DEVELOPMENT OF MICRO-ENCAPSULATED SYNBIOTIC

STREPTOCOCCUS SALIVARIUS K12 AND MUSA ACUMINATA

EXTRACT (OROSYN©) AND ITS EFFECT ON CANDIDA
ALBICANS BIOFILM ISOLATED FROM SPECIAL NEEDS
CHILDREN
NURUL AQILAH BINTI AMRAN
MASTER IN SCIENCE OF PHARMACY
DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY, KULLIYAH OF PHARMACY

SUPERVISOR:
ASSISTANT PROFESSOR TS. DR. MOHD HAFIZ BIN ARZMI
DEPARTMENT OF FUNDAMENTAL DENTAL AND MEDICAL SCIENCES, KULLIYAH OF DENTISTRY

CO-SUPERVISORS:
ASSISTANT PROFESSOR DR. ABD ALMONEM DOOLAANEA
DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY
ASSISTANT PROFESSOR DR. NOORHAZAYTI BINTI AB HALIM
DEPARTMENT OF PUBLIC DENTAL HEALTH
ASSISTANT PROFESSOR DR. NORZAITI BINTI MOHD KENALI
DEPARTMENT OF PAEDIATRIC SCIENCE
 Content
1.0 INTRODUCTION
- PROBLEM STATEMENT
- RESEARCH OBJECTIVES
- SIGNIFICANCE OF STUDY
2.0 LITERATURE REVIEW
3.0 METHODOLOGY
4.0 EXPECTED OUTCOMES
5.0 PROJECT MILESTONE
6.0 CURRENT PROGRESS
7.0 REFERENCES
 1.0 Introduction
Special needs children can be defined as individuals under the age of 18 who have
either physical or cognitive disabilities, including intellectual disability (ID), Down
Syndrome (DS), autism spectrum disorder (ASD), cerebral palsy (CP), and
attention deficit hyperactivity disorder (ADHD) (Ningrum et al., 2021)

Synbiotic is a dietary supplement

that comprises of probiotic and
prebiotic (Hemarajata and
Versalovic, 2013).

Streptococcus salivarius K12

and Musa acuminata skin
extract
 1.1 Problem Statement
A learning disability: a state of functional Performing microencapsulation
limitation that is below the general average in any to increase its biocompatibility,
of the areas of human functionality and most biodegradability, and
importantly in the adaptation to their mucoadhesive properties.
surroundings.

Encapsulated
Oral health of special Synbiotic therapeutic
synbiotic as
needs children effects
antibiofilm agent

Nowadays, people have consumed probiotics and prebiotics as a supplement and treatment
for a variety of ailments. However, it is not known whether synbiotic could provide any
therapeutic effect towards oral infections despite the fact that people have used them for a
multitude of diseases.
 1.2 Research Objectives
To determine the association between parent’s/guardian’s
knowledge, attitude, and practice in the oral health
management of special needs children

To develop OroSYN extract micro-capsules by performing

encapsulation using sodium alginate.

To assess the anti-biofilm activity of micro-encapsulated

OroSYN towards Candida albicans isolated from special
needs children.
 1.3 Significance
To investigate the challenges in maintaining oral health for
special needs children from the parent’s perspectives

To determine the ability of microencapsulated synbiotic in

dental caries inhibition
 2.0 Literature Review
Many difficulties could lead to postponement or
denial of care as a study proven that the
SPECIAL NEEDS behavior of special needs children to be a major
CHILDREN AND ORAL barrier during dental care or treatment whereby
HEALTH children with major outbreaks or tantrums have
higher odds of having unmet dental needs
(Alkhabuli et al., 2019).

Although halitosis could be caused by a multitude

of reasons, most of the cases are related to oral
cavities such as poor oral hygiene, periodontal
disease, tongue coat, food impaction, unclean
HALITOSIS
dentures, faulty restorations, oral carcinomas, and
throat infections (Aylıkcı & Colak, 2013).
 2.0 Literature Review
....t hey defi ned synbi ot i c s a s “ a mi xtur e of pro bio tic s
and prebi o ti c s t ha t benefi t ing t he ho st by impr ovi ng
the su rvi va l a nd i mpla nt a t i o n o f l i ve mic ro bi al di etary
supple ment s i n t he ga st ro int e st inal tr ac t thro ugh ORAL SYNBIOTIC
sel ec ti ve ly st imu la t i ng t he gro w t h and/o r a c tivati ng
the met a bo li sm o f one o r a li mit ed number of heal th-
pr omo ti ng ba c t eri a (Gi bso n & Ro berf roi d, 1995)

. .Th e eff ect of S . sa liva rius K 1 2 on ora l h e a lth wa s previously

Streptococcus d iscusse d (Is h ijim a et a l. , 2 0 1 2 ; M oon , Moon , L ee & Ch o,
salivarius K12 2 0 1 6 ; Zup a n cic, K riks ic, K ova cevic, & K ova cevic, 2 0 1 7 ).

