Abstract of SciFair2019

The development of electrochemical sensor for simultaneous
detection of uric acid and dopamine using poly(glutamic acid)
modified screenprinted electrode

Phatcharakon Woranuch, Pichayanin Hirunpalanon and Weena Siangproh *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email : [email protected], [email protected]

Biomarker is a measurable indicator of some biological state or condition. Uric
acid (UA) and dopamine (DA) are biological markers that commonly coexist in
human body fluid and play an important role in the physiological processes. The
abnormal level of these markers can cause the several diseases such as gout,
kidney diseases, hypertension, cardiovascular, and Parkinson’s disease. Therefore,
the detection of these biomarkers is considerably necessary for indicating symptom
of those mentioned diseases. This research focuses on the development of a new
electrochemical sensor for sensitive detection of UA and DA. However, the
oxidation of UA occurs at a similar determination of UA and DA in a presence of
ascorbic acid (AA). The concentrations potential to DA. Thus, differentiation of their
signals becomes challenging. To overcome this limitation, an electrochemical sensor
based on poly( glutamic acid) modified screen-printed graphene electrode was
developed for simultaneous of target analytes were investigated by differential pulse
voltammetry (DPV). All experimental parameters were symmetrically studied to
obtain the optimal conditions. Furthermore, the developed sensor was successfully
applied to detect UA and DA in real samples with reliable results. This finding could
be an alternative platform for practical applications.

Keywords: Uric acid, Dopamine, Simultaneous detection, Screen-printed
electrode, Poly(glutamic acid)

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An application of carbon quantum dots as fluorescent sensor
probe for melamine determination

Thitiwut Sriharun, Puthita Singwongsa and Kriangsak Songsrirote *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Melamine is an organic compound containing 66% nitrogen by mass. Therefore,
it is illegally used as a contaminant in the food production of human and animal
to inflate the apparent protein content of food products. If consumers receive high
dose of melamine into the body, adverse effects can occur to their health. This
research aims to study a simple method for melamine determination based on the
fluorescence properties of carbon quantum dot (CDs) with high stability and low
toxicity. The CDs were synthesized from citric acid and urea using microwave-
assisted hydrothermal method resulting in the CDs with an emission wavelength
of 543 nm and excitation wavelength of 276 nm. The developed method offers
simplicity, high sensitivity, high selectivity, and short analysis time for melamine
detection. Linearity range for melamine measurement was in the range of 1 ppm
to 250 ppm (R2 = 0.9906).
Keyword: Melamine, Carbon quantum dot, Fluorescence, Quenching

- 38 -

Computational study of novel multitarget inhibitors against
Alzheimer’s Disease

Pawanrat Phuntee, Ploychompoo Deemark and Pornthip Boonsri *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Multitargeted hybrids of donepezil-coumarin derivatives (C1-C26) as dual
acetylcholinesterase (AChE) / monoamine oxidase type B (MAO-B) inhibitors
were designed and prepared based on the molecular docking in order to find the
lead optimization for anti-Alzheimer’s disease (AD). The docking results showed
all compounds posed their structures to the active site of AChE / MAO-B enzymes,
suggested that they shared similar mode of binding to the reference drugs.
Furthermore, the substituent effect on benzene ring at donepezil part was explored
to improve the binding affinity. The results indicated that compounds have the
substituted with electron-withdrawing groups showed higher binding affinity than
the electron-donating groups. For AChE binding, most compounds exhibited tight
binding interactions to the enzyme with -10.21 to -12.44 kcal/mol, which bind even
more stronger than the drug donepezil (-11.03 kcal/mol). In addition, those hybrids
compound oriented their structures by pointing the coumarin moiety to bind with
the peripheral active site (PAS) while the donepezil moiety was posed in the
catalytic active site (CAS) of AChE. The obtained results revealed that the hybrids
could be served as a dual-binding mode for AChE. Among them, the meta-phenyl
substituted group (C21) was identified as the most potent inhibitor to both AChE
and MAO-B with the binding energy values at -12.16 and -12.28 kcal/mol,
respectively. Moreover, C21 formed the hydrophobic interactions and strong
hydrogen bonding to the key amino acids located in the PAS and CAS of the
AChE which confirmed as the dual-binding inhibitor. For MAO-B binding, C21
formed the importance hydrophobic interactions to the entrance cavity toward the
flavin adenine dinucleotide (FAD) binding domain of the MAO-B which predicted
that the potent inhibitor could be obtained. Therefore, the increasing of aromaticity
at donepezil moiety influenced to the better binding affinity of the hybrid
molecules. Taken into account, a new series of donepezil-coumarin hybrids can be
highlighted as the designing novel anti- AD drugs.

Keywords: Coumarin, Acetylcholinesterase, Monoamine oxidase type B,
Multitargeted, Dual-binding inhibitor

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The study of binding interaction between quinoline derivative
with serum albumins by spectroscopy and molecular docking

Natchaphon Ngueanngam, Atikan Boonwan and Apinya Chaivisuthangkura *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

The intermolecular interaction between quinoline derivative with serum albumins
(SAs) under imitated physiological conditions was investigated using UV-Vis,
flunder imit spectroscopy and molecular docking. The results revealed that the
fluorescence quenching of bovine serum albumin (BSA) and human serum
albumin (HSA) by quinoline derivatives resulted from the formation of quinoline
derivatives – BSA and HSA complexes. The number of binding sites (n) for ligand
binding on protein targets was approximately equal to 1. The experimental and
docking results revealed that after binding ligand to BSA and HSA, the binding
process of quinoline derivatives with SAs is spontaneous, and ligand could be
inserted into the hydrophobic cavity of both proteins (Site I). The enthalpic change
(ΔH0) and entropic change (ΔS0) in the process of interaction of quinoline
derivative with BSA and HSA were -55.8568 kJ mol–1, –99.1528 J mol–1 K–1and –
108.057 kJ mol–1, –262.257 J mol–1 K–1, respectively. The obtained results
indicated that the main interaction forces of quinoline derivatives with BSA and
HSA were Van der Waals and hydrogen bonding interactions. Additionally, it can
be suggested from the molecular docking results that the flexibility of quinoline
derivative plays an important role in increasing the stability of the whole system
upon association of quinoline derivatives with BSA and HSA.

Keyword: Quinoline derivative, Serum albumin, UV-Vis spectroscopy,
Fluorescence spectroscopy, Molecular docking

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Development of a portable PEDD detector for paper-based
analytical device to determine total phenolic compound.

Pilawan Chaiyatan, Praeploy Porapattanachan and Thitirat Mantim *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

This work proposes a simple portable PEDD detector coupled with paper-based
device (PAD) for determination of total phenolic compound content in tea
sample. The PAD device was made of Whatman no.4 filter paper, a square paper
(26×26 mm) with circular hydrophobic pattern (11 mm inner diameter) fabricated
by stam- ping method using permanent ink. Folin-Ciocalteu (FC) method was
used for analysis of total phenolic compound. Aliquots of FC reagent, gallic acid
standard solution and alkaline solution were sequentially pipetted onto the center
of the PAD device to form a blue color product. which was detected using the
portable PEDD detector at 660 nm within 1 min. The develop method provides a
rapid analysis with good precision. Linear calibration of gallic acid was obtained
in the range of 10-150 mgL-1 (y = 2.37×10-4) X + 0.0050, R2 = 0.9991). The
developed method will be applied to tea samples for quantification of total
phenolic compound.
Keywords: Folin-Ciocalteu method, Paper-based device, Portable PEDD
detector

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Chemical constituents of Gymnopetalum Chinese (Lour.) Merr.

Peradon Phonglumjeakngam, Aunchiya Pitaksa and Nuttapon Apiratikul *
Department of Chemistry, Faculty of Science, Srinakharinwitrot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Kadom or Khi-ka-dong (Gymnopetalum chinense (Lour.) Merr.) is a plant from
Cucurbitacae family that mainly found in Thailand and have been used in
traditional medicine since the emergence of ancient cultures, mainly in Asia. In
addition, it has many utilization such as food medicine and detoxification.
However, recent studies showed that Cucurbitacae family exhibit interesting
biological activities such as antitumer, anticancer and antibacterial activity. In
this work we isolate a constituent from dry ripe Kadom in hexane extract, separate
by normal phase column chromatography technique using hexane/ dichloromethane/
methanol as eluents, follow the separation result by Thin layer Chromatography
(TLC), detect by ultraviolet light at 254 (green color) and 365 (blue color) nm.
and characterize by Nuclear Magnetic Resonance Spectroscopy (NMR).
Keywords: Gymnopetalum Chinense (Lour.) Merr, Cucurbitacin

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Synthesis of sulfonamide derivatives as aromatase inhibitor

Thiranai Sarinyawat, Thanatorn Sititeerapan and Ratchanok Thongnum *
Department of Chemistry, Faculty of Science, Srinakharinwitrot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Breast cancer is one of the leading causes of cancer-related mortality among
women worldwide. The majority of breast cancers are deriving from estrogens
production. Inhibition of the aromatase is a crucial strategy to prevent the growth
stimulation effect of estrogens in breast cancer. So, aromatase inhibitors (AIs)
have been developed and used as anti-breast cancer agents. Sulfonamide is
pharmacophore found in a diverse range of biologically active molecules including
anticancer, antimicrobial, antimalarial and anti-aromatase agents. The potent
biological activities of sulfonamides have drawn much attention from various
research groups in designing and synthesizing the pharmacophore. This work
presents the synthesis of sulfonamide derivatives by reductive amination of 4-
methoxyaniline with 4-methoxybenzaldehyde followed by N-sulfonation with
various benzene sulfonylchloride derivatives. Structures of the desired sulfonamides
were confirmed based on their NMR spectra. This work provides a variety of
sulfonamide derivatives which will be further investigated for their anti-aromatase
activity.
Keywords: Sulfonamide, Reductive amination, Sulfonation, Aromatase
inhibitor

