Paccon2012 FSC 2

GLASS TRANSITION TEMPERATURE OF PROTECTIVE AGENT
THAT AFFECTING THE SURVIVAL OF LACTOBACILLUS

PLANTARUM FT35 DURING SPRAY DRIED
Mathuros Pradeamchai1, Cheunjit Prakitchaiwattana2 and Sumate Tantratian1*
2
1
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
Author for correspondence; E-Mail: sumate.t@chula.ac.th, Tel. +66 22 187661, Fax. +66 22 187662
Abstract: The objectives of this study were to

investigate protective agents with various glass transition
temperature (Tg) that affecting the survival of L.
plantarum
FT35
during
spray
drying
and
stroage. The protective agents used in this study were
glucose, sucrose, lactose, maltodextrin DE 10, and
soluble starch. The Tg of these protective agents were
35.33 oC , 75.13 oC, 119.34 oC, 160 oC and 243 oC
respectively. The protective agents were mixed with
cell of L.plantarum FT35 to give the mixture with
viscosity of 1.6-1.7 Cp. and contained 1011cfu of viable
cells/ml. The mixtures were spray dried with lnlet
temperature at 185oC, feed rate of 20 ml/min and outlet
temperature
of 85+5 oC. The survival of
L. plantarum FT35 after dried were 48.2%, 65.4%,
89.3%, 91.8% and 93.4% respectively. The powderproducts were stored in laminate aluminum foil
(PP/PE/AluPE/PP) bags at room temperature (30-35oC)
for 4 week. The stored products contained viable cell of
L.plantarum FT35 of stored products were at 56.0%,
52.6% 78.7%, 79.4% and 85.6% of the initial amount ,
respectively.
1. Introduction
Spray drying is one of the methods for preserving
microbial
culture
using in
fermented food
industries. The removal of water during drying may
cause changes in the structure of bacterial membrane
and died. The damage of membrane during drying is
based on the stability of phospholipid bilayers,
maintained by balance of mutual forces between van
der Waals attractive and interactions among the
hydrocarbon chains. Consequently, phospholipid
bilayer structures are thermodynamically unstable and
are easily perturbed [1]. Drying may cause bacterial
cell decrease during processing. A common method
for improving the viability of dried cultures is the
addition of protective agent. The protective agents
prevent the damage of membrane by depressing
the membrane phase transition, which is the main
mechanism
of protecting
macromolecules
in
organism against moderate water loss [2]. According
to the water replacement hypothesis, protective agent
depress the membrane phase transition by specifically
interacting with phospholipid headgroup. The use of
sugar as protectants of bacterial cell can alternatively
be explained by the water replacement hypothesis,
which envisages the function of sugar as water
substitutes when the hydration shell of proteins as

well as water molecules around polar residues in
membrane
phospholipids are removed.
The
replacement leads to maintenance of phospholipids
bilayer at their hydrated spacing, which in turn
preserves the structure of the membrane, Thereby
preventing damage during drying [3].
The different protective ability of protective
agents was believed to be related to the difference in
their glass-forming tendencies, which is reflected in
their Tg [4] and indicated the efficacy of protective
agent to protect the bacterial cell during drying [5]. As
already reported by Ananta et al.
that skimmilk,
polydextrose and raftilose P95 with glass transition
temperature of 109oC , 108 oC and 102 oC
respectively were used as protectant of Lactobacillus
rhamnosus GG after drying there were 65% 62%
and 55%, respectively, survived [6]. However there
was no study on carbohydrates with different Tg
related to protective ability. The objectives of this
study were to investigate the Tg of carbohydrate
protective agents that affecting the survival of
L.plantarum FT35 during spray drying and storage.
2. Materials and Methods
2.1 Determination of glass transition temperatures
of protective agents
The glucose, sucrose, lactose, maltodextrin DE 10,
and soluble starch were
determined Tg
by
Differential Scanning Calorimeter (DSC8000 Model,
Perkin Elmer). Measurements were made at a linear
heating rate of 20oC/min using an aluminum pan and
an empty pan as a reference material. Samples of 1020 mg were initially heated up linearly to 150 oC to
remove moisture from the sample. Prior to initiation
of second scan, the heated, water-free samples were
rapidly cooled down to -30oC with nitrogen flush.
The Tg was determined during the second run and
was defined as the midpoint value of the shifted in
specific heat observed as an endothermic shift in the
baseline of the DSC signal [6].
2.2 Spray drying of L. plantarum FT35
2.2.1 Viscosity of protective agent
The solution of glucose, sucrose, lactose,
maltodextrin DE 10, and soluble starch in water were
PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)
809
adjusted to have viscosity of 1.6-1.7 Cp. The viscosity

was determined
using an Cannon capillary
viscometer size 75 A442 at 25oC.
2.2.2 Microorganism and cultivation
L. plantarum FT35, isolate from commercial
fermented fish, was transferred into coconut broth
medium [7] 10 ml and incubated at 37oC for 24 h,
transferred 2% (w/v) into coconut broth 100 ml. The
culture was incubated at 37oC to exponential phase and
transferred 2% (w/v) into 3 L coconut broth medium
which contained in a 5 L fermenter. The culture was
incubated at 37oC with 100 rpm/min agitation. When
growth of organism reached stationary phase about 24
h. Cell were harvested by centrifugation (7500 rpm for
5 min at 4 oC) and washed once with 0.1 M phosphate
buffer pH 6.5-7.0. After washing, the pellet was
resuspended in the protective agent solution to give
the cell concentration of 1011 CFU/ml.
2.2.3 Spray drying
The protective agents were mixed with cell
of L.plantarum FT35. The feed solution contained
1011cfu/g dry weight was undertaken in a spray dryer
(model DV-2, NIRO, Germany). Spray drying
condition was the inlet temperature of 185oC, feed rate
of 20 ml/min and outlet temperature of 85+5 oC.
2.2.4 Determination of the survival of L.plantarum
FT35 after spray drying
The survival rate was determined by the 0.1 g
spray-dried powders were rehydrated with 9.9 ml
sterile peptone water (Merck, Germany) to obtain
1:100 serial dilution [8] and spread plate on MRS
agar, thus incubated at 37oC for 48 h. Survival rate
were calculated as follows: % Survival = N/N0 100,
where N0 represented the number of bacterial before
drying and N was the number of bacteria after drying.
2.2.5 Sensitivity test of spray dried products
Dried powder were plated on MRS agar
supplemented separately with 5% NaCl and
incubated at 37oC [9]. The plates were examined
after 2-6 days and viable numbers were compared
with numbers obtained on MRS plates without salt
addition.
2.2.6 Determination of moisture content and water
activity
The moisture content of powder samples was
determined according to method absorbed AOAC [10].
The water activity was determined using an Aqualab
Model series 3 (Decagon, Pullman, Washington) 25oC.
3. Results and Discussion

3.1 The Tg of carbohydydrate protective agents
Table. 1 show The Tg of carbohydrate protective
agents were glucose, sucrose, lactose and soluble
starch with glass transition temperature of 35.33 ,
75.13 , 119.34 and 243 oC respectively. The Tg of
matodextrin DE 10 was unable to determine, 160oC
was the Tg reported by Roos et al. (1993) [11]
Table 1 The glass transition temperature of protective
agents
Protective agent
Glucose
Sucrose
Lactose
Maltodextrin DE10
Soluble starch
Glass transition
temperature (Tg)
35.33 oC
75.13 oC
119.34 oC
160 oC [11]
243 oC
3.2 The survival of L.plantarum FT35 after spray

drying
In this study glucose, sucrose, lactose,
maltodextrin DE 10, and soluble starch were used as
protectant, viable cell of L. plantarum FT35 after spray
dried were 48.2%, 65.4%, 89.3%, 91.8% and 93.4%
respectively (Fig 1). It was found that protective agent
with high Tg provide the survival rate of
L. plantarum FT35 was greater than protective agent
with lower Tg. The lactose, maltodextrin DE 10 and
soluble starch as protectant provide the highest on
survival rate. However, when compare the viable cell
and salt sensitive cells after drying it was also found
that soluble starch protected the bacterial cells better
than maltodextrin. The replacement of protective
agents leads to maintenance of phospholipids bilayers
at their hydrated spacing, which in turn preserves the
structure
of the membrane, thereby preventing
damage during drying [12]. On the other hand, the
ability of sugar protective agent to stabilize cell
membrane proteins during drying is due to the ability
of the sugar to form hydrogen bonds with the
protein when water was removed, and prevent protein
denaturation by maintaining the protein structure[13].
Furthermore, polysaccharide, such as maltodextrin DE
10 and soluble starch, formed cross linked with the
proteins and might interact with head groups of
phospholipids and depress
membrane
phase
transition [14]. This interaction of polysaccharides
with phospholipids greatly depends on the flexibility
of the structures. The maintenance of the structural of
bacterial cell membrane and
survival
rate of
L.plantarum FT35 >90%.
2.3 Determination of the survival of L.plantarum

FT35 during storage
The powder-products were stored in laminate
aluminium foil (PP/PE/AluPE/PP) bags at room
temperature (30-35oC) for 4 week. The data were
expressed as logarithmic value of survival cells. The
moisture content and water activity were determined.
810
protection on the L.plantarum FT35 during storage

under room temperature. The high moisture content
and water activity of the product could also be
factors of low survival rate. Glucose and sucrose
provide the spray dried products with high moisture
content, 4.5 and 3.6%, respectively, when compared to
the rest of protective agents. This agreed with
Champagne et al, [15] who reported that the powder
products with high moisture content found to have
high percentage of cell death during storage. It is
recommended that the moisture content of 4% was a
good-quality parameter for dried products. The high
in moisture content could cause the oxidation of lipid.
The lipid oxidation in the cell membrane caused in
changes in membrane function and structure[16].
Fig 1. The effect of glass transition temperature on the
survival of L platarum. The survival rate during
drying ( ) and survival to NaCl ( ). Initial viable
cell count of the feed solution was 1011cfu/ml with
viscosity about 1.6-1.7 Cp.
10
log (CFU/g)
Table 2. Comparisons of viable cell and salt sensitive

cells of L plantarum FT35 with different protective
agents after spray dried
12
8
6
4
Protective
agent
Viable cell
log CFU/g
Salt sensitive cell

log CFU/g
glucose
5.49 a+0.35
1.65a+0.12
sucrose
7.46 b+0.37
1.56b+0.14
Storage time (week)
lactose
10.20 c+0.25
1.42c+0.23
maltodextrin
DE 10
10.48 c +0.23
0.90d+0.31
Fig.2 The survival of L.plantarum FT35 with different

protective agents; glucose (
), sucrose (
),
lactose( ), matodextrin () and soluble starch ( ),
stored at room temperature (30-35oC) for 4 weeks.
2
0
0
10.65 c +0.31
0.38e+0.22
soluble
starch
note; means in the same column, with different superscript
are statistical different (p< 0.05)
6
% moisture content
Table 2 shows the salt sensitive cells increase in

the
follow
order
glucose>sucrose>lactose>
maltodextrin DE 10>soluble starch , respectively. It
was found that protective agent with low Tg had poor
ability to protect bacterial cell. The highest amount of
salt sensitive cell was 1.65+0.12 log CFU/g when
glucose was as protective agent while soluble starch
which had the highest Tg provide the best of protective
activity showed lowest amount of sensitive cell was
0.38+0.22 log CFU/g.
5
4
3
2
1
0
0
Storage time (week)
3.3 The survival of L.plantarum FT35 during storage

The survival of L.plantarum FT35 spray dried
with glucose, sucrose, lactose, maltodextrin and
soluble starch stored for 4 weeks were 3.08 , 3.93, 8,
8.33 and 9.12 log CFU/g , respectively (Figure 2).
The rates of survival of were 56.0, 52.6, 78.7, 79.4 and
85.6% , respectively. The data showed that the
protective agents with low Tg provided poor
Fig.3 The residual moisture content of L. plantarum

FT 35 powder products with different protective agents;
glucose ( ), sucrose ( ), lactose ( ), matodextrin ()
and soluble starch (
), stored at room temperature
(30-35oC) for 4 weeks.
811
0.7
water activity (aw)
0.6
0.5
0.4
0.3
0.2
0.1
0
Stroage time (week)
Fig. 4 Water activity (aw) of L. plantarum FT 35

powder products with different protective agents; were
glucose ( ), sucrose ( ), lactose ( ), matodextrin ( )
and soluble starch (
) , stored at room temperature
(30-35oC) for 4 weeks.
4. Conclusions
[7] S. Permpong, Production and storage of lactic acid

bacte bacteria used as dietary supplements in the form of
pow powder pigs, Master's Thesis, Kasetsart University,
(1981(1981).
[8] G.E. Gardiner, E. OSullivan, J. Kelly, M.A.E.
Auty, G.F. Fitzgerald and J.K. Collins, Appl. and Env.
Microbiol 66 (2000) 2605-2612.
[9] E.O. Sunny-Rob D.C, and D. Knorr, Int Dairy 19
(2009) 209-2149
[10] Washington, A.O.A.C. 16 (1995) 1-2-214
[11] Y. Roos ,Carbohydrate. Research 238 (1993) 39-48.
[12] S.B. Leslie, E. Israeli, B. Lighthart, J. H. Crowe and
L.M. L.M. Crowe, Appl. Enviton. Microbiol 61 (1995)
3592-3592-3597.
[13] J.H. Crown, F.A. Hoekstra, K.H.N. Nguyen and L.M.
Crowe, Biochim Biophys. 1280 (1996) 187-196.
[14] I.J. Vereyken,V. Chupin, R.A. Demel, S.C. Smeekens
and and B. de e Kruijff, Biochim. Biophys 1510 (2001) 307307-320.
[15] C.P. Champagne, N. Gardner, E. Brochu and Y.
BeaulBeaulieu, Canadian. Inst. Food Sci. Technol 24
( 199(1991) 118-28.
[16] G. In t Veld, A.J.M. Driessen and W.N. Konings,
Bioch Biochim. Biophys 1108 (1992) 31-39.
The protective agents used in this study were

glucose, sucrose, lactose, matodextrin DE 10 and
soluble starch. The Tg of these protective agents were
35.33 oC , 75.13 oC, 119.34 oC, 160 oC and 243 oC
respectively. It was found that protective agents with
high Tg provide the survival rate of L.plantarum FT35
greater than protective agents with low Tg. The
protective agent with high Tg provide the good
protective activity and increase resistance to heat of
bacterial cell. It was found that soluble starch provide
the best survival rate at 93.4% and lowest amount of
salt sensitive cell at 0.387+0.22 log CFU/g. The low
Tg protective agents, glucose and sucrose show the
poor survival of L.plantarum FT35 during the 4
weeks of storage period at room temperature.
Acknowledgements
This work was supported by the Graduate School
of Chulalongkorn University, Thailand. Furturemore,
The author would like to thank Perkin Elmer Thailand
Company Ltd. and her persernals for assisting in the
determination of glass transition temperature (Tg) by
Differential Scanning Calorimeter (DSC).
References
[1] C. Santivarangkna, B. Higl
and P. Foerst, Food
micr microbiol 25 (2008) 429-441.
[2] C. Desmond, R Ross, E. OCallaghan,G. Fitzgerald
and and C. Stanton, Appl.Microbiol 93 (2002) 1003-1011.
[3] J.H. Crowe, L.M. Crowe, J.F. Carpenter and C.
AurAAurell- Wistrom , Biochem 242 (1998) 1-10.
[4] R.T. Sinha and B. Ranganathan, Food Sci 39 (1974)
641- 6641-642.
[5] L.J.M. Linders, G. Meerdink and K. Vantriet, Appl.
MicroMicrobiol 82 (1997) 683-688
[6] E. Ananta, M. Volkert and D. Knorr, Int. Dairy 15
(2005(2005) 399-409.
812
IDENTIFICATION OF MICROFLORA IN MILK KEFIR IN THAILAND

Montira Tangsangasaksri1, Thapapatch Punarnunnon1, Pattaraporn Yukphan2,
Winai Chaipitakchonlatarn2 and Siwarutt Boonyarattanakalin1*
1
School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University,
Pathum Thani 12121, Thailand
2
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development
Agency, 113 Thailand Science Park (TSP), Paholyothin Road, Klong Nueng, Klong Luang, Pathum Thani 12120, Thailand
*
Author for correspondence; E-Mail: siwarutt@siit.ac.th, siwarutt.siit@gmail.com Tel. +66-2-986-9009 ext 2305, Fax. +66-2986-9112
Abstract: Kefir culture contains bacteria and yeasts

staying together in symbiotic relationships. Kefir has
been cultured in milk by fermenting kefir grains in cowmilk to generate a kefir culture. Microorganisms in kefir
culture are naturally probiotic and thus, consumption of
kefir culture gives several health benefits. Developments
of kefir culturing on commercial scales are important to
several aspects of food industry including nutrition
productions, additive products, and therapeutic foods.
Under the different conditions for culturing kefir such as
types of cultivating media, temperature or level of
humidity, types and species of bacteria and yeast
presence in those culture conditions would be different.
In this report, milk kefir in Thailand is evaluated for its
microorganism components. The microorganisms are
isolated by using a cultural method. DNA sequences of
16S rDNA or D1/D2 domain of 26S rDNA of the
isolates were determined and compared similarity
against DNA database by using BLAST and megaBLAST
programs to identify the microorganisms. The result
revealed four species of bacteria and one species of yeast.
The yeast found in Thai kefir grains was Candida
tropicalis while types of bacteria found in the kefir grains
were classified into two categories; gram positive and
gram negative bacteria. Gram positive bacteria strains
found are Staphylococcus cohnii, Lactobaciilus paracasei,
and gram negative bacteria found are Acetobacter
orientalis and Acetobacter fabarum.
1. Introduction
Kefir, knowing as a natural probiotic food, is a
fermented dairy milk product. The kefir presents in the
form of grains which differentiates it from other
fermented milk products. The grains, having
cauliflower-like structure, are white or yellow in
colour containing groups of microorganisms namely
lactic acid bacteria, acetic acid bacteria, and yeasts [13]. Kefir grains, cultivating in different conditions
such as media, temperature and period of time,
presents different microflora composition [2, 4, 5]. The
media for cultivating kefir is the vital variable that
affects the taxonomic nomenclature of microflora
species in the grains. The characteristic of survived
species must fit to that growth stimulating source and
can use bioproducts produced by other survived
species as an energy source for themselves. This
shows a symbiotic relationship between bacteria and
yeasts [2, 6].
Lactic acid bacteria, producing lactic acid, inhibit

the growth of spoilage bacteria by lowering pH in
fermented milk, resulting in a longer shelf life of the
products. Besides, kefir is used as probiotic food for
alleviating ailments associated with digestive and
immune systems [7]. The aim of this study was to
isolate and identify species of microorganisms present
in Thai kefir grains using a culture-dependent method.
The identification of bacteria and yeasts in kefir grains
would enable the study of specific products from the
fermentation which allow utilization of these
symbiotic microorganisms in many applications such
as in food and therapeutic purposes [8].
2.1 Culture of Thai kefir grains
Thai kefir grains were collected from the central
part of Thailand. The grains were cultured in sterilized
milk at room temperature (25 28 C). The culturing
media was replaced every two days to maintain its pH.
2.2 Isolation of microorganisms
Kefir grains were disrupted by pestle and mortar.
Kefir grains (1mL) were homogenized in 9 mL of
sterile saline solution (0.85% sodium chloride
solution) for 1 min. Serial dilutions were used for
microbial enumerations. MRS (Difco Laboratories,
Detroit, MI, USA) and GECA (2% glucose, 0.3%
peptone, 0.3% yeast extract, 0.5% EtOH, 0.7% CaCO3,
1.2% agar) agar was used for bacterial isolation under
aerobic condition. Cycloheximide (Sigma, St. Louis,
MO, USA 50 mg/mL) was added for both MRS and
GECA agar to inhibit the growth of yeasts. For yeasts
isolation, YM agar (10g/L glucose, 3 g/L malt extract,
5g/L peptone, 3 g/L yeast extract, 15 g/L agar) was
used under aerobic conditions with 0.1 g
chloramphenical (Sigma) in ethyl alcohol and 2 g
sodium propionate (Sigma) in 9 mL distilled water
added to inhibit the growth of bacteria. The plates
were incubated at 30 C for 2 days. The resulting
colonies were counted before further purification.
813
2.3 Identification of bacteria

DNA templates for PCR amplification were
prepared by using Genomic DNA mini kit
(Blood/culture cell) (Geneaid Biotech Ltd., Taiwan).
DNA coding for 16S rRNA regions was amplified by
means of PCR with Taq polymerase. A PCR product
for sequencing 16S rDNA regions was prepared by
using the following two primers, 20F (5- GAG TTT
GAT CCT GGC TCA G-3, positions 9-27 on 16S
rDNA by the E. coli numbering system) and 1500R
(5-GTT ACC TTG TTA CGA CTT-3, position 15091492 on 16S rDNA by the E. coli numbering system).
The PCR product was analyzed by 0.8% (w/v) agarose
gel electrophoresis and purified with a QIAquick
PCR purification kit (QIAGEN GmbH, Hilden,
Germany). Direct sequencing of the single-banded and
purified PCR products (ca. 500 bases) was carried out.
Sequencing of the purified PCR products was carried
out with an ABI PRISM BigDye Terminator
Ready Reaction Cycle Sequencing Kit (version 3.1,
Applied Biosystems, Foster City, California, USA) by
Macrogen Inc., Korea. The primers 27F (5-AGA GTT
TGA TCM TGG CTC AG-3) and 800R (5-TAC
CAG GGT ATC TAA TCC-3) for partial sequencing,
and additional 1492R (5-TAC GGY TAC CTT GTT
ACG ACT T-3) and 518F (5-CCA GCA GCC GCG
GTA ATA CG-3) for full length sequencing, were
used for sequencing of 16S rDNA. The nucleotide
sequences obtained from all primers were assembled
using the Cap Contig assembly program, an accessory
application in BioEdit (Biological sequence alignment
editor) program. The identification of phylogenetic
neighbors was initially carried out by the BLAST and
megaBLAST programs against the database of type
strains with validly published prokaryotic. The 50
sequences with the highest scores were then selected
for the calculation of pairwise sequence similarity
using global alignment algorithm, which was
implemented at the EzTaxon server.
2.4 Identification of yeasts
Isolation of DNA was carried out by boiling of
cells with lysis buffer. The divergent D1/D2 domain of
26S rDNA was amplified with primers NL-1 (5- GCA
TAT CAA TAA GCG GAG GAA AAG-3) and NL4
(5-GGT CCG TGT TTC AAG ACG G-3). The
amplified DNA was purified with QIAquick PCR.
Visualization of the purified amplified DNA was
performed by electrophoresis using 0.8% agarose gel
in 1X TBE buffer and stained with ethidium bromide
(8x10-5 g/mL) and observed under a UV illuminator.
The nucleotide sequences of D1/D2 domain of 26S
rDNA were directly determined using PCR products.
Cycle sequencing of the D1/D2 domain was employed
with forward primer NL1 (5-GCA TAT CAA TAA
GCG GAG GAA AAG-3), and reverse primer, NL4
(5-GGT CCG TGT TTC AAG ACG G-3) by
Macrogen Inc., Korea. The sequences of D1/D2
domain of 26S rDNA were compared by BLASTn

Homology Search.
3.1 Enumeration values
Kefir grains were cultured in different media for
selectively culture microorganisms. Viable lactic acid
bacteria were estimated on the plate of MRS agar
while viable acetic acid bacteria were counted on the
plate of GECA agar. For the plate of YM agar, the
number of yeasts was determined. The result of
enumeration values in three different media is given in
Table 1.
Table 1: Enumeration values from kefir grains in three
different media
Media
CFU/mL
MRS
1.75 x 108
GECA
3.6 x 108
YM
5 x 105
The three different media show values ranging

from a minimum of 5 x 105 to a maximum of 3.6 x 108
CFU/mL kefir grains. The difference in numbers
indicated the variation of microbial species in the kefir
grains since each media is selective for microbial
communities. For enumeration values, the numbers
covered only part of microorganisms that can be
cultured and/or are metabolically active.
3.2 Identification of isolates
The result of isolated microflora is shown in Table
2. Bacteria genera isolated were Staphylococcus,
Lactobacillus and Acetobacter. Yeast isolated was
Candida tropicalis. The data were compared with the
database in BLAST and megaBLAST programs. The
percent similarity indicates the reliability of data with
the database. Based on gram staining, the bacteria in
kefir grains could be grouped into two categories:
gram positive bacteria and gram negative bacteria.
Gram positive bacteria including Staphylococcus and
Lactobacillus showed opaque colonies with smooth
edges. For Acetobacter classified as gram negative
bacteria, colonies were yellow in color with smooth
edges.
The species of microflora in kefir grains were not
limited to the data presented above. The culturedependent method revealed a part of microorganisms
that was viable which mostly were the dominant
species. The metabolisms from symbiotic relationships
among different species of bacteria and yeast led to the
acidity and viscosity of kefir. Staphylococcus cohnii
presents a carbohydrate reduction pattern which can
produce acid from glucose, fructose, and maltose from
milk [9]. For Lactobacillus paracasei supsp. tolerans,
814
the acids are produced from galactose, D-glucose,

lactose, and D-mannose [10]. Acetobacter orientalis
are capable of acetate oxidation and lactate oxidation
while A. fabarum are specifically able to produce
gluconic acid from D-glucose [11, 12]. For yeast,
Candida tropicalis shows lactose-negative but are
positive to the fermentation glucose, maltose, and
galactose [13].
Table 2: Identification of Microflora in Thai kefir
grains
Microflora
%Similarity
% Similarity
of 16s r DNA
Staphylococcus cohnii subsp. urealyticus
100
Lactobacillus paracasei subsp. tolerans
100
Bacteria
Acetobacter orientalis
Acetobacter fabarum
Yeast
Candida tropicalis
temperature and humidity. Although the culturedependent method could reveal only part of
microorganisms which can be cultured, several
identified bacteria were the predominant strains in
kefir grains that play an important role in kefir
fermentation. The types of microorganisms found in
the Thai kefir confirms the symbiotic relationship
among them.
Kefir is believed to possess important biological
activities including anti-bacterial and anti-fungal
activity, and cytotoxicity against cancer cell lines [1].
Knowing the species of microorganisms facilitates our
progress toward validating and understanding the
health benefits from kefir culture that have been
suggested by regular kefir household users.
Acknowledgements
This work was supported by the National Research

University
Project of Thailand Office of Higher
100
Education Commission, and Thammasat University
99.85
Research Fund. MT thanks the Young Scientist and
%Similarity of Technologist Program (YSTP) scholarship from
National Science and Technology Development
26s rDNA
Agency (NSTDA).
100
References
Different species of microorganisms were found in

Thai kefir grains when compared to other sources [5,
6]. The specification of species based on the ambient
conditions, type of milk sources, period of culture
time, and also the origin of kefir grains. Although the
species of microorganisms were different, the
properties of kefir fermentation are not very different.
The symbiotic relationship between bacteria and
yeast suggests the complex interactions among them.
For example, the bioproducts produced by lactic acid
bacteria such as galactose from galactose-exporting
lactic acid bacteria are used as an eneygy source for
yeasts that are lactose-negative. The increase in kefiran
production rates are shown in the co-culture of bacteria
and yeasts in kefir [14]. The found micro-organsisms
can digest components in cow milk and benefit the
human digestion system. The predigest of protein and
the fermentation of lactose from microorganisms in
kefir allows better performance in absorption of
protein and improves symptoms of lactose intolerance
[1].
4. Conclusions
In this study, a culture-dependent method was used
to identify microorganisms present in Thai kefir
grains. The number of bacteria could be successfully
identified. Four bacteria and one yeast species were
revealed. It is interesting that all of microorganisms
found in kefir grains in different regions have some
microorganisms in common which are Lactobacilli
and Acetobactor [5, 6]. Some strains are varied due to
the difference in ambient conditions such as
[1] S. Otles and O. Cagindi, Pak. J. Nutr. 2 (2003) 54-59.

[2] R. C. Witthuhn, T. Schoeman and T. J. Britz, Int. J.
Dairy Technol. 57 (2004) 33-37.
[3] E. Simova, D. Beshkova, A. Angelov, T. Hristozova, G.
Frengova and Z. Spasov, J. Ind. Microbiol. Biotechnol.
28 (2002) 1-6.
[4] G. L. Garrote, A. G. Abraham and G. L. De Antoni, J
Dairy Res 68 (2001) 639-52.
[5] M. Motaghi, M. Mazaheri, N. Moazami, A.
Farkhondeh, M. Fooladi and E. Goltapeh, World J.
Microbiol. Biotechnol. 13 (1997) 579-581.
[6] J. Zhou, X. Liu, H. Jiang and M. Dong, Food
Microbiol. 26 (2009) 770775.
[7] H. C. Chen, S. Y. Wang and M. J. Chen, Food
Microbiol. 25 (2008) 492-501.
[8] S. Y. Wang, H. C. Chen, J. R. Liu, Y. C. Lin and M. J.
Chen, J. Dairy Sci. 91 (2008) 3798-3805.
[9] K. H. Schleifer and W. E. Kloos, Int. J. Syst. Bacteriol.
25 (1975) 50-61.
[10] M. D. Collins, B. A. Phillips and P. Zanoni, Int. J. Syst.
Bacteriol. 39 (1989) 105-108.
[11] A. Seearunruangchai, S. Tanasupawat, S. Keeratipibul,
C. Thawai, T. Itoh and Y. Yamada, J. Gen. Appl.
Microbiol. 50 (2004) 47-53.
[12] I. Cleenwerck, . Gonzalez, N. Camu, K. Engelbeen, P.
De Vos and L. De Vuyst, Int. J. Syst. Evol. Microbiol.
58 (2008) 2180-2185.
[13] E. Azoulay, F. Jouanneau, J. C. Bertrand, A. Raphael, J.
Janssens and J. M. Lebeault, Appl. Environ. Microbiol.
39 (1980) 41-47.
[14] F. Lopitz-Otsoa, A. Rementeria, N. Elguezabal and J.
Garaizar, Rev. Iberoam. Micol. 23 (2006) 67-74.
815
ISOLATION AND CHARACTERIZATION OF EXOPOLYSACCHARIDE

PRODUCING-LACTIC ACID BACTERIA FROM THAI FERMENTED
VEGETABLES
Wichuda Wilairussamee1,*, and Suthep Thaniyavarn1
1
Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

*
Author for correspondence; E-Mail: kati_narak@hotmail.com
Abstract: Exopolysaccharide (EPS) producing-lactic acid

bacteria (LAB) were isolated from Thai fermented
vegetables. Eight isolates (L01 to L08) were found
capable of producing EPSs in MRS medium containing
sucrose as carbon source. The highest amounts of EPS
production were found in strain L06 and L07 at 11.20
and 11.40 g/l, respectively. Chemical and physical
properties of EPSs from these isolates were characterized
by chemical composition, total carbohydrates and
proteins, solubility test, water holding capacity and
emulsifying activity. All EPSs consisted of glucose, 71-99
% of total carbohydrates, 5-9 % of total proteins. The
EPS from L07 was well soluble in water but failed to do
so in organic solvents (methanol, acetone, isopropanol
and n-butanol). All EPSs possessed low water capacities
while demonstrated good emulsifying activities in
soybean oil and olive oil.
polymers and offer an alternative source of microbial

polysaccharides for use in food and/or other industries
[10]. EPSs from LAB have potential application in the
improvement of the rheology, texture and mouthfeel of
fermented milk products including yoghurt and cheese
[11]. Most of the EPS-producing LAB have been isolated from different fermented foods such as
sourdoughs, sausages, table olives, cheeses, yoghurt,
kefir, other fermented dairy products and some
traditional foods from non-industrialized countries
[12-13]. The aim of the present study was to isolate
EPS-producing LAB and characterize of their physical
and chemical properties.
1. Introduction
2.1 Isolation and screening for EPS production

Thai fermented vegetables were collected from
Nakhonpathom province, Thailand. Bacterial isolation
was performed by streaked on MRS agar and
incubated at 30C for 24 hr. The obtained colonies
were then streaked onto MRS agar containing
bromocresol purple and incubated at 30C for 24 hr.
The colonies which show yellow area around were
selected and then streaked onto MRS containing 4%
(w/v) sucrose and incubated at 30C for 24 hr. Strains
which produced slimy colonies were collected for
further screen for their EPS production.
Natural polymers have received an increased

demand for various industrial applications and led to a
development of exopolysaccharide (EPS) production
in recent years. Microbial EPSs are extracellular
polysaccharides which are either associated with the
cell surface in the form of capsules or secreted into the
extracellular environment in the form of slime [1].
Bacteria and microalgae have a better excreting ability
than yeasts and fungi [2]. The term EPS may refer to
homopolysaccharide (HoPS), composed of a single
type of sugar monomer [3], or heteropolysaccharides
(HePS), containing several types of sugar monomers
[1]. The EPSs have special physiochemical and
rheological properties, such as viscosifying,
stabilizing, gelling or emulsifying which enabling
them as potential food additives [4-6]. Several
microbial EPSs have been used in industrial level due
to the similarities of their physiochemical properties to
those of polysaccharides from plant (cellulose, pectin
and starch) and seaweed (alginate and carrageenan).
Examples of these are xanthan, acetan and gellan,
produced by the gram negative bacteria Xanthomonas
campestris, Acetobacter xylinum, and Sphingomonas
paucimovilis respectively [7]. Polysaccharides are also
believed to protect bacterial cells from desiccation,
penetration of toxic metals, antibiotics, phagocytosis,
phage attack and as means to produce biofilms [8-9].
Lactic acid bacteria (LAB) are generally recognized
as safe (GRAS) microorganisms. Thus, the EPS
excreted by LAB can be regarded as safe biologicaly
2.2 Partial purification of EPS

