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Figure 1 Standard curve of the Bovine Serum Albumin solution
RESULT & CONCLUTION:
Regarding the study, the effect of feed formulations with local agricultural residues and products. The
study found that the larvae fed the mixed soybean meal formula (MIX SB) had the best specific growth rate
(31.45%), during the larvae fed the jasmine rice (WR) had the highest protein concentration and were followed by
the larvae that ate mixed riceberry (MIX RB) (36.96%) and (34.31%). While larvae fed the mixed Black glutinous
rice it has a protein concentration of 9.88%, which is less than others. Therefore, the selection of larvae that ate
jasmine rice had the highest amount of protein per body weight.
Figure 2 Comparison chart of protein concentration in different treatments
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Table 1 Daily single larva weight and daily growth rate
ACKNOWLEDGEMENT:
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abstract is not for citation.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:
ประสิทธ์ิ มานะพมิ พ.์ การศกึ ษาเปรยี บเทียบวธิ ีการหาปริมาณโปรตีนในแมงป่องช้างโดยวธิ เี จล์ดาหล์ และไบยูเรต .หลกั สูตรปรญิ ญาวิทยาศาสตร
มหาบณั ฑิต มหาวิทยาลยั อุบลราชธานี; 2549.
อไุ รวรรณ นิลเพช็ ร,์ ชนะ สรอ้ ยศร.ี การศกึ ษาปรมิ าณโปรตีนและไขมนั ของหนอนไหมวัย 5. มหาวทิ ยาลยั เกษตรศาสตร์ วิทยาเขตกำแพงแสน
นครปฐม; 2550.
Jaichansukkit, T, Sittigool, P, Moonsrikeaw, S, Somjaipang, A, Bootsee, P. A raise of black soldier fly
(Hermetia illucens) larvae feed by waste treatment for use as alternative protein source in animal feed. Faculty of
Agricultural Technology and Ago-industry Raja Mangala university of Technology Suvanarbhumi; 2020
Jittima Munkit. Using Black Soldier Fly Larval Meal as Ingredients in Common Climbing Perch (Anabas
testudineus Bloch) Dietary. The Journal of Institute of Agricultural, Sisaket College of Agriculture and
Technology ;2020
Liya Yi. A Study on the Potential of Insect Protein and Lipid as a Food Source. Technology, Agrobiotechnology,
Nutrition and Health Sciences, Graduate School VLAG; 2015.
Spranghers, T, Ottoboni, M, Klootwijk, C, Ovyn, A, Deboosere, S, Meulenaer, B.D, Michiels, J, Eeckhout, M,
Clercqb, P.D & Smeta, S.D. Nutritional composition of black soldier fly (Hermetia illucens) prepupae reared on
different organic waste substrates. J Sci Food Agric; 2017, P. 97: 2594–2600
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Title : Identification of extracellular enzyme-producing bacteria OB2_10_05
isolated from sediment of shrimp cultivation ponds
Field : Biology and Biodiversity
Author : Miss Manassikan Kiettichonkan
Miss Nattanicha Netamporn
Miss Nuttaya Lawpattarakasem
School : Kasetsart University Laboratory School Kamphaeng Saen Campus Educational Research and
Development Center
Advisor : Dr. Ratchanee Mingma, Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng
Saen Campus, Nakhon Pathom
Abstract : Shrimp farming in Thailand is one of the most popular aquacultures in many provinces. The
accumulation of various organic substances derived from waste (feces) and residual feeds can cause shrimp
disease and water pollution. The propose of this study was to investigate the identifiable extracellular enzyme-
producing bacteria from shrimp pond sediments. A total of 20 bacterial isolates were obtained and 19 isolates
had the ability to produce extracellular enzyme. There were 18, 11, 6, 10 and 6 isolates capable of producing
enzyme amylase, lipase, caseinase, gelatinase and cellulase, respectively. Amylase activities were frequently
found in bacterial strains, followed by lipase and gelatinase activities. Three bacterial isolates, KUSK64-04,
KUSK64-10 and KUSK64-15 had all five enzymatic activities. All bacterial isolates were identified by 16S
rRNA sequence analysis. The results revealed that they were classified to 4 genera within 10 different species.
Seven isolates belong to Pseudarthrobacter sp., 6 isolates belong to Bacillus sp., 5 isolates belong to
Sinomonas sp. and 2 isolates belong to Methylobacterium sp. These isolates can be further studied in
application in waste-water treatment in shrimp ponds.
Keywords : Extracellular enzyme; bacteria; shrimp pond sediment
Introduction
Shrimp farming in Thailand is an economically important aquacultures industry. Thailand is now one
of the world’s leading exports. High demand for shrimp leads to intensive farming, which can lead to bacterial
disease problems. High organic load due to residual feed, debris and fecal matter on pond bottom may also
cause water problems and diseases outbreaks such as Yellow Head disease, White Spot Disease (WSD) ,
Early Mortality Syndrome (EMS) , etc. To prevent bacterial disease and promote growth, antimicrobial
drugs are used such as tetracyclines, oxolinic acid and trimethoprim (1). The used of antimicrobial drugs
creates several negative effective effects on environment including bacterial antibiotic resistance and residue
present in seafood (2).
Bacteria have the ability to produce the extracellular enzymes that degrade organic waste, known as
heterotrophic bacteria. The decomposition process of the organic material consists of various types of bacteria
such as proteolytic, cellulolytic, amylolytic, lipolytic, nitrification and denitrification. The sediments from the
farmed shrimp came from the remaining of feed and feces which can stimulate the growth of heterotrophic
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bacteria. The Objective of this study was to isolate and identify bacteria found in the shrimp pond sediments.
The production of extracellular enzyme, including protease, amylase, lipase and cellulase were investigated.
Methodology
1. Sample collection and isolation of bacteria
Soil samples were collected from shrimp pond at Nakhon Pathom in November 2021. The samples
were air dried (not expose to sunlight) for a week before isolation of actinobacteria. One gram of soils was
serial diluted with sterile distilled water (10-3 - 10-6) and 0.1 mL of each dilution were spread on Nutrient agar
(NA) plate and incubated at room temperature for 2-3 days. The bacterial colonies were selected and streaked
on NA plates until the single and purified colony were obtained. The Gram status of the isolated bacteria were
determined by the microscopic examination of Gram-stained isolates.
2. Extracellular enzyme production
For detection of extracellular enzyme activity, the bacterial isolates were spot inoculated on tested
medium and incubated for 2-3 days. Skim milk agar (1%, w/v) and gelatin agar (0.5%, w/v) were used for
detection of proteolytic enzyme activity. The presence of clear zone surrounding the colonies on skim milk
agar were considered positive for caseinase production. For gelatinase detection, the colonies were flooded
with HgCl2 solution and the presence of clearing halo around the colonies were considered positive.
Amylolytic activity was tested on starch (1%, w/v) agar plates. Positive isolates were confirmed by
the presence of clearing halo around the colony after flooding with iodine solution.
Lipolytic activity was assessed on Tween80 (1%, w/v) agar plates. Development of precipitation zone
around the colonies were considered as positive for lipase production.
Carboxymethyl cellulose (CMC 0.5%, w/v) agar plates were used for screening of cellulase
producing isolates. After incubation, the colonies were stained with 1% Congo red solution for 15 minutes and
washed with 1 M NaCl. Presence of clear zone surrounding the colonies were considered positive.
3. Identification of isolates by 16s rRNA sequence analysis
Genomic DNA was extracted using i-genomic BYF DNA extraction mini kit (iNtRON
Biotechnology, Korea). Universal primers 27F and 1525R (3) were used for amplification of 16S rRNA gene.
The purified PCR products were sequenced commercially at Bionics company (Korea). The 16S rRNA gene
sequence was compared to other microbial sequences in Eztaxon-e database (4).
Results
1. Isolation and identification of bacteria
A total of 20 bacterial isolates were obtained and given the coded KUSK64-01 to KUSK64-21. After
Gram staining and observing under light microscope at 100X objective lens found that 18 isolates were rod-
shaped Gram-positive bacteria and classified to different 3 genera consisting of Bacillus sp. (6 isolates),
Sinomonas sp. (5 isolates) and Pseudarthrobacter sp. (7 isolates) based on 16S rRNA gene analysis. The
remaining 2 isolates (KUSK64-17 and KUSK64-18) were Gram-negative bacteria with rod-shaped cells and
classified to the genus Methylobacterium sp. within 2 different species (Figure 1).
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3 Isolates 1 Isolate
6 Isolates
Sinomonas halotolerans Sinomonas flava
Sinomanas mesophile Pseudarthrobacter enclensis
Pseudarthrobacter phenanthrenivorans Bacillus xiamenensis
Bacillus albus Bacillus altitudinis
Methylobacterium gregans Methylobacterium longum
Figure 1 Number of bacterial genera isolated from shrimp pond sediments.
2. Extracellular enzymes production
Out of 20 isolates, there were 19 isolates were found to produce at least one type of extracellular
enzyme. Amylase activities were frequently found in 18 bacterial isolates, followed by lipase (11 isolates) and
gelatinase activities (10 isolates). There were 10 isolates had abilities to produce gelatinase and cellulase
enzyme and 6 isolates had ability to produce enzyme caseinase. The enzyme production activities are shown
in Figure 2.
Figure 2 The clear zone of enzyme caseinase (a) gelatinase (b) cellulase (c) and precipitation zone of lipase
activity (d).
Conclusions
A total of bacteria 20 isolates were isolated from shrimp pond sediments. All of them had rod-shaped
cell with Gram-positive 18 isolates and Gram-negative 2 isolates. Most isolates except KUSK64-17 were able
to produce extracellular enzyme. There were 18, 11, 6, 10 and 6 isolates capable of producing enzyme amylase,
lipase, caseinase, gelatinase and cellulase, respectively. Based on 16S rRNA gene sequence analysis, 20
isolates were identified within 4 genera including Sinomonas sp. (5 isolates), Pseudarthrobacter sp. (7
isolates), Bacillus sp. (6 isolates) and Methylobacterium sp. (2 isolates) in different 10 species. Three isolates,
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KUSK64-04, KUSK64-10 and KUSK64-15 which were identified in the genus Bacillus sp. had all five
enzymatic activities. These isolates can be further studied in application in waste- water treatment in shrimp
ponds.
Acknowledgment
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. Holmström K, Gräslund S, Wahlström, A, Poungshompoo S, Bengtsson B-E, Kautsky N. Antibiotic use
in shrimp farming and implications for environmental impacts and human health. International Journal of
Food Science & Technology 2003;38: 255-266.
2. Binh VN, Dang N, Anh NTK, Ky LX, Thai PK. Antibiotics in the aquatic environment of Vietnam:
sources, concentrations, risk and control strategy. Chemosphere 2018;197: 438-450.
3. Lane DJ. 16S/23s rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds). Nucleic acid techniques in
bacterial systematics Wiley, New York, pp 115-175; 1991.
4. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. Introducing EzBioCloud: A taxonomically
united database of 16S rRNA and whole genome assemblies. International Journal of Systematic and
Evolutionary Microbiology 2017;67: 1613-1617.
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Title : Study on the antioxidant efficacy of crude extracts 12th SCiUS Forum
from some selected blue - green algae (Cyanobacteria)
OB2_03_02
Field : Biology
Author : Miss Kangsadan Chananithithum
Miss Pathita Phatharasiwawechkul
School : Naresuan University Secondary Demonstration School
Advisor : Assistant Professor Dr. Wittaya Tawong
Abstract
Nowadays, humans are facing with unhealthiness and environmental pollution. This has led people
turn to health care, such as choosing natural foods and supplemented with antioxidants. The purpose of this
study was to determine pigment characterization and content as well as to investigate the antioxidant activities
from four cyanobacterial species including Nostoc, Scytonema, Leptolyngbya and Arthrospira platensis. All
four studied species were extracted using freeze-thaw method. The crude extracts were determined pigment
characterizeation and DPPH inhibition (0.05 mM). The results showed that the pigments contained in each
crude extract of studied cyanobacteria were chlorophyll a, chlorophyll b, carotenoids, and phycobilins
(phycocyanin, allophycocyanin and phycoerythrin). Each species had different pigment content. The maximum
of total chlorophyll (1 2 2 . 2 mg/g), carotenoids (6 . 5 4 mg/g) and phycobilins (4 2 4 . 0 5 mg/g) content were
obtained Arthrospira, while Nostoc showed the lowest pigment content. The result of the antioxidant activities
of each crude extract from studied cyanobacteria showed that the best activity and IC50 values were obtained
in A. platensis with 7 5 % inhibition at 1 . 0 7 mg/ml when compared with vitamin C. This study indicated that
crude extracts from cyanobacteria had a significant potential to inhibited free radicals and can develop for
industries of natural algae products to reduce the import of synthetic substances in the future.
:Keywords blue – green algae, antioxidant
Introduction
Nowadays, human beings are faced with health problems caused by unhealthy food and
environmental pollution. When the body receives toxins, there will be an oxidative reaction in the body that
causes free radicals and cause various diseases such as cancer, heart disease, diabetes, premature aging, or
Alzheimer's. This has made people more aware of how to take care of themselves. Start consuming food from
nature and reduce the use of chemicals to prevent free radicals. But if chemically synthesized, safety must be
taken into account therefore, safe and non-toxic antioxidants are increasingly in demand.
Blue-green algae are prokaryotic organisms without organelles. They are very small and must
be studied under a microscope. It is classified in the phylum Cyanophyta Kingdom Monera. Blue-green algae
are among the best sources of nutrients and contain up to 70% protein by dry weight. And up to 18 amino acids
that the human body cannot produce by itself. It has a higher protein content than meat with valuable vitamins.
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Blue-green algae can also produce phycocyanin, the blue pigment that is the main component of
phycobiliproteins. Studies have shown that Phycocyanin is very useful in industry and medicine. It is used as
a natural color. It also has an antioxidant effect. Anti-inflammatory and prevent brain cells from being
destroyed (neuroprotective), for this reason, various types of blue-green algae are extracted for use in industrial
plants as a dietary supplement to prevent cancer-causing free radicals. Alzheimer's disease inflammation of the
body, including cosmetics industry.
In this study, four blue-green algae species were extracted by freeze-thaw methods to assess their
antioxidant efficacy. It will eliminate free radical DPPH. The results obtained from this study will be used as
the basis for the development of natural products from blue-green algae for food safety.
Methodology
The experiments were devided into 3 parts as follows,
Part 1: Preparation of Starting Material and Extraction Method
1.1 Prepare the starting material
Bring four species of blue-green algae Nostoc, Scytonema,
Leptolyngbya and Arthrospira platensis which were cultured in
the laboratory and dried at 40 °C for 24 hours and then placed in a
desiccator to keep the algae free from moisture. Weighed 5 mg algae
sample into a test tube and stored at a temperature of -20 ° C for further
experimentation.