- 43 -

Extraction of chlorogenic acid from coffee plup

Nuntaporn Poonpao, Niranya Sarawong and Siritron Samosorn *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Nowadays, coffee is the most popular beverage around the world and the most
popular type of coffee is arabica. Arabica coffee has unique smell and taste, the
need of arabica as a material is increasing. In the production of arabica, coffee
plup will be left to decompose naturally. However, after the decompose process
is done, the soil will become acidic and it will lead to environmental issues. Green
coffee has a higher level of chlorogenic acid compared to regular. Chlorogenic
acid is an ester of caffeic acid and quinic acid. Chlorogenic acid is the major
polyphenolic compound in coffee. This compound has been known for a long
time as an antioxidant. The objective of this research is to study the method of
extraction of chlorogenic acid and other compounds from green coffee plup. The
final objective was to find an easy way to extract high-value molecules from a
complex mixture, avoiding as much as possible the use of toxic solvents and
finding guideline of adding value to coffee waste production process, which can
reduce environmental problems. The use of ethanol extraction method was tested
in combination with three isolation methods using activated carbon, different
solvents and magnetite (Fe3O4).
Keywords: Antioxidant, Arabica, Chlorogenic acid, Green coffee plup

- 44 -

Development of pretreated screen-printed graphene electrode by
overoxidation technique for determination of Vitamin B9

Nattida Yomkwang, Nuntiya Khomprasert and Weena Siangproh *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email : [email protected], [email protected]

Vitamin B9 (VB9), also known as folic acid, is one of the water-soluble B vitamins
and is an essential component for consideration of biological significance for
human health, particularly during period of rapid cell division and growth. The
deficiency of VB9 can cause numerous severe diseases such as macrocytic
anemia, psychiatric disorders, and carcinogenic processes. Accordingly, a practical
and reliable analytical assay is required for the determination of VB9. Herein, we
first reported on the development of electrochemical sensor for the sensitive
detection of VB9 using overoxidized screen-printed graphene electrode (O-
SPGE). To prepare this sensor, pH5 phosphate buffer solution (PBS) was used
as an over-oxidizing reagent to convert a bare screen-printed graphene electrode
(SPGE) surface to an O-SPGE by electrochemical pretreatment. For measurement,
Britton Robinson buffer solution (BRBS) was served as a suitable supporting
electrolyte for VB9 detection. In comparison to a bare SPGE, it was found that an
O-SPGE provides a higher oxidation peak current at a lower oxidation potential.
This implies to the electro-catalytic property of O-SPGE for VB9 detection. From
the preliminary results obtained, all experimental parameters will be investigated
to obtain the optimal conditions for the determination of VB9 and results will be
discussed.
Keywords: Electrochemically Pretreatment, Overoxidized Screen-Printed
Graphene Electrode, Overoxidation, Vitamin B9

- 45 -

Synthesis and study interaction of metal complex nanoparticles
with phenolic acid in natural

Thanathorn Kunanuphanchai, Veerapattra Chamnionphat, Sawanya Homwong and Sujitta Srisung *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Nowadays, silver nanoparticles (AgNPs) are widely applied to biomedical
application, nanotechnology and nano-electronics leading to a release of
nanoparticles into the environment, especially in soil and water resulting in
danger to organism. Human receives nano-metal into the body by various
methods such as breathing, touching and eating. Ferulic acid and vanillic acid are
as phenolic compound being interested, because they are found naturally in many
plants such as vegetables, fruits and grains. They are able to remove free radicals
and metal ions that can accelerate the oxidation reaction of fats and other
molecules. The objective of this research is to develop a method for synthesis and
investigate the structure of AgNPs. The interaction of nanoparticles with the
phenolic compound ligands were also determined in method offers simplicity and
short analysis time. To characterize the nanoparticles, UV-visible spectrometry
was used. AgNPs are synthesized from reduction method using sodium
borohydride at 10°C for 25-30 minutes. This method produced yellow solutions
and the spectrum showed maximum absorption at wavelengths of approximately
392.8-394.2 nm which indicates the development of AgNPs. The study of the
interaction of AgNPs with phenolic compound was performed using UV titration
method. The results showed the effectiveness of the phenolic compound for
absorption of these nano-metal to be a 1: 1 interaction.

Keywords: Interaction, Metal complex, Phenolic compound, Silver
nanoparticles, Synthesis

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Synthesis of modified sliver nanoparticles for Mn2+ detection

Nattakamon Deemoonmang, Waraporn Hongwaingchun, Pan Thongraung *
Department of chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

The purpose of this study was to use a facile colorimetric sensor based on the 4-
MBA-CT-MA stabilized silver nanoparticles (AgNPs) for detection of Mn2+ ion
in aqueous solution. The 4-MBA-CT-MA-AgNPs was synthesized from 4-
mercaptobenzoic acid (4-MBA), sodium citrate (CT) and melamine (MA). The
UV-Vis spectroscopy was used to characterize AgNPs and examine the
complexation of 4-MBA-CT-MA-AgNPs with various cations (Cd2+, Co2+, Cr3+,
Cu2+, Fe2+, Fe3+, Mn2+, Ni2+, Pb2+ and Zn2+). The result of this technique shows a
strong surface plasmon resonance (SPR) at 398 nm. The cooperative metal-ligand
interacted with coordination bonds between Mn2+ion and functional group on
surface AgNPs, so Color of AgNPs changed from yellow to orange-brown .Under
conditions, the linearity given a positive coefficient correlation (R2 > 0.99).
Therefore, the synthesized silver nanoparticles are alternative method for
Mn2+measurement and applied to determination Mn2+ in water sample.
Keywords: Colorimetric sensor, Modified silver nanoparticles, Mn2+

- 47 -

Domestic microwave assisted synthesis of carbon dots from
tartaric acid and triethylenetetramine for detecting metal ions

Khunracha Koonkery, Vorakamon Kasorn, Ngamjit Pri-ngam * and Pornpimol Prayongpan *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected], [email protected]

This study focused on synthesis fluorescence two carbon dots (CDs) from tartaric
acid ( TA) with triethylenetetramine ( TETA) and only TETA with domestic
microwave- assisted hydrothermal method for Cr6+ ion detection based on
quenched fluorescence intensity of CDs in aqueous solution. CDs synthesized
from TA with TETA and only TETA emitting blue light ( em 468 nm, ex 390
nm) and ( em 434 nm, ex 350 nm) respectively. Fourier transform infrared
spectrometer was used to analyses functional group on the surface of both CDs
which represented amino group, C-H, -C-N and hydroxyl groups meanwhile CDs
prepared from TA and TETA carbonyl group has also observed. Fluorescence
spectra exhibited fluorescence quenching for Cr6+. A linear relationship between
the concentration of Cr6+ and relative fluorescence intensity was obtained at 0.1-
1 mM (R2 = 0.9037) and (R2 =0.9158) with a limit of detection at 1.19 and 1.18
mM for CDs from TA with TETA and only TETA respectively and % recovery
of both CDs are acceptable value.
Keyword: Carbon dots, Cr6+, Domestic microwave-assisted hydrothermal
method, Tartaric acid, Triethylenetetramine

- 48 -

Investigation of bioactive compounds stimulated breast milk from
herbs

Supana Poopaitoon and Chatchadaporn Pinthong *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Breast milk is the most important nutrient for infant. Some most postpartum
mothers had insufficient milk to feed babies from many psychological factors
resulted in stressed. Recently, there are several ways to help mothers increase
their breast milk such as milk pumps, the milk supplement, and herbs. In
Thailand, there were some reports using herbs to increase breast milk by adding
herbal ingredients such as curry and stir-fried ginger, but still lack of the data to
support on how it increase the breast milk. Therefore, this research aimed to
investigate of bioactive compounds from herbs like ginger, moringa, banana
blossom for their function of stimulate breast milk. The results not complete yet,
but we propose that there are some bioactive compounds in herbs that can be used
to stimulate breast milk.
Keywords: Ginger, Moringa, Banana blossom, Stimulate milk

- 49 -

Gelatin-Tannin modified filter membrane for preconcentration of
Lead

Vanlada Suksomphot and Nuanlaor Ratanawimamwong *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

This research aims to develop a gelatin-tannin modified filter membrane for
preconcentration of lead before electrochemical detection. The circular shape of
a paper-based device was fabricated on a filter paper by stamping method. In
order to develop a gelatin-tannin modified filter membrane, gelatin and tannin
was dissolved in water and then adjusted pH to 8 . The mixture solution was left
in 5 8 oC in water bath for 3 h. Then small aliquot of the mixed solution (20 L)
was dropped into a circular pattern of the paper-based device and keep in room
temperature for 5 min. The gelatin-tannin modified filter membrane was washed
with water before using as an adsorbent for lead. For preconcentration of lead ion,
pH of a standard lead solution was adjusted to 10 before loading into the gelatin-
tannin modified filter membrane by using vacuum filtration apparatus. Elution
was taken placed by using 800 L of 0.1 M HNO3. The eluate was added with
200 L acetate buffer pH 4.5. Detection of lead was carried out using differential
pulse anodic stripping voltammetry (DPASV), involving the use of screen-
printed electrodes (SPEs). Glassy carbon modified with mercury salt was
employed as a working electrode. The lead signal occured at -0.752 V and peak
current of 4.495 A was obtained for 1 ppm Pb solution. Gelatin-Tannin modified
filter membrane provides good potential to adsorb lead ions for preconcentration
of lead.