The bacterial strains were inoculated in 250 ml arm
flask containing 50 ml of MRS medium plus 4% (w/v)
sucrose (pH 6.5) and incubated at 30C in a rotary
shaker (200 rpm) until the culture OD550 reached 0.81.0. Cell suspension was then transfered into MRS
medium and further incubated at 30C in a rotary
shaker (200 rpm) for 18 hr. Trichloroacetic acid (TCA)
solution (40% w/v) was added to the culture broth to
give a final concentration of 4%. The precipitates were
removed by centrifugation (8,000 rpm for 20 min at
4C). The supernatant was added with two volumes of
absolute ethanol, stored at 4C for 24 hr. The
precipitated EPS was then centrifuged at 8,000 rpm at
4C for 20 min and dissolved in distilled water. EPS
was re-precipitated with ethanol twice followed by
lyophilized to dryness. After 24 hr of drying in
desiccator, the dry weight of the precipitated EPS was
determined.
816
2.3 Monosaccharide analysis

The sugar compositions of EPS were determined via
the modified method of Kiratiphibun et al. [14]. EPS
was hydrolyzed with 1M H2SO4 at 121C for 2 h. The
pH was brought up to 7.0 and filtered through nylon
filter (0.2 m). Quantitative determination of the
monosaccharides was carried out by HPLC at 60C on
a carbohydrate (aminopropyl) column eluted with a
mixture of acetonitrile and water (8 : 2) at a flow rate
of 1 ml/min and monitored by differential
refractometer.
2.4 Determination of total carbohydrate and protein
The total concentration of carbohydrate of EPS was
determined by phenol sulfuric acid method [15] using
glucose as a standard. The total concentration of
protein of EPSs was determined by protein dye
binding method [16].
2.5 Solubility of EPS
Solubility of EPS was determined in distilled water
and solvents including methanol, acetone, isopropanol
and n-butanol [17].
2.6 Measurement of water-holding capacity
Water-holding capacity of EPS was determined by
ascending paper chromatography using guar gum,
carrageenan and xanthan gum (food grade) as
standards. One end of the filter paper (2400 mm) was
dipped into 0.5% polysaccharide and the
chromatography was allowed to run for an hour. Rates
of syneresis of all polysaccharides were determined as
percentage of the developed length on paper
chromatography against full length of the paper [18].
2.7 Emulsifying activity
Emulsifying activity of the polysaccharide was
performed via the modified method [19]. EPS in
distilled water (0.1% w/v) was mixed with equal
volume of each of the hydrophobic substrate (soybean
oil and olive oil) in test tubes and vigorously vortexed
followed by shaking at 150 rpm for 10 min until an
emulsion of lipid was formed. Emulsion was further
centrifuged at 2000 rpm for 10 min, height of the
emulsified layer was measured. Lipid emulsifying
activity of the polysaccharide was expressed as
percentage of the height of emulsified layer per height
of whole layer using xanthan (food grade) as control
3.1 Isolation and screening for EPS production
Eight isolates were found capable of producing EPS
based on slimy appearance of their colonies on MRS
agar containing sucrose. Strains L01, L02, L03, L04,
L05, L06, L07 and L08 could produce 5.40, 6.05, 1.73,
5.97, 7.41, 11.20, 11.40 and 5.45 g/l of EPS. Strains
L06 and L07 showed the highest EPS yields of 11.20
and 11.40 g/l, respectively. Previous study reported
Lactobacillus reuteri LB121 was able to produce EPS
up to 10 g/l [20]. The amount of EPS produced by
LAB can be influenced by the composition of the

culture medium, growth conditions and incubation
time [21].
3.2 Components of partially purified EPSs
Acid hydrolysates of exopolysaccharide produced
by strains L01, L02, L03, L04, L05, L06, L07 and L08
were analyzed by HPLC. Results showed that all EPSs
contained glucose as their major component. Hence,
all EPSs were classified as homopolysaccharide which
contain a single type of monosaccharide, mainly
glucose termed glucan [22]. Glucose was reported as
constituent of EPS from LAB produced by members
of the genera Leuconostoc, Lactobaciilus and Weisella
[23]. The total carbohydrate and proteins content of
EPSs produced by strains L01, L02, L03, L04, L05,
L06, L07 and L08 are presented in Table 1.
Table1: The total carbohydrate and protein contents of
EPSs
EPS from
strains
L01
L02
L03
L04
L05
L06
L07
L08
Total carbohydrate
(%)
99.61
77.61
81.83
71.83
65.06
73.39
88.61
77.61
Total protein
(%)
5.35
5.55
8.63
5.63
9.55
5.63
6.55
5.63
3.3 Solubility of EPSs

The EPSs from strains L01 to L08 were partially
soluble in water at room temperature except L07 was
completely soluble. All EPSs were insoluble in all
organic solvents tested
(methanol,
acetone,
isopropanol and n-butanol). The results suggested that
the hydroxyl groups of EPSs are favor to hydrogen
bonding with one or more water molecules which in
turn led to the swelling of gel as well as partially or
completely dissolve in water [24]. While the
insolubility of these polysaccharides in organic
solvents are due to the increase number of hydroxyl
groups in polymer couple to the increase of
crystallinity of the polymer that are difficult to break
by organic solvents [25].
3.4 Measurement of water-holding capacity
Syneresis rate of 0.5% polysaccharide solutions of
strains L01, L02, L03, L04, L05, L06, L07, L08,
xanthan gum, guar gum and carrageenan are presented
in Table 2. Rate of syneresis of all polysaccharide
showed high value in comparison to the control
polysaccharides which indicated low water-holding
capacity. This is because the percentage of syneresis is
of reversion to the rates of water-holding capacity
[18].
817
Table 2 : Rate of syneresis of polysaccharides

EPS from strains
Rate of syneresis (%)
L01
L02
L03
L04
L05
L06
L07
L08
xanthan gum
guar gum
carrageenan
11.11
73.82
68.30
67.62
82.85
87.29
80.91
73.26
9.15
46.14
35.73
Table 3: The emulsifying activity of polysaccharides
L01
L02
L03
L04
L05
L06
L07
L08
xanthan gum
Emulsifying activity (%)

Soybean oil
47
46
47
47
48
47
46
47
47
H.M. the Kings 72nd Birthday Scholarship from

Graduate School, Chulalongkorn University, National
Research University Project of Thailand, Office of the
Higher Education Commission (FW 653A),
Innovations for the improvement of food safety and
food quality for new world economy, the government
research budget of Thailand, and Chulalongkorn
University are acknowledged for the support.
References
3.5 Emulsifying activity

Microbial gum, plant gum and animal proteins are
known to exhibit emulsifying activity. Xanthan gum
from microbial origin showed numerous applications
in food industry due to its high emulsifying activity
[26]. The emulsifying activity of 0.1% polysaccharide
of strains L01, L02, L03, L04, L05, L06, L07, L08 and
xanthan gum against vegetable oils (soybean oil and
olive oil) are shown in Table 3. When compared to
commercial
emulsifier
(xanthan),
all
EPSs
demonstrated good emulsifying activity against
soybean oil and olive oil with similar value to that of
xanthan. From these results, EPSs from strains L01,
L02, L03, L04, L05, L06, L07 and L08 are expected to
have potential use in food industry due to their good
emulsifying activities.
EPS from
strains
Acknowledgements
Olive oil
47
48
49
48
48
48
49
49
48
4. Conclusions
This study reported microbial exopolysaccharide
produced by strains L01, L02, L03, L04, L05, L06,
L07 and L08 isolated from Thai fermented vegetables.
Strains L06 and L07 showed the highest EPS yields of
11.20 and 11.40 g/l, respectively. The major
component of all EPSs was glucose. All EPSs showed
potential useness in food industry as they are GRAS
bacteria and have good emulsifying activities.
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from lactic acid bacteria. FEMS Microbiology Reviews,
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Ryukyus, (1982),Vol. 29, 79-86.
[19] Yun, U.J. and Park, H.D. Physical properties of an
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819
METHOD DEVELOPMENT FOR EVALUATION OF TOTAL

ANTI-OXIDANT CAPACITY OF TAGETES ERECTA LINN. FLOWER
EXTRACT
Sontaya Phanrudee1,*, Nathawut Choengchan2, and Patchanee Charoenying1
1
Department of Chemistry, Faculty of Science, King Mongkuts Institute of Technology Ladkrabang, Bangkok 10520,
Thailand
2
Flow Innovation-Research for Science and Technology Laboratories (FIRST Labs),
Department of Chemistry, Faculty of Science, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520,
Thailand
*
Author for correspondence; E-Mail: Pha.sontaya@hotmail.com, Tel. +66 11 523690
Abstract: In this research, a fluorescence quenching

method has been developed for evaluation of total
antioxidant capacity of T. erecta flower extract. The CdS
quantum dots (QDs) were synthesized in a mixed solution
between methanol and dimethyl formamide at room
temperature. Maximum emission wavelength (em) of
QDs was found at 730 nm. Prior to applying to the crude
flower extract, it was tested with ascorbic acid. Results
revealed that the fluorescence intensity of the QDs was
linearly decreased with increasing acid concentration, up
to 1000 mgL-1(y = 0.159x + 0.787; r2 = 0.9970). The
solution of T. erecta was prepared by dilution (of a stock
extraction fraction) with methanol, then the QDs were
applied to the solution. The results showed that the
intensity was linearly decreased with increasing
concentration of the extract, ranging from 20 to 100
mgL-1(y = 0.032x + 0.834; r2 = 0.9940).
1. Introduction
Antioxidants from natural product extracts have
attracted increasing interest, due to human awareness
and perception of the danger of side effects and the
toxicity of synthetic antioxidants in food. Extracts of
fruits [1-3], vegetables [4], flowers [5-8] and their byproducts, such as wheat and soybean by-products [910], mango peel [11], and peel, pulp and seed of fruits
(guava, kiwifruit, purple mulberry, strawberry, white
pomegranate, lukan and honey tangerine pulps and
etc.) [12], all revealed effective antioxidant activity in
a trial model. Natural antioxidants were also tested in
real food systems. For example, carob fruit extracts
importantly in decelerated lipid oxidation in cooked
pork meat [13].
Flowers are natural products and major sources of
bioactive compounds, which have been abundantly
reported on describing successful therapies for
conditions such as cardiovascular, cancer, jaundice and
hepatitis diseases [14]. Tagetes erecta belongs to the
family of Compositae, a prevalent garden plant and is
commonly known as Marigold. It is a native of the
Americas, especially in Mexico, and has been very
popular in South Asia, Southwest Asia and is
cultivated in Thailand and India for religious
ceremonies. Recently the T. erectra flower extract has
been reported to be revealing the presence of active
compounds such as antimicrobial, antiwrinkle and

antioxidant agents [15].
Several methods have been reported for the
determination of antioxidant activity. The most
common method has been generally based on the
inhibition of radicals by the presence of antioxidants.
Oxidizing reagents could be organic radical producers.
Free radical reagents are reduced by antioxidants and
the absorbance at certain wavelengths is decreased,
such
as
2,2'-azino-bis-(3-ethylbenzthiazoline-6sulphonic acid) or Trolox equivalent antioxidant
capacity (TEAC), ferric reducing/antioxidant power
(FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
oxygen radical absorbance capacity (ORAC) [16-17].
These methods have advantages in that they are
accurate, rapid, comfortable, repeatable and
inexpensive. However, these methods have some
drawbacks of the stability of the color agents.
Therefore, this research focuses on the development of
the fluorophore agents. In recent years, the
applications of water-soluble QDs in the fields of
analytical chemistry have attracted a growing interest
because of their advantages over organic fluorescent
dyes. Currently, Quantum dots (QDs) were used in
numerous works such as luminescence probe for
detection of heavy and transition metal ions [18] and
sensor for the detection of glucose [19]. The QDs had
good optical properties and stability. The objectives of
this research are to focus on the synthesis of CdS QDs
using the fluorophore agents, and used for evaluation
of total antioxidant capacity from flowers extract of T.
erecta.
2.1 Chemicals and apparatus
Cadmium nitrate (four hydrate) (Cd )NO3(24H2O),
and sodium sulfide (nine hydrate) (Na2S9H2O) were
purchased from Lab Systems Co., Ltd.. All other
reagents and solvents analytical grade were used. The
absorption spectra were acquired on a SHIMADASU
UV-1800 spectrophotometer. All fluorescence
measurements were made with a JASCO FP-6300
fluorescence spectrophotometer equipped with a
plotter unit and a 1 cm quartz cell.
820
2.4 Extraction
Ground dry flowers of T. erecta (20 g) sample
were extracted with methanol (300 mL) for 7 days at
room temperature. The supernatant and the sediment
were separated by filtration through cheesecloth and
once through Whatman No. 1 filter paper. The residue
was extracted two times as described as the first
extraction. The total extraction of the solutions was
evaporated under reduced pressure to give a crude
methanol extract (12.85 g, 64.18% w/w).
2.5 Evaluation of anti-oxidant activity
2.5.1 DPPH assay
The antioxidant activity of the crude methanol
extracts was measured in terms of hydrogen-donating
or radical scavenging ability, using the DPPH method
[22]. Crude methanol extract (10 mg/mL) was mixed
with 2.9 mL of DPPH radical solution in ethanol
(4.5%v/v). The reaction mixtures were shaken
vigorously and incubated in the dark for 30 min. The
absorbance of solution was measured at 517 nm.
Butylated hydroxytoluene (BHT) was used as a
standard antioxidant.
2.5.2 Fluorescence quenching method
The method development, fluorescence quenching
of CdS QDs by antioxidants was measured in crude
methanol extract while ascorbic acid was used as
standard test. One milliliter of QDs colloids solution
was aliquoted into a test tube, then 9 mL of crude
methanol extract (of a stock concentration up to 1000
mgL-1) was added into the test tube and shaken
vigorously. The mixed solutions were then measured
by fluorometer. Under the excitation wavelength of
370 nm, the fluorescence spectra of QDs were
recorded at wavelength of 730 nm.
1.5
-1
- 0.5
0.5
-0
0
300
500
700
900
Normalized PL intensity (a.u.)
2.3 Preparation and characterization of CdS QDs

The CdS Quantum dots were prepared at room
temperature by applying from the procedures of Saha
[20] and Shozo [21]. Cadmium nitrate and sodium
sulfide were used as precursors. Dimethyl formamide
(DMF) and methanol were used as co-solvents. A 100
mL of 3.2 mM Cd(NO3)2 solution was mixed with 100
mL of 3.2 mM Na2S solution. Yellow CdS colloidal
solution was rapidly obtained. The mixed solutions
were stirred for 3-5 mins. And then, the CdS colloidal
solution was characterized for optical absorption by
using a UV-Vis spectrophotometer (SHIMADASU
UV-1800) with co-solvent of methanol and DMF in
1:1 ratio as reference solution. The photoluminescence
studies were also carried out on the colloidal solution
using a JASCO FP-6300 spectrafluorometer.
The synthesis of CdS QDs has been achieved by

several groups [22]. Here in this work, the mixed
solvents between methanol and DMF were easily
synthesized without stabilizing agent. The UV/vis
absorption and PL emission spectra of synthesized
CdS QDs are shown in Fig. 1. The obtained QDs
exhibited a broad absorption spectra and symmetrical
emission peaks with the excitation wavelength at 370
nm.
Absorbance
2.2 Plant sample

The flowers of T. erecta were collected from the
Department of Plant Production Technology, King
Mongkuts Institute of Technology Ladkrabang.
Wavelength (nm)
Figure 1. UV/vis absorption and PL emission spectra
of synthesized CdS QDs.
3.2 Total antioxidant capacity (TAC)
3.2.1 Determination of TAC by DPPH method
The DPPH assay is a preliminary test to investigate
the antioxidant potential of extract. The results found
that the crude methanol extract of T. erecta flower
showed an antioxidant potential with EC50 value with
9.5 g/ml. This result was in good agreement with the
study of Pratheesh et al. (2009) and their report of
xanthophylls antioxidant activity of the crude extract
of T. erecta. The results of this study provided
evidence that T. erecta has antioxidant potential.
3.2.2 Determination of TAC by CdS QDs method
The proposed method was applied to the
determination of antioxidant activity in crude methanol
extract of T. erecta flower. L-ascorbic acid was used
as a standard test before being applied to real sample.
The ratio of the initial CL intensity I0 of CdS QDs
system to the CL intensity I at a given concentration of
L-ascorbic acid, I0/I, was proportional to the
concentration of L-ascorbic acid. The L-ascorbic acid
concentration dependence of the CL intensity was
coincident with the fluorescence quenching described
by a SternVolmer equation Eq. 1.
I0/I = 1 + Ksv[Q]
(Eq. 1)
Ksv was found to be 1.5910-1 mol1L. This large

value provided a sensitive CL detection of L-ascorbic
acid (Fig. 2a and 2b). Under the optimized
experimental conditions, the relative CL intensity
decreased linearly in the concentration range of 200
1000 mg L1 for L-ascorbic acid (R2 = 0.9970).
3.1. Optical property of CdS QDs

821
blank
200
ppm
400
ppm
1000
900
725
775
825
Wavelength (nm)
Figure 2a. PL spectra representing the quenching
effect of L-ascorbic acid with different concentrations
on CdS QDs PL intensity.
y = 0.159x + 0.787
R = 0.9970
n=3
2.5
2
I0/I
1000
900
800
700
600
500
400
300
200
100
0
-100 675
CdS QDs. The strategy was actualized through the

optimization of the experimental conditions. Stern
Volmer equation Ksv was found to be 3.2010-2
mol1L. This large value provided a sensitive CL
detection of crude methanol extract (Fig. 3a and 3b).
Under the optimized experimental conditions, the
relative CL intensity decreased linearly in the
concentration range of 20100 mg L1 for crude
methanol extract (R2 = 0.9940).
800
700
Blank
600
20
ppm
40
ppm
500
400
300
200
100
0
-100 675
725
775
825
Wavelength (nm)
Figure 3a. PL spectra representing the quenching
effect of crude methanol extract of T. erectra with
different concentrations on CdS QDs PL intensity.
1.5
1
0.5
0
200
400
600
800 1000
Concentration (ppm)
Figure 2b. The relationship between I0/I and the
concentration of L-ascorbic acid detection using CdS
QDs.
In order to achieve the sensitive detection of Lascorbic acid, the quenching of CdS QDs by Lascorbic acid was investigated. Results showed that the
QDs PL intensity decreased with the increase of acid
concentration, and it was found that linear relationship
existed between the PL intensity decreased with
increasing acid concentration. After that The CdS QDs
colloids solution was applied to T. erectra flower
extract. As shown in Fig. 3a and 3b, the result
displayed the effect of the concentration of crude
methanol extract solution on CdS QDs. There was a
linear relationship between the fluorescence intensity
of CdS QDs and extract solution concentration. As a
result, a spectrofluorometric method for methanolic
solution would be developed based on the quenching
effect of methanolic solution on the fluorescence of
I0/I
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
y = 0.032x + 0.834
R = 0.9940
n=3
50
100
Concentration (ppm)
Figure 3b. The relationship between I0/I and the
concentration of crude methanol extract of T. erectra
detection using CdS QDs.
The results indicated that the proposed CL system
has good linearity, relatively high sensitivity and
precision and acceptable reproducibility.
822
4. Conclusions
A fluorescence quenching method has been
developed as a novel and convenient technique for
antioxidant determination based on the quenching of
the fluorescence of CdS QDs. The possible quenching
mechanism is due to the change of the surface of the
CdS QDs. Under optimum conditions, the method
achieved a good linear relationship between relative
fluorescence intensity ratio of the system and
concentration of L-ascorbic acid in the range of 200 to
1000 mgL1. It is prospective to apply this technique
to determine the antioxidant activity in biological
samples. The method provides a fairly practical and
economical approach.
Further study will be required for isolation of crude
extract by solvent partitioning technique before testing
with the QDs. In addition, investigation of an
antioxidant activity of each fraction by using this
method will be also studied.
Acknowledgements
Authors acknowledge the Department of
Chemistry, Faculty of Science, King Mongkuts
Institute of Technology Ladkrabang for financial
support and we also thank Associate Professor Dr.
Chamroon Laosinwattana for kindly providing the
plant materials.
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823
MOLECULAR IDENTIFICATION OF ENDOPHYTIC

STREPTOMYCES SP. P4 STRAIN
Julaluk Tang-um and Hataichanoke Niamsup*
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science,
Chiang Mai University, Chiang Mai 50200, Thailand
*
Author for correspondence; E-Mail: hataichanoke.n@cmu.ac.th, Tel. +66 53 943341, Fax. +66 53 892277
Abstract: Endophytic actinomycete P4 strain was

previously isolated from a sweet pea root. In our
previous study using the dual culture method, it
antagonistically inhibited the growth of Fusarium
oxysporum f.sp. lycopersici., a phytopathogen of tomato
(Lycopersicon esculentum). Thus, this actinomycete has a
potential to be used as a biocontrol agent to reduce the
use of chemical fungicide. In addition, the P4 strain was
found to possess extracellular chitinase and amylase. In
this study, its identity was confirmed to be Streptomyces
sp. by 16S ribosomal RNA full sequencing (GenBank
accession no. JN102356, 1436 nucleotide long). The Basic
Local Alignment Search Tool was used to search
homology within non-redundant DNA sequences in the
GenBank database. The highest similarity of 99.7%
between 16S rRNA sequences of P4 strain and
Streptomyces griseoflavus, based on a pairwise alignment,
was found. When multiple alignments among 16S rRNA
sequences of the P4 strain and the other 9 species of
Streptomyces were performed with T-coffee program and
their phylogenetic relationship was illustrated with
Treeview, the P4 strain was also placed in the same clade
as S. griseoflavas.
1. Introduction
Streptomyces belonging to the taxonomic
lineage, Bacteria; Actinobacteria; Actinobacteridae;
Actinomycetales; Streptomycineae;Streptomycetaceae,
is comprised of 376 approved species [1].
Streptomycetes have been found to be endophytic,
marine and terrestrial. Many species have been proved
to be beneficial such that they produced antimicrobial
compounds [2]. Streptomyces strain P4 was previously
isolated from sweet pea root [3]. In dual culture, this
endophytic actinomycete could inhibit the growth of
pathogenic fungi, Fusarium oxysporum f.sp.
lycopersici, causing a wilt disease in tomato [4].
Analysis of 16S ribosomal RNA (16S rRNA)
sequences is currently the most powerful method for
determining higher taxonomic relationships of
actinomycetes [5]. To date, there are 15,229
Streptomyces 16S rRNA sequences submitted to
GenBank database. Of these, 6,722 sequences are from
known species [6]. In order to molecularly characterize
the P4 strain, its 16S rRNA gene was amplified by
polymerase chain reaction (PCR) and sequenced. The
obtained nucleotide sequence was analyzed by
bioinformatic tools to find phylogenetic relationship
with other Streptomyces species.

2.1 16S rRNA sequence analysis
The actinomycete isolate P4 was cultured in
IMA-2 broth (5 g glucose, 5 g soluble starch, 1 g beef
extract, 1 g yeast extract, 2 g N-Z-case, 2 g NaCl and
1 g CaCO3 per liter) for 7 days at 37 C with rotary
shaking at 160 rpm. Genomic DNA of the endophytic
actinomycete was extracted using CTAB method. A
1500-bp PCR product coding 16S rRNA region was
amplified from Streptomyces P4 genomic DNA using
two primers, 20F (5-GAG TTT GAT CCT GGC TCA
G-3) and 1500R (5-GTT ACC TTG TTA CGA CTT3) under the following condition: an initial
denaturation step at 94 C for 3 min, 25 cycles of 94
C for 1 min, 50 C for 1 min, 72 C for 2 min,
followed by a final extension at 72 C for 3 min. The
purified PCR product was sequenced by an ABI
Prism BigDye Terminator Ready Reaction Cycle
Sequencing Kit (Applied Biosystems, USA) on an ABI
Prism 3730XL DNA Sequence (Applied Biosystems,
USA).
2.2 Bioinformatic analysis
The obtained 1438-bp DNA sequence has been
submitted to GenBank database under the accession
no. JN102356. The homology was analyzed using
BLAST (Basic Local Alignment Search Tool)
(http://blast.ncbi.nlm.nih.gov/) for highly similar
sequences
(megablast)
against
non-redundant
nucleotide sequence database (on August 30, 2011).
Top 9-hit sequences from different species
along with the P4 strain were multiply aligned using a
freeware T-Coffee ver. 8.99 available at
http://tcoffee.crg.cat/ [7]. Phylogenetic tree was drawn
with TreeView ver.1.6.6 program downloadable from
http://taxonomy.zoology.gla.ac.uk/rod/treeview.html.
After PCR amplification of the P4 genomic
DNA using primers specific to bacterial 16S rRNA
gene, expected 1500 bp product was obtained as
revealed by the agarose gel electrophoresis (Figure 1).
824
Table 1: Nine Streptomyces species with the most

similarity with Streptomyces sp. P4
Next, the purified PCR product was sequenced.

The 1436-bp 16S rRNA nucleotide sequence data was
submitted to the GenBank database with the accession
number JN102356, as demonstrated in first line of
each row in Figure 2.
The 16S rRNA sequence of the P4 strain was
blasted against non-redundant DNA sequences of
Genbank database. BLAST result showed that it had
the highest homology to Streptomyces griseoflavus
(accession number: EU741217, EU570501) with the
99.70% identity score, followed by 99.50% equal
scores when aligned with S. variabilis (DQ442551,
AB184884), S. vinaceus (AB184763, AB184186) and
S.
griseoincarnatus
(AB184207,
AJ781321),
respectively (as shown in Table 1). Many species of
the genus Streptomyces are closely related.
Consequently, it cannot be definitely concluded which
species of strain P4 belonged to or if it should be
designated as a new species.
The rRNA gene sequences of P4 strain and the
9 Streptomyces species of the highest similarity scores
were performed multiple alignments using T-coffee
program. The alignment after manual edition, shown
in Figure 2, reveals differences in some of single
nucleotide substitutes. The full sequence of
Streptomyces P4 strain showed that each single
nucleotide at the positions C-222 and G-533 was
similar to those of S. griseoflavus, S. malachitofuscus
and S. heliomycini (high-lighted in blue in Figure 2). In
addition, each single nucleotide at the positions A-39
and T-48 was similar to those of S. althioticus, S.
almquistii and the aforementioned 3 Streptomyces
species (high-lighted in yellow in Figure 2).
Accession
No.
Pairwise
similarity (%)
136670
EU741217
99.70
173668
EU570501
99.70
NRRL B3984(T)
DQ442551
99.50
NBRC
12825(T)
AB184884
99.50
NBRC 3406
AB184763
99.50
NBRC
12840
AB184186
99.50
NBRC
12871
AB184207
99.50
LMG 19316
AJ781321
99.50
LMG 19406
AJ781328
99.40
S. malachitofuscus LMG 20067
AJ781347
99.00
Streptomyces
species
S. griseoflavus
Figure 1. Agarose gel electrophoresis of PCR product

from Streptomyces sp. P4 genomic DNA amplified
using primers directed to 16S rRNA gene. Lane M:
1kb DNA ladder, Lane C: no template control, Lanes 1
and 2: with 1 and 2 l genomic DNA of the
Streptomyces P4 strain, respectively. Arrow indicates
an expected product.
Strain
Rank
S. variabilis
S. vinaceus
S. griseoincarnatus
S. erythrogriseus
S. heliomycini
LMG 19960
AJ781343
99.00
S. althioticus
NRRL B3981
AY999791
98.90
S. almquistii
NRRL B1685
AY999782
98.80
However, the nucleotide sequence of the P4

strain exhibited a unique single nucleotide deletion
between positions A-914 and G-915 (high-lighted in
red in Figure 2). This deletion that is absent in the
other 9 Streptomyces species might result from a
unique genetic evolution of the P4 strain. But an error
in sequencing was also possible.
A phylogenetic tree was constructed from the
multiple alignments of 10 Streptomyces species and
illustrated with Treeview as a rectangular cladogram in
Figure 3. The P4 strain was placed in the same clade as
S. griseoflavas. Potentially, these two Streptomyces
species have a hypothetical common ancestor.
825
P4
1 ------------------------GGCGGCGTG
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
10
29
34
19
20
20
9
20
29
29
CTTAACACATGCAAGTCGAACGATGAACCACCT
CTTAACACATGCAAGTCGAACGATGAACCTCCT
CTTAACACATGCAAGTCGAACGATRAACCTCCT
CTTAACACATGCAAGTCGAACGATGAACCAC-T
42
61
66
51
52
52
41
51
60
60
cons
34 ************************ **** * *
66
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
43
62
67
52
53
53
42
52
61
61
75
94
99
84
85
85
74
83
92
92
cons
TCGGGTGGGGATTAGTGGCGAACGGGTGAGTAA
TCGGGAGGGGATTAGTGGCGAACGGGTGAGTAA
TCG-GTGGGGATTAGTGGCGAACGGGTGAGTAA
67 *** * ***************************
76
95
100
85
86
86
75
84
93
93
CACGTGGGCAATCTGCCCTGCACTCTGGGACAA
CACGTGGGCAATCTGCCCTTCACTCTGGGACAA
108
127
132
117
118
118
107
116
125
125
cons
100 ******************* *************
132
P4
109 GCCCTGGAAACGGGGTCTAATACCGGATACTGA
141
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
142
161
166
151
152
152
141
150
159
159
165
184
189
174
175
175
164
173
182
182
cons
166
**
198
P4
166 GAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGC
198
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
199
218
223
208
209
209
198
207
216
216
231
250
255
240
241
241
230
239
248
248
cons
******
CTATCAGCTTGTTGGTGAGGTAACGGCTCACCA
CTATCAGCTTGTTGGTGAGGTAATGGCTCACCA
CTATCAGCTAGTTGGTGAGGTAATGGCTCACCA
CTATCAGCTAGTTGGTGAGGTAATGGCTCACCA
232 ********* ************* *********
529
548
553
538
539
539
528
537
546
546
TGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAAC
TGTCACGTCGGTTGTGAAAGCCCGGGGCTTAAC
561
580
585
570
571
571
560
569
578
578
cons
562 **** ****************************
594
P4
562 CCCGGGTCTGCAGTCGATACGGGCAGGCTAGAG
594
P4
595 TTCGGTAGGGGAGATCGGAATTCCTGGTGTAGC
627
P4
628 GGTGAAATGCGCAGATATCAGGAGGAACACCGG
660
P4
661 TGGCGAAGGCGGATCTCTGGGCCGATACTGACG
693
P4
694 CTGAGGAGCGAAAGCGTGGGGAGCGAACAGGAT
726
P4
727 TAGATACCCTGGTAGTCCACGCCGTAAACGGTG
759
P4
760 GGCACTAGGTGTGGGCGACATTCCACGTCGTCC
792
P4
793 GTGCCGCAGCTAACGCATTAAGTGCCCCGCCTG
825
P4
826 GGGAGTACGGCCGCAAGGCTAAAACTCAAAGGA
858
P4
859 ATTGACGGGGGCCCGCACAAGCGGCGGAGCATG
891
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
892
911
916
901
902
902
891
900
909
909
TGGCTTAATTCGACGCAACGCGA-GAACCTTAC
TGGCTTAATTCGACGCAACGCGAAGAACCTTAC
923
943
948
933
934
934
923
932
941
941
cons
925 *********************** *********
957
P4
924 CAAGGCTTGACATACACCGGAAAGCATCAGAGA
956
P4
957 TGGTGCCCCCCTTGTGGTCGGTGTACAGGTGGT
989
P4
990 GCATGGCTGTCGTCAGCTCGTGTCGTGAGATGT 1022
P4
1023 TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGT 1055
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
1056
1076
1081
1066
1067
1067
1056
1065
1074
1074
cons
1090 *************** ****** * **** *** 1122
P4
1088 GGACTCACGGGAGACCGCCGGGGTCAACTCGGA 1120
P4
1121 GGAAGGTGGGGACGACGTCAAGTCATCATGCCC 1153
P4
1154 CTTATGTCTTGGGCTGCACACGTGCTACAATGG 1186
99
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
ACCG--CTTGGGCATCC----AGGCGGT---TC
ACCG--CTTGGGCATCC----AGGCGGT---TC
CCCG--CTTGGGCATC---C-AAGCGGT---TC
CCTT--CACGGGCATC---CRT----GARGKTC
TCCG--CCTGGGCATCC----AGGCGGT---TC
TCMT--CTTGGGCATCYWRGM-------KGWTC
--YCMKCTTRGGCATCYWRGM-------KGWTC
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
CCCGTGTTGCCAGCAGGCCCTT-GTGGTGCTGG
CCCGTGTTGCCAGCAAGCCCTTCGGGGTGTTGG
1087
1107
1112
1097
1098
1098
1087
1097
1105
1105
264
P4
232 AGGCGACGACGGGTAGCCGGCCTGAGAGGGCGA
264
P4
1187 CCGGTACAATGAGCTGCGATACCGCGAGGTGGA 1219
P4
265 CCGGCCACACTGGGACTGAGACACGGCCCAGAC
297
P4
1220 GCGAATCTCAAAAAGCCGGTCTCAGTTCGGATT 1252
P4
298 TCCTACGGGAGGCAGCAGTGGGGAATATTGCAC
330
P4
1253 GGGGTCTGCAACTCGACCCCATGAAGTCGGAGT 1285
P4
331 AATGGGCGAAAGCCTGATGCAGCGACGCCGCGT
363
P4
1286 CGCTAGTAATCGCAGATCAGCATTGCTGCGGTG 1318
P4
364 GAGGGATGACGGCCTTCGGGTTGTAAACCTCTT
396
P4
1319 AATACGTTCCCGGGCCTTGTACACACCGCCCGT 1351
P4
1352 CACGTCACGAAAGTCGGTAACACCCGAAGCCGG 1384
P4
1385 TGGCCCAACCCCTT-GTGGGAGGGAGCTGTCGA 1416
P4
1417 AGGTGGGACTGGCGATTGGG------------- 1436
P4
griseoflavus
variabilis
vinaceus
griseoincarnatu
erythrogriseus
malachitofuscus
heliomycini
althioticus
almquistii
397
416
421
406
407
407
396
405
414
414
TCAGCAGGGAAGAAGCGAAAGTGACGGTACCTG
TCAGCAGGGAAGAAGCGCAAGTGACGGTACCTG
429
448
453
438
439
439
428
437
446
446
cons
430 ***************** ***************
462
P4
430 CAGAAGAAGCGCCGGCTAACTACGTGCCAGCAG
462
P4
463 CCGCGGTAATACGTAGGGCGCGAGCGTTGTCCG
495
P4
496 GAATTATTGGGCGTAAAGAGCTCGTAGGCGGCT
528
Figure 2. Multiple alignments of the P4 strain and the

other 9 Streptomyces species, S. griseoflavus, S.
variabilis, S. vinaceus, S. griseoincarnatus, S.
erythrogriseus, S. malachitofuscus, S. heliomycini, S.
althioticus, and S. almquistii. Conserved sequence of
the ten Streptomyces species is deduced. Only
sequence of the P4 strain is shown wherever all 10
aligned sequences are identical.
826
We would like to acknowledge Asst. Prof. Dr. Ampan

