J Life Sci Res 2011; 9:7-11Ⓒ 2011 Osaka Prefecture University

Journal of Life Science Research
Online ISSN 2186-5809

Original

Effects of Intake of Puffed wx ae Double Mutant Brown Rice on Human Blood Glucose
and Lipid Levels
Shigeyo HORINO,1,2 Makoto NAKAYA,3 Aiko SHOJO,3 Hiroaki HIRAI,3 Shinich KITAMURA,3 Akemi KINOSHITA,1 and Shuichi
MIYATANI†1
1

Graduate School of Comprehensive Rehabilitation, Osaka Prefecture University, 3-7-30 Habikino, Habikino-shi 583-8555, Japan;

2Department of Development Nutrition, Faculty of human development, Soai University, 4-4-1 Nanko-naka Suminoe-ku, Osaka-shi 559-

0033, Japan; 3Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuencho Naka-ku, Sakai-shi
599-8531, Japan.
Received 28 September 2011; accepted 11 November 2011

The amylopectin in waxy amylose-extender (wx ae) double mutant rice characteristically has a small
number of branch chains and long side chains. In this study, we conducted single intake trials and middle
term intervention trial to puffed non-glutinous Uruchi and wx ae brown rice. Postprandial blood glucose
response to a single intake of puffed Uruchi and wx ae brown rice were measured with 3 men and 4 women(47.1 ± 12.7 years of age (mean ± standard deviation [SD])). Subjects who were fasted for 10 to 12 h, has
ingested 20 g of the puffed sample. Incremental blood glucose levels of wx ae at 45 and 60 min (p < 0.01),
and its peak (30 min) were much lower than for Uruchi. Middle term intervention trial had assigned 14
healthy men and women to eat either puffed brown rice of wx ae or Uruchi for 15 days to compare possible
effects on blood glucose and serum lipid levels. Each group consumed 60 g of puffed brown rice a day, at
any time of day, in addition to their ordinary meals. Those in the experimental diet (ED) group (4 men and3 women; 45.1 ± 16.8 years of age) ate wx ae brown rice, while those in the control diet (CD) group (5 men
and 2 women; 45.1 ± 15.1 years of age) ate Uruchi rice. Fasting blood samples were obtained before andafter the experiment to examine and compare clinical chemical test values. The results showed that 1,5anhydroglucitol (1,5-AG) declined significantly in the CD group and total cholesterol levels declined in the
ED group, while triglyceride values rose in the CD group. Thus we expect that wx ae brown rice can beused as a functional food superior to Uruchi brown rice.
Key words: wx ae double mutant rice; resistant starch; blood glucose; serum lipid

1 Introduction
In recent years, lifestyle diseases have accounted for
an increasing percentage of deaths. It is therefore important to improve the content of meals to prevent lifestylerelated diseases such as type 2 diabetes or dyslipidemia.
Consequently, many attempts have been made to use thefunctionality of various foods, including functional
rice.1−3
A Japonica rice (Oryza sativa) mutant, the waxy
amylose-extender (wx ae) double mutant, produces aunique starch of pure amylopectin, with branched chains
longer than those of normal rice amylopectin, the result of
defective starch branching enzyme IIb and granule-boundstarch synthase I.4 The wx ae starch shares a feature ofresistant starch (RS) and represse increase of the post†

Corresponding author. E-mail: miyatani@rehab.osakafu-u.ac.jp

prandial glucose level in mice.5 RS cannot be digested in
the small intestine, 6 and is effective against hypo glycaemia,7, 8 hypocholesterolaemia,9, 10 and fat accumulation.11
To make functional food part of the regular diet, wemust think about how to cook it. We used puffed wx aebrown rice in this study because an elevated intake ofmilled rice seems to be associated with an increased riskof lifestyle-related diseases,12, 13 while brown rice seems todecrease the risk of lifestyle-related diseases.14−16 We
were able to give a roasted flavor to the wx ae rice andeliminate a smell of bran by puffing it.
Based on preliminary animal testing results, weplanned a 15-day study using human subjects with theaim of verifying whether puffed wx ae brown rice inhibits
postprandial blood glucose elevation and improves serum
lipid levels.