Keywords: Gelatin, Gelatin-tannin modified filter membrane, Lead,
Preconcentration, Tannin acid

- 50 -

Study of extracellular enzymes activities of bamboo mushroom on
culture media containing ground coffee

Kittiyaporn Saranyupong, Arunothai Thiansuan and Supakan Rattanakon *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Ground coffee (GC) is coffee scraps that has been roasted and then brewed for
drinking. It is discarded after extraction into waste that can affect the
environment. However, there still remain nutrients in ground coffee, especially,
cellulose that can be used as a food source for mushroom culture. Therefore, the
purpose of this research was to study the enzyme activity of bamboo mushroom
mycelium containing ground coffee as an ingredient in comparison with potato
dextrose agar (PDA) and carboxymethyl cellulose (CMC). The enzyme activity
produced from white bamboo mushroom mycelium was measured by gram
iodine stain method. The result showed that the bamboo mycelium mushroom
cultured on CMC media had the highest growth of mycelium and releasing of
enzymes, followed by GC and PDA, respectively. In addition, this work also
studied culture media in bottle containing sorghum and ground coffee mixed
with sorghum. The result showed that sorghum with 80% moisture had the best
mycelium growth, followed by ground coffee mixed with sorghum (50:50). This
study will be useful for interested researcher because the data about enzymes of
bamboo mushroom has not been studied yet and ground coffee can be used for
maximum benefit.
Keywords: Bamboo mushroom, Enzyme activities, Gram iodine stain, Ground
coffee

- 51 -

Synthesis of carbon dots from tartaric acid and urea using
microwave-assisted hydrothermal method as a metal ion detector

Tidawan Ngamyoo, Tusawan Namwong, Ngamjit Praingam * and Pornpimol Prayongpan *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected], [email protected]

Carbon dots (CDs) has recently emerged and gained much interest as a new class
of the carbon nanomaterials especially suited for biological applications owing to
their characteristic advantages such as non-toxicity, bio-compatibility and
element abundance. In this study, the CDs were synthesized by Tartaric acid and
urea with microwave-assisted hydrothermal method. Property and ability of
luminescence are to detect metal ions such as Cr6+, Fe3+, Fe2+ were studies.
Characterization of the synthesized carbon dots including the investigation of
their functional groups and optical properties were performed by Fourier-
transform infrared spectroscopy, UV-vis spectroscopy, and Fluorescence
spectroscopy. The results show that CDs’ fluorescence was quenched by metal
ions. The fluorescence intensity quenched significantly with Cr6+, Fe3+, Fe2+
respectively. A good linear relationship between the concentration of Cr6+, Fe3+
and Fe2+ with the relative fluorescent intensity was obtained (R2 = 0.9897, 0.9941
and 0.9999) with a limited of detection at 1.45 mM, 16.47 mM and 0.145 M,
respectively. In addition, quenching of fluorescence intensity depends on pH
values, and response is most selective and sensitive to Cr6+, Fe2+ at pH7 and to
Fe3+ at pH5.
Keywords: Carbon dots (CDs), Tartaric acid, Urea, Fluorescence, Quench,
Microwave, Metal ion

- 52 -

Design and development of tuberculosis inhibitors by molecular
docking

Papawarin Panpala, Pratsaneeya Janta, Thitisuda Suwannahong and Mayuso - Kuno *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Tuberculosis (TB) is a serious contagious disease caused by bacteria mycobaterium
tuberculosis which, can be contacted via air, by entering to the alveoli therefore
causing disease. Currently, antibiotics have been widely used to treat TB for
patient. The effect of market drugs with the target enzyme, causes tuberculosis,
found that some drugs can inhibit TB with high potency. However, there are
some drugs that have not been able to inhibit well and causing many side effects.
Therefore, this research was performed to search new inhibitors targeting TB
enzyme using molecular docking approach with reverse docking procedure. The
selected enzymes associated with TB were obtained from the Protein Data Bank
(PDB) with, code 4nni, 2x22, 2wt9, and 5nr3. The derivatives that were used
to dock into those proteins were 13, 7a, 5c, 5d, 8c and 8d. The result showed that
compounds 5c, 5d, 8c and 8d had both inding energy and number of
conformations higher than compound 13 and, 7a. Therefore, all derivatives can
be further developed as a target for TB treatment.
Keywords: Tuberculosis, Molecular docking

- 53 -

Synthesis of polycarbonate from carbon dioxide and biomolecules
using metal oxide catalyst

Yodsathorn Duangrat, Koranis Sillapachai and Sucheewin Chotchatchawankul *
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Carbon dioxide is a greenhouse gas that is abundant in the atmosphere resulting
in global warming. Utilization of carbon dioxide using natural materials without
energy-consuming reduction is a promising solution. This research aims to couple
D-glucose, a ubiquitous polyol from a nature, with carbon dioxide to form
polycarbonate. With CeO2 catalyst and 2-cyanopyridine promotor under 50 bar
CO2 at 120 – 160°C, the reaction produced dark brown liquid probably from
thermal decomposition of glucose. Interestingly, it showed FT-IR signal at 1580
cm -1, which was in the region of anionic C=O experiment without D-Glucose.
Our best hypothesis is that an oxalate salt formed from the coupling of carbon
dioxide under the conditions. Although the formation of the expected polycarbonate
was not observed, we might have a new route toward other carbon dioxide utilization.
Keywords: CO2, Polycarbonate, Oxalate, Polyol, D-glucose

- 54 -

Commercial biodegradable plastic degradation by whole cell of
Actinomadura keratinilytica strain T16-1 in 3-L stirrer fermenter
and under composting condition

Suparat Chuenprapai and Sukhumaporn Krajangsang *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Polylactic acid (PLA) is a biodegradable polyester made up of lactic acid as
building blocks. It can be degraded by various microorganisms. In the previous
study, the researcher has focused on bioprocessing of PLA degradation by a
microbial enzyme. However, it has not been reported by using whole cell of
bacteria to degrade these plastics. Therefore, this work aims to evaluate PLA
degradation process by whole cell of Actinomadura keratinilytica strain T16-1
using 3-L fermenter and under composting condition. The enzyme activity of
248.8 U/mL produced by strain T16-1 was obtained after incubation for 48 hours
using 5-L fermenter. The percentage of 100% PLA powder weight loss was
99.79%. Moreover, two types of commercial plastic bags (20-30% PLA and 15-
25% PLA) were used for the determination of PLA degradation by whole cell of
strain T16-1. The result indicated that the maximum enzyme activity and weight
loss (%) were received with 7.80 U/ml and 80.95%, respectively, after incubation
for 24 hours using 20-30% PLA bag. Moreover, PLA plastic with 15-25% PLA
was also determined. However, the degradation efficiency was lower than 20-
30% PLA with a weight loss of 41.43%. While, lactic acid was not detected
during fermentation process. Commercial PLA bags and 100% PLA film were
subject to evaluate the degradation by whole cell under the composting condition.
The results showed that weight loss was 66.67% by spraying inoculum on pieces
of 100% PLA film after incubation for 28 days. Unfortunately, 15-25% PLA and
20-30% PLA bag were not degraded by whole cell of strain T16-1.

Keywords: Actinomadura keratinilytica, Biodegradation, Fermenter, Plastic waste,
Weight loss

- 55 -

Elucidating the mechanisms of DNA repair via homologous
recombination in Bacillus subtilis

Wasanakorn Satang and Ponlkrit Yeesin *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Homologous recombination (HR) is a DNA repair pathway in many organisms.
In bacteria, there are many proteins involved in homologous recombination,
Such as RecA, which is a central protein in HR. However, RecA needs accessories
proteins for DNA substrate preparation. A group of such proteins that has not
been fully characterized is the AddAB complex. This work aims to study the
function of AddA protein in HR. Bacillus subtilis recombination-deficient
mutants were constructed by deleting ΔaddA, resulting in no production of
AddAB complex. We then tested ultraviolet (UV) sensitivities of the mutant
compared to wild types. Because UV irradiation can induce DNA damage, we
found that ΔaddA mutant haves less ability to withstand UV radiation than wild
types. Were also measured the growth rates of ΔaddA mutant and found that the
mutant grew slower than wild types. Altogether, these data indicate that AddA
proteins are is required for homologous recombination
Keyword : Bacillus subtilis, addA , Homologous recombination, DNA repair

- 56 -

Isolation and antimicrobial susceptibility test of bacterial isolated
from hospital wastewater treatment

Yanika Sue-nguan, Putthipong Chansanit and Pichapak Sriyapai *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Water pollution is a major problem throughout the country. The wastewater in
the hospital consists of chemicals and is contaminated with pathogenic
microorganisms. Enterobacteriaceae is a large family of Gram-negative bacteria
found in hospital wastewater. These bacteria can be both pathogenic and
opportunistic pathogenic microbes in immunocompromised individuals and
often cause food poisoning and gastroenteritis if contaminated food and water
are consumed. This study was conducted from the wastewater treatment pond at
the HRH Princess Maha Chakri Sirindhorn Medical Center, Nakhon Nayok
Province. Our study aims to isolate the bacterial pathogens and to evaluate the
antimicrobial resistance patterns from hospital wastewater treatment. We
carried out the bacterial competence screening on seventy bacterial isolates.
About twenty-two isolates were resistant to third-generation cephalosporins.
Species identities were analyzed by 16S rDNA sequence analysis and
biochemical characterization. The 16S rDNA sequences of these strains were
100% similar to Escherichia coli (15 strains), Klebsiella pneumoniae (6 strains)
and Pseudomonas aeruginosa (1 strain).
Keywords: Antimicrobial susceptibility test, Enterobacteriaceae, 16S ribosomal
RNA gene, Wastewater treatment

- 57 -

Commercial biodegradable plastic degradation using the enzyme
produced by Actinomadura keratinilytica strain T16-1 in 3-L stirrer
fermenter and under composting condition

Kirana Atthawichien, Orapan Innate and Sukhumaporn krajangsang *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Polylactic acid (PLA) or PLA is biodegradable aliphatic polyester and can be
degraded by various enzymes produced by microorganisms. However, the
product of PLA degradation process is lactic acid, it inhibited enzyme activity
since lactic acid-binding to the active site of the enzyme. The dialysis method is
the best method of PLA-degradation, it can reduce the concentration of lactic acid
during the degradation process. Therefore, this study focuses on the scale-up of
commercial PLA degradation by the enzyme produced by Actinomadura
keratinilytica strain T16-1 in bioreactor using the dialysis method and evaluates
the degradation under the composting condition. PLA powder degradation
efficiency of the control condition (without dialysis) was lower than the dialysis
method in 3-L stirrer bioreactor. The highest degradation of PLA powder weight
loss was 33.33% under the dialysis condition for 2 days. Therefore, the dialysis
method was subjected to use for commercial PLA degradation testing. Two
commercial PLA, 15-25% PLA in T-shirt bag and 20-30% PLA in T-shirt bag
were used as substrate in this study. Weight loss of 15-25% PLA and 20-30%
PLA in T-shirt bag PLA were 5% and 10%, respectively. Furthermore, all
commercial PLAs were evaluated the degradation by using the enzyme produced
by Actinomadura keratinilytica strain T16-1 under composting condition for 28
days at 60 °C. The result indicated that 100% PLA film was completely degraded.
While 20-30% T-shirt bag had a few cracks.

