Nghiên cứu sự thay đổi cấu tử thơm 2-AP và các chất bay hơi khác trong qui trình chế biến bún từ gạo

J. Sci. & Devel., Vol. 10, No. 5: 730 - 737 
Tạp chí Khoa học và Phát triển 2012 Tập 10, số 5: 730-737
www.hua.edu.vn 
RESEARCH ON THE CHANGE OF 2-AP AND OTHER VOLATILE COMPOUNDS 
IN PROCESSING BUN FROM RICE 
Phan Phuoc Hien1, J.D. Park2, Truong Thi Bich Lieu1 
1Nong Lam university HCMC Vietnam; 2Korea Food reseach Institute 
Email: pphien@gmail.com 
Received date: 29.05.2012 Accepted date: 15.09.2012 
ABSTRACT 
Vermicelli is the traditional dish of Vietnam which is the main material to prepare “Hue Beef rice vermicelli” (Bun 
bo Hue), a very famous specialty originated from Hue city, the former imperial capital of Vietnam. Flavor and taste are 
important attributes of vermicelli. This study was carried out to determine the change in 2-AP and other volatile 
compounds as influenced by different durations of soaking rice in water during vermicelli processing. In order to 
identify and quantify the amount of 2-AP and other volatile compounds 2-AP extracted from pandan leaves was used 
as standard. Results indicated that 2-AP and other volatile compounds clearly changed in the vermicelli processing 
process and soaking for 12 hours was recommended. 
Keywords: Rice vermicelli, 2- Acetyl - 1 Pyrroline (2-AP), volatile compounds. 
Nghiên cứu sự thay đổi cấu tử thơm 2-AP và các chất bay hơi khác 
trong qui trình chế biến bún từ gạo 
TÓM TẮT 
Bún là nguyên liệu chính để chế biến “Bún Bò Huế”, đây là một món ăn đặc sản của Việt Nam có nguồn gốc từ 
thành phố Huế, trước đây là kinh đô của Việt Nam. Hương vị đặc trưng của sợi bún quyết định tới chất lượng bún 
sản phẩm. Để làm rõ những sự thay đổi này, đề tài nghiên cứu đã tiến hành chiết xuất cấu tử chính tạo nên mùi 
thơm 2 Acetyl- 1 Pyrroline từ lá dứa và sử dụng nó như là chất chuẩn để định tính và định lượng sự thay đổi này bao 
gồm cấu tử chính 2-AP và các cấu tử bay hơi khác trong gạo ngâm nước theo quy trình chế biến bún truyền thống. 
Kết quả chứng tỏ rằng cấu tử thơm 2-AP và những cấu tử bay hơi khác đã biến đổi một rất rõ rệt trong quá trình chế 
biến này. Trên cơ sở đó đưa ra khuyến cáo ngâm gạo trong 12 giờ quy trình chế biến bún. 
Từ khóa: Bún, cấu tử bay hơi, 2 Acetyl - 1 Pyrroline (2-AP). 
1. INTRODUCTION 
In Vietnam, there are many rice varieties in 
that the most interesting is aromatic rice (Phan 
Phuoc Hien et al., 2009). The key aromatic 
constituent 2-Acetyl-1-Pyrroline (2 - AP) in 
aromatic rice was found out in pandan leaf 
(Pandanus amaryllifolius), also existed in white 
bread, and flowers (Vallaris glabra) (Varaporn 
Laksanalamai et al., 1993). Due to pandan leaf 
contains this specific constituent with very high 
content as compared with aromatic rice (Phan 
Phuoc Hien, 2011), it is often used to enhance 
the appealed flavor of foods in many countries 
such as Indonesia, Philippines, Malaysia, 
Thailand, Vietnam and Burma, especially in 
rice cooked and sweet cakes (Varaporn 
Laksanalamai and Sarath Ilangantileke, 1993). 