8

2 Subjects and Methods
2.1 Materials
We used a waxy amylase-extender mutant line
AMF18 which is a double-recessive mutant for waxy (wx)and amylase-extender (ae). It was derived from a crossbetween the EM21 and EM16 lines. The wx mutant line
EM21 and the ae mutant line EM16 are geneticallydefective in granule bound starch synthase I and starchbranching enzyme IIb, respectively. Those mutants weregenerated by treating fertilized egg cells of japonica rice
(Oryza sativa) cv Kinmaze with N-methyl-N-nitrosourea.17
We used Uruchi rice cv Kinmaze as a control to measure
postprandial blood glucose response to a single intake,and Uruchi rice cv Hinohikari as a control in a middle
term intervention trial. Hinohikari and wx ae rice plants
were grown in the summer of 2009 in Nara, Japan andKinmaze rice plants were grown in the summer of 2007 in
an experimental field at Osaka Prefecture University.
We puffed the rice using a puffing machine SL(Tachibana Kiko, Fukuoka, Japan). After puffing, the rice
was kept dry until the intervention trial.
2.2 Puffed Uruchi and wx ae brown rice for trials
We measured the postprandial blood glucose
response to a single intake of puffed Uruchi and wx aebrown rice. Seven healthy men and women (3 men and 4
women; 47.1 ± 12.7 years of age) who work at Osaka
Prefecture University were recruited for single intake
trials for puffed Uruchi and wx ae. Subjects fasted for 1012 h before the trials, and then ingested 20 g of the puffedsample with 80 mL water. Blood samples were takenfrom the tip of the finger after 0, 15, 30, 45, 60 and 120
min. Blood glucose levels were determined by the glucose oxidase method using a NIPRO Freestyle Freedom
blood glucose monitoring system (NIPRO Co., Osaka,
Japan). The single intake trials of puffed Uruchi and wx
ae were repeated with the same subjects a week later.
2.3 Middle term intervention trial with puffed rice
We recruited 14 healthy men and women (aged 2068 years) comprising teachers, staff and students of Osaka
Prefecture University.
The ED consisted of 60 g of puffed wx ae brown
rice, while the CD comprised 60 g of puffed Uruchi. It is
the maximum volume that we can intake without unreason able ness consecutively. The experiment was per formed for 15 consecutive days in March 2010. The subjects were randomly assigned to either the CD (5 men and2 women; 45.1 ± 15.1 years of age [mean ± SD]) or theED (4 men and 3 women; 45.1 ± 16.8 years) group. They
were asked to consume their quota of puffed rice at any
time of the day in addition to their usual meals. The study

was designed as a parallel group comparison.
Before the experiment, we investigated the normal
dietary habits of the participants. During the experiment,
we used diet records to examine the influence of the newdiet on their meals. Calculations were made with Excel"Eiyokun" software Ver. 5.0 and Food Frequency
Questionnaire (FFQg Ver. 2.0, Kenpakusha). We also
recorded the amount and hardness of their stools, presence or absence of abdominal wind and status of otherdigestive organs. The amount of energy consumed wasdetermined with a portable pedometer (PW-900, Yamasa
Tokei Co., Ltd). To determine physical status, wemeasured the participants' weight and body fat percentage
using the Body Planner DF-800 (Yamato-Scale Co., Ltd).
Fasting blood samples were obtained in the earlymorning before and after the experiment to determine
levels of blood glucose, glycoalbumin and 1,5-AG. In
addition, we measured levels of serum triglyceride, totalcholesterol, high density lipoprotein (HDL) cholesterol
and low density lipoprotein (LDL) cholesterol. LDL
cholesterol concentrations were calculated with theFriedewald formula.
Informed consent was obtained from each of thesesubjects. The study was approved by the Research Ethics
Review Committee of Osaka Prefecture University ofComprehensive Rehabilitation (approval number: 08-307,
2009-05) and conducted observing the terms set by thecommittee. Blood tests were performed in cooperation
with the Houtokukai Medical Association Houwa
Hospital.
Data were expressed as mean value ± SD. Statisticalprocessing was performed with the Wilcoxon t-test withingroups, while group comparisons were made with theMann-Whitney U-test. We used Dr. SPSS II software(IBM) for the analyses. Data were considered significant
when p < 0.05.
3 Results
Fig. 1 shows the subjects' postprandial incremental
blood glucose response. Incremental blood glucose levels
were lower after ingesting puffed wx ae brown rice (p <0.01; 30, 45 and 60 min) than after ingesting puffed
Uruchi. Blood glucose concentration at its peak (30 min)was much lower for wx ae than for Uruchi. Based onthese results and given that puffed rice is an easy-to-eat
food with a roasted flavor, we decided to conduct a
middle term intervention trial using puffed Uruchi and wx
ae brown rice.
Throughout the middle term intervention trial, both
CD and ED groups showed no significant change inphysical status, including weight, body fat percentage,

9and the amount of energy intake and amount of energy
consumed (Table 1). Also, no adverse events, including
diarrhea, were reported during the period.
Table 2 indicates changes in clinical blood test

Fig. 1

Postprandial blood glucose response to puffed Uruchi
and wx ae brown rice.
Puffed Uruchi brown rice group (n = 8), Puffed wx aebrown rice group (n = 7).
＊Significant difference groups within the time wereanalyzed by the Wilcoxon t-test at p < 0.01. Data aremean ± SD.