Keywords: Commercial biodegradable plastic, Dialysis method, PLA

- 58 -

Efficiency of synthetic dye decolorization by white-rot fungi and
ligninolytic enzyme study

Bancha Tamprapaporn, Pitchapa Thongsuwan, Pisit Thamvithayakorn and Nuttika Suwannasai *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Synthetic dyes are extensively used in the textile industry and are difficultly
degraded into the environment due to their complex structures. White-rot fungi
(WRF) are found to be capable of degrading lignin which has aromatic structures
similar to most synthetic dyes. Therefore, this study aimed to screen the
efficiency of WRF in the decolorization of synthetic dyes and to study the
ligninolytic enzyme production. For the screening results, all nineteen fungal
isolates showed varying degrees of decolorization against four different synthetic
dyes by using agar plate assay. Most fungal isolates could decolorize Remazol
Brilliant Blue R (RBBR) > crystal violet > safranin O > malachite green. The best
decolorization belonged to Pseudolagarobasidium sp. (PP17-33) followed by
Lentinus squarrosulus (PP17-02) and Coriolopsis retropicta (PP17-134). The
percentages of decolorization were then measured by using a spectrophotometer
and the results revealed that PP17-33 was the most effective isolate by 83.65 –
91.99 % decolorization of all synthetic dyes within 8 days. In addition, the
ligninolytic enzymes of laccase, manganese peroxidase (MnP) and lignin
peroxidase (LiP) were analyzed by using RBBR as a substrate in submerged
culture. The maximum activity of the enzyme was MnP (19.95 U/mL) in 16 days
under shaking condition at 150 rpm and 30°C. MnP was partially purified from
500 mL crude extract contained 13,000 U of total activity and 70.97 mg of total
protein. After precipitation with 65% ammonium sulfate and dialysis, the total
activity of MnP and total protein were 10,600 U and 5.78 mg, respectively.

Keywords: Decolorization, Ligninolytic enzymes, Manganese peroxidase,
Synthetic dyes, White-rot fungi

- 59 -

Biocontrol of postharvest diseases in various fresh products by
using volatile compound of Steptomyces sp. strain AT14898

Nutsada Seangdao and Sukhumaporn Krajangsang *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Postharvest diseases are one of the main causes in agricultural product losses. In
the recent decades, use of synthetic fungicides has been the primary choice for
reducing postharvest losses, resulting in a large number of toxic residues which
are harmful to human health and the environment. Therefore, the use of biological
agents is an effective method of disease control that is safe and free of toxic
residues. The aim of this work was to determine the effect of volatile compound
for inhibiting postharvest diseases by endophytic Streptomyces sp. strain AT-
14898. The strain was grown on mung bean supplemented with ammonium sulfate
as nitrogen source. The pathogenic fungi used in this study were isolated from
longan, mango, mangosteen and rambutan. The results of in vitro trials
demonstrated that Streptomyces sp. strain AT-14898 still had high efficiencies to
inhibit the fungal growth after keeping at room temperature and 44°C until 30
days. The highest mycelial growth inhibition was found Trichoderma sp. with
66.35% under aerobic condition at 4°C. The in vivo trials were tested in the
rambutans, and demonstrated that the percentage of severity and disease
incidence of pathogenic fungal strain RBT-F03 were 100% and 22%,
respectively. The percentage of severity and disease incidence of pathogenic
fungal strain RBT-F05 was 0%. Finally, the fumigant effects of the volatile
compounds on postharvest diseases at natural condition was studied. The results
indicated that after incubation of the 30 rambutans with volatile compound for 5
days the percentage of severity and disease incidence were 74.74% and 30.70%,
respectively.

Keywords: Biocontrol, Volatile compound, Streptomyces sp., Postharvest
disease

- 60 -

Natural dye production from Penicillium sp. PK17-44 for dyeing
fabrics

Damrongrit Wongprakarnsanti a, Sirinun Kaenthong b and Nuttika Suwannasai a,*
a Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
b Department of Home Economics, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Natural dyes from fungi and mushrooms have been proposed as an eco-friendly
alternative to synthetic dyes. They are biodegradable and less hazardous to
health. Therefore, the present study aimed to extract colour pigments from
fungi, and to evaluate the dyeing capacity on textile fabrics. Colour pigments
from 4 fungal species: Penicillium sp. PK17-44, Boletus sp. (NP19-03), Russula
virescens (NP19-21) and Russula rosacea (NP19-24), were extracted by hot
distilled water (80°C). The pigments extracted were reddish brown, orange,
green and pink in colour, respectively. After dyeing Multifibre fabrics (wool,
acrylic, polyester, nylon, cotton, cellulose acetate and silk) with the fungal
pigments, all extracted pigments could dye nearly all fabrics in different colours,
except for cotton. Because the pigments produced by PK17-44 exhibited the
strongest colour on nylon fabric as analysed by spectrophotometry, it was then
selected for a scale-up colour production and evaluation of the dyeing capacity
on nylon fabric. The optimum conditions for dyeing were 3% owf (on-weight-
fabric) of colour pigments at 130°C for 30 minutes. For molecular identification
of PK17-44, the internal transcribed spacer (ITS) was amplified, sequenced and
analysed. BLAST result revealed the highest % identity to Penicillium
chermesinum (100%) and P. gerundense (100%). The results obtained from this
study showed that the pigments produced by Penicillium sp. PK17-44 could
serve as a source of natural dye for nylon fabrics.

Keywords: Penicillium, Fungi, Natural dye, Nylon, Fabrics

- 61 -

Isolation and characterization of lytic bacteriophage against
Pseudomonas aeruginosa

Napasorn Kamolvathin and Onanong Pringsulaka *
Department of Microbiology, Faculty of Science, Srinakarinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Pseudomonas aeruginosa is one of the nosocomial pathogens, particularly in
immunocompromised patients. Infections caused by P. aeruginosa are being
treated with different antibiotics; however, the extensive use of antibiotics has
resulted in the emerging of multidrug resistance (MDR). Phage therapy becomes
an acceptable alternative therapy to treat and control the MDR infection. This
study aims to isolate and characterize lytic phages from sewage and river water
for the inactivation of P. aeruginosa. Cetrimide agar was used as a selective
medium for the isolation of P. aeruginosa. The five suspected isolates were
further identified by 16S rDNA sequence analysis. Among these, only isolate
PM2 showed 99% similarity to P. aeruginosa. P. aeruginosa PM2 was employed
as a host for phage isolation. Two different phages, P2 and P0.1 were selectively
isolated based on their ability to produce large and clear plaques in plaque assays.
Electron micrographs revealed that both phages belong to the Siphoviridae family
in the order Caudovirales. Host-range determination demonstrated that these
phages were capable of infecting other strains of P. aeruginosa. These phages
were relatively stable at the temperature ranging from 4 to 45°C, especially phage
P2 that can survive even at 100°C for up to 1.5 h. In vitro study revealed that
phage cocktail P2/P0.1 was able to reduce the titre of P. aeruginosa by
approximately 2 log10 CFU.mL-1 during 12 h of incubation.
Keywords: Bacteriophage, Phage therapy, Pseudomonas aeruginosa

- 62 -

Encapsulation of canine probiotic bacteria in sodium alginate and
sodium alginate-goat milk matrices

Ngamlak Foongsawat and Onanong Pringsulaka *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

The use of probiotics in canine has been of growing interest. For successful use
as a probiotic, the bacterial species should be of canine intestinal origin since
these species exhibit host specificity. Moreover, probiotic bacteria must endure
high acidic condition in the stomach, and hydrolytic enzymes and bile salt in the
small intestine. In our previous study, 6 lactic acid bacteria (LAB) strains, isolated
from faecal microbiota of healthy dogs with potential probiotic properties, were
selected. The aim of this study was to microencapsulate these probiotic bacteria
using the extrusion method in sodium alginate (SA) and sodium alginate-goat
milk (SAGM), and to evaluate the survival of free and encapsulated bacterial cells
under simulated gastrointestinal conditions, as well as under refrigeration. The
encapsulation yield of six strains in SA and SAGM varied from 92.23-98.97%.
Lactobacillus fermentum Shi1 encapsulated in SAGM showed the maximum
survival rates in simulated gastric juice (7.94 log cfu g−1) and in simulated
intestinal juice (8.20 log cfu g−1). The survival of encapsulated cultures and free
cells of these strains during storage at 4°C for 28 days were evaluated. Among
these, 4 strains (Enterococcus hirae Pom4, Lactobacillus salivarius Pom5, L.
fermentum Shi1 and Pediococcus pentosaceus Chi8) encapsulated in SAGM
demonstrated higher survival capabilities, and L. salivarius Pom5 showed the
highest cell viability rate during storage. This study showed that SAGM has a
potential to be used as a new encapsulation material for encapsulating canine
probiotic bacteria.

Keywords: Encapsulation, Probiotics, Sodium alginate, Sodium alginate-goat milk

- 63 -

Study of the immune response to seasonal influenza vaccine in
end-stage renal disease patients

Nattaya Kamchompoo a, Pichapak Sriyapai a,* and Kobporn Boonnak b,*
a Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
b Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol
University, Bangkok, 10400
* Project Advisor Email: [email protected], [email protected]

End-stage renal disease patients are immunocompromised patients caused by
uremia, which is a risk factor for infections and leads to increase mortality.
Uremia causes inflammation and reduces immune system function. This study
aims to study immune responses in end-stage renal disease patients who have
been administered a high dose seasonal influenza vaccine and booster compared
to the standard dose. A prospective, open-label, randomized study was conducted
in Bhumibol Adulyadej hospital with a total of 100 samples collected from end-
stage renal disease patients and was randomized into 2 groups. Group I (n=50)
received one dose of seasonal influenza vaccine while Group II (n=50) received
two doses and booster. Blood samples were collected in both groups. After that,
antibodies were detected by Hemagglutination Inhibition Assay (HAI) against
A/Chanthaburi/128/2018 (H1N1), A/Thailand/TM-8453_54/2018 (H3N2),
B/Ubonrachthani/203/2019 and B/Phuket/287/2013. For Group I and II, a
significant difference was observed for the seroconversion rate against H3N2
were 60% and 84%, respectively (p-value <0.01). For other strains, no significant
difference in the seroconversion rates. The GMT (95% CI) had a significant
difference against H3N2 as well as the seroconversion rate (p-value=.0471). In
conclusion, the seroconversion rate and GMT of 3 strains had no differences in
both groups, except for H3N2. As end-stage renal disease patients are
immunocompromised, they have a deficient immune cell function resulting in
low immune responses. Therefore, both end-stage renal disease patients who
received a high dose seasonal influenza vaccine with a booster and those who
received standard dose had no differences in immune responses.