In order to develop aromatic rice production 
in Viet Nam, reliable and practical methods to 
assess 2-AP and other volatile compounds in 
aromatic rice are required to evaluate and select 
the better varieties. In response to this demand, 
during the past 7 years, two modern methods 
have been fitted up and operated at the 
Physiochemical laboratory in Nong Lam 
University, Ho Chi Minh City, Vietnam. The first 
is Solid Phase Micro-Extraction coupling with 
Gas Chromatography (SPME/GC) and Mass 
Spectrometry (SPME/MS), and the second is SDE 
(Simultaneous Distillation Extraction) also 
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Research on the Change of 2-AP and Other Volatile Compounds in Processing Bun from Rice 
coupling with GC and GCMS. SPME/GC enables 
for estimation of 2-AP low concentration like 
aromatic rice. The SDE method is suitable for 
extraction of the 2-AP high concentration 
materials like Pandan leaf (Phan Phuoc Hien, 
2011). Based on the two methods we studied for 
extraction and quantitative analysis of 2-AP in 
the pandan leaf and used it as the standard for 
qualitative and quantitative analysis of 2-AP in 
aromatic rice and other medicinal plants such as 
Thien Nien Kien Homalomena aromatica (Phan 
Phuoc Hien et al., 2011). 
In the process of vermicelli strands (Bun) 
prepared from rice, step of soaking rice in water 
with different duration definitely influences on 
quality and flavor for the end-product “Bun”. In 
reality, this step made change biochemical 
properties of rice material leading to finally 
change 2-AP and other volatile compounds in 
rice. In order to demonstrate clearly these 
changes, SDE and SPME coupling with GC-FID 
and GCMS were used to identify, quantify and 
presented in this paper. 
2. MATERIALS AND METHODS 
2.1. Materials 
The pandan (Pandanus amaryllifolius) 
leaves (Fig. 1) were used to extract 2-AP as 
standard. Rice materials include two varieties 
from Vietnam, OM 6162, Khao Dawk Mali 
(KDM) and two varieties from Korea, 
Chucheong variety (milled rice), and Black rice 
(mixture of several varieties). 
Pandan leaves (collected from Di An District, Binh Duong, 
Vietnam) are classified into 3 types: 
old leaf (a), young leaf (b), and mature leaf (c) 
Fig 1. Three types of Pandan leaves: old 
leaf (a), young leaf (b), and mature leaf (c) 
For the identification and quantification 
of change in 2-AP and other volatile 
compounds, milled rice of KDM was soaked in 
water for 12 and 48 hours; non-soaked rice 
served as control. 
2.2. Extraction methods 
a. Simultaneous Distillation-Extraction (SDE) 
The steam distillation-solvent extraction 
was used as a reference for 2AP and other 
volatile compounds quantification. Extraction 
was performed using Godefroot apparatus 
(Godefroot et al., 1981) on 20g of brown rice 
with dichloromethane as solvent and collidine 
as internal standard. Duration of extraction was 
30 minutes from apparition of the first drop of 
water in the bottom of the condensed tube. 
Volatile compound extracts were then 
concentrated to 0.3 ml by drying under a 
nitrogen flow at room temperature and stored 
at -18°C prior to GC/FID and GCMS analysis 
(Phan Phuoc Hien et al, 2009; 2010; 2011). 
b. Solid Phase Micro Extraction (SPME) 
Extraction of volatile fractions in rice was 
performed by using a Supelco® 
VB/Carboxen/PDMS (divinylbenzène/ Carboxen/ 
polydiméthylsiloxane) fiber 3.5 g of milled rice 
with 500 µl of water were placed in a 10 ml vial. 
As for rice samples analysed by SPME-GC, 
collidine was added as an internal standard. 
The solution was equilibrated at 80°C for 5 
minutes then the fiber was introduced in the 
headspace surrounding rice at the same 
temperature for 15 minutes (Phan Phuoc Hien, 
2009; 2010; 2011). 