Bhromsiri for providing Streptomyces P4 and Miss
Jenjira Somkeaw for technical help on PCR
experiment.
References
Figure 3. Cladogram showing genetic relatedness of

the P4 strain and the other 9 Streptomyces species.
[1] V.B.D. Skerman, V. McGowan, P.H.A. Sneath,

Approved Lists of Bacterial Names (Amended). ASM
Press,Washington (DC), (1989).
[2] G. Sykes, F. A. Skinner, Actinomycetales:
Characteristics and Practical Importance, Academic
Press, London (1973), pp. 64-75.
[3] P. Thapanapongworakul, Characterization of endophytic
actinomycetes capable of controlling sweet pea root rot
diseases and effects on root nodule bacteria, Master's
thesis, Chiang Mai University, (2003).
[4] J. Tang-um and H. Niamsup, Maejo Int. J. Sci. Technol.
6 (2012) 95-104.
[5] P. Nimnoi, N. Pongsilp and S. Lumyong, World J.
Microbiol. Biotech. 26 (2010) 193-203.
[6] http://www.ncbi.nlm.nih.gov/ (retrieved October 19,
2011).
[7] P.D. Tommaso, S. Moretti, I. Xenarios, M. Orobitg, A.
Montanyola, J.-M. Chang, J.-F. Taly and C. Notredame,
Nucleic Acids Res. 39 (2011), Web Server issue W13
W17.
[8] B. Lanoot, M. Vancanneyt, B. Hoste, K.
Vandemeulebroecke, M. C. Cnockaert, P. Dawyndt, Z.
Liu, Y. Huang, and J. Swings, Res. Microbiol., 156
(2005), 755-762.
[9] F. M. Atta and A. A. Zohri, J. Basic Microbiol., 35
(1995), 3-7.
It is worth noting that 16S ribosomal RNA

sequences are not highly variable among Streptomyces
species and the Streptomyces systematics is rather
complex. From 16S ribosomal RNA sequence, the
actinomycete P4 was identified to be Streptomyces
with the highest similarity to Streptomyces
griseoflavus.
However,
more
morphological,
physiological, and chemotaxonomic analyses are
needed in order to confirm that P4 strain is S.
griseoflavus. Lanoot et al. [8] suggested that 16S-ITS
RFLP fingerprinting has a higher taxonomic resolution
than 16S rDNA sequencing. Transformation reaction
of progesterone was also proposed to be used in
Streptomyces taxonomic classification [9].
4. Conclusions
Endophytic actinomycete P4 strain, previously
isolated from a sweet pea root, is most likely to be
identified as Streptomyces griseoflavus P4 by the
methods of 16S rRNA sequencing and bioinformatics.
Acknowledgments
This work was financially supported by the
Center of Excellence for Innovation in Chemistry
(PERCH-CIC), Lanna Products Company, and the
Graduate School of Chiang Mai University, Thailand.
827
PASTING PROPERTIES AND GEL TEXTURE OF STARCH BLEND

BETWEEN MANGO SEED KERNEL STARCHES AND THAI RICE
STARCH (CHIANG PHATTHALUNG CULTIVAR)
Kwankeaw Saeaurng1 and Daris Kuakpetoon1*
1
Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*Author for correspondence; E-mail: kdaris@gmail.com, Tel. +66 2 2185521
Abstract: The influences of mango seed kernel (MSK)

starch isolated from three mango cultivars (Namdokmai,
Kaew, Chokanun) on pasting and gel texture properties
of Thai rice starch (Chiang Phatthalung cultivar) were
investigated. MSK starch was mixed with rice starch at 3
different percentages (25%, 50%, and 75%). Amylose
contents of MSK and rice starches were in the range of
31.50-33.12% with no significantly different. MSK
starches exhibited lower in swelling power and solubility
than rice starch. For pasting properties, MSK starch
pastes were significantly higher in peak viscosity (PV)
and breakdown (BD) but lower in final viscosity (FV),
setback (SB) and pasting temperature (PT) than rice
starch paste. Increasing in MSK starch made the riceMSK starch paste exhibited higher in PV but lower in
PT. There was synergistic effect to the rice-MSK starch
paste which showed significantly higher in BD, SB, and
FV than the unblended starch paste. Texture profile
analysis showed that rice starch gel had significantly
lower in hardness, gumminess, and chewiness than the
gel of MSK starches. Hardness, gumminess, springiness
and chewiness of the rice-MSK starch gel proportionally
increased with the amount of MSK starch. Among all
MSK starches, Kaew MSK starch had the most similar in
its pasting and gel texture properties to rice starch. As a
result, it could be blended with rice starch up to 50%
without significant change in the hardness of rice gel.
This study showed the possibility to partly replace rice
flour with MSK starches; especially, from Kaew cultivar
in rice-based product such as rice noodle.
1. Introduction
Mango (Mangifera indica L.) is one of the major
economic fruits produced in Thailand. Only the pulp is
utilized as food; while, peels and seeds are disposed as
waste. In Thailand, there are about 1,063 kg/week of
mango seeds disposed from mango processing plants
[1]. Mango seeds which hardly degrade are generally
disposed by landfilling. Ultimately, the mango
processors face a problem to dispose mango seeds due
to a deficient in land. Mango seed kernel (MSK) is a
source of many nutrients and phytochemicals such as
oil, starch, polyphenolics, phytosterols, antioxidants,
and antimicrobials [2]. These compounds make MSK
had more applications and increase its value. MSK
flour has a potential to partly used in flour-based food
products such as biscuit without significant changes in
their texture and sensory properties [3].
In Thailand, rice is a staple food and is mainly
consumed as cooked rice. However, rice is also
produced as noodle which is the second major meal for
Thai people after cooked rice. Generally, rice noodle is

a gel of starch molecules; especially, amylose; a linear
molecule which easily retrogrades during cooling and
trap water molecules in its gel network. This
phenomenon promotes a short gel texture required
for rice noodle. Therefore, a high-amylose rice cultivar
is preferred in noodle production [4].
Nowadays, the price of rice keeps increasing due to
a high ratio of demand to supply [4]. Partial replacing
rice flour with other flours or starches which have
similar physicochemical properties to rice flour might
help to reduce the production cost of rice noodle.
This study investigated the influence of MSK
starches from three mango cultivars (Namdokmai,
Kaew, Chokanun) grown in Thailand on pasting and
gel texture properties of Thai rice starch (Chiang
Phatthalung cultivar). The findings of this study could
be used to evaluate the possibility to partially
substitute rice flour with MSK starch in rice flourbased product such as noodle.
2.1 Materials
MSK starches from three mango cultivars; namely,
Namdokmai, Chokanun and Kaew were isolated by the
method of Benjapalakorn [5]. Rice starch from Chiang
Phatthalung cultivar was isolated by the method of
Wang and Wang [6].
2.2 Apparent amylose content (%)
Apparent amylose content of starch was
determined by iodine colorimetry method [7 and 8].
2.3 Swelling power and water solubility index
Swelling power and solubility index of starch at
60C, 70C, 80C and 90C was determined by the
method of Wang et al. [9].
2.4 Pasting properties of starches
Rice starch was blended with MSK starch at three
different percentages; 25, 50, and 75%. The pasting
properties of unblend and blended starches were
determined with a Rapid Visco Analyzer (RVA,
Newport Scientific, Warriewood, Australia) according
to AACC method 76-71 [10].
2.5 Gel texture properties of starches
828
The starch paste prepared from the RVA test was

transferred into a small plastic cup and stored at 4 C
overnight for gel setting. The set gel was evaluated its
texture properties by Texture Analyser (TA-XT. Plus,
Stable Micro Systems, Surrey, UK) using the profile of
Sandhu and Singh [11] with some modifications. The
gel was compressed at a speed of 0.5 mm/s to a
distance of 10.0 mm with a cylindrical plunger (6.0
mm in diameter). The pause between the first and
second compressions was 0.5 seconds. Texture
parameters, hardness, cohesiveness, chewiness,
springiness and gumminess were obtained from the
force-time curve of the texture profile.
proposed by Tester and Morrison [15]. They suggested

that amylopectin contributes to swelling of starch
granules, whereas amylose and lipid restrict the
swelling. Although there was no significant different
in amylose content among MSK and rice starches, the
differences in their SP and WSI might be due to a
variation in their lipid content, molecular weight of
amylose, or amylopectin fine structure.
2.6 Statistical analysis

The data were subjected to one-way analysis of
variance (ANOVA) using SPSS Software version 16.
3.1 Apparent amylose content
The amylose content of rice starch (Chiang
Phattalung cultivar) and all types of MSK starch were
not significantly (p 0.05) different (Table 1).
Figure 1. Swelling power of rice and MSK starches.
Table 1: Amylose content of rice and mango seed

kernel (MSK) starches.
Source of
starch
Cultivar
Amylose content (%)
Rice
Chiang Phatthalung
33.08a 0.79
MSK
Namdokmai
Kaew
Chokanun
33.12a 2.03
31.63a 1.90
31.50a 2.33
The values are the mean SD of three independent observations. The values
with same superscripts in a column did not differ significantly (p0.05).
Figure 2. Water solubility index of rice and MSK

starches.
Due to its linear structure, amylose is an important

factor affecting the texture of noodle [12]. Tam et al.
[13] reported that normal corn starch which has
amylose content about 28% were successfully used for
noodle making; in contrast, waxy corn starch (0.23.8% amylose) and high amylose corn starch (> 40%
amylose) failed to produce noodles with desired
characteristics [14].
3.3 Pasting properties

Pasting properties by RVA of rice starch blended
with MSK starch at different percentages are presented
in Table 2. Rice starch slurry from Chiang Phatthalung
cultivar started to develop a viscous paste at a
significantly (p0.05) higher temperature than MSK
starch slurry. Moreover, the rice starch paste showed a
significantly (p0.05) lower in peak viscosity (PV)
than the MSK starch paste. These results suggested
that rice starch granules were harder to swell and swell
to a lower extent than MSK starch granules. The
differences in pasting temperature (PT) and PV of
starches could be due to the variation in lipid content
and fine structure of amylose and amylopectin as
previously discussed in swelling power (Fig.1). When
rice starch was blended with MSK starch, the PT of
blended starch proportionally decreased with the
increase in amount of added MSK starch. On the other
hand, the more MSK starch in the starch blend, the
higher the peak viscosity (PV) of the blended starch
paste was. Kaew MSK starch had the lowest effect on
the change in PT and PV of rice starch; while,
Namdokmai MSK starch gave the highest effect.
According to their significantly (p<0.05) higher in
breakdown (BD) (Table 2), the pastes of Namdokmai
3.2 Swelling power and water solubility index

MSK starches exhibited significantly (p0.05)
higher swelling power (SP) and water solubility index
(WSI) than rice starch at all heating temperature steps;
particularly, at 90C (Fig.1 and 2). All MSK starches
from three mango cultivars were not significantly
different in their SP and WSI. However, starch from
Namdokmai MSK swelled at a lower extent than
starches from Chokanun and Kaew MSK. The higher
in SP and WSI of MSK starches over rice starch
suggested that the MSK starch granule could absorb
more water and expand to a greater extent; as a result,
more starch molecules could leach out. These
differences in SP and WSI among rice and MSK
starches might be related to the roles of amylopectin,
amylose, and lipid in the swelling of starch granule
829
Table 2: Pasting properties of rice starch blended with MSK starch at different percentages
Pasting properties
MSK starch
content (%)
Pasting
Temperature (C)
Peak
viscosity (cP)
Breakdown (cP)
Final
viscosity (cP)
Setback (cP)
Namdokmai
0
35
50
75
100
87.89d 0.43
82.82c 0.40
81.68b 0.03
81.13b 0.49
80.32a 0.46
3,930.25a 28.72
4,166.67b 30.66
4,586.33c 21.08
5,125.67d 60.04
5,087.67d 55.32
764.75a 63.66
1,030.00b 42.57
1,483.00c 56.40
1,830.00d 24.27
1,519.00c 27.51
Kaew
0
35
50
75
100
87.89d 0.43
88.10d 0.05
86.00c 0.44
84.95b 0.09
83.50a 0.40
3,930.25a 28.72
4,171.00b 111.95
4,191.33b 248.13
4,445.33c 37.44
4,274.25bc11.44
764.75a 63.66
1,013.67b 55.97
1,241.00c 132.01
1,370.33d 13.32
865.50a 14.57
4,728.00b 85.37 1,562.50b 137.18

4,945.67c 78.01 1,788.67c 12.22
4,804.67b 115.21 1,854.33c 36.94
5,156.33d 11.50 2,081.33d 18.04
4,568.00a 47.87 1,159.25a 48.46
Chokanun
0
35
50
75
100
87.89d 0.43
84.88c 0.03
84.03b 0.04
82.52a 0.03
81.92a 0.51
3,930.25a 28.72
4,084.67b 7.77
4,347.00c 45.25
4,662.33e 9.07
4,514.00d 32.19
764.75a 63.66
968.00b 52.37
1,340.00c 28.28
1,623.67d 24.58
1,349.00c 18.52
4,728.00bc 85.37 1,562.50b 137.18

4,805.00c 26.89 1,688.33b 48.99
4,925.50d 94.50 1,918.50c 65.76
4,660.00b 30.44 1,621.33b 52.32
4,348.33a 37.58 1,183.33a 29.40
4,728.00b 85.37
4,982.00c 24.64
5,052.33c 67.83
4,946.67c 126.47
4,478.33a 61.50
1,562.50b 137.18
1,845.33c 41.93
969.00c 62.86
651.00b 80.18
909.67a 1.53
The values are the mean SD of three independent observations. The values with same superscripts in a column did not differ significantly (p0.05).
and Chokanun MSK starches were more susceptible to

shear thinning than those of rice and Kaew MSK
starches. Generally, starch paste displays thixotropic
rheology behavior [16]. The resistance to shear force of
starch paste depends on both the amount and fine
structure of amylose and amylopectin [17]. A long
linear molecule as of amylose and highly long
amylopectin chain contribute to shear resistance of
starch paste. Although all three MSK starches were not
significantly (p0.05) different in their amylose
content (Table 1), the higher resistance to shear force
of Kaew MSK starch paste over the other two MSK
starch pastes might be due to the different in their
amylose and amylopectin fine structure. The paste of
rice-MSK starch blend was significantly (p0.05)
(Table 2) higher in BD than that of unblended starch;
especially, when rice and Kaew MSK starches were
blended. This synergistic effect might be because rice
and MSK starch molecules interfered among each
other, resulting in a weak molecular association.
Rice starch paste displayed a significant (p0.05)
higher in final viscosity (FV) and setback (SB) than
MSK starch paste (Table 2). This can be implied that;
after cooling, gelatinized rice starch molecules could
form a stronger molecular network which trapped
more water molecule inside than gelatinized MSK
starch molecules. Generally due to its linear structure,
amylose tends to associate with each other better than
highly branch-structure amylopectin [18]. Therefore,
the paste of high amylose starch should have a high
SB. Although rice and MSK starches contain no
significant in their amylose content (Table 1), but the
significant difference in their SB could be due to the
molecular weight of their amylose [18]. Similarly to
the synergistic effect on BD, the SB of blended starch

paste were significantly (p0.05) higher than those of
unblended starch pastes. After cooling, amylose or
amylopectin from MSK starch might have an optimum
molecular weight which increase the number of
junction points for rice amylose reassociation,
resulting in a higher paste viscosity. Lin et al. [19]
suggested that in contrast to a single starch system,
starch blends generally exhibit unique viscoelastic or
physical properties that cannot be directly interpreted
by the final apparent amylose content or mixing ratio.
This may be because of the uncertainties involved in
intermolecular association.
3.4 Gel texture properties
Gel of MSK starches showed significantly (p0.05)
higher in hardness, springiness, gumminess and
chewiness than that of Chiang Pathalung rice starch;
while their gel cohesiveness were not significantly
different. Excluding cohesiveness, the gel texture
properties of rice-MSK starch blend proportionally
increased with the amount of MSK starch. The gel
hardness is mainly depended on the degree of starch
retrogradation [20]. Starch with high in amylose and
long-chain amylopectin tends to retrograde easily,
resulting in a hard gel [21]. The contradiction between
the lower in SB but higher in gel hardness of MSK
starch than rice starch suggested that, at a prolonging
storage, MSK starch molecules were more susceptible
to retrograde than rice starch molecules. This might be
due to a variation in their amylopectin fine structure.
Moreover, there were water droplets on the surface of
stored MSK starch gel. This syneresis confirmed that a
high degree of starch retrogradation occurred in the
830
Table 3: Gel texture properties of rice starch blended with MSK starch at different percentages
MSK starch
content (%)
Hardness (g )
Cohesiveness
Texture properties
Springiness
Namdokmai
0
35
50
75
100
49.87a 1.76
53.69a 1.45
64.11b 8.51
75.49c 2.43
118.38d 7.66
0.45ab 0.05
0.50b 0.03
0.42a 0.06
0.47ab 0.04
0.44ab 0.04
0.88a 0.05
0.90a 0.01
0.99ab 0.09
0.94a 0.05
1.14b 0.18
22.21a 2.22
27.03a 2.03
26.93a 5.02
35.39b 4.18
54.41c 3.13
20.13a 1.82
24.43ab 1.74
26.49b 3.89
33.47c 4.62
56.12d 4.79
Kaew
0
35
50
75
100
49.87a 1.76
41.38a 2.52
46.09a 4.35
66.85b 7.50
110.46c 8.97
0.45a 0.05
0.52 0.02
0.49ab 0.02
0.44a 0.04
0.44a 0.06
0.88a 0.05
0.92ab 0.08
0.94ab 0.04
0.98ab 0.03
1.05b 0.14
22.21a 2.22
21.66a 1.53
22.63a 2.20
29.38b 3.39
51.67c 2.31
20.13a 1.82
19.87a 2.61
21.25a 1.80
28.71b 2.19
50.13c 4.43
Chokanun
0
35
50
75
100
49.87a 1.76
52.95a 3.48
60.60a 4.82
85.57b 9.37
115.01c 9.39
0.45ab 0.05
0.52b 0.50
0.46ab 0.05
0.43ab 0.04
0.36a 0.18
0.88a 0.05
0.93a 0.50
0.95a 0.05
0.97a 0.04
1.17b 0.18
22.21a 2.22
27.21b 0.59
27.53b 0.82
36.17c 1.93
45.30d 2.76
20.13a 1.82
25.27b 1.92
26.00b 0.71
35.78c 1.01
51.67d 3.64
Gumminess
Chewiness
The values are the mean SD of three independent observations. The values with same superscripts in a column did not differ significantly (p0.05).
MSK gel. When water molecules are squeezed from

the gel network, the starch molecules will be more
closely packed. As a result, the starch gel will need
more force to press (harder) and easier to recover to
the same height (more springiness). Gel hardness is
considered to be a dominant parameter related to the
texture of noodle [22]. If the hardness of rice starch gel
is the optimum hardness for noodle, Kaew MSK starch
might be used to replace rice flour up to 50% without
significantly change in noodle hardness, springiness,
gumminess, and chewiness.
4. Conclusions
MSK starch significantly influenced the pasting
and gel texture properties of rice starch (Chiang
Phattalung cultivar). At a prolonging storage, MSK
starch molecules retrograded at a higher degree than
rice starch, resulting in a harder gel. However, Kaew
MSK starch could be blended with rice starch up to
50% without significant change in gel hardness. This
study showed the possibility to partially replace rice
flour with MSK starch in rice-based product such as
rice noodle.
Acknowledgements
This work was supported by the
scholarship of Chulalongkorn University.
research
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832
PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF

NEPHELIUM LAPPACEUM L. FRUIT RESIDUES: EFFECTS OF DRYING
TREATMENTS AND EXTRACTION CONDITION
Asleena Hayeedoloh1, Ngamsupa Charoenpong1, Ampaiwan Chainan1,
Phanuphong Chaiwut1,2, and Nont Thitilertdecha1,*
1
School of Cosmetic Science, Mae Fah Lunag University, Chiang Rai 57100, Thailand
Excellent Center of Cosmetic and Wellness, Mae Fah Luang University, Chiang Rai 57100, Thailand
*Author for correspondence; E-Mail: nont.thi@mfu.ac.th Tel. +66 53 916834, Fax. +66 53 91 6831
Abstract: Nowadays, there is an increasing interest in

utilizing bio-waste as natural sources of bioactive
compounds. Rambutan (Nephelium lappaceum L.) fruit
residues are a great source of phenolic antioxidants. This
research aims to study the phenolic content and
antioxidant activities of rambutan peel and seed affected
by sample drying treatments and extraction condition
(weight ratio of sample:solvent). The rambutan peel or
seed were freeze-dried under high vacuum or hot air
dried at 50 C and the weight ratio of sample:solvent for
extraction were 1:5, 1:10 and 1:20 (w/v). The antioxidant
activities were determined by DPPH radical scavenging
assay, ABTS radical scavenging assay and ferric
reducing antioxidant power (FRAP) assay and the
amount of phenolic compounds was estimated by FolinCiocalteu assay. The phenolic contents and antioxidant
properties of freeze dried rambutan peel and seed were
higher than hot air dried samples (P < 0.05). By varying
of the weight ratio of sample:solvent found that the ratio
1:10 (w/v) was the best weight ratio that the amount of
phenolic compounds and antioxidant activities were
potential. The rambutan peel contained higher amount of
phenolic compounds and exhibited stronger antioxidant
properties than rambutan seed (P < 0.05). This research
suggests that freeze-dried N. lappaceum L. peel could be
considered for being utilized in value-added applications
as both a medicine and food industry.
1. Introduction
Vast quantities of agricultural-food waste are
produced annually worldwide. Those disposals of
agricultural-food waste can have detrimental effect to
environment because of their high content of
biological components which make them susceptible
to modification by microorganisms causing leachate
formation and gas emission. Therefore in recent years,
there is increased interesting in the evaluation of the
bio-waste materials in possible value-added
applications. Various agricultural wastes, such as seeds
and peel of grapes, pomegranate, longan and/or litchi
have been evaluated as rich sources of natural
antioxidants [1-6].
Nephelium lappaceum L., commonly known as
rambutan and belonging to the Sapindaceae, is an
attractive tropical fruit widely grown in South-East
Asia, especially in the eastern and southern regions of
Thailand. The approximate annual harvest quantity of
rambutan in Thailand is half a million tons. Its fruit is

consumed fresh, canned or processed. Its consumption
produces main by-products (peels and seeds)
representing up to half of the fruit weight [7].
Rambutan fruit especially its peel is rich in bioactive
compounds particularly phenolic compounds, geraniin,
corilagin and ellagic acid responsible for potential
antioxidant property [8-9].
In the processing of rambutan fruit residues, drying
method might play an important procedure to lower
the microorganism growth rate as well as inactivate
enzymes which are responsible for degrading bioactive
components. Nevertheless, drying temperature and
time affect the stability of bioactive compounds and
their
biological
properties
through
enzyme
degradation, oxidation acceleration or thermal
decomposition. As far as our literature survey could
ascertain, no studies on bioactive compounds stability
and antioxidant property of N. lappaceum L. have been
previously reported. This study will focus on the effect
of drying method (freeze-drying and hot-air drying)
and then the extraction condition (the weight ratio of
sample:solvent) on the amount of phenolic compounds
and antioxidant activities through various assays;
DPPH radical scavenging activity, ABTS radical
scavenging activity and ferric reducing antioxidant
power (FRAP).
2.1 Chemicals
2,2-diphenyl-1-picryl-hydrazyl radical (DPPH),
gallic acid, 2,2-azino-bis(3-thylbenzothiazoline-6sulfonic acid) (ABTS), 2,4,6-tri(2-pyridyl)-s-triazine
(TPTZ) and trolox were purchased from SigmaAldrich (Steinheim, Germany). All other chemicals of
analytical grade were purchased from Merck
(Darmstadt, Germany).
2.2 Plant materials
The fresh fruits of rambutan (Rongrien cultivar)
were purchased from local market at Chiang Rai,
Thailand. The fruit samples were separated into peel
833
Table 1: The amount of phenolic compounds and antioxidant activities of Nephelium lappaceum L. fruit peel and
seeds dried with different methods: freeze drying (FD) and hot air drying (HD)
Samples
N. lappaceum peel
FD
HD
N. lappaceum seeds
FD
HD
Total phenolic contents

(mg gallic acid
equivalent/g sample)
Antioxidant activity (mg trolox equilvalent/g sample)

DPPH
ABTS
FRAP
92.37 1.615a
48.32 1.560b
926.9 111.4a
207.4 5.078b
425.6 15.78a
255.7 11.33b
68.51 3.112a
36.42 1.235b
2.401 0.094c
2.692 0.064c
0.192 0.002c
0.697 0.056c
1.662 0.020c
1.819 0.020c
0.258 0.033c
0.707 0.106c
2.3 Sample extraction

The dried samples (10 g) were extracted (3) by
using 95% ethanol (100 mL) as extracting solvent. The
extraction process was done by shaking for 24 h. The
extracts were filtered, evaporated under reduced
pressure and stored until further analysis. Each
extraction process was performed in triplicate.
2.4 Determination of the total phenolic contents
The amounts of phenolic compounds in the
samples were determined according to the method of
Waterman and Mole (1994) [10] using Folin-Ciocalteu
method and gallic acid was used as reference phenolic
compound. Briefly, sample solution (0.1 mL) was
added to 7.9 mL of distilled water. After that, 0.5 mL
of Folin-Ciocalteu reagent was added and the resulting
mixture was then added by 200g/L sodium carbonate
(1.5 mL) in three min later. The absorbance at 760 nm
of the mixture was then measured after incubated at
room temperature for 1 h. The total phenolic contents
were expressed as gallic acid equivalents.
2.5 Determination of antioxidative activity
2.5.1 DPPH scavenging activity
The DPPH scavenging activity was determined by
the method of Gln et al. (2003) [11] with slight
modification. The trolox was used as reference
antioxidant. Briefly, a 0.1 mmol/L DPPH solution in
ethanol (3 mL) was mixed with 1 mL of sample
solution. The absorbance of the mixture was measured
at 517 nm after the mixture was allowed to stand for
30 min at room temperature in dark place. The DPPH
scavenging activity was expressed as trolox
equivalents.
2.5.2 ABTS+ radical scavenging activity
The ABTS+ scavenging activity was performed as
reported previously by Re et al. (1999) [12]. Briefly,
stock solution of ABTS cation chromophore was
prepared by the reaction between 7 mM ABTS
solution in distilled water and 2.45 mM potassium
persulfate in the dark for 16 h. The mixture was
diluted with phosphate buffer (50 mM, pH 7.4) to an
absorbance of 0.70 0.02 at 734 nm. An aliquot of
samples (200 L) was added to 3 mL of ABTS reagent
and the mixture was incubated for 30 min at room
temperature prior to being measured the absorbance at

734 nm. The ABTS+ scavenging activity was
expressed as trolox equivalents.
2.5.3 FRAP assay
The procedure of FRAP assay was performed
according to the method of Thaipong et al. (2006) [13]
with some modifications. Briefly, the FRAP reagent
contained 100 mL of 10 mM TPTZ (2,4,6-tripyridy-striazine) solution in 40 mM hydrochloric acid and 100
mL of 20 mM ferric chloride was prepare freshly and
warmed at 37C. An aliquot of samples (100 L) was
allowed to react with FRAP reagent (1,500 L) and
1,400 L of acetate buffer (300 mM, pH 3.6). The
mixture was then measured at 593 nm after incubated
at room temperature for 30 min. The FRAP value was
expressed as trolox equivalents.
All experimental data were expressed as means
S.D. Analysis of variance was performed by ANOVA
procedures. Correlation coefficient (R) was used to
determine two variables. SPSS software was used for
statistical analysis.
The phenolic contents and antioxidant activities
based on DPPH ABTS and FRAP assays of the
extracts were evaluated and presented in Table 1. The
N. lappaceum peel extracts not only possessed higher
level of phenolic compounds but also exhibited greater
antioxidant properties than N. lappaceum seeds (P <
0.05). Although all determined parameters of N.
lappaceum seeds dried with two different methods
were not statistically different (P > 0.05), it was
obvious that freeze drying procedure was appropriate
for N. lappceum peel drying treatment as its content of
phenolic and antioxidant activities. Higher degradation
of phenolic compounds was observed at higher
temperatures due to chemical, enzymatic, thermal, and
oxidative decomposition [14]. Besides freeze drying
method could lower the incidence of bioactive
component degradation at low temperature, it may
release phenolics bound to cellular structure to make
them more accessible for extraction [15].
834
Table 2: The amounts of phenolic compounds and antioxidant activities of freeze-dried Nephelium lappaceum L.
fruit peel and seeds extracted by conventional method at various ratios of sample:solvent
Ratio of
sample:solvent
N. lappaceum peel
1:5
1:10
1:20
N. lappaceum seeds
1:5
1:10
1:20
Total phenolic contents

(mg gallic acid
equivalent/g sample)
Antioxidant activity (mg trolox equilvalent/g sample)

DPPH
ABTS
FRAP
60.85 1.348b
92.37 1.615a
93.46 0.889a
354.0 10.36c
926.9 111.4a
770.8 34.64b
332.5 7.006c
425.6 15.78b
449.6 4.058a
29.14 1.702c
68.51 3.112a
59.18 1.859b
1.845 0.057c
2.401 0.094c
2.313 0.035c
0.186 0.004d
0.193 0.002d
0.154 0.001d
2.725 0.037d
1.662 0.020d
1.671 0.012d
0.197 0.012d
0.258 0.033d
0.178 0.001d
Compared to other fruits, ethanolic extract of N.

lappaceum peel has been reported that exhibited
greater antioxidant property (3.07 mM TEAC/mg
extract) compared with Cocos nucifera peel (1.53),
Garcinia mangostana peel (3.00), Hylocereus undatus
peel (0.685), Lansium domesticum peel (0.507), Musa
sapientum peel (1.80) and Passiflora foetida peel
(0.591) [16].
The freeze dried N. lappaceum peel and seeds were
then selected for being optimized the ratio of
sample:solvent (1:5, 1:10 and 1:20) for extraction.
Table 2 exhibited the contents of phenolic compounds
and antioxidant activities of freezed-dried N.
lappaceum peel and seeds after extraction by using
various weight ratios of sample:solvent. The ratio of
1:10 and 1:20 provided better amount of phenolic
contents and antioxidant activities than the ratio of 1:5
for N. lappaceum peel (P < 0.05) while the ratio of
sample:solvent did not affect to for phenolic
antioxidant extraction in N. lappaceum seeds (P >
0.05). Higher antioxidant properties based on DPPH
and FRAP assays (P < 0.05) were observed in the
rambutan peel extract with the ratio of 1:10 except for
ABTS scavenging activity which was lowered slightly.
However, the phenolic contents were not different
between the ratio of 1:10 and 1:20 (P > 0.05).
Generally, increasing in solvent ratio could provide
higher content of phenolic compounds and also
antioxidant properties. From the result, the phenolic
content of rambutan peel extract with the ratio of 1:20
might be the highest content as it was not different
compared with the ratio of 1:10. The evaporation
process during extract preparation might affect
bioactive component through degradation or
hydrolysis because of the major phenolic constituents
in rambutan peel is geraniin which could be
hydrolyzed to give corilagin, ellagic acid and gallic
acid [17,18]. These hydrolyzed products were reported
that exhibited lower DPPH radical scavenging
property. There were many studies reported that
antioxidiant properties of phenolic compounds not
only depended on their structures [19] but also also the
method for antioxidant determinations [20]. However,
lack on literature data relating to antioxidant activities

of geraniin based on ABTS and FRAP assays.
From these results, the ratio of 1:10 was more
preferable due to low solvent consumption
accompanying high content of phenolics and
antioxidant activities.
According to the results, the antioxidant properties
increased proportionally with the content of phenolics.
Correlations between the amount of phenolics and
antioxidant activities were analyzed. It was found that
the amount of phenolic compounds was potentially
correlated versus DPPH radical scavenging activity (R2
= 0.970), ABTS radical scavenging activity (R2 =
0.995), and ferric reducing antioxidant power (R2 =
0.982). Therefore, it can be noted that the phenolic
compounds contained in N. lappaceum L. fruit
residues are responsible for antioxidant properties.
This was in agreement with previous published studies
that have stated that phenolic compounds were
responsible for antioxidant properties [7-9].
4. Conclusions
The drying process of N. lappaceum L. fruit
residues suitable to maintain phenolic compounds and
antioxidant activities was freeze drying method. The
ratio 1:10 (w/v) was the most appropriate weight ratio
of sample:solvent for conventional extraction to obtain
potential phenolic antioxidants from N. lappaceum
peel. In summary, N. lappaceum peel was suitable for
being an alternative source of natural antioxidants to
be utilized in both food and medicine industry. We are
continuing to improve extraction efficiency of
phenolic antioxidants from this plant material by
comparison of various extraction techniques.
Acknowledgements
This research was financially supported by Mae
Fah Luang University through research grant, 2012
from the Division of Research Services, and from
School of Cosmetic Science, Mae Fah Luang
University.
835
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836
PHYSICOCHEMICAL PROPERTIES OF FLOUR

FROM DIFFERENT NATIVE RICE CULTIVARS IN THAILAND
Jitranut Leewatchararongjaroen and Jirarat Tattiyakul*
Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*
Correspondence Author; E-Mail: Jirarat.t@chula.ac.th, Tel. +66 2 2185515, Fax. +66 2 2544314
Abstract: Rice flours from nine different native cultivars

in Thailand (Ayutthaya 1, Khao Dawk Mali 105, Plai
Ngahm Prachin Buri, Prachin Buri 1, Prachin Buri 2, RD
41, RD 45, RD 47 and Shaw Lung 97) prepared by dry
and wet milling processes were studied for their
physicochemical properties. The flours contained 8.21%12.02% (wb) moisture content, 0.20%-1.01% (db) fat,
5.30%-9.16% (db) protein, 0.19%-0.92% (db) ash and
78.57%-86.36% (db) carbohydrate. The starch consisted
of 14.65%-41.05% apparent amylose. The result from
Differential Scanning Calorimetry (DSC) revealed that
the gelatinization enthalpy of dry-milled flours were in
the range of 2.47 to 5.87 J/g and they were significantly
(p<0.05) lower than those of wet-milled flours which
varied in the range of 7.61 to 12.12 J/g. Dry-milled flours
showed significantly (p<0.05) lower peak viscosity and
final viscosity comparing to those of wet-milled flours.
Starch granules of both dry-milled and wet-milled flours
still showed birefringence. The values of crystallinity
degree of wet-milled flours that varied in the 20.10 to
24.23% range were significantly (p<0.05) higher than
those of dry-milled rice flours that varied in the 13.01 to
14.71% range.
1. Introduction
Rice is a staple of over approximately one-half of
the world population. Nowadays, Thai rice has been
exported to more than 160 countries, which is about 7
million tons per year. Rice production in Thailand has
a spectacular increase in the recent years and is
approximately 30% of the total rice production of the
world.
According to the research by Juliano (1993) [1],
the chemical composition of rice consists of protein
(6.3 to 7.1 g), fat (0.3 to 0.5 g), fibers (0.2 to 0.5 g),
ash (0.3 to 0.8 g) and carbohydrates (77 to 89 g). Since
carbohydrates are the major component in rice, rice
grains are usually processed into flour.
Physicochemical properties need to be determined
before flour use as they will affect the way to control
qualities, consistency and also lead to the good
consumer satisfactory. Some studies have investigated
the properties of rice flour and have found many
factors which influence the physicochemical properties
of rice flour, such as rice cultivars [2], amylose content
[3], protein [4] and milling methods [5].
The objective of this research was to investigate
the physicochemical properties of flour from different
native rice cultivas in Thailand. Additional database
will lead to the appropriate and more diverse industrial
applications and processes.

