Extended Abstract : Biology and Biodiversity

12th SCiUS Forum

Title : Effect of Hydroxyapatite Nanoparticles on the OB5_18_03
Growth of Green Oak (Lactuca scariola) in
Field : Hydroponic System.
Author : Biology and Biodiversity
Nachaphon Maleewong
School : Burathat Khongsuk
Advisor : Kanon Sichan
Surawiwat School Suranaree University of Technology
Asst. Prof. Dr. Duangkamol Maensiri

Abstract
Eutrophication or algal bloom is one of the environmental problems that was caused by phosphorus

and nitrogen from agricultural fertilizers. Nowadays, nanoparticles are used in planting, which can promote
plant growth because they are very small and can be easily absorbed by plants. Hydroxyapatite nanoparticles
contain phosphorus and calcium, which are essential nutrients for plants. Perhaps we chose these nanoparticles
to study their effects on green oak growth and water quality after the experiment to track the likelihood of
eutrophication. We planted green oaks in a hydroponic system to remove the limitation of water management
in 2 weeks. We divided the green oaks into 4 groups, and each group was fed with a nutrient solution at a
difficult concentration of Hydroxyapatite 500 mg/L, 400 mg/L, 300 mg/L, and the control group used a SUT
solution that does not contain Hydroxyapatite. The result of our experiment was evaluated by dry weight, fresh
weight, length of roots and shoots, water quality based on nutrients left after the experiment, dissolved oxygen,
and biochemical oxygen demand. In conclusion, we found that the hydroxyapatite group with 500 mg/L of
Hydroxyapatite group is the best group that can significantly increase plant growth insignificant level. The
water quality result also indicates that the SUT solution group that does not contain hydroxyapatite has a
greener color and more nutrients remain than the group that contains hydroxyapatite. However, the water
quality results do not indicate that hydroxyapatite could significantly reduce the likelihood of eutrophication.
However, they suggest that it could reduce the nutrients remaining after feeding because the green oak could
absorb the hydroxyapatite more than the SUT solution because the hydroxyapatite nanoparticles are small and
well absorbable. Overall, hydroxyapatite nanoparticles can increase the growth of green oak and decrease the
remaining nutrients. The limitation of the experiment is that the green oak tissue was not studied, which could
reveal the mechanism of the effect of hydroxyapatite on the development of green oak tissue. In addition, we
cannot follow the promotion of plant hormones related to the root and shoot growth of the plant.

Keywords : Hydroxyapatite nanoparticles, Hydrophonic system, Green oak, Eutrophication.

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Introduction
Eutrophication is one of the major environmental problems caused by the release of phosphorus

from agricultural fertilizers. Currently, nanoparticles are used to promote plant growth because they are small
and can be easily absorbed by plants. Hydroxyapatite nanoparticles contain calcium and phosphate as
components. We know that calcium and phosphate are the most important nutrients for plants. Therefore, the
aim of this study was to investigate the effect of hydroxyapatite nanoparticles on green oakleaf lettuce growth
and water quality in a hydroponic system.

Methodology
Green oak seeds were planted in planting material (sponge) on irrigated trays for 1 week. Then,

seedlings were planted in troughs containing different nutrient solutions as follows: HA500, HA400, HA300,
and SUT for 12 days, the planting procedure is shown in Figure 1. After 12 days, the growth of green oakleaf
lettuce was measured by monitoring the fresh weight, dry weight, root length, stem length, and a number of
leaves.

Sow seed 7 day old seedlings Transfer to the seedlings Waiting for harvest
into the planting trough

Figure 1 planting process

Results

The experimental results will be measured and processed as follows.

a. The growth of green oakleaf lettuce.

The growth of green oak leaf lettuce was measured by fresh weight and dry weight, crown width,
and root length. The results of the experiment are shown in the following table.

Picture 2 The color of each green oak lettuce

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Table 2 fresh weight, dry weight, crown width, root length of green oak lectuce.

Nutrients Fresh weight Dry weight Root length Stem length Number
of leaves
Control (mg) (mg) (cm) (cm)
group 6
nHAP500 231.78 17.26 8.7 3.98 ±0.63
± 53.95 ±7.22 ±2.58 ±0.54
nHAP400 219.26 20.88 6
± 57.49 ±4.43 9.8 4.1 ±0
nHAP300 177.56 16.24 ±0.75 ±0.2
±41.00 ±3.56 3.76 6
123.26 11.92 7.8 ±0.73 ±0.63
±19.72 ±2.09 ±1.29
3 6
6.5 ±0.44 ±0
±0.89

Mean ± SD

From Table 2, it can be seen that the measurement of various data on the growth of green oak
Lactuce showed that nHAP 500 mg/L gives results close to those of the control group. However, the results of
nHAP 300 and 400 mg/L are inferior to those of the control group in all respects except for the number of
leaves, which is similar to that of the control group. According to Figure 2, the color of the leaves of the control
group is greenish-purple. For the others, the color of the leaves is green. Water quality measurement

Figure 3 water quality

In Figure 3, the color after the experiment of about 2 weeks shows that the control group has a
darker color than the nHAP fertilizer, which means that the control group has more residue than the others.

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Discussion and Conclusion
The results of the experiment showed that culturing plants in Green oak salad using a formula

containing nanohydroxyapatite particles was most effective at concentrations of 500, 400, and 300 mg/L,
respectively. Conventional formulations have been shown to replace these particles. In addition to water quality
and plant leaf color, the formulas containing hydroxyapatite nanoparticles also showed a better effect, namely
that the color of the water was not as intense as in the control group. This indicates better water quality after
planting and less nutrient residues. The leaf color of the control group was colored. Greenish-purple different
from the leaf color of the nanoparticles, indicating the characteristics of slow release of phosphorus, causing
the plants to have a steady uptake of phosphorus and not like the group The controls used a formula with larger
phosphorus particles.

Acknowledgment
This research was sponsored by the Science Classrooms in University-Affiliated School Project

( SCIUS) under the Suranaree University of Technology and Surawiwat School. The funding of SCIUS is
provided by the Ministry of Higher Education, Science, Research, and Innovation. This extended abstract is not
for citation

References
1. Bala N, Dey A, Das S, Basu R, Nandy P. Effect of hydroxyapatite nanorod on chickpea (Cicer
arietinum) plant growth and its possible use as nano-fertilizer. Iranian Journal of Plant Physiology.
2014;4(3):1061-9.
2. Hydroponic NCISU. Effects of different cultivation media on vegetative growth, ecophysiological
traits and nutrients concentration in strawberry under hydroponic and aquaponic cultivation systems.
Advances in Environmental Biology. 2012;6(2):543-55.
3. Szameitat AE, Sharma A, Minutello F, Pinna A, Er-Rafik M, Hansen TH, et al. Unravelling the
interactions between nano-hydroxyapatite and the roots of phosphorus deficient barley plants. Environmental
Science: Nano. 2021;8(2):444-59.
4. Yuvaraj M, Subramanian K. Different Types of Hydroponics System. Biotica Research Today.
2020;2(8):835-7.

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Title : Antibacterial efficacy of silver nanoparticles OB5_15_01
Field : Biology and Biodiversity

Author : Miss Pawanrat Naphaphongsuriya and Miss Pemika Kesornsawat

School : PSU. Wittayanusorn school, Prince of Songkla University

Advisor : Asst. Prof. Dr. Suchera Thananimit, Mr. Surawut Saengmanee and Miss Apinya Boonkoom

Abstract
Silver nanoparticles have the ability to inhibit bacteria. The difference in reducing agents in silver nano

synthesis affects the ability of bacteria to be inhibited. Therefore, this research aims to study the efficiency of
silver nano in inhibiting and killing three types of bacteria: Bacillus subtilis, Staphylococcus aureus, which are
gram-positive bacteria, and Salmonella Typhimurium, which is a gram-negative bacteria. Wherewith the
researchers compared the efficiency of silver nano with various reducing and stabilizing agents. It is classified
into two types: chemical silver nanoparticles synthesis and biological silver nanoparticles synthesis. Chemical
silver nanoparticles synthesis has two kinds which are silver nanoparticles are synthesized using NaBH4 and
adding Polyvinylpyrrolidone (AgNPs-PVP) and silver nanoparticles are synthesized using chitosan (AgNPs-
CS). Biological silver nanoparticles synthesis has a kind which is silver nanoparticles are synthesized using
Phenolic-rich extract (AgNPs-GPRE). When comparing the efficiency of AgNPs-PVP, AgNPs-CS, and
AgNPs-GPRE in inhibiting and killing bacteria, it was discovered that AgNPs-GPRE inhibits and kills three
types of bacteria at a lower concentration than AgNPs-PVP and AgNPs-CS.

Keywords : silver nanoparticles, chemical synthesis, biological synthesis, antibacterial activity

Introduction
Nanotechnology involves manipulating materials at the atomic, molecular, or micro-scale of around 1-

1 0 0 nm, resulting in unique new material or device properties. Which can add value to the economy
(NANOTEC, 2011) by applying in various industries seen in daily life such as electronics, consumer products,
medical products, and so on. Silver nanoparticles are classified as one of the sciences in Nano-biotechnology
that has the ability to resist the growth of microorganisms that has a mechanism to interfere with biomolecules
and the fluid balance within the bacterial cells, resulting in disruption of cell processes and a hole in the cell
wall. Until the bacteria stop growing and eventually die. (Srinya, 2014; Sininat & Khamchan, 2019)

The difference between chemical and biological nanoparticle synthesis is a reducing agent and a
stabilizing agent. Instead of using chemicals, biosynthesis employs natural substances derived from the
synthesis of bacteria, fungi, and plants. Contributes to differences in the size and shape of silver nanoparticles,
which affects the ability of bacteria to be inhibited. (Paveena et al., 2016).

The researchers are therefore interested in the synthesis and study of the efficacy of silver nanoparticles
on bacterial growth by comparing chemically synthesized silver nanoparticles, namely AgNPs-PVP (NaBH4-
synthesized silver nanoparticles with added Polyvinylpyrrolidone), AgNPs-CS (Chitosan-added silver

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nanoparticles), and biologically synthesized silver nanoparticles, namely AgNPs-GPRE (Phenolic-rich extract
synthesized silver nanoparticles). For further research and development in order to add value and benefits to
users.

Methodology
The experiments were divided into 4 parts as follows,

Part 1 : Silver nanoparticles synthesis using sodium borohydride that adds Polyvinylpyrrolidone
1.1 Take 2 millimolar sodium borohydride solution 30 milliliter and put it on the magnetic stirrer and
stir at a speed of 1400 rpm.
1.2 Drop 1 millimolar silver nitrate solution 10 milliliter.
1.3 Add 0.3% weight by volume of Polyvinylpyrrolidone solution 4 milliliters.
1.4 Notice the color of the solution. When the reaction has completed the color of the solution changes
from colorless to yellow. Then, place the solution at room temperature for 1 hour.
1.5 Measured the absorbance value of silver nano solution at a wavelength between 200 and 800
nanometres with the UV-Visible spectrophotometer.
1.6 Keep the silver nano solution in the reagent bottle by keeping it at 4°C.

Part 2 : Preparation of bacteria including Salmonella Typhimurium, Bacillus subtilis, and Staphylococcus
aureus.

2.1 Separate a single colony of bacteria and put it in a microcentrifuge tube that has Tryptic soy broth
1 milliliter. Then, incubate it with a shaking incubator at 37 degrees celsius according to the Log
phase of that species.

2.2 Transfer bacteria suspension 20 microlitres to a test tube that has 0.85% w/v sodium chloride 2
milliliters. Then measure bacteria suspension turbidity with a densitometer and adjust it until
bacteria suspension turbidity is 0.5 McFarland.

2.3 Diluted the bacteria suspension to 106 CFU/ml with Mueller Hinton broth.

Part 3 : Study minimum inhibition concentration of silver nano solution by Microdilution broth test
The microdilution method was created in a 96-well plate by diluting the silver nanoparticles in a 2-fold

dilution with distilled water, with each well containing 250 µL. The positive control was distilled water, while
the negative control was distilled water with bacteria added.

Part 4 : Study minimum bactericidal concentration of silver nano solution by spot assay
The minimum bactericidal concentration (MBC) was determined from the research of the minimum

inhibitory concentration (MIC) by diluting the solution in the well at a concentration where no bacterial growth
occurred using a 10-fold dilution with distilled water. After that, the diluted solution was dropped on the
Mueller Hinton Agar 5 µL per drip. It was then incubated at 37 °C for 16-18 hours. and read the results.

Results and Discussion

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The researchers study the MIC values of AgNPs-PVP (NaBH4-synthesized silver nanoparticles with
added Polyvinylpyrrolidone) compared with AgNPs-CS (Chitosan-added silver nanoparticles) and AgNPs-
GPRE (Phenolic-rich extract synthesized silver nanoparticles). The MIC values can be specified as shown in
Table 1.

Table 1 shows the MIC of silver nanoparticles synthesized by various methods.

Bacteria AgNPs-PVP MIC AgNPs-GPRE
(µg/ml) (µg/ml)
Bacillus subtilis AgNPs-CS
Staphylococcus aureus - (µg/ml) 0.1053
Salmonella Typhimurium - 0.7355 0.8427
1.5313 1.4711 0.2107
0.7355

- mean not found the concentration that is MIC.

The researchers study the MBC values of AgNPs-PVP (NaBH4-synthesized silver nanoparticles with
added Polyvinylpyrrolidone) compared with AgNPs-CS (Chitosan-added silver nanoparticles) and AgNPs-
GPRE (Phenolic-rich extract synthesized silver nanoparticles). The MBC values can be determined as indicated
in Table 2.

Table 2 shows the MBC of silver nanoparticles synthesized by various methods.

Bacteria AgNPs-PVP MBC AgNPs-GPRE
(µg/ml) (µg/ml)
Bacillus subtilis AgNPs-CS
Staphylococcus aureus - (µg/ml) > 0.4214
Salmonella Typhimurium - 2.9422 3.3709
6.125 5.8844 0.8427
> 2.9422

- mean not found the concentration that is MBC.

From the table above, it can see that the researcher did not find the MIC and MBC values of AgNPs-PVP
inhibit Bacillus subtilis and Staphylococcus aureus. Because the highest concentration the researchers used in the
experiment is 6.125 µg/ml. It has to use a concentration of more than 6.125 µg/ml to inhibit these two types of
bacteria. Gram-positive bacteria has a thicker cell wall with more peptidoglycan and polysaccharide than gram-
negative bacteria. As a result, a high concentration of silver nano solution is required.