1.2 Algae Extraction
The algae samples were extracted with two different solvents, phosphate-buffered saline, and
methanol under a 1:10 ratio (w/v) of the biomass to solvent. Then, the freeze-thaw method was used where the
extracted algae samples were frozen under -20 °C for 24 hours, then the extract was dissolved at room
temperature for 24 hours then the extraction was carried out with an ultrasonic machine for 20 min. After that,
the extract was precipitated with a centrifuge at 13,000 rpm for 5 minutes. Then the translucent was placed in
the test tube for further experimentation.
Part 2: Estimation of Phycobiliproteins, Chlorophyll and Carotenoids
2.1 Measure the composition and pigment content of algae extract.
A crude extract of algae was measured for absorbance at a wavelength of 250 to 750 nm using a
Spectrophotometer model UV-1900 UV-VIS. The absorbance values were then calculated for
phycobiliproteins, chlorophyll and carotenoids.
Part 3: Determination of antioxidant activity
3.1 Prepare a 0.05 mM DPPH solution.
Weighed 2 mg of DPPH, dissolved in
methanol. Adjust volume until 100 ml.
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3.2 Preparation of blue - green algae extract
Extract 1 ml of algae extract obtained from 1.2 with a micro-pipet, drop it into a test tube containing
1 ml of 0.05 mM DPPH solution, shake for 3 min, and store in a dark place for 30 min at room temperature,
and absorbance was measured at a wavelength of 517 nm using a microplate reader. Using methanol Replace
the sample solution with a blank solution. Then, the absorbance values were calculated to calculate the
antioxidant DPPH value compared to the standard vitamin C (positive control) and calculate the IC50 value.
Results
From the pigment characterize analysis of blue-green algae extracts to look at the absorbance
wavelength range and compare them with the absorbance wavelengths of different functional groups to
determine the product type. (Scytonema (A), Leptolyngbya (B), Nostoc (C) and Arthrospira (D))
The absorbance of chlorophyll a, b and total carotenoids The absorbance of Phycobiliprotein
It was found that pigment of Chlorophyll a, b, Carotenoid, Phycocyanin, Allophycocyanin and
phycoerythrin from the all 4 extracted. Arthrospira algae having the highest pigment content, contains total
chlorophyll carotenoids and phycobilin.
Chlorophyll content and the sum of carotenoids Phycobillin content of 4 algae species of 4 of
4 species
For antioxidant efficacy of extracted. The tables show the inhibitory concentration of 50% (IC50)
of 4 crude extracts and compared with the standard Ascorbic acid (vitamin C)
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Table 1 : Free radical inhibition DPPH extracted with methanol solvent.
Strains % Inhibition
%
Conc. (mg/mL) IC50 (mg/mL)
Scytonema 1.54 54.08 1.42
Arthrospira 1.61 75.49 1.07
Leptolyngbya 242.25 94.08 128.74
Nostoc 46.11 55.49 41.55
Ascorbic acid 0.005 84.06 0.003
It was found Arthrospira had the highest antioxidant activity at 75% when compared with vitamin C.
Conclusion
The characteristic pigment of blue-green algae extracted contained chlorophyll a, chlorophyll b,
carotenoids, phycocyanin allophycocyanin and phycoerythrin. It was found that all 4 algae species had different
pigment content, Arthrospira had the highest pigment content and also had the highest antioxidant activity at
75% when compared with vitamin C. Therefore, it was concluded that algae extracts contain important pigment
components that can inhibit free radicals.
Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS) under
Naraesuan University and Naresuan University Secondary Demonstration 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 . Eduardo Jacob-Lopes, Maria Isabel Queiroz, Leila Queiroz Zepka. Pigments from Microalgae Handbook.
Switzerland: Springer, Cham; 2019.
2 . Kim In Sung. Blue-green algae extract [Internet]. 2020. [ cited 2021 Sep 21] Available from :
www.zenbiotech.co.th
3. Machu et al. Phenolic Content and Antioxidant Capacity in Algal Food Products. [Internet]. 2015.
[cited 2021 Sep 21] Available from : https://www.researchgate.ne
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List of Science Projects 12th SCiUS Forum
Oral presentation
Biology and Biodiversity Group 3
Saturday August 27, 2022
No. Code Title Author School
1 OB3_16_03 Antioxidant and Antibacterial Mr. Sippakorn Petsirasan Demonstration School
Activities of Edible Flowers Prince of Songkla
Extracts and Flower Tea University, Pattani
Processing Campus
2 OB3_18_07 The effects of Mr. Thitikorn Hengsoontorn Surawiwat School,
Oligosaccharides on the Miss Nichkamol Klaitrakool Suranaree University of
growth of bacteria. Mr. Suphawit Manthongkaw Technology
3 OB3_09_03 Analysis of antioxidant Miss Pawarisa Ueramnoypornchok Engineering Science
capacity and inhibition of Miss Nutthapatsorn Jumroonjareet Classrooms
pathogenic bacteria growth in Mr. Pochara Dokprom (Darunsikkhalai School)
digestive system of
watermelon wine, pineapple
wine and orange wine from
free cell fermentation
4 OB3_06_05 Investigations of the solution Miss Chanapa Rachatasomboon Rajsima Witthayalai
structure of MurI for the Miss Tharnmattanee School
development of bacterial cell Thirathanabudsri
wall biosynthesis inhibitor Miss Itsaree Meksaengaroonrung
5 OB3_16_04 Production and adsorption Mr. Tanachai Jiaranai Demonstration School
properties of bacterial Miss Jarawee Rangsansarit Prince of Songkla
cellulose by University, Pattani
Komagataeibacter oboediens Campus
TISTR 2294 using tomato
juice medium
6 OB3_10_01 Screening of Thai red holy Mr. Nutjirapat Jirapimuggul Kasetsart University
basil cultivars for a Mr. Ratchakarn Jandorn Laboratory School
nutraceutical by bioactive and Miss Kusalin Somritchinda Kamphaeng Saen
Eugenol synthase gene Campus Educational
expression analysis Research and
Development Center
7 OB3_01_04 The Study of Antioxidant Mr. Rachata Yenglam Chiang Mai University
activity in Citrus Peel crude Mr. Pongsakorn Promming Demonstration School
extracts in UV B-induced
human skin keratinocytes
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No. Code Title Author School
8 OB3_04_05 Isolation and probiotics Miss Natchaya Panalikul Demonstration School
properties of lactic acid Miss Praechomphoo Phitaktim of Khon Kaen
bacteria from fermented University
foods
9 OB3_03_09 Study on prebiotic properties Miss Kankamol Thoopmongkol Naresuan University
of some mushrooms in Mr. Phatcharaphon Taksee Secondary
promoting probiotic growth Demonstration School
compared to Jerusalem
Artichoke
10 OB3_15_02 Comparison of the efficacy of Mr. Nuttakorn Na Thong PSU.Wittayanusorn
bacteriophages OPA17 and Mr. Ponrakun Tritsanawasuntra School
OTA22 for the removal of
Vibrio campbellii biofilm
11 OB3_17_01 The study of antimicrobial Miss Pathitta Charoenrak PSU Wittayanusorn
activity of turmeric for the Miss Sunruthai Boonchuay Surat Thani School
microorganisms causing
dermatitis
12 OB3_03_04 Study on antifungal efficacy of Miss Jiratchaya Moonsri Naresuan University
Senna alata (L.) Roxb. extract Miss Taraporn Netlawan Secondary
Demonstration School
13 OB3_10_04 Inhibitory Action of Thai Miss Muthita Pratuksakul Kasetsart University
Herb on The Growth of Mr. Thanabodee Charoenvuttitham Laboratory School
Pathogenic Bacteria Mr. Peerawat Phetsuth Kamphaeng Saen
Campus Educational
Research and
Development Center
14 OB3_01_02 Analysis of chemical Miss Nutravee Chullanan Chiang Mai University
compositions and antioxidant Miss Jiratthaya Phoomphant Demonstration School
activity in the extracts of Miss Thanisara Thongthong
curly kale crops harvested at
different time
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Title: Antioxidant and Antibacterial Activities of Edible OB3_16_03
Flowers Extracts and Flower Tea Processing
Field: Biology and Biodiversity
Author: Mr. Sippakorn Petsirasan
School: Demonstration School Prince of Songkla
University, Prince of Songkla University, Pattani Campus
Advisor: Dr. Somrak Panphon, Asst.Prof.Dr. Weeraya Khummueng, Department of Science, Faculty
Science and Technology, Prince of Songkla University, Pattani campus
Abstract
Edible flowers are potential sources of bioactive substances beneficial to human health. This
study aimed to determine the antioxidant and antibacterial properties of edible flowers and prepare them as
flower tea for healthy drinking. Dried flowers of Jasminum sambac, Rosa damascena, Mimusops elengi,
Osmanthus fragrans, Hibiscus rosa-sinensis, Ixora chinensis, Nelumbo nucifera, and Bougainvillea
hybrid were extracted with a 50% ethanol solution. The highest percentage yield of the extracts (29.87%) was
obtained from the Rosa damascene flower. The antioxidant profiles of flower extracts were determined using
the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and the highest one was found in Osmanthus fragrans with
161.88 ±1.17 mg TEAC/g extract. The antibacterial activity of flower extracts was tested against Bacillus
subtilis, Salmonella sp., Staphylococcus aureus, and Escherichia coli using the disc diffusion method. The
most active antibacterial effect was observed in Rosa damascene extract with an inhibition zone of 12.33 ±
3.38 mm on the Salmonella sp. plate. Based on antioxidant and antibacterial profiles, Rosa damascena,
Osmanthus fragrans, and Nelumbo nucifera were selected to make flower tea recipes and tested for their
antioxidant activity. Rosa damascena tea had the highest antioxidant activity of 36.62 ±0.39 mg TEAC/g tea.
Sensory evaluation performed using a 7 - point hedonic scale showed that the combination flower tea of Rosa
damascena and Osmanthus fragrans had the highest overall liking score (5.5). In addition, it had the L*, a*,
and b* color values of 97.11±0.02,-4.45 ±0.01, and 10.82 ±0.04, respectively.
Keywords: Antioxidants, Antibacterial, Flowers, Tea
Introduction
In recent times, more people are paying attention to their physical health and trying to maintain
a healthy lifestyle. Therefore, many activities are intended for good health, such as exercising and eating
healthy fruits and vegetables containing many antioxidants. Edible flowers have been known as a rich source
of valuable bioactive substances, which benefit human health. Therefore, they can be processed into various
flower products, especially flower tea. There are many varieties of flower tea products at present, but there
are little data on those tea products regarding their antioxidants or antibacterial profiles. Thus, the objectives
of this study were to investigate the antioxidant and antibacterial activities of edible flowers and prepare them
as flower tea. Moreover, antioxidant activity, color analysis and satisfaction test of tea recipes were also
evaluated.
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Methodology
Flower extraction and antioxidant and antibacterial profile testing
The flowers, including jasmine (Jasminum sambac), hibiscus (Hibiscus rosa-sinensis), damask rose
(Rosa damascene), osmanthus (Osmanthus fragrans), Spanish cherry (Mimusops elengi), sacred lotus
(Nelumbo nucifera), west Indian jasmine (Ixora chinensis), and bougainvillea (Bougainvillea hybrid) were
used in this study. Flowers were purchased at a flower shop in Chiang Mai, Thailand or via online markets.
Hibiscus and west Indian jasmine were picked on the Pattani campus of Prince of Songkla University. Dried
flowers were extracted using a 50% of ethanol solution in a 1:10 (w/v) ratio and stored in a dark place at 4°C
for two days. To obtain crude extracts, the extraction solvent was evaporated.
The DPPH assay was used to assess the antioxidant activity of flower extracts dissolved in methanol
at concentrations of 10, 25, 50, 75, and 100 ppm. The extract solution (300 μl) was mixed with 0.00008
mg/ml 2,2-diphenyl- 1-picrylhydrazyl (DPPH), adjusted to a volume of 1000 μl. The samples were stored in
the dark for 30 minutes before analyzing using a spectrophotometer at 515 nm. The % radical scavenging
activity was calculated as follows: % radical scavenging activity = [1-(Abs.sample/Abs.control)] x100, where
methanol is used as the control. The DPPH radical scavenging activity was then assessed. The disc diffusion
method was used to investigate the antibacterial potential of flower extracts against Bacillus subtilis,
Staphylococcus aureus, Salmonella sp. and Escherichia coli. The turbidity of the 24-hour old bacterial culture
in Mueller- Hinton medium was adjusted to the 0.5 McFarland standard and diluted to a concentration of 107
CFU/ml. After that, the bacterial suspension (100 μl) was swabbed all over it. Next, the paper disc (6 mm in
diameter) containing the extract at a concentration of 100 mg/ml (20 µl) was placed on the surface of each
inoculated plate. Chloramphenicol disc (30 μg) and 10% DMSO were used as positive and negative controls,
respectively. The plates were kept at 4°C for 1 hour and incubated at 37°C for 24 hours. The diameter of the
transparent zone of inhibition was measured in millimeters.
Preparation of flower tea from flowers with high antioxidant and antibacterial profiles and
satisfaction test
Damask rose, osmanthus, sacred lotus, and west Indian jasmine were chosen as flowers with
antioxidant and antibacterial properties. To prepare the tea, the dried flowers were blended first, and then 1 g
of the blended flowers was placed in teabags. Different tea recipes were made from these flowers. For tea
blend recipes, the flowers were blended in a 1:1 ratio. A tea sample was prepared by soaking a flower teabag
in a cup of hot water (150 ml) for 5 minutes. The antioxidant profiles of all the tea samples were then evaluated.
The L*, a*, and b* color values of teas were determined using a colorimeter. Satisfaction testing was done to
measure the color, aroma, flavor, and overall liking of tea samples. The hedonic scale was employed, with 1
indicating strong dislike, 2 indicating moderate dislike, 3 indicating minor dislike, 4 indicating indifference, 5
indicating slight liking, 6 indicating moderate liking, and 7 indicating strong liking. The tea product
satisfaction test included 15 untrained participants.