Keywords: End-stage renal disease, Hemagglutination inhibition Assay, Immune
response, Seasonal influenza vaccine

- 64 -

Optimization of lipid production by Lipomyces tetrasporus
ADrSe9-2 from hydrolyzed agriculture wastes using crude enzyme

Arthittaya Khaykham, Phitchamon Thongthirach and Wanlapa Lorliam *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project advisor Email: [email protected]

Microbial oil is lipid from an oleaginous microorganism such as bacteria, mold
and yeast. This microorganism can accumulate lipid more than 20% of their
biomass. Recently, much attention has been paid to the development of microbial
oils, especially yeast oils that are now believed to be a promising potential
feedstock for biodiesel production due to their composition of fatty acids being
similar to vegetable oils. Microbial lipid production from agriculture wastes is
considered as a potential alternative feedstock for large scale production of
biodiesel at low cost and also to prevent a shortage of feedstock. Therefore, this
study aimed to optimize conditions for lipid production of Lipomyces tetrasporus
ADrSe9-2 from cassava chip hydrolysate by using crude amylase and cellulase
from Actinomadura keratinitilytica T16-1. The hydrolysate, supplied with various
of nitrogen sources such as yeast extract, malt extract, corn steep liquor, casein,
peptone, ammonium nitrate and ammonium sulfate was used in 50 mL batch
cultivation for lipid production. Biomass, lipid production, and lipid content were
analyzed. The result suggested that the cassava chip hydrolysate supplemented
with 2% ammonium sulfate as nitrogen source was the most suitable for the high
concentration of lipids. The biomass, lipid production and lipid content achieved
from batch fermentation were 4 g/L, 3.58 g/L, and 89% of lipid content,
respectively. Thus, L. tetrasporus ADrSe9-2 has the potential to be a lipid
production from cassava chip hydrolysate by using crude enzymes from A.
keratinitilytica T16-1.

Keywords: Actinomadura sp., Amylase, Cellulase, Lipomyces sp., Oleaginous
yeast

- 65 -

A study of washing methods to reduce contaminated
microorganisms on fresh vegetables

Watcharaphorn Khongsimahachok and Wanlapa Lorliam *
Department of Microbiology, Faculty of science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Customer demands for fresh vegetables and their ready-to-eat (RTE) salads
have increased since people changed their eating habits because of healthier
lifestyle interests. However, the number of foodborne outbreaks associated with
fresh produce has also increased, with Escherichia coli being the most common
pathogen. Especially, E. coli O157: H7, which causes diarrhea, hemorrhagic
colitis, hemolytic uremic syndrome, is of concern. Vegetables can be
contaminated with E. coli at any point from pre- to post-harvest. Therefore, this
study was to determine the efficacy of different cleaning methods in reducing
bacterial contamination on fresh vegetables. Lettuce and cabbage samples were
inoculated with E. coli (approximately 108 CFU/ml). Each sample was then
subjected to one of the following cleaning procedures: either (I) soak for 10 min
in 5% salt solution, Veggie Wash solution, 2% vinegar solution, 2% calcium
hydroxide solution, or 0.1% potassium permanganate solution, or (II) soak for
30 min in water from washed rice, 15% lime juice, or 1% sodium bicarbonate.
After that, the samples were rinsed under running tap water. The results
revealed that the best washing method was soaking in 15% lime juice for 30
minutes and then rinsed with tap water. The number of E. coli after treatment
was 4.12-4.15±0.51-0.65 log CFU/g. Additionally, using hot water treatment for
15 minutes reduced the number of E. coli by less than 1 log CFU/g. This study
indicates that 15% lime juice can be used as a household washing agent for the
reduction of E. coli on Lettuce and cabbage.

Keywords: Fresh vegetables, Washing methods, E. coli, Lime juice

- 66 -

Isolation and characterization of lactic acid bacteria and yeasts
involved in coffee fermentation during wet processing of Coffea
arabica

Napisa Wanichayachat, Pornporm Pongsarai and Wanlapa Lorliam *
Department of Microbiology, Faculty of science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Coffee is one of the preferred beverages consumed worldwide. Thailand is the
third largest coffee producer in Asia. Arabica coffee (Coffea arabica) is grown
widely throughout northern Thailand. Wet processing is used mainly for Arabica
coffee. The process involves removal of the outer skin and part of the mucilage
by machines. The remaining mucilage is then removed by fermentation in water
for 24–48 h, followed by drying. During wet processing, the ripe coffee fruits
undergo a spontaneous fermentation, carried out by a complex microbiological
process that involves the actions of microorganisms like yeasts, bacteria and
filamentous fungi. Therefore, this study investigated the microbial communities
involved in coffee fermentation during wet processing of C. arabica. Coffee
cherries, de-pulped coffee and liquid from fermentation samples were collected
during fermentation. A total of 132 microbial isolates were obtained from 21
samples: 32.6 % lactic acid bacteria and 66.67 % yeasts. Lactic acid bacteria were
grouped by phenotypic characterization as cocci (34.9 %) and bacilli (65.12 %).
Yeasts were grouped by the ability to assimilate different carbon sources. As a
result, 5 groups of yeasts were characterized by carbon sources utilization as
followed: only glucose (75%); glucose and galactose (1.14%); glucose, galactose
and maltose (1.14%); glucose and sucrose (27.28%); and glucose, sucrose and
maltose (1.14%). Further research should include identification of the isolated
yeasts and lactic acid bacteria by genotypic characterization and screening for
suitable strains for coffee fermentation.

Keyword: Arabica coffee, Lactic acid bacteria, Yeasts, Wet processing

- 67 -

Effect of bacterial antagonist on the induction of defense-related
gene expression and reactive oxygen species accumulation in Pan
– lime

Konkamon Sujijun, Natthida Sudyoung, Siriruk Sarawaneeyaruk *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

In Thailand, citrus canker caused by Xanthomonas citri subsp. citri (XCC),
bacterial infection that affects the production of Pan-lime (Citrus aurantifolia
cultivar Pan). Typically, plants have evolved a variety of defense mechanisms
including the production of reactive oxygen species (ROS) in respose to
pathogenic bacterial invasion. The previous biocontrol study reported that
Pseudomonas aeruginosa SWUC02 (SWUC02) can inhibit XCC efficiently.
Hence, this research aims to (1) evaluate the expression levels of defense-
related genes in Pan-lime inoculated with XCC and/or SWUC02 by reverse
transcription PCR (RT-PCR), and (2) to study the effect of XCC and SWUC02
co-infection on ROS accumulation in Pan-lime. Firstly, we inoculated the
bacteria to Pan-lime seedlings grown under the tissue culture condition.
Seedlings were divided into 4 treatments as follows: (1) non-inoculation, (2)
inoculation with XCC only, (3) SWUC02+XCC, and (4) SWUC02 only. Next,
total RNA from leaves of each treatment was extracted and used as the template
in RT-PCR using primers specific to Pt14, LRR8, PR1 and LOX genes. The
results showed that SWUC02 inoculation was able to stimulate Pt14 and LRR8
gene expression in the seedling, while XCC inoculation was able to stimulate
PR1 gene expression. Finally, ROS accumulation in the seedling was evaluated
by the potassium iodide method. We found that XCC-inoculated seedling had
lower ROS accumulation than the non-inoculation, whereas the seedling that
co-inoculated with SWUC02 followed by XCC infection showed a comparable
level of ROS accumulation to the control. In conclusion, the antagonist
SWUC02 can induce the expression of plant defense-related genes, Pt14 and
LRR8. Decreasing ROS accumulation may be a mechanism of XCC to invade
plant cells. However, the co-inoculation of SWUC0 prior to XCC infection can
recover ROS accumulation level to uninoculated seedlings.

Keywords: Citrus canker, Defense-related genes, ROS accumulation, Xanthomonas
citri subsp. citri (XCC), Pseudomonas aeruginosa SWUC02 (SWUC02)

- 68 -

Efficacy of culture filtrate of Pseudomonas aeruginosa SWUC02
against bacterial pathogen

Kotchanat Srisangchun a, Natthida Sudyoung a, Kwannan Nantavisai b and
Siriruk Sarawaneeyaruk a,*
a Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
b Department of Microbiology, Faculty of Medicine, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Novel medicines for controlling pathogenic bacteria are valuable nowadays.
From previous study, the culture filtrate obtained from Pseudomonas aeruginosa
SWUC02, was used as a biocontrol agent to inhibit Xanthomonas citri subsp.
citri. Therefore, the culture filtrates of SWUC02 may be capable for inhibiting a
variety of bacterial pathogens. This study aimed to evaluate the efficacy of the
culture filtrate as an antibacterial and to test the toxicity of the culture filtrate on
human normal and cancer cell lines. Firstly, we investigated the optimum
incubation time to produce culture filtrate with high antibacterial activity and
evaluated the thermal stability of culture filtrate. From well diffusion method, the
highest antibacterial activity was found in 12 hours of incubation at 30 ˚C with
agitation. The culture filtrate that was incubated at 35, 37, 40, or 45 ˚C for 1 and
2 hours, exhibited the same antibacterial activity (P<0.05). For inhibition test, the
pathogenic bacteria were inhibited by the culture filtrate. The minimum
bactericidal concentration (MBC) against Staphylococcus aureus and Bacillus

subtilis was 7.0× 102 μg/ L. The MBC of the gram-negative bacteria including

Enterobacter sp., Escherichia coli, Salmonella sp., Shigella sp., Klebsiella
pneumonia and Acinetobacter baumannii was 2.8×103 μg/L which is lower than
that of the gram-positive bacteria. The minimum inhibition concentration (MIC)
of Enterobacter sp. and E.coli was 87.5 μg/L. In conclusion, the culture filtrate
from SWUC02 was a broad antibacterial agent inhibiting the growth of bacterial
pathogens. In the future, I will study the toxicity test on normal and cancer cell
lines.