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In addition, the inhibition efficiency of bacteria was directly related to the zeta potential of the surface charge
of silver nanoparticles and the bacterial cell wall. Silver nanoparticles surrounded by positive charge may interact
more with the negatively charged gram-negative bacteria than the gram-positive ones. Since the gram-negative
bacterial cell wall has a higher negative charge than the gram-positive bacterial cell wall (Sarinya, 2014). As a result,
the abilities in inhibit gram-positive and gram-negative bacteria in each silver nano solution are different. And from
the experiment, found that AgNPs-GPRE was used at the lowest concentration to inhibit and kill three types of
bacteria when compared with others.

Conclusion
From the experiment, it can see that AgNPs-GPRE was used at the lowest concentration to inhibit and kill

three types of bacteria when compared with others. Both chemical reagents and biological reagents can be used in
silver nano synthesis. Silver nano can inhibit well or not depending on several factors such as the size of silver nano,
type of bacteria and the zeta potential, etc.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS) under

Prince of Songkla University and PSU. Wittayanusorn school. The funding of SCiUS is provided by Ministry
of Higher Education, Science, Research and Innovation. This extended abstract is not for citation.

References

1. Paweena Porrawatkul, Montakarn Thongsom, Pornpailin Khaosuk, Toyibah Dolohmi. Green
Synthesis of Silver Nanoparticles Using Garcinia mangostana Linn. for Antibacterial. Wichcha
Journal 2016;35:26-40.

2. Sarinya Poadang. Synthesis of Silver Nanoparticle From Fruit Peels [thesis]. Nakhon Pathom:
Silpakorn University; 2014.

3. Pichayabha Sorsiw. Inhibition of Fungal Contamination in the Male Flower of Borassus flabellifer
using Silver Nanoparticles [thesis]. Songkhla: Prince of Songkla University; 2018.

4. Silver Nano's Properties and Benefit [internet]. 2020 [cited 2021 Aug 13]. Available from:
https://marumothai.com/article/%E0%B8%8B%E0%B8%B4%E0%B8%A5%E0%B9%80%E0%B8
%A7%E0%B8%AD%E0%B8%A3%E0%B9%8C%E0%B8%99%E0%B8%B2%E0%B9%82%E0
%B8%99-%E0%B8%84%E0%B8%B8%E0%B8%93%E0%B8%AA%E0%B8%A1%E0%B8%9A
%E0%B8%B1%E0%B8%95%E0%B8%B4/

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Title : Investigating Clinical Correlation of Calcium Activities in OB5_18_01
Field : Liver Cancer
Author : Biology and Biodiversity

School : Miss Issaree Kittisupaset
Advisor : Miss Tanyarat Anotaipaiboon
Mr. Pantath Jaengbunjurdwong
Surawiwat School, Suranaree University of Technology
Dr. Pishyaporn Sritangos, Suranaree University of Technology

Abstract :

Liver hepatocellular carcinoma or liver cancer is the most commonly found type of cancer in
Thailand and the first leading cause of death from cancer among men. Liver cancer is hard to detect in its early
stage. Therefore, this variant of cancer undergoes a diagnostic process unobserved, correlating with poor patient
survival. A trend of abnormal mRNA expression in specific types of protein can be detected in cancerous
tumors. These proteins are responsible for calcium ion signaling, which regulates cellular pivotal activities such
as apoptosis, cell movement, and mRNA translation. When these activities are disturbed, there are chances of
cancer cells occurring. Data of abnormal mRNA expression were analyzed through statistical methods to find
the correlation between mRNA expression and patient survival rate. Data mining revealed that ATP2A2, MCU,
MCUR1 , and MCUB have the potential to be used in developing more accurate treatment in pharmaceutical
products. ATP2 B4 , ATP2 A2 , MCU, MCUR1 , and MCUB have the potential to be used in diagnosing liver
cancer.

Keywords : Liver Cancer, Gene Expression, Calcium ion, Calcium Signaling Protein

Introduction
In the present day, liver cancer is the sixth most commonly found type of cancer and the third

leading cause of cancer death globally. In Thailand, it is the most common cancer and the major cause of death
among Thai male patients. Difficulty in diagnosing is usually encountered when the tumor is in its earlier stage
leading to delayed treatment and a low survival rate.

Calcium ions (Ca2+) are one of the substances used in intracellular communication and regulating
various cellular activities such as regulate cell function, exocytosis, cell movement, apoptosis and mRNA
translation. Instead of normal cell cycle, cells that undergo error in Ca2+ signaling triggers cancer cycle.
Moreover, a trend of abnormal mRNA expression in specific types of protein can be detected in cancerous
tumors. The lack of understanding in the regulation of intracellular Ca2+ signaling is a great obstacle.
Consequently, fewer data were analyzed. A better understanding of Ca2+ signaling regulators will lead us to
more accurate treatment and lessen the detriment to other cells.

For internal use in the 12th SCiUS Forum only. Not for citation.

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This project was developed to dwindle a notable obstacle of identifying liver cancer in its early
stage and to find a new potential target for drugs development purposes. By studying and investigating the Ca2+
abnormalities that can be observed as the initiator of cancer in the human body.

Methodology
Data mining and statistics

The proteins that play an important role in Ca2+ signaling were sourced from www.genecards.org
and www.proteinatlas.org. mRNA expression data of each calcium signaling protein isoforms in tumor and
normal tissues were gathered from www. firebrowse. org. TCGA- LIHC Kaplan– Meier survival data were
obtained from www.proteinatlas.org. All raw data obtained from data mining were re-plotted (mRNA expression
based on the Log2 median-centered intensity of each calcium signaling protein isoforms and Kaplan-Meier
survival curves correlating the survival of LIHC patients mRNA expression) and analyzed using GraphPad
Prism version 9 to find the significance. Statistical significance is defined as p < 0.05.

Results

Data mining from two different open- source databases, www. firebrowse. org and

www. proteinatlas. org, was performed to determine whether alteration in PMCA, MCU and SERCA gene

expression in LIHC tumor are correlated with tumor tissue and normal tissue.

PMCA B. C. D.
A.

Figure A-D. PMCA mRNA expression based on the Log2 median-centered intensity of ATP2B1 (P= 0.1292),

ATP2B2 (P= 0.6167), ATP2B3 (P= 0.6769), and ATP2B4 (P= 0.0382) are individually presented as box and

whisker plots. E.

Figure E. Kaplan-Meier survival curves correlating the survival of LIHC patients to the low (black) or high (red)
expression of ATP2B1 (P=0.0047).

The mRNA expression based on the Log2 median-centered intensity data showed that ATP2B1,
ATP2 B2, ATP2 B3 and ATP2 B4 were expressed in tumors more than normal tissue (Figure A-D). ATP2B4

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was significantly expressed in tumors (P < 0.05) (Figure D). Moreover, the survival graph showed that patient
with high expression of ATP2B1 had significantly lower survival (P < 0.05) (Figure E).

MCU G. H.
F.

Figure F-H. MCU mRNA expression based on the Log2 median-centered intensity of MCU (P= 0.0000014),
MCUB (P= 0.00914) and MCUR1 (P= 0.000000214) are individually presented as box and whisker plots.

I. J. K.

Figure I-K. Kaplan-Meier survival curves correlating the survival of LIHC patients to the low (black) or high
(red) expression of MCU (P=0.000061), MCUB (P=0.017), and MCUR1 (P=0.0020).

The gene expression graph showed that MCU, MCUB and MCUR1 are significantly expressed
in tumors more than normal tissue (P < 0.05) (Figure F-H). Additionally, patient with high MCU, MCUB and
MCUR1 expression had significantly decreased chance of survival (P < 0.05) (Figure L-K).

SERCA M. N.
L.

Figure L-N. SERCA mRNA expression based on the Log2 median-centered intensity of ATP2A1 (P=0.6936),
ATP2A2 (P=0.0004), and ATP2A3 (P=0.1104)are individually presented as box and whisker plots.

O. P.

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Figure O-P. Kaplan-Meier survival curves correlating the survival of LIHC patients to the low (black) or high
(red) expression of ATP2A2 (P=0.0037) and ATP2A3 (P=0.0146).

The expression graph showed that ATP2A1, ATP2A2 and ATP2A3 were expressed in tumors
more than normal tissue (Figure L-M). Interestingly, ATP2A2 is significantly expressed in tumors more than
normal tissue (P < 0.05) (Figure M). By the same time, the high expression of ATP2A2 had significantly lower
survival of patient (P < 0 .0 5 ) (Figure O) whereas patient with high expression of ATP2A3 had significantly
higher survival (P < 0.05) (Figure P).

Discussion
Additional cell properties should be investigated in future research through cancer cell culture to

compare the results of tissue culture and the data set to be examined from the database. This will help to round
out the data.

Conclusion
The abnormalities of calcium signaling proteins have been detected compared to normal tissue.

Through data mining of available databases, it was discovered that ATP2B4, ATP2A2, MCU, MCUR1 and
MCUB were the proteins that had statistical significance in the mRNA expression graph, and hence they could
potentially be utilized to diagnose liver cancer. Furthermore, ATP2A2, MCU, MCUR1, and MCUB were
discovered as potential proteins for drug development due to statistical significance in the mRNA expression
and survival graphs. This implies that these proteins are related to patient survival.

Acknowledgements
This project would not have been possible without the support of many people. This project was

supported by Science Classroom in University Affiliated School (SCiUS). The funding of SCiUS is provided
by Ministry of Higher Education, Science, Research and Innovation. This extended abstract is not for citation.

Futhermore, we would like to appreciate our adviser, Dr. Pishyaporn Sritangos, Suranaree University
of Technology, who helped make some sense of the confusion and encouraged with a perfect blend of insight
and humor. Also thanks to our teacher, Dr. Napaporn Sriden, who offered guidance and support.

References
1. Fongchan, S.; Vorapongsathorn, S.; Bhavabudananda, P.; Chooratna, K., Liver Cancer Prevention and

Control. Thai Cancer Journal 2019, 2, 64-74.
2. Islam, M., S., Calcium Signaling: From Basic to Bedside. Advances in Experimental Medicine and

Biology 2020. 1131, 1-6.
3. Puri, B., K., Calcium Signaling and Gene Expression. Advanced in Experimental Medicine and Biology,

2020, 1131, 537-545

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Title : Development of Instant Rice on Quality an OB5_12_02
Extension of Shelf Life with Chitosan
Field :
Author : Biology and Biodiversity

School : Miss Waranya Thongkam
Advisor : Miss Warinyupa Weaweerakupt

Princess Sirindhorn’s College, Silpakorn University

Assoc. Prof. Dr. Boonsri Jongsareejit (Department of Microbiology Faculty of Science
silpakorn University)

Abstract :

The purpose of this research was to study the effect of chitosan on quality and extension of shelf life of
rice. The experiment were performed in rice with chitosan concentration of 0%, 0.33% and 0.67% (g/ml) The
results showed that rice without chitosan has the greatest microbiological count and rice in every concentrations
could decrease microbial count. The results showed that the number of microbial after 6 hours rice without chitosan
is 1x103 cfu/ml. Meanwhile, 0.33% and 0.67% chitosan rice is <30x10 cfu/ml. At 12 hours rice without chitosan
is 2.2x104 cfu/ml. Meanwhile, 0.33% and 0.67% chitosan rice is <30x102 cfu/ml. At 18 hours rice without chitosan
is 1.7x104 cfu/ml. Meanwhile, 0.33% chitosan rice is <30x102 cfu/ml, 0.67% chitosan rice is 5.5x102 cfu/ml. In
conclusion, of the rice containing chitosan could extend to 18 hours since it was discovered that after 18 hours, the
number of microbial count of the addition of chitosan concentration is about as much as without chitosan. At 24
hours rice without chitosan is 6.3x103 cfu/ml. Meanwhile, 0.33% chitosan rice is <30x102 cfu/ml and 0.67%
chitosan rice is 5.1x103 cfu/ml. At 30 hours rice without chitosan is 3x104 cfu/ml. Meanwhile, 0.33% chitosan rice
is 1.4x104 cfu/ml, and 0.67% chitosan rice is < 30x102 cfu/ml. At 36-48 hours rice without chitosan is 104-105
cfu/ml. Rice with chitosan after 18 hours is highly harmful and has a higher chance of finding a pathogens. Rice
with chitosan has a lower amount of pathogen, which means that the addition of chitosan could decrease microbial
count in rice.

Keyword : rice, chitosan, extension

Introduction
Rice is considered a principal food that gives us energy. But Situations such as a Covid-19 pandemic

affected the global economy and unprecedented rapid rises in unemployment in many countries. Many volunteers

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gave lunch boxes to each other but the shelf life of rice can keep in no time. Moreover, It can be a little hard and
troublesome to visit some places. Consequently, the development of rice on quality and extension of shelf life with
Chitosan is one option that can use in real life and is safe for consumers’ health.

Since the resulting rice is important to export foods of Thailand but today many counties produce more
rice thus have high competition and lower rice prices in the global market are seen as important to developing rice.
Many solutions to extend rice such as freezing, Filling vinegar in rice, and Using preservatives or chemicals can
make the property of rice change. Todays have more solutions to extend rice that save the environment and remain
fully nutrients.

Chitosan is a natural polymer produced by the deacetylation of chitin with high concentration. It is often
found in shrimp shells, crab shells and octopus cores. Currently, there are many applications of chitosan. In nature
and can be striped itself, therefore it is safe and does not cause any harm to the world. Nowadays, chitosan has
been utilized in various designs, especially the food aspect. Chitosan is used in food preservation to extend shelf
life. The preservation mechanism of chitosan was inhibition of microorganisms, found that the amino acids of
chitosan were able to absorb food and metal ions. that is beneficial to growth of microorganisms. It can form a
complex compound on the surface of the cell wall and microorganism. Interfering with the microorganism's
nutrition make the structure of the cell wall. Microorganisms caused malignancy. Chitosan, which has a possitive
charge, binds to the cell membrane of negatively charged microorganisms, causing other toxins and proteins to
leak.

For the aforementioned reasons, the project organizers were keen to think of antimicrobial activity by
chitosan, which will be able to keep your rice for longer. It also responds to the countless demands that are counted
as food and energy sources.

Methodology
1. Prepare rice without chitosan, rice with 0.33% chitosan, and rice with 0.67% chitosan.