Musa acum inata has be e n re co gnise d as a preb iotic that m ay hav e

the potential to induce o ral he alth ((Nazre n e t al., 2014). Musa
....h ave been sh own to possess antim ic robial an d antioxidant
pr operties (Gonzále z-Mon te lon go, Lob o, & Gonzalez. 2010; Ch abuc k acuminata
et al., 2013; Fadhilah , Jalan i, Moham ad, Nazatul, & Shahidan, 2014).
 2.0 Literature Review
..Algina tes a re heteropo lymer ca rbo xylic
ENCAPSULATION USING a cids linked by1 ,4 -glyco sidic bonds o f p-
SODIUM ALGINATE D- ma nnu ronic ( M) and a -L- gulu ronic
a cid( G) units ( Hamed et a l., 20 1 7 ).

CANDIDA ALBICANS
 3.0 Methodology
3.1 QUESTIONNAIRE ON THE ORAL
HEALTH OF SPECIAL NEEDS
CHILDREN.

3.2 ENCAPSULATION OF OROSYN.

3.3 THE ANTI-BIOFILM ACTIVITY OF

OROSYN TOWARDS CANDIDA
ALBICANS
 3.1 Questionnaire on the oral health of special needs children

&
Dental polyclinic of Kuliyyah of Dentistry,
Telegram
International Islamic University Malaysia Example of
(IIUM).
Group
questionnaire
Where?

Parents with special needs children

under the age of 18 years old

Who?

One month

Duration?

Spearman correlation
coefficient

how to
analysis?
Chi-square Test
T-test
 3.2 Encapsulation of Orosyn.

RAW MATERIAL FABRICATION OF OROSYN

ALGINATE MICROPARTICLES
GROWTH OF BACTERIA 2% sodium alginate
Streptococcus salivarius K12 will be sub-cultured on Ionic gelation method will be
Brain Heart Infusion (BHI) agar (Difco, USA) and carried out utilising the
aerobically incubated for 24 hours at 37 C°. 1x106 cells/mL of S. 100 mg/mL M.
electrospray technique to
salivarius K12 acuminata skin extract
encapsulate OroSYN in alginate
MUSA ACUMINATA EXTRACTION microparticles using a method
M. acuminata skin will be cut into small pieces, adapted from research by
oven-dried at 50°C for three days and ground to a Alallam et al., (2020).
fine powder using a mechanical blender (Philips,
Malaysia).
ALGINATE MIXTURE

MICROCAPSULES TESTING
FORMULATION OF
PARTICLE SIZE AND ZETA POTENTIAL ANALYSIS
Particle size will be measured using laser diffraction technique and zeta potential will
MICROPARTICLE POWDER
be determined by dynamic light scattering (Zetasizer Nanoz, Malvern Instrument,
UK) After microparticle preparation, the
filtered microparticle will be converted
TRANSMISSION ELECTRON MICROSCOPE (TEM)
The microstructure (size and surface morphology) of the microparticle will be into powder to increase the storage
observed by the Libra 120 Transmission Electron Microscope (Zeiss, germany). stability. The microparticles will be added
with mannitol as lyopreservatives then
ATR-FTIR
ATR-FTIR spectra of lyophilised blank alginate microparticle and the loaded alginate they will be subjected to freeze-drying
microparticle will be obtained by scanning from 400 to 4000 cm-1 at 4 cm-1 resolution
at room temperature using Perkin Elmer's spectrophotometer (L1600401 Spectrum
two DTGS, llantrisant, UK).
 3.2 Encapsulation of Orosyn