Keywords: Antibacterial activity, Culture filtrate, Pathogenic bacteria,
Pseudomonas aeruginosa

- 69 -

Isolation and characterization of lytic bacteriophages against
Aeromonas hydrophila

Supreeya Ketlekha, Orathai Wetchaphurksapitak and Onanong Pringsulaka *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Aeromonas hydrophila is responsible for the disease known as Motile
Aeromonad Septicemia (MAS) in fish. The disease causes high mortality rates in
fish farming especially when fish are held under stress. To reduce MAS
incidences, one approach has been the use of bacteriophages in aquaculture
operations. However, so far, isolation and characterization of lytic bacteriophages
against A. hydrophila in Thailand have not been studied extensively. The
objective of this study is to isolate lytic phages specific to A. hydrophila for the
control of this disease-causing pathogen in aquaculture. Nine river and canal
water samples in Bangkok were used to isolate both A. hydrophila and phages.
Two different phages sGY and DL, were isolated using strain AHGX5 as the
host. AHGX5 was identified as A. hydrophila by biochemical testing and 16S
rRNA gene sequencing. Transmission electron microscopy analysis
revealed that both phages belonged to family Siphoviridae. Host range
determination showed that these isolated phages were highly specific against
A. hydrophila. Both sGY and DL were relatively stable over a wide range of
temperatures (4-75 oC) and pH (4-12). An in vitro study of the efficiency of phage
cocktails in an inactivation of A. hydrophila revealed a remarkable reduction in
bacterial approximately 2 log CFU/ml. These findings illustrate the potential for
using phages as an effective method to control A. hydrophila in fish farms.

Keyword: Aeromonas hydrophila, Lytic bacteriophages, Temperature stability,
pH stability, Phage therapy

- 70 -

Comparison of enzyme kinetics of two commercial cellulases in
presence of potassium chloride

Chanikant Chiarakrai and Prapakorn Tantayotai *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

The enzymatic hydrolysis of cellulose is one of the main limiting steps of the
conversion of lignocellulosic biomass into biofuels. The remaining residues from
pretreatment processes, such as acids. ionic liquids, and inorganic salts, can
increase efficiency of enzymatic hydrolysis. Therefore, this research aimed to
study the effect of potassium chloride (KCl) pretreatment residue on Celluclast
and Accellerase1500 hydrolysis kinetics. Three types of substrates: CMC, Avicel,
and bagasse were hydrolyzed with Celluclast and Accellerase1500 separately at
50 °C, and reducing sugars were determined as hydrolysis products by DNS
assay. Then the hydrolysis reactions were challenged with the presence of 0.5 and
1.0 M KCl in the same manner. Without KCl, the highest Vmax of Celluclast was
obtained at 0.109 mg/ml•min and the lowest Km was obtained at 0.584 mg/ml•min
when using CMC as a substrate. Compared to Celluclast, Accellerase1500 had
the better hydrolysis performance by having Vmax at 0.201 mg/ml•min and Km at
1.418 mg/ml•min when using CMC as a substrate. Under influence of 1.0 M KCl,
both enzymes had lower in hydrolysis efficiency as the reducing sugars decreased
to 76.49- 93.68% compared to no-KCl condition. Interestingly, when using 0.5
M KCl, the hydrolysis kinetics of Celluclast and Accellerase1500 on sugarcane
bagasse were improved with Km value at 0.394 and 0.300 mg/ml•min and Vmax at
0.031 and 0.044 mg/ml•min, respectively. These results suggested the important
of process optimization of pretreatment and hydrolysis and the knowledge could
be applied to consolidate processing in biorefining industry.

Keywords: Enzymatic hydrolysis, Kinetics, Celluclast, Accellerase1500, Potassium
chloride

- 71 -

Study of weedy rice elimination method using Bacillus subtilis
strain OK1101

Yanin Prain, Nuttanicha Sungsirichaikul and Sukhumaporn Krajangsang *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Weedy rice, or red rice, is one of the major weeds in rice fields of Thailand that
adulterates with cultivated rice. Due to weedy rice contamination, the cultivated
rice productivity and quality have been decreasing. Usually, farmers use chemical
treatment to kill weedy rice, but elimination efficiency is very low. The pesticide
also affects the farmer health. Therefore, this study aims to (1) evaluated Bacillus
subtilis strain OK1101 for elimination of weedy rice in lab scale, ( 2) to scale up
of strain OK1101 production in 100L fermenter, and (3) to apply strain OK1101
for elimination of weedy rice in the rice field. B. subtilis strain OK1101 showed
the highest cellulase and amylase activity with 2,597 and 300 U/ ml in CMC
medium, respectively. Next, we test for the elimination efficiency of weedy rice
by strain OK1101 in lab scale. The results demonstrated that after spraying 200
ml of strain OK1101 on weedy rice, percentage of seed germination was
decreased to 27.5% compared to the control condition. Moreover, the scale up of
seed culture production was achieved by using 100L fermenter. The maximum
cell density measured at OD 600 nm was 2.82 using the working volume of 20L.
Finally, strain OK1101 was tested in the rice field and showed high weedy rice
inhibition (70%). Shelf life of strain OK1101 as seed culture was also determined.
Cell growth in broth and freeze dry condition at room temperature and 4C was
not significantly different.
Keywords: Weedy rice, Bacillus subtilis, Elimination method, Scale up.

- 72 -

Medium optimization for cultivation of carotenoid producing yeasts

Sasiwimon Jansri and Prapakorn Tantayotai *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Carotenoid produced by red yeasts plays a major role in food and feed industries
as an important antioxidant and a source of provitamin-A. The potential of
carotenoid production of selected yeast strains is mainly limited by culture
medium composition. Therefore, this study aimed to optimize medium for
carotenoid production of three yeast strains: Rhodotorula rubra TISTR5134,
Rhodotorula mucilaginosa TISTR5927 and Sporobolomyces pararoseus
TISTR5213. Carbon, nitrogen, and supplement of the medium were varied. After
incubation, microbial carotenoid was extracted with acetone and DMSO and
determined by spectrometry. The results suggested that the optimal medium for
R. rubra TISTR5134 was consisted of 10 g/L glucose, 10 g/L yeast extract and 6
g/L dipotassium phosphate. The optimal medium for R. mucilaginosa
TISTR5927 was contained 15 g/L glucose, 15 g/L yeast extract and 4 g/L
dipotassium phosphate, and the optimal medium for S. pararoseus TISTR5213
comprised 10 g/L glucose, 5 g/L yeast extract and 2 g/L ferrous sulfate.
Employing these optimized media, the highest amounts of carotenoids from R.
rubra TISTR5134, R. mucilaginosa TISTR5927 and S. pararoseus TISTR5213
were 2.64, 3.08, and 1.75 µg/L, respectively. Then, investigation on 250-mL
submerge fermentation using 5% (v/v) inoculum with 10% (v/v) cassava pulp as
potential cattle feed was performed. It was found that the obtained carotenoids
were less than those from the adjusted media in all strains. In short, media for
culturing the red yeasts were optimized, and to utilize the red yeasts for animal
feed further study is required.

Keywords: Red yeast, Carotenoids, Medium optimization, Cassava pulp

- 73 -

Antimicrobial susceptibility and genetic characterization of
Salmonella spp. isolated from environment samples

Seatthanan Pewla-oo, Kanokwan Ngokngam and Pichapack Sriyapai *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Salmonella serovars are among the foremost bacterial pathogens implicated in
food-borne gastroenteritis worldwide. Salmonella infection, known as
Salmonellosis, is one of the most important public health problems in many
countries. Disease outbreaks are usually caused by multidrug-resistant (MDR)
Salmonella strains that are related to the presence of antibiotic resistance genes.
The present study was carried out to determine the antimicrobial resistance
patterns and antibiotic resistance genes of Salmonella isolated from environment
samples between 2017 and 2020. Out of 322 isolates of Salmonella spp., 9 isolates
were resistant to third-generation cephalosporin drugs (cefotaxime, cefpodoxime,
ceftazidime and ceftriaxone). All cephalosporin-resistant isolates were ESBL
(extended-spectrum beta-lactamase) positive. All of ESBL-producing isolates
were screened for resistance genes by PCR and DNA sequencing. The results
showed that ESBL genes (blaCTX-M-15 and blaTEM-1b) were found on plasmids from
the ESBL-producing Salmonella isolates.
Keywords: Salmonella, Antibiotic resistance, Antibiotic resistance genes, third-
generation cephalosporin drugs, Food, Environment

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Development of probiotic carriers: dried bacon and dried pork
liver, for dog snack with the impregnation of probiotic bacteria

You Chonghan and Onanong Pringsulaka *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Probiotics are defined as living microorganisms that are beneficial to health if
they are taken in sufficient numbers into the body via foods. The effects of
supplementing food products with probiotics have been studied extensively in
humans. However, combinations of probiotics with dietary fibers have not been
adequately explored in dogs. Coating liver and bacon with probiotics is a good
option for passing probiotics to dogs. The aim of this study was to produce dried
bacon- and dried pork liver- based novel probiotic carriers. The bacon and pork
liver slices were impregnated with 3 probiotic strains including Lactobacillus sp.
Pom1, Lactobacillus sp. Pom2 and Lactobacillus salivarius Pom5, dried with
convectional drying at 45 °C, and then stored at 4 °C for 7 days. The probiotic
counts on pork liver slices were greater than those on bacon slices under storage
conditions.Therefore, impregnated pork liver slices were used as a carrier matrix
for further study. SEM images demonstrated well adaptation of the bacterial cells
on pork liver surfaces. Survival of probiotics in dried pork liver was evaluated
over the period of 28 days of storage at 4 °C. Probiotic viabilities ranged from
6.16 to 9.18 log CFU/g during the 28 days of storage. Water activity values of
dried pork liver were between 0. 7 and 0. 9. Simulated gastro-intestinal (GI)
digestion suggested the ability of the strains to survive the digestive process.
Coating dried pork liver with alginate microcapsule of probiotics, probiotic
bacteria encapsulated in alginate showed slower log reduction than free probiotic
bacteria.