2. Take a sample of rice from step one at 0, 6, 12, 18, 24, 30, 36, 42, 48 hrs.

3. Bring rice from step two to make serial dilution for fine microbial count. The step is as follows.

3.1) Bring 10 g of rice inserted into 0.85% Sodium chloride and shake the solution (stock solution) to
disperse molecules of rice.

3.2) Make serial dilution as follows, at 0-24 hrs. making 1 by 100th dilution, at 30-36 hrs. making 1 by
1,000th dilution and at 42-48 hrs. making 1 by 10,000th dilution

4. Apply the solution obtained from the sequence dilution onto the petrifilm sheet by using a pipette to absorb the
solution from the experimental dilution sequence. Take 1 ml. and put it in the petrifilm sheet at 0-24 hrs, use
dilution at 10-1-10-2 at30-36 hrs.use dilution at 10-2,10-3 at 42-48 hrs. use dilution at 10-2,10-3,10-4

5. Put the prteifilm sheet into the incubator at 35 degrees Celsius for 2 days.
6. Collect the results and count the number of microorganisms growing on the petrifilm sheet.

7. Measure the pH by bringing the nectar into the sodium chloride solution to measure the pH by using a pH meter.

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Result
The study of rice on quality and extension. After serial dilution, the results showed that the microbial

count of rice without chitosan and rice with chitosan is trending up as times passes. Using chitosan solution could
decrease microbial count of rice, as shown in Table 1

Table 1 : The results of the analysis of the total number of microorganisms. In the rice samples cooked with chitosan solution at
concentrations of 0, 0.33 and 0.67%, then preserved at room temperature for 0, 6, 12, 18, 24, 30, 36, 42 and 48 hours.

By studying the pH value of rice to see acidification. Because acid rice is an opportunity to induce
bacteria. This experiment measured the pH value 4 times The results showed that the pH value that is derived each
time is different. Every pH value is at 5-7 which means weak acid to base. Anyhow measuring pH-value can’t tell
that microorganism can make acid which maybe there is not enough microorganism that can make acid or this
microorganism can’t change starch to glucose and using chitosan solution didn’t affect pH-value, so the result of
pH-value is unstable. (see Figure 1).

pH-
value

Number of hours rice samples are collected (hours)

Figure 1 : The average pH of cooked rice was achieved in four experiments.

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The study of physical whiteness, from observing the appearance of
the eye after 72 hrs, found the position on every side still have
normal. After 96 hrs. starts to meet at no chitosan. At 42, 48 hrs, the
flooding began, but did not cross at 0.33% and 0.67%, remained
normal after 108 hrs. Normal rice have black mold at the sides of
36, 42, 48 hrs. Concentration 0.33% and 0.67% remained normal
after 140 hrs. The chitosan-free at 0-24 hrs. remained. It likes
normal. but 30 hrs, have unpleasant smells should last 36-48 hrs. The mold that used to be black There are darker.
The cross section at 0.33% concentration at 0-30 hrs. is still the normal rice at 36-48 hrs. There is mucus, there is
green mold. The cross section at 0.67% intensity at 0-36 hrs. is still here as it is 42-48 hrs. has mucus.

Conclusion
The study of rice on quality and extension. After serial dilution, the results showed that rice without

chitosan is more likely to increase microbial count, and the use of chitosan solution will decrease microbial count
in rice. This experiment also says the use of chitosan solution can extend rice life by 18 hours by putting chitosan
at concentrations of 0.33 and 0.67%. And according to the study of pH values of rice to see acidification, the results
showed that the pH value that is derived each time is different. Every pH value is at 5-7. So chitosan intake does
not affect acidity. And physical aspiration results were found after 72 hrs. All rice were normal after 96 hrs. Rice
without chitosan at 42, 48 hrs. started to change. After 108 hrs, the rice without chitosan were found to be at 36,
42, 48 hrs. After 140 hrs., the rice without chitosan were found at 30 - 48 hrs. There was a change in intensity.
0.33% 0-30hrs normal, 36-48 hrs. change 0.67% 0-36hrs normal 42-48 hrs. change.

Acknowledgement
This project was supported by Science Classroom in University Affiliated School (SCiUS) under

Silpakorn University and Princess Sirindhorn’s College. The funding of SCiUS is provided by Ministry of Higher
Education, Science, Research and Innovation, which is highly appreciated. This extended abstract is not for
citation.

References
[1] Disthai.com. ไคโตซาน.[อนิ เทอร์เนต็ ]. 2559. [เขา้ ถงึ เมอื่ 18 ก.ค. 2564] เขา้ ถงึ ไดจ้ าก :
https://www.disthai.com/16488248/
[2] สบาบนั วิจยั และพฒั นาผลิตภณั ฑอ์ าหหาร.การใชป้ ระโยชน์จากไคโตซานในการยืดอายอุ าหาร.[อนิ เตอร์เน็ต]. 2564. [เขา้ ถึงเมื่อ 9 ก.พ. 2565] เขา้ ถึงจาก :
https://li01.tci-thaijo.org/index.php/anres/article/view/251033/171674

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Title : Analysis of aflatoxin B1 in herbs and spices and OB5_03_07

Field : exposure assessment
Author :
Biology and Biodiversity
School :
Advisor : Miss Rasita Soypetcasem

Miss Chonlada Mueangsong

Naresuan University Secondary Demonstration School, Naresuan University

Dr. Siriwan Wichai, Department of Microbiology and Parasitology, Faculty of Medical Science

Naresuan University

Abstract
Herbs and spices are at risk of mold and mycotoxin contamination, which can cause harm to

consumers, especially liver cancer. The preliminary study of this research was to determine aflatoxin B1 in
spices and herbs from the markets in Muang District, Phitsanulok by using the ScreenEZ® test kit and then to
assess exposure to aflatoxin the consumer using the developed website. The results showed that spices samples,
cayenne pepper, dried chili, curry powder, coriander seeds, pepper, cumin, cinnamon, cloves, nutmeg, and
anise, were the aflatoxin contents at 13.41, 1.22, 11.26, 2.48, 1.98, 21.38, 69.58, 77, 7.42 and 92.82
nanograms/gram, respectively. Herb samples, garlic, shallots, turmeric, ginger, galangal, kaempferia, onion,
holy basil, sweet basil, and lemongrass were found with the aflatoxin contents at 0.74, 1.06, 1.82, 0.12, 0.83,
0.54, 0.534, 0.22, 0.48 and 0.72 nanograms/gram, respectively. There were four samples, cumin, cinnamon,
cloves, and anise, containing aflatoxin contaminants higher than the Thai standard contamination criteria. The
AnaAfla website was designed to assess aflatoxin exposure from the consumption of 14 menus: spicy fried
with basil leaves, stir-fried mixed vegetables, Thai pork curry, egg and pork in sweet brown sauce, stir-fried in
curry powder, massaman curry, steamed fish with curry paste, pork stir-fried with ginger, chicken Biryani,
chicken in coconut soup, stir-fried spicy with fingerroot, chicken green curry, pork in-ground peanut-coconut
cream curry, and Thai boat noodle. When choosing a menu to eat and entering the weight of the consumer, it
showed the risk of eating that menu at high, medium, or low-risk levels based on the MOE calculated. If there
is more information about aflatoxin of spices and herbs, it can develop various menus and extend this website
for more valuable.

Keywords : Aflatoxin, Exposure assessment, Mycotoxin, Herbs and Spices

Introduction
The fungal toxins are secondary metabolites produced by several fungi belonging to Aspergillus,

Penicillium, Fusarium, Claviceps, Alternaria, and Stachybotrys. These toxic substances such as aflatoxin,
ochratoxins, trichothecenes, zearalenone, fumonisins, and patulin are produced on various agricultural
products. Toxic substance contamination can occur during planting, storage, and transportation. Of these
toxins, aflatoxin is the most important and the most dangerous to humans. The Ministry of Public Health,
Thailand has set the amount of aflatoxin contamination in Brazilian nuts, almonds, hazelnuts, and pistachios
to not exceed 15-20 micrograms per kg (ppb), Fig and Dried guava not more than 10 micrograms per kg of

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food (ppb), and other food (other than the above) not more than 20 micrograms per kilogram of food (ppb) to
avoid the occurrence of health hazards (Ministry of Public Health, 2019).

Herbs and spices are natural products of plants, animals, and minerals that have been used as
medicine or in combination with other substances to cure diseases and promote health (Royal Academy, 2002).
Some herbs and spices are used in cooking to provide a specific smell, color, and taste, especially in Thai foods.
Essential oils from spices act as antioxidants and preservatives in food. To extend shelf life and be convenient
to use, spices are preserved in the form of powders or extracts (Pimpen Pornchaloempong and Nithiya
Ratnapnon, n.d.).

Herbs and spices are at risk of contaminating mold and mycotoxins. Aflatoxin contamination in
herbs and spices in Thailand was reported in 4 years of data collection, it was found that dried chili and cayenne
pepper were the spice types that should be monitored for aflatoxin incidence because they were continually
contaminated and there was a high amount of aflatoxin contamination. Naphuasoong (Warapa
Mahakarnchanakul et al., 2017)

Aflatoxins are classified according to their chemical structure into two groups. The first group is
aflatoxins B1 and B2. The second group is the aflatoxin is aflatoxins G1 and G2. When aflatoxin enters the
body, it can eliminate Up to 95 percent of aflatoxin is excreted in the urine within 24 hours, but absorption also
occurs at a faster rate. Aflatoxin B1 will enter the blood system by passive diffusion method and bind to plasma
protein or macromolecule by covalent bonds. Then it will enter the metabolic process in the liver. The
metabolites are catalyzed and reacted with DNA. It affects carcinogenesis due to DNA abnormalities resulting
in transcription errors, causing the abnormal formation of another DNA strand (Warapa Mahakarnchanakul et
al., 2017)

Therefore, exposure assessment of aflatoxin from the consumption of herbs and spices is an
interesting issue. Consumer estimate exposure (EDI) and margin of exposure (MOE) are based on the amount
of aflatoxin in food (herbs and spices), the amount consumed, and the consumer’s body weight. The experiment
will be conducted to analyze the total aflatoxin found in herbs and spices and assess consumers’ exposure to
aflatoxin as well as the risk assessment. Using the ScreenEZ® method for measuring the aflatoxin was based
on the analytical principle, Enzyme-Linked Immunosorbent Assay (ELISA), which is a method to measure the
competitiveness of antigen and aflatoxin binding to antibodies at the well surface. The quantitative result was
obtained by reading the color intensity in the test well with a Micro ELISA Reader. The increased color
intensity was directly related to the reaction and was inversely related to the toxin content contaminated in the
sample.

Methodology
The experiments were divided into 3 parts as follows,

Part 1: Method for the sampling of the samples
21 samples of herbs and spices were purchased from markets and stores in Mueang Phitsanulok. After

that, bring the samples in a sterile bag then covered with a zip-lock plastic bag, labeled the sample code, time,
and place. Lastly, the zip-lock plastic bags were stored in a foam box with cooling gel before being transported
to the lab. This procedure complies with the standard of Biosafety and Biosecurity.
Part 2: Method for the preparation of aflatoxin extraction

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The samples had to prepare for the aflatoxin extraction according to the test kit requirement. The
samples were ground by using a blender and then weighed the sample 20 grams in a flask. The flasks were
added 100 ml of 70% methanol and covered with parafilm. Next, the flask was shaken at 200 rpm in the shaker
bath for 30 min. The flasks had to leave for about 5-10 minutes to separate the clear parts. Afterward, the
filtered part which was filtered by using filter paper No. 4 was kept in a tea-colored bottle. The filtered fraction
was diluted with a 1:3 washing buffer.
Part 3: Method for the aflatoxin

50 microlites of the sample were added to a microplate, then the same value of the conjugate was to
the same plate. The plate was incubated in a dark place at 37 degrees Celsius for 30 minutes, then washed 3
times. Lastly, 100 microlites of the substrate, then incubate the plate in a dark place at 37 degrees Celsius for
7 minutes. 100 microlites of stop solution were added to stop the activity. The amount of aflatoxin in a
microplate was read with a microplate reader and calculated by the software. Geaph 1 and 2 are the standard
curve that compared the amount of aflatoxin in the samples.

graph 1: the aflatoxin standard from the first day experiment graph 2: the aflatoxin standard from the second day experiment

Results and Discussion
The results showed that spices samples, cayenne pepper, dried chili, curry powder, coriander seeds,

pepper, cumin, cinnamon, cloves, nutmeg, and anise, were the aflatoxin contents at 13.41, 1.22, 11.26, 2.48,
1.98, 21.38, 69.58, 77, 7.42 and 92.82 nanograms/gram, respectively. Herb samples, garlic, shallots, turmeric,
ginger, galangal, kaempferia, onion, holy basil, sweet basil, and lemongrass were found with the aflatoxin
contents at 0.74, 1.06, 1.82, 0.12, 0.83, 0.54, 0.534, 0.22, 0.48 and 0.72 nanograms/gram, respectively as shown
in graph 3 and 4. There were four samples, cumin, cinnamon, cloves, and anise, containing aflatoxin
contaminants higher than the Thai standard contamination criteria.

graph 3: the amount of aflatoxin in spices graph 4: the amount of aflatoxin in herbs

The optimum pH for the test must be neutral under the test kit requirements (approximately 7). On

the other hand, the 4 samples of cayenne pepper, dried chili, cloves, and star anise which have lower pH than

7 may have inaccuracies in the test results, as shown in table 1.

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Table 1: pH values of samples

The AnaAfla website was designed to assess
aflatoxin exposure from the consumption of 14 menus:
spicy fried with basil leaves, stir-fried mixed vegetables,
Thai pork curry, egg and pork in sweet brown sauce, stir-
fried in curry powder, massaman curry, steamed fish with
curry paste, pork stir-fried with ginger, chicken Biryani,
chicken in coconut soup, stir-fried spicy with fingerroot,
chicken green curry, pork in-ground peanut-coconut cream curry, and Thai boat noodle. When choosing a
menu to eat and entering the weight of the consumer, it showed the risk of eating that menu at high, medium,
or low-risk levels based on the MOE calculated. The website is stable, provide the correct answer and can be
used on both phone, iPad, and computer, as shown in figure 1 and figure 2.

figure 1: calculating exposure assessment page figure 2: result of the consumer exposure page

Conclusions
The sample of cumin, cinnamon, clove, and star anise was found to be higher in aflatoxin B1 than the

regulation, 20 ppb. However, the sample of cayenne pepper, dried chili, cloves, and star anise has acidic pH,
which may have inaccuracies in the test results. This project creates the website and selects 14 menus, for
evaluating the risk of consumers to aflatoxin exposure. When choosing a menu to eat and entering the weight
of the consumer, it showed the risk of eating that menu at high, medium, or low-risk levels based on the MOE
calculated.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS). The

funding of SCiUS is provided by The Ministry of Higher Education, Science, Research and Innovation. This
extended abstract is not for citation.