Results, Discussion and Conclusion
The highest yield crude extract (29.87%) was obtained from Rosa damascena while the lowest yield
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(20.52%) was found in the extract from Hibiscus rosa-sinensis. The results of antioxidant profiles from
flower extracts, as shown in Table 1, the highest value was found in osmanthus (161.88 ±1.17), followed by
west Indian jasmine (161.15 ±1.44), sacred lotus (160.26 ±3.27), and damask rose (159.57±1.70), which has
the same high antioxidant profiles as the other results (Mao et al., 2017). Statistical analysis revealed that no
significant difference was observed in group of Rosa damascena , Ixora chinensis, Nelumbo nucifera and
Osmanthus fragrans. Table 2 shows the results of the antibacterial profile analysis. None of the flower
extracts had any inhibitory effects on Escherichia coli growth. As a result, it is more resistant to the extracts,
but if the flower extracts are concentrated enough, it may be inhibited. Other antibacterial inhibitory profiles,
such as the antibacterial tests of Rosa damascena extract, are also consistent with previous research results
(Ramdan et al., 2021). Table 3 shows the antioxidant profiles, pH, and color of the tea preparations. In groups
of samples, there were significant differences (P<0.05). Damask rose tea (36.62 0.39) had the highest
antioxidant activity, followed by a floral tea blend of three types of flowers (35.81 0.96), sacred lotus tea
(35.38 0.51), and osmanthus combined with sacred lotus tea (35.18 0 29). The color values of tea samples
were significantly different in L* a* and b* values (P< 0.05). The tea samples had higher L* values,
indicating that they were lighter. A 7-point hedonic scale was used to assess the preference of seven tea
samples prepared from three flowers: damask rose, osmanthus, and sacred lotus. According to a notification
from Thailand's Ministry of Public Health, West Indian jasmine is not on the list of plants that can be used
to make tea. The 15 participants were divided into two age groups: 9 were between the ages of 20 and 29,
and 6 were between the ages of 30 and up. Results showed that osmanthus mixed with sacred lotus tea,
damask rose tea, and osmanthus mixed with sacred lotus tea were the teas with the highest preference in
color, aroma, and flavor, with scores of 6.75, 5.88, and 5.75, respectively, in the first group. The teas with
the highest preferences in color, aroma, and flavor among the six participants aged 30 and up were damask
rose tea (5.80), tea blend of three flowers (6.20), and osmanthus tea (6.00). Moreover, damask rose mixed
with osmanthus tea received the highest overall liking from the participants (n =15), with a score of 5.5.
Statistical analysis results were expressed as mean ± SD from triplicate samples. The results were analyzed by
ANOVA (one-way), followed by Duncan. Statistical significance was defined as P < 0.05.
In conclusion, edible flowers are natural sources of antioxidant and antibacterial properties.
Osmanthus fragrans, Rosa damascena, Ixora chinensis, and Nelumbo nucifera have high antioxidant activity.
These flowers also have antibacterial activity. Floral tea recipes with antioxidants can be created to meet the
needs of people in a healthy way. These results support the use of edible flowers as functional foods or
healthy drinks. In addition, research into other edible flowers should also be investigated.
Table 1 Antioxidant activity of flower extracts
Flower mg TEAC /g extract Flower mg TEAC /g extract
Jasminum sambac 66.17 ± 4.82e Hibiscus rosa-sinensis 153.96 ±0.64b
Rosa damascena 159.57 ±1.70a Nelumbo nucifera 160.26 ±3.27a
Mimusops elengi 90.64 ± 2.02d Bougainvillea hybrid 136.92 ±5.89c
Ixora chinensis 161.15 ±1.44a Osmanthus fragrans 161.88 ±1.17a
Different letters in the same column indicate significant difference (P <0.05).
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Table 2 Antibacterial activity of flower extracts
Flower extracts Inhibition zone size (mm)
Bacillus subtilis Salmonella sp. Staphylococcus aureus Escherichia coli
N/A
Rosa damascena 9.75 ±0.66 12.42 ±0.63 12.33 ±0.38 N/A
N/A
Ixora chinensis 7.92 ±0.63 8.75 ±0.50 7.58 ±0.14 N/A
29.6
Nelumbo nucifera N/A N/A 8.38 ±0.53 N/A
Osmanthus fragrans N/A N/A 10.63 ±0.53
Chloramphenicol (C) 21.5 31.6 33.5
10% DMSO N/A N/A N/A
*N/A ( Not available)
Table 3 Antioxidant activity, pH and color parameter of flower tea recipes
Tea recipes mg TEAC/g tea pH L* a* b*
17.11 ±0.02e
Damask rose 36.62 ±0.39ab 5.740.05 b 95.32 ±0.01f -0.62 ±0.01d 20.49 ±0.03c
3.67 ±0.01h
Osmanthus 35.09 ±0.84c 5.590.05 c 98.56 ±0.19b -7.36 ±0.01g 28.54 ±0.04b
10.82 ±0.02f
Sacred lotus 35.38 ±0.51c 6.280.02 a 98.96 ±0.10a -0.17 ±0.01b 23.68 ±0.03b
18.64 ±0.01d
Damask rose + Osmanthus 34.77 ±0.45c 5.790.04 b 97.11 ±0.02g -4.45 ±0.01f
4.46 ±0.01g
Damask rose + Sacred lotus 34.85 ±0.80c 5.600.05 c 97.58 ±0.02d -0.51 ±0.00c
Osmanthus + Sacred lotus 35.18 ±0.29c 5.730.05 b 97.58 ±0.03c -7.61 ±0.01h
Damask Rose + Osmanthus + 35.81 ±0.96bc 5.580.04 c 96.09 ±0.01e -3.06 ±0.01e
Sacred lotus 4.530.07 d 93.98 ±0.01h 9.52 ±0.01a
West Indian jasmine 37.27 ±0.27a
Different letters in the same column indicate significant difference (P <0.05).
Acknowledgements
First of all, I would like to pay our gratitude to my advisor, Dr. Somrak Panphon, and Asst.Prof.Dr.
Weeraya Khummueng for guidance and lots of support to complete the project. Notably, there would not
have been this project without the opportunity they had given to do this project. Furthermore, this project
would not have been completed if there had not been financial support from Science Classrooms in
University-Affiliated School supported by Ministry of Science and Technology.
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. Ramdan B, Mrid R.B, Ramdan R, Karbane M.F,Nhiri M. Promising effects of Rosa damascena
petal extracts as antioxidant and antibacterial agents. Pakistan Journal of Pharmaceutical
Sciences.2021; 34,001-008.
2. Mao S, Wang K, Lei Y, Yao S, Lu B, Huang W. Antioxidant synergistic effects of Osmanthus
fragrans flowers with green Tea and their major contributed antioxidant compounds. Scientific
Reports. 2017;7(1).
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Title The effects of Oligosaccharide on the growth of OB3_18_07
bacteria
Field Biology and Biodiversity
Author 1 Thitikorn Hengsoontorn
2 Nichkamol Klaitrakool
School 3 Suphawit Manthongkaw
Advisor Surawiwat School, Suranaree University of Technology
Prof Pharm Dr Montarop Yamabhai
Abstract
Oligosaccharides containing 2 10 single molecule sugar are beneficial for the growth of good
microorganisms and inhibit the growth of pathogenic microorganisms In this study, we studied the effects of
chitooligosaccharides CHOS and manno oligosaccharide MOS on the growth of such pathogens and lactic
acid bacteria CHOS was produced by enzymatic digestion and analyzed by Thin layer chromatography TLC,
it was found that CHOS was produced after 48 hours The effects of CHOS and MOS on the growth of bacteria
on solid mediums were investigated A disc diffusion method was used to determine the effect of CHOS and
MOS on bacterial growth Escherichia coli and Lactobacillus plantarum were tested on LB Agar and MRS
agar Oligosaccharides concentrations between 10 mg ml to 200 mg ml, ampicillin and sterile distilled water
were used and incubated at 37 °C for 20 h The results showed that there are no effects on stimulating or
inhibiting the growth of bacteria on the solid medium Therefore, the effect of CHOS and MOS on bacterial
growth in the liquid medium were studied E coli was cultured in LB Broth and M9 medium, while L plantarum
was cultured in MRS broth CHOS and MOS were used as a carbon source at concentrations of 2, 4 and 8 ,
and cultured at under anaerobic conditions The turbidity of the cell was measured using a microplate
reader every hour for 24 h The results showed that CHOS and MOS were not able to inhibit E coli growth but
able to stimulate L Plantarum growth
Keywords Chitooligosaccharide, Mannooligosaccharide, bacterial growth, Escherichia coli, Lactobacillus
plantarum
For internal use in the 12th SCiUS Forum only Not for citation
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Introduction
Nowadays, people are increasingly interested in health care and wellness By studying the
application of bioactive substances derived from nature for health care benefits It is one of the topics that many
researchers are interested in today This has resulted in the study of various bioactive compounds as well as the
research team with interest in the oligosaccharide group In this project The researcher is interested in studying
the bioactive properties of chitooligosaccharides and manno oligosaccharides Due to the literature review, it
was found that previous studies reported that Both substances have prebiotic properties and can inhibit the
growth of certain groups of bacteria But in the production of such substances which normally uses acid to
produce and extract substances As a result, the substance has unstable properties Until there was a study of
production by using enzymes to digest polysaccharides into chichitooligosaccharidesnd specific mannose
glycosaccharides instead of the acid making the substance have more stable properties and can be applied in
more different dimensions The research team was interested in studying the activity of Chito
oligosaccharides and Manno oligosaccharides extracted from enzymes on the growth of gram positive and
gram negative bacteria trying to understand effects of COS and MOS on the growth of bacteria
Methodology
1 CHOS production
Chitosan was dissolved in 1 Hydrochloric acid then, adjust the pH with 5 M NaOH to get a pH of
5 5 The purification chitosanase enzyme was added to the chitosan solution to the final concentration of 5 U ml
The samples were incubated at 37 and 200 rpm then, collected at 0,6,24 and 48 hours and visualized by thin
layer chromatography TLC After 48 hrs the samples were centrifuged at 4,000 rpm for 10 minutes and freeze
dried Yamabhai, Buranabanyat, Jaruseranee, & Songsiriritthigul, 2011 Pechsrichuang et al , 2018
2 The growth of bacteria on agar media
10 microliter of COS and MOS concentrate between 10 200 mg ml, ampicillin, and sterilized water
werdroppedop on sterile filter DISC The DISC were plated on LB agar that had already spread with E coli
and MRS agar with L plantarum for 20 hours The diameter of the clear
zone was measured
3 The growth of bacteria in liquid media
The probiotic bacteria L plantarum growth was grown in MRS basal media under aerobic
overnight The cell was The cell pellet was
resuspended in 0 85 normal saline containing OD600 0 50 20 microliters of the resuspended cell were
added into 180 MRS with 2 , 4, and 8 w v MOS and COS and incubated at 37ºC under anaerobic
conditions The pathogenic bacteria E coli grown in LB broth and M9 minimal media with the similar
condition The turbidity of the cells was measured using the microplate reader every 1 h for 24 h
Results, Discussion, and Conclusion
Result
1 Result of CHOS production
Analyzing products by thin layer chromatography TLC using chitosan substrates was performed
to determine the hydrolytic activity of purified chitosanase The samples were collected at 0, 6, 24, and 48
hours The major CHOS products are disaccharides and trisaccharides Fig 1
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Figure 1 TLC analysis of hydrolytic products at different time points
2 Result of The growth of bacteria on agar media
The effect of CHOS and MOS on the growth of E coli and L plantarum were tested on LB agar
and MRS agar, respectively 10 uL of CHOS and MOS concentrations between 10 to 200 uL mL were dropped
on the sterilized filtered disc and placed on agar plates Ampicillin and sterilized deionized water were used as
the positive and negative control, respectively The results
showed that there is no effect on the growth of E coli Fig 2A and L plantarum Fig 2B and the clear zone
inhibition of oligosaccharides were measured Table 1
Table 1 Clear zone diameter on agar media
3 Result of The growth of bacteria in liquid media
The effect of COS and MOS on the growth of bacteria in a liquid medium E coli was cultured in
LB Broth and M9 medium L plantarum was cultured in MRS Broth medium be normal medium, non
carbonized medium, and COS and MOS are added to be carbon source at different concentrations
Inoculation growth was monitored by turbidity measurements at 595 nm every hour, it was found that E coli
was unable to grow in all M9 mediums Fig 3 The growth of E coli in the LB medium showed that COS and
MOS were not inhibiting the growth of E coli Fig 4 and it was found that COS and MOS were able
topromote the growth of L Plantarum in the MRS medium Fig 5
Figure 4 The h E coli in LB media with and without interested oligosaccharides
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Figure 5 The growth of L plantarum in MRS media with and without interested oligosaccharides
Discussion
The oligosaccharides studied were CHOS and MOS It inhibited the growth of E coli and stimulated
the growth of L plantarum from the experimental results It was found that from the second experiment, the
effect of oligosaccharides on the growth of bacteria on solid media was found Both CHOS and MOS were
found to have no incidence or inhibition of bacterial growth on solid media since oligosaccharides are often
used by bacteria as carbon sources However, testing on the solid medium is therefore not appropriate to
study the activity of oligosaccharides on bacterial growth
The effect of oligosaccharides on bacterial growth in liquid medium It was found that CHOS and
MOS did not inhibit the growth of E coli, but had a stimulating effect on the growth of L
plantarum because L plantarum was able to use oligosaccharides as a carbon source when compared to the
negative control
This is consistent with previous research that has used MOS to test the growth of pathogens and
probiotics The samples were cultured with Salmonella and L rueteri Ariandi, Yopi, & Meryandini, 2015
Therefore, it can be said that COS and MOS have prebiotic properties
Conclusion
Chitooligosaccharide was produced for testing the growth of bacteria Since COS and MOS were
unable to inhibit the growth of E coli however, CHOS and MOS at a concentration of 8 is the appropriate
concentration to promote the growth of L plantarum We would like to culture E coli and L plantarum
together to see the effects of synbiotics in the future
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
References
1 Ariandi, Yopi, & Meryandini, A 2015 Enzymatic Hydrolysis of Copra Meal by Mannanase from
Streptomyces sp BF3 1 for The Production of Mannooligosaccharides HAYATI Journal of
Biosciences, 22 2 , 79 86 doi https doi org 10 4308 hjb 22 2 79
2 Pechsrichuang, P , Lorentzen, S B , Aam, B B , Tuveng, T R , Hamre, A G , Eijsink, V G H , &
Yamabhai, M 2018 Bioconversion of chitosan into chito oligosaccharides CHOS using family
46 chitosanase from Bacillus subtilis BsCsn46A Carbohydr Polym, 186, 420 428
doi 10 1016 j carbpol 2018 01 059
3 Yamabhai, M , Buranabanyat, B , Jaruseranee, N , & Songsiriritthigul, C 2011 Efficient E coli
mannanases and other bacterial extracellular
enzymes Bioengineered Bugs, 2 1 , 45 49
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Title : Analysis of antioxidant capacity and inhibition of OB3_09_03
pathogenic bacteria growth in digestive system of
Field :
Author : watermelon wine, pineapple wine and orange wine
School : from free cell fermentation
Advisor :
Biology and Biodiversity
Ms. Pawarisa Ueramnoypornchok
Ms. Nutthapatsorn Jumroonjareet
Mr. Pochara Dokprom
Darunsikkhalai School Engineering Science Classroom ( KMUTT )
Mr. Witsanu Supandee
Abstract :
Wine is a type of alcoholic beverage made by fermenting various kinds of fruits. Nowadays, the
wine gains popularity among alcoholic beverages due to its fine taste and awesome fragrance. Wine is also
beneficial to your health if drank in a moderate amount. This study has an objective to studies and compares
the amount of antioxidants and inhibition of pathogenic bacteria growth in digestive system of watermelon
wine, orange wine and pineapple wine from free cell fermentation. The study on the amount of antioxidants
and inhibition of pathogenic bacteria growth in digestive system of watermelon wine, orange wine and
pineapple wine was obtained by culturing bacteria E. coli O157:H7, Salmonella Typhi and Shigella dysenteriae
and using the agar well diffusion method to test. As a result, all these three wine can not inhibit the pathogenic
bacteria growth in digestive system. Whilst, the amount of antioxidants that used DPPH radical scavenging
assay to determine, found that the antioxidants trend of the sample was increasing. According to studies and
comparisons, the highest antioxidant was orange wine, because orange has the highest vitamin C content among
the fruit that we have chosen. In summary, the knowledge we gained from this study can be further developed
to help farmers and gardeners earn more from agriproduct processing and to find the most efficient way to use
the available resources.