Keywords: Dog snack, Gastrointestinal conditions, Lactobacillus, Pork liver,
Probiotics, Probiotics viability

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Extraction of beta-glucan from edible mushrooms and
Amauroderma subresinosum (NP17-12) and their antioxidant activity

Kawin Chantima a, Cherdchai Phosri b and Nuttika Suwannasai a,*
a Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
b Department of Biology, Faculty of Science, Nakhon Phanom University, Nakhon Phanom, 48000
* Project Advisor Email: [email protected]

Beta-glucan is a polysaccharide in the fungal cell wall and has effects on immune
system activation including antimicrobial, antioxidant, antiviral and antitumor
effects. In this study, the total glucans (beta- and alpha-glucans) were extracted
from six different edible mushrooms of Amauroderma subresinosum (NP17-
12), Boletus sp. (NP19-02), Boletus sp. (NP19-22), Flammulina velutipes (BK19-
02), Lentinula edodes (BK19-01) and Russula rosacea (NP19-14) and then crude
extracts were evaluated for their antioxidant activity in term of % DPPH radical
scavenging activity, the results of beta-glucan contents from all mushroom crude
extracts were 1.91, 2.24, 2.86, 2.63, 1.62 and 2.34 % w/w. For antioxidant
activity, the highest activity belonged to NP19-14 (69.7 %) followed by NP17-
12 (69.5%) and BK19-01 (57.5%). According to these results and mycelium
growth rate, NP17-12 was selected to be cultured in the large scale of 8-L potato
dextrose broth for mycelium production. The fungal mycelium was filtrated,
dried and ground to powder by using liquid nitrogen. The optimum ratio of
mycelium powder to extraction solvent (12M sulfuric acid) to obtain the
maximum yield of beta-glucan content was 0.4: 1 (g: ml). Then, twenty-eight
grams of mycelium powder was extracted and partially purified by 95% ethanol
precipitation at 4°C overnight. The total glucans obtained was 1.24 % w/w and
the antioxidant activity was 96.12 % scavenging activity at the concentration of
0.125 g/mL.

Keywords: Antioxidants, Beta-glucan, Total glucan, Polysaccharide, Mushroom

- 76 -

Detection of mosquito-borne diseases in Aedes aegypti and Aedes
albopictus mosquitoes in Phetchabun province

Vichapon Tiacharoen a, Pornsawan Leaungwutiwong b, Narin Thippornchai b,
Siriporn Chettanadee b, Akanitt Jitmittraphap b,* and Siriruk Sarawareeyaruk a,*
a Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
b Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol
University, Bangkok, 10400
* Project Advisor Email: [email protected], [email protected]

Dengue and Chikungunya fevers are mosquito-borne diseases spreading by
infected mosquitoes, Aedes aegypti or Aedes albopictus. The diseases usually
propagate during the rainy season in Thailand. There was reportedly an outbreak
diagnosis of dengue fever at Phetchabun province in 2 0 0 4 . Dengue and
Chikungunya fevers are caused by dengue virus (DENV) and chikungunya virus
(CHIKV) which are in the genus Flavivirus and Alphavirus, respectively. Those
viruses are positive-sense single-strand RNA viruses. The symptoms of them
include fever, joint pain, headache, and rash. In this study, we focused on the
detection of DENV and CHIKV in infected A. aegypti and A. albopictus which
were collected in the field from Phetchabun province, Thailand. The total RNA
of 96 pooled mosquito samples were extracted and subjected to two steps
reverse transcription PCR. The cDNA samples were detected by PCR and
followed by gel electrophoresis. The positive PCR results were found in 21
pooled samples (21.88%) of DENV and 8 pooled samples (8.33%) of CHIKV.
These results show that mosquito-borne diseases were circulated in Phetchabun
province. Therefore, residents should receive the information and education
about how to prevent mosquito-borne diseases. Future studies will include
sequencing data and phylogenetic analysis of DENV and CHIKV.

Keywords: Dengue virus, Chikungunya virus, Mosquito borne diseases,
Phetchabun province

- 77 -

Screening of red yeasts and medium modification for carotenoid
production

Kiratika Chidkoksung and Prapakorn Tantayotai *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Carotenoids are important natural pigments used in many industrial applications,
such as food supplements and cosmetic additives owing to their antioxidant
properties and pro-vitamin A function. In the feed industry, chemically
synthesized carotenoids are used to increase growth and productivity. Those
carotenoids are unstable and require high-cost substrates. Therefore, this research
aimed to screen and identify high-carotenoid-producing yeasts and to optimize
the medium for carotenoid production to be used as a feed supplement. Eleven
red yeasts were isolated from soil samples by using dilution technique. The
carotenoid concentrations, produced by these isolates, ranged between 0.50 –
2.89 µg/mL. Three isolates, KH4-2, JB1-1 and RF14-1, demonstrated the highest
amount of total carotenoids at 2.89, 2.88 and 1.59 µg/mL, respectively. Hence,
those three isolates were identified and used for further studies. Based on
nucleotide sequence, KH4-2 and JB1-1 were Sporidiobolus pararoseus, and
RF14-1 was Rhodotorula mucilaginosa. Medium optimization was performed by
varying carbon and nitrogen sources of YM medium. Among the various nitrogen
and carbon sources examined, maximum pigmentation was observed in the
culture medium containing peptone as a nitrogen source and sucrose as a carbon
source. The obtained carotenoid concentrations of KH4-2, JB1-1 and RF14-1
were 3.50, 3.40 and 1.21 µg/mL, respectively. Solid-state fermentation of
selected strains with sterile cassava pulp revealed that KH4-2 and JB1-1 produced
observable carotenoids on the fifth day of incubation, whereas RF14-1 showed
limited growth. Altogether, KH4-2 and JB1-1 yeasts have shown the promising
potential for being used as sources of carotenoids in feed supplement.

Keywords: Carotenoid, Red yeast, Medium optimization, Solid-state
fermentation, Cassava pulp

- 78 -

Detection of Felis catus gammaherpesvirus 1 (FcaGHV1) in
domestic cats in Bangkok, Thailand

Chattanan Tangjirawattana a, Erngsiri Kaewkhunjob b, Nichapat Rojjananavin b,
Navapon Techakriengkrai b,c,d, Wanlapa Lorliam a,* and Prawat Aungpraphapornchai a,*
a Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok,
Thailand
b Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn
University, Bangkok, Thailand
c Diagnosis and Monitoring of Animal Pathogens Research Unit (DMAPRU)
d Center of Excellence in Emerging Infectious Diseases in Animals, Chulalongkorn
University (CU-EIDAs), Bangkok, Thailand
* Project Advisor Email: [email protected], [email protected]

Felis catus gammaherpesvirus 1 (FcaGHV1), a novel Gammaherpesvirus of
domestic cats (Felis catus), first identified in the USA in 2014, has since been
reported in several countries, including Australia, Germany, Japan, Brazil and
Italy. Risk factors for FcaGHV1 DNAemia include being adult (> 2 years old)
male cats and coinfection with feline immunodeficiency virus (FIV). Although
FcaGHV1 has long been proposed to possess an oncogenic potential, however
the bona fide pathogenic role of FcaGHV1 in domestic cats is currently unknown.
To date, FcaGHV1 infection has never been reported in domestic cats in
Thailand. The objective of this study was to investigate the presence of FcaGHV1
DNA in domestic cats in Bangkok and to identify potential risk factors. Presence
of FcaGHV1 DNA was screened by conventional PCR specific to a conserved
360 bp region of the glycoprotein B gene (gB), and positive PCR amplicons were
subjected to direct Sanger sequencing. Sequences were aligned and compared
with those of other FcaGHV1 and various gammaherpesviruses available from
Genbank using Bioedit and MEGA X softwares. Among 100 samples tested,
FcaGHV1 gB was detected in 34 samples, giving an unexpectedly high
prevalence of 34%. The prevalence was higher in male than female, and in FIV
positive cats than FIV negative cats. This is the first study reporting the presence
of FcaGHV1 in domestic cats in Thailand, and confirming that FcaGHV1 is
indeed widely distributed throughout the world. However, the prevalence of 34%
reported in this study is surprisingly the highest among other FcaGHV1
epidemiological studies. This might be due to the limited sample size.

Keywords: Bangkok, FcaGHV1, Felis catus gammaherpesvirus 1, Thailand

- 79 -

Rapid diagnosis of Sporothrix schenckii from clinical samples by
PCR and MALDI-TOF MS.

Ratchnida Kamwa ª, Peechapack Somyoonsap ª,* and Nuvee Prapasarakul b,c,*
ª Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok,10110
ᵇ Department of Veterinary Microbiology, Faculty of Veterinary science, Chulalongkorn

University, Bangkok,10330

ͨ Diagnosis and Monitoring of Animal Pathogens Research

* Project Advisor Email: [email protected], [email protected]

Sporothrix schenckii is a thermally dimorphic fungus, meaning it can take on one
of two forms depending on the temperature at 37C presented in yeast form and at
25C presented in mold form. There are cause of Sporotrichosis. It is a zoonosis
that can be transmitted from animals to humans. In this study, it presents in a
domestic short-haired (DSH) cat in Bangkok, Thailand. This study aimed to
explore the rapid method for extraction DNA for PCR reaction and application
for diagnosis by MALDI-TOF MS. Fifteen clinical isolates of S. schenckii were
used in this study. The clinical samples were kept from different types of samples,
such as a swab, feline tissue, blood, and sabouraud dextrose agar (SDA) slant.
The test strains were subcultured twice on SDA slant for checking colony
examination first. After that, one sample was used to extract DNA by using
phenol-chloroform and PCR. For MALDI-TOF MS, the sample was prepared on
SDA slant. For the protein extraction for analysis. We must use EtOH and DI
water for resuspend colony. Using 70% formic acid and extracted in acetonitrile.
In conclusion, this study has a benefit for the person who wanted to extract the
DNA of dimorphic fungi without liquid nitrogen and kit, and we sent sample
sequencing to the National Center of Biotechnology Information (NCBI) for
approve accession number. Finally, NCBI approve accession number as
MN2186221 and MN2186222.