2.1 Materials
Nine native rice cultivars in Thailand were used in
this study. They were Ayutthaya 1, Plai Ngahm
Prachin Buri, Prachin Buri 1, Prachin Buri 2, RD 45
(from Prachin Buri Rice Research Center, harvested
during March, 2011) Khao Dawk Mali 105 (from
Pathum Thani Rice Research Center, harvested during
December, 2010) Shaw Lung 97 (from Pattani Rice
Research Center, harvested during March, 2011) RD
41 and RD 47 (from Phitsanulok Rice Research
Center, harvested during November, 2010). All of the
samples were kept at 4oC before analyses.
2.2 Preparation of rice flour
Dry milling process
The polished rice kernels were ground by using a
vertical disc mill. Flour samples were passed through a
100-mesh sieve, sealed in plastic bags and stored in a
desiccator at room temperature until further analyses.
Wet milling process
Rice (1 kg) was soaked overnight in NaHSO3
solution (1.25% w/v) at rice:solution of 1:2 and ground
with a hammer mill under continuous addition of
water to obtain a rice slurry. The slurry was filtered
through a filter bag to obtain a rice cake. The cake was
dried overnight in a tray dryer at 40C. The dried rice
flour was ground and sieved through a 100 mesh sifter
to obtain rice flour. Flour samples were packed in
plastic bags and stored in desiccator at room
temperature for further used.
2.3 Chemical Composition
Moisture (AOAC 32.1.03), fat (AOAC 32.1.13),
protein (AOAC 32.1.22), ash (AOAC 32.1.05) and
carbohydrate of the rice flour samples were
determined using the AOAC Official Methods
(AOAC, 2005) [6]. The conversion factor (N5.95)
was applied to convert nitrogen content to the crude
protein content.
Amylose content was determined by using an
iodine binding method. One millilitre of ethanol (95%)
and 9 ml of 1 N NaOH were added to 100 mg of flour.
After mixing, the samples were heated for 10 min in a
boiling water bath to gelatinize the starch. Samples
were cooled down and transferred to a 100 ml
volumetric flask, and 5 ml of flour solution and 1 ml
of 1 N acetic acid were added. After addition of 2 ml
iodine solution the volume was adjusted to 100 ml
837
with distilled water, mixed and let stand for 20 min.

The absorbance was measured at 620 nm using a UV
spectrophotometer. The amylose content was
determined from a previous standard curve of potato
amylose.
2.4 Thermal Properties
Thermal Properties of rice flours were examined
by a Differential Scanning Calorimeter (Perkin-Elmer,
model Diamond DSC, USA). Flour samples were
dispersed in water to give a flour:water ratio of 3:7.
The suspension was then transferred to an aluminum
pan and hermetically sealed. After equilibration at
room temperature overnight, the sample was heated
from 30 to 95C at a rate of 10 C/min. An empty pan
was used as reference. The onset (To), peak (Tp) and
conclusion (Tc) temperatures of gelatinization, and the
gelatinization enthalpy (H in J/g) were recorded.
2.5 Pasting Properties
Pasting properties were measured with a Rapid
Visco Analyser, RVA model 4 (Newport Scientific,
Australia). First, 25 ml of distilled water was added
directly to a metal RVA canister. Then 3.00 g of rice
flour was weighed, added to the water, and
immediately measured by RVA. The slurry was held at
50oC for 1 min, heated to 95oC within 3 min 42 s and
then held at 95oC for 2 min 30 s. It was subsequently
cooled to 50oC within 3 min 48 s and held at 50oC for
2 min, while maintaining a rotation speed of 160 rpm.
Viscosity parameters (peak, trough, final, breakdown,
and setback viscosity) were expressed in centipoise
(cP).
2.6 Microscopy
Light microscopy and scanning electron
microscopy (SEM) were used to examine the starch
granules. Samples were observed using a polarized
light microscope (Olypus model CH30RF200, Japan).
For SEM, all samples were mounted on aluminum
stubs using double-sided tape, sputter-coated with gold
and investigated using SEM (JEOL model JSM-5800
LV, Japan) at an accelerated voltage of 20 kV.
2.7 X-ray Diffraction
X-ray diffraction analysis was performed using an
X-ray diffractometer (Bruker AXS model D8
Discover, Germany) operated at 40 kV voltage and 40
mA current, on a copper target. Rice flour were packed
tightly into the aluminum sample holder and
diffraction data were collected over an angular range
from 5 to 30 degree with a scanning speed of 0.5
sec/step. Relative crystallinity degree (%) was
estimated from the ratio of the peak area to the total
area of a diffractogram.
(DMRT) were used for comparing differences in the

mean values at the 0.05 confidence level.
3.1 Chemical Composition
The flours contained 8.21%-12.02% (wb) moisture
content, 0.20%-1.01% (db) fat, 5.30%-9.16% (db)
protein, 0.19%-0.92% (db) ash and 78.57%-86.36%
(db) carbohydrate. The starch consisted of 14.65%41.05% apparent amylose. Due to the preparation of
wet-milled flour, rice kernels had to be soaked and
ground with water. Soluble protein, such as albumin
was removed, resulting in lower amounts of protein
components in wet-milled rice flours [5]. The rice
flour samples could be classified into two groups: RD
45 and Khao Dawk Mali 105 as low amylose content
(14.65-21.00%), RD 41 RD 47 Shaw Lung 97
Prachin Buri 1 Prachin Buri 2 Plai Ngahm Prachin
Buri and Ayutthaya 1 as high amylose content (31.1441.05%).
3.2 Thermal Properties
The thermal parameters from endotherms are
shown in Table 1. The To of the rice flour samples
varied from 61.68C for dry-milled rice flour RD 47 to
75.44C for dry-milled rice flour Shaw Lung 97,
whereas the Tp range was 68.32C for wet-milled rice
flour RD 47 to 80.46C for wet-milled rice flour Shaw
Lung 97 and the Tc range was 74.01C for dry-milled
rice flour RD 47 to 85.63C for dry-milled rice flour
Shaw Lung 97. The gelatinization enthalpy of drymilled flours were in the range of 2.47 to 5.87 J/g and
they were significantly (p<0.05) lower than those of
wet-milled flours which varied in the range of 7.61 to
12.12 J/g. The crystalline structure of dry-milled rice
flours were disrupted due to the grinding process. The
mechanical force caused starch damage during milling,
with a lower gelatinization enthalpy in dry-milled rice
flour [7].
Gelatinization temperature and H are indicators
of the overall crystallinity of amylopectin, which is
directly related to the structure of amylopectin.
Yoenyongbuddhagal et al 2002[8] demonstrated that
starch with a greater amount of long-chain
amylopectin and smaller amount of short-chain
amylopectin exhibited higher To, Tp and H than
starch with fewer long branched chains. This is
because the longer chains have a greater ability to
form double helices, which required greater thermal
energy to dissociate.

Completely Randomized Design (CRD) was used
as the experimental design. SPSS for Windows
program, version 17.0, was employed for analyzing the
results obtained from three replications. Analysis of
variance (ANOVA) and Duncans multiple range test
838
Table 1: Thermal properties of flours

Cultivars
To
Tp
Tc
RD41(D)
63.23d
71.26e
77.07e
5.22d
RD41(W)
64.60f
70.51d
75.83c
9.64h
RD45(D)
64.38f
71.69f
77.91f
5.28d
RD45(W)
65.13g
70.50d
75.49c
9.93hi
RD47(D)
61.68a
69.94c
75.80c
5.32d
RD47(W)
62.11b
68.32a
74.01a
8.59g
SL97(D)
75.44n
80.46m
85.63j
4.18c
SL97(W)
75.39n
79.48l
83.71i
11.42j
PB1(D)
62.66c
70.14c
76.65d
5.87e
PB1(W)
63.79e
69.20b
74.82b
10.26i
PB2(D)
73.03j
78.94k
83.56i
3.28b
PB2(W)
73.99kl
78.08j
82.38h
12.12k
PNG(D)
74.25
lm
78.89
83.53
5.05
PNG(W)
73.18
77.53
82.13
11.20j
K105(D)
67.22i
74.14h
79.90g
3.94c
K105(W)
66.32h
72.52g
77.76f
11.45j
AY1(D)
74.54m
79.31l
83.53i
2.47a
Pasting properties of starch have been reported to be

affected by amylose and lipids contents and by branch
chain-length distribution of amylopectin. Amylopectin
contributes to swelling of starch granules and pasting,
while amylose and lipids inhibit the swelling. Also, the
amylopectin chain-length and amylose molecular size
produce synergistic effects on the viscosity of starch
pastes [9].
Moreover, the milling process influences pasting
viscosity properties by the mechanical damage of
starch granules. The result showed that dry-milled rice
flours provided significantly (p<0.05) lower peak
viscosity (1010 to 2624 cP) than those of wet-milled
rice flours (2062 to 3818 cP) (Figure 1). The lower
peak viscosity was caused by a higher starch damage
of dry-milled rice flours, similar to the result reported
by Yoenyongbuddhagal and Noomhorm (2002) [10].
Final viscosity was significantly (p<0.05) lower for
dry milled rice flours (1650 to 4680 cP) than those of
wet-milled rice flours (2532 to 4960 cP).
RD41(W)
AY1(W)
73.70k
78.00j
82.03h 7.61f
Means in the same column follow by the same
lowercase superscript letter are not significantly
different at P < 0.05
(D) = flour from dry milling method,
(W) = flour from wet milling method
SL97 = Shaw Lung 97, PB1 = Prachin Buri 1,
PB2 = Prachin Buri 2,
PNG = Plai Ngahm Prachin Buri,
K105 = Khao Dawk Mali 105, AY1 = Ayutthaya 1
To = Onset temperature (oC),
Tp = Peak temperature (oC),
Tc = Conclusion temperature (oC), H = enthalpy (J/g)
3.3 Pasting Properties
Pasting properties differed significantly among
different rice flours. Pasting temperature of different
rice flours ranged from 76.22 to 91.85 oC. The increase
in viscosity with temperature may be attributed to the
removal of water from the exuded amylose by the
granules as they swell. Trough viscosity among
different rice flours ranged from 684 to 2892 cP. Final
viscosity increases upon cooling which might be due
to the aggregation of the amylose molecules. Final
viscosity and setback varied from 16504960 to 836
2687 cP, respectively. Setback value is the recovery of
the viscosity during cooling of the heated starch
suspension. High setback may be due to the amount
and the molecular weight of the amylose leached from
the granules and the remnants of the gelatinized starch.
RD41(D)
Figure 1. Selected RVA pasting profiles of flours from

RD41.
(D) = flour from dry milling method,
(W) = flour from wet milling method
3.4 Microscopy
Polarized light micrographs of flours are shown in
Figure 2. Wet-milled and dry-milled rice starch
granules from nine different native cultivars showed
similar birefringence. The refraction of polarized light
by the intact crystalline regions in starch gives
characteristic "Maltese cross" patterns on each
granule.
The scanning electron micrographs in Figure
3show examples of dry-milled and wet-milled rice
flours. The result indicated that wet-milled and drymilled rice starch granules are polygonal but irregular
in shape. Dry milled rice flour contained significantly
larger flour particles in the form of these aggregated
starch granules compared to the separated starch
granules of wet-milled samples. Suksomboon and
Naivikul (2006) [5] stated that during the soaking
process of wet-milling rice flour, protein matrix and
other substances were leached out from the surface of
starch granules, causing the structure of starchy
endosperm to become loosen which resulted in the fine
particles and less damaged starch.
839
viscosity than those of wet-milled flours. The XRD

patterns of rice flours were A type, the values of
crystallinity degree of wet-milled flours were
significantly (p<0.05) higher than those of dry-milled
rice flours.
Acknowledgements
Figure 2. Polarized light micrographs of RD 45 wetmilled rice flours.

a
This research was partly supported by H.M. King

Bhumibol Adulyadejs 72nd Birthday Anniversary
Scholarship, Graduate School, Chulalongkorn
University and the Ratchadapisek Somphot En
dowment Fund # R-028-2553 for Development of
Rice Products for the Agriculture Coorperation under
the Chaipattana Foundation Patronage and Faculty of
Science Chulalongkorn University.
References
Figure 3: SEMs of starch granules from a) RD 47 drymilled rice flours and b) RD 47 wet-milled rice flours.
3.5 X-ray Diffraction
X-ray diffractions of rice flours showed a typical
A-pattern and similarly strong reflections at 2 = 15,
17, 18, and 23, which were the same as that of most
ordinary rice flours [2]. The values of crystallinity
degree of wet-milled and dry-milled rice flours are
varied in the 13.01 to 24.23% range. These differences
in crystallinity degree of rice flours maybe attributed
to rice cultivars, starch structure, the distribution of
amylose and amylopectin in starch granule, and other
components such as protein and lipid. These results
were similar to the earlier report of Singh et al. (2007)
[11] who reported that the difference in crystallinity in
different rice starches may be attributed to difference
in proportions of amylose to short and long side-chain
amylopectin. Moreover, other components might
influence the granule structure and crystallinity of
grains [12].
In addition, the effect of milling also affects the
value of crystallinity degree. The result showed that
crystallinity degree of wet-milled rice flours were
significantly (p<0.05) higher than those of dry-milled
rice flours. This result is corresponding to the amylose
contents since amylopectin is the predominant
crystalline component in granules.
[1] B.O. Juliano, FAO Food and Nutrition, Vol.26, Food

and Agriculture Organization of the United Nation
(FAO), Rome (1993).
[2] L. Iturriaga, Food Research International 37 (2004)
439447.
[3] S. Varavinit, Starch/Strke 55 (2003) 410415.
[4] C. Marco, Journal of Food Engineering 84 (2008)
132139.
[5] A. Suksomboon, Kasetsart Journal 40 (2006)
125 134.
[6] AOAC, Official Methods of Analysis, 17thed,
Association of Official Analysis Chemists. Washington,
D.C. (2005).
[7] J. J. Chen, Cereal Chem. 76 (1999) 796799.
[8] S. Yoenyongbuddhagal, Cereal Chemistry 79 (2002)
481485.
[9] N. Singh, Food Hydrocolloids 20 (2006) 532542.
[10] S. Yoenyongbuddhagal, Starch/Strke 54 (2002)
534539.
[11] N. Singh, Starch/Strke 59 (2007) 1020.
[12] A.A. Ibez, Journal of Agriculture and Food
Chemistry 55 (2007) 67616771.
4. Conclusions
In this study, we found that rice variety and milling
process profoundly affected the physicochemical
characteristics of rice flours. Dry milled rice flours
contained greater amounts of protein than those of wet
milled rice flours. The flour samples prepared from
these rice cultivars exhibited differences in
gelatinization and pasting properties. Moreover, drymilled flours showed significantly (p<0.05) lower
gelatinization enthalpy, peak viscosity and final
840
PRELIMINARY STUDY ON SEPARATION OF CATECHINS FROM

GREEN TEA USING A MINI-PILOT SCALE COLUMN PACKED WITH
AMBERLITE XAD-7HP RESIN
Chalida Attarun, Piyuda Seangariyanan, and Theerapong Theppakorn*
Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand
* Author for correspondence; E-Mail: theerapong@mfu.ac.th, Tel. +66 53 916750, Fax. +66 53 916738
Abstract: A preliminary study on separation of catechins

from green teas using a created separation unit consisting
of a 1-L column packed with Amberlite XAD-7HP was
investigated. Green tea was produced from young fresh
shoots and determined for total polyphenols content
(TPC), caffeine (CF) and total catechin content (TCC),
including (-)-epicatechin (EC), (+)-catechin (C), (-)epicatechin gallate (ECG), (-)-epigallocatechin (EGC), ()-epigallocatechin gallate (EGCG), (+)-gallocatechin
(GC), (+)-gallocatechin gallate (GCG) and (+)-catechin
gallate (CG). A green tea had 15.60.04% of TPC and
2.610.48% of CF (dry weight basis). It composed of 8
catechins including GC, EGC, C, EC, EGCG, GCG,
ECG and CG at the concentrations of 0.88, 2.98, 0.47,
0.61, 3.65, 0.43, 0.57, and 0.07%, respectively. These
account for 9.49% of TCC. The 1-L column was created
and filled with an Amberlite XAD-7HP resin. The
column was holed in a supporting unit which was
designed and built up in order to set as a separation unit.
The extract green tea solution was loaded into a column
at a flow rate of 74 ml/min. A step gradient elution of
water-ethanol was then sequentially loaded into the
column. Each fraction was collected and assayed for CF
and catechin contents. The majority of tea catechins were
efficiently eluted by the eluent ranged from 30 to 60%
ethanol with 58.77% recovery yield. This technique
allows fractionation of tea catechin extracts by the minipilot plant scale which is efficient, relatively inexpensive,
and easy to scale up into industrialized usage.
1. Introduction
The tea plant is an evergreen of the Camellia family.
It has two main varieties, Camellia sinensis and
Camellia assamica. Based on tea processing, tea can
be classified into 3 main types, fermented tea (black
tea), semi-fermented tea (oolong tea) and nonfermented tea (green tea). Nowadays, green tea is a
popular drink and it has been interested by many
researches for its benefit. It is well known that green
tea contains phenolic compounds having healthpromoting properties. Numerous studies on green tea
have demonstrated the antimutagenic [1], anti-obesity
[2], anti-cardiovascular diseases [3] and antioxidant
properties [4] of the phenolic compounds which form
the major portion of the soluble tea constituents.
Among these, the catechins are the major fraction in
unfermented tea. The catechins include (-)-epicatechin
(EC), (+)-catechin (C), (-)-epicatechin gallate (ECG),
(-)-epigallocatechin (EGC), (-)-epigallocatechin gallate
(EGCG), (+)-gallocatechin (GC), (+)-gallocatechin

gallate (GCG) and (+)-catechin gallate (CG). These
catehcins make up to 10% of the dry weight basis [5].
Green tea also contains methylxanthines: caffeine and
two minor isomeric dimethylxanthines, theobromine
and theophylline, which are responsible for mildly
stimulant effects of the tea [6]. Due to the benefits of
compounds in green tea, catechins and caffeine are
isolated from green teas. Current procedures for the
isolation tea catechins mainly include liquid-liquid
extraction [7, 8], high speed counter current
chromatography (HSCCC) [9] and adsorption
separation [10]. These methods have recently reviewed
[11, 12]. Among these methods, resin adsorption has
proven to be one of the most efficient techniques for
the selective enrichment and recovery of polyphenolic
plants. Resin adsorption might also be a useful tool to
fractionate the crude extracts of green tea. As found
previously that the resin Amberlite XAD-7 provided
the highest adsorption capacity to catechins [10]. It
was used to separate the catechins from green teas in a
created 1-L column in separation unit.
The aim of this research was to preliminary study
on the fraction of catechins from green teas (Camellia
sinensis L.) using a created separation unit composed
of a mini-pilot scale column packed with Amberlite
XAD-7HP resin.
2.1 Chemicals
FolinCiocalteus phenol reagent and gallic acid
was purchased from Fluka (Buchs, Switzerland).
Anhydrous sodium carbonate was purchased from
Merck (Darmstadt, Germany). The standard catechins
which
include
(-)-gallocatechin
(GC),
(-)epigallocatechin (EGC), (+)-catechin (C), (-)epicatechin (EC), (-)-epigallocatechin gallate (EGCG),
caffeine (CF), (-)-gallocatechin gallate (GCG), (-)epicatechin gallate (ECG) and (-)-catechin gallate
(CG), were purchased from Sigma-Aldrich (St. Louis,
Missouri, USA). Acetonitrile, trifluoroacetic acid
(TFA) and methanol (HPLC-grade) were purchased
from Fluka (Buchs, Switzerland). Critic acid, ascorbic
acid and ethanol (99.8%) were purchased from Unilab
(Auckland, New Zealand). Amberlite XAD-7HP
polymeric adsorbent was purchased from Rohm and
Haas (Philadelphia, PA, USA).
841
2.2 Green tea preparation

Fresh tea leaves (Camellia sinensis var. sinensis)
were collected from the tea garden of Mae Fah Luang
Univerisity, Chiang Rai, Thailand. Leaves were out
door withered and turned 2-3 times. Leaves were fired
using pan-firing machine for 5 min at 250-300C, and
then rolled by rolling machine for 5 min. The rolled
tea was finally dried using a hot-air oven at 70C for
10 hours. The green tea was milled using a miller to
produce a green tea powder.
2.3 Column packing and building a separation unit
Amberlite XAD-7HP resin (625 g) was dissolved
in 12 L of 10%v/v ethanol. The mixture was slowly
filled in a 1-L plastic column (polypropylene, 35 cm
long and 6.2 cm diameter). A separation unit
composed of a packed column and 7 elution tanks was
set up.
2.4 Separation of catechins
Green tea powder (10 g) was extracted with 1L
reverse osmosis water pH 3.0 (adjust with citric acid)
containing 0.002% ascorbic acid. The extraction was
carried out at 70oC for 7 minutes. The solution was
then filtered through a cheesecloth and a filter paper
(Whatman no. 1). The clear solution was loaded into a
packed column at a flow rate of 74 ml/min and then
desorbed with 0, 10, 20, 30, 40, 50, 60, 70, and100%
ethanol for volumes of 5, 2, 2, 2, 2, 2, 2, 2 and 3 L,
respectively. Each fraction (250 mL) was collected and
tested the caffeine and catechin contents by HPLC.
2.5 Analytical Procedure
2.5.1 Determination of moisture content
Tea samples (5 g weighed to the nearest 0.001 g)
were placed in a moisture can and heated in an oven at
1032C for at least 16 h to a constant weight. The
percentage of moisture and dry matter (DM) in the
sample were then calculated. All tests were performed
in duplicate.
2.5.2 Determination of total polyphenols content
The total polyphenols content was determined by
spectrophotometry, using gallic acid as standard,
according to the method described by the International
Organization for Standardization [13]. Briefly, 1.0 mL
of the diluted sample
extract (50-fold dilution)
was transferred in duplicate to separate tubes
containing 5.0 mL of 10%v/v dilution of FolinCiocalteus reagent in water. Then, 4.0 mL of a
sodium carbonate solution (7.5% w/v) was added. The
tubes were then allowed to stand at room temperature
for 60 min before absorbance at 765 nm was
measured. The concentration of polyphenols in
samples was derived from a standard curve of gallic
acid ranging from 10 to 100 g/mL. The TPC was
expressed as gallic acid equivalents in g/100 g dry
basis (%GAE). All tests were performed in duplicate.
2.5.3 Determination of caffeine and 8 catechins and

caffeine content
Caffeine (CF) and 8 catechins were determined
by ISO method [14]. The individual standard solutions
of CF, GC, EGC, C, EC, EGCG, GCG, ECG, CG were
prepared by dissolving them in a small volume of
methanol, to generate a stock concentration of 999.0,
313.6, 412.0, 880.0, 911.8, 1,036, 1,000, 469, 832.0
and 514.8 g/ml respectively. The mixed stock
standard solution was prepared by mixing an equal
volume of each stock standard. Working standard
solutions were prepared by dilution of the mixed stock
solution and then filtered through a-0.45 m PTFE
filter. HPLC analysis of standards and samples was
conducted on Water 966 high performance liquid
chromatography comprising vacuum degasser,
quaternary pump, auto-sampler, thermostatted column
compartment and photo diode array detector. The
column used was a Platinum EPS C18 reversed phase,
3m (53x7mm). Mobile phase eventually adopted for
this study was water/acetonitrile (87:13) containing
0.05% (v/v) trifluoroacetic acid (TFA) with the flow
rate of 2 ml/min. Absorption wavelength was selected
at 210 nm. The column was operated at 30C. The
sample injection was 20 l. Caffeine and 8 catechins
were identified by comparing their retention times and
UV spectra in the 190-400 nm range with standards.
They were quantified by external standard method and
the results were expressed as g/100 g dry basis
(%w/w). Total catechin content (TCC) was calculated
from the addition of individual catechin. All tests were
performed in duplicate.
All analysis was performed in duplication. Data
was expressed as mean + standard deviation.
3.1 Chemical compositions in green tea
Table 1 shows compositions in of the prepared
green tea sample. Green tea had 3.91+0.37 %w/w of
moisture content. Total polyphenol content (TPC)
was15.61+0.04 %w/w. This value was in the range of
TPC content found in green tea [15, 16]. HPLC
analysis revealed that green tea had 2.61+0.48%
caffeine. A mean TCC in green tea was 9.49%w/w. Of
this value, EGCG was present at the highest
(3.65+0.71%w/w)
followed
by
EGC
(2.98+0.48%w/w). These two catechins were found as
major catechins in the prepared green teas.
842
Table 1: Some compositions in green tea

(%w/w)
Catechins
Moisture
3.910.37 GC
0.880.38
CF
2.610.48 EGC
2.980.48
TPC
15.610.04 C
0.470.11
TCC
9.460.38 EC
0.610.07
EGCG
3.650.71
GCG
0.430.01
ECG
0.570.10
CG
0.070.02
3.2 Separation of catechins from green tea

In this study, green tea extract was adsorbed onto
the surface of resin. The desorption was performed by
varying the gradient elution with ethanol-water from
0-100% with a constant flow rate of 74 ml/min. The
effluent fractions from the adsorption, and each of the
elution step was collected for compositional analysis.
The dynamic elution curve of TCC and CF were
shown in Figure 2. It shows that the majority of CF
was efficiently eluted by the eluent ranged from
fraction no. 14-19 (0-20%EtOH). The catechin fraction
was eluted after CF fraction by the eluent ranged from
fraction no. 20-34 (30-60%EtOH). Figure 3 shows the
dynamic elution profiles of CF and 2 major catechins,
EGCG and EGC. The elution was in order of first CF,
second EGC and last EGCG. This order is in
accordance with the polarity order reported by Lai et
al. [10]. EGC was present at the highest concentration
in the fraction ranged from 22 to 30 (30-50%EtOH),
whereas EGCG was mainly found in the fraction
ranged from 27-34 (50-60%EtOH). It indicated that
EGC and EGCG did not efficiently separate with an
elution condition used in this study.
Amount (mg)
60
(%w/w)
Amount (mg)
Component
70
50
40
EGC
30
EGCG
20
CF
10
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Fraction
Figure 3. Elution curves of EGC, EGCG, CF and EC

in each fraction.
Table 2 summarizes a separation of CF and TCC in
each gradient elution. It can be seen that the majority
of CF (61.35%) was eluted in the fraction of
20%EtOH. Total catechin content (TCC) was mainly
found in the fraction of EtOH ranging from 30-60%.
Under the separating conditions used in this study, the
recovery yields of CF and TCC were 79.05 and
58.77%, respectively. Although, it has been reported
that the Amberlite XAD-7HP resin provided the
highest adsorption capacity to catechins [9], we found
that it also showed a fairly good adsorption capacity to
CF as well. However, Amberlite XAD-7HP was fairly
good to be an adsorption resin that can be used to
separate TCC from CF in green tea extracts.
Table 2: Recovery yield of total catechin content
(TCC) and caffeine in each step of operations
Operation
Step
volume
Caffeine
Recovery
TCC
(L)
Mass
(mg)
Adsorption
261
0% EtOH
0.48
0.19
2.86
0.30
10%EtOH
1.76
0.67
5.52
0.58
80
20% EtOH
160.13
61.35
1.53
0.16
70
30% EtOH
13.47
5.16
43.65
4.60
40%EtOH
8.21
3.14
161.31
17.00
50% EtOH
1.01
0.39
186.42
19.64
60% EtOH
7.76
2.97
65.21
6.87
70% EtOH
10.49
4.02
32.36
3.41
100%EtOH
3.01
1.15
58.89
6.21
Total
23
206.33
79.05
557.75
58.77
(%)
Mass
(mg)
Recovery
(%)
949
60
50
40
30
CF
20
TCC
10
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44
Fraction
4. Conclusions
Figure 2. Elution curves of caffeine (CF) and total
catechin content (TCC) in each fraction.
This preliminary study indicated that a 1-L column

packed with an Amberlite XAD-7HP can be used to
separate caffeine and major catechins (EGC and
EGCG) from green tea extracts. In order to optimize
the recovery yield, further studies based on the elution
conditions (flow rate, elution volume, concentration of
tea extracts) should be investigated in the future.
843
Acknowledgements
The authors thank School of Agro-Industry and
Tea Institute, Mae Fah Luang University.
References
[1] S.-C. Wu, G.-C. Yen, B.-S. Wang, C.-K. Chiu, W.-J.
Yen, L.-W. Chang and P.-D. Duh, LWT--Food Sci.
Technol. 40 (2007) 506-512.
[2] T.M. Rains, S. Agarwal and K.C. Maki, J. Nutr.
Biochem. 22 (2011) 1-7.
[3] A. Deka and J.A. Vita, Pharmacol. Res. 64 (2011) 136145.
[4] Y. Kim, K.L. Goodner, J.-D. Park, J. Choi, S.T. and
Talcott, Food Chem. 129 (2011) 1331-1342.
[5] T. Yamamoto ,L.R. Juneja LR, D.C. Chu and M. Kim,
Chemistry and applications of green tea, CRC Press ,
LLC (1997).
[6] A.L. Anaya, R. Cruz-Ortega R, and G. R. Waller,
Front. Biosci. 11(2006) 2354-2370.
[7] K. H. Row and Y. Jin, Bioresour. Technol. 97 (2006)
790-793.
[8] G. Rusak, D. Komes, S. Liki, D. Hori, M. Kova,
Food Chem. 110 (2008) 852-858.
[9] D. Baumann, S. Adler and M. Hamburger, J. Nat. Prod.
64 (2001) 353-355.
[10] S.-M. Lai, W.-L. Lee, Y.-M. Chang and W.-C. Liaw,
J. Liq. Chromatogr. Relat. Technol. 32 (2008) 293311.
[11] Q.V. Vuong, J.B. Golding, M. Nguyen and P.D. Roach,
J. Sep. Sci. 33 (2010) 3415-3428.
[12] Q.V. Vuong, C.E. Stathopoulos, M.H. Nguyen, J.B.
Golding and P.D. Roach, Food Rev. Int. 27 (2011)
227-247.
[13] ISO 14502-1, Determination of substances
characteristic of green and black teaPart 1: Content of
total polyphenols in teaCalorimetric method using
Folinciocalteu reagent, Switzerland (2005).
[14] ISO 14502-2, Determination of substances
characteristic of green and black tea: Part 2: Content of
catechins in green tea: method using high-performance
liquid chromatography, Switzerland (2005).
[15] K.M. Alam, M.S. Uddin, M.A.M. Chowdhury and M.
A. Motalib, Plant Achieves 11 (2011) 173-177.
[16] S. Jayasekera, A.L. Molan, M. Garg and P.J. Moughan,
Food Chem. 125 (2011) 536-541.
844
QUALITY MONITORING OF FRESH-CUT PHULAE PINEAPPLE