:Keywords Free Cell / DPPH / Antioxidant / Inhibition of pathogenic bacteria growth in digestive system
Introduction
Wine is a type of alcoholic beverage originated in 7,000 BC. Wine is a type of alcoholic
beverage made by fermenting between various kinds of fruits and yeast. Wine was originally fermented using
rice, honey and fruit for the purpose of preserving food. After the Greek civilization expanded in the territory,
wine was used as an important part of various religious ceremonies as well as an important commodity that
facilitates the dissemination and exchange of culture and knowledge. Nowadays, the wine gains popularity
among alcoholic beverages due to its fine taste and awesome fragrance. Wine is also beneficial to the health if
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drank in a moderate amount, such as the amount that help antioxidants and inhibit the growth of pathogenic
bacteria in digestive system. Nowadays, the most common and widely available wine is grape wine. Therefore,
we are interested in trying to use different ways and more different fruits to fermenting wine to studies and
compares the amount of antioxidants and inhibition of pathogenic bacteria growth in digestive system.
For the fruits that we choose to use are watermelons, pineapples and oranges, since we have an
objective to further developed to help farmers and gardeners earn more from agriproduct processing and to
find the most efficient way to use the available resources.
Methodology
Part 1: Wine fermentation
1.1 Clean watermelon, orange and pineapple and peel it. Then blend the watermelon, orange and
pineapple and filter it by thin cloth. Add water to watermelon juice and pineapple juice in 1 : 1 ratio each and
add to orange juice with 1:2 ratio. Then heat watermelon juice, orange juice and pineapple juice at 63°c for 30
minutes to sterile.
1 . 2 Culture Saccharomyces cerevisiae on yeast malt agar(YMA). Then incubate at 2 5 ° c for 2 4
hours. Scrape a colony. Put in watermelon juice flask, orange juice flask and pineapple juice flask. Incubated
at 25°c for 24 hours. Then find absorbance by spectrophotometer at 600 nm with absorbance value at 1.000
1.3 Adjust sugar in watermelon juice, orange juice and pineapple juice to 20° Brix and adjust pH
to 6.0-6.5. Keep watermelon juice, orange juice and pineapple juice for 1500 ml each and separate 5% of each
juice (75 ml) in small flasks.
1.4 Scrape 1 loop of Saccharomyces cerevisiae colony from YMA into each small flasks. Incubate
at 25°c for 24 hours then measure the absorbance to OD=1.0000 at 600 nm. Mix back together with the 1500
ml flask of its batch.
1.5 Devied each juices into 500 ml flask for 3 flasks each. Incubated at 2 5 ° c and collect sample
on day 0, day 4, day 8 and day 12 to compare %Brix, %alcohol and pH.
Part 2: Antibacterial test
Cultured bacteria E. coli O1 5 7 : H7 , Salmonella Typhi and Shigella dysenteriae in Brain Heart
Infusion Broth (BHI broth). Incubated at 3 7 ° c for 1 8 - 2 4 hours. Adjust the amount of bacteria to be turbid
equivalent to mcfarland no. 0 . 5 standard solution. Then use sterile cotton swab sticks Dip into cultured tube.
Swap it on BHI Agar and wait until dry. Drilled BHI Agar for 5 holes with 5 cm diameter. Drop watermelon
juice, orange juice and pineapple juice in each hole. Add sterile water and antibiotic gentamicin concentration
10 ml/mL into the other two holes to be controle set. Incubated at 37°c for 24 hours. Recorded the results by
measure the clear zone diameter of the inhibition.
Part 3: Antioxidant test
Prepare 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) concentration 0.1 mM by dilute in 99.9%
methanol as solvents and prepare 0.5 ml of watermelon wine, orange wine and pineapple wine. Mixed diluted
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DPPH 1.5 ml with each wine in test tube. Incubated at 25°c for 20 minutes. Then measure of absorbance at
517 nm and Calculate DPPH inhibition (%)
Results and DiscussionBrix (°)
From the experiment, wine made from 3 selected fruits which are watermelon, pineapple and orange
has a different quality from wine fermenting based on the juice that was used.
The result we have obtained shows that the Brix trend has decreased in each wine. On day 12 , the
finished day of the fermenting period, the wine that has the highest Brix degree is orange and pineapple wine which
is 6 degrees Brix. On contrary, the wine that has the lowest Brix degree is watermelon wine which is 5.83 degrees
Brix.
20
10
0
0 4 8 12
Fermentation time (days)
Watermelon Pineapple Orange
Figure 1 : Trend of %Brix in fermenting watermelon juice, pineapple juice and orange juice
The pH trend of these wines is also decreasing. On day 12, the wine that has the highest pH is orange
wine which is 3.5 and the wine that has the lowest pH are watermelon and pineapple wine which is 3.4.
On the alcohol concentration part, we found that the result contradicts the theory. On day 0, the wine
has a 50-60% alcohol concentration. In contrast to the theory, which is supposed to have no alcohol concentration.
This made the alcohol concentration trend decrease too. On day 1 2 , the wine that has the highest alcohol
concentration is pineapple wine which is 15% (v/v). On contrary, the wine that has the lowest pH are watermelon
and orange wine which is 14% (v/v).
6 100
4
2 50
0
0
0 4 8 12 0 4 8 12
Fermentation time (days)
Fermentation time (days)
Watermelon Pineapple Orange
Watermelon Pineapple Orange
pH
%Alcohol (%, v/v)
Figure 2 : Trend of pH in fermenting watermelon juice, Figure 3 : Trend of %Alcohol in fermenting watermelon
pineapple juice and orange juice juice, pineapple juice and orange juice
Antibacterial activity result shows that none of the 3 wine can inhibit the growth of pathogenic bacteria in the
digestive system. The alcohol concentration in our wine that has only 14-15%, while inhibiting the bacterial growth
requires up to 70%.
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E E E
A A AD C
D D (3) Shigella dBysenteriae
(1) E. CoBli O15C7:H7 (2) SalmBonella TCyphi
From sample of fruit wines fermented by Saccharomyces cerevisiae for 12 days
A = watermelon wine B = orange wine C = pineapple wine
D = gentamicin 0.1 mg/ml E = distilled water
Figure 4 : Characteristics of the formation of clear zone,
inhibition of the growth of
Antioxidant capacity in orange wine is higher than in watermelon wine coinciding with vitamin C
capacity in the fruit. Among the selected fruits, orange has the highest vitamin C capacity at 53.2 mg, followed by
pineapple at 47.2 mg and watermelon at 8.1 mg. Which in line with the research of Earl (2021) that studies the
relation between antioxidant capacity and vitamin C capacity.
Conclusion
As a result of the experiment, it was concluded that when the three fruits, watermelon, pineapple,
and orange, were fermented, and then fermented wine was completed on time for quality inspection, the ethanol
content of the three wines showed an abnormal decreasing trend, and %Brix and pH showed a consistent
decreasing trend.
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. Panitta Punyadilok et al. Wine fermentation process. KNOLEDGE GUIDE 2013;6:3-26.
2. Pornphan Puaphaiboon. Conduct research on wine. ANALYSIS OF ANTIOXIDANT CAPACITY OF
WINE PRODUCED FROM RINDS AND CORES OF THE FRUITS 2006;1:21-43.
3. Supranee Lauhkitikul et al. Yeast strains and appropriate ratio for making Jambolan plum wine [Internet]. date
unknown [cited 21 Jun 2021]. Available from: http://herp-nru.psru.ac.th/file/O54534_11.pdf
4 . Anu Appaiah. All about fruit wines. DEVELOPMENT OF INDEGIENOUS YEAST FOR WINRE
PRODUCTION 2010;11:3-24.
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Title : Investigations of the solution structure of MurI for the OB3_06_05
development of bacterial cell wall biosynthesis inhibitor
:Field Biology and Biodiversity
:Author Chanapa Rachatasomboon, Itsaree Meksaengaroonrung and Tharnmattanee Thirathanabutsri
:School Ratchasima Witthayalai School, SCiUS-Suranaree University of Technology
:Advisor Dr. Sakesit Chumnansilpa, School of Biochemistry, Suranaree University of Technology
Abstract
Multidrug-resistant bacteria are one of the most serious threats to human health. Antibiotics used to
treat bacteiral infections are relatively toxic and frequently cause unwanted side effects. The bacterial cell
wall is a highly cross‐linked polymeric structure and it has been known that the Mur-complex enzymes are
crucial components of the bacterial cell wall biosynthetic pathway. Inhibiting these enzymes is expected to
cripple this process leading to weakening of the cell wall and subsequent bactericidal activity. Therefore,
Mur-complex enzymes are attractive targets for the development of new antibiotics. It is the aims of this
study to investigate the solution structure of MurI for paving the way to the development of bacterial cell
wall biosynthesis inhibitor. In this study, the murI gene from P. aeruginosa ATCC27853 was cloned into the
modified pET-21d(+) expression vector. MurI was expressed in E. coli BL21 (DE3). Immobilized metal
affinity chromatography was used to purify the protein, which was then followed by gel filtration
chromatography. We tried protein crystallization in various crystallization conditions. Unfortunately, the
protein crystals diffracted poorly in X-rays. So, we used SAXS to investigate quaternary structural data.
After exposing the protein to X-rays, it was shown that the protein was heavily aggregated in standard
protein purification buffer. Aggregation was not reduced by glycerol or Triton X-100. Metal ion (Na+)
supplementation, pH (7.0), and salt concentration (2 mM). We found that a 4 mM concentration of L-
glutamate reduced protein aggregation. To determine the oligomeric state, the molecular mass of MurI in
solution by SAXS reveals and supports that MurI is a monomer with a MW of 34 kDa, Rg = 2.71 nm,
Dmax = 8.78 nm, and a predominantly globular shape. Furthermore, PaMurI exhibits conformational changes
in solution, as shown by CRYSOL analysis. The structural characterization of MurI as oligomerization and
conformation can support researchers in the discovery and development of specific bacterial cell wall
biosynthesis inhibitors, reducing bacteria's ability to evolve drug resistance.
Keywords: Multi drug resistant (MDR), MurI (Glutamate racemase), Small angle X-ray scattering (SAXS),
Bacterial cell wall biosynthesis
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Introduction
The use of antibiotics to treat microbial infectious diseases represents one of the most important
advances in modern medicine. However, bacterial resistance has become a serious issue as a result of the
obvious, widespread use and abuse of antibiotics. The spread of multidrug-resistant bacteria has posed a very
serious threat to human health. As a result, establishing antibacterial resistance measures and developing
new antibacterial medications is becoming increasingly important to better managing infections.
Pseudomonas aeruginosa is a gram-negative bacterium that becomes one of multidrug resistant
bacterias. Additionally, antibiotics have a difficult time penetrating the layer of its peptidoglycan. This study
was therefore, has a pressing need to identify a new target for againts its peptidoglycan, a major component
of their cell walls.
Glutamate racemase or MurI is a cofactor independent enzyme responsible for the racemization of
L-glutamate to D-glutamate which is an importance component of the peptidoglycan biosynthesis of the
bacterial cell wall. Certain bacteria synthesise D-glutamate exclusively via glutamate racemase, and the
structural gene for this enzyme, murI, is required for growth. Importantly, MurI is absent in humans, hence,
the approach in this study was to investigate the solution structure of MurI, for paving the way to the
development of bacterial cell wall biosynthesis inhibitor.
Methodology
The murI gene from P. aeruginosa ATCC27853 (PaMurI) was cloned into modified pET 21d(+)
vector (pSY5), to be expressed inframe with N-terminal 8xhis-tag. The cultures were grown and collected by
centrifuging. Then, Cell pellets containing PaMurI disrupted by sonication. The cell was removed by
centrifugation and the supernatant was incubated with pre-equilibrated nickel-NTA metal affinity resin. The
non-specifically bound protein was removed by washing buffer and eluted by using elution buffer. Then
purified and analyzed the homogeneity by gel filtration chromatography. The eluent fractions corresponding
to the major peak observed in chromatogram were collected, pooled and concentrated in protein buffer. Each
concentrated protein fraction was analyzed molecular weights by SDS-PAGE. The initial crystallization
screening was performed by the microbatch under-oil method. The crystal growing was observed under the
microscope for every 7 days. Finally, SAXS data from solution of PaMurI were performed at Synchrotron
Light Research Institute (SLRI). The scattering data was analyzed by the program ATSAS. Scattering pattern
of the crystal structures of MurI in different stages was calculated by program CRYSOL and molecular weights
were determined using PRIMUS.
Results and Discussion
Cloning and expression
PaMurI construct were overexpressed in E. coli as 28 kDa
His-tagged protein. The protein was purified by affinity 31 kDa
chromatography, PaMurI were highly soluble.
Purification Fig.1 Fusion protein map of PaMurI
Initial SEC on an S75 10/300 GL column indicated that PaMurI showed a single peak from the
chromatogram of greater than 95% purity protein (Fraction 29-33) as determined by SDS-PAGE.
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M
M B
M
Fig.2A Gel filtration chromatography of PaMurI. Fig.2B SDS-PAGE analysis of eluted fractions from Gel filtration chromatography.