Keywords: Feline cat disease, Fungemia diagnosis, Sporothrix schenckii

- 80 -

Detection of Hepatitis B and Hepatitis C virus in Tha Song Yang,
Tak province

Anon sae-oung a, Akanitt Jitmittraphap b, Narin Thippornchai b, Siriporn Chettanadee b,
Pornsawan Leaungwutiwong b,* and Siriruk Sarawareeyaruk a,*
a Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
b Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol
University, Bangkok, 10400
* Project Advisor Email: [email protected], [email protected]

Hepatitis B and Hepatitis C are major diseases in Thailand, especially in Tha
Song Yang, Tak province. Clinical manifestation of these diseases are liver
cirrhosis, hepatocellular carcinoma, hepatic fibrosis and steatosis. Transmission
of Hepatitis B virus (HBV) and Hepatitis C Virus (HCV) usually occurs through
blood, secretion and serum. HBV is DNA virus belonging to family
Hepadanviridae, genus Othohepadnavirus and HCV is RNA virus belonging to
family Flaviviridae genus Hepacivirus. This research focuses on the detection of
HBV and HCV in serum obtained from patients with fever in Tha Song Yang
hospital, Tak province. Total DNA and RNA were extracted from 145 pooled
samples and used to detect for HBV and HCV using nested PCR and RT-PCR
method, respectively. The size of the PCR products of HBV and HCV were 802
and 244 bp. Only 17 (11.7%) of the pooled samples were positive for HBV while
all the pooled samples were for HCV. These results indicate that the Hepatitis B
disease is circulated in this area. Therefore, residents should be educated and take
medical advice for Hepatitis B disease.
Keywords: Hepatitis B virus, Hepatitis C virus, Tha Song Yang

- 81 -

Isolation and selection nitrogen-fixing bacteria to be used as
biofertilizer in hydroponic system

Sasita Boonphu, Nopparuj Meemusor and Prapakorn Tantayotai *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

The hydroponically-grown vegetable is one of popular choices for healthy diet.
However, providing an excess of chemical fertilizer in hydroponic system may
result in high-nitrate content in the vegetable which is related with the risk of
several cancers. To solve this problem, biological nitrogen fixation by
microorganisms can play a vital role as a substitution to commercially available
N-fertilizer. The aims of this study were to isolate and select nitrogen fixing
bacteria for applying in hydroponic system. Using Jensen’s medium, nitrogen-
fixing bacteria were isolated from the samples obtained from rhizospheric soils
and roots of vegetables planting conventionally, organically and hydroponically.
All isolates were evaluated for nitrogen fixing potential in Burks medium, and
fixed nitrogen was determined by using Nessler’s reagent. Among 170 isolates,
10 potential isolates, fixing nitrogen, as amount of ammonium, ranging from 2.57
to 10.04 mg/l, were selected. Next, the selected potential isolates were inoculated
into green oak seeds and percent germination was determined. The results showed
that the treatments with isolates: ISL-16, ISL-17 and ISL-22, had percent
germination similar to uninoculated control, and the presence of the treated
bacteria on the seedling roots was confirmed. Finally, this study provides some
of potential nitrogen fixing bacteria and suggests the method for applying in
hydroponic system.

Keywords: Hydroponics, Biofertilizer, Nitrogen-fixing bacteria, Seed
inoculation

- 82 -

Antioxidant activity and alpha-glucosidase inhibitory effect of
edible mushroom teas

Danaipong Khumwong a, Cherdchai Phosri b and Nuttika Suwannasai a,*
a Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
b Department of Biology, Faculty of Science, Nakhon Phanom University, Nakhon Phanom, 48000
* Project Advisor Email: [email protected]

Edible mushrooms are good sources of bioactive compounds containing highly
diverse biological activities, such as anticancer, antimicrobial, antioxidant and
antidiabetic activities. This study aims to evaluate the antioxidant activity and
alpha-glucosidase inhibitory effect of edible mushroom teas. Twenty-five species
of edible mushrooms obtained from local markets were dried, ground and packed
into tea bags (1 g per bag). Each mushroom tea bag was soaked in 100 mL of
boiling water for 15 minutes. The antioxidant activity (% scavenging activity),
total phenolic compound (µg GAE/ mL) and alpha-glucosidase inhibitory effect
(% inhibition) of different tea were determined. We found antioxidant activities
in all mushroom teas, and the highest activity belonged to Boletus sp. NP19-09
(96.12%), followed by Boletus sp. NP19-19 (41.64%). The highest total phenolic
contents were found in Boletus sp. NP19-03 (163.53 µg GAE/ mL) and Boletus
sp. NP19-18 (122.39 µg GAE/ mL). For alpha-glucosidase inhibitory effect,
Astraeus odoratus MP19-14 exhibited the strongest % inhibition (89.28%) to
alpha-glucosidase, followed by Boletellus endodensis NP19-11 (64.71%),
Pulveroboletus sp. NP19-17 (59.22%) and Craterellus aureus NP19-23
(54.39%). Due to strong odors of mushroom teas, two samples containing the
highest antioxidant activity, NP19-09 and NP19-19, were selected to mix with
two different Thai herbs (Pandanus leaves and lemongrass). The mixture of
NP19-09 and p andanus leaves or lemongrass in different ratios (4:0, 3:1, 2:2
w/w) revealed higher antioxidant activities than Pandanus leaves or lemongrass
alone (0:4 w/w). We obtained similar results when mixing NP19-19 with both
Thai herbs.

Keywords: Antioxidant activity, Total phenolic compound, Alpha-glucosidase
inhibitor

- 83 -

Comparative kinetics of two commercial cellulase cocktails in
hydrolysis of lignocellulosic materials with presence of sodium
chloride

Jadesada Ouncharoen and Prapakorn Tantayotai *
Department of Microbiology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Lignocellulosic biomass is a promising renewable biomaterial for biofuel
production. Hydrolysis of lignocellulosic biomass releases reducing sugars which
can be used for bioethanol fermentation. Pretreatment step, such as using acids or
salts, is necessary to increase the efficacy of enzymatic hydrolysis. However, the
study of hydrolysis with the presence of pretreatment residue is limited. In this
study, we aimed to compare the kinetics of two commercial cellulase cocktails:
Celluclast and Accellerase1500, with a presence of sodium chloride, an inorganic
salt using in pretreatment. The experiment was conducted on 3 substrates: CMC,
Avicel and sugarcane bagasse. The performance of enzymes was monitored in
terms of reducing sugar yields and the enzyme kinetics were calculated in terms
of Km and Vmax. Most of the cases, except hydrolysis of Avicel, reducing sugar
yields released from CMC and sugarcane bagasse increased under the influence
of 0.5 M and 1.0 M of NaCl. Compared to no-NaCl, sugarcane bagasse hydrolysis
by using Celluclast showed 24.15% increases of reducing sugars in the presence
of 1 M NaCl. Similarly, in Accellerase1500, reducing sugar yields released from
CMC and sugarcane bagasse were increased ranging from 12.47 to 61.48%, under
influence of NaCl. Moreover, under NaCl treatment, the kinetics of sugarcane
bagasse hydrolysis by using Accellerase1500 were significantly improved
compared to Celluclast, by reducing Km to 0.065 mg/ml and increasing Vmax to
0.248 mg/mlmin. These results indicated the impact of inorganic salt residues on
chemical and physical structures of different types of lignocellulosic biomass to
be vulnerable to enzymatic hydrolysis.

Keywords: Lignocellulose, Enzyme kinetics, Celluclast, Accellerase1500,
Sodium chloride

- 84 -

Anesthetic efficacy of vanilla essential oil for juvenile red tilapia
(Oreochromis niloticus-mossambicus) transportation

Benchapon Loima, Thirada Panukornpimol and Nalena Praphairaksit *
Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Transporting live fish from hatchery to pond or live harvested fish to the market
is a very important process of fish culture. In order to maintain its value, fish are
often anesthetized during the transportation process to reduce stress, pain and
mortality rate. In this study, the efficacy of vanilla essential oil was investigated
as an anesthetic for red tilapia (Oreochromis niloticus mossambicus). The fish
were exposed to concentrations of vanilla extract between 500-700 µL/L compared
with a chemical agent (MS-222) to evaluate the efficacy in inducing deep anesthesia
(stage 3) in red tilapia. The results showed that the optimal concentration of
vanilla essential oil was 700 µL/L which can induce anesthesia within 5 minutes
and the fish recovered within 10 minutes. In another experiment, fish transportation
was simulated for 2 hours to determine the effective concentration for fish
transportation. The results showed that the effective concentration of vanilla
essential oil to induce stage 1 anesthesia was 300 µL/L. There was no fish
mortality in all experiments. Moreover, there were no significant changes in water
quality, including DO, pH and temperature before and after the experiments.
Therefore, vanilla essential oil is an effective anesthetic agent for transportation of
juvenile red tilapia and can be recommended for transport of fish.
Keyword: Anesthetic, Vanilla essential oil, Red tilapia, Fish transportation

- 85 -

Genus diversity of phytoplankton in Koh Sichang, Chonburi

Apitchaya Chaijaroen, Surapich Ngernsri and Wisrutta Atthakor *
Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, 10110
* Project Advisor Email: [email protected]

Koh Sichang is an island that is located in Chonburi province. This island is one
of the most popular tourist attractions and commercial aquaculture in Chonburi.
Phytoplankton are organisms that live near the water surface and unable to
swim against a current. They serve as a primary producer at the base of aquatic
food webs in marine ecosystems. The impact on the phytoplankton can affect
diversity and ecosystem. Therefore, the objective of this study was to
investigate the genus diversity and density of surface phytoplankton around Koh
Sichang. It was carried out from October 2019 to January 2020. Phytoplankton
and coastal water samples were collected monthly from three stations for
analysis of phytoplankton diversity and physico-chemical parameters. Station 1
(13°09'01.6"N 100°49'01.0"E) was affected by wastewater from aquaculture.
Station 2 (13°08'44.2"N 100°48'24.3"E) was the most popular tourist attraction
in Koh Sichang while station 3 (13°07'40.6"N 100°48'42.9"E) had the lowest
level of anthropogenic activities. The results revealed that a total of 45 genera of
phytoplankton were encountered. All three stations were dominated by
Oscillatoria sp. The highest number of phytoplankton (91,783 cell/l) was
recorded at station 2 and the lowest number of phytoplankton (34,600 cell/l)
was recorded at station 1. The greatest value of species richness was 3.39 at
station 1. The lowest value of Shannon-Wiener diversity index and evenness
index were 1.66 and 0.45, respectively, at station 2. Stations 1 and 2 had the
greatest similarity index (0.94) and stations 2 and 3 had the lowest value of
similarity index (0.90).

Keywords: Diversity, Koh Sichang, Marine ecosystem, Phytoplankton

- 86 -