References
1. M. W. Trucksess & P. M. Scott. Routledge and Iqra Naeem. Mycotoxins in botanicals and dried fruits: A
review [dissertation]. [place unknown]: College Park; 2008
2. Amir Ismail, Saeed Akhtar, Muhammad Riaz, Yun Yun Gong, Michael N. Routledge and Iqra Naeem.
Prevalence and Exposure Assessment of Aflatoxins Through Black Tea Consumption in the Multan City of
Pakistan and the Impact of Tea Making Process on Aflatoxins [dissertation]. [place unknown]: University of
Leeds; 2020

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Title : Antifungal and antibiofilm activity of denture soft OB5_19_03
Field : liner incorporated with ethanol extract of Melastoma
malabathricum leaf
Biology and biodiversity

:Author Mr. Arnas Naengdam

Ms. Nada Beraheng

:School Islamic Science Demonstration School, Prince of Songkla University, Pattani Campus

:Advisor Dr.Udomlak Matsathit (Prince of Songkla University, Pattani Campus)

Asst.Prof.Subhan Salaeh (Prince of Songkla University, Pattani Campus)

Abstract
Background: Soft denture liners are extensively used in dentistry for patients who are not able to

tolerate denture-induced stresses. However, these liner surfaces provide a favorable environment for adhesion
and colonization of several microbes, especially pathogens like Candida albicans, thereby causing denture
stomatitis. A Melastoma malabathricum leaf is a traditional herbal medicine with pharmacological such as
antibacterial and antifungal. This study aims to investigate the antifungal and antibiofilm activities of ethanolic
extract of M. malabathricum leaf and investigate the mechanical properties of ethanolic extract of M.
malabathricum leaf incorporated with a soft liner. Methods: M. malabathricum leaf was extracted with 99.99%
ethanol using ultrasonic extraction. Susceptibility testing of Candida albicans by disc diffusion method.
Determination of Minimum Inhibitory Concentration (MIC), Minimum Fungicidal Concentration (MFC), and
antibiofilm activity using Resazurin-based 96-well plate microdilution method. Test mechanical properties of
denture soft liner material incorporated with M. malabathricum leaf extract by using a Universal testing
machine. Results: Planktonic C. albicans were susceptible to M. malabathricum leaves extract with an MIC50
and MFC of 62.5 mg/mL and 125 mg/mL, respectively. The percentage of biofilm reduction was 9.40 ± 6.9 to
56.40 ± 9.70 dose-dependent manners. Therefore, Soft liner incorporated with ethanol extract of M.
malabathricum leaf showed high elongation at break. The materials showed no significant changes in tensile
strength compared with the controls. M. malabathricum may be useful as promising agent management for
antifungal properties of denture soft lining materials.

:Keywords Melastoma malabathricum leaf, Candida albicans, Denture soft liner material

Introduction
Denture relining materials are widely used to prevent the irritation and inflammation of tissues

that are affected by the hard denture materials (Chladek et al., 2014). However, these liner surfaces produced
a conducive environment for infection by the adhesion, and colonization Candida albicans (C. albicans) that

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result in biofilm formation and denture stomatitis (Chincholikar et al., 2019). The biofilms are characterized
by a complex network of the yeast cell and hyphae intensely insert into an imperfections surface of the denture
leading to the therapeutic failure of antifungal agents (Ryalat et al., 2011). Inhibition of biofilms formation
plays a crucial role in preventing the development of denture stomatitis. Therefore, denture liner incorporated
with antifungal agents has been attentive. A Melastoma malabathricum leaf is a traditional herbal medicine
typically use for toothache and showed pharmacological properties such as antibacterial and antifungal
(Sarbadhikary et al., 2015). This study aims to investigate the antifungal and antibiofilm activities of ethanolic
extract of M. malabathricum leaf and investigate the mechanical properties of ethanolic extract of M.
malabathricum leaf incorporated with a soft liner.

Methodology
Part 1: Preparation of extract using ultrasonic extraction

Dried M. malabathricum leaves in the oven and grinds into a course. The coarse powder of dried
M. malabathricum leaves were soaked in 99.99% ethanol in the ratio of 1:10 for 24 hours then extracted in an
ultrasonic bath at a temperature of 40-50ºC for 30 minutes. Next, the ethanol supernatant was filtered using
Whatman No.1 filter paper. Concentrated the extract with Rotary Evaporator at 40ºC speed at 60 rpm and store
crude extract at 4ºC until use.
Part 2: Susceptibility testing of Candida albicans by disc diffusion method

Two-fold serial dilution of M. malabathricum extract was prepared to range from 31.25 mg/mL to
500 mg/mL in 30% ethanol. Preparing 0.5 McFarland of C. albicans solution then spread over the petri dish
containing Mueller Hinton agar, glucose 2% with Methylene blue (MHA) using a sterile cotton swab and leave
it for 5 minutes to dry the agar surface. The sterilized paper disc containing M. malabathricum ethanolic leaf
extract in various concentrations was placed onto the agar surface. The 30% ethanol and 0.06% Chlorhexidine
were used as negative and positive control, respectively. All tested plates were incubated at 37ºC for 24 and 48
hours then measure clear zone with Vernier Calipers.
Part 3: Determination of MIC and MFC using Resazurin-based 96-well plate microdilution method

The determination of minimum inhibitory concentration (MIC) of M. malabathricum extract was
evaluated using the Resazurin-based 96-well plate microdilution method. Preparing 2X of two-fold dilution
series of M. malabathricum leaf extract in broth. Briefly, a 96-well plate was filled with 100 µl of C. albicans
culture in Muller Hinton broth (MHB) which had a final density of 0.5 McFarland standard. Then, a 100 µl of
extract of various concentrations was added to the well and incubated at 37ºC for 24 hours. After incubation,
60 µl of 0.02% Resazurin solution was added and then the plate was incubated for 24 hours. The MIC was
determined visually in color changed from blue to pink. The minimum fungicidal concentration ( MFC) was
evaluated by subculturing well with no color change onto the MHA agar plate and then incubated at 37ºC for
24 hours. After incubation, the lowest concentration that did not show any visible growth of C. albicans was
represented as MFC.
Part 4: Determination the antibiofilm activity using Resazurin-based 96-well plate microdilution method

Biofilms were formed by pipetting cell suspensions (100 µl of a suspension containing 107 cells/ml
in RPMI-1640) into selected wells of 96-well plates and incubated at 37 ℃ for 24 hours. Wells were washed
with sterile PBS to remove non-adhered cells and 100 µl of RPMI-1640 was added to adhered cells. To test

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susceptibility to biofilm development, medium containing various concentrations of the leaf extract were added
immediately after the adhesion phase. The plates were incubated for 24 hours at 37 ℃.

Biofilm formation was quantitated using the Resazurin-based 96-well plate microdilution method.
The wells containing biofilm were washed with sterile PBS to remove non-adhered cells and add 200 µl of
RPMI-1640 to each well plate. 60 µl of resazurin at 0.02% (w/v) were added to plates containing RPMI-1640
and incubated at 37 ℃ for 24 hours in dark, at 100 rpm. The absorbance was measured at 570 and 600 nm as
a reference by using a microtiter plate reader. The percentage of biofilm inhibition was then calculated using
the following formula: % of biofilm inhibition = [ (controlOD570nm /Treated OD570nm)/ controlOD570nm]× 100.
Part 5: Mechanical properties of denture soft liner filled incorporated with M. malabathricum leaf extract

GC Reline Soft was used in this study. The soft liner was prepared by mixing powder and liquid
parts with the ratio of 5.5 g/4.5g according to manufacturer’s constructions. Then, the addition of 125,250
mg/ml of leaf extract, 0.06% chlorhexidine and 15% ethanol into the liner material, were investigated. After
mixing, the soft-liner films were pressed with a compression molding machine at 35 ℃ for 15 minutes. All
specimens were cut to the appropriate standard sizes ( ASTM D412 - die C) and mechanical properties were
tested using a universal testing machine.

Results
Ethanolic extract of Melastoma malabathricum leaf at concentrations 31.25 to 500 mg/ml showed a

mean inhibition zone ranging from 7.75 ± 0.35 to 16.25 ± 0.35 mm. at 24 hours and 7.38 ± 0.53 to 14.63 ±
0.88 mm. at 48 hours (Figure.1A). Planktonic C. albicans were susceptible to M. malabathricum leaves extract
with an MIC50 and MFC of 62.5 mg/mL and 125 mg/mL, respectively (Figure.1A). The percentage of biofilm
reduction was 9.40+6.9 to 56.40+ 9.70 in dose-dependent manners (Figure.1B). Therefore, soft liner
incorporated with ethanol extract of M. malabathricum leaf this result indicates that 125 mg/mL of M.
malabathricum is suitable concentration for soft liner exhibited excellent in flexibility. Meanwhile, the
materials showed no significant changes in strength compared with the controls (Figure 1C).

Figure1: Mean inhibition zone at 24h and 48h (A), percent reduction of biofilm for 24 h of C. albicans after
incubated with ethanol extract of M. malabathricum leaf at various concentrations (31.25-500 mg/ml) (B) and

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mechanical properties of soft liner incorporated with 125 and 250 mg/mL of ethanolic extract of M.
malabathricum leaf (C).

Discussion

Melastoma malabathricum Linn. is a traditional herbal medicine that has been reported to display
potent antibacterial and antifungal activities. Results of the present study suggested that M. malabathricum leaf
showed significant antifungal efficacy against C. albicans, and this agreed with Sarbadhikary et al., (2015)
showed that leaf extracts of M. malabathricum had moderate activity against human pathogenic fungi C.
albicans. The previous study by Mita et al., (2017) showed methanol extract of M. malabathricum leaf contains
saponins, flavonoids, and tannins, these constituents may work as antifungals.

Conclusion
The ethanolic extract of M. malabathricum leaf exhibited antifungal and antibiofilm activities against

C. albicans. Soft liner incorporated with extract exhibited mechanical properties like reference material suggesting
the possibility of this M. malabathricum leaf for antifungal management on denture soft lining materials.

Acknowledgements
Acknowledgment This project was supported by Science Classroom in University Affiliated

School (SCiUS) under Prince of Songkla University and Islamic Science Demonstration School. The funding
of SCiUS is provided by the Ministry of Higher Education, Science, Research, and Innovation, which is highly
appreciated.

References
Chincholikar, S., Sridevi, J., Kalavathy, N., Singh, S., Kapoor, A. and Saumya, S. Comparative evaluation of

two antifungal agents incorporated in auto polymerizing denture base resin, heat polymerizing denture
base resin and permanent silicon soft liner-an in vitro study. J Clin Diagn Res 2019;13(1):49-54.
Chladek, G., Zmudzki, J. and Kasperski, J. Long-term soft denture lining materials. Materials (Basel)
2014;7(8): 5816-5842.
Mita, N., Ardana, M., Arifuddin, M. and Priastomo, M. Determination of extract quality parameters of
Sepabang (Melatoma malabathricum L.) leaves from Dayak bahau and Abai ethnics in Borneo and its
antibacterial and antifungal activity. International Seminar of Natural Product, Sekoloh tinggi ilmu
farmasi makassar; 2017. pp. 17-21.
Sarbadhikary, S.B., Bhowmik, S., Datta, B.K. and Mandal, N.C. Antimicrobial and antioxidant activity of leaf
extracts of two indigenous angiosperm species of Tripura. Int J Curr Microbial App Sci 2015;4(8):
643-655.
Ryalat, S., Darwish, R. and Amin, W. New form of administering chlorhexidine for treatment of
dentureinduced stomatitis. Ther Clin Risk Manag 2011;7:219-225.

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Title : Effect of Chitin-Binding Proteins from Para Rubber Seeds on OB5_16_01
Field : Phytopathogenic Fungi and Bioactivity Enhancement
Author : Biology and Biological Diversity
School :
Advisor : Ms. Nihusna Nadaman
Ms. Sasita Seh

Demonstration School Prince of Songkla University, Prince of Songkla
University, Pattani campus

Dr. Apichai Bourchookarn1 and Asst. Prof. Dr. Walairat Bourchookarn2
1Department of Agriculture and Fishery Science, Faculty of Science and Technology,
Prince of Songkla University, Pattani Campus
2Department of Science, Faculty of Science and Technology, Prince of Songkla
University, Pattani Campus

Abstract

Chitin binding proteins (CBPs) have specific biological properties to bind to chitin. This protein
family has high potential to inhibit fungal growth. CBPs can be obtained from several biological sources
including plants and other organisms. This project aimed to extract and enrich CBPs from rubber seed
kernels (RSK) using column containing colloidal chitin prepared from squid pens and to evaluate for
antifungal activity against Phytophthora botryoza and Schizophyllum sp. Extraction of RSK-CBPs was
performed by grinding of RSK in extracting buffer (Tris-HCl 0.05 M, pH 8, NaCl 0.15 M). The resulting
suspension was filtered through Muslin cloth and centrifuged at 8,000 rpm at 4°C for 30 min. The obtained
cleared solution was incubated with squid-pens colloidal chitin for overnight and 4°C. After successively
washed with washing buffer, the CBPs were then eluted with extracting buffer containing 1 M of N-acetyl-
D-glucosamine (NAG). CBPs concentration was estimated with Bradford assay reagent. By this method,
CBPs of 5.39 mg per 100 g of RSK was obtained. Antifungal activity was carried out using agar well
diffusion method. The synergism antifungal activity between CBPs and graphene oxide (GO) at a ratio of
75%:25%, 50%:50% and 25%:75% were also performed against P. botryoza and Schizophyllum sp. CBPs at
a concentration of 1 mg/mL affected the growth of P. botryoza, but not Schizophyllum sp. However, it was
still not able to observe any clear inhibitory effect of GO including combination between CBPs and GOs at
selected concentration ratios on growth of those phytopathogenic fungi. The results from this project
introduced the basis insight to the utilization of rubber seed constituents, which are considered as
agricultural waste, that support the bio-based economy and also support approaches to the use of
nanomaterials in agriculture.

Keywords : Chitin-Binding Protein, Para Rubber Seeds, Graphene Oxide, Phytophthora botryoza,
Schizophyllum sp.