values. In both groups, fasting blood glucose levels
tended to decline slightly. Glycoalbumin did not fluctuate
in either group before or after the experiment. A significant decline in 1,5-AG was observed in the CD group (p= 0.040).
Triglyceride levels were significantly elevated in the
CD group (p = 0.022), while total cholesterol values
declined significantly in the ED group (p = 0.042). HDL
and LDL cholesterol concentrations tended to decrease inboth CD and ED groups.
4 Discussion
Uruchi brown rice inhibits the postprandial incremen tal blood glucose response better than white ricedoes. 16, 18 Our results suggest that wx ae brown rice
inhibits this response better still. We recruited subjects
for middle term intervention trials for wx ae and puffed
Uruchi. We measured the participants' body weight, bodyfat percentage, amount of energy intake, amount of energy consumed, blood glucose and serum lipid level.
No significant change was observed in the amount ofTable 1

Changes in physical status of subjects

Table 2

Changes in clinical chemical test values

10energy intake or amount of energy consumed in eithergroup. We concluded that these factors did not influence
blood test results.
1,5-AG is the most abundant sugar in healthy humanblood after glucose. It is contained in a wide variety offoods, but only in minute amounts. Normally, it is reabsorbed into the kidney after excretion. If its blood concentration exceeds the threshold value of renal excretion,
renal reabsorption is inhibited, 1,5-AG is discharged into
the urine and its blood concentration declines,19 which
serves as an indicator of hyperglycemia after meals. The
1,5-AG concentration value is used mainly for improving
high blood sugar after meals or for determining the beneficial effects of diet or exercise therapy.
Recently, 1,5-AG has been introduced into theguidelines on the management of postprandial hyperglycemia implemented by the International DiabetesFederation (IDF).20, 21 In our study, the 1,5-AG levels of
the subjects were all 14 μg/mL or above, falling within
the normal range. A significant decrease was observed in
the CD group from Day 0 through Day 16. This canprobably be explained by leakage of sugars into urine due
to transient hyperglycemia after meals. This phenomenon
was not observed in the ED group.
Diagnostic criteria for metabolic syndrome include
triglycerides at 150 mg/dL or above and HDL cholesterolat less than 40 mg/dL. It is essential to maintain normal
serum lipid levels to stay healthy. In our experiment, theCD subjects showed negative effects, including decreased
HDL cholesterol and elevated triglyceride levels. Ueno etal. reported that total cholesterol and LDL cholesterol
dropped in subjects who consumed unpolished brown rice
for a month,22 and dietary fiber improves serum cholesterol levels.23−25 In our study, total cholesterol levels
decreased significantly in the ED group, which is consistent with these results. Lowered total cholesterol in the
ED group may be explained by the fact that wx ae brownrice contains as much as four times the dietary fiber ofUruchi brown rice.
Triglyceride synthesis increases after carbohydraterich meals, because such foods provide sufficient levels ofacetyl-CoA, a component of fatty acid synthesis, andnicotinamide adenine dinucleotide phosphate (NADP), a
coenzyme necessary for the synthetic reaction. Highcarbohydrate consumption is associated with hyper triglyceridemia.26 Suzuki et al. divided subjects into twogroups and asked them to eat either Uruchi white rice orbrown rice. Both groups consumed a large amount (50g/day) of sugar. As a result, the white rice group showed
higher elevation of triglycerides and cholesterol than thebrown rice group. 27 Similarly, triglyceride levels

increased significantly in the CD group in our study. Thiswas presumably because Uruchi rice starch was quickly
digested and absorbed into sugars, resulting in synthesisof triglycerides in the liver. In contrast, probably because
wx ae rice starch, rich in RS, is digested slowly and less isabsorbed, triglyceride levels did not increase in the ED
group in our study.
5 Conclusion
We reconfirm that puffed wx ae brown rice improves
both carbohydrate and lipid metabolism more effectively
than puffed Uruchi brown rice in middle term inter vention trial. Our results suggest that puffed wx ae brownrice has a different functionality from that of Uruchi, andwx ae may be useful as a new dietary ingredient for preventing lifestyle-related diseases. Now we plan to do along-term crossover study.
Acknowledgements
We are deeply grateful to the volunteers who agreed
to participate in this study. We thank Dr. Hikaru Satoh at
Kyusyu University for providing rice seeds.
This study was partly supported by the Iijima
Memorial Foundation for the Promotion of Food Scienceand Technology as part of its academic research fundingin 2008 and a grant of Ministry of Agriculture, Forestry
and Fisheries of Japan.
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