2.3. Analysis methods 
a. Quantification of 2AP concentration by 
GC-FID 
a b c 
The extracts obtained by the SDE and 
SPME were analysed by using a Hewlet 
Packard 5890 Series II gas chromatograph with 
a flame ionisation detector (GC-FID). The 
column was a non-polar DB-5 (J&W Scientific) 
capillary column (length 60m, 0.32mm, film 
731 
Phan Phước Hiền, J.D. Park, Trương Thị Bích Liễu 
732 
thickness 0.25 µm). Helium was used as carrier 
gas at a flow rate of 1.9 ml/min at 250C. The 
injection was performed in splitless mode first 
(5 min for SPME and 2 min for SDE), then in 
split mode to the end of the cycle (38.5 min for 
SPME and 70 min for SDE). After warming the 
column at 400C for 5 minutes, the following 
temperature programs were applied: 
- For SDE: from 400C to 2200C at a rate of 
30C/min and finally maintained at 2200C for 5 min; 
- For SPME: from 40°C to 115°C at a rate of 
3°C/min then from115°C to 220°C at 30°C/min 
and finally maintained at 220◦C for 5 min. The 
detector port was maintained at 2500C. 
Concentration of 2-AP in samples s is identified 
and quantified in the 3.1 section in this paper. 
b. Volatile compounds analysis by SPME 
coupling with Mass Spectrometry (SPME-
MS) 
Fig 3. Adsorption phase in SPME extraction 
injector temperature were respectively 
maintained at 260°C and 250°C. He at 2 ml/min 
was the carrier gas. The column was 
maintained at 220°C for 15 min. Source 
temperature was 150°C and the mass spectra 
were scanned at 70 eV in the m/z range from 40 
to 200 at 8.17 scans/second. The global signal 
registered between 2.8 and 10 minutes was 
transformed by using the Pirouette®software. 
SPME fiber was directly introduced in the 
GC/MS injector operating with splitless mode 
for 4 minutes at 250°C. An Agilent 6980 gas 
chromatography equipped with a DB-WAX 
fused silica capillary column (60 m 0.25 mm 
d.i.; film thickness = 0.25 µm) coupled with a 
Agilent 5973N mass spectrometer was used for 
the GC/MS analysis. The transfer line and the 
3. RESULTS AND DISCUSSION 
3.1. Extraction, identification, and 
quantification of 2-AP in Pandan leaf 
In this experiment, response factor (RF) of 
collidine has been used to identify and quantify 
2-AP in pandan leaf that was extracted by SDE 
and then analyzed by GC-FID. By this method, 
retention time (Rt) of collidine and 2-AP in 
pandan were detected at 12.936 minute and 
9.498 minute respectively wherein this 2-AP 
will be used as a standard to identify and 
quantify 2-AP in aromatic rice (Phan Phuoc 
Hien, 2009; 2010; 2011). 
Quantification of 2-AP in pandan leaf: 
Content of 2 - AP in pandan leaf extracted 
by SDE was calculated as follows: Fig 2. System of the SPME extraction 
Research on the Change of 2-AP and Other Volatile Compounds in Processing Bun from Rice 
Figure 4. GC-FID chromatograph of 2-AP and other volatile compounds in pandan leaf 
[2 - AP]SDE (g/kg) = , in which: 
- A: Area of the 2 - AP peak 
- RF: Response factor under the external 
standard collidine 
- d: diluted concentration of sample 
- m: sample mass analyzed (kg) 
The peak areas were quantified as table 1. 
RF of 2 - AP was calculated under the external 
standard collidine as follows: 
RFcollidine = 01.1
14009069
= 1400906900 (pA*s/µg). 
Whereby collidine mass injected into 
GCFID was 0.01 µg. By this way, 2-AP content 
of the pandan leaves was quantified (Table 2). 