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Introduction

Nowadays, plant pathogenic fungi are one of the main causes of damage to agricultural products.
This results in a decrease in quality and quantity of produce. It is, therefore, necessary to use fungicides to
prevent these fungi. But these substances are toxic to human health and the environment. Seeking of a method
without negative impact and environmental-friendly is therefore an attractive. Rubber seeds are biologically
important by-products and are often wasted or useless. Preliminary studies in the laboratory have shown that
the kernel of the rubber seed contains a group of proteins called Chitin-Binding Proteins (CBPs). It is well
known that these proteins from several sources including plant seeds have specific biological activity against
a wide variety of plant pathogenic fungi. However, the use of protein substances in biological methods for
plant disease prevention still has several limitations and then loss of bioactivity during use. So we thought of
using the method of meeting each other halfway, which is a synergistic method. Graphene Oxide (GO) is now
widely reported to have a good fungicidal effect at the laboratory level. We are therefore interested in using
GO to enhance the bioactivity of CBPs against some selected plant pathogenic fungi.

Methodology

Preparation of chitin
Squid pens were cleaned with water, dried and then grounded into powder. Demineralized

by soaking Squid pens powders in 1M HCl at a ratio between the squid pens to HCl is 1:10 (g/ml)
at room temperature overnight. Then deproteinization by soaking in 4% NaOH solution at a ratio of
1:10 (g/ml) at room temperature for one night, the solution was filtered out. Then washed with
distilled water to neutral pH and dried until dried-white powder chitin was obtained.

Preparation of colloidal chitin
2g of chitin powder was soaked in 100 ml of 85% phosphoric acid. Stirred and placed in

the refrigerator at 4°C overnight. Filtered through 3 layers of filter paper in a Buchner funnel.
Finally, washed with distilled water to neutral pH.

Extraction of chitin-binding proteins
Extraction of CBPs was performed by method described by Gifoni et al.1 Rubber seed

kernels was separated from seed coat then grounded. The powder was then soaked in extracting buffer
(Tris-HCl 0.05 M, pH 8, NaCl 0.15 M) at a ratio between the rubber seed kernels to buffer is 1:10
weight by volume. The resulting suspension was filtered through muslin cloth and centrifuged at
8,000 rpm at 4°C for 30 min. Crude extract protein was obtained. The supernatant was used for
extraction of chitin-binding proteins by affinity chromatography using colloidal chitin from squid
pens as a chitin column. The chitin column was equilibrated with extracting buffer then loaded crude
extract protein. Finally, the chitin-binding proteins were eluted with extracting buffer containing 1M
of N-acetyl d glucosamine.

Analysis of chitin-binding proteins concentration
The total proteins content was determined by Bradford assay at 595 nm with a spectrophotometer.

Synthesis of graphene oxide
GO was performed by method described by Marcano et al.2 A mixture of 3g of graphite

and 18g of KMnO4 were added slowly into a 9:1 mixture of concentrated sulfuric acid and
phosphoric acid. Experiments were carried out in ice bath all the time. Continuous stirred for 72 h
until the color changed from dark green to dark brown. Then added distilled water into the mixture
and the oxidation reaction was stopped by slowly adding 30% of hydrogen peroxide (H2O2) into the
mixture with continuous stirred until the color of the substance turned bright yellow. Filtered

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through filter paper and washed with 5 % w/w of HCl, distilled water and ethanol by centrifuged at
8,000 rpm for 30 min, respectively. The filtrate was dried at 50°C and black brown graphite oxide
powder was obtained. Then exfoliation in a sonication bath until brown graphene oxide suspension
was obtained.

Analysis of Graphene oxide

Analysis was performed successfully using Fourier Transform Infrared Spectroscopy
(FTIR) and Ultraviolet-Visible Spectroscopy (UV-Vis)

Antifungal activity assay

Antifungal activity was carried out by agar well diffusion method. Placed the fungi at the
center of the PDA agar plates. Then incubated for 2-3 days. The wells were set using cork borer.
Pipetted test substances, that were CBPs, GO, in the ratio between CBPs and GO at 75:25, 50:50
and 25:75. Incubated the plate at 30°C for 24-48 h then observed the inhibition zone.

Results, Discussion and Conclusion

Preparation of colloidal chitin from the squid pens and FT-IR of chitin.
Chitin can be prepared from the squid pens as white powder in Figure 1A and the colloidal

chitin is a white viscous slurry in Figure 1B. The FTIR spectra of chitin was shown in Figure 2A.
Chitin presented some characteristic peaks: the peak at 3279 cm-1 is attributed to the hydroxyl
group (O-H stretching). Moreover, observed the peak of the vibration of amide I (C=O stretching),
Amide II (N-H bending) and Amide III (C-N stretching) groups, at 1627, 1554 and 1315 cm-1,
respectively.

Preparation of chitin-binding proteins from rubber seed.
Be able to prepare crude extracted protein from rubber seed kernels in Figure 1C and be able

to extract CBPs from crude extracted protein using chitin prepared from squid pens. The CBPs can
be eluted out as a clear solution as shown in Figure 1D. CBPs concentration was estimated with
Bradford assay reagent, the result is blue color indicating the presence of proteins in Figure 1E, which
are CBPs. By this method, CBPs of 5.39 mg per 100 g of RSK can be obtained.

Synthesis of Graphene Oxide (GO), FTIR and UV-VIS.
Graphene Oxide was successfully synthesized using the Tour method in Figure 1F, It was

found that the colors and characteristics were changed in the same manner as described by Marcano
et al.2 FTIR was measured in a comparison between the synthesized GO and graphite in Figure 2B.
The presented of IR peak at web number 3363 cm−1 corresponded to the O–H stretching vibrations.
The band observed at 2104 cm−1 was assigned to -C≡C- stretching and the band observed at 1729
cm−1 is attributed to the presence of C=O stretching. Moreover, the vibrational mode of C=C
stretching and C-O stretching peak also appeared in the FTIR spectra of GO. This shows that the
functional group of graphite has been changed to GO. GO was also tested with UV-Vis spectroscopy
and compared with the graphite spectra in Figure 2C. The spectrum of graphene oxide has an
absorption maximum at 230 nm. The absorbance characteristics are consistent with the research by
Marcano et al.2 who reported that the synthesized GO had a peak absorption at a wavelength of 227-
231 nm. It was found that the absorbance spectra of GO and the graphite were different, indicating
that the graphite was oxidized and the functional group appeared in the structure of graphite.

Antifungal activity assay.
CBPs at a concentration of 1 mg/mL affected the growth of Phytophthora botryoza, but not

Schizophyllum sp. However, it was still not able to observe any clear inhibitory effect of GO including
combination between CBPs and GO at selected concentration ratios on growth of those

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phytopathogenic fungi in Figure 1G and 1H. This may be due to the use of the tested substances at
inappropriate concentrations and needed to be confirmed by other tests.

AB CD E

F GO CBPs G GO CBPs H

NE NE

CBPs:GO CBPs:GO CBPs:GO CBPs:GO
25:75 75:25 50:50 75:25

CBPs:GO CBPs:GO
50:50 25:75

Figure 1: (A) Chitin, (B) colloidal chitin, (C) crude extracted protein from rubber seed kernel, (D) eluted
chitin-binding proteins, (E) Bradford assay of CBPs (Left: CBPs ; Right: eluted solution), (F) dispersed
graphene oxide suspension, (G) well diffusion assay with Phytophthora botryoza and (H) well diffusion
assay with Schizophyllum sp.

Transmittance [%]
75 80 85 90 95

3279
1627
1554

1375
1315

1063
1030

3500 3000 2500 2000 1500 1000 500
Wavenumber cm-1

D:\IR spectrum\2565\Sasita\Chitin.0 Chitin Instrument type and / or accessory 1/10/2022

D:\IR spectrum\2565\Sasita\Chitin.0 Chitin
Chitin
Figure 2: (A) FTIR spectra of chitin, (B) FTIR spectra of graphite (black) GO (blue) and (C) UV-VISD:\IRspectrum\2565\Sasita\Chitin.0
Instrument type and / or accessory 1/10/2022
Instrument type and / or accessory 1/10/2022

Page 1/1

spectra of graphite (black) GO (blue)

Acknowledgements

This project was supported by Science Classroom in University Affiliated School (SCiUS). The
funding of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation. This
extended abstract is not for citation.

References

1. Gifoni JM, Oliveira JTA, Oliveira HD, Batista AB, Pereira ML, Gomes AS, et al. A novel chitin-
binding protein from Moringa oleifera seed with potential for plant disease control. J Pept Sci 2012;
98:406-15.

2. Marcano DC, Kosynkin DV, Berlin JM, Sinitskii A, Sun Z, Slesarev A, et al. Improved Synthesis
of Graphene Oxide. ACS Nano 2010;4;4806-14.

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Title : Development of edible coating from OB5_17_06
Gracilaria fisheri and tannin of banana

peel extracts for extending shelf life of
‘Nam Dok Mai’ mango fruit

Field : Biology and Biodiversity

Authors : Ms. Warissara Nateetorn and Ms. Witsuta Noosing

School : PSU. Wittayanusorn Surat Thani School, Prince of Songkla University, Surat Thani Campus

Advisors : Asst. Prof. Dr. Nittaya Ummarat (Prince of Songkla University, Surat Thani Campus)

Ms. Usa Petbanna (PSU. Wittayanusorn Surat Thani School)

Abstract
‘Nam Dok Mai’ mango is an economically important fruit in Thailand. However, there are some

postharvest problems limiting its shelf life, such as high respiration and ethylene production, as well as some
postharvest diseases. Therefore, this study aims to investigate the efficiency of some edible coatings to prolong
shelf life of ‘Nam Dok Mai’ mango fruit. The coating treatments including Gracilaria fisheri extract
(GE 20 g/L), banana peel tannin extracts (TE 40 % (v/v)) and the combination (GE + TE) were applied to
‘Nam Dok Mai’ mango fruits at 80% ripeness. The results showed that changes of peel color (L and a values),
weight loss and pulp firmness reduction tended to delay in all coated fruits. In addition, the mango fruit coated
with GE + TE treatment showed the highest TSS comparing with other treatments, while their pulp firmness
was maintained and DI was delayed up to day 6 of the storage time. These results suggest the potential of GE
and TE coating for maintaining the postharvest quality of ‘ Nam Dok Mai’ mango fruit. However, further
investigation is still needed to optimize the most suitable coating formula.

Keywords: Edible coating; tannin; Gracilaria fisheri; ‘Nam Dok Mai’ mango, postharvest quality

Introduction
‘Nam Dok Mai’ mango is one of the most economically important fruit in Thailand. However, mango

is a climacteric fruit that rapidly ripens and softens after harvest because of its high respiration and ethylene
production (Acosta et al., 2000). Moreover, anthracnose caused by Colletotrichum gloeosporioides (Penz.) is
a major postharvest disease in mango that can reduce shelf life of the fruits. According to postharvest
technologies, fruit coating is becoming one of the most popular methods to preserve the postharvest quality of
fruits by delaying ripening, water loss, and decay caused by pathogens (Baldwin et al., 1997; Amarante and
Banks, 2001). As nowadays, consumers are more interested in healthy food, many researches have conducted
on environmentally friendly methods for postharvest treatments such as the application of chitosan, shellac and
tannic acid in mango fruits (Jitareerat et al., 2007; Jongsri et al., 2016; Zahedi et al., 2019; Ma et al., 2021) and
seaweed extract in banana fruit (Ziedan et al., 2018).

Since the red seaweed Gracilaria fisheri contains high content of agar and can be widely grown in
Southern of Thailand, this research aims to develop an edible coating from its agar extract. Moreover, tannin
extract from banana peel will be used as a natural additive of coating to find the optimum condition for
maintaining postharvest quality and extending the shelf life of 'Nam Dok Mai' mango fruit.

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Methodology
Preparation of G. fisheri and tannin extracts: Samples of G. fisheri (10, 20 and 40 g) were pretreated with
6 % NaOH and 0.025 % H2SO4 as modified method by Wang et al. (2017). The pre-treated seaweed samples
were cut into 1- 1. 5 cm length, added to 1,500 mL of distilled water ( pH 6. 2 - 6. 5) and boiled for 1. 5 h with
intermittent stirring. The seaweed extracts were filtered with a filter cloth, adjusted volume to 1 L, and then
used for coating treatment of 10, 20 and 40 g/L. For tannin extraction, samples of banana peel were completely
dried in hot air oven (60 °C), ground into powder, extracted with distilled water (1:30 w/v), incubated in water
bath ( 50 °C) for 3 h and in incubator shaker ( 40 °C) for 3 h. The supernatant of tannin extract was collected
after centrifuged at 6,000 g for 5 min (Phanpanat, 2019). Then, the tannin extract was used as coating treatment
of 30, 40 and 50 % concentrations (v/v).
Coating treatment: All coating treatments were mixed well with glycerol 1 % ( w/ v) . Then, samples of
‘Nam Dok Mai’ mango fruits at 80 % ripeness were dipped into each coating treatment for 3 min, let the fruit
dried at room temperature for 30 min and packed into a clamshell (1 fruit/clamshell, 10 fruits/treatment).
Fruit quality evaluation: Changes in postharvest quality including percentage of weight loss, peel color ( L,
a, b and hue values) , percentage of disease incidence ( DI) , pulp firmness and total soluble solids ( TSS) were
examined every 2 or 3 days during storage at 25 °C until the end of shelf life (6 - 8 days).
Statistical analysis: Data were subjected to analysis of variance ( ANOVA) by using the SPSS program
(P < 0.05). All data were expressed as Mean ± S.E. (n = 10).