Jeeranuch Jatturaspaibool1, Chompoonoot Kankompeck1, Sutthiwal Setha2 and
Phunsiri Suthiluk1*
1
Food Technology Program, School of Agro-Industry, Mae FahLuang University,Chiang Rai 57100, Thailand
2
Technology Management of Agricultural Produces and Packaging Program, School of Agro-Industry,
Mae Fah Luang University, Chiang Rai 57100, Thailand
*
Author for correspondence; E-Mail: phunsiri.s@mfu.ac.th, Tel. +66 53 916738, Fax. +66 53 916739
Abstract: Fresh-cut Phulae pineapple becomes popular

in the market. However, mechanical operations during
minimal processing damages tissue of fruit, which
elevated respiration and promoted rapid deterioration.
Therefore, this study aims to control quality of fresh-cut
Phulae pineapple during storage. Fresh-cut Phulae
pineapple was treated in various solutions including
0.5% citric acid (CA), 0.5% ascorbic acid (AA), 2.0%
calcium chloride (CaCl2) and 2.0% sodium chloride
(NaCl) for 1 min and stored at 5+1 C, 95% RH. Quality
changes in terms of color, firmness, pH, titratable acidity
(TA), total soluble solid (TSS), vitamin C, total plate
count, coliform, yeast and mold and sensory evaluation
were determined during 0 14 days of storage. The pH,
TA and TSS value did not significant change over time
(P>0.05). Sample treated with 2.0% CaCl2 or NaCl for 1
min had highest overall acceptability score and firmness
when compared to other treatments. Moreover, fresh-cut
sample treated with 2.0% CaCl2 or NaCl for 1 min were
packed in polypropylene anti-fog bag (OTR: 3,685 cc
/m2.day) providing modified atmosphere condition. The
treatment of 2% NaCl for 1 min obtained the best visual
appearance and highest score of overall acceptability.
Juice leakage in all treatments could not be observed.
The shelf life of fresh-cut Phulae pineapple was limited
to 12 days at 5C storage judged by bacterial population
and sensory score.
1. Introduction
Pineapple (Ananas comosus L. Merr) is an exotic
fruit accepted by its aroma, juiciness and flavor.
Phulae pineapple is an economical plant and be a
geographical indicator (GI) of Chiang Rai province,
Thailand. It contains vitamin, mineral and bioactive
compounds such as bromelain, vitamin C and betacarotene and be a good source of dietary fiber [1]
which are beneficial to human health. However,
Phulae pineapple has small size and it is not
convenience for consumer to peel so the need of freshcut product is highly in demand.
Fresh-cut
processing is rapidly becoming popular and addresses
all of these issues by making products available in a

convenient, easy-to-use format with minimal waste.
Quality of fresh-cut fruit products is a combination of
attributes, properties or characteristics including
appearance, texture, flavor, nutritional value and
determines their value to consumers. However,
mechanical operations during minimal processing
cause wounding, increases metabolic activities and
delocalization of enzymes and substrates. This may

lead to deterioration such as browning, softening and
decay and off-flavor development [2]. Thus, various
chemical substances have been used to minimize
visual deterioration in fresh-cut products. Reducing
agents such as citric acid and ascorbic acid [3] have
been investigated to prevent browning while calcium
treatment can maintain or improve tissue firmness and
crispness of fresh-cut product [4]. In addition, sodium
chloride has been used for fresh-cut apple and lettuce
production to control microbial and browning and the
concentration at 300 is the optimal dose for
maintaining quality of apple slices [5]. Nevertheless,
only chemical treatment could not extend the shelf life
of fresh-cut products. It needs to combine with cold
storage and modified atmosphere packaging (MAP)
which can slow down undesirable quality changes and
increase the shelf life of fresh-cut pineapples [6]. The
development of techniques for preserving fresh-cut
fruit needs to overcome some of hurdles to successful
commercial distribution of product.
The objective of this study was to determine the
effects of chemical treatments and modified
atmosphere packaging on quality of fresh-cut Phulae
pineapple during storage at 5C.
2.1 Materials
Phulae pineapples (Ananas comosus L. Merr)
harvested in Tumbol Nang Lae, Amphur Muang,
Chiang Rai Province at the commercial maturity stage
(20-40% yellow color of fruit peel) were used as a raw
material in this experiment. Fruits were pre-cooled at
13+1C overnight prior to processing. An 0.5%
scorbic acid (AA), 0.5%citric acid (CA), 2.0%sodium
chloride (NaCl) and 2.0%calcium chloride (CaCl2)
were used to maintain the quality of fresh-cut
Phulae pineapple. Polypropylene bag (PP) and
polypropylene anti-fog bag (PP-AF) which has the
oxygen transmission rate (OTR) at 1,064 and 3,685
cc.m-2.day, respectively were used in this experiment.
2.2 Methods
Fresh-cut Processing: Working area and other
utensils used during processing were cleaned and
sanitized with 70% ethanol. Fresh Phulae pineapple
was washed with 200 ppm chlorine solution for 2 min
845
and drain. After that crown leaves, peel and eyes were
removed. Fresh pineapple was cut vertically into 4
pieces per 1 fruit. Fresh-cut Phulae pineapple was
dipped in each of above chemical solution.
Experimental Design: The experiment was divided
into 2 parts. The first part, fresh-cut Phulae
pineapple was dipped in various solution; AA (0.5%),
CA (0.5%), NaCl (2.0%) or CaCl2 (2.0%) for 1 min
and drain. A 200-220 g was packed in PP tray, put into
PP bag and sealed. Samples were stored at 5 + 1C,
95% RH and randomly taken at 0, 2, 4, 6, 8, 10, 12 and
14 days for quality determinations. Treated sample in
distilled water was used to be a control. The second
part, fresh-cut Phulae pineapple was dipped in two
selected conditions from part 1 which provided the
best result. A 200-220 g was packed in PP tray, put in
PP-AF bag and sealed. Samples were stored at 5 +
1C, 95% RH and randomly taken at 0, 2, 4, 6, 8, 10,
12 and 14 days for quality determinations.
Determination of pH, TSS, TA and Vitamin C:
Fresh-cut sample was homogenized and filtered. The
pH was directly measured using a pH meter (Eutech,
pH 510) and total soluble solids content (TSS) was
measured using a hand refractometer (ATAGO, Japan)
and expressed as % TSS. The pineapple juice was
titrated with 0.1N NaOH and titratable acidity (TA)
was expressed as percentage of citric acid (AOAC
official method 2000). For vitamin C content, the juice
was titrated with indophenols solution and expressed
as milligram per 100g fresh weight (FW) of pineapple
(AOAC official method 2000). All of the
determinations were done in triplicate.
Color Measurement: Color was measured directly
by colorimeter (Color Quest XE Hunter Lab, USA)
using the hunter scale (L*, a* and b*). Three readings
were obtained for each replicate by changing the
position of the pineapple piece.
Juice Leakage: Juice leakage from sample was
measured according to the method of Marrero and
Kader (2006) with some modifications. It was assayed
by tilting the packages at a 20 angle for 5 min and
recovering an accumulated liquid. Results were
reported as liquid volume recovered per 200-220 g of
sample in the package.
Firmness Evaluation: The center of each piece of
fresh-cut pineapple was measured by texture analyzer
TA-XT2 (Stable Micro System, Surrey, UK) at room
temperature and used knife blade (HDP/BS) probe
with force compression and expressed as Newton (N)
unit.
Headspace Gas Analysis: The internal atmosphere
of each package was analyzed using Head space gas
analyzer (CheckMate II, PBI-Dansensor America Inc.,
USA) and expressed as percentage of O2 and CO2.
Three trays from each treatment were randomly
selected for gas analysis at each sampling date, during
the 14 days storage time.
Determination of Microbiological Quality: Total
Plate Counts (TPC), coliform and yeast and mold in
sample were enumerated according to the BAM: 2001
guideline. Analyses were carried out in randomly
sampled taken at 0, 2, 4, 6, 8, 10, 12 and 14 days.
Sensory Evaluation: Sensory evaluation was

conducted in the Sensory Laboratory, Scientific and
Technological Instruments center, Mae Fah Luang
University, Chiang Rai, Thailand. A total of 3 trained
panelists participated in the study. Sensory attributes
were color, sourness, sweetness, softness fermented
odor and overall acceptability. Hedonic rating using 9point scales were used (1, dislike very much and 9,
like very much).
Statistical Analysis: Data were subjected to
statistical analysis using Statistical Package for the
Social Science (SPSS) version 11.5. Duncan test was
used to determined differences among means with a
level of significance of P< 0.05.
3.1 Effect of Chemical Treatment on Quality of Freshcut Phulae Pineapple
The pH, TSS and TA of all treatments showed
slightly changed during storage at 5C. The values for
pH, TSS and TA were in the range of 3.79-4.17,
12.20-14.80% and 0.31-0.43%, respectively. MonteroCaldern et al. (2008) and Gil et al. (2006) found
similar results for fresh-cut pineapple (Gold cultivar)
stored under modified atmosphere condition. Several
factors such as the maturity stages and the position in
side the fruit may cause the variability of pH, TSS and
TA found in this study. Vitamin C content of all
treatment was significant decreased (P<0.05)
throughout the storage and was in the range of 5.636.80 mg/100ml at 10 days storage conformed to the
study by Raiputta et al. (2011) for the same cultivar.
The reduction of vitamin C could be explained by
minimal processing may induce the nutrient loss
especially vitamin C [8].
Changes in color parameters L* (luminosity) and
b* (-blue to + yellow) among four chemical treatment
conditions were not significant differences through
storage time (Table 1). The differences in L* and b*
were mostly attributed to observed changes in
browning reaction appearance on the surface fresh-cut
pineapple, which changed from a yellow color to a
brown color. Fresh-cut sample treated with 0.5% CA
for 1 min obtained the highest of L* and b* value.
This result showed that the chemical treatments
especially CA treatment could prevent the color
change of fresh-cut pineapple during storage at 5C.
Results of accumulated juice leakage inside the
package of fresh-cut Phulae pineapple during
storage at 5C are shown in Table 1. The liquid inside
the package was firstly observed for all treatments at 8
days storage and ranged from 1.24 to 3.12 ml per
package. Fresh-cut sample treated with 2.0% CaCl2
for 1 min had juice leakage per fruit weight less than
other treatments and also obtained the highest value of
firmness as 11.82 N. These results confirmed that
calcium treatments can maintain or improve tissue
firmness and crispness of fresh-cut products [4, 10].
Table
1
shows
the
development
of
microorganisms in terms of TPC and yeast and mold
on fresh-cut sample during storage at 5C. Microbial
846
Table 1: Changes in color (L* and b*), firmness, juice leakage, TPC and yeast and mold of fresh-cut Phulae
pineapple during storage at 5+1 C, 95% RH
Quality/
Treatment
Color L*
Control
0.5% CA
0.5% AA
2.0% CaCl2
2.0% NaCl
Color b*
Control
0.5% CA
0.5% AA
2.0% CaCl2
2.0% NaCl
Firmness (N)
Control
0.5% CA
0.5% AA
2.0% CaCl2
2.0% NaCl
Juice leakage
(ml)
Control
0.5% CA
0.5% AA
2.0% CaCl2
2.0% NaCl
TPC (log CFU/g)
Control
0.5% CA
0.5% AA
2.0% CaCl2
2.0% NaCl
Yeast & mold
(log CFU/g)
Control
0.5% CA
0.5% AA
2.0% CaCl2
2.0% NaCl
Day(s) of storage
4
6
10
68.50 + 2.35*a
68.28 + 2.26a
68.73 + 2.57a
68.46 + 3.81a
68.09 + 2.39a
70.13 + 4.88a
69.00 + 3.43a
69.60 + 1.03a
69.63 + 7.62a
72.26 + 1.91a
71.12 + 0.87a
70.60 + 0.27a
70.64 + 3.31a
71.15 + 0.55a
71.01 + 0.96a
71.42 + 0.82a
69.69 + 1.09a
70.19 + 0.34a
70.34 + 1.13a
66.07 + 0.91b
66.23 + 3.82a
67.70 + 2.44a
68.66 + 0.89a
67.55 + 2.60a
66.41 + 3.42a
65.83 + 2.02a
67.22 + 1.54a
66.78 + 0.39a
63.77 + 1.87a
66.30 + 2.70a
36.92 + 0.75a
38.29 + 1.47a
37.24 + 2.53a
37.69 + 6.83a
35.21 + 4.46a
33.50 + 1.95a
36.23 + 4.41a
34.78 + 3.02a
31.06 + 9.80a
34.68 + 1.73a
38.85 + 3.53a
38.49 + 3.17a
31.30 + 3.05b
32.39 + 2.54b
39.35 + 0.59a
36.56 + 1.64ab
38.98 + 3.52a
31.28 + 1.41c
33.71 + 0.61bc
39.92 + 2.13a
34.90 + 0.36a
35.96 + 2.04a
34.11 + 0.87a
34.28 + 1.81a
34.06 + 3.25a
38.44 + 7.05a
45.14 + 1.06a
37.98 + 1.39a
39.14 + 5.88a
37.29 + 4.84a
15.44 + 3.12a
14.65 + 2.66a
15.25 + 2.60a
15.37 + 2.25a
14.94 + 3.76a
14.71 + 3.25a
12.55 + 0.56a
15.10 + 2.76a
15.62 + 1.94a
13.90 + 2.61a
14.53 + 0.26a
12.49 + 3.23a
14.74 + 3.39a
14.26 + 0.03a
12.82 + 0.24a
11.98 + 1.02a
12.05 + 0.50a
13.89 +1.44a
13.82 + 2.06a
12.70 + 3.36a
11.76 + 2.83a
11.51 + 1.17a
12.71 + 0.77a
12.02 + 1.32a
11.28 + 2.44a
8.73 + 1.42b
9.29 + 1.09b
10.12 + 0.27ab
11.82 + 0.84a
10.42 + 2.10ab
ND**
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.12 + 0.12a
1.96 + 0.70b
1.79 + 0.21bc
1.24 + 0.28c
1.36 + 0.04bc
3.49 + 0.29a
2.07 + 0.60b
2.05 + 0.65b
1.82 + 0.20b
1.98 + 0.30b
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.16 + 0.01a
3.06 + 0.02b
3.03 + 0.00c
2.98 + 0.00d
2.85 + 0.01e
4.33 + 0.01a
4.31 + 0.00b
4.29 + 0.01c
4.32 + 0.01a
4.29 + 0.01c
5.34 + 0.01a
5.26 + 0.00b
5.24 + 0.00c
5.21 + 0.02d
5.19 + 0.00e
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.65 + 0.03a
2.56 + 0.02b
2.45 + 0.02c
2.39 + 0.03d
ND
3.19 + 0.03a
3.10 + 0.00b
3.05 + 0.02c
2.92 + 0.01d
2.75 + 0.02e
4.12 + 0.00a
4.10 + 0.00b
3.98 + 0.01c
3.87 + 0.02d
3.79 + 0.04e
Means + SD (n = 3), within a column followed by the same letter are not significantly different (P>0.05).
ND means not detected.
**
growth was firstly observed after 6 days storage in all

treatments.
Significant differences (P<0.05) were found
among chemical treatments. TPC ranged from 2.85 to
3.16 log CFU/g at 6 days of storage and reached 5.19
to 5.34 log CFU/g at 10 days of storage. Similar
increased was observed for yeast and mold. However,
coliform was not found in all treatments sample
during storage. A sign of undesirable changes and
degradation processes such as off-flavors and offodors of fresh-cut sample was after 10 days of
storage at 5C. Good manufacturing practices were
used during fresh-cut Phulae pineapple preparation
in this experiment so microbial counts could not
observed in day 0, 2 and 4 of storage.
From sensory evaluation score (data not shown),
fresh-cut samples treated with 2% NaCl and CaCl2
obtained 10 days shelf life with the acceptable overall
acceptability score (acceptable score = 5). Therefore,
the 2% NaCl and 2% CaCl2 treatment for 1 min and

stored at 5C were used for next experiment.
3.2 Effect of Chemical Treatment and Modified
Atmosphere Packaging on Quality of Fresh-cut
Phulae Pineapple
Changes in head space gas composition inside
containers are shown in Figure 1. Modified
atmosphere condition in packaging occurred after 3
days storage. Headspace O2 content significantly
decreased up to 6 days and was gradually constant
until the end of storage. In the other hand, the CO2
level significantly increased during storage (P<0.05)
for all treatments. From the results, modified
atmosphere of approximately 2%O2 and up to
17%CO2 will be recommended for fresh-cut
Phulae pineapple to achieve 12 days storage life at
5C. Marrero and Kader (2006) recommended the
modified atmosphere of 8%O2 and 10%CO2 for
847
fresh-cut Smooth Cayenne pineapple to obtain 12

days storage life at 5C and more than 15 days at 2.2
and 0C.
Figure 1 Internal O2 and CO2 contents in PP-AF bag

during 14 days storage at 5C: Control (a), CaCl2 (b)
and NaCl (c).
Moreover, Montero-Caldern et al. (2008)
suggest that the headspace composition in the range
from 2% to 40%O2 and up to 15%CO2 is suitable for
fresh-cut Gold pineapple to achieve 14 days
storage at 5C and the concentration of O 2 should not
be lower than 2% to avoid anaerobic conditions and
possible formation of off-odors and off-flavors.
However, in this study, fresh-cut Phulae pineapple
storage life was limited by microbial population and
sensory score after 12 days of storage, as will be
discussed below.
The pH, TSS and TA of all treatment also
showed little changes during storage at 5C. The
values for pH, TSS and TA were in the range of 3.804.16, 14-15% and 0.34-0.57%, respectively. Santos et
al. (2005) also observed little changed in fresh-cut
pineapple (Perola cultivar) during storage under
several modified atmosphere conditions at 8C. The
pattern of vitamin C content reduction was similar to
the previous experiment. At the 14 days storage,
fresh-cut sample treated with 2.0% NaCl for 1 min
had the highest vitamin C content as 4.61 mg/100 ml
while the control and 2.0% CaCl2 treatment had only
1.41 and 1.95 mg/100 ml, respectively.
Significant differences for both L* and b* were found

among all treatment conditions over time as shown in
Table 2. Because of a complex fruit anatomy and
maturity pattern, fruit flesh is non-uniform in color
and texture, this explains why L* and b* value of
flesh are very variable among fresh-cut pineapple
pieces [6]. It may conclude that modified atmosphere
condition in this study did not play the important role
in prevention of color changes for fresh-cut Phulae
pineapple when compared to chemical treatment.
The juice leakage was not observed in all
treatment. Absence of liquid inside packages was
attributed to the property of PP-AF bag which
prevent the droplet formation on the surface of the
bag. Calcium treatment still obtained the highest
firmness value of 13.43 N at the 14 days storage
while the control and 2.0% NaCl treatment had 10.98
and 12.16 N, respectively. The result suggested that
the firmness affect to the juice leakage and PP-AF
bag used in this study properly protected the integrity
of fresh-cut Phulae pineapple pieces.
The number of microorganism presented in
fresh-cut sample is shown in Table 2. TPC and yeast
and mold were firstly observed at 6 days storage of
all treatments. Treated sample with 2.0% NaCl for 1
min combined with PP-AF bag obtained the lowest
number of TPC (6.93 log CFU/g) and yeast and mold
(6.11 log CFU/g) until the end of the storage at 14
days. However, coliform was not found in all
treatments sample during storage. Thai regulation for
microbiological criteria of foods and food contact
container (Notification of Department of Medical
Sciences, 2010) include a maximum limit for
mesophilic bacteria of 6 log CFU/g for ready to eat
food prepared form raw fruit and vegetable. TPC in
fresh-cut sample treated with 2.0% NaCl reached that
level on the 14th days of storage. NaCl is a traditional
chemical food preservative that has been used to
prevent the growth of undesirable microorganism in
fruit and vegetable products. Combination of NaCl
treatment and modified atmosphere packaging in this
study extend the shelf life of fresh-cur Phulae
pineapple when compared to the use of only chemical
treatment.
Sensory evaluation results are shown in Table 3.
Fresh-cut samples treated with 2.0% NaCl for 1 min
combined with PP-AF bag had the highest overall
acceptability score of 5.67 at 12 days of storage
(acceptable score = 5).
4. Conclusions
Treatment of 2.0% NaCl solution for1 min
combined with PP-AF bag is the most effective
condition to maintain quality and prolong shelf life of
fresh-cut Phulae pineapple during storage at 5C.
The shelf life of fresh-cut Phulae pineapple was
limited at 12 days of storage judged by the population
of bacterial growth and sensory score.
848
Table 2: Changes in color (L* and b*), firmness, juice leakage, TPC and yeast and mold of fresh-cut Phulae
pineapple treated with 2.0% NaCl or CaCl2 combined with PP-AF bag during storage at 5+1 C, 95% RH
Day(s) of storage
6
8
Quality
Color L*
Control
A
B
Color b*
Control
A
B
Firmness
Control
A
B
TPC
Control
A
B
Yeast &
mold
Control
A
B
10
12
14
66.40 + 4.38*a
66.48 + 3.06ab
67.14 + 2.70a
66.23 + 0.91a
66.11 + 4.08ab
63.69 + 2.09a
63.39 + 1.84a
64.60 + 3.60ab
64.48 + 1.18a
62.70 + 3.72a
62.99 + 2.89ab
64.48 + 1.35a
63.99 + 2.70a
63.25 + 3.58ab
65.90 + 3.38a
62.41 + 3.41a
68.88 + 3.14a
64.82 + 0.68a
61.38 + 2.37a
61.59 + 2.89bc
64.70 + 0.92a
61.50 + 4.82a
57.17 + 0.30c
58.54 + 6.07b
40.70 + 3.64a
39.86 + 2.76a
38.07 + 0.04ab
34.41 + 5.77a
27.38 + 0.65b
31.13 + 6.01c
41.62 + 1.72a
36.37 + 2.80a
40.16 + 2.16ab
38.02 + 3.05a
35.85 + 7.45a
34.53 + 2.70bc
37.60 + 1.31a
40.87 + 1.12a
37.57 + 2.24ab
38.34 + 1.61a
36.05 + 1.77a
36.91 + 2.55ab
38.15 + 1.74a
36.43 + 0.71a
38.50 + 2.09ab
34.66 + 6.92a
35.20 + 0.32a
41.92 + 3.45a
17.41 + 2.03a
19.53 + 2.47a
18.99 + 2.86a
15.78 + 1.18c
17.90 + 0.63a
16.84 + 0.86ab
14.49 + 1.38b
17.20 + 0.49a
16.61 + 0.69a
13.35 + 3.40a
16.72 + 0.30a
16.44 + 3.30a
12.59 + 2.13b
16.56 + 1.23a
15.70 + 0.51a
11.95 + 1.80b
15.42 + 0.69a
14.39 + 0.45a
11.70 + 1.70b
14.32 + 0.15a
13.65 + 1.14ab
10.98 + 0.68c
13.43 + 0.37b
12.19 + 1.30ab
ND**
ND
ND
ND
ND
ND
ND
ND
ND
2.94 + 0.04a
2.91 + 0.02ab
2.88 + 0.03b
4.32 + 0.01a
4.25 + 0.02b
4.25 + 0.02b
6.10 + 0.01a
5.46 + 0.58ab
5.10 + 0.02b
7.34 + 0.01a
6.18 + 0.01b
5.92 + 0.01c
8.35 + 0.01a
7.26 + 0.01b
6.93 + 0.02c
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.87 + 0.06a
2.48 + 0.11b
2.39 + 0.03b
3.19 + 0.03a
2.90 + 0.03b
2.89 + 0.03b
4.25 + 0.01a
3.89 + 0.03b
3.87 + 0.01b
6.24 + 0.03a
5.20 + 0.03b
4.76 + 0.01c
7.33 + 0.01a
6.40 + 0.04b
6.11 + 0.01c
Means + SD (n = 3), within a column followed by the same letter are not significantly different (P>0.05).
ND means not detected.
A means fresh-cut sample treated with 2.0% CaCl2 for 1 min combined with PP-AF bag.
B means fresh-cut sample treated with 2.0% NaCl for 1 min combined with PP-AF bag.
**
Table 3: Sensory evaluation score of fresh-cut Phulae pineapple treated with 2.0% NaCl or CaCl2 combined with
PP-AF bag during storage at 5+1 C, 95% RH
0
Day(s) of storage
6
10
12
8.70 + 0.58a
8.70 + 0.58b
8.70 + 0.00b
8.67 + 0.58b
9.00 + 0.00a
9.00 + 0.00a
6.33 + 0.58c
7.67 + 0.58c
7.67 + 0.58c
5.00 + 0.00d
7.67 + 0.58c
7.67 + 0.58c
4.00 + 0.00e
6.67 + 0.58d
6.33 + 0.58d
3.33 + 0.58f
4.67 + 0.58e
6.00 + 0.00e
3.00 + 0.00g
3.00 + 0.00f
5.00 + 0.00f
8.70 + 0.58a
8.70 + 0.58a
8.70 + 0.00a
7.33 + 0.58b
8.33 + 0.58b
8.67 + 0.58b
6.00 + 0.00c
7.00 + 1.00c
8.00 + 0.00c
5.67 + 0.58d
7.00 + 0.00c
7.67 + 0.58d
3.67 + 1.15e
6.00 + 0.00d
6.67 + 0.58e
3.00 + 0..00f
5.33 + 0.58e
6.00 + 1.00f
3.00 + 0.00g
3.67 + 0.58f
5.33 + 0.58g
8.70 + 0.58a
8.70 + 0.58a
8.70 + 0.58b
8.67 + 0.58b
8.33 + 0.58b
9.00 + 0.00a
7.00 + 0.00c
7.67 + 0.58c
7.67 + 0.58c
5.33 + 0.58e
7.67 + 0.58c
7.33 + 1.15d
3.67 + 0.58d
6.67 + 0.58d
7.33 + 0.58d
3.67 + 0.58f
4.33 + 0.58e
5.33 + 0.58f
3.33 + 0.58g
3.33 + 0.58f
5.33 + 0.58g
8.70 + 0.58a
8.70 + 0.58a
8.70 + 0.58a
7.00 + 0.00b
8.00 + 0.00b
8.33 + 1.15b
4.33 + 0.58d
8.00 + 0.00b
8.00 + 0.00c
5.00 + 0.00c
7.00 + 0.00c
6.67 + 1.53d
3.00 + 0.00e
6.33 + 0.58d
6.67 + 0.58d
3.00 + 0.00e
4.67 + 0.58e
6.67 + 0.58d
3.00 + 0.00e
3.33 + 0.58f
5.67 + 0.58f
8.33 + 0.58a
9.00 + 0.00a
9.00 + 0.00a
7.67 + 0.58b
8.67 + 0.58b
9.00 + 0.00a
6.00 + 0.00c
8.00 + 0.00c
8.00 + 0.00b
4.67 + 0.58d
7.67 + 0.58d
7.67 + 0.58c
3.67 + 0.58e
6.67 + 0.58e
7.00 + 0.00d
3.33 + 0.58f
5.00 + 0.00f
6.33 + 0.58e
3.00 + 0.00g
3.67 + 0.58g
5.67 + 0.58f
Attribute
Color (+)
Control
A
B
Softness (-)
Control
A
B
Sweetness (+)
Control
A
B
Fermented
odor (-)
Control
A
B
Overall
acceptability
Control
A
B
*
Means + SD (n = 3), within a row followed by the same letter are not significantly different (P>0.05).
A means fresh-cut sample treated with 2.0% CaCl2 for 1 min combined with PP-AF bag.
B means fresh-cut sample treated with 2.0% NaCl for 1 min combined with PP-AF bag.
(+) means positive attribute, score is direct variation with the attribute.
(-) means negative attribute, score is reverse variation with the attribute.
849
Acknowledgements
This work was financial supported by National
Science and Technology, Development Agency,
Northen Network Program. School of Agro-Industry
and Scientific and Technological Instruments Center,
Mae Fah Luang University, Chiang Rai, Thailand are
acknowledged for the support.
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[7] A. Merrero and A.A. Kader. Posthavest Biol. Technol.,
39(2006), 163-168.
[8] M.I. Gill, E. Aguayo and A.A. Kader. J. Agr. Food
Chem., 54 (2006), 4284-4296.
[9] J. Raiputta, S. Setha, S. Chaiwong and P. Suthiluk.
Agriculture Sci. J., 42:3 (Suppl.)(2011), 673-676.
[10] I. Luna-Guzmn and D. M. Barrett, Postharvest Biol.
Technol., 19 (2000), 61-72.
[11] J.C.B. Santos, E.V. de Barros, B. Vilas Boas, M.E.
Torres Prado and A.C. Marques Pinheiro. Cinc.
Agrotec., Lavras, 29 (2005), 353-361.
850
SIMPLE AND RAPID DETECTION OF PINEAPPLE MEALYBUG WILTASSOCIATED VIRUS BY RT-LAMP ON A CHIP SYSTEM
Najwa Yanya1, Titima Maturos 2, Adisorn Tuantranont2, Masato Saito3, Eiichi Tamiya3,
and Piyasak Chaumpluk1*
1
Laboratory of Plant Transgenic Technology and Biosensor, Department of Botany, Faculty of Science, Chulalongkorn
University, Bangkok, 10330, Thailand
2
Nanoelectronics and MEMS Lab, National Electronics and Computer Technology Center, 112 Thailand Science Park,
Phahon Yothin Rd., Klong 1, Klong Luang, Pathumthani 12120, Thailand
3
Department of Applied, Graduate School of Engineering, Osaka University, 2-1 Chulalongkorn University, Bangkok,
10330, Thailand
*
Author for correspondence; E-Mail: piyasakcha@gmail.com, Tel. +66 22185494, Fax+66 22185494
Abstract: Pineapple Wilt Disease (PWD) is one of the most

serious diseases which cause a major loss to Cayenne
pineapple production. Several studies related to PWD have
been addressed to understand and identify the cause of
disease. A specific detection method relied on reverse
transcription loop-mediated isothermal amplification (RTLAMP) on a chip system for rapid detection of specific coat
protein gene of Pineapple Mealybug Wilt Assciated Virus-1
(PMWaV-1), is generated in this study. A complete chip
consists of reaction zones for extraction and amplification of
RNA sample, and microchannels for sample and reagents
injection. The total volume of reaction zone was estimately
10 l. A PMWaV-1 coat protein region was amplified by
RT-LAMP at 63C and DNA signal was detected by visual
observation of green fluorescent response under UV light.
This fluorescent detection can differentiate the positive
amplification from negative amplification. No cross reaction
was detected from other types of plant viruses. The whole
process in single chip is really simple and rapid; the result
can be obtained within 40 minutes with limit of detection at
100 copies of viral RNA. An accurate and efficiency
detection method which relies less on laboratory facilities for
PMWaV-1 is reported in this study.
1. Introduction
Pineapple wilt disease (PWD) caused by pineapple
mealybug wilt associated-virus (PMWaV) is one of the
most serious pineapple diseases that have an effect on
cayenne cultivar distributed in many parts of the world
[1-4]. In Thailand the cayenne type had export values
more than US$ 853.53 million [5]. Since crown, and
slip parts are commonly used as starting materials for
pineapple cultivation. Thus, monitor of plant
propagules for virus free plays a crucial role in control
and prevention of the disease. Recently, many
applications of molecular methods have been demon
strated [6-10], however, most of them rely heavily of
laboratory facilities and have more time for post
analysis. Thus, Micro-total analysis system (TAS)
for plant virus detection is developed in this study. It
combines ferrous oxide resin for nucleic acids
extraction, reverse transcription loop mediated
isothermal amplification (RT-LAMP) for better
nucleic acid amplification, and fluorescent signal
detection for fast visual observation. This fabricated
simple -TAS device or chip makes all detection

processes complete in a single chip platform with
simple and low cost manipulation.
2.1 Device design and fabrication.
The microfluidic device (Figure 1.) consists of the
multi channel inlet for fluid injection, a reaction zone
for RNA extraction, cDNA amplification and DNA
signals detection and an outlet for fluid outlet. It was
designed to have microchannel dimension 200x100
m width and depth with lining of microchannel
throughout the chip. It was fabricated by standard softlithography techniques [11] using polydimethyl silo
xane (PDMS) (Dow Corning, USA). Holes of 500m
diameter were accessed for the inlet and the outlet of
the liquid reagent solutions. Fluorinated ethylene
propylene (FEP) tube (0.150.05-mm i.d.) (BAS Inc.,
Japan) was inserted into these holes, and sealed with
small amounts of PDMS to prevent liquid leaks. The
PDMS device was hermetically bonded to the glass
substrate via plasma process by PDC-210 Plasma
Cleaner (Yamato, Japan). Operation of the microfluid
in the device was by manual micromanipulator
(Figure1) using 1 mL syringe and 20Gx1 needle
(Terumo, Japan).
Figure 1. The device structure consists of 5 inlet

channels, a reaction zone and outlet. Magnetic field is
set at rare part of reaction zone (left) and manual
micromanipulator (right).
2.2 Viral specimen
PMWaV-1, PMWaV-2, Tobacco mosaic virus
(TMV), Cucumber mosaic virus (CMV), Cymbidium
mosaic virus (CYMV), Papaya ring spot virus (PRSV)
specimens, were collected from both experimental
fields at Agricultural Research and Development
Division 5, Petchaburi, Thailand and Virus Collection
at Laboratory of Plant Transgenic Technology and
851
Biosensor, Department of Botany, Faculty of Science,

