cell viability and proliferation inhibition evaluated using

a) Trypan blue dye exclusion assay
b) Phenazine methosulfate/ 3-(4,5-dimethyl thiazole-2-yl)]-2,5-
diphenyl tetrazolium bromide (MTS) colorimetric assay
 ANTICANCER ACTIVITY OF RICE BRAN PEPTIDE 
HYDROLYSATES ON COLON CANCER CELLS

90
a,b
80
a,b
70
% inhibition

60 a,b
50
40 HCT‐116
30 Caco‐2
20
10
c
0
Positive Negative <5 kDa 5‐10 kDa
Peptide fractions

MTS confirmatory assay s for anti-cancer activities of rice bran peptide fractions on HCT-116, Caco-2 (Colon) cancer
cells. HCT-116 cells cause colorectal carcinoma; Caco-2 cells cause colorectal adenocarcninoma. Sources of two cell lines are
different.
Values are means of two trials. Values not connected by same letters are significantly different (P<0.05).
MTS - (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
Negative: saline
Positive: Genistein (200µM).
 ANTICANCER ACTIVITY OF RICE BRAN PEPTIDE 
HYDROLYSATES ON BREAST CANCER CELLS

90
a,b
80 a,b
70
a,b
% inhibition

60
50
40 MCF‐7
30 HTB‐22
20
10 c
0
Positive Negative <5 kDa 5‐10 kDa
peptide fractions

MTS confirmatory assay s for anti-cancer activities of rice bran peptide fractions on MCF-7, HTB-22 (Breast) cancer
cells. Both cell types cause breast adenocarcinoma but the sources are different.
Values are means of two trials. Values not connected by same letters are significantly different (P<0.05).
MTS - (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
Negative: saline
Positive: Genistein (200µM).
 ANTICANCER ACTIVITY OF RICE BRAN PEPTIDE 
HYDROLYSATES ON LUNG AND LIVER CANCER CELLS

100
90
a,b
80 a,b
a,b
70
%  ihibition

60
50 A‐549
40
30 HepG2
20
10 c
0
Positive Negative <5 kDa 5‐10 kDa
pepide fractions

MTS confirmatory assay s for anti-cancer activities of rice bran peptide fractions on A-549 (Lung), and HepG2 (Liver)
cancer cells. A-549 cells cause lung carcinoma; HepG2 cells cause hepatocellular carcinoma.
Values are means of two trials ± SE. Values not connected by same letters are significantly different (P<0.05).
MTS - (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
Negative: saline
Positive: Genistein (200µM).
 RICE BRAN PEPTIDES

¾ Invention disclosure and filing patent

¾ Nutraceutical Innovations, LLC’s interest in

Licensing
 VISIBILITY OF OUR RESEARCH ON ANTICANCER RICE 
BRAN PEPTIDES
PHARMACY NEWS ARTICLE

1/27/09 - Research on liver cancer described by scientists

at University of Arkansas

2009 JAN 27 - (NewsRx.com) -- "Rice bran, an economical, underutilized coproduct of rough rice milling, was used to
produce peptide hydrolysates, which were investigated for anticancer activity (see also Liver Cancer). Protein
hydrolysates prepared by Alcalase hydrolysis under optimized conditions were treated further to obtain gastrointestinal
(GI)-resistant peptide hydrolysates," scientists in the United States report.
"They were fractionated into > 50, 10-50, 5-10, and <5 kDa sizes and evaluated for inhibitory activity on proliferation of
human colon (Caco-2) and liver (HepG2) cancer cell lines by Trypan blue dye exclusion assay. GI-resistant <5 and 5-10
kDa sized peptide fractions inhibited growth of Caco-2 cells by 80%, and the <5 kDa fraction inhibited growth of HepG2
cells by similar to 50% compared to controls and nonresistant fractions. An MTS cell titer assay confirmed antiproliferative
effects of the peptide fractions," wrote A. Kannan and colleagues, University of Arkansas.
The researchers concluded: "The results demonstrated that 5-10 and <5 kDa sized GI-resistant fractions promoted
significant (p < 0.05) inhibitory activities on both cancer cell lines compared to controls, More investigations are needed to
show such value-added effects on the technofunctional and sensorial properties of the food protein and peptide matrices."
Kannan and colleagues published their study in the Journal of Agricultural and Food Chemistry (Human Colon and Liver
Cancer Cell Proliferation Inhibition by Peptide Hydrolysates Derived from Heat-Stabilized Defatted Rice Bran. Journal of
Agricultural and Food Chemistry, 2008;56(24):11643-11647).
For additional information, contact N. Hettiarachchy, University of Arkansas, Dept. of Food Science, Fayetteville, AR
72704, USA.