Results and Discussion
Based on the screening of effective concentration by measuring peel color and weight loss, 20 g/L GE

and 40 % ( v / v ) TE coating treatments were selected for determining their effects on 'Nam Dok Mai' mango
fruit quality, comparing with the combination treatment (GE + TE).
Peel color: The results of peel color showed that GE and GE + TE tended to have lower L and a values
compared to other treatments (Figure 1A & 1B) up to day 8 of the storage. A delay of fruit ripening had been
reported in carrageenan treated fruit (Dwivany et al., 2020), which can be a result of limiting oxygen and then
affecting on a reduction of peel color change (Sirikan et al., 2012)
Weight loss: All coated fruits tended to have lower weight loss than control fruit, which TE treated fruits
showed the lowest weight loss, followed by those of GE + TE, on day 8 after storage (P > 0.05) compared to
other treatments ( Figure 1D) . Ma et al. ( 2020) have reported that tannic acid ( TA) combined with shellac
coated fruits showed lower weight loss compared to shellac coated fruits, which was related to the thicker of
TA-shellac film, resulting in a reduction of respiration rate and ripening process.
Pulp firmness and TSS: Fruits treated with TE and GE + TE showed higher pulp firmness than other
treatments (Figure 2A). Moreover, GE and TE tended to have lower TSS, while GE + TE treated fruits showed
the highest TSS, compared to other treatments (Figure 2B). The maintained of fruit firmness was indicated in
TA-shellac treated mango (Ma et al., 2020). In lime fruit, the higher TSS in banana peel extract incorporated
with alginate coated fruits compared to control fruits had been observed (Phanpanat, 2019).
Disease incidence (DI): Fruits treated with GE and GE + TE showed less DI than other treatments up to day
6 of the storage (Table 1), however, there was similar DI on day 8. The improvement of the antifungal effect
also had been reported in mango treated with shellac combined with tannic acid (Ma et al., 2020).

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Figure 1. Changes of peel color including L value ( A) , a value ( B) and , b value ( C) , weight loss ( D) of
'Nam Dok Mai' mango treated with G. fisheri extract (GE), tannin extract (TE) and the combination (GE + TE)
during storage at 25 °C for 8 days. Different letters indicate significant differences (P < 0.05) for at each storage
time.

Figure 2. Pulp firmness (A) and total soluble solids (TSS; B) of 'Nam Dok Mai' mango treated with G. fisheri
extract (GE), tannin extract (TE) and the combination (GE + TE) after storage at 25 °C for 8 days.

Table 1. Disease incidence (DI) of 'Nam Dok Mai' mango fruits treated with Gracilaria extract (GE), Tannin
extract (TE) and the combination (GE + TE) during storage at 25 °C for 8 days.

Treatment Disease incidence (DI, %)

Control Day 0 Day 2 Day 4 Day 6 Day 8
GE 20 g/L 0 0 0.2 0.2 0.3
TE 40% 0 0.2
GE 20 g/L + TE 40% 0 000 0.2
0 0 0 0.2 0.3

000

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Conclusion
Regarding our results, the edible coating from agar extract of G. fisheri and Tannin extract of banana

peel showed some tendencies for maintaining postharvest quality of ‘Nam Dok Mai’ mango fruit by delaying
changes of peel color, weight loss, firmness, TSS and the occurrence of disease. However, more investigation
will be needed to develop a higher efficiency edible coating from G. fisheri agar and tannin extracts. These
edible coatings can be an alternative postharvest treatment since they are extractable from natural and local
products, which are environment-friendly and cost-effective materials.

Acknowledgments
This project was supported by Science Classroom in University Affiliated School (SCiUS). The

funding of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation. This
extended abstract is not for citation. Also, the funding by 24th Young Scientist Competition, National Science
and Technology Development Agency, which is highly appreciated.

References
1. Acosta, R. M., Neito, A. D., Mena, N. G. V., Vaquera, H. H., Teliz, O. D., Nieto, A. R., et al. Effect
of post-harvest temperatures on the development of internal darkening in mango fruits (Mangifera
indica L.) cv. Haden and their quality. Acta Horticulture 2000; 509: 401-12.
2. Amarante, C., Banks, N.H. Postharvest physiology and quality of coated fruits and vegetables.
Horticultural Reviews 2001; 26: 161-238.
3. Baldwin, E.A., Nisperos, M.O., Hagenmaier, R.H., Baker, R.A. Use of lipids in edible coatings for
food products. Food Technology 1997; 51: 56-62.
4. Dwivany, F.M. et al. Carrageenan edible coating application prolongs Cavendish banana shelf life.
International Journal of Food Science 2020.
5. Jongsri, P., Wangsomboondee, T., Rojsitthisak, P., Seraypheap, K. Effect of molecular weights of
chitosan coating on postharvest quality and physicochemical characteristics of mango fruit. LWT
Food Sci Technol 2016; 73: 28-36.
6. Ma, J. et al. Novel edible coating based on shellac and tannic acid for prolonging postharvest shelf
life and improving overall quality of mango. Food chemistry 2021; 354: 1-12.
7. Wang, L. et al. Impact of alkali pretreatment on yield, physico-chemical and gelling properties of
high quality agar from Gracilaria tenuistipitata. Food Hydrocolloids 2017; 70: 356-62.

8. พรรณพนัช แช่ม. ผลของสารสกัดหยาบจากเปลือกกล้วยน้ำว้าและอัลจเิ นทตอ่ คุณภาพของ ผล
มะนาวหลงั การเก็บเกี่ยว. วทิ ยานิพนธป์ ริญญาวิทยาศาสตรมหาบณั ฑติ , มหาวทิ ยาลัยเทคโนโลยี
ราชมงคลธัญบุรี 2562; 42-84.

9. ศิรกานต์ ศรีธัญรันต์ และคณะ. ผลของสารเคลือบผิวบางชนิดต่อคุณภาพของมะม่วงพนั ธ์ุ
น้ำดอกไม้ เบอร์ 4 ระหว่างการเกบ็ รกั ษา. วิทย์ กษ. 2555; 101-4.

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Oral presentation
Biology and Biodiversity Group 5

Sunday August 28, 2022

No. Code Title Author School

1 OB5_09_01 The Neurodevelopment of Miss Nantanat Wongsamut Engineering Science

Attention and Working Miss Kewalee Thirasak Classrooms

Memory Miss Prempavee Treevijitpaisan (Darunsikkhalai School)

2 OB5_15_06 Biosynthesis of silver Miss Kamonchanok Thirawut PSU.Wittayanusorn

nanoparticles using corncob Miss Tichada Jokloy School

extract and evaluation of

antibacterial activity against

Vibrio parahaemolyticus

3 OB5_06_01 Potential evaluation of Mr. Kritin Prajonsant Rajsima Witthayalai

Amycolatopsis oliviviridis Miss Treerat Potchanapong School

strain SCM_MK2-4 for Miss Rujirada Bucharam

PLA-bioplastic degradation

and lactic acid recovery

4 OB5_02_08 Geographical tracking and Miss Tareetip Chanwikkarn Demonstration School,

mapping of coronavirus Miss Tanyavee Tawbunyuen University of Phayao

disease COVID-19 of Phayao

5 OB5_18_06 Coccoid forms of Miss Thannumtip Pitchayasopanan Surawiwat School,

Helicobacter pylori related Mr. Potsathon Phaktiyanuwat Suranaree University of

with false negative Rapid Miss Peerapan Khumkrong Technology

Urease Test

6 OB5_03_10 Development of edible Mr. Phummiphat Promnoi Naresuan University

coating combined with bitter Secondary

gourd extract on fruit peel Demonstration School

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12th SCiUS Forum

Title : OB5_09_01The Neurodevelopment of Attention and Working Memory
Field :
Author : Biology and Biodiversity

School : Ms. Kewalee Thirasak
Advisor :
Ms. Nantanat Wongsamut

Ms. Prempavee Treevijitpaisan

Darunsikkhalai Science School

Dr. Sirawaj Itthipuripat (King Mongkut's University of Technology Thonburi)

Dr. Kanda Lertladaluck (King Mongkut's University of Technology Thonburi)

Ms. Jintana Wongta (Darunsikkhalai Science School)

Abstract

Several studies show the relationship between age and the development of visual working memory
(VWM) and focused attention. However, it is still unclear if the development would continue along with age.
Therefore, the research aims to study the development of VWM and focused attention across age groups and
explain the development with neuro indexes: prefrontal bias signa, and contralateral delay activity. The visual
working memory capacity and focused attention of healthy participants (6-36 years old) were measured using
the delayed match-to-sample task which required participants to remember the targets and ignore distractors
in 150 ms and 500 ms time interval. At the same time, an electroencephalogram (EEG) was applied to record
brain electrical signals. Possible related factors were controlled by giving participants some questionnaires:
Demographic Information, IQ, EQ, Mindset, Behavior assessment, Economic status, and Game addiction. The
EEG results, behavioral data, and questionnaires will be analyzed to detect possible factors related to the
developments. The results show that the development of VWM capacity and focused attention tend to continue
from childhood into teenagers, as teenagers can remember more targets than children. Teenagers are likely to
be able to remember more targets than adults, but when distractors appear in 500 ms time interval, adults can
remember targets more than teenagers. In other words, the development of focused attention and visual
working memory is obviously increasing from childhood (6-12 years old) to adolescence (13-17 years old).
Indicated from prefrontal bias signal and contralateral delay activity, when distractors appear, the filtering
process starts the exclusion of unnecessary information in VWM capacity which has maximum limit at 3
objects.

Keywords : Visual working memory, Focused attention, Electroencephalogram, Development

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Introduction

Focused attention and visual working memory play an important role in learning, as focused attention
helps focusing on interested information uninterrupted, then the gathered information is filtered and sent to
the brain as a short-term memory which allows VWM to process a limited amount of visual information
promptly.

Previous studies indicated that people at different ages have differences in VWM capacity and general
development, but it is still controversial how VWM capacity develops and how it is related to focused
attention. Therefore, we aim to study the development of focused attention and VWM capacity. Since there
are no studies using neuro index to explain neurodevelopment yet, in this study, the development was
considered with the research unfamiliar neuro indexes: contralateral delay activity, and prefrontal bias signal
in order to study the relationship between brain mechanism and the development. In addition, the same task is
applied in all age groups for more accurate results.

Methodology

The experiments were divided into 3 parts as follows,
Part 1: Completing questionnaires and using them as controlled variables.

Questionnaires include Demographic information, Relational dimension (IQ), Emotional intelligence (EQ),
Implicit theories of intelligence (Mindset), Behavior assessment (SNAP-IV), Socio-Economic Survey (SES),
and Game Addiction Screening Test (GAST).

Part 2: Electroencephalogram (EEG) waves detecting and doing computer task ‘delayed match-to-
sample'. Participants need to memorize the targets and ignore distractors.

2.1 EEG preparation: EEG cap is put on the
participants’ head, followed by filling the gel in the
channel and attaching electrodes to the scalp.
2.2 EEG recording and delayed match-to-sample task:
Examining prefrontal bias signal and Parietal delay
activity. Those are detected by electrodes with wires
and amplifiers, to the BioSemi’s monitor while doing
the computer task run by MATLAB.
Part 3: Data analysis using Excel, PowerBI and EEGLAB: Data from Questionnaire, delayed match-
to-sample task, and EEG was analyzed together to find the relationship between them.

Results

From collected behavioral data at 150 and 500 ms time intervals, it illustrates that children have the
least VMW capacity. VWM capacity increases rapidly from children to teenagers and reaches the peak at 3
objects. In adults, the capacity slightly drops; however, there is no difference of the maximum VWM capacity
between teenagers and adults.

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Figure 1: Graphs of participants’ responses to delayed-match-to sample task in 500 and 150 ms time interval.

Figure 2: Graphs of EEG waves from frontal cortex
The graphs illustrated the larger amplitude of frontal EEG signals at the encoding time when
distractors were added to visual working memory capacity which confirms the release of prefrontal bias signal
as a sign to start the filtering process.

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Figure 3: Graphs of EEG waves from parietal cortex
The detected contralateral delay activity amplitude from EEG shows the maximum number of objects
held in VWM which is no more than 3, evidenced from the highest amplitude which is shown in setsize 3.
Even distractors appear, they have no effect on the amplitudes if the capacity reaches maximum limit.

Discussion and Conclusion
The results illustrate age-related development of VWM capacity: the least VWM capacity is found

in children, then it reaches the peak in teenagers and slightly decreases in adults. Indicated from contralateral
delay activity and prefrontal bias signal from parietal cortex and prefrontal cortex, they both share the
maximum of 3 objects held in VWM capacity. The filtering process starts the detection and exclusion of
unnecessary information from a limited VWM capacity when distractors are shown.

Because of the limitation of VWM capacity, when distractor was detected in VWM capacity,
prefrontal bias signal from prefrontal cortex was released to start filtering unnecessary information out of
VWM capacity in order to process interested information efficiently. The contralateral delay activity from
parietal cortex proved that the filtered information was memorized and collected in VWM capacity with the
maximum capacity of 3 objects. Consequently, development of the prefrontal cortex may affect the ability to
filter out unnecessary information.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS) under

King Mongkut’s University of Technology Thonburi, Neuroscience Center for Research and Innovation,
Learning Institute, and Darunsikkhalai Science School. The funding of SCiUS is provided by the Ministry of
Higher Education, Science, Research and Innovation. This extended abstract is not for citation.

References
Cowan N., 2017, Working Memory Maturation: Can We Get At the Essence of Cognitive Growth?,

Perspect Psychol Science, 11(2), 239-264.
Plebanek, D. J., & Sloutsky, V. M., 2019, Selective attention, filtering, and the development of

working memory, Developmental Science, 22(1), e12727.
Liesefeld et al., 2014, Intercommunication Between Prefrontal and Posterior Brain Regions for

Protecting Visual Working Memory From Distractor Interference, Association for Psychological Science,
25(2), 325-333.

Isbell et al., 2015, Visual working memory continues to develop through adolescence, Frontiers in
Psychology, 6(May), Doi: 10.3389/fpsyg.2015.00696.

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Title : Synthesis of silver nanoparticles using corncob OB5_15_06
Field : extract and evaluation of antibacterial activity against
Vibrio parahaemolyticus

Biology and Biodiversity

:Author Miss Kamonchanok Thirawut

School : Miss Thichada Jokloi
PSU.Wittayanusorn School, Prince of Songkla University

:Advisor Dr.Wilanee Chunglok

Abstract
In the present study, cost-effective use of non-toxic materials and environmental fabrication of

silver nanoparticles by using an extract of waste corn cob. The experiments were to study the temperature, time,
and extract/volume ratio of AgNO3 suitable for synthesis. The synthesized silver nanoparticles were examined
to confirm the formation of silver nanoparticles by measuring the absorbance with a UV-Vis spectrophotometer
at a wavelength of 300 – 750 nm. It was found that the temperature at 90 °C, 45 min, and the extract/volume
ratio of AgNO3 at 1: 10 were the suitable conditions for the synthesis. The maximum absorbance is at a
wavelength of 415 nm, which is the spectral range of silver nanoparticles. For a study on the ability to inhibit
Vibrio parahaemolyticus bacteria pathogenic in shrimp. The agar well diffusion method was tested for efficacy
in inhibiting bacteria. It was found that V. parahaemolyticus ATCC and V. parahaemolyticus PSU513 had
inhibition zone values of 10.19 and 9.28 mm, respectively.