Chulalongkorn University, Bangkok, Thailand. Some
of viral-infected pineapple plants were also collected
from the fields in Rayong, Chonburi, and Prajuab
khirikhan Provinces in Thailand.
2.3 RNA extraction
RNA extraction on microfluidic device starts from
crushing of 100 mg of basal part of leaf with apparent
lesions in 200 L extraction buffer containing 100 mM
Tris HCl pH 7.5, 1 mM EDTA, 300 mM NaCl, 2 M
guadinidium thiocyanate, 0.1 mM DTT and 0.1%
bentonite in 1.5 mL microcentrifuge tube using micropestle. After spinning down at 12,000 rpm, supernatant
containing viral RNA were transferred to a new tube
and mixed with 7L of ferous oxide resin (Promega,
USA) to ensure target RNA capture and injected into
the device using manual micro manipulator. RNA,
once binded to resin surfaces, was trapped by magnet
located at rare part of reaction zone. The washing
buffer (80% ethanol) was subsequently flowed through
the reaction zone. Further air dry was carried out by
flow air slowly into the reaction zone. These processes
allowed viral nucleic acid to be viable and ready for
use in amplification assay.
2.4 Primer, probe and oligonucleotide design.
The RT-LAMP specific primers set for PMWaV-1
coat protein was designed based on the information
given in NCBI Database (accession No. AF414119,
NC_010178, and EU7691140) using Primer Explorer
version4 [12]. Sequences were aligned and compared
with those of other PMWaVs. The selected primers
were showed in Table 1.
2.5 cDNA synthesis and TA cloning for control
nucleic acid.
The total RNAs from PMWaV-1 specimens were
extracted and amplified through RT-PCR [15] using
F3 and B3 primers from Table 1. The DNA products
using TA cloning approach (Invitrogen, USA). This
vector was transfer to E.coli by trans formation [13].
After that selection nucleotide sequence confirmation
was carried out. For control RNA, the selected clone
was cleaved and RNA transcripts were made in vitro
via T7 transcription protocol (Ambion, USA). These
PMWaV-1 transcripts were used as a positive
reference and a standard template of known copy
number RNAs.
2.6 Confirmation of PMWaV-1 in samples by RTPCR.
The total of RNAs extracted from the leaf bases of
viral-infected pineapple plant were amplified by RTPCR as previous described [7] in order to confirm the
presence of PMWaV-1.
2.7 Reverse Transcription Isothermal DNA
amplification assay.
The RT-LAMP was performed on chip by flowing
20 l of RT-LAMPs reagent containing 1.6 M each
of FIP and BIP, 0.2 M each of F3 and B3, 16 U of the

Bst DNA polymerase large fragment (New England
Biolabs Inc., USA), 100U AMV reverse transcriptase
(Finnzymes, Finland), 1.4 mM dNTP, 0.8 M Betaine
(SigmaAldrich, USA), 20 mM Tris-HCl (pH 8.8), 10
mM KCl, 10 mM (NH4)2SO4, 16 mM MgSO4, 0.1%
Tween 20, 0.1% Triton-X 100 and 0.1 x EvaGreen
(Biotium Inc, USA) into the reaction zone in the chip.
The RT-LAMP reactions were incubated at 63C for
40 min using Accu BlockTM (Labnet, USA). The
results were compared to that of RT-PCR which is
used as standard reference for this study [7].
2.8 Specificity of RT-LAMP assay
The RNAs from PMWaV-1 and PMWaV-2 and
RNA derived from other types of common plant
viruses; TMV, CMV, CYMV, PRSV) were amplified
using the specific RT-LAMP primer as described. The
cross reaction of primers with other plant viruses was
determined to make sure that the designed RT-LAMP
primer is only specific to PMWaV-1. The virus-free
pineapple plant samples were used as a negative
control for this test.
2.9 Sensitivity of RT-LAMP assay.
The detection limit of the assay was conducted
using equivalent copy numbers of RNA of PMWaV-1
coat protein gene at 10-fold dilutions. This DNA
samples covering from 108 to 100 copies were then
amplified by RT-LAMP with condition at 63C for 40
minutes. After the amplification had been done on
chip, the solution mixture from chip was analyzed for
the sensitivity via gel electrophoresis and fluorescence
detection.
2.10 DNA Signals detection after DNA amplification
by RT-LAMP assay.
Normal DNA signals detection was performed via
both agarose gel analysis and UV light observation.
On chip DNA signals detection was performed under
UV light (=365nm). The fluorescence signals
response from both negative and positive
amplifications was recorded.
3. Result and Discussion
PMWaV-1 is one of the plant distinct viruses of
pineapple (cv. Smooth Cayenne) leading to the PWD.
In Thailand, it causes not only the decrease in
pineapple production but also the wilting and dying of
pineapple plant [14]. Therefore, the effective method
for detection of PMWaV-1 in propagated materials is
highly concerned. In this study, the application of
nucleic acid extraction protocol, using ferrous oxide
resin in combination with the nucleic acid ampli
fication using an isothermal amplification with
fluorescence binder, allows the PMWaV-1 to be
detected on single chip with simple operation, rapid
reaction, low cost and easy detection. The isolation of
viral RNA from sample is an important step.
Introduction of magnetic separation technique allow
quick, simple and efficient way to separate nucleic
852
acids and a far less rigorous method than traditional

techniques [13]. Rapid diagnosis of enterovirus infec
tion [15] and a system for purifying SARS coronavirus
RNA [16] using magnetic bead had been reported.
Commercial applications for total RNA isolation were
available [17]. Since most of magnetic separations
involve a non-phenol lytic step and a quick magnetic
separation without the use of alcohol precipitation, so
it is suitable for chip application. Simple chip structure
was then designed based on steps of ferrous oxide
resin use. It was comprised of five inlets, which joined
together to a single micro channel at fore part. Those
inlets are for loading sample solution, washing
solution, air drying, amplification solution and extra
channel, not in use. A single microchannel then folded
close together to form a reaction zone (8x11mm2).
After folding, micro channel separated to single line
until it reached outlet part. This differed from most
microfluidic devices [18-20] since the use of ferrous
oxide resin and isothermal nucleic acid amplification
helped to avoid any needs of micro valves and pumps,
all of which increased the cost and complexity in
fabrication and manipulation.
Plant viral RNA
extraction was never been demonstrated on chip
before. Lower pH buffer maintained condition
preferable for RNA extraction while bentonite helped
to interfere with ribonuclease activity. Lysing the cells
was by strong detergent of guadinidium thiocyanate.
Pretreatment of sample by crushing of the specimen in
buffer using micro-pestle, disrupted the tissues and
cells and released viral nucleic acid into buffer
solution. Pretreated sample was clarified by
centrifugation to get rid of any trace of debris within
microchannel. Supernatant was transferred, mixed
with ferrous oxide resin and injected into No. 1. inlet
(Figure 2.). Extraction of RNA on chip was then
carried out based on the binding of the RNA onto the
silica surface at high-ionic-strength salt, followed by a
trapping it under magnetic field. After two times of
washing step with ethanol via inlet No. 2 and drying
via inlet No. 3, the viral RNA was eluted and
amplified simultaneously by amplification reagent and
primer set designed as a main system detection via
inlet No. 4.
Figure 2. Chip and the diagram of flow direction of

solution into microchannel inlets on chip
Most of the PWD assays was based on HSP70
region [7][8][10]. However, the cDNA amplification
system in this study relies on the PMWaV-1 coat
protein region due to its significant roles for virus
pathogenicity and morphology.
Table 1: The details for RT-LAMP primer set used for

PMWaV-1 coat protein detection.
Sequence(5 to 3)
Target(position)
41-239 (198 nt)
F3 Primer
B3 Primer
FIP Primer
BIP Primer
ctctgggcctaagacagt
cctatcaccttgtgtgca
cgacacagtagtcctacctccggaagaccaagtcaaggaa
acgtttgaagatttgatagcagccgatcggaacgttaatcatgc
The feasibility of DNA amplification relied on the

specificity of the primers and also the optimum
temperature for incubation. In this study, the primers
were designed and the optimum temperature required
for best RT-LAMP reactions was at 63C. The
standard for RT-LAMP assay was carried out using a
set of four primers designed as described. The
detection of RT-LAMP products was later analyzed.
Agarose gel electrophoresis of amplified products
displayed the ladder-like DNA pattern of 198 nucleo
tides. This result was correlated with the RT-PCR
assay [7] (Figure 3). The obtained results indicated
that the designed primer set was validated for RTLAMP amplification.
Fluorescen
ce
Figure 3. The RT-LAMP standard via 3% gel

electrophoresis with comparison to RT-PCR and
visual observation of fluorescence. Lane M, 1-kb DNA
ladder; Lane N, non-template control (distilled water);
Lane P, positive control (Plasmid containing the
PMWaV-1 coat protein gene).
The specificity of the RT-LAMP was also
determined by amplifying other types of common
plant viruses (PMWaV-2, TMV, CMV, PRSV, and
CYMV) in order to observe any cross reactions. The
result obtained from RT-LAMP products running on
agarose gel showed that only lane 2 gave a ladder-like
DNA pattern on the gel (Figure 4). There were no
RNA fragments from other type of plant viruses that
could be amplified by PMWaV-1 RT-LAMP primer
set. These also corresponded to RT-PCR results using
primers which specific to HSP 70 gene as previously
described [7] (Figure 4). In the study, the limit of
detection (LOD) of the assay was carried out using
known copy number of transcripts. The result obtained
from chip revealed LOD at 100 copies of viral
template. Moreover, the concentrations in the range of
108 to 100 copies of template were able to be detected
under UV light (Figure 4). This result demonstrated
that the sensitivity of fluorescent dye detection was
comparable to RT-LAMP amplified products.
Moreover, the ladder-like DNA patterns from RTLAMP products showed absolutely clear banding even
at LOD while the clearness of DNA banding from RTPCR product was dramatically decreased with pro
portional to the decrease of template concentration
level. This revealed the excellent performance of RTLAMP even when the template concentrations were
neared to LOD.
853
Figure 4. The specificity and sensitivity tests of RT-LAMP on chip.

The specificity test was performed by comparing 3 different assays;
RT-LAMP, RT-PCR and fluorescence detection (left). The
amplified product was found only from the RNA sample from
PMWaV-1. Lane M, 1kb DNA ladder; lane 1, non-template control
(NTC); lane 2, PMWaV-1; lane 3, PMWaV-2; lane 4,TMV; lane 5,
CMV; lane 6, CYMV; lane 7, PRSV. Sensitivity test was performed
by comparing RT-LAMP with RT-PCR and Fluorescence dye
detection. Lane M, 1kb DNA ladder; lane 1, non-template control
(NTC); lane 2 to lane 8, 108 100 copies of PMWaV-1 DNA; lane
9, 10 copies of PMWaV-1 DNA; lane 10, 5 copies of PMWaV-1
DNA.
The DNA signals detection system here was based

on DNA intercalator, EvaGreen (Figure 5). It is a
newly developed low-cost dye, and has been employed
in PCR detections [21-22] due to its much lower in
amplification inhibition. Mixing this dye to the
reaction allow an on chip detection of DNA signals in
term of green fluorescence light upon UV illumination
with similar absorption and emission spectra to that of
SYBR Green.
Negative without fluorescence
Figure 5. VisualizationPositive
of chip
UV light with
with under
fluorescence
close capture.
In this study, the feasibility of using RT-LAMP
and fluorescent detection on chip for detection of
PMWaV-1 from samples was validated by evaluating
20 of pineapple plant propagules and the results were
compared to RT-PCR approach(Table 2). According to
the result, 8 out of 20 pineapple plant propagules were
found to be infected with PMWaV-1.
Table 2: Comparison of RT-PCR with RT-LAMP for
the detection of PMWaV-1. N=20
Specimens
RT-PCR
Positive
Negative
False positive
False negative
sensitivity
specificity
8
12
-
RT-LAMP with
fluorescence detection
8
11
1
100%
91.6%
Sensitivity= true positive/(true positive+ false negative)

Specificity= true negative/(false positive+ true negative)
The usage of RT-LAMP amplification was for the

increasing specificity of the product outcome and
applying only one isothermal condition which is an
ideal for on chip temperature control.
4. Conclusions
An accurate and efficiency on chip detection
method which relies less on laboratory facilities for
PMWaV-1 is established. The amplified products by

RT-LAMP at 63 C with DNA patterns of the target
sequence of 198 nucleotides. No cross reaction was
detected from other types of plant viruses. The entire
process is really simple and rapid; the result can be
obtained within an hour with limit of detection at 100
copies of viral RNA.
Acknowledgements
We would like to express our gratitude to the
Integration Project: Innovations for the Improvement
of Food Safety and Food Quality for New World
Economy, Chulalongkorn University, and the National
Electronics and Computer Technology Center,
Thailand for the support in the project.
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855
SIMPLE DNA-ON-A-CHIP SYSTEM FOR DETECTION OF

CRUSTACEAN ALLERGENIC RESIDUES IN FOOD MATRIX
Nisamanee Charoenchon1, Piyasak Chaumpluk1*
1
Laboratory of Plant Transgenic Technology and Biosensor, Department of Botany,

Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand
Author for correspondence; E-Mail: piyasakcha@gmail.com, Tel. +66 2 185494, Fax. +66 2 185494
Abstract: Food allergy is important health problem

found in wide range of world population. Especially
crustacean allergy, a major allergy group, has easily
recognized among allergenic patients due to famous
edible meat in family of Crustacae. Hence detection of
crustacean through allergenic gene is proper way to
assure food safety in the products. In our study, a simple
and complete lab-on-a-chip system had been developed to
detect trace of crustacean species via a rapid assay on,
allergenic Pen gene. The chip has a design structure
comprised of three separated zones, an inlet, a reaction
zone and an outlet linked together with 100 L
microchannels. The whole processes containing DNA
extraction, Pen gene amplification, and DNA signals
detection occurred in reaction zone in a single chip with
15 l in total volume. DNA extraction was based on
chaotropic effect using trapped ferrous oxide resin under
magnetic field set in portal part of the reaction zone.
DNA was immediately amplified in steady temperature
using primer set highly specific to six domains of Pen
gene. Amplified DNA signals were detected by
fluorescence illumination using minorgroove DNA
binder. The operation time of the system was less than 1
hour. This chip performed at high sensitivity with limit of
detection (LOD) at 100 copies of DNA/reaction.
Moreover, it revealed no cross-reactivity of either other
edible meats or associated species. This chip provided a
basis for a complete assay suitable for field application
requiring less operation time and demanding only small
scale equipments such heat block and UV illuminator for
the detection.
1. Introduction
Food allergy has been recognized as a crucial issue
among all races in the world. Nowadays no certain
treatment can be used to cure this ailment completely
[1].Many countries thus require the food manufactures
to provide the allergenic ingredient labels [2].The rules
of allergenic ingredient label were established to be the
standard controller of import and export food products,
one example of the official regulations is the
Commission Directive 2008/100/EC of 28 October
2008 [3]. The delectable seafood such as crustacean, is
the famous edible meat. These foods contain the major
allergen. The major allergen protein identified in
various shrimps is tropomyosin, i.e., Pen a 1 [4], Pen l
1 [5], Pen o 1 [6], Met e 1 [7] and Pen m 2 [8].
Because crustacean residue can probably contaminate
in food matrix, inspecting crustacean allergen in food
is crucial for health safety and essential following the
directive of national tradition.
Scientific approach to detect allergic residues had

focused on target protein or DNA. However, a cooking
process particularly heat condition is the major cause
of a protein denature [9]. Consequently, allergen
detection based on DNA has been wildly developed
[10]. Currently, an extensive detection based on DNA
method is polymerase chain reaction (PCR)[11].
Previously, the food allergen detection was performed
via real time PCR with primers specific to allergen
gene [12]. Nevertheless, such techniques require
sophisticated laboratory equipments. This method is
irreconcilable with the point of care.
The small device called chip or micro-total analysis
systems (TAS) has been developed to to detect the
target molecule in small scale comfortably. It was
comprised of micro channels or chambers as main
operative area. The chemical reaction in chip system
was resulted from movement of various reagents. The
system can be started or stopped depended on function
diversify of the reaction. To illustrate, sample
preparing solution mixing, target molecule amplifying
and residue detecting had been applied for this device
in micro or nano scale [13].
We have developed the DNA-on-a-chip for rapid
detection crustacean gene in food matrix. Nucleic acid
extraction on device was based on ferrous oxide resin
method. Detection system was took merit of loop
mediated isothermal amplification (LAMP) for single
temperature control [14]. This chip can be used to
check target gene conveniently in all in one process.
Using LAMP for DNA amplification in chip propels
the reactio n co mp letely. T he d emand ing fo r
complicated and expensive equipments to perform
with this chip is less than others methods, such as,
PCR.
2.1 Sample for DNA extraction
Samples used for sensitivity and specificity of
LAMP assay, including six edible crustaceans, i.e.,
shrimp, prawn, flower crab, black crab, lobster and
mantis shrimp including four various fish, namely, nile
tilapia fish, puffer fish, snakehead fish and walking
fish, were collected from Aquatic Animals
Development Center, Ministry of Agriculture and
Cooperative, Thailand. Other edible meat such as pig ,
chicken including ten mercantile food products and
fruit fly are supplied from Food Research and Testing
856
Laboratory, Faculty of Science, Chulalongkorn

University, Thailand. Mouse's genome was supported
by Antibody Production Research Unit, The Institute
of Biotechnology and Genetic Engineering,
Chulalongkorn University.
2.2 Chip design and invention
The microfluidic device in Fig. 1 consisting of the
multi channel inlet part for fluid inletting, a reaction
zone for DNA extraction, DNA amplification and
DNA signals detection and an outlet part for fluid
outlet, was designed to have microchannel dimension
200x100 m channel width and depth with lining of
microchannel troughout the chip. It was fabricated by
standard soft-lithography techniques [15] using
polydimethylsiloxane (PDMS) (Dow Corning,
Midland, Michigan). Holes of 500m diameter were
accessed for the inlet and the outlet to inject and
dispose of the liquid reagent solutions. Fluorinated
ethylene propylene (FEP) tube (0.150.05-mm i.d.)
(BAS Inc., Tokyo, Japan) was inserted into these
holes, and sealed with small amounts of PDMS to
prevent liquid leaks. The PDMS device was
hermetically bonded to the glass substrate via plasma
process by PDC-210 Plasma Cleaner (Yamato, Japan).
Pretreatment of the chip was by flowing 1 g/L of
BSA into microchannel and soaking before use.
Operation of the microfluid in the device was by
manual micromanipulator in Fig. 1 using 1mL syringe
and 20Gx1 needle (Terumo, Japan).
Flow direction
magnetic field
outlet
6
reaction zone
0.81.1cm
inlet
4
1cm
micromanipulator. DNA, once binded to resin

surfaces, was trapped by magnet located at rear part of
reaction zone. The washing solution (80% ethanol)
was subsequently flowed through the reaction zone.
Further air dry was carried out by flowing air slowly.
These processes allowed nucleic acid to be viable and
ready for use in amplification assay.
2.4 Primer design
Primer sets for PCR based on the report from the
previous study [8] were able to amplify Pen m 2. To
establish novel primer sets for LAMP, Pen m 2
sequences were chosen. Selected data of Pen m 2
sequences from GenBank (accession no. GO080505,
GO081310, GO078951, GO078946, GO080195,
GO080171, GO080368, GO079876, GO078320,
GO080541) were input to Primer Explorer V4 (Fujitsu,
Japan) for primer design and primer sequences were
shown in Table 1.
Table 1: Designed LAMP primers to detect Pen m 2
Location
165- 369
(204 bp)
Primer
Pen M 2
FIP
BIP
F3
B3
Sequences (5 to 3)
ctgtggcggtgtagaaggagttgg
acttcatccgtta
ccacccatagggggttgagagga
gaaggagatgtaagaa
ctcactccatccatccggtacg
ggtatccttgagctcatcaa
2.5 DNA amplification and control nucleic acid

augmentation by TA cloning
The whole genome from shrimp (Penaeus
merguiensis) were extracted by Genomic DNA
Extraction Kit (Bioscience, USA) and amplified
through PCR [15] using F3 and B3 primers from Table
1. All DNA products corresponding to crustacean
allergenic gene were cloned into TA vector using TA
cloning approach as described in manufacturer,
protocol (Invitrogen, Madison, USA). This vector was
transfer to E.coli by transformation technique [16].
After that selection nucleotide sequence confirmation
was carried out. For control DNA, the selected clone
was used as a positive reference and a standard
template of known copy number DNA.
Figure 1. Chip structure was composed of 5 injection

channels then joined together at fore part and
connected to reaction area which has magnetic field.
The outlet is solutions exit.
2.6 PCR for crustacean allergenic gene confirmation in

food residue
The total of DNAs extracted from the specimens
were amplified by PCR as previous described in order
to confirm the presence of Pen.
2.3 DNA preparation and extraction

DNA extraction on microfluidic device starts from
crushing of 100 mg of sample in 200 L extraction
buffer containing 100 mM Tris HCl pH 8.0, 1 mM
EDTA, 300 mM NaCl, 2 M guadinidine
hydrochloride, 0.1 mM DTT in 1.5 mL
microcentrifuge tube using micro-pestle. After
spinning down at 12,000 rpm, supernatant having
DNA were transferred to new tube and mixed with
7L of ferrous oxide resin (Promega, Madison USA)
to ensure target DNA capture from the specimen and
injected
into
the
device
using
manual
2.7 LAMP assay

LAMP assay was carried out in a final reaction
volume of 10 l on chip. This total volume was
comprised of 5.05 l of distilled water, 1.25 l of
betaine, 1 l of 10 buffer, 0.4 l of dNTP, 0.3 l of
MgSO4, 0.5 l of LAMP primer set, 0.5 l of Bst
DNA polymerase and 0.1 Evagreen (Biotium Inc,
Hayward, CA, USA). This LAMP solution was
injected to reaction area in chip then incubated at
constant temperature which is 63 C for 40 min by
using Accu BlockTM(Labnet, USA) [14]. LAMP
857
products were analyzed in 3% agarose gel and soaked

in ethidium bromide solution 10 min prior to
visualization on UV illuminator.
2.8 LAMP for sensitivity assay
Shrimp (Penaeus merguiensis) DNA concentration
at 1-106 copies were amplified and analyzed as mention
in 2.7. Chip detection sensitivity was also carried out
using known copies number of DNA from the above.
2.9 LAMP for specificity assay
Shrimp, prawn, flower crab, black crab, lobster,
mantis shrimp, four various fish, namely, nile tilapia
fish, puffer fish, snakehead fish and walking fish were
used as specimen for specificity assay to measure cross
reactivity of designed LAMP primers.
2.10 DNA signal detection on a chip
Normal DNA signals detection was performed via
both agarose gel analysis on 3% of agarose followed
by ethidium bromide staining and UV light
observation. On chip DNA signals detection was
performed under UV light (=365nm). The
fluorescence signals development of samples from
both negative and positive amplifications was
recorded.
3.1 Chip design and invention
Simple chip structure here was designed based on
simplified steps of ferrous oxide resin based nucleic
acid extraction. It was comprised of five inlets, each
of which had 100 x 200 m, wide x depth (No. 1-5),
joined together at fore part, leading to a single
microchannel. Those inlets are for loading sample
solution, washing solution, air drying, amplification
solution and extra channel not in use. A single
microchannel then folded close together (14 folds
here) to form a reaction zone (8x11mm2) where
magnetic field from magnet was located in rare part of
this zone. After folding, microchannel refolded to
single line until it reached to the outlet part. This
differed from most microfluidic devices [17] since the
use of ferrous oxide resin and isothermal nucleic acid
amplification helped to avoid any need of microvalves
and micropumps, all of which increased the cost and
complexity in fabrication and manipulation.
Crustacean allergenic DNA extraction was never
been demonstrated on chip. Lysing the cells was by
strong detergent of guadinidium hydrochloride.
Pretreatment of sample by crushing of the specimen in
extraction buffer using micro-pestle, disrupted the
tissues and cells and released nucleic acid into buffer
solution. Pretreated sample was clarified by
centrifugation to get rid of any trace of debris that
might interferred with flowing capacity within
microchannel. Supernatant was transferred, mixed
with ferrous oxide resin and injected into No. 1. inlet
in Fig. 2 (A). Extraction of DNA on chip was then
carried out based on the binding of the DNA onto the
silica surface at high-ionic-strength chaotropic salt,
followed by a trapping it under magnetic field, set at

rare part of reaction zone. After two times of washing
step with ethanol via inlet No. 2 and drying via inlet
No. 3, the DNA was eluted and amplified
simultaneously by amplification reagent and primer set
designed as a main system detection via inlet No. 4.
In detection part, EvaGreen was used as
intercalator. Mixing this dye to the reaction allow an
on chip detection of DNA signals in term of green
fluorescence light upon UV illumination in fig. 2(B).
magnetic field
outlet
3
reaction zone
inlet
1cm
B
Figure 2. A Reagent transfer onto chip starting from
pre treatment sol. in inlet No. 1, to washing in inlet
No. 2, drying by air flow via inlet No. 3 and detection
solution in No. 4. B The DNA signals detection
system here was visualized based on DNA binding
fluorescence dye.
3.2 Sensitivity test
Standard band of present Pen m 2 was shown in
fig. 3(A). Limit of detection (LOD) of Pen m 2 primer
is at 100 copies of DNA in Fig. 3(B). Thus Pen primer
has high sensitivity. In addition, observation the result
of chip under UV illuminator was valid from DNA
concentration 108-100 copies.
3.3 Specificity test and applicant to real food products
Specificity test of presence Pen m 2 in various
edible meats in fig. 4 (A) and detection Pen m 2 in 11
food products in fig. 4 (B).
Figure 3. A (a) Isothermal amplification product

running on 3% agarose gel, lane M, 100 bp ladder
sizes markers , lane P, amplified Pen m 2 was detected
in Penaeus merguensis genome as standard sample, in
contrary, lane N, no DNA template condition had no
detecting (b) PCR product running on 1.5 % in agarose
gel, 204 bp and sentitivity test B (a) Isothermal
amplification product running on 3% agarose gel, lane
M, 100 bp ladder sizes markers; lane N, negative
control; lane 1, 108 copies; lane 2, 107 copies; lane 3,
858
106 copies; lane 4, 105 copies, lane 5, 104 copies; lane

6, 103 copies; lane 7, 500 copies; lane 8, 100 copies;
lane 9, 50 copies; lane 10, 10 copies; lane 11, 5
copies; lane 12, 1 copies; lane M, 100 bp ladder sizes
markers; amplified Pen m 2 gene products were
detected at concentration of 100-108 DNA copies
(lane 1 -9) and lower concentration of DNA from 50
copies was no detected (lane 10-12). (b) PCR product
running on 1.5 % in agarose gel, 204 bp .
lane 5, f5; lane 6, f6; lane 7, f7; lane 8, f8; lane 9, f9;
lane 10, f10; lane 11, f11; amplified Pen gene products
were detected in food no 1, 2, 4 and 6. In the other
modified foods were not detected. (b) PCR product
running on 1.5 % in agarose gel, 204 bp.
4. Conclusions
An accurate and efficiency on chip detection
method which relies less on laboratory facilities for
Pen is established. The amplified products by LAMP
at 63 C from designed primer set displayed the
ladder-like DNA pattern with multiple loops consisting
of several repeats. No cross reaction was detected from
other types of animals. The entire process is really
simple and rapid; the result can be obtained within an
hour with limit of detection at 100 copies of DNA. In
this study, the application of fluorescent biosensing
which is based on LAMP and fluorescent dye
detection for rapid detection of Pen in crustacean is
reported.
Acknowledgements
Authors would like to thank the Development and
Promotion of Science and Technology talents project
(DPST) for the support. Thanks also go to the
Integration Project: Innovations for the Improvement
of Food Safety and Food Quality for New World
Economy, Chulalongkorn University, Bangkok,
Thailand. All chip design and use had been submitted
to the Patent Registration Off ice, Ministry of
Commerce, Kingdom of Thailand.
References
[1]
Figure 4. (A) Specificity test of presence Pen m 2 in

various edible meats (a) Isothermal amplification
product running on 3% agarose gel, lane M, 100 bp
ladder sizes markers; lane N, negative control; lane 1,
Penaeus merguiensis (shrimp); lane 2, Litopenaeus
vannamei (prawn); lane 3, Portunus pelagicus(flower
crab); lane 4, Scylla serrata (black crab) ; lane 5,
Homarus sp. (lobster); lane 6, Squilla empusa (mantis
shrimp) lane 7, Oreochromis niloticus (nile tilapia
fish); lane 8, Lagocephalus lunaris (puffer fish); lane
9, Channa striata (snakehead fish); lane 10, Clarias
macrocephalus (walking fish); lane 11, Sus domesticus
(pig); lane 12, Gallus gallus (chicken); lane 13, Mus
sp.(mouse); lane 14, Drosophila melanogaster (fruit
fly); lane M, 100 bp ladder sizes markers; amplified
Pen gene products were detected in shrimp, prawn,
crabs , lobster and mantis shrimp (lane 1-6). In the
other edible meats were not detected (lane 7-14). (b)
PCR product running on 1.5 % in agarose gel, 204 bp
(B) Detection Pen m 2 in 11 food products (a)
Isothermal amplification product running on 3%
agarose gel, lane M, 100 bp ladder sizes markers; lane
N, negative control; lane P, Litopenaeus vannamei;
lane 1, food no1 (f1); lane 2, f2; lane 3, f3; lane 4, f4,
[2]
[3]
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K. Watanabe, N. Amino and T. Hase, Nucleic Acids
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Kobayashi, S.R. Rao, Y. Yonezawa, K. Nakano, A.
Hino, S.Yamamura, Y. Takamura and E. Tamiya,
Anal. Bioanal. Chem. 386 (2006) 13271333.
[16] J. Sambrook, E.F. Fritsch and J. Maniatis, Molecular
cloning: a laboratory manual, Cold Spring Harbor
Laboratory Press, New York, USA.
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Actuators B 63 (2000) 138-146.
860
SIMULTANEOUS DETERMINATION OF TOTAL SOLUBLE