light PC

camera
1% CaCl2 with
magnetic stirrer

Alginate
voltage supply mixture

syringe pump
 3.3 The anti-biofilm activity of OroSyn towards Candida albicans
C. albicans American Type Cell Culture
(ATCC) MYA-4901, and three yeast strains
from different patients (randomly isolated
from special needs children) will be used.
The suspensions will be
mixed thoroughly using
a vortex mixer for 30
seconds.
The cell suspensions, together with
OroSYN microcapsules will be
pipetted into a sterile 96-well plate and
incubated for 72 hours at 37 C°. The
medium will be replenished aseptically
every 24 hours.
C. albicans strains will be sub-cultured on
yeast peptone dextrose (YPD) agar (Difco,
USA) and will be incubated at 37◦C
aerobically for 24 hours.
C. albicans will be re-suspended in RPMI-1640
and standardized to give a final cell density of
105 cells/ml, which equivalent to OD620
nm0.5.
Then, the plates will be subjected to
Crystal Violet Assay according to the
protocol outlined by Alnuaimi et al.
4.0 Expected Outcome
Findings from the focus groups will describe barriers
in maintaining oral health for their special needs
children

Successful microencapsulation of OroSYN and

increase its biocompatibility for it to be used in dental
products.

OroSYN microcapsule could help in reducing oral infections

by performing a significant anti-biofilm activity against C.
albicans isolated from special needs children.
 5.0 Project Milestones
2022 2023
Projects
Activities FEB APR JUNE JULY AUG SEPT OCT DEC JAN FEB MAC
Proposal writing and
defence
IREC application

Literature review
Sample collection

OroSYN
microencapsulation
Biofilm study

Data analysis

Report preparation

Thesis submission
 6.0 Progress Reports

GROWTH OF MICROENCAPSULATION
Attended Hands-on: QUESTIONNAIRE OF OROSYN
Systematic Review and BACTERIA
IREC submission Optimizing the
Meta-analysis Workshop for C. albicans American Type
(24th Feb 2022) parameters
Cell Culture (ATCC) MYA-
ICOMOI 2021
4901 and S. Salivarius K12 (March 2022)
(FACILITATOR)
(Feb 2022)
6.0 Progress Reports
 6.0 Progress Reports
TEST RUN 1 TEST RUN 2
• 1% Alginate solution • 1% Alginate solution
• 1% Calcium Chloride • 1% Calcium Chloride
• 800mg/ml banana extract • 800mg/ml banana extract
• Voltage: 5.0kv • Voltage: 6.2kv
• Flow Rate: 5.0 ml/h • Flow Rate: 5.0 ml/h
TEST RUN 3
• 2% Alginate solution
• 1% Calcium Chloride
• 100mg/ml banana extract
• Voltage: 7.0kv
• Flow Rate: 2.0 ml/h
 7.0 References
1. Ningrum, V., Bakar, A., Shieh, T-M. & Shih, Y-H. (2021). The Oral Health Inequities between Special Needs Children and Normal Children in Asia: A Systematic Review and Meta-
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2. Hemarajata, P., & Versalovic, J. (2013). Effects of probiotics on gut microbiota : mechanisms of intestinal immunomodulation and neuromodulation. Therapeutic Advances in
Gastroenterology, 6(1), 39–51. https://doi.org/10.1177/1756283X12459294
3. Alkhabuli, J. O. S., Essa, E. Z., Al-Zuhair, A. M., & Jaber, A. A. (2019). Oral Health Status and Treatment Needs for Children with Special Needs: A Cross-Sectional Study. Pesquisa
Brasileira Em Odontopediatria e Clínica Integrada, 19(1), 1–10. doi:10.4034/pboci.2019.191.127
4. Aylıkcı, B. U., & Colak, H. (2013). Halitosis: From diagnosis to management. Journal of natural science, biology, and medicine, 4(1), 14–23. https://doi.org/10.4103/0976-9668.107255
5. Gibson, G. R., & Roberfroid, M. B. (1995). Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. The Journal of Nutrition, 125(6), 1401–1412.
https://doi.org/10.1093/jn/125.6.1401
6. Ishijima, S. A., Hayama, K., Burton, J. P., Reid, G., Okada, M., Matsushita, Y., & Abe, S. (2012). Effect of Streptococcus salivarius K12 on the In vitro growth of Candida albicans and its
protective effect in an oral candidiasis model. Applied and Environmental Microbiology, 78(7), 2190–2199. https://doi.org/10.1128/AEM.07055-11
7. Moon, Y., Moon, J., Lee, M., & Cho, J. (2016). Antibacterial Effects of Streptococcus Salivarius K12 on Oral Bacteria. International Journal of Clinical Preventive Dentistry, 12(4),
209–219.
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Thank you!