:Keywords corncob extract, silver nanoparticles, Vibrio parahaemolyticus, antibacterial activity.

Introduction

Silver nanoparticles (AgNPs) are of great interest because of their remarkable antibacterial and
localized surface plasmon resonance properties, making them unique in broad-spectrum antimicrobial
properties. However, several nanoparticle synthesis methods, including silver nanoparticles, often lead to
producing several undesirable wastes that are harmful to the environment. Therefore, the search for more
effective and environmentally friendly methods is ongoing. These methods are called "green synthetic
methods" and are efficient since they do not use hazardous reagents for the environment.

Several plant extracts have been used as substitutes for reducing agents and stabilizers. For example,
chemicals containing phenol and flavonoid groups can reduce silver ions (Ag+) to silver zero (Ag0) and form
silver nanoparticles. In addition, plant-derived polysaccharides can stabilize nanoparticles and be suspended
in water.

This study shows the green synthesis of silver nanoparticles using corncob extract as a reducing agent
and the antibacterial activities of synthesized silver nanoparticles. In addition, the utilization of corncob makes
up the utilization of agricultural waste prior to its removal for decomposition or combustion. Therefore, it is
also an alternative to maximizing the use of agricultural waste from plants.

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Methodology
The experiments were divided into 4 parts as follows,

Part 1 : CornCobs extract preparation
Corncobs are collected and washed thoroughly, then dried and cut into small pieces. Next, bake and

grind to a fine powder. After that, we weighed corncob powder to 10 g, put it in a beaker, added 100 ml of
distilled water, and then boiled it for two hours. Then, they filtered the mixer and stored it at 4–10 degrees
celsius.

Part 2 : Study the factors affecting the synthesis of AgNPs
silvernitrate solution to a volume of 20 ml and then heated on a magnetic stirrer. Then, the synthetic

reaction times were 15, 30, 45, and 60 minutes. Next, study synthetic temperatures at 70, 80, and 90 degrees
celsius and the ratio between corncob extract and AgNO₃ solution at 1:5, 1:10, and 1:20. The last step is to
measure the silver nanoparticle absorbance at a 300–750 nm wavelength with a UV-Vis spectrophotometer.

Part 3 : Bacterial preparation
Vibrio parahaemolyticus ATCC 17802 and Vibrio parahaemolyticus PSU513 were cultured in TSA

and incubated at 37 °C for 18–24 h. Then it picked 2 or 3 colonies of bacteria to be cultured on MHB containing
1% NaCl and incubated at 37 °C in a shaking incubator at 150 rpm for 4 hours. Then, bacteria were adjusted
at 0.5 Mcfarland turbidity and diluted to 106 CFU/millilitre before studying antibacterial activity.

Part 4 : Antibacterial activity at the of AgNPs against V. parahaemolyticus.
Vibrio parahaemolyticus ATCC 17802 and Vibrio parahaemolyticus PSU513 were swabbed onto

MHA agar containing 1% NaCl with a sterile cotton swab. Then, a well of 6 mm in diameter was punctured
using sterile pipette tips. Next, 50 μL of synthetic silver nanoparticle solution was added to each well. The
positive controls were gentamicin discs. On the other hand, the negative control was corncob extracts. Then, V.
parahaemolyticus ATCC 17802 was incubated on the plate at 35°C and V. parahaemolyticus PSU513 at 30°C.
The inhibition zones were measured after 24 h.

Results
In the study of synthetic time, temperature, and the ratio of corncob extract to AgNO₃ solution, it was

found that the synthesized has a maximum absorbance of a wavelength of 415 nm, and the suitable synthetic
time is 45 minutes, as shown in Figure 1. The maximum absorbance temperature is 90 degrees celsius, as
shown in Figure 2 and the highest absorbance ratio is 1:10 because at the ratio of 1:5, the graph is unstable, as
shown in Figure 3.

Figure 1 Figure 2 Figure 3

Figure 1 : spectrum of synthetic silver nanoparticles at 15, 30, 45 and 60 minutes.
Figure 2 : spectrum of synthetic silver nanoparticles at temperatures of 70, 80 and 90 degrees celsius.
Figure 3 : spectrum of silver nanoparticles synthesized using extract ratio corncobs per AgNO₃ 1:5, 1:10

and 1:20 by volume.

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In to study of the efficacy of AgNPs against V. parahaemolyticus ATCC 17802 and V.
parahaemolyticus PSU513 by agar well diffusion method show that AgNPs are effective in inhibiting bacteria
which can see from the size of clear zone in plate, as shown in Figure 4 and 5. T h e r e s u l t o f c l e a r z o n e
measurement with vernier caliper show in Table 1 can explain that V. parahaemolyticus ATCC 17802, corn
cob extract doesn’t have a clear zone that mean doesn’t inhibit bacteria, average clear zone of AgNPs is 10.19
mm and average clear zone of gentamicin is 19.81 mm. V. parahaemolyticus PSU513, corn cob extract doesn’t
have a clear zone that mean doesn’t inhibit bacteria, average clear zone of AgNPs is 9.28 mm and average clear
zone of gentamicin is 15.53 mm.

Figure 4 Figure 5

Figure 4 : Antibacterial effect of V. parahaemolyticus ATCC 17802.
Figure 5 : Antibacterial effect of V. parahaemolyticus PSU513.

Table 1 : Average antibacterial of AgNPS, corncob extract and gentamicin when tested by Agar well
diffusion.

*0 means no clear zone
Conclusion

The ideal condition for the synthesis of silver nanoparticles is to synthesize silver nanoparticles from
corncob extract is 45 min at 90 °C with a ratio of corncob extract to silver nitrate solution 1:10 by volume.

Silver nanoparticles synthesized from corncob extract are effective in inhibiting growth of V.
parahaemolyticus ATCC 17802 and V. parahaemolyticus PSU513.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCIUS) under

Prince of Songkla University and PSU.Wittayanusorn School. The funding of SCiUS is provided by Ministry
of Higher Education, Science, Research and Innovation. This extended abstract is not for citation.

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References

Doan V. D. Luc V. S. Nguyen T. L. H. and Nguyen T. D. Utilizing waste corn- cob in biosynthesis of noble
metallic nanoparticles for antibacterial effect and catalytic degradation of contaminants. Springer

Link. 2019 [cited 2021 Oct 25];27:[about 14 p.]. Available from:

https://link.springer.com/article/10.1007/s11356-019-07320-2

Hemlata Meena P.R. Singh A.P. and Tejavath K.K. Biosynthesis of Silver Nanoparticles Using Cucumis
prophetarum Aqueous Leaf Extract and Their Antibacterial and Antiproliferative Activity Against
Cancer Cell Lines. Pubmed. 2020 [cited 2022 Apr 10];5(10):[about 14 p.]. Available from:
https://pubmed.ncbi.nlm.nih.gov/32201844/

Chen Q. Liu G. Chen G., Chen T. and Mi T. Green Synthesis of Silver Nanoparticles with Corn Straw for the
Preparation of Antibacterial Paper. BioResources. 2017 [cited 2022 Apr 10];12( 4) :[about 11 p.].
Available from https://bioresources.cnr.ncsu.edu/resources/green-synthesis-of-silver-nanoparticles-
with-corn-straw-for-the-preparation-of-antibacterial-paper/

Phunsuk A. “Antioxidant activity and preparation of Butea monosperma flowers extract nanoparticles = Anti-
oxidation effect and preparation of nanoparticles of pueraria flower extract”.. 2554 [cited 2022 Apr
3]. Available from: http://164.115.28.48/2558/?page=result_search&record_id=10393598

Udomlak S., Uraiwan D., Prapassorn R., Siriporn S. and Potjaman P. Extraction and antimicrobial activity of

mangosteen peel extracts. 2006 [cited 2022 Apr 2]. Available from:

https://kukr2.lib.ku.ac.th/kukr_es/index.php?/BKN/search_detail/result/9845

Patchara P. and Orawan J. Effect of grapefruit peel extract on inhibition of Vibrio harveyi and Vibrio
parahaemolyticus bacteria . 2014 [cited 2022 Mar 30]. Available from:
http://www.sure.su.ac.th/xmlui/handle/123456789/14458

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Title : The degradative evaluation of OB5_06_01
Field : Amycolatopsis oliviviridis strain SCM_MK2-4T
for PLA-bioplastic degradation and lactic acid recovery
Biology and Biodiversity

Author : Mr. Kritin Prajonsant

Miss Rujirada Buchram

Miss Treerat Potchanapong
School : Ratchasima Wittayalai School, SCiUS-Suranaree University of Technology
Advisor : Dr. Watsana Penkhrue and Dr. Sirilak Chumkiew, Institute of Science, Suranaree

University of Technology

Abstract
The use of petroleum-based plastics affects the environment with carbon dioxide emissions, especially

in plastic production and waste management. This research aims to study polylactic acid (PLA) bioplastic film
and cup degradation using Amycolatopsis oliviviridis strain SCM_MK2 - 4 T, which is proposed for a novel
actinobacteria species. The casted PLA film and commercial BIO cup Ingeo of Inthanin Coffee were tested in
basal medium containing cocoon silk for PLA degradative evaluation within 14 days. The result showed PLA
film was degraded higher and faster than PLA cup, with 95. 4% weight loss, 8. 22 U/ mL of PLA- degrading
enzyme activity, and 5,666.29 mg/L of lactic acid recovery. PLA film surface was observed under the scanning
electron microscope and showed many holes on PLA film and cup due to PLA- degrading enzyme activity. It
was also indicated that the actinobacteria strain SCM_MK2 - 4 T can be a PLA bioplastic degrader, and lactic
acid can be recovered at a high concentration from the culture medium after 12 days of incubation

Keywords : Polylactic acid, Actinobacteria, Silk cocoon, Lactic acid, Bioplastics, Biodegradable polymers

Introduction
During the COVID-19 pandemic, the amount of plastic used has increased enormously. World plastics

production in 2020 was about 367 million metric tons, which packaging was the dominant user of petroleum-
based plastic and accounts for about half of the world’s plastic waste (Plastics Europe, 2021). Moreover, the
production processes of petroleum-based plastic generate a high level of carbon dioxide and the refinement
process of petroleum could be toxic. Therefore, in order to cater this problem, biodegradable polymers were
introduced. Bioplastics are alternatives of conventional petroleum-based plastics. Bioplastics are defined as
plastic deriving from biological sources, renewable feedstocks, or microbes, owing to the ability to reduce the
environmental effect (Coppola et al., 2021). They can be divided into 3 types: biobased-nonbiodegradable,
biobased-biodegradable, and fossil-based-biodegradable plastics (Garrido et al., 2021).

Polylactic acid (PLA) is a biobased and biodegradable bioplastic, specifically made from renewable
resources with low toxicity and their industrial usage and production have grown in the latest years. PLA is an
aliphatic polyester and it is obtained from the ring-opening polymerization of lactic acid, which has L-form,
D-form, and DL-form as monomer. It has a melting range of 173-178°C and a molecular mass of approximately
60000 g/mol (Li et al., 2016). There are three main applications for PLA bioplastics, which are domestic,

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medical, and packaging and 3D printing applications. It can be hydrolyzed by various enzymes (protease,
lipase, esterase, and PLA-degrading enzyme) produced from microorganisms (Panyachanakul et al., 2019).
Several groups of bacteria and actinobacteria are able to hydrolyze PLA e.g., Amycolatopsis sp., Bacillus
smithii, Actinomudura sp., Laceyella sacchari, Chryseobacterium sp., Sphingobacterium sp., Pseudomonas
aeruginosa (Bubpachat et al., 2018)

Our hypothesizes are Amycolatopsis oliviviridis strain SCM_MK2-4T can degrade commercial PLA
film and cup and generate byproduct as lactic acid, which can be used as monomer to re-polymerize into PLA.
This study investigated the effect of PLA type on lactic acid recovery and PLA-degrading enzyme production
by Amycolaptopsis oliviviridis strain SCM_MK2-4T under liquid fermentation.

Methodology

Bioplastics and chemicals
PLA pellets (4043D grade) with a ̅ n of 1.30 x105 g/mol and ̅ w of 1.50 x105 g/mol was supplied

by NatureWorks LLC (Minnesota, USA). PLA film (thickness 0.01 mm) was prepared (Penkhrue et al., 2017)
and commercial PLA cup Ingeo ( thickness 0. 20 mm) was purchased from Inthanin Coffee. Silk cocoon of
Bombyx mori were purchased from the Queen Sirikit Department of Sericulture, Loei province, Thailand. All
other chemicals used were of the highest reagent grade that is commercially available.
Culture media

The International Streptomyces Project Medium 2 (ISP2) broth containing malt extract (1%, w/v),
yeast extract ( 0. 4% , w/ v) and glucose ( 0. 4% , w/ v) was used for the preparation of the seed inoculum. The
optimized basal medium for PLA-degrading enzyme production was prepared as follows: (NH4)2SO4 (0.1%,
w/v), K2HPO4 (0.1%, w/v), KH2PO4 (0.01%, w/v), MgSO4·7H2O (0.01%, w/v), trace element (2%, v/v) and
silk cocoon (0.39%, w/v) (pH 8.0) (Penkhrue et al., 2017).
Microorganism and inoculum preparation

The actinobacterium, Amycolatopsis oliviviridis strain SCM_MK2-4T has
been reported as a novel PLA-degrading actinobacterium that was isolated from paddy
soil in Thailand ( Penkhrue et al. , 2018) . The strain was cultivated in a 250 mL
Erlenmeyer flask containing 50 mL of the ISP2 broth and incubated at 30°C with

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shaking at 150 rpm. After 3 days, cells were harvested under aseptic conditions, washed, and resuspended in
50 mL sterile distilled water and used as seed inoculum. A total of 6% (v/v) of the inoculum was transferred
for the PLA-degrading enzyme production medium.

PLA film preparation
The PLA film was prepared by dissolving 2 g of PLA pellets in 100 mL of dichloromethane. After

the pellets had completely dissolved, the liquid solution was poured into a Petri dish and then dried overnight
at room temperature. The PLA film was cut into sample sizes of 2 cm x 2 cm and used to investigate the
degradation ability of the PLA-degrading enzyme production by A. oliviviridis SCM_MK2-4T.