Protein molecular weight marker with 29-33 kDa are eluted fractions corresponding to the major peak
Crystallization screening of PaMurI
Precrystallization test suggested that 4 mg/ml
PaMurI is suitable for crystallization screening. The small
crystal protein was able to crystallize in 22% PEG
5000MME, 100 mM HEPES, pH 7.0 (Fig.4A) and in 27%
PEG 5000MME, 100 mM Mes-NaOH, pH6.2 (Fig.B). Those
crystals were too small for the X-ray diffraction. Fig.3 Initial screening of crystallization conditions of
PaMurI, with equal volumes of protein and precipitation
Small angle X-ray scattering (SAXS)
The protein was severely aggregate in standard protein purification buffer (Fig.A), suggested that
the protein was unstable and very sensitive to the X-ray. Glycerol, Triton X-100, and supplement of metal
ions (Na+) did not reduce the protein aggregation (Fig.B). However, we found that L-glutamate
concentration (4 mM) reduced the protein aggregation (Fig.C) as observed in Guinier region (red square).
A BC
Fig.4A An X-ray scattering profile of PaMurI pooled of fraction 29-33 from gel filtration chromatography. PaMurI in protein
buffer. Fig.4B PaMurI in protein buffer and difference additives. Fig.4C PaMurI in protein buffer and 10X L-glutamate.
Oligomeric state determination of PaMurI by SAXS
The protein samples (4, 6.5, and 10 mg/ml) used to determine whether there is any concentration-
dependent intermolecular interaction. PaMurI gave normal scattering profiles with no sign of aggregation.
Additionally, a Kratky analysis was conducted PaMurI exhibits a bell shape curve, indicating compact globular
fold and flexibility with all samples. As a result, we used a sample concentration of 10 mg/ml for this research
because it has the lowest noise. Guinier approximation (Fig.C) showed that PaMurI had Rg = 2.62 nm and
Dmax = 8.78 nm (Fig.D). The molecular weight of PaMurI was calculated as 34 kDa rather than 28 kDa.
A B CD
Rg = 2.62 nm
Dmax= 8.78
nm
Fig.5A Scattering patterns of PaMurI of 3 concentrations in 20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM D,L glutamate,
10% glycerol; Fig.5B Kratky plot. Fig.5C Guinier plot of 10 mg/ml. Fig.5D Pair distribution functions of 10 mg/ml.
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Structural analysis by CRYSOL A BC
showed that the scattering curve of PaMurI
was not well fit to any calculated scattering I(q) (a.u.) I(q)
curves of MurI from the different crystal
(a.u.
0°
structures. However, it shows the most /
resemblance with substrate free MurI of q (Å-1) q (Å-1) Z 90°
Aquifex pyrophilus (ApMurI) with Chi^2
Fig.6A Superimposition of the scattering model of the known crystal structures.
value of 0.99. The molecular Fig.6B CRYSOL-derived models for MurI (PDB:1b73).
Fig.6C Averaged and filtered DAMMIF model overlaid with the crystal.
model calculated by DAMMIF is shown as
ab initio bead reconstruction (gray surface). Superimposition of the crystal structure of MurI (PDB:1b73, pink)
demonstrated that the molecular envelope (gray surface) was much larger than the single molecule of MurI.
Conclusion
The glutamate racemase encoding gene or murI of P. aeruginosa amplified by PCR was successfully
cloned into an expression vector, pSY5. The SEC revealed a single peak chromatogram with a purity of 95%
and a molecular weight of approximately 31 kDa, according to SDS-PAGE. Crystallization screening of
PaMurI showed that the small crystal protein was able to crystallize in 2 conditions. However, the X-ray
diffraction quality is insufficient for structure determination. SAXS data showed that PaMurI displayed
monomer in solution with the Rg = 2.62 nm and Dmax = 8.78 nm. The structural analysis demonstrated the
different conformation between substrate bound (PaMurI) and substrate free (ApMurI). The results suggest
that conformation changes of the MurI upon substrate binding is a part of enzyme activity, this may restrict
other substrates (such as drug) accessing to the active site. The finding of this work which revealed structural
characteristic of MurI as oligomerization and conformation can support researchers in the discovery and
development of specific bacterial cell wall biosynthesis inhibitors
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. Fisher, S.L. (2008). “Glutamate racemase as a target for drug discovery.” Microb Biotechol 1(5): 345–360.
2. Waldemar, V., Didier, B., Miguel A. (2008). “Peptidoglycan structure and architecture.” FEMS
Microbiology Reviews 32(2): 149–167.
3. Hansa D. Bhargava., et al. (2020). “Pseudomonas Infection.” Clin Microbiol 22(4): 582–610.
4. Franke, D., et al. (2017). ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from
macromolecular solutions, Journal of Applied Crystallography 50(4): 1212-1225.
5. Svergun, D., Barberato, C. & Koch, M. H. J. (1995) CRYSOL– a Program to Evaluate X- ray Solution.
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Title : Production and adsorption properties of bacterial OB3_16_04
cellulose by Komagataeibacter oboediens TISTR 2294
using tomato juice medium
Field : Biology and Biodiversity
Author : Mr. Tanachai Jiaranai
Mrs. Jarawee Leelapaiboon
School : Demonstration School Prince of Songkla University, Prince of Songkla University, Pattani
Campus
Advisor : Dr. Somrak Panphon and Asst. Prof. Dr. Jaruwan Maneesri, Faculty of Science and Technology,
Prince of Songkla University, Pattani Campus
Abstract
Bacterial cellulose is a biodegradable material produced by bacteria. It has a high purity because
it is free of lignin and hemicellulose. This research aimed to study bacterial cellulose production from
Komagataeibacter oboediens TISTR 2294 using a tomato juice medium and determine its adsorption property.
Different production media, including tomato juice medium, tomato juice with the additional nutrient, and
diluted tomato juice medium supplemented with nutrient, were used. It was found that bacterial cellulose
obtained from autoclaved tomato juice medium with an additional nutrient, without pH adjustment, had the
highest percentage of water adsorption of 720% and 910%, at 13 hours and 33 hours, respectively. Bacterial
cellulose production was scaled up using a selected culture medium in plastic boxes. Bacterial cellulose was
purified using NaOH and dried at 45 °C for 48 hours. Two forms of bacterial cellulose were prepared: powdered
form and sheet form. The adsorption properties of distilled water and vegetable oil on bacterial cellulose were
determined. The result showed that water adsorption on bacterial cellulose sheets was 463.81%. At the same
time, rice oil and palm oil adsorption on bacterial cellulose were 203.31% and 201.75%, respectively. In
addition, distilled water, rice oil, and palm oil adsorption on powdered bacterial cellulose were 904.93%,
561.68%, and 144.08%, respectively. The morphological structure of bacterial cellulose observed under a
scanning electron microscope showed that the diameter of bacterial cellulose fibrils ranged from 40.47 to 102.7
nm. This study shows that K. oboediens TISTR 2294 can produce bacterial cellulose using a tomato juice
medium. Furthermore, the adsorption properties of bacterial cellulose make it suitable for further application
in various fields.
Keywords : Bacterial cellulose, Komagataeibacter oboediens TISTR 2294, Adsorption properties
Introduction
Cellulose is the main structure of plant cell walls. Generally, plant cellulose contains
Hemicellulose, lignin and many other substances. which, before being used, they must go through a chemical
process first. Bacterial cellulose does not contain any of the substances like plant cellulose. It is purer and has
a higher water holding capacity than plant cellulose. The property of bacterial cellulose is a biological polymer
with special properties. It has a unique nanoscale structure. High purity, crystallinity, stability and chemical
stability. It is compatible with body tissues. It has no toxicity and good water holding capacity due to its
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structure. and can be degraded naturally. Therefore, it can be applied in a variety of applications such as the
textile industry. agricultural industry audio industry pharmaceutical and medical industries, etc.
However, the industrial production of bacterial cellulose is still quite expensive so we looking for
an alternative ingredient that is cheap and readily available, which is tomato due to its short shelf life and
nutrients that are useful in large quantities. There is the tomato production all year and can be found in the
general market. Bacterial cellulose has also reported to be produced from a culture medium that made from
spoiled tomato juice (Fatima et at., 2021), making tomatoes suitable as a raw material for the production of the
bacterial cellulose and as a waste material that can be reused. Komagataeibacter oboediens TISTR 2 2 9 4 is
able to produce bacterial cellulose, a sticky membrane. Its main component is cellulose. non-pathogenic, grow
in air and being a bacteria that can grow well in the pH range of 4 - 6 . Tomato juice has a pH in this range, so
this bacteria is selected to produce bacterial cellulose.
Our objective is to study the production of bacterial cellulose from Komagataeibacter oboediens
TISTR 2294 and apply it to adsorp water and oil as sheet and powder form and we also expected to learn
optimal conditions for the bacterial cellulose production and more alternative adsorpbent materials.
Methodology
The experiments were divided into 3 parts as follows,
Part 1: Production of bacterial cellulose by Komagataeibacter oboediens TISTR 2294 using different tomato
juice medium
1.1 Prepare the starter by using coconut water medium by mixing 5% sucrose, 0.5% acetic acid,
0.3% ammonium dihydrogen phosphate with the coconut water and inoculate 10% Komagataeibacter
oboediens TISTR 2294 then incubate for 10 days
1.2 Prepare the culture medium by blending and filtrating the tomato then divide them to 7 parts
to produce different culture mediums including: Pure tomato juice at pH 6, tomato juice with additional
supplement at pH 6, 50% diluted tomato juice with additional supplement at pH 6, Pure tomato juice at original
pH (both separated autoclaved and boiled) and tomato juice with additional supplement at original pH (both
separated autoclaved and boiled)
The additional supplement is including: 5% sucrose and 0.5% ammonium sulfate, the pH was
adjusted by using 0.1 M NaOH, separate autoclaving at 121 ℃ for 15 minutes and boiling for 15 miutes to
compare the differences
1.3 Inoculate 10% of the incubated starter into each culture medium and incubate for 10 days
1.4 Purify and measure the thickness, wet weight, dry weight and water adsorption capacity of the
bacterial cellulose.
Part 2 : Scale-up the production of bacterial cellulose
2.1 Prepare the tomato in large quantity and then repeat the process from Part 1 but using only the
medium that gave highest water adsorption capacity.
2.2 Prepare the bacterial cellulose into sheet and powder form by blending and sieving at 0.50 mm
2.3 Testing the liquid adsorption capacity. The liquids are including: distilled water, rice bran oil
and palm oil.
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Part 3 : Observing the surface morphological structure of bacterial cellulose under scanning electron
microscope.
Results
Bacterial cellulose from pure tomato juice orginal pH (autoclaved) produced the highest thickness and
weight. The average thickness is 0.616±0.030 cm, average wet weight is 17.6797±2.6129 g, average dry weight is
0.4163±0.1157 g and the water adsorption capacity at 13 hours and 33 hours is 310% and 340%, respectively.
Production of bacterial cellulose show as Table 1.
Table 1 Production of bacterial cellulose by Komagataeibacter oboediens TISTR 2294 using different mediums
Mediums Bacterial cellulose production Productivity
Thickness (cm) Wet weight (g) Dry weight (g) (g/L/d)
Pure tomato juice pH 6 0.37 11.01 0.20 7.34
Tomato juice with additional 0.38 12.47 0.24 8.31
supplement pH 6
50% Diluted tomato juice with 0.44 12.99 0.29 8.66
additional supplement
Pure tomato juice 0.62 17.68 0.42 11.79
Pure tomato juice (boiled) 0.39 12.06 0.24 8.04
Tomato juice with additional 0.31 9.29 0.34 6.20
supplement
Tomato juice with additional 0.42 14.00 0.25 9.33
supplement (boiled)
Bacterial cellulose from tomato juice with additional supplement at original pH (autoclaved) gave the
highest water adsorption capacity. Average thickness is 0.311±0.042 cm, average wet weight is 9.2934±0.2649 cm,
average dry weight is 0.3409±0.0413 and water adsorption capacity at 13 hours and 33 hours is 720% and 910%,
respectively. Water adsorption capacity of bacterial cellulose from different mediums show as Table 2.
Table 2 Water adsorption capacity of bacterial cellulose from different mediums
Mediums Water adsorption capacity (%)
13 Hours 33 Hours
Pure tomato juice pH 6 250 270
Tomato juice with additional supplement pH 6 470 510
50% Diluted tomato juice with additional supplement 420 420
Pure tomato juice 310 340
Pure tomato juice (boiled) 330 360
Tomato juice with additional supplement 720 910
Tomato juice with additional supplement (boiled) 580 560
Bacterial cellulose from the scaled-up production has average thickness at 0.170±0.053 cm, average
wet weight at 51.3165±14.8037 g, dry weight at 0.5017±0.0931 g and liquid adsorption capacity of powder form
was more than sheet form. The liquid adsorption capacity show as Table 3
Table 3 Liquid adsorption capacity of bacterial cellulose in sheet and powder form
Forms of bacterial cellulose Liquid adsorption capacity (%)
Sheet Distilled water Rice bran oil Palm oil
Powder 201.75
463.81 203.31 144.08
904.93 561.68
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Bacterial cellulose morphological structure was observed under scanning electron microscope and
found that the cellulose fibrils size was in the range from 40.47 to 102.7 nm and the average size was 71.41 nm and
also can adsorp water and oil. The morphological structure at different magnifying power of bacterial cellulose that
adsorp water, rice bran oil and palm oil show as Figure 1, 2 and 3, respectively.
Figure 1 Morphological structure of dried bacterial cellulose Figure 2 Morphological structure of bacterial cellulose that Figure 3 Morphological structure of bacterial cellulose that
adsorped rice bran oil adsorped palm oil
Conclusion
Komagataebacter oboediens TISTR 2294 produces nano bacterial cellulose with 0.17 cm
thickness using tomato juice medium in plastic box scale at 10 days. The width of dried bacterial cellulose
fibrils is ranging from 40.47 to 102.7 nm and the average size is 71.41 nm. Bacterial cellulose has liquid
adsorption capacity in descending order as follows: water and oil. Bacterial cellulose in both sheet and
powder form does not have structural differences but may different in terms of surface area.
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
Fatima A, Yasir S, Khan MS, Manan S, Ullah MW, Ul-Islam M. Plant extract-loaded bacterial cellulose
composite membrane for potential biomedical applications. Journal of Bioresources and Bioproducts. 2021
Feb 1;6(1):26-32.
Khemacheewakul J. The Production of Bacterial Cellulose from Acetobacter xylinum and Application in
Industry. Rajabhat Agric. 2016;15(2):25-33.
Maneesri J, Longsawang S, Artnarong S. Isolation of Cellulose-Producing Bacteria from Toddy Palm. The
16th Food Innovation Asia Conference 2014; 2014 June 12-13; Bangkok, Thailand; 2014.