Table 1. Peak areas of collidine and 2-AP in pandan leaves 
recorded by GC-FID and GCMS 
Samples Peaks’areas (pA*s) 
Collidine 14009069 
2-AP in young pandan leaf 58157862 
2-AP in mature pandan leaf 20672313 
2-AP in old pandan leaf 31776315 
Table 2. Content of 2 - AP (ng/kg) in the pandan leaves quantified by SDE-GCFID 
Pandan leaves Content 2 - AP (ng/kg) 
Young pandan leaf 2.07572 
Mature pandan leaf 737.818 
Old pandan leaf 1.134134 
Collidine 
12.936min 
2 – AP 
9.498 min 
733 
Phan Phước Hiền, J.D. Park, Trương Thị Bích Liễu 
3.2. Identification of 2-AP in Korea rice 
varieties 
By SPME coupling with GC-FID, rice 
samples from Korea were extracted and analyzed 
by the same conditions as described above. 
The analytical results showed that there is 
no 2-AP peak in the Korea rice samples at the 
Rt (9.498 minute) as the 2-AP peak of Pandan 
leaf. It means that the Korea rice varieties are 
not aromatic (Fig 5). 
No peak 2-AP 
at the 9,498 mins 
Fig 5. Volatile compounds in Chucheong rice from Korea recorded 
by GC-FID showed that it has no peak 2-AP at the Rt 9.498 minute 
3.3. Identification of 2-AP in OM rice from 
Cuulong Rice Research Institute, Viet Nam 
GC-FID chromatograph of OM 6162 variety 
recorded in Figure 6 showed that OM 6162 is 
an aromatic rice variety because its peak 2-AP 
was identified clearly at the Rt 9.678 minute. 
2-AP 
Fig 6. SPME/GC-FID chromatograph of OM 6162 
exposed the peak 2-AP at the Rt 9.678 minute 
734 
Research on the Change of 2-AP and Other Volatile Compounds in Processing Bun from Rice 
3.4. Investigating the changes of 2-AP and other volatile compounds of rice in Bun 
processing 
Table 3. The volatile compounds in non-soaking and 12 hours soaking 
of Khao Dawk mali recorded by GCMS 
N0 12 hours water soaking Non-soaking 
1 1 - butanol 0 
2 hexanal hexanal 
3 1- hexanol ethanone, 1-(2-methyl-1-cyclopenten-1-yl)- 
4 2- heptanone ethylbenzen 
5 heptanal 1-hexanol 
6 2- acetyl -1- pyrroline 1-nonanol 
7 benzaldehyde 2- acetyl -1- pyrroline 
8 1- heptanol 1- heptanol 
9 1-octen-3-ol 1-octen-3-ol 
10 2 -pentyl-furan 2 -pentyl-furan 
11 butanoic acid, butyl ester 5-hepten-2-ol,6-methyl- 
12 octanal octanal 
13 2-heptenal tetradecane 
14 benzeneacetaldehyde benzeneethanol, -dimethyl- 
15 butanoic acid, 3-methylbutyl ester 1-hexanol,2-ethyl- 
16 2-octenal 2-octen-1-ol 
17 ethanone, 1-(1H-pyrrol-2-yl)- ethanone, 1-(1-cuclohexen-1-yl)- 
18 2-octen-1-ol 0 
19 1-octanol 1-octanol 
20 2-nonanone 
21 propanoic acid, 2-methyl-, pentyl ester 5,9-undecadien-2-one,6,10-dimethyl- 
22 2-nonanol tetradecane,2,6,10-trimethyl- 
23 nonanal nonanal 
24 2,4-pentanedione, 3-butyl- 0 
25 3-nonen-1-ol 0 
26 cyclohexanone, 5-methyl-2-(1-methylethyl)- cyclohexanol, 1-methyl-4-(1-methylethyl)- 
27 2-nonenal 2-undecanone,6,10- dimethyl- 
28 1-nonanol 
29 not available in NIST libray of GCMS Not available in NIST libray of GCMS 
30 dodecane dodecanal 
31 decanal decanal 
32 phenol,4-ethyl-2-methoxy- 0 
33 2-decenal 2-decenal 
34 butanoicacid, heptyl ester 0 
35 2-undecanone 2-undecanone 
36 undecanal undecanal 
37 pentadecanone, 6,1,14-trimethyl- 2-pentandecanone,6,10,14-trimethyl- 
38 n-hexadecanoicacid 0 
735 
Phan Phước Hiền, J.D. Park, Trương Thị Bích Liễu 
Notes: 
(1) Blue line: (control treatment)
(2) Green line: soaked 1 night 
(3) Red line: soaked 2 days 
(1) 
(2)
(3) 
Fig 7. GC-FID chromatograph found out the change of 2-AP and other volatile compounds 
in three treatments: KDM not to be soaked, to be soaked for 8 hours, and for 2 days. 