Batch fermentation
Each 25 mL of optimized basal medium containing 1. 62% (w/v) PLA film and commercial PLA

cup was prepared in a 125 mL flask with 4% ( v/ v) inoculum. Both were incubated at 30°C on the 150 rpm
shaker for 14 days. Samples were collected every 2 days and assayed to record PLA-degrading enzyme activity,
lactic acid concentration, film weight loss and pH value.

Results and Discussion
The effect of PLA thickness between PLA cup ( thickness 0. 20 mm) and PLA film ( thickness 0. 01

mm) on the percentage degradation of PLA film and cup were investigated to compare lactic acid yield, with
the results shown in Figure 1. The maximum degradation at 95. 4% was obtained from PLA film degradation
with 5. 66 g/ L of lactic acid, which was higher than fermentation using PLA cup Ingeo of Inthanin Coffee.
Lomthong et al. (2021) reported that the hydrolysis of PLA polymer in a 2 L fermenter using Laceyella sacchari
LP175, the highest percentage of weight loss was 68% when incubated at 50°C for 48 h.

a 10 3.5 b 10 120
9 3 9 100
8 2.5 8
7PLA-degrading enzyme activity (U/mL), pH,
Lactic acid (mg/L)
Wieght loss (%)

PLA-degrading enzyme activity (U/mL), pH,
Lactic acid (g/L)
Wieght loss (%)
62 7 80
5 6
4 1.5
5 60

31 4
3 40
2
0.5 2 20
1
1

00 00
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

Incubation time (Days) Incubation time (Days)

Figure 1: Time course of batch fermentation for PLA-degrading enzyme activity (), Weight loss (), Lactic acid
() and pH () by A. oliviviridis SCM_MK2-4T in optimized basal medium containing PLA cup (a) and film (b)

The thickness of PLA polymer was performed at different PLA packaging types for PLA cup and PLA
film, as described above. The results showed that the PLA film degradation produced the highest enzyme production
at 8.77 U/mL (Figure 1b) for 6 days incubation and 8.22 U/mL (Figure 1a) for 14 days in PLA cup degradation. In
batch fermentation of PLA film, lactic acid was detected as 5.66 g/L at 12 days. Furthermore, at high lactic acid
concentration, lactic acid decreased the pH in the reaction and enzyme activity decreased (Lomthong et al., 2017).

Stereo and scanning electron micrograph revealed that the PLA film and cup were affected by loss of

rigidity, with the appearance of holes and fractures in PLA film and cup compared to without degradation

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( Figure 2; Day 0) . This showed the feasibility of upscaling PLA degradation at an industrial level using A.
oliviviridis SCM_MK2-4T.

Figure 2: Stereo (A1 and B1) and scanning electron micrograph (A2 and B2) of PLA cup (A1-2) and PLA film
(B1-2) by PLA-degrading enzyme produced by A. oliviviridis SCM_MK2-4T after incubation at 30°C for 14
days

Conclusion
The findings from this work can be concluded as follows:
1. Amycolatopsis oliviviridis strain SCM_MK2- 4T can degrade both commercial BIO cup Ingeo of Inthanin
Coffee and PLA film. However, the degradation time of PLA cup with the thickness more than 0. 2 mm was
longer than PLA film degradation.
2. Lactic acid (5.66 g/L) can be recovered from 0.405 g of PLA film degradation
3. Production of PLA- degrading enzyme ( 8. 77 U/ mL) using A. oliviviridis strain SCM_MK2- 4T was
significantly dependent upon PLA film and silk cocoons as the enhancers in the basal medium composition.
4. Actinobacteria A. oliviviridis strain SCM_MK2- 4T can be used to reduce the environmental accumulation
of PLA and recycle lactic acid into the repolymerization process of PLA as a biomodel for sustainable green
technology.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS). The

funding of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation. We thank
Suranaree University of Technology for providing the proper laboratory equipment for this work. This
extended abstract is not for citation.

References
1. Penkhrue W, Kanpiengjai A, Khanongnuch C, Masaki K, Pathom- Aree W, Punyodom W, Lumyong S.

Effective enhancement of polylactic acid- degrading enzyme production by Amycolatopsis sp. strain
SCM_MK2- 4 using statistical and one- factor- at- a- time approaches. Preparative Biochemistry and
Biotechnology. 2017 Aug 9;47(7):730-8.

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Title : Geographical tracking and mapping of coronavirus OB5_02_08

disease COVID-19 of Phayao
Field : Biology and Biodiversity
Author : Mrs.Tanyavee Tawbunyuen

Mrs.Tareetip Chanwikkarn
School : Demonstration School, University of Phayao
Advisor : Dr.rer.nat Chatchawal Wongchai, University of Phayao

Abstract

COVID-19 is a respiratory infection caused by the coronavirus. The positive sense of single-stranded RNA shows
a high ability to infect human and animal which disrupt the social community, economy, and health, especially in
nine districts of Phayao, Thailand. The reopening of local academic institutes, tourism businesses, and social
activities need big data and various methods to maintain people's behavior and health concern. Since 2019, records
of local COVID-19 clusters in Phayao have been reported when the people entered the public area and shared their
activities in pubs, bars, nightclubs, public parties, and gamecock communities. Local quarantine, organization
quarantine, and screening checkpoints are incorporated to control and limit the area of contamination.
Nevertheless, geographic tracking and mapping of infected populations are critical to basic communication and
necessary for early quarantine guidelines. This work summarizes the cluster and number of the infected population
in each area during the critical period of March – December 2021. The data show that the funeral cluster is the
biggest group of spreaders, especially in Muang and Pong districts. University and school clusters show a high
number of infected people in the Muang district while Ice factory and Temple clusters show a very low number of
infected population (Ice factory cluster and Temple cluster). Interestingly, Gamecocks, the Probation office, and
Socialize cluster present a non-different number of infected people (Gamecocks cluster, Probation office cluster
and Socialize cluster) but the Restaurant cluster shows a similar tendency to the Funeral cluster. Finally, Data,
Geographical tracking, and mapping are necessary tools for community communication and supporting local
quarantine, managing social activities, and building up the structure of a platform for reopening universities and
schools in Phayao.

Keywords: COVID-19, Public health system, Reopening, Timeline check

Introduction

Currently, there are many epidemics occurring around the world but the epidemic of special attention
around the world is COVID-19, which first outbreak in Wuhan, China in 2019, The disease, officially known as
SARS-CoV-2 comes from a virus that found in bats (1). A mutation that can be transmitted from animals to humans
until eventually leading to infection from person to person which can be transmitted by receiving secretions from
the patient's respiratory system, such as coughing and sneezing including contact with secretions of infected
people. Causing the outbreak of COVID-19 to spread to almost every country in the world (4). That have a sick
population and a large number of deaths people from COVID-19 and also has a huge impact on the world especially
in the economy in trade travel and the lifestyle of the population. Thailand is another country that has been affected
more and more severely, so each province has to set up various measures to prevent and reduce the spread of
COVID-19 and to reduce the number of infected people (4). However, the population still needs to have the basic

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knowledge about COVID-19 both in terms of preventive measures against COVID-19, vaccinations, basic
protection, How to detect the Covid-19, How to behave when we are COVID-19 patient, medicines and tools
related to COVID-19 to have more knowledge and understanding of COVID-19. In the future, this disease may
be just an endemic disease like a common flu that can be cured and then can return to normal life. Because when
we cannot make this disease disappear, we have to try to live with it, prevent and treat ourselves as best as possible
(2). Therefore, the researcher wants to study the epidemiology of Geographical tracking and mapping of
coronavirus disease COVID-19 of Phayao in each wave of COVID-19, different clusters and where it originated,
What clusters are there. Since 2019 Until 2021 within each district of Phayao (3).

Methodology
The purpose of this project “Geographical tracking and mapping of coronavirus disease COVID-19 of

Phayao” was To study about the epidemiology of COVID-19 in Phayao, Thailand and the trend of the situation
of COVID-19 in the future.
The experimental steps are as follows:

1. Study the information on the spread of coronavirus in Phayao the Phayao Public Health
website for the year 2020-2021.
2. Gather the cluster in ripples using the month as a separate criterion, popping up for each month how
many times in the cluster the ripples occur in that ripple and how many clusters.
3. Use the collected data to analyze the results using GIS.
4. Summarize the results of the outbreak and the trend of the future covid-19 situation in Phayao province
in a model preparation.
Result and Discussion
For epidemiological data collected the COVID-19 situation in 2020 in Phayao showed that the total
number of COVID-19 cases wasn’t have until June when the number of COVID-19 cases has begun, but there are
few. The COVID-19 situation in 2021, in April, the number of COVID-19 cases began to increase rapidly. Multiple
clusters occur in this ripple as shown in the chart.

Figure 1. The wave of Covid-19 in March 2021 – December 2021 in Phayao.

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From collecting data, the number of people infected COVID-19 is increasing steadily. The picture
showing the wide spread of infection throughout Phayao. The number of population density and the central area
of the province was related to the number of infections. This can be seen in Muang District, Phayao an area with
a high population and to increase in the number of infected people as well. There is information as shown in the
graph as follows:

October 2021 - January 2022

400
350
300
250
200
150
100

50
0

Figure 2. The information collected from the summary of the meeting No. 3/2021 The cluster ripple from
October 2021-January 2022

Also, timeline check is important to show the risk of a place and who has been to that place. Citizens can
save their timeline through various applications on electronic devices such as use Google map to tracking timeline
by function location history.

Measures of Phayao Province
1. A coordination center has been established to bring Phayao people back home. It will accept covid

infected from high-risk provinces and domiciled in Phayao province to return for treatment within the province.
2. There is news about the COVID-19 situation. Through the website and Facebook of the Ministry of

Public Health regularly.
3. There is a surveillance measure for people entering the province who come from risk areas.
4. Curfew measures are enforced as announced by the government.
5. There is public relations and campaign for people in the province to vaccinate against COVID-19 at

hospitals or health centers near their homes.
6. Patients are screened according to severity of symptoms. The patients with mild symptoms can be

treated by home isolation.

Conclusion
Covid-19 is a contagious disease caused by Coronaviruses that contain spike or S protein, a protein that

binds to receptors and allows the infection to enter the body. It can mutate in the body and multiply, so a wide
variety of vaccines are being produced, performance varies.

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Due to the spread of COVID-19 situation in April 2021, there are many clusters and violence. The
Ministry of Public Health, Phayao, has established a coordination center to get people back home from Phayao. In
the beginning, the rate of infection was low. Most of them are infected from high-risk provinces. Since July, there
are more infected people, causing the Phayao Provincial Public Health Department to set various measures
according to the situation to reduce the number of infections and inviting people to vaccinate at a hospital. There
is news and public relations about COVID-19. through social media regularly and use Google map to tracking
timeline by function location history. The current situation shows that the future trend of the COVID-19 situation
will be less risky and less violence because there are ready countermeasures and people have more immune that
from vaccination.

Tracking and mapping by GIS system shows the distribution of corona virus in Phayao. This will be an
aid in decision making in controlling the COVID-19 situation. Traveling and touring various places, the awareness
of epidemiological information will make the population aware and active in protecting themselves and their
families. It can also help avoid increasing the number of infected people. It is an information system that can be
easily accessed and distributed via social media. which will lead to dealing with the epidemic of COVID-19
effectively.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS). The funding

of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation, Jointed by
Demonstration School and School of Science, University of Phayao. This extended abstract is not for citation.

References
1. Amorn leelarasamee. 2020. Interesting facts about COVID-19 infection caused by SARS-CoV-2, 5-15.
Retrieved from https://tmc.or.th/covid19/download/pdf/tmc-covid19-19.pdf
2. Department of disease control. 2020. Coronavirus Disease (COVID-19). Retrieved from
https://ddc.moph.go.th/viralpneumonia/eng/index.php
3. Phayao Provincial Public Health Office. 2020. Covid-19 situation report in Phayao. Retrieved from
https://www.pyomoph.go.th/index_sub.php?id_type=23
4.World Health Organization Department of Disease Control. 2021. Coronavirus disease 2019 (COVID-19).
Retrieved from https://www.who.int

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Title: Coccoid forms of Helicobacter pylori related with OB5_18_06

Field: false negative Rapid Urease Test
Author: Biology and Biodiversity
Thannumtip Pitchayasopanan
School: Potsathon Phaktiyanuwat
Peerapan Khumkrong

Surawiwat School, Suranaree University of Technology

Advisor: Assoc. Prof. Taweesak Thongtawee, Helicobacter pylori Research Unit, Institute of Medicine

Dr. Wareeporn Wattanawongdon, Helicobacter pylori Research Unit, Institute of Medicine
Abstract:

The Rapid Urease Test (RUT) is one of the reliable methods for diagnosed H. pylori infection.
However, some patients were diagnosed with H. pylori infection by RUT was negative, but the Scanning
Electron Microscope (SEM) showed infection. Therefore, the aim of this study was to evaluate the relationship
between high resolution endoscopy (NBI) pictures and SEM in gastritis patients infected with H. pylori and find
the cause of false negative result in the RUT. Thirty gastritis patients were included. Gastric mucosal
morphologic patterns related with infection was analyzed first by NBI. The infection was then analyzed by
RUT and SEM, respectively. The relationship between RUT, NBI and SEM were assessed using chi-square
test. H. pylori infection was identified in 19 (63.33%) cases by using RUT and NBI, while detected by SEM was
26 (86.67%) cases. Statistical analysis showed that the RUT results were associated with NBI and SEM (P<0.005).
Some patients were diagnosed infection by RUT was negative, but the SEM showed coccoid forms. RUT results
were associated with NBI and SEM. Present of coccoid forms were associated with false negative RUT. SEM
can be used to identify H. pylori infection that would not be detected by RUT and/or NBI.

Keywords: H. pylori, Endoscopy, Scanning Electron Microscope, Rapid Urease Test

Introduction
Helicobacter pylori is a gram-negative, spiral-shaped bacterium that lives in the stomach and causes

gastric inflammation. Commonly, the stomach is an organ that doesn't get much infection because the condition
is highly acidic. Therefore, it is not suitable for the growth of germs and bacteria. However, H. pylori contains
urease enzymes that can convert urea to carbon dioxide and ammonia, which are alkaline. It can be neutralized
with stomach acid to be suitable for bacterial growth. After that, it will secrete virulence factors such as
cytotoxin associated gene (cagA) and vacuolating toxin (vacA), to induce inflammation of the epithelial cells of
the stomach. The transmission of infection can be transmitted from person to person through several ways.

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