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Title : Screening of Thai red holy basil cultivars for a OB3_10_01
Field : nutraceutical by bioactive and Eugenol synthase gene
Author :
expression analysis
School :
Advisor : Biology and Biodiversity
Mr. Ratchakarn Jandorn
Mr. Nutjirapat Jirapimuggul
Miss Kusalin Somritchinda
Kasetsart University Laboratory School Kamphaeng Saen Campus Center for Educational
Research and Development.
Asst. Prof. Dr. Siriporn Sripinyowanich
Abstract
Red holy basil (Ocimum sanctum linn.) is an aromatic perennial herb with medicinal properties for
treating ailments ranging from colds to chronic diseases, such as cancers and diabetes. The purple color on
anthocyanin-enriched leaves of red holy basil varies on cultivars widely grown throughout the different regions
of Thailand, where a lack of information on key bioactive substances profiles of each cultivars exists
nowadays. In the current study, we provided this information by elucidation the quantity of eugenol and
anthocyanins from leaves of the nine red holy basil cultivars (OS01-OS09) in comparison to the three white
holy basil (OS10-OS12) to screen the cultivars high potentially for nutraceutical usages. Bioactive substances
were profiled by using Agilent headspace sampler and GC-MS. Eugenol, ß-caryophyllene, and ß-elemene
were major constitutes from dried leaves of most red holy basil cultivars, while methyl eugenol had more
detected in white holy basil. OS05 had significantly more eugenol than other cultivars. In addition, total
anthocyanins were also analyzed by using pH differential technique from water extracts. From the results, the
contents of total anthocyanins were found in the range of 2.12-22.76 mg/L and the highest is detected in OS03
red holy basil. Additionally, we confirmed the results by using real-time PCR to evaluate expression level of
the genes regulated in eugenol and anthocyanin biosynthesis. OsEGS1 and OsANS were most expressed
consistent with eugenol and anthocyanin content, respectively. Taken together, the top five cultivars of Thai
holy basil with the high bioactive accumulation were red basil cultivars no. OS05, OS08, OS03, OS04 and
OS07, respectively. There was no significant correlation between eugenol and anthocyanin content. Finally,
this study provides information on phytochemistry and related gene expressions that are useful for medical
and pharmaceutical applications such as dietary supplement and medicine developments in the future.
Keywords : Anthocyanin; Eugenol; Gene expression; Holy basil; Red holy basil
Introduction
Red holy basil is a member of the genus Ocimum within the Lamiaceae family, a group of culinary
and medicinal aromatic plants, and originated from South-East Asia including Thailand. [1] Leaves of Thai
red holy basils have various purple-green colored characters that potential human health benefits regarding
their accumulation of many biomolecules. [2] One important is eugenol which has an antioxidant and anti-
inflammatory effect and helps with cancer treatment. [1] Anthocyanin can reduce the risk of disease related to
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the heart and blood vessels and resist cancer and free radicals. [1],[3] These two important biomolecules found
within red holy basil leaves are noted for pharmaceutical properties.
Eugenol and anthocyanin are classified as polyphenols and synthesized from the same precursor
which is phenylalanine. [1],[3] It is possible that these two main biomolecules may be linked in red holy basil.
However, there is no previous research reported. Therefore, we investigated the relationship of eugenol with
anthocyanins by determining the eugenol and anthocyanin contents in leaves of various Thai red holy basil
cultivars. If such a relationship is found, the color of red basil leaves can be used as an indicator of the amount
of eugenol. Our research aims to screen the potential Thai red holy basil with the highest eugenol and
anthocyanin contents by using GC/MS and a spectrophotometer method determines eugenol and anthocyanin
content in the leave tissue, respectively. In this study, we also confirmed the accumulations of eugenol and
anthocyanin in Thai red holy basil by qRT-PCR expression analysis of the key genes regulated in eugenol and
anthocyanin biosynthetic pathways.
Methodology
1. Tissue and extract preparation
1.1 Sample preparation: Nine cultivars of red
holy basil from Thai genetics source namely OS01-
OS09 and three cultivars of white holy basils namely
OS10-OS12 (Figure 1) were used in the experiment.
1.2 Quantitative analysis of eugenol and
methyl eugenol: Dried leaves tissue (0.1 g) were tested
by using Headspace solid-phase microextraction gas
chromatography mass spectrometry (HS-SPME/GC-
MS). The experiment was performed by using the
Agilent 7890A gas chromatography interfaced with a
detector Agilent 5975C mass spectrometry. The column
used was fused silica capillary (30 mm × 0.25 mm i.d.,
0.25 μm film thickness: Mega-5MS). The oven
temperature was increased from 60-270℃ at a rate of 4 Figure 1: Red (OS01-OS09) and white (OS10-
℃/min. Then post run for 5 minutes. The carrier gas was OS12) holy basils from Thai genetics source
Helium with a flow rate of 1 mL/min. Injector and investigated in this study.
detector temperatures were 200℃ and 270℃, respectively. The chemical constituents of red holy basils were
identified by comparing the results of the chromatogram and reference retention time using The National
Institute of Standards and Technology (NIST 2011) database library (GC/MS system).
1.3 Extraction and quantitative analysis of total anthocyanins: Take 10 g of dried leaves tissue, add
30 ml of distilled water into titration flask, shake at room temperature for 48 h, then filter with filter paper size
11 μm. The extracts were distilled by a rotary evaporator at 60℃, and the crude extracts were collected and
weighed to calculate the percentage of results. Then, prepare a solution of pH 1 (KCl-HCl buffer) and pH 4.5
(CH3COONa-HCl), and pipette 0.3 ml of 0.1 mg/ml of extract into a 2.7 ml of pH buffer solution, and leave
for 10 min. The absorbance was measured at 510 and 700 nm wavelengths
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2. Validation of gene expression involved in eugenol and anthocyanin biosynthesis
The mRNA was extracted from leaves plant tissue using RNeasy® Plant Mini Kit (Trizol) volumes at
50 μl. The extracted mRNA was analyzed by agarose gel electrophoresis. The total RNA content was assessed
by spectrophotometer at 260/280 nm. To calculate by [RNA] = OD260 × 40 × dilution × 10-3. To remove
DNA contaminated by DNase (Sigma-Aldrich Co., Ltd., USA). The cDNA synthesis reacts in RNA 500 ng
with primer Oligo dT12-18 (Invitrogen, USA) RiboLock RNase Inhibitor (Thermo Scientific, USA) and
RevertAid Reverse Transcriptase (Thermo Scientific, USA) at 42℃ within 90 minutes. Then, to select the
objective gene and design primers for checking gene expression by Real-time quantitative PCR (CFX
managerTM). First-stand cDNA synthesis was performed using iTaqTM Universal SYBR® Green Supermix
(BIO-RAD, USA). The specific primer conditions are 95℃ with 3 s, 95℃ with 10 s, 52℃ with 20 s and 72℃
with 10 s. Then, to analyze gene expression by 2-CT.
3. Screening of the potential Thai red holy basil accessions
Consulting data of anthocyanin and eugenol contents by ranking top five cultivars of red holy basil. In
order to find our the relationship between anthocyanin and eugenol, the results were submitted to multivariate
principal component analysis (PCA).
4. Statistical Analysis
The variance was analyzed according to the complete randomized design trial and the mean was
compared by Duncan's multiple range test at 95% confidence level.
Results Table 1: Eugenol and methyl eugenol content (% relative
Cultivar had a significant effect on measured
gDW-1) in dried leaves of red and white holy basil
eugenol contents as shown in Table 1. The highest
eugenol content was detected in OS05 at 7.56% gDW-1, Samples % relative gDW-1
followed by OS08 and OS07 with eugenol content 5.55%
and 5.54% gDW-1, respectively (Table 1). While, Eugenol Methyleugenol
eugenol was lower in OS03, OS04 and OS12 ranged
from 2.78 to 3.25% gDW-1. OS05, OS07 and OS08 were Red OS01 0.4 1.04
classified into the group of high eugenol content. The red
(OS06 and OS09) and white (OS10 and OS11) holy basil holy OS02 0.29 4.2
cultivars were non-detected eugenol but detected find
methyl-eugenol instead (Table 1). basil OS03 3.25 NA
The red holy basil accumulated total OS04 3.7 NA
anthocyanins higher than the white holy basil. It was
found that OS03 and OS07 of red holy basil had high OS05 7.56 NA
OS06 NA 3.16
OS07 5.54 NA
OS08 5.55 NA
OS09 NA 3.79
White OS10 NA 3.36
holy OS11 NA 3.22
basil OS12 2.78 NA
total anthocyanins, 22.76 and 22.25 mg cyanidin L-1, respectively. The detection of purple-leaved holy basil
experimental plants was accompanied by a significant increase in the anthocyanin content in all variants
compared with that of green-leaved holy basil due to the principle that anthocyanin compounds are pigments
that give red, purple, and blue colors.
To confirm biomolecules content, we carried out Real-time quantitative PCR for five genes encoding
OsEGS1 for eugenol and OsCHS, OsCHI, OsANS and OsDFR for anthocyanin biosynthesis
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Our finding revealed OsEGS1 and OsANS gene expression positively correlated wih eugenol and
anthocyanin contents, respectively (Figure 2). The highest level of OsEGS1 gene expression was detected in
OS07 that 19.10 times higher than that of the control OS10, followed by OS05, OS03 and OS08 that higher
18.45, 17.77 and 14.64 times, respectively. However, there was no signal detection of OsEGS1 in the cultivars
that had no estimation on GC/MS. In addition, red holy basil (OS03 and OS07) with high total anthocyanins
content were also detected the expression level of OsANS significantly higher than that of other cultivars.
Figure 2: Relationship between the contents of eugenol, Figure 3: Principal component analysis (PCA)
total anthocyanins and gene expression of OsEGS1, displaying clustering of red holy basil cultivars
OsCHS, OsDFR, OsANS and OsCHI in Thai holy basil in this study based on individual eugenol and
showed by Hierarchical cluster analysis. anthocyanins contents.
Conclusion
Hierarchical cluster analysis revealed that there is no significant relationship between eugenol and
anthocyanin content in red holy basil (Figure 2). This result indicates that the top five red holy basil cultivars
potentially for phytochemical usages are OS05, OS03, OS04, OS07 and OS08 (Figure 2 and 3). There is no
significant relationship between eugenol and anthocyanin content in red holy basil (Figure 2).
The leaves of Thai red holy basil are particularly rich in eugenol and anthocyanins, leading to the
strong potential for medical and pharmaceutical applications of these cultivars. These basil properties may
therefore be desirable for plant breeding programs in which growers seek to biomolecules. This study could
aid in understanding the relationship between bioactive compounds and their purple-leaved color.
Acknowledgement
This project was supported by Science Classroom in University Affliated School (SCiUS) under
Kasetsart University Kamphaeng Saen Campus and Kasetsart University Laboratory School Kamphaeng Saen
Campus Educational Research and Development Center. 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. Hock Eng Khoo, Azrina Azlan, Sou Teng Tang and See Meng Lim, 2017. Anthocyanidins and
anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food and
nutrition research. 61(1): 1361779.
2. N. Sarrami, M.N. Pemberton, M.H. Thornhill and E.D. Theaker, 2002. Adverse reactions associated with
the use of eugenol in dentistry. British dental journal. 193, 257-259
3. Pavithra, 2014. Eugenol-A review. Journal of Pharmaceutical Sciences and Research. 6(3):153-154.
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Title : The Study of Antioxidant activity in Citrus Peel crude extracts in UV B-induced human skin
Field : keratinocytes
Author :
Biology and Biodiversity OB3_01_04
School : Mr. Pongsakorn Promming
Advisor :
Mr. Rachata Yengram
Chiang Mai University Demonstration School, Chiang Mai University
Asst. Prof. Dr. Supachai Yodkeelee Department of Biochemistry, Faculty of Medicine,
Chiang Mai University
Abstract : Nowadays, fruits and vegetables are the general sources of vitamins and nutrients that also maintain
the body system. The fruits that are high in vitamin C like citrus fruits such as orange, tangerine, lime, pomelo,
etc. Citrus fruits contain many other functional bioactive phytochemicals such as flavonoids and phenolic acids
that are beneficial to human health. Citrus fruits are mainly found in Thailand, but fruit peels are mostly inedible.
Therefore, the bark is a large amount of waste left over. Tangerines, pomelo and lime are also rich sources of
phenolic acids and flavonoids. They have various properties, including antioxidants. However, the efficiency of
these measures has not yet been examined. To investigate the phytochemical and antioxidant properties of
tangerine, pomelo and lime. We did phenolic compound analysis, flavonoids compound analysis, DPPH and ABTS
analysis. In phenolic compounds analysis, the result showed that the tangerine extract had the highest amount of
phenolic compounds. There was 80.64 mg of gallic acid equivalent (GAE) per gram of extract, followed by pomelo
and lime. Flavonoid analysis showed that the tangerine extract had the highest concentration of flavonoids. There
was 24.41 mg of citric acid equivalent (CAE) per gram of extract, followed by lime and pomelo. In ABTS radical
,the result showed that the tangerine extract had the highest antioxidant activity of 50%. The IC50 value was 56
μg/mL, followed by lime and pomelo, respectively. In the DPPH radical scavenging assay, the results showed that
the value tends to be the same as in the ABTS assay. Moreover, only tangerine peel extract exhibited a protective
effect on UVB-induced keratinocyte cell death, but not pomelo and lime peel extract. Overall, it can be concluded
that tangerine has the highest antioxidant activity, flavonoid compounds, and phenolic compounds. Which is
shown to be cytoprotective in UVB-induced skin cell damage. In the future, this study could lead to the
development of anti-aging skincare products.
Keywords : Citrus group, Phenolic acid, Flavonoids, Antioxidant, UVB-induced keratinocyte cell
Introduction
The effect of UV rays on destroying human skin is being considered worldwide in the present community.
Although the main benefit of UV radiation is promoting the synthesis of vitamin D from precursors in the skin,
UV irradiation induces physiological and morphological skin damage, resulting in skin dryness, wrinkle formation,
and loss of elasticity. Nowadays, fruits and vegetables are the general sources of vitamins and nutrients that also
maintain the body system. Citrus fruits contain many other functional bioactive phytochemicals such as flavonoids
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and phenolic acids that are beneficial to human health. They are mainly found in Thailand, but their fruit peels are
mostly inedible. Therefore, the bark is a large amount of waste left over. Dietary flavonoids have been revealed to
reduce the damage caused by UV exposure. Tangerines are known to contain high concentrations of flavonoids
and phenolic content. In this study, the effects of citrus fruits on photoaged skin were demonstrated using UVB
irradiated human skin kerationocytes.