The publisher's contact information for the Journal of Agricultural and Food Chemistry is: American Chemical Society,
1155 16th St., NW, Washington, DC 20036, USA.

Copyright © 2009 Cancer Weekly via NewsRx.com

 References
• Abayomi P. Adebiyi, A. P., Ayobamitale O. Adebiyi, A. O., Tomohisa Ogawa, T., and Koji Muramoto,
K. 2006. Purification and characterisation of antioxidative peptides from unfractionated rice bran
protein hydrolysates. Int. J. Food Sci. Tech. 43(1):35-43
• Collin, P., Thorell, L., Kaukinen, K., and Mäki, M. 2004. The safe threshold for gluten contamination
in gluten-free products. Can trace amounts be accepted in the treatment of coeliac disease?
Aliment. Pharmacol. Ther. 19: 1277–1283.
• Daenzer, M., Petzke, K. J., Bequette, B. J., and Metges, C. C. 2001. Whole-body nitrogen and
splanchnic amino acid metabolism differ in rats fed mixed diets containing casein or its
corresponding amino acid mixture. J. Nutr. 131:1965–1972.
• Eggum, B.O., Juliano, B.O., and Maniñgat, C.C. 1982. Protein and energy utilization of rice milling
fractions by rats. Qual. Plant. Plant Foods Hum. Nutr., 31: 371 -376.
• Juliano, B.O. 1985. Factors affecting nutritional properties of rice protein. Trans. Natl. Acad. Sci.
Technol. (Philipp.), 7: 205-216.
• Kannan, A., Hettiarachchy, N., Johnson, M.G., and Nannapaneni, R. 2008. Human colon and liver
cancer cell proliferation inhibition by peptide hydrolysates derived from heat-stabilized defatted
rice bran. J. Agric. Food Chem. 56(24):11643-7.
• Lang, V., Bellisle, F., Oppert, J., Craplet, C., Bornet, F. R. J., Slama, G., and Guy-Grand B. 1998.
Satiating effect of proteins in healthy subjects: a comparison of egg albumin, casein, gelatin, soy
protein, pea protein, and wheat gluten. Am. J. Clin. Nutr. 67:1197–204.
• Matoba, T., Yoshida, H., and Yonezawa, D. 1982. Changes in casein and egg albumin due to
reactions with oxidizing methyl linoleate in dehydrated systems. Agric. Biol Chem. 46(4): 979-986.
• Paul L. Hurst, P., and Chris J. Lovell-Smith, C. J. 1981. Optimized assay for serum angiotensin-
converting enzyme activity. Clin. Chem. 27(12):2048-2052.
• Pedersen, B. and Eggum, B.O. 1983. The influence of milling on the nutritive value of flour from
cereal grains. IV. Rice. Qual. Plant. Plant Foods Hum. Nutr., 33: 267-278.
• Rackis, J. J., Anderson, R. L., Sasame, H. A., Smith, A. K., and Van Etten, C. H. 1961. Soybean
amino acids, amino acids in soybean hulls and oil meal fractions. J. Agric. Food Chem. 9 (5): 409-
412.
• Rainer, H., and Meisel, H. 2007. Food-derived peptides with biological activity: from research to
food applications. Curr. Opin. Biotech. 18(2):163-169.
• Thompson, T. 1997. Do oats belong in a gluten-free diet? J. Am. Diet Assoc. 97:1413-1416.
• Usui, Y., Nakase, M., Hotta, H., Urisu‡, A., Aoki, A., Kitajima, K., and Matsuda, T. 2001. A 33-kDa
Allergen from rice (Oryza sativa L. Japonica): cDNA cloning, expression, and identification as a
novel glyoxalase. J. Biol. Chem. 276(14):11376–11381.
Thank You

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