PHENOLIC COMPOUNDS AND ANTIOXIDANT CAPACITY IN RICES
Kittitat Tanta1, 2, Kate Grudpan1, 2, Ponlayuth Sooksamiti3, Malyn Chulasiri4, and
Somchai Lapanantnoppakhun1, 2*
1
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University,
Chiang Mai, 50202, Thailand
2
Center of Excellence in Innovation for Analytical Science and Technology, Chiang Mai University, Chiang Mai 50200,
Thailand
3
The Office of Primary Industries and Mines Region 3, Amphur Maung, Chiang Mai, 50202, Thailand
4
Research & Development Division, SJI, Bangkok, 10120, Thailand
*
Author for correspondence; E-Mail: chai40200@gmail.com, Tel. +66 53 941908, Fax. +66 53 941910
Abstract: We report here a preliminary study on the

determination of total soluble-phenolic compounds in
rice samples by employing modified method involving
reduction of iron(III) and complexation with 1,10phenanthroline. This can be monitored for absorbance at
510 nm. Ten rice varieties which distinct in their
characteristics were prepared and extracted with warm
water (55oC) for 20 min. It was found that the
concentrations of total soluble-phenolic compounds
expressed as gallic acid were 2.75 to 89.93 mg gallic
acid/100g rice and caffeic acid were 2.17 to 70.29
mg(CA)/100g rice. Antioxidant capacity values involving
iron (II) generated from the redox reaction were 0.05 to
1.93 mmol Fe(II)/100g rice.
1. Introduction
Rice is a cereal that serves as a staple food in many
countries of Asia, serving either directly as human
foods or indirectly as animal feeds [1-3] and also as a
rich source of natural phenolic compounds and
antioxidants such as phenolic acids, phenylpropanoids,
flavonoids, and so forth. Phenolic compounds exhibit
antioxidant, antimutagenic and anticancer properties
and thus play an important role in maintaining health
[4]. Major phenolic compounds in rice are ferulic acid
and p-coumaric acid and they exist in free, soluble
conjugated and soluble bound forms [5]. Soluble forms
of phenolic compounds usually exhibit as free acid of
a small molecule or conjugate with the carboxyl group
esterified with sugar, quinic, and other organic
acids[6]. The forms can then dissolve a in polar
solvent such as water and methanol.
The analytical techniques for determination of total
phenolic compounds in rice may involve
chromatography, such as high performance liquid
chromatography
(HPLC)
and
gas-liquid
chromatography (GC), and spectroscopy, such as
fourier transform infrared spectroscopy (FTIR),
nuclear magnetic resonance (NMR) and UV-Visible
spectrophotometry [7]. UV-Visible spectrophotometric
methods using Folin-Ciocalteau [8,9], Prussian blue or
1,10-phenanthroline [7] as reagents are widely used for
total
phenolic
compounds
assay.
Sufficient
information for the evaluation of contents of total
phenolic compounds may be obtained by
spectrophotometric measurements employing specific

analytical reagents. In addition, Gonzalez et al. [7]
reported the use of 1,10-phenanthroline complex for
the assay of total phenolic compounds.
The orange-red color of iron(II)-phenanthroline
(Phen) complex is applied in the classical
spectrophotometric method for determination of iron.
However, to the best of our knowledge, there are few
references on the application of the reaction between
iron(II) ions and 1,10-phenanthroline for determination
of phenolic compounds and antioxidant capacity of
edible oils and the other foodstuffs [10]. This reaction
is based on the reduction capacity of phenolic
compounds with iron(III) ions as a reagent to ferrous
ion product. However, larger amounts of regents are
consumed and more chemical wastes are produced.
This report presents the use of iron (III) reduction
and complexation with 1,10-phenanthroline for the
determination of soluble phenolic compounds and
antioxidant capacity in water rice extracts. The method
was modified to reduce the consumption of chemicals.
In addition, iron ore waste (iron (III) source) from iron
ore mines in Northern Thailand was used as a natural
reagent.
2.1 Reagents and solutions preparation
All chemicals were analytical reagent grade.
Deionized water was used to prepare all aqueous
solutions. 0.5 % 1,10-phenanthroline (Fisher
Chemical) was prepared by dissolving 0.5 g in 100 mL
of 10% (v/v) Ethanol in water. Sodium acetate (0.1 M,
pH 4; Carlo Erba) was prepared by dissolving 27.22 g
in 1000 mL of deionized water and adjusted to pH 4
with 0.1 M HCl or 0.1 M NaOH. 0.03 M iron(III)
solution was prepared by dissolving 0.12 g hematite
(70.31 % Fe(III)), which was separated from iron
mining waste by using a magnet, and heating the
solution with 10 mL of concentrated HCl, then
reaching a 100 ml final volume with deionized water.
Stock standard solution of gallic acid GAE (1000
g/mL) was prepared as follows: 0.1106 g of gallic
acid monohydrate (Sigma-Aldrich) was dissolved in
100 mL of 1% (v/v) ethanol in water. The stock
861
solutions of the tested phenolic compounds were

prepared by deionized water in the concentration range
of 0.5000-7.0000 g (GAE)/mL.
Stock standard solution of caffeic acid CA (1000
g/mL) was prepared as follows: 0.1031 g of caffeic
acid (Fluka) was dissolved in 100 mL of 1% (v/v)
Ethanol in water. The stock solutions of the tested
phenolic compounds were prepared by deionized water
in the concentration range of 0.5000-7.0000 g (CA)/
mL.
Stock standard solution of iron(II) (0.1 M) was
prepared as follows: 2.7801 g of iron(II) sulphate
heptahydrate (Sigma-Aldrich) was dissolved in 100
mL of deionized water. The stock solutions of the
tested antioxidants were prepared by deionized water
in the concentration range of 0.0050-0.3000 mM.
2.2 Sample Preparation of rice extracts
Rice samples (5-10 g) were extracted with 50 ml
of water for 20 minutes at 55oC, filtered through filter
paper and centrifuged at 70 rpm for 10 min. The clear
aqueous extracts were analyzed for phenolic
compound contents and antioxidant capacity.
2.3 Total phenolic compounds assay
2 ml of standard or sample solutions was pipetted
into 25 mL of volumetric flask. 0.5 mL of 0.03M
iron(III) and 0.5 mL of 0.5% 1,10-phenanthroline
solution were added until a 25 mL final volume with
0.1 M CH3COONa buffer (pH 4) was obtained. The
solution was incubated at 37oC for 3 h. The orange-red
solution was measured at 510 nm by UV-Vis
spectrophotometer (Shimadzu
UV-1800). The
absorbance result of the sample was compared with the
standard calibration curve of gallic acid and caffeic
acid.
either of the 1,10-phenanthroline procedures, iron(III)

forms a 1:3 chelate with the phenanthroline ligand (the
mixtures
were prepared to contain three
phenanthroline molecules per ferric ion), this redox
potential is shifted to higher values due to selective
stabilization of iron(II) ion with these ligands
(e.g., E0 = 1.06V for the iron(III), iron(II)-phen
couple). Since Fe(phen)32+ has a higher conditional
stability constant than Fe(phen)33+ for both
phenanthroline ligands, these reagents have higher
potentials than 0.77V. These analytical methods were
used to determine the antioxidant capacities of
vegetables oils [13]. The orange-red chelate
[Fe(phen)3]2+ (max,510 nm, 510= 1.12 x 104 L.mole1
.cm-1) is well known as a very stable complex cation
[13-14]. However, a larger amount of reagents and
more analytical time are consumed. In this work
analytical parameters such as concentration of ferric
ion, pH of buffer, temperature and reaction time were
studied for reducing the amount of reagent and
analytical time.
3.1 Effect of iron(III) concentration
The concentration of iron(III) ion can affect the
degree of oxidation of phenolic compounds such as
garlic acid. 2.0 mM iron(III) ion is recommended in
the literature. That concentration is in excess for
reaction. But transition metal waste is produced to the
environment. Thus, iron(III) ion concentrations (0.22.0 mM) were studied by using 0.5000-5.0000 g/mL
of gallic acid. The results are shown in Figure 1 and
Table 1. The figure shows that the iron(III) ion
concentration of 0.2-2.0 mM provided good linear
calibration and the same sensitivity was obtained.
Then, 0.6 mM iron(III) ion was selected for further
studies.
2.4 Total antioxidant capacity [11, 12]

2 ml of sample solutions was pipetted into a 25 mL
volumetric flask. 0.5 mL of 0.03M iron(III) and 0.5
mL of 0.5% 1,10-phenanthroline solution were added
until a 25 mL final volume with 0.1 M CH3COONa
buffer (4 pH) was obtained. The solution was
incubated at 37oC for 20 min. The absorbance of an
orange-red solution was measured at 510 nm by UVVis spectrophotometer. The absorbance result of the
sample was compared with the standard calibration
graph of iron(II) ion and 1,10-phenanthroline complex.
The relevant chemical reaction of the Phen
methods should be presented with a reducing agent A
as an analyte, as shown in the following reaction
equation:
Fe(III)L + Are Fe(II)L + Aox
Figure 1. Effect of iron(III) ion concentration on the

calibration curve of gallic acid; : 2.0 mM; : 1.0
mM; : 0.6 mM; : 0.2 mM; : 0.1 mM.
Aox is the oxidized form of the analyte A in

reduced form such as phenolic compound or
antioxidant etc., where L is the iron(II)-selective
chromogenic ligand (1,10-phenanthroline). The redox
potential of the Fe3+/Fe2+ couple is 0.77V, but since in
862
Table 1: The linear calibration of gallic acid for

different concentrations of iron(III) ions
Concentration of
Fe(III) (mM)
0.1
0.2
0.6
1.0
2.0
Linear calibration
R2
y = 0.3036x - 0.0047
y = 0.3120x - 0.0041
y = 0.2871x + 0.0143
y = 0.2872x + 0.0451
0.9985
0.9998
0.9987
0.9978
3.2 Effect of pH
The iron(III)-phen complex may be various species
at different pH of the solution and the redox reaction
between iron(III)-phen and phenolic compounds is
also dependent on pH. Therefore, the effect of pHs on
reduction of iron(III) ion and gallic acid were
examined over pH range of 1-6. The results are
presented in Figure 2 and Table 2. It was found that
good sensitivity was obtained on pH 3-5. Thus, the pH
of 4 was selected for further studies.
Figure 2. Effect of pH on the calibration curve of

gallic acid for: pH 1; : pH 2; : pH 3; : pH 4; +: pH 5;
o: pH 6.
Figure 3. Effect of temperature

temperature (23oC); : 370C.
for:
room
3.4 Effect of reaction time

The effect of reaction times (20min, 1, 2, 3, 6, 12
and 24 h) was studied by using 1 g/mL of gallic acid
and caffeic acid. The results are shown in Figure 4. It
was found that the absorbance reached a plateau at 3 to
24 hours. It means that the reaction is constant after 3
hour. So 3 hours was selected for this reaction and was
used for total phenolic compounds in rice water
extract.
Figure 4. Effect of reaction time on phenolic

compounds with iron(III)-phen for: gallic acid; :
caffeic acid.
Table 2: The linear calibration of gallic acid for pH

pH
1
2
3
4
5
6
Linear calibration
y = 0.0833x - 0.0113
y = 0.3270x - 0.0003
y = 0.3716x + 0.0216
y = 0.3915x + 0.0025
y = 0.3552x + 0.0219
y = 0.3111x + 0.0093
R2
0.9986
0.9995
0.9987
1.0000
0.9991
0.9999
3.3 Effect of temperature

Room temperature and 24 h for this method are as
recommended in the literature. The rate of reaction can
be increased by temperature. Room temperature
(23oC) and 37oC, which was the same temperature for
antioxidant capacity determination, were both studied.
The results are shown in Figure 3. The results indicate
that the temperature at 37oC provided better sensitivity
than at room temperature. A temperature of 37C was
selected for use in this method.
3.5 Summarized analytical parameters

The suitable parameters for total phenolic
compounds and total antioxidant capacity of the
proposed method were studied. Those parameters are
summarized and shown in Table 3 and Figure 5. It
indicated that the proposed method used less chemical
and time consumption than reference method [7, 12].
3.6 Working range of the standard calibrations
The working ranges of standard gallic acid and
caffeic acid calibrations were studied. It was found
that the standard calibrations in the range of 0.50007.0000 g/mL of gallic acid and caffeic acid were
obtained as shown in Figure 5. The linear calibrations
are y = 0.4398x + 0.0470; R2 = 0.9994 for gallic acid
and y = 0.4843x + 0.1345; R2 = 0.9961 for caffeic
acid. The detection limits are 0.0347 g/mL of gallic
acid and 0.0959 g/mL of caffeic acid.
The working ranges of iron(II) calibrations were
studied. It was found that the standard calibrations in
the range of 0.0050-0.3000 mM of iron(II) were
863
obtained as shown in Figure 6. The linear calibrations

is y = 11589x + 0.0421; R2 = 0.9997 for iron(II).
Figure 6. Working range for 0.0050-0.3000 mM of

iron(II).
Figure 5. Working range for 0.5000-7.0000 g/mL
of: gallic acid; : caffeic acid.
Table 3: Summarized parameter of proposed method and compared with reference method
Parameter
Total phenolic compound
Total antioxidant capacity
Proposed method
Proposed method
Ferric ion concentration

1,10-phenentholine concentration
pH
Referee method[7]
Referee method[12]
0.6 mM
2 mM
0.6 mM
2 mM
0.5% w/w
0.5% w/w
0.5% w/w
0.5% w/w
3-4
Room temperature
(23oC)
24 h
37 C
370C
20 min
20 min
Temperature
37 C
Time
3h
Table 4: Total water soluble-phenolic compounds in rice samples

Total soluble-phenolic compounds
Sample
Proposed method
Referee method[7]
Proposed method
Referee method[7]
mg (GAE)/100g
mg (GAE)/100g
mg (CA)/100g
mg (CA)/100g
Organic black rice
46.67
37.31
36.57
28.18
Purple brown rice
89.93
81.10
70.29
65.57
Red fragrant rice
42.04
36.68
32.86
29.48
Organic pink jasmine rice
65.06
49.97
50.81
40.83
Organic red jasmine rice
51.64
44.74
40.35
36.36
Pounded red rice
44.71
37.76
34.95
30.40
Thai jasmine rice
2.75
2.52
2.17
1.54
Japanese rice
5.75
4.96
4.51
3.63
Basmati rice
6.69
6.51
5.25
4.95
Glutinous rice
4.35
3.82
3.42
2.65
3.7 Determination of total water soluble-phenolic compounds and total antioxidant capacity in rice
This proposed method was applied to determine
total water soluble-phenolic content and total
antioxidant capacity in ten rice varieties, as results
shown in Table 4 and Table 5, respectively. It was
found that the contents of total water soluble-phenolic
compound expressed as gallic acid were 2.75 to 89.93
mg (GAE)/100g rice and caffeic acid were 2.17 to
70.29 mg (CA)/100g rice. Antioxidant capacity values
involving ferrous generating from the redox reaction
were 0.05 to 1.93 mmol Fe(II)/100g rice. These results

were close to the values determined from the referee
method [7, 12].
4. Conclusions
This proposed method could be applied for total
water soluble-phenolic compounds and total
antioxidant capacity in rice samples. The advantages
of this method are that it is simple, convenient, precise,
accurate and fast. The proposed method could be used
864
for simultaneous determinations of total phenolic

compounds and antioxidant capacity.
Table 5: Total antioxidant capacity in rice samples
[12] S. Koch, G. Ackermann and P. Lindner, Talanta 39

(1992) 693-696.
[13] K.I. Berker, K. Guclu, I. Tor and R. Apak, Talanta 72
(2007) 1157-1165.
[14] K. Chvatalova, I. Slaninova, L. Brezinova and J.
Slanina, Food Chem. 106 (2008) 650-660.
Total antioxidant capacity

Sample
Proposed method
mmol Fe(II)/100g
Organic black
rice
Purple brown rice
Referee method[12]
mmol Fe(II)/100g
0.83
0.86
1.93
2.08
Red fragrant rice

Organic pink
jasmine rice
Organic red
jasmine rice
Pounded red rice
0.88
0.93
1.20
1.11
1.05
0.96
0.87
0.92
Thai jasmine rice
0.05
0.07
Japanese rice
0.11
0.11
Basmati rice
0.16
0.17
Glutinous rice
0.08
0.09
Acknowledgements
Agricultural Research Development Agency, The
Center of Excellence for Innovation in Chemistry:
Postgraduate Education and Research Program in
Chemistry (PERCH-CIC), The Office of Primary
Industries and Mines Region 3, Chiang Mai and The
Center of Excellence for Innovation in Analytical
Science and Technology (I-ANALY-S-T) are
acknowledged for the support.
References
[1] M. Bunzel, J. Ralph, J.M. Marita, R.D. Hatfield and H.
Steinhart, J.Sci. Food Agric. 81 (2001) 653-660.
[2] O. Pourali, F.S. Asghari and H.Yoshida, Chem. Eng. J.
160 (2010) 259-266.
[3] J. Vichapong, M. Sookserm, V. Srijesdaruk, P.
Swatsitang and S. Srijaranai, LWT - Food Sci. Technol.
43 (2010) 1325-1330.
[4] C.K. Zhai, C.M. Lu, Q. Zhang, G.J. Sun and K.J.
Lorenz, J. Food Compos. Anal. 14 (2001) 371-382.
[5] F. Sosulski, K. Krygier, and L. Hogge, J. Agric. Food
Chem. 30 (1982): 337.
[6] L. Bravo, Nutr Rev. 56(1998) 317-333.
[7] M. Gonzalez, B. Guzman, R. Rudyk, E. Romano and
A.A. Molina, Lat. Am. J. Pharm. 22 (2003): 243-248.
[8] M. Yam-Tak and Peter C. K. Cheung, J. Agric. Food
Chem. 55 (2007) 4222-4228.
[9] K. Leamsomrong, M. Suttajit and P. Chantiratikul,
Asian J. Applied Sci. 2 (2009) 184-190.
[10] R. Apak, K.Guclu, B. Demirata, M. Ozyurek, S.E.
Celik, B. Bektasoglu, K.I. Berker and D. Ozyurt,
Molecules 12 (2007) 1496-1547.
[11] A. Szydlowska-Czerniak, C. Dianoczki, K. Recseg,G.
Karlovits and E. Szlyk, Talanta 76 (2008) 899-905.
865
SUPPLEMENTATION OF PREBIOTIC FROM KLUAI NAMWA Musa (ABB group)

AND Bacillus S11 PROBIOTIC IN PACIFIC WHITE SHRIMP Litopenaeus vannamei
Ratchana Boonmee1 and Sirirat Rengpipat1,2*
1
Program in Biotechnology, 2 Department of Microbiology, Faculty of Science, Chulalongkorn University,

Bangkok 10330, Thailand
*
Author for correspondence; sirirat@sc.chula.ac.th, Tel. +66 22 185081, Fax. +66 22 527576
Abstract: Practicing probiotic(s) and prebiotic(s)

approach is known as Synbiotic(s) which denotes a
mutual benefit on shrimp health. Regular feed
supplemented
with
Bacillus
S11(BS11)-probiotic
bacterium and Musa (ABB group) Kluai Namwa (BN)
of Pacific white shrimp Litopenaeus vannamei were
prepared and used for cultivation in closed-recirculating
high-density polyethylene (HDPE) tanks (~200 Litres) for
90 days. Eight treatments as follows: regular (as a
control),
BS11-,
10%BN-,
20%BN-,
30%BN-,
BS11&10%BN-, BS11&20%BN-, and BS11&30%BNsupplemented feeds were conducted. Average daily gain
of shrimp fed BS11&10% BN-feed was significantly
higher (P<0.05) than that of control. Survival and
production of shrimp fed any BS11- feeds were higher
than the one of control significantly at P<0.05. After 3
days of immersion challenge tests by the luminescent
bacterium Vibrio harveyi 639, cumulative death (%) of
90, 40, 33.33, 40, 40, 33.33, 43.33, and 56.67 were detected
in shrimp fed regular, BS11-, 10%BN-, 20%BN-,
30%BN-,
BS11&10%BN-,
BS11&20%BN-,
and
BS11&30%BN- supplemented feeds, respectively. Thus,
the results from this study confirm possibility to use
BS11 and/or BN as alternatives to antibiotics in farming
process which will lead to organic industry and benefit in
marketing aspect by adding more value to shrimp
products.
1. Introduction
A new definition of probiotics was proposed as:
Live microorganism that survive passage through the
gastrointestinal tract and have beneficial effect on the
host by FAO/WHO (2002). It has been known that
Bacillus S11 is one of the probiotic bacteria isolated
from healthy Black tiger shrimp Penaeus monodon
could enhance shrimp immunity against shrimp
pathogen Vibrio harveyi [1, 2]. Whereby, Kluai
Namwa [Musa (ABB group)] was hybrided from the
merging between Musa acuminata Colla and Musa
balbisiana Colla, these two are typical fruits which
contain considerable amounts of inulin as a prebiotic
that help promoting the growth of bacteria within
small intestine reach the colon, thus proving to be an
advantageous initiative for health improvement[3].
Chemically, inulin (IN) and oligofructose (OF) can be
categorized as polydisperse fructans. These certain
fructans are typically linked by (21)fructofuranosyl bonds consisting of a glucose moiety
that typically resident at the end of most fructose
chain, whereas a small branched-chain fraction was
also comprised within the structure as well (12% of
-2,6-linkages; [4]). Additionally, OF is always
present in IN as a blend of fructose oligomers and

polymers, which differs from IN as based on the
degree of polymerisation (DP; 2 DP 10 for OF; 2
DP 60 for IN). OF conversion from IN is normally
processed through controlled partial enzymatic
hydrolysis, which resemble the chicory roots
processing[5]. The amendment of Inulin-derived
Fructooligo-Saccharide into regular animal feed has
illustrated a broad inhibiting effect on Escherichia
coil, Salmonella spp., Clostridium spp., Eubacteria,
Enterobacteria and Coliforms [6]. Briefly, this
protecting effect is attributed to each unique
characteristic of inulin and FOS. In this case, the inulin
itself functions as water absorber that balances the
humidity within the microenvironment. In addition,
owning to short fatty acid chain of the FOS that
facilitates the absorption of mineral. In addition,
owning to short fatty acid chain of the FOS that
facilitates the absorption of mineral; thereby, this
characteristic help preventing osteoporosis in animals
and humans. Furthermore, as the fat synthesis in liver
and vascular wall was reduced by the effect of this
molecule, therefore, the level of fat and cholesterol in
blood will also be affected as well, thus minimizing
the risk of heart disease [7].
Taken together, these approaches improve shrimp
health by making it more tolerable to pathogen thereby
resulting in a significant increase in shrimp size and
survival rate. As for Pacific white shrimp farming, its
economically considered as one of a major industry in
Thailand. Thus, were aiming on an improvement of
Pacific white shrimp culturing technique while
ensuring food safety along with environmental
friendly process. This work will employ both of the
probiotic BS11 and prebiotic Kluai Namwa as a
supplement to a regular shrimp dietary feed instead of
using synthetic agent.
2.1 Bacterial strains
Bacillus S11 was cultured in Tryptic soy broth with
a shaking incubator at 200 rpm, 30C. After 24 h,
washing with normal saline solution and centrifuging
three times at 8,000 rpm, 4C for 15 min. V. harveyi
639 was cultured in Tryptic soy broth, agar with 2%
NaCl (w/v), and Thiosulfate citrate bile salt sucrose
agar, and used for inducing infection during challenge
test in the experiment.
2.2 Banana extracts preparation
866
The 10g banana was obtained by peeling, slicing,

and homogenization, diluted with 100 ml of hot water
at 100 C for 10 minutes and centrifuged at 8,000 rpm,
4C for 10 min to remove suspended particles. The
banana extraction liquid was stored at 4C for further
use.
was collected and recorded daily. For every dead

shrimp, the hepatopancreas-intestine was dissected and
examined for the presence of V. harveyi 639 initially
by isolation on TCBS agar and followed by
identification as V. harveyi as described by Bauman
and Schubert [11].
2.3 Analysis of inulin

The banana extraction liquid (BN) was diluted
before determination. Total carbohydrate was
determined by the phenolsulphuric acid method of
Dubois et al. using inulin as standard [8]. Reducing
sugar was determined by the dinitrosalicylic acid
method using D(-)-Fructose as standard[9]. The inulin
content was measured with the difference between
total carbohydrate and reducing sugars. Inulin
extraction yield (%) = (inulin content x volume of
extraction liquid/mass of powder) x 100[10].
2.6 Statistics
All data are presented as meansSE (standard error
of the mean) of supplementation with prebiotic from
Kluai Namwa Musa (ABB group) and Bacillus S11
probiotic on shrimp growth, survival and disease
resistance in respective trials were compared using
analysis of variance (ANOVA)and Duncans multiple
range tests (SAS software). A significant level of p <
0.05 as the confident level for significant differences
between the tested treatments.
2.4 Shrimp feed preparation

Commercial shrimp fed a commercial (CPF feed,
Thailand) was used as regular Pacific white shrimp
dietary feed for the supplementation of probiotic and
prebiotic. Shrimp feed was mixed with banana
extraction liquid at 10%, 20%, and 30% (as a
10%BN-, 20%BN-, and 30%BN-supplemented feeds,
respectively). Fresh Bacillus S11 cells were
thoroughly mixed with regular shrimp feed and BNsupplemented feed at 1:3 ratio (as a BS11,
BS11&10%BN-, BS11&20%BN-, and BS11&
30%BN-supplemented feeds, respectively), the
mixture was spread out and dried in an oven for 12 h
at 37C. Fresh Feed was then stored in clean, plastic
bags at 4C until used. Each batch was analyzed for
Bacillus S11 and total bacterial count.
2.5 Experimental diets and feeding
Litopenaeus vannamei were obtained from a
shrimp farm in Pathumthain Province, Thailand and
grew to 12 g in an aerated, closed-recirculating water
system at 30C and 25% salinity. Shrimp age was
PL-20 at the first day of culture. Shrimp were placed
in 200-l high-density polyethylene (HDPE) tanks.
Eight treatments as follows: regular (as a control),
BS11-, 10%BN-, 20%BN-, 30%BN-, BS11&10%BN-,
BS11&20%BN-, and BS11&30%BN-supplemented
feeds. Each treatment was performed in triplicate, each
containing 30 shrimp. Shrimp were fed three times
daily at 10% body weight per day. The following
measurements were made every 30 days for 90 days:
shrimp live weight and survival; total bacteria,
Bacillus S11, and Vibrio spp. counts from rearing tank
water, and shrimp intestines. After feeding for 90
days, shrimp in each treatment were exposed to
pathogenic Vibrio harveyi 639 in tank water at ~107
CFU ml1. No water was exchanged thereafter for the
duration of the 5 days trial. During challenge tests,
water quality parameters and shrimp survival were
measured every 24 h as in the field trials. Shrimp
samples and 100 ml water from the challenge tanks
were randomly collected for detection of V. harveyi
639 and BS11. The cumulative number of dead shrimp
3.1 Inulin extraction yield and growth profile of

Bacillus S 11
Average inulin yield extraction from Kluai Namwa
[Musa (ABB group)] were summarized in Table 1.
The most content of inulin (%w/w) was found in ripe
BN. Thereby, inulin from ripe BN were prepared as a
mass extraction product from the same batch of ripe
BN and kept at 4C before use throughout our
experiment.
Table1: Contents of BN extraction*
Contents of BN
Inulin extraction yield

Raw BN
Mature
BN
Ripe BN
Total
169.07.80 148713.5 188326.4
carbohydrate (mg)
Reducing
7.6260.67 581.68.19 625.114.0
sugar(mg)
Inulin (%w/w)
1.6140.07 9.0030.18 12.580.33
*Values represent from triplicate analysis

Bacillus S11 a probiotic strain was cultured under
the same experimental conditions in different media:
TSB, TSB without dextrose, TSB without dextrose
supplemented with inulin, and TSB without dextrose
supplemented with ripe BN extraction. Viable cell
counts vs time were monitored for 24 h at 37 oC.
Different growth profiles of Bacillus S11 were shown
in Figure 1. Highest growth rate of cells was detected
when cultured in TSB without dextrose supplemented
with inulin. No difference of growth rate of Bacillus
S11 cultured in TSB and TSB without dextrose
supplemented with ripe BN extraction. The lowest
growth rate was found in cells cultured in TSB without
dextrose. However, the same total cell counts after 10
h of culture were determined. Therefore, ripe BN
extraction could replace dextrose in TSB as carbon
source for Bacillus S11 culture which may due to ripe
BN extraction is composed of inulin and certain sugar.
Concurrently, our results also confirmed that inulin
can be used and clearly supported our probiotic
867
bacteria growth. BN extraction supplemented

feed in this study was prepared from ripe BN.
into
Figure 1. Growth profiles of Bacillus S11 with viable

cell number vs time under conditions: TSB, TSB
without dextrose, TSB without dextrose supplemented
with inulin, and TSB without dextrose supplemented
with BN extraction.
3.2 Shrimp growth and survival
Average ranges of water quality in the eight feed
treatments during shrimp culture were as follows:
dissolved oxygen (4-6 mgl-1), pH (7.5-8.4), ammonia
(0-0.50 mgl-1), nitrite (0-0.50 mgl-1). Which of these
values were safe for shrimp culture. After shrimp fed
regular feed and BS11 and/or BN supplemented feeds
for 90 days, live weights of shrimp fed BS11-or BNsupplemented
feeds
were
not
significantly
different(p>0.05) from shrimp fed regular feed while
significant difference (p<0.05) were found in shrimp
fed both BS11 and BN-supplemented feeds especially
in shrimp fed with BS11&10%BN-supplemented feed.
Furthermore, the outcome of average daily gained
were the same trends as mentioned above were
determined (Figure 2A&2B).
Production of shrimp fed any BS11-supplemented
feeds were significantly greater (P<0.05) than control
group whereas no significant difference (P<0.05) of
shrimp fed BN-supplemented feeds among those
groups. The most production of shrimp (380.8736.22
g) fed BS11&10%BN- supplemented feeds was found
but only 247.1149.95 g of shrimp yields from
feeding with regular feed (Figure 3). Shrimp survival
after 90 days was significantly greater (P<0.05) in the
regular feeds supplementation with BS11 (Figure 4).
Figure 2. Live weights (A) and average daily gain (B)

of L. vannamai during 90 days of culture fed with:
regular feed (control), and BS11 and/or BN- feed
supplementation.
Figure 3. Productions of L. vannamai during 90 days

of culture fed with: regular feed (control), and BS11
and/or BN- feed supplementation.
Figure 4. Survivals of L. vannamei reared with and

without supplementation treatment groups.
A
3.3 Survival (%) of shrimp after challenged with V.
harveyi 639
Shrimp collected from previous culture with
different feed conditions were collected for
challenging tests. After challenging with V. harveyi
639 (~107 CFU ml -1) by immersion for 3 days,
cumulative death (%) of 90, 40, 33.33, 40, 10, 33.33,
43.33, and 56.67 were determined in shrimp fed
regular,
BS11-, 10%BN-, 20%BN-, 30%BN-,
BS11&10%BN-, BS11& 20%BN-, and BS11&
30%BN- supplemented feeds, respectively. Besides,
disease resistance was clearly found in shrimp fed
868
BS11- and/or BN supplemented feed due to survival

after 5 days of challenging was 60%, 56.67%, 46.67%,
for shrimp fed BN-supplemented, 50%, 36.67%, 26.67
% for shrimp fed BS11&BN-supplemented, and
46.67% for shrimp fed shrimp fed BS11-supplemented
while shrimp fed regular feed all died within 4 days
(Figure 4). Accordingly, Bacillus S11 may produce
some anti-microbial such as bacteriocins or byproducts negatively affected V. harveyi 639[1, 2].Or,
Bacillus S11 can act as residential flora in shrimp
intestines and protect L.vannamei against bacterial
infection by competitive exclusion [2].
References
[1] S. Rengpipat, W. Phianphak, S. Piyatirativorakul, and P. Menasveta, Aquaculture. 167
(1998) 301-313.
[2] S. Rengpipat, S. Rukpratanporn, S. Piyatirativorakul, and P. Menasveta, Aquaculture. 191
(2000) 271-288.
[3] G.R. Gibson and M.B. Roberfroid, J. rtuN. 125
(1995) 14011112.
[4] J. Van Loo, P. Coussement, L. De Leenheer, H.
Hoebregs and G. Smits, Crit. Rev. Food Sci.
Nutr.35(1995) 525-552.
[5] E.A . Flickinger, J. Van Loo and G.C. Fahey,
Crit. Rev. Food. Sci. Nutr. 43(2003) 1960.
[6] E.J. Vandamme and D.G. Derycke, Adv. Appl.
Microbiol. 29(1983) 139-176.
[7] L.S. Boeckner, M.I Schnepf and B.C. Tungland,
Adv. Food .Nutr. Res. 43(2001) 1-63.
[8] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A.
Rebers and F. Smith, Anal. Chem. 28(1956) 350
356.
[9] G.L. Miller, Anal.Chem. 31(1959) 420-428.
[10] W. Lingyun and other, J. Food. Eng. 79(2007)
1087-1093.
[11] P. Baumann and R.H.W. Schubert, in: N.A.
Krieg, J.G. Holt, (Eds.), Bergeys Manual of
Systematic Bacteriology vol. 1, William &
Wilkins, Baltimore, (1984), pp. 516-549.
Figure 5. Survival (%) of L. vannamei during 5 days of

challenging by Vibrio harveyi 639.
4. Conclusions
This study demonstrates the supplementation of
probiotic and prebiotic in regular feed can significantly
improve disease resistance in Pacific white shrimp by
enhancing immunity, as well as presumably
modulating microflora in the Pacific white shrimps
digestive tracts. The present work is the first study
demonstrated that Bacillus S11 and FOS had a
synergistic effect on enhancing the growth and disease
resistance of L. vannamei. This may be of great
interest to those involved in aquaculture research and
the shrimp farming industry.
Acknowledgements
This research was supported by
the Thai
Government Stimulus Package 2 (TKK 2555)
under
the
Project
for Establishment
of
Comprehensive Center for Innovative food, Health
Products and Agriculture
(PERFECTRA);
the
Higher Education Research Promotion and National
Research University Project of Thailand, Office of the
Higher Education Commission
(FW643A) and
partially granted from the 90th Anniversary of the
Chulalongkorn University fund (Ratchadaphiseksomphot Endowment Fund).
869
THE DIRECTED EVOLUTION OF ALPHA-AMYLASE GENES BY USING