Table 4. The change of 2-AP content in KDM rice 
with different soaking durations in Bun processing 
No 
Treatment 
(KDM rice sample) 
Rice sample mass (g) Area of 2-AP (pA*S) 2-AP content (ppb) 
1 Rice not soaked in water 1.5030 21.8000 2.439731 
2 Rice soaked in water 12 hours 1.5029 14.7000 1.664187 
3 Rice soaked in water 48 hours 1.0500 0.0000 0.000000 
The purpose of this study is to demonstrate 
the change of 2-AP and other volatile 
compounds in vermicelli processing from rice. 
Aromatic rice variety Khao Dawk Mali as 
confirmed by analysis results with SPME-
GCMS was used in the experiment and three 
treatments were employed as follows: 
(1) Rice KDM is not soaked in water 
(Control treatment) 
(2) Rice KDM is soaked in water for 12 hours 
(3) Rice KDM is soaked in water for 48 hours 
 The amount of 2-AP was identified and 
quantified in both control and treatments. The 
key aromatic constituent 2-AP still retained 
after 12 hours soaking of rice but slightly 
decreased. 29 volatile compounds were 
detected in the control treatment as compared 
to 38 volatile compounds when soaked for 12 
hours (Table 3).. It means that after 12 hours 
soaking rice in water 9 new volatile compounds 
were produced, viz. 1 - butanol, 2-octen-1-ol, 
2,4-pentanedione, 3-butyl, 3-nonen-1-ol, 
phenol, 4-ethyl-2-methoxy, butanoic acid, 
heptyl ester, n-hexadecanoic acid, 1-nonanol, 
and 2-nonanone (Table 3). These changes 
influenced by duration of soaking created new 
flavor for the end-product. 
In contrast to soaking for 12 hours, soaking 
KDM in water for 48 hours (or two days) , 
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Research on the Change of 2-AP and Other Volatile Compounds in Processing Bun from Rice 
resulted in nearly complete loss of 2-AP and 
other volatile compounds. The qualitative 
results recorded by GCMS and quantitative 
results are presented in Figure 7 and table 4, 
respectively. This might be attributable to the 
accompanied fermentation process due to long 
duration soaking. 
4. CONCLUSION 
By SDE extraction method and use 
response factor of collidine, 2-AP from pandan 
leaf was extracted, identified and quantified by 
GC-FID, GCMS and used as a standard to 
identify and quantify 2-AP in aromatic rice and 
its change during vermicelli processing. Based 
on this method, only cultivar OM 6162 from 
Cuu long Delta Rice Research Institute and 
KDM from the institute of agriculture south 
were identified as aromatic rice. 
Different soaking durations clearly change 
2-AP and other volatile compounds. Soaking 
rice for 12 hours increased the volatile 
compounds (38) as compared to the control (29) 
The 9 new volatile compounds were 1 - butanol, 
2-octen-1-ol, 2,4-pentanedione, 3-butyl, 3-
nonen-1-ol, phenol,4-ethyl-2-methoxy, butanoic 
acid, heptyl ester, n-hexadecanoic acid, 1-
nonanol, 2-nonanone and these produce new 
flavor and specific attribute to the end product. 
The 2-AP amount was 1.664.187 ppb when 
soaked for 12 hours and decreased by 31.79% 
as compared to the control However, if rice 
soaked for 48 hours 2- AP and many other 
volatile compounds were reduced or completely 
lost. These might be explained by biochemical 
fermentation process in natural conditions. 
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