The objective of this study is to study the flavonoid and phenolic contents contained in the peels of citrus
fruits and the antioxidant properties of crude extracts from peels of citrus fruits and to study the antioxidant activity
of the citrus peel crude extracts in UVB-induced human skin keratinocytes.
Methodology
Protocol Phenolic Contents
Preparing the standard Gallic acids by dissolving Gallic acid 10 mg in 1,000 μL of DMSO (Dimethyl
Sulfoxide) at the concentration 10,000 μg/mL and for the reaction part, dilute it into 20, 10, 5, and 2.5 μg/mL via
deionized water (DI) as a solvent and the final concentration is 0 μg/mL which is pure DI and preparing the sample
by dissolve crude extracts of Citrus peels 20 mg in 1,000 μL of DMSO and dilute for the reaction part, dilute into
the concentration at 100 and 50 μg/mL. For the reaction to find the contents of the Phenolic compound in the peels
by using 400 μL of each concentration of Gallic acid (as well as the samples) into the microtube, add 300 μL of
10% volume by volume of Folin-Ciocalteau and incubate for 3 minutes in a room-temperature and a dark place,
add 300 μL of 7.5% weight by volume of Na2CO3 and incubate for 30 minutes in room-temperature and dark place
and read the absorbance by using a UV spectrometer at the wavelength of 765 nm.
Protocol Flavonoid Contents
Preparing the standard Catechin by dissolving Catechin 10 mg in 1,000 μL of DMSO at a concentration
of 10,000 μg/mL and for the reaction part, diluting it into 20, 10, 5, and 2.5 μg/mL via deionized water as a solvent,
until the final concentration is 0 μg/mL, which is pure DI. Preparing the sample for the reaction part by dissolving
crude extracts of citrus peels (20 mg) in 1,000 μL of DMSO and dilute for the reaction part. Dilute it in the
concentration at 200 and 100 μg/mL. For the reaction part, using 250 μL of each concentration of catechin (as well
as the samples) into the microtube, add 125 μL of 5% weight by volume of NaNO2 and incubate for 5 minutes in
a room-temperature and dark place, add 125 μL of 10% weight by volume of AlCl3 6H2O and incubate for 5
minutes in a room-temperature and dark place, add 1,000 μL of 1 N of NaOH and incubate for 5 minutes in room-
temperature and dark place , and read the absorbance by using a UV spectrometer at the wavelength of 510 nm.
ABTS assay
In the ABTS assay, prepare ABTS (radical form) and potassium persulfate at 13.2 mg by dissolving it in
1 mL of deionized water and diluting 100 μL of potassium persulfate solution with 900 μL of deionized water.
Next, mix it together in a dark place. This solution has a life expectancy of 12-16 hours. In the reaction part,
prepare the ABTS (radical form), then incubate 10 mL of the sample in 990 microliters of ABTS (radical form)
for 6 minutes in a dark place at room temperature and measure the absorbance.
DPPH assay
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In the DPPH assay, prepare the DPPH. Then prepare the DPPH for use in reacting with the samples. Next,
test DPPH with a spectrometer at a wavelength of 540 nm. The value must be less than 2.0. In the reaction part,
mix 20 μL of the sample and 180 μL of DPPH in a 96-well plate for 20 minutes, then read the absorbance from
the Elisa Reader at a wavelength of 540 nm.
Cell Viability assay
Test citrus peel extract (50 or 100 μg/mL) with keratinocyte cells. Next, irradiated the cell with UVB (15
mJ/cm2) and incubate it for 24 hours. So, the cell survival will be determined by the SRB assay.
Result
In total Phenolic compounds in Citrus group samples, the result has showed that tangerine extract has the
highest contents of Phenolic acids which is 78.7 mg/g extract and followed by pomelo extract around 67.5 mg/g
extract and the least contents of Phenolic acids in the extracts is 61.9 mg/g extracts that consists in the lime crude
extract and the total Flavonoid contents in the samples of Citrus peel , The total quantity of Flavonoid contents is
highest in tangerine crude extract about 24.4 mg/g extract following by 13.9 mg/g extract in lime extract and the
least Flavonoids content is 8.4 mg/g extract which is found in pomelo crude extract.
For the free radical scavenging of Citrus peel extracts was determined into two methods; ABTS assay
and DPPH assay. The radical inhibition is determined by IC50 value, the IC50 value is the least concentration
that decreased the free radical by 50%. The IC50 value (determined by ABTS assay) has the least concentration
in tangerine (56.7 μg/mL) following by lime (74.7 μg/mL) and pomelo (139.0 μg/mL) respectively. However,
the efficacy of Citrus peel extracts to inhibit the radical in each concentration determined by DPPH assay, the
IC50 value of the samples will be able to consider via DPPH assay at a higher concentration
Figure 5 Cytoprotective effect of citrus extracts on UVB-irradiated human
skin keratinocyte cells.
The UV test with human skin keratinocytes showed that testing human skin keratinocytes with UV-B 20
mJ/Cm2. As you can see, tangerine at 50 ug/mL reduced the cell viability from UVB to 59.8%. Only tangerine
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peel extract at 50 ug/mL increased cell viability to 138.6% in keratinocytes exposed to UVB but not pomelo or
lime.
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 the citation.
Conclusion
The quantity of phenolic and flavonoid compounds is richest in tangerine peel extract, respectively.
Whereas the pomelo peel extract was the lowest in the phenolic content and lime peel extract showed the lowest
in flavonoid contents. In terms of an antioxidant properties, it was found that the lowest concentration of those
extracts to decrease the radical by 50% was the extracts from tangerines, That shows the tangerine extract is the
best choice to use as a radical inhibition substance because it uses the least concentration compared to the others.
Moreover, only tangerine peel extract exhibited a cytoprotective effect on UVB-induced skin keratinocyte cell
death. Take the results together, and tangerine peel extract showed the highest content of phenolics and flavonoids
and exhibited the best antioxidant properties. which is associated with UVB-induced cell death protection. This
would be indicative of the potential to establish a promising strategy to prevent skin photoaging.
References
Abd Elmageed ZY, Gaur RL, Williams M, Abdraboh ME, Rao PN, Raj MHG, et al. Characterization of
coordinated immediate responses by P16INK4A and p53 pathways in UVB-irradiated human skin cells.
Journal of Investigative Dermatology. 2009;129(1):175–83.
Abe S, Hirose S, Nishitani M, Yoshida I, Tsukayama M, Tsuji A, et al. Citrus peel polymethoxyflavones,
Sudachitin and nobiletin, induce distinct cellular responses in human keratinocyte HaCaT cells.
Bioscience, Biotechnology, and Biochemistry. 2018;82(12):2064–71.
Oboh G, Ademosun AO. Characterization of the antioxidant properties of phenolic extracts from some citrus
peels. Journal of Food Science and Technology. 2011;49(6):729–36.
Sir Elkhatim KA, Elagib RA, Hassan AB. Content of phenolic compounds and vitamin C and antioxidant
activity in wasted parts of Sudanese citrus fruits. Food Science & Nutrition. 2018;6(5):1214–9.
ZHANG H, YANG Y-fei, ZHOU Z-qin. Phenolic and flavonoid contents of Mandarin (citrus reticulata blanco)
fruit tissues and their antioxidant capacity as evaluated by DPPH and Abts Methods. Journal of
Integrative Agriculture. 2018;17(1):256–63.
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Title : Isolation and probiotics properties of OB3_04_05
lactic acid bacteria from fermented foods
Field : Biology and Biodiversity
Authors : Miss Natchaya Panalikul
Miss Praechomphoo Phitaktim
School : Demonstration School of Khon Kaen University
Advisor : Assoc. Prof. Dr. Sophon Boonlue
Department of Microbiology, Faculty of Science, Khon Kaen University
Abstract
Probiotics are lactic acid bacteria which can be found in fermented foods and have the ability to inhibit
pathogenic bacteria in digestive system. This study is focused on isolated probiotics from fermented foods and
determined the efficiency of isolated probiotics for inhibiting growth of pathogenic bacteria. The morphology and
antibiotic activities of isolated lactic acid bacteria were examined. The results showed that 13, 7 and 5 isolates of
lactic acid bacteria were isolated from drinking yogurt (FM1–FM13), sour pork (SP1–SP7) and fermented
mushrooms (SM1–SM5) respectively with catalase-negative properties. According to antibiotics properties, the 2
isolates of lactic acid bacteria FM5 and FM7 were revealed to have the most effective antimicrobial against
Escherichia coli (11.05 and 9.60 mm of inhibition zone) and Shigella sonnei (9.55 and 10.48 mm of inhibition zone),
respectively. The two isolates were survival in MRS broth at pH 2-9. The isolate FM7 was showed higher acid
tolerance than FM5 and also had survival rate up to 50% at pH 6-9. Moreover, they were gram-positive bacteria
which appearance of Streptococcus sp. on microscopic identification. As the results, lactic acid bacteria isolated from
fermented foods were showed the probiotic properties of pathogenic inhibition and particularly interesting for further
developed to enhance the survival rates in digestive system by bio-polymer encapsulation.
Keywords : Probiotics, Lactic acid bacteria, Fermented food, Antibiotic
Introduction
Nowadays, diarrhea is the disease with the highest morbidity rate in Thailand. Especially, in the Northeastern
which was become a pressing problem of local healthcare. The cause of this disease is in gastrointestinal tract that
contains of both beneficial and harmful bacteria. Recently, beneficial bacteria, which is also called “Probiotic bacteria”,
is used in food supplement industry and plays a significant role in prevention of this disease. Probiotics are produced as
dietary supplements, which are defined as any live microorganism that has medical benefits for using as an alternative
to conventional therapies such as antibiotics for the treatment of intestinal infections (e.g. Escherichia coli, Salmonella
sonnei, etc.). Additionally, probiotics used in supplement production should have a resistance to acids and alkaline to
increase the survival rate in gastrointestinal tract. Probiotics are found in a variety of fermented products such as drinking
yogurt, sour pork and fermented mushrooms. The aim of the present work is to isolate probiotic cells from different
fermented food samples from local markets nearby Khon Kaen University then determine the efficiency of isolated
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probiotic cells for inhibiting growth of pathogenic bacteria and investigate the capacity of acid to alkaline pH tolerance
to simulate the condition of Probiotic living in digestive system.
Methodology
1. Isolation and selection of lactic acid bacteria from fermented foods
Lactic acid bacteria were collected from drinking yogurt, sour pork, fermented mushrooms, yogurt and
kimchi. 25 g of each sample was first mixed with 0.85% NaCl solution 225 ml and put into stomacher machine to
make it homogeneous. The dilution from 10-4 to 10-6 were spread directly onto the surface of MRS agar with
CaCO3 and incubated at 37 °C for 48 h. The colony that has clear zone were picked up by streak plate. Isolates
were tested with catalase, and only negative result bacteria were kept in freezing medium in -40 °C.
2. Inhibition of pathogenic bacteria by probiotics
The determination of pathogenic bacteria inhibition was consisted of preparing cell free supernatant and
inhibitory activity testing. The process of preparing cell free supernatant the isolates lactic acid bacteria were
cultured in MRS broth and incubated at 150 rpm at 37 °C for 24 h and centrifugation at 10,000 rpm at 4 °C for 10
min. The process of inhibitory activity testing by swab-paper discs method. Pathogenic bacteria included E. coli
and S. sonnei were cultured in nutrient broth (NB) incubated at 150 rpm at 37 °C for 24 h and swabbed on nutrient
agar (NA). Paper discs were placed on NA then 20 μl cell free supernatant of lactic acid bacteria were dropped on
paper discs and incubate at 37 °C for 24 h.
3. Acid and alkali tolerance
1 ml of isolated probiotics (108 CFU/ml) were cultured in 9 ml of MRS broth at pH 2 to 9 at 37 °C for 6
h. and were spread on MRS agar with CaCO3 and incubated at 37 °C for 24 h.
4. Bacteria identification
The pure cultures from each sample which selected from MRS agar were added few drops of 3% H2O2
on the slide and were used in gram-staining method for morphology observation under microscope.
Results
1. Isolation and selection of lactic acid bacteria from fermented foods
The results of this study revealed 13, 7 and 5 strains of lactic acid bacteria isolated from drinking yogurt
(FM1-FM13), sour pork (SP1-SP7), and fermented mushrooms (SM1-SM5). All strains of lactic acid bacteria have
clear zone except isolate SP2 (Figure 1).
AB
Figure 1. The clear zone of lactic acid bacteria, (A) Lactic acid bacteria with clear zone (B) Lactic acid bacteria
without clear zone.
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2. Inhibition of pathogenic bacteria by probiotics
The inhibition of pathogens was determined. The results showed that out 23 isolates the 10 isolates
effected on E. coli inhibition and FM5 was showed significantly difference for highest inhibition activity as 11.05
mm diameter of inhibition zone (Figure 2). For S. sonnei inhibition activity was not significantly difference
obtained on FM7, FM2, FM9 and FM5 for 10.48, 10.08, 10.00 and 9.55 mm, respectively (Table 1).
Table 1. Inhibition activity of probiotic bacteria against the tested human pathogens.
AB
Figure 2. The inhibition zone of probiotic
bacteria. (A) isolate FM5, (B) isolate FM7.
The red circle was represented for the size of
diameter.
3. Acid and alkali resistant
The acid to alkaline pH tolerance test was studied under pH 2-9. The cell count was obtained at 6h then
cell count was enumerated after 48h of incubation on MRS agar. The probiotic isolate FM5 and FM7 was survival
at pH 2-9 for 6h. The isolate FM7 showed higher acid tolerance than FM5 which showed survival rate 41.28,
34.14, 32.91 and 49.26% at pH 2,3,4 and 5, respectively. However, only isolate FM7 had survival rate up to 50%
at pH 6-9. (Figure 3)
.
Figure 3. In vitro tests of tolerance to pH 2-9 of
probiotic bacteria isolated from fermented food.
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4. Lactic Acid Bacteria identification
The lactic acid bacteria strain FM5 and FM7, which has high inhibition activity, was identified by
morphological. The colony of strain FM5 and FM7 were white to light yellow with smooth surface. The
microscopic morphology appearance of streptococci cells with gram-positive. Isolates FM5 and FM7 were
identified as Streptococcus sp. (Figure 4) and all lactic acid bacteria isolated were showed negative catalase.
AB
Figure 4. Gram stain identification, (A-B) gram-positive bacteria of isolate FM5 and FM7 appearance of streptococci
cells, respectively.