DNA SHUFFLING TECHNIQUE
Sirima Sukasem1*
*
1
Innovative Learning Center, Srinakharinwirot University, Bangkok 10110, Thailand
Author for correspondence; E-Mail: jewysukasem@hotmail.com, Tel. +66-2204-2704, Fax. +66-2204-2704
Abstract: DNA shuffling is a powerful method of directed

evolution to improve the property of enzymes at gene
level. Two alpha-amylase genes from two Bacillus
licheniformis strain DSM13 and DSM 8785 were used as
a model. They were cloned into pET-21d(+) and pFLAGCTS. The library construction of shuffled alpha-amylase
was created by recombination of error prone PCR and
followed by DNA shuffling. Multiple alignment analysis
showed that shuffled no. 2a11h had the highest mutation
points on nucleotide sequence. The termination codon
(TAA) was also occurred. The alteration of nucleotide
sequence was occurred by changing between each purine
or purimidine base, or by replacing of pyrimidine into
purine base. One to six points of amino acid mutation
were presented. The shuffled alpha-amylase no.2a11h,
no.2b8b, and no.4d2d contained shuffled amino acids
between two alpha-amylases. The phylogenic tree showed
that sequences of shuffled alpha-amylases no. 1b9a, 2e2a,
9g7d, 2b7g, 1c5b, and 7c12h were not closely related to
their native alpha-amylase B. licheniformis DSM 8785 or
DSM13. The shuffled no.1b9a, no.2e2a, no.9g7d and
no.2b7g had high evolution. Therefore, DNA shuffling
generates the diversity of gene that makes the high
evolution of protein. This method can be applied for
other proteins in various applications.
1. Introduction
Directed evolution is powerful tool for modification
of protein properties. DNA shuffling was developed by
Stemmer [1]. The key of DNA shuffling is not only
recombine DNA fragments, but also introduce point
mutation at low rate [2] or create multiple crossover in
reassembled sequences [3].
Alpha-amylase (E.C.3.2.1.1) or alpha-1,4-glucanglucanohydrolase is endo-hydrolyzing enzyme which
hydrolyzes starch, amylose, amylopectin, and various
maltodextrin by randomly cleaved alpha-1,4glycosidic linkage. The primary structure is TIM barrel
or parallel (/)8, which is in Domain A. Domain A
forms the core of structure and contains the active site.
Domain B is formed between 3rd strand and 3rd helix of
TIM-barrel and it forms a large part of the substrate
binding cleft [4]. Domain C is C-terminal part which
contains Greek key motif, beta-sandwich structure [5].
Alpha-amylase has four highly conserved regions
(region I, II, III and IV) [4, 6]. These regions are
related to the catalytic and substrate-binding site of
enzyme.
2.1 Bacterial strains and cultivation conditions
B. licheniformis DSM 8785 and DSM 13 were

cultured on M1 culture medium at 30C and 37C
respectively, at 200 rpm where as E. coli DH5F and
E. coli BL21(DE3) were grown in LB medium at
200 rpm and 37C.
2.2 Construction of alpha-amylase expression vector
The genomic DNA of Bacillus strains were boiled
at 95C for 5 min and put on ice immediately. Two
primer sets (3-BspHI and 5-XhoI or 3-EcoRI and 5XhoI) were designed based on sequence no. X03236
from Genbank to create 1.5 kb of alpha-amylase gene
with/without signal peptide. PCR reaction consisted of
1x pfu DNA polymerase buffer, 2.9 mM MgCl2, 0.25
mM of each dNTP, 0.25 M of primer, and 3 U of pfu
DNA polymerase. Thirty cycles of PCR were steps of
95C for 45s, 55C for 1 min, and 72C for 4 min. The
alpha-amylase gene with signal peptide were cloned
into pET21d(+) to create rpET13, rpET8785 whereas
genes without signal peptide were inserted into
pFLAG-CTS to generate rpFL13 and rpFL8785.
2.3 Sequence analysis
DNA sequences were analyzed by DNA sequencing
service unit of Macrogen Inc (World Meridian Venture
Center, Seoul, Korea). Multiple alignment and
phylogenic tree were analyzed by vector NTI program.
The similarity was determined by ClustalW2 from
EMBL-EBI software on-line. The 2nd structure was
done by Espript 2.2 program.
2.4 Library construction of shuffled alpha-amylase
2.4.1 Error prone PCR
DNA sequences from rpET13, rpET8785, rpFL13
and rpFL8785 were used for PCR. The reactions
contained 6.24 ng of DNA, 1xTaq DNA polymerase
buffer, 0.2 mM of each dATP and dGTP, 1.0 mM of
each dCTP and dTTP, 2.9 mM MgCl2, 0.5 mM MnCl2,
5 U of Taq DNA polymerase and 0.3 M of primers.
The 45 cycles were steps of 95C for 45s, 55C for 1
min and 72C for 2 min.
2.4.2 DNaseI digestion
The random fragments size of DNA was
performed at 15C for 7 min. The reaction consisted of
DNaseI at 0.1-1 U/g DNA (error prone PCR),
50 mM Tris-HCl pH 7.4 and 10 mM MnCl2.
Fragments size from 50 bp to 250 bp were cut and
removed by 3 M ammonium acetate at 37C for18 hr.
The purification was performed by phenol:choloform
extraction and ethanol precipitation.
2.4.3 Reassembled PCR into full length
870
Amplification without primers was undertaken.

The 50 l contained 30 l of fragments, 1x pfu DNA
polymerase, 25 mM of each dNTP and 2.5 U of pfu
DNA polymerase. Amplification was applied by steps
of 96C for 2 min and followed by 30 cycles of 95C
for 45 s, 55C for 1.30 min and 72C for 2 min+5
s/cycle, and 72C for 10 min.
2.4.4 Amplification of shuffled alpha-amylase
The 50 l consisted of 1 l of reassembled DNA,
1x pfu DNA polymerase buffer, 0.25 mM each of
dNTP, 2.9 mM MgCl2, 3 U of pfu DNA polymerase
and 2.5 U of Taq DNA polymerase and 0.25 M
primers. The amplification was done by 35 cycles of
95C for 45 s, 55C for 1 min, and 72C for 4 min.
2.5 Library screening of shuffled alpha-amylases
2.5.1 Primary screening. The rpET-SH and rpFLSH libraries were screened on LB agar plus 1%(w/v)
soluble starch and 100 g/ml ampicillin. Vector
pET21d(+) and pFLAG-CTS were negative. The
positive control was rpET8785 and rpFL8785.
2.5.2 Secondary screening of rpET-SH library.
The screening was performed at pH 3.5 and pH 9.5.
The control activity was done at pH 6.5. Reaction (200
l) consisted of 20 l sample and 180 l of 0.025%
(w/v) cassava at 90C for 10 min. The reaction was
stopped by 20 l of 0.5 M acetic acid. Remaining
cassava was determined at 595 nm after I2 addition
(20 l).
2.5.3 Secondary screening of rpFL-SH library. The
screening was done at (1)37C, 30 min, 0.025% (w/v)
cassava at pH 3, (2) 0.5%(w/v) cassava at pH 11, (3)
37C, 30 min and 0.5% (w/v) cassava at pH 7, (4)
stability test (pH7) at 60C, 1 hr + 10 mM EDTA, and
(5)75C, 2 hr and 0.025%(w/v) cassava at pH 7. The
positive was 0.025% (w/v) cassava at pH 7. Remaining
cassava was determined at 595 nm.
B. licheniformis NH1 alpha-amylase [7] and well

known alpha-amylase (pdb code; 1BLI.pdb) [8]. There
were 5 different points between alpha-amylase
DSM8785 and DSM13. First point was in Bacillus
signal peptide. Four points were in mature enzymes of
alpha-amylase (Table 1).
Figure 1. Maps of recombinant vectors.

3.1 Construction of recombinant expression vector
The rpET13, rpET8785, rpFL8785 and rpFL13
were shown in Fig. 1. The expression of rpET13 and
rpET8785 vectors were controlled by T7 promoter on
pET21d (+) and promoter on DE3 domain of E. coli
BL21(DE3). Vector rpET13 and rpET8785 carried
signal peptide resulted in secretion of alpha-amylase
(Fig 2). The expression of pFLAG-CTS vector was
controlled by tac promoter. The fusion of E. coli
OmpA signal peptide on vector with mature alphaamylase gene showed the secretion of alpha-amylase
enzyme. The pFLAG-CTS vector is leaky plasmid.
Thus, the expression was detected without IPTG
induction (Fig. 2).
3.2 Alpha-amylase sequence analysis
The sequences of rpET13 and rpFL13 (without
signal peptide) show 100% of similarity to native
alpha-amylase B. licheniformis DSM13. The alphaamylases had 512 amino acids including signal peptide
(29 amino acids) and mature alpha-amylase (483
amino acids). These structures were similar to
Figure 2. The alpha-amylase activity on 1 %(w/v)

soluble starch plate.
3.3 Library construction of shuffled alpha-amylase
The rpET-SH and rpFL-SH libraries were shown
in Fig 3. The gene diversity was introduced by error
prone PCR with addition of MnCl2 that led to increase
rate of mutation. The diversity was success under
presence of MgCl2 [9] or both MgCl2 and MnCl2 [10].
The fragments (50 to 300 bp) were efficient to create
full length of gene [9-10]. However, the small
fragments (<50 bp) were done [11]. The shorter
fragments acted as primers by using longer fragments
as template. Reassembly reaction required pfu DNA
polymerase to avoid additional A at 3end. The
871
reassemble PCR by pfu DNA polymerase was better

than reassemble reaction by using only Taq DNA
polymerase [11]. The additions of specific primers (3BspHI and 5-XhoI, 3-EcoRI and 5-XhoI) were
performed to create the corrected size of shuffled
alpha-amylases (1.5 kb).
Table 1: The different points of amino acid sequences
on recombinant alpha-amylase DSM13 and
DSM8785
Name
DSM13
rpET13
rpET8785
rpFL13
rpFL8785
Concensus
>50
Different point of amino acid

Mature alpha-amylase sequence
30
160
270
|
|
|
AANLKG
GEERIK
KLSFL
AANLKG
GEERIK
KLSFL
AANLKG
GEELIK
KFSFL
.ANLKG
GEERIK
KLSFL
.ANLKG
GEELIK
KFSFL
.ANLkG
GEHrIK
Kl SFL
350
|
KAV
KAV
KSV
KAV
KSV
KaV
3.4 The screening of shuffled alpha-amylase library

The 230 clones of 5,000 clones from rpET-SH
library showed activity (5% of positive clones)
whereas 1,100 clones of rpFL-SH which showed clear
zone were collected. The 2nd screening was the most
critical step in directed evolution [12]. Some shuffled
enzymes from rpET-SH had activity at pH 3.5 and 9.5.
While rpFL-SH library, only control experiment was
found the activity. Thus shuffled alpha-amylases
which showed lower, equal, or higher activity than
wild type were collected.
between alpha-amylase DSM13 and DSM8785

(Table 2). The shuffled no. 2a11h showed the highest
mutations points and termination codon (TAA) (data
did not show). The mutation by error prone PCR was
success by altering MnCl2 concentration coupled with
an unbalanced nucleotide concentration [9].
The DNA shuffling created 1-6 points of amino
acid mutation. One point mutation was found on
shuffled no. 1b8b, 2d11h, 7c12h, and 7c4h. Five points
were occurred on 2e2a and 9g7d. The shuffled no.
2a11h, no. 2e2a, no. 2d11d, and no. 2e5e had mutation
in domain B or catalytic and substrate binding site that
led to change substrate specificity and stability [6,13]
and decrease the activity in shuffled no. 9d7g (K251E)
and no. 2e2a (T252M), which were 30% and 67%
relative activity to rpFL-8785, respectively.
Phylogenic tree was analyzed by deleting signal
peptide and 6xHis (Fig. 4). The shuffled no.1b8b was
similar to rpFL13 or rpFL13. The shuffled no.1b9a,
2e2a, 9g7d, 2b7g, 1c5b, and 7c12h were not closely
related to rpFL13 or rpFL8785, especially shuffled no.
1b9a, 2e2a, 9g7d and 2b7g. They had high evolution
of amino acid sequences. Sequences no. 2d11h, 7b3a
had closely related to rpFL8785. The shuffled no.
1b9a, 2e2a, and 9g7d had the mutation in conserved
region led to high evolution level because the
spontaneous mutation was not often occurred by
natural evolution. The shuffled no. 2b8b had 2 amino
acids (N6 and L136) from DSM 8785 and 2 amino
acids (L240 and A320) from DSM13.
Figure 3.Construction of shuffled alpha-amylase.

3.5 Multiple sequence alignment of shuffled
alpha-amylase
The 2nd structure showed domain A (3-111 and
207-384), domain B (112-206 and 385-482) and
domain C (385-482). Three conserved amino acid
were D233, E257, and D330. The 1 st calcium binding
sites (N106, D196, D202, and H237), the 2 nd calcium
binding sites (D163, A183, D185, D294, and D206),
the 3rd calcium binding sites (G302, Y304, H408,
D409, and D423), and sodium binding sites (D163,
D185, D196, D202, and I203) were conserved
sequences, except shuffled no.1b9a which mutations
were occurred in conserved regions (H237P, M258N,
and F259V) and at 1st calcium binding site (H237P).
Shuffled no. 2b8b, 2a11h, and 4d2d from
rpFL-SH library showed successfully base shuffling
Figure 4. Phylogenic tree of shuffled alpha-amylase.

4. Conclusions
The construction of shuffled alpha-amylase genes
by DNA shuffling was success to create the diversity
of alpha-amylase genes. The mutation was occurred
in conserved region, calcium and substrate binding
site. Moreover, the shuffled amino acids between
alpha-amylase strain DSM13 and DSM8785 were
presented. The results suggested that DNA shuffling
created the evolution of gene. DNA shuffling led to
change in protein property not only enzymes but also
other interested proteins. The next round of DNA
shuffling may be required for the next evolution to get
more designed protein property.
872
Table 2: Different point of shuffled alpha-amylases

Different point of amino acid
Name
rpFL13
rpFL8785
1b8b
1b9a
1c5b
2b7g
2b8b
2e2a
2d11h
7b3a
7c4h
7c12h
rpET13
rpET8785
SHM43
SHM154
SHM197
SHM199
Consensus>5
0
rpFL13
rpFL8785
4d2d
2a111h
2e5e
2d11d
2a2b
1c12b
Consensus>
50
Complete sequences
1
20
70
|
|
|
EFANLK DGQH FHQKG
EFANLK DGQH FHQKG
EFANLK DGQH FHQKG
EFANLK DGQH FHQKG
EFANLK DGQH FHQKG
EFANLK DGRH FHQKG
EFANLK DGQH FRQKG
EFANLK DGQH FHQKG
EFANLK DGQH FHQQG
EFANLK DGQH FHQKG
EFANLK DGQH FHQKG
EFENI K DGQH FHQKG
AAANLK DGQH FHQKG
AAANLK DGQH FHQKG
AAANLK DGQH FHQKG
AAANLK DGQH FHQKG
AAANLK DGQH FHQKG
AAANLK DGQH FHQKG
e f a N l n DGqH F hQkG
Non complete sequences

1
30
70
|
|
|
EFANLK DSA EFH
EFANLN DSA EFH
EFANLN DAA EFH
EFANLK DSA ESH
EFANLN DSA EFH
EFANLN DSA EFH
EFANLN DSA EFH
EFANLN DSA EFH
EFANLn DsA EfH
140
|
RIKAW
LIKAW
RIKAW
LIKAW
LIKAW
LIKAW
LIKAW
LIKAW
LIKAW
LIKAW
RIKAW
LIKAW
RIKAW
LIKAW
LIKAW
LIKAW
LIKAW
LIKAW
l IKAW
136
|
HRI
HLI
HRI
HPI
HLI
HLI
HLI
HLI
H lI
150
170 190
240 248
|
| |
|
|
GST WDES SNE KHIKLS VNH
GST WDES SNE KHIKFS VNH
GST WDES SNE KPIKLS VNH
GST WDES SNE KHIKFS VKH
GST WDES SI E KHIKFS VNH
GST WDES SNE KHIKFF VNH
GNT WDES SNE KHIKLS VNH
GST WGES SNE KHIKFF VNH
GST WDES SNE KHIKSS VNH
GsT Wd ES SnE KhIKfs VnH
265
|
YWQ
YWQ
YWQ
YGQ
YWQ
YWQ
YWQ
YWQ
YwQ
273
|
ENY
ENY
ENY
ENY
ENY
ENY
ENY
ETY
EnY
283
|
NHS
NHS
NHS
NLS
NHS
NHS
NHS
NHS
NhS
Acknowledgements
[8]
School of Biotechnology, Institute of Agricultural

Technology, Suranaree University of Technology,
Thailand, University of Natural Resources and
Applied Life Science, Austria, University Mobility
in Asia and the Pacific program, ASEAN-EU
University network program and National Science
and Technology Development Agency scholarship
are acknowledged for the support
[9]
References
[13]
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[10]
[11]
[12]
300
|
STQ
STQ
STQ
SSQ
STQ
STQ
STQ
STQ
StQ
260 320
457 473
| |
| |
EKTGKEMFT LKAV TGN FHVN
EKTGKEMFT LKSV TGN FHVN
EKTGKENVT LKSV TGN FHVN
EKTGKEMFT LKSV TGN FRVN
EKEGKEMFT LKSV TGN FHVN
EETGKEMFT LKSV TGI FHVN
E kt GKE mfT LKsV TGn FhVN
316 320
| |
VVS PLKAV
VVS PLKSV
VDS PLKAV
VVS PLKAV
VVS PLKAV
VVS PLKAV
VVS PLKAV
VVS PLKAV
VvS PLKsV
396
|
REGD
REGD
REGD
R .GD
REGD
REGD
REGD
REGD
ReGD
450
|
GETW
GETW
GETW
GETW
GEKW
GETW
GETW
GETW
GEtW
M. Machius, N. Declerck, R. Huber and G. Wiegand,

Structure 6 (1998b) 281-292.
J. Aubrey, L. Michael, P.F. Torben, S. Tina, H.
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M.R. Parikh, and I. Matsumura, J Mol Biol. 352
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F.H. Arnold and J. C. Moore, Adv. Biochem. En.g
Biotechnol. 58 (1997) 1-14.
B. Krammer, K.Rumbold, M. Tschemmernegg, P.
Pochlauer and H. Schwab, J Biotechnol 129 (2007)
151-161.
B. Svensson, Plant. Mol. Biol. 25 (1994) 141-157.
W.P. Stemmer, Nature 370 (1994) 389-391.

H. Zhao and F.H. Arnold, Nucleic Acids Res. 25
(1997) 1307-1308.
G.L. Moore, C.D. Maranas, S. Lutz, and S.J.
Benkovic, Proc Natl Acad Sci USA. 98 (2001) 32263231.
J.E. Nielsen, L. Beier, D. Otzen, T.V. Borchert,
H.B.Frantzen, K.V. Andersen and A. Svendsen, Eur.
J. Biochem. 264 (1999) 816-824.
J.E.Nielsen and T.V.Borchert, Biochim. Biophys.
Acta 1543 (2000) 253-274.
T. Kuriki and T. Imanaka, J. Biosci. Bioeng. 87
(1999) 557-565.
N. Hmidet, A. Bayoudh, J.G.Berrin, S. Kanoun, N.
Juge, and M. Nasri, Process Biochemistry 43 (2008)
499-510.
873
TOTAL PHENOLIC CONTENT AND ANTIOXIDANT EFFECT OF

COCONUT OIL WITH EXTRACTED GINGER
Pensri Penprapai1*, and Pongtep Nokkaew1
1
Faculty of Science and Technology, Rajamangala University of Technology Srivijaya, Nakronsri Thammarat 80010, Thailand
*Corresponding Author : pensir_srivub@hotmail.com, Tel. +66 899706698, Fax. +66 75773338
Abstract: Ginger had phenolic compounds which are a

powerful antioxidant for stabilization of coconut oil and
prevent deleterious effect of free radicals. Coconut oil
enriched with phenolic compound can be used for
healthy food, skin and hair care. This study aimed to
produce coconut oil with extracted ginger. The total
phenolic compounds and the antioxidant effect of
coconut oil with extracted ginger were studied. The total
phenolic content was found to be 209 mg/100g oil and 51
mg/100g oil (expressed as Gallic acid) for coconut oil with
extracted ginger and virgin coconut oil, respectively. 2,2Diphenyl-1-picril hydrazyl radical (DPPH) scavenging
reached 58.9% at 2.30 hr and exceeded that of virgin
coconut oil (4.5%). The antioxidant activity in a linoleic
acid/water emulsion system of coconut oil with extracted
ginger was determined as compared with virgin coconut
oil and -tocopherol. It was found that antioxidant
activity of coconut oil with extracted ginger (94.0%)
showed a higher antioxidant activity than virgin coconut
oil (91.3%), but slightly lower than -tocopherol (98.8%).
Results revealed coconut oil with extracted ginger, which
enriches phenolic compounds, was more potent
antioxidant than virgin coconut oil.
1. Introduction
Coconut oil is one of vegetable oil that is rich in
saturated fatty acids (93%). It contains medium chain
fatty acids (60%), especially lauric acid (C12:0)
(45%), which are burnt for energy rather than stored
in the body. Lauric acid, found in coconut oil in
major amounts, are known for their unique antiviral,
antibacterial and antiprotozoal properties. Moreover,
coconut oil lead to a normalization of body lipids,
protect against alcohol damage to the liver and
improves the immune sytems anti-inflammatory
response. However, coconut oil is deficient in
antioxidant content [1-2]. Synthetic antioxidants such
as butylated hydroxyl toluene (BHT) were used as
food additives. But synthetic antioxidants may be
implicated in many health risks, including cancer,
carcinogenesis [3]. Due to these safety concerns, the
natural antioxidants such as polyphenol compounds in
plant have been used in food to replace these synthetic
antioxidants. Ginger is one of the plants which was
found to contain polyphenol compounds with high
antioxidant activity [4]. Coconut oil enriched with
phenolic compounds can be used for healthy food, skin
and hair care. This work aimed to produce coconut oil
with extracted ginger (COG). The total phenolic
compounds and the antioxidant effect of coconut oil
with extracted ginger as compared with virgin coconut

oil (VCO) were studied.
2.1 Materials
Coconut milk was extracted from 10-12 month old
coconuts which were purchased from local markets in
Thongsong, Nakron Sri Thammarat, Thailand.
2.2 Preparation of VCO
Coconut milk was prepared by hand pressing
scraped coconut endosperm (5 kg of coconut
endosperm : 5L of water) and filter the viscous slurry
from hand pressing scraped coconut endosperm
though a cheesecloth to obtain coconut milk. This
coconut milk was stored at 40 oC for 24 hr and then the
oil layer was separated from the coconut cream and
water. VCO was kept in brown flask.
2.3 Preparation of COG
1kg of fresh ginger and 100 mL of water were
mixed and blended in blender. This mixture slurry was
filtered though a cheesecloth to obtain solution from
fresh ginger. Then the solution of ginger was added
into a flask (10 L) containing coconut milk (5 kg of
coconut endosperm: 5L of water). This mixture was
stored at 40 oC for 24 hr to allow some compounds
from the solution of ginger to be extracted into the oil
layer. The oil layer was separated from the cream and
water. COG was kept in brown flask.
2.4 Extraction of pheholic compounds
The procedure reported by Kapila (2009) was
slightly changed [5]: Five grams of oil sample was
weighed, dissolved in 25 ml hexane and transferred to
a separatory funnel. Twentyfive milliliters of the
methanol-water mixture (80:10 v/v) was added. After
2 min of shaking the lower methanol-water layer was
removed. The extraction was repeated twice and the
methanol-water phase was combined. The methanolwater extract was condensed in rotary evaporatory
under vacuum at 40 oC. The dry residue was then
diluted in 1 ml of methanol.
2.5 Determination of total phenolic content
The content of total phenolic compounds in the
VCO and COG was determined by Folin-Ciocalteau
reagent using a slightly modified method by Gutfinger
874
(1981)[6]. The reaction mixture contained 1 ml of

methanol-water extract, 1 ml of freshly prepared
diluted Folin-Ciocalteau reagent and 8 ml of sodium
carbonate solution. The mixture was kept in the dark at
ambient conditions for 30 min to complete reaction.
The absorbance at 725 nm was measured on
spectrophotometer (Biochrom S22). Gallic acid was
used as standard. Results are expressed as mg of gallic
acid per kg of oil.
2.6 Radical scavenging activity (RSA) toward DPPH
radicals
Radical scavenging activity in VCO and COG were
examined by reduction of DPPH radical in ethyl
acetate. The procedure reported by Lee (2007) was
slightly changed : 1 g of oil (exactly weighted) was
dissolved in ethyl acetate in 10 ml volumetric flask,
then 1 ml of this solution was transferred into the
second 10 ml volumetric flask containing DPPH
radical solution, which was freshly prepared in ethyl
acetate at a concentration of 10-4 M. Reaction flask
was shaken for 10 s in vortex apparatus and it was
allowed to stand in the dark for 30 minutes. The
absorption of this mixing was measured with
spectrophotometer (Biochrom S22) in a 1 cm quartz
cell at 0, 30, 60, 120 and 150 min against a blank of
pure ethyl acetate without DPPH radicals. %Inhibition
was calculated from differences in absorbance of
DPPH solution with or without sample (control) using
the following equation:
Acontrol Asample
100
% Inhibition
Acontrol
where Acontrol and Asample are absorbance of control and

sample, respectively.
2.7 Antioxidant activity
Antioxidant activity (AA) of extract was evaluated
via -carotene-linoleate assay. One milliliter of carotene solution (0.2 mg/mL in chloroform) was
pipetted into an erlenmeyer flask (250mL) containing
0.02 mL of linoleic acid and 0.2 mL of 100%Tween
20. This mixture was slowly diluted with 100 mL of
distilled water and agitated vigorously to form an
emulsion. Five milliliters of this emulsion was
transferred into different test tubes containing 0.2 mL
of samples in solvents (final concentration of solution
at 1 mg/mL). The mixture was then gently shaken and
placed in water bath at 45 0C for 2 h. The absorbance
of the samples was measured at 470 nm using a
spectrophotometer (Biochrom S22) at initial time (t=0)
against a blank, consisting of an emulsion without carotene. Standard -tocopherol of the same
concentration of samples was used for comparison. A
0.2 mL of methanol was added in above emulsion and
used as a control. The antioxidant activity was
determined by using equation 1.
o o
AA(%) = [1-(Ao-At)/(Ao-At)]x100
Where Ao and Ao are absorbance values measured at

the initial time for sample and control, respectively,
o
while At and At were the absorbance values measured
in the samples and control at t=120 min, respectively.
3.1 Total phenolic content (PC)
Total phenolic content was calculated from linear
regression
equation of
standard
curve.
(y=1.0539x+0.025; r2= 0.9985). Where x and y are
concentration of gallic acid as standard solution and
absorbance value measured at 725 nm. We found that
total phenolic content in VCO and COG are 51 and 209
mg/100 g oil, respectively (Table 1). This indicated
that the total phenolic content from ginger (158 mg)
could dissolve in coconut oil.
Table 1: Total phenolic content of COG and VCO
Sample
COG
VCO
Total phenolic content (mg /100g oil)

209
51
3.2 Radical scavenging activity (RSA) toward DPPH

radicals
The antioxidant activity of VCO and COG was
determined by DPPH radical-scavenging activity
assay. The DPPH radical-scavenging activity is shown
in Table 2 and figure 1. The DPPH radical-scavenging
activity of coconut oil with extracted ginger increased
with time, whereas the DPPH radical-scavenging
activity of virgin coconut oil was stable. The DPPH
radical-scavenging activity of COG reached 58.89%
at 150 min and was higher than VCO (4.5%). Results
revealed that the COG, which enriches phenolic
compounds, was more potent antioxidant than VCO.
Table 2: %inhibition of COG and VCO for 150 min
Time (min)
0
30
60
90
120
150
%inhibition of
COG
30.18
41.06
46.78
51.45
55.74
58.89
%inhibition of
VCO
3.54
4.79
4.67
4.92
4.54
4.54
(1)
875
70.00
%inhibition of COG
%inhibition of VCO
60.00
%inhibition
50.00
-tocopherol (98.8%). This concluded that the COG

enriched with phenolic compound shows high
antioxidant activity and %inhibition. It can be useful to
be healthy food and product of skin and hair care
40.00
Acknowledgements
30.00
20.00
This work was supported with grants from the

office of the National Research Council of Thailand
(budget 2554); the Faculty of Science and Technology,
Rajamangala University of Technology Srivijaya.
10.00
0.00
0
50
100
150
200
Time (min)
References
Figure 1. %inhibition of COG and VCO for 150 min.
3.3 Antioxidant activity
The antioxidant activity in a linoleic acid/water
emulsion system of COG was determined as compared
with VCO and -tocopherol (Table 3, Figure 2). It was
found that antioxidant activity of COG (94.0%)
showed a higher antioxidant activity than VCO
(91.3%), but slightly lower than -tocopherol (98.8%).
This result indicated that the COG, which enriches
phenolic compounds, was higher antioxidant activity
than VCO.
Table 3: Total antioxidant activity (AA) of COG and
VCO
% AA
Sample
-tocopherol
COG
VCO
[1] A.S. Bhatnagar, P.K. Kumar, J. Hemavathy and

A.G. Krishna, J. Am. Oil. Chem. Soc. 86 (2009)
991-999.
[2] A.M. Marina, Y.B. Che Man, S.A.H. Nazimah,
Food chem. 83 (2009) 301-307.
[3] K. Imadia, S. Fukushima, T. Shirai, M. Ohtani, K.
Nakanish, and N. Ito, Carcinogensis. 4 (1983) 895899.
[4] I. Stoilova, A. Krastanov, A. Stoyanova, P. Denev,
S. Gargova, Food chem. 102 (2007) 764-770.
[5] K.N., Seneviratne, C.D., Hapuarachch I., S.
Ekanayake. Food Chem. 114(2009) 14441449.
[6] T. Gutfinger, J. Am.Oil. Chem. Soc. 1(1981) 966968.
[7] J. M. Lee, H. Chung, P. Chang and J. Lee, Food
Chem. 103(2007) 662-669.
AA (%)
94.0
91.3
68.8
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
- tocopherol
COG
VCO
Figure 2. Total antioxidant activity of COG and VCO.

4. Conclusions
The total phenolic content was found to be 209 and
51 mg/100g oil (expressed as Gallic acid) for COG
and VCO, respectively. DPPH scavenging activity of
COG is higher than VCO at 150 min. Moreover, the
antioxidant activity in a linoleic acid/water emulsion
system of VCO and -tocopherol. It was found that
antioxidant activity of coconut oil with extracted
ginger (94.0%) showed a higher antioxidant activity
than virgin coconut oil (91.3%), but slightly lower than
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GLASS TRANSITION TEMPERATURE OF PROTECTIVE AGENT

THAT AFFECTING THE SURVIVAL OF LACTOBACILLUS

Abstract: The objectives of this study were to

substitutes when the hydration shell of proteins as

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

adjusted to have viscosity of 1.6-1.7 Cp. The viscosity

3. Results and Discussion

3.2 The survival of L.plantarum FT35 after spray

2.3 Determination of the survival of L.plantarum

protection on the L.plantarum FT35 during storage

Table 2. Comparisons of viable cell and salt sensitive

Salt sensitive cell

Storage time (week)

Fig.2 The survival of L.plantarum FT35 with different

Table 2 shows the salt sensitive cells increase in

Storage time (week)

3.3 The survival of L.plantarum FT35 during storage

Fig.3 The residual moisture content of L. plantarum

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

Stroage time (week)

Fig. 4 Water activity (aw) of L. plantarum FT 35

[7] S. Permpong, Production and storage of lactic acid

The protective agents used in this study were

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

IDENTIFICATION OF MICROFLORA IN MILK KEFIR IN THAILAND

Abstract: Kefir culture contains bacteria and yeasts

Lactic acid bacteria, producing lactic acid, inhibit

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

2.3 Identification of bacteria

domain of 26S rDNA were compared by BLASTn

The three different media show values ranging

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

the acids are produced from galactose, D-glucose,

This work was supported by the National Research

Different species of microorganisms were found in

[1] S. Otles and O. Cagindi, Pak. J. Nutr. 2 (2003) 54-59.

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

ISOLATION AND CHARACTERIZATION OF EXOPOLYSACCHARIDE

Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

Author for correspondence; E-Mail: kati_narak@hotmail.com

Abstract: Exopolysaccharide (EPS) producing-lactic acid

polymers and offer an alternative source of microbial

2.1 Isolation and screening for EPS production

Natural polymers have received an increased

2. Materials and Methods

2.2 Partial purification of EPS

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

2.3 Monosaccharide analysis

LAB can be influenced by the composition of the

3.3 Solubility of EPSs

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

Table 2 : Rate of syneresis of polysaccharides

Rate of syneresis (%)

Table 3: The emulsifying activity of polysaccharides

Emulsifying activity (%)

H.M. the Kings 72nd Birthday Scholarship from

3.5 Emulsifying activity

[1] De Vuyst, L. and Degeest, B. Heteropolysaccharides

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

bacterium strains and their biopolymers. Appl. Environ.

PURE AND APPLIED CHEMISTRY INTERNATIONAL CONFERENCE 2012 (PACCON 2012)

METHOD DEVELOPMENT FOR EVALUATION OF TOTAL

Author for correspondence; E-Mail: Pha.sontaya@hotmail.com, Tel. +66 11 523690

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562 ************************

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