Conclusion
Isolation and selection of lactic acid bacteria from drinking yogurt, sour pork, and fermented mushrooms
resulted in 13, 7, and 5 isolates of lactic acid bacteria, respectively which found the probiotic property of catalase-
negative. The two lactic acid bacteria isolates FM5 and FM7 showed the most effective antimicrobial activity
against E. coli and S. sonnei, with 11.05 and 9.60 mm of inhibition zone and 9.55 and 10.48 mm of inhibition
zone, respectively and also survival at pH 2-9 for 6 h. Both were gram-positive bacteria with the microscopic
morphology of Streptococcus sp. Therefore, these 2 isolates have probiotic qualities and are appropriate for
bio-polymer encapsulation to improve the survival rates in the digestive system and production as a health food
supplement.
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. นนทพร รัตนจักร์. การคัดแยกและศึกษาคุณสมบัติการเป็นโพรไบโอติกของแบคทีเรียกรดแลคติกที่คัดแยกจากกล้วยนํ้าว้าดิบในเขต
จังหวัด พษิ ณโุ ลก. วารสารวิทยาศาสตร์และเทคโนโลยี มหาวิทยาลยั อบุ ลราชธานี 2563;2:50-57.
2. ศรีสรรค์ ปูพบุญ. การแยกแลคติกแอสิดแบคทีเรียจากอาหารหมักเพื่อนํามาพัฒนาเป็นตัวนําดีเอ็นเอ. วารสารวิทยาศาสตร์และ
เทคโนโลยี มหาวิทยาลยั อบุ ลราชธานี 2559;1:21-29.
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:Title Study on prebiotic properties of some mushrooms in
promoting probiotic growth compared to Jerusalem OB3_03_09
Field : Artichoke
Biology
Author : Ms. Kankamol Thoopmongkol
School : Mr. Phatcharaphon Taksee
Naresuan University Secondary Demonstration School, Naresuan University
: ,Advisor Dr. Songkran Chuakrut Naresuan University
Abstract
Colorectal cancer (CRC) is the third most common mortality among all cancers in the global
population and is the second-largest in the population in Thailand. The incidence of CRC increases year by
year in the elderly, with risk factors related to genetics and long-term dietary habits of fatty foods and eating
foods with less fiber. CRC has been associated with an imbalance in the amount of microbial in the
gastrointestinal tract (dysbiosis). Probiotics play an essential role in maintaining the balance of beneficial and
harmful microorganisms. This study aims to examine the prebiotic properties of some mushrooms that can
promote probiotic growth compared to the standard prebiotic inulin and Jerusalem Artichoke extract. Two
probiotics bacteria reported as anti-colon cancer strains, Streptococcus thermophilus TISTR 894 (ATCC
19258) and Bifidobacterium bifidum TISTR 2129 (ATCC 29521), were inoculated in MRS broth that
supplemented with 2% (w/v) prebiotic extract from each type of mushrooms. There were 11 types of mushroom
samples: Yanagimatsutake, White beech mushrooms, Brown beech mushrooms, White ear mushroom, Black
ear mushroom, Sajor-cajun mushroom, Straw mushroom, White Lingzhi mushroom, Black Lingzhi mushroom,
Lentinus mushroom, Bamboo mushroom, and Jerusalem Artichoke as a plant control sample. The cultures
were incubated at 37 °C for 48 hours for monitoring the growth of probiotics at 0, 24, and 48 hours by
measurement of the optical density (OD) at 600 nanometers. The results found that the Bamboo mushroom
extract (BME) could significantly stimulate the growth of S. thermophilus at 24 and 48 hours more than other
mushroom extracts. Particularly, it could stimulate the growth of S. thermophilus better than the Jerusalem
Artichoke extracts. For the results from B. bifidum, it was found that the BME still stimulated the growth of
the probiotic better than other mushroom extracts, especially at 48 hours. In addition, its stimulative effect on
growth of B. bifidum was similar to that of Jerusalem Artichoke extract. After comparing the growth-
stimulating effect of BME on the two probiotics, it revealed that S. thermophilus was more responsive to BME
than B. bifidum. The results of this experiment showed that BME had prebiotic properties in which were better
than or equal to the Jerusalem Artichoke extract, depending on the probiotics tested.
:Keywords Prebiotic, Probiotic, Mushroom,Jerusalem Artichoke
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Introduction
Nowadays, many foods are harmful to health, such as fast food, foods high in fat and high in
cholesterol, and low-fiber foods. It has been reported that continued and prolonged intake of these foods can
lead to dysbiosis, a condition in which microbiota, a probiotic in the body, is out of balance and can affect
human health. It can lead to diseases such as allergies (allergy), cholesterol tumors, and cancer. Currently, the
disease that is receiving the most attention is colorectal cancer. CRC is the 3rd most common cancer
mortality among all cancers. Around the world and there are more than 600,000 deaths per year.
This type of cancer mainly occurs in the elderly and has many causes such as eating habits, genetics, obesity,
drinking alcohol, or smoking for a long time. Several studies have shown that due to dysbiosis, nutritious
food intake reduces the risk of CRC and prebiotic consumption, contributing to the increase and maintenance
of beneficial microorganisms (probiotics). Studies have shown that mushrooms have prebiotic properties.
Carbohydrates, a and b-glucose, xylose, mannose, galactose, and inulin stimulate probiotic growth.
Methodology
The experiments were divided into 3 sections as follows.
Section 1 : Preparing MRS-broth
1.1 Prepare 800 mL. of water in a beaker and mix chemicals from Table 1 on a magnetic stirrer
,and then adjust the water volune to 1000 mL.
Number Chemicals g/L
1 Proteose peptone 10.0
2 10.0
3 Beef extract 4.0
4 Yeast extract 10.0
5 Carbon source 5.0
6 Sodium acetate 2.0
7 Potassium hydrogen phosphate 2.0
8 Triamonium citrate 0.2
9 Magnesium sulphate 7H2O 0.05
10 Magnesium sulphate 4H20 0.5
11 1.0
L.cysteine
Polysorbate 80 (Tween80)
Table 1 : Table showing Chemicals used for MRS-broth
Section 2 : Mushroom extraction
2.1 Dissolved with water mixed in a ratio of 1 gram of mushroom to 10 mL of water (1:10).
2.2 Take extract incubated in a Shaking water bath at 60 celsius for 4 hours.
2.2 After incubation filtrate the extract and centrifuge at 1000 rpm for 10 minutes.
2.3 Take the extract without light sludge to evaporated by vacuum condition at 50 celsius.
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Section 3 : Bacterial culture and record the results
3.1 Dissolved the extract with prepared MRS-broth.
3.2 Measured pH and directed autoclave at 121 celsius for 15 minutes.
3.3 Culture by used MRS-broth with extract and measured Optical density by Spectrophotometer
at 0 24 and 48 hours recorded and compared.
Results
Measurement of probiotic growth in MRS broth supplemented with prebiotic extracted from the
11 type of mushrooms by the optical density; OD from Spectrophotometer 600 nm. Due to making 3 tubes
per sample, the mean and standard deviation (SD) must be determined in each sample. There are Negative
control (without glucose), Positive control (inulin), Control medium (with glucose) and Comparative sample
(Jerusalem Artichoke).
Figure 1: Average OD of Streptococcus thermophilus at 24 and 48 hours
Figure 2: Average OD of Bifidobacterium bifidum at 24 and 48 hours
Conclusion
The results found that the Bamboo mushroom extract (BME) could significantly stimulate the
growth of S. thermophilus at 24 and 48 hours more than other mushroom extracts. Particularly, it could
stimulate the growth of S. thermophilus better than the Jerusalem Artichoke extracts.
The results from B. bifidum, it was found that the BME still stimulated the growth of the
probiotic better than other mushroom extracts, especially at 48 hours. After comparing the growth-
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stimulating effect of BME on the two probiotics, it revealed that S. thermophilus was more responsive to
BME than B. bifidum. The results of this experiment showed that BME had prebiotic properties in which
were better than or equal to the Jerusalem Artichoke extract, depending on the probiotics tested.
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. C.H. Stuelten, C.A. Parent,D.J. Montell, Cellmotility in cancer invasion and metastasis: insights from simple
simple model organisms, Nat. Rev. Cancer 2018; 296-312.
2. C. Fitzmaurice, C. Allen, R.M. Barber, L. BArregard, Z.A. Bhutta, H. Brenner, et al., Global, regional, and
national cancer incidence, mprtality, year of life lost, years lived with disability, and disability-adjusted
life-years for 32 cancer groups, 1990 – 2015: a systematic analysis. For the global burden of disease study,
JAMA Oncol 2017; 524-548.
Ofir E, Oren Y, Adin A. Electroflocculation: the effect of zeta-potential on particle size. Desalination 2007;204:
33-38.
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Title: Comparison of efficacy of bacteriophages OB3_15_02
OPA17 and OTA22 for the removal of
Vibrio campbellii biofilm
Field: Biology and Biodiversity
Authors: Mr. Nuttakorn Na Thong and Mr. Ponrakun Tritsanawasuntra
School: PSU. Wittayanusorn school, Prince of Songkla University
Advisors: Assoc. Prof. Dr. Pimonsri Mittraparp-arthorn, Miss Apinya Boonkhum
and Mr. Surawut Saengmanee
Abstract
Vibrio campbellii is one of the leading causes of luminous vibriosis, especially in post larvae shrimp
cultured in hatcheries. With the proper environment, this bacterium can form a complex structure known as a
biofilm to attach to the surface of the environment. These situations led bacteria and their biofilm to become
more resilience and difficult to remove. This research aimed to study the efficiency of lytic bacteriophages
OPA17 and OTA22 at different multiplicities of infections (MOIs) for the inhibition and removal of
V. campbellii PSU3280 biofilm. The results from this research show that phages OPA17 and OTA22 at the
MOI 1 was the most effective in reducing the number of V. campbellii PSU3280. For the inhibition of biofilm,
phages OPA17 and OTA22 at the highest MOI (10,000) was the most effective in biofilm inhibition when
treated for 4 hours. For biofilm removal, the best efficiency was found in biofilm treated with phage OPA17
at MOI 1 for 4 hours. No differences were found in biofilm treated with phage OPA17 at different MOI and
periods and the biofilm removal efficiency was independent of MOI and treated time. Similar results were
found in phage OTA22, and the results were consistent with the results from the scanning electron microscope
(SEM). In conclusion, both phages at the concentrations of MOI 1 were the most effective in reducing the
planktonic V. cambellii PSU3280. Both phages exhibited good activity to inhibit or remove V. campbellii
PSU3280 biofilm. However, further study about physiological characteristics of these phages is still needed
for further applications.
Keywords: Vibrio campbellii, Bacteriophage, Biofilm removal
Introduction
Vibrio campbellii is one of the economically important microorganisms in cultured shrimp. Many
reports support that this infection can cause diseases in cultured shrimp, such as luminous disease, causing an
increased in mortality rate of shrimp [1]. In addition, V. campbellii is considered as one of the opportunistic
marine pathogens which can form biofilms to persist on shrimp and marine environments. The removal of the
biofilm can be done in several ways, for example by using chemicals or using probiotics, which have many
limitations. Making the use of bacteriophages to remove biofilms is another interesting method because it has
been reported that phages can produce enzymes that can interfere with biofilm formation. Another advantage
is the absence of side effects on endemic microorganisms or animals. The use of phages for the removal of
biofilms of human pathogenic Vibrio species such as V. cholerae, V. parahaemolyticus, and V. harveyi has
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also been reported [2]. However, there is no report regarding the ability of phages to remove biofilms of V.
campbellii. Thus, this study aimed to investigate and compare the efficacy of V. campbellii biofilm removal
by phages OPA17 and OTA22. This study paves the way to the feasibility of applying phages for biofilm
removal and further application in the aquaculture industry.
Methodology
Part 1: Enrichment of V. campbellii lytic phage
V. campbellii PSU3280 was cultured in 5 ml of Tryptic Soy Broth (TSB)+1%NaCl, then
shaken at 150 rpm at 30 °C for 4 hours. After that, 200 microliters (at 108 CFU/ml) of bacteria and 100
microliters (at 107 PFU/ml) of phages were mixed into 3 ml soft agar (TSB + 1%NaCl + 0.7% agar), then
poured onto TSA+1%NaCl, incubated at 30 °C for 16 hours, and observed for plaques (clear zones). Phages
were collected by pouring 3 ml of SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl, pH 7.5) on the
surface of the medium, incubated at 30 °C for 6 hours with shaking at 120 rpm. Then, the phage-containing
SM buffer was harvested, transferred into a test tube, and centrifuged at 10,000 rpm for 10 minutes to
precipitate the bacteria. The supernatant was filtered using a 0.22 µm membrane filter and phages were
enumerated by the double-layer agar method.
Part 2: Investigation of the killing efficacy against planktonic V. campbellii PSU3280
V. campbellii PSU3280 was cultured as described in part 1. Then, cells were adjusted to 0.5
McFarland, diluted to 105 CFU/ml, and 180 µl of bacteria was drawn into a 96 well plate. A total of 20 µl each
of the OPA17 or OTA22 phage at the MOIs 1, 10, 100, 1,000, or 10,000 was added to the 96-well plate,
whereas the untreated group was served as control. The absorbance at OD600 nm was measured with the
microtiter plate reader every 20 minutes for 16 hours.
Part 3: Biofilm inhibition assay
V. campbellii PSU3280 was cultured in marine Luria Bertani (mLB) broth using 96 well plate with
the same condition as described in part 2. Phage OPA17 or OTA22 at the MOIs 1, 10, 100, 1,000, or 10,000
was added to the plate and incubated at 30 °C for 4, 8, and 12 hours. At indicated time, plates were washed
and stained with the 0.1% Crystal Violet (CV). After washing, the OD570 nm were measured using the
microtiter plate reader.
Part 4: Biofilm removal assay
In this part, cells were allowed to form biofilm on 96 well plate prior to phage treatment. Briefly, 200
µl of V. campbellii PSU3280 grown on mLB were grown on 96 well plate (at 105 CFU/ml) and incubated for
48 hours. Then, the medium was removed and the 20 µl of phage in 180 µl of mLB were added to a preformed
biofilm, whereas only the mLB was used in the control group. The plate was incubated at 30 °C for 4, 8, and
12 hours, and the absorbance was measured at OD600 nm. The biofilm was determined by the CV assay at
OD570 nm using the microtiter plate reader.
Part 5: Biofilm visualization by Scanning Electron Microscope (SEM)
To develop preformed biofilms, 1 ml V. campbellii culture was applied to coverslips immersed in a
24-well plate and incubated at 30°C for 48 hours. Phage OPA17 or OTA22 at MOI 10,000 was added in to
the well and incubated as described in part 4. The coverlips were washed and subjected to SEM analysis at the
Office of Scientific Instrument and Testing, Prince of Songkhla University.
OB3_15_02/2
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