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International Journal of Nanomaterials and Biostructures
Universal Research Publications. All rights reserved
ISSN 2277-3851
Original Article
Synthesis of Gold and Silver Nanoparticles from Fermented and Non Fermented
Betel Leaf
Ananda D1, Suresh Babu T V2, Chandrashekhar G Joshi3, Manjula Shantaram4
Research Scholar1, Post Graduate Student2, Assistant Professor3, Professor4
Post Graduate Department of Biochemistry, PG Center, Chikka Aluvara 571232, Somwarpet Taluk,
Kodagu District, Karnataka
Author for correspondence: Dr. Manjula Shantaram
manjula59@gmail.com
Received 12 January 2015; accepted 24 March 2015
Abstract
Piper betel leaves are extensively used in India and the neighboring Southeast Asian countries. Wines are made from other
sources besides grapes. In Kodagu district of Karnataka in India, wines are made from betel leaves too. Alcoholic
beverages act as an important adjuvant to the diet by increasing satisfaction. In the present study, White wild yeast was
isolated from spoiled grape and betel leaf wine was prepared by using the isolated yeast (White wild yeast) and
commercially available yeast. Wine was filtered, separated and the remaining fermented leaves were used to check the gold
and silver nanoparticles. The synthesis of gold and silver nanoparticles from fermented and nonfermented betel leaf extract
was confirmed by UV-Visible spectroscopy.
© 2015 Universal Research Publications. All rights reserved
Key words: Betel leaf, Fermentation, Gold nanoparticles, Silver nanoparticles. .
1. Introduction
Piper betel leaves are widely used as a post meal mouth
freshener and the crop is extensively grown in India, Sri
Lanka, Malaysia, Thailand, Taiwan and other Southeast
Asian countries. Due to strong pungent aromatic flavor,
betel leaves are used as masticators by the Asian people. Its
common names are betel (English) and paan (in Hindi).
The leaves of this plant are economically and medicinally
important and have been traditionally used in India and
China. In Thailand leaves are used to prevent oral malodor
since it has an antibacterial activity against obligate oral
anaerobes responsible for halitosis [1]. The leaves of P.
betel have a strong pungent and aromatic flavor and are
used as a mouth freshener, in wound healing as a digestive
and pancreatic lipase stimulant [2], antioxidant [3]
antifungal,
antibacterial
[4,5]
anti-inflammatory,
bioprotective [6] and antidiabetic [7] agent.
Nanotechnology is mainly concerned with a
synthesis of nanoparticles of variable sizes, shapes,
chemical compositions and the potential use for human
benefits. Production of nanoparticles can be achieved
mainly through three methods such as, chemical, physical
and biological. Since noble metal such as gold, silver and
platinum nanoparticles are widely applied to human
20
contacting areas, a large variety of possible biomedical
applications have been examined, ie, drug and gene
delivery [8,9] protein and pathogens detection,
deoxyribonucleic acid labeling, fluorescent labeling, tissue
engineering, photothermal ablation and contrast agents for
magnetic resonance imaging and other imaging
methods[10].There is a growing need to develop
environmental friendly processes of nanoparticle synthesis
that do not use toxic chemicals. Biological methods of
nanoparticle synthesis using microorganism, enzyme, and
plant or plant extract have been suggested as possible ecofriendly alternatives to chemical and physical methods [11].
Annamalai et al, have synthesised and characterized silver
and gold nanoparticles using aqueous leaf extract of
Phylanthus amarus Schum and Thonn [12]. Memecylon
edule leaf extract showed intense peak at 560 nm and also
for silver nanoprticles at 475nm in UV visible spectroscopy
[13]. In Amaranthus spinosus characterization of gold
nanoparticles was done with UV-Vis study which exhibited
the typical surface plasmon resonance property of the
colloidal solution which shows intense peak at 535-565nm
[14].
However, there are no reports so far on the gold
and silver nanoparticles from fermented and nonfermented
International Journal of Nanomaterials and Biostructures 2015; 5(1): 20-23
betel leaves. Therefore, this study was aimed at
synthesizing the gold and silver nanoparticles in the
fermented and nonfermented betel leaves prepared with
different concentrations of betel leaves using White wild
yeast and Commercial yeast.
2. Materials and Methods
colony was picked, streaked on YEPDA slant to obtain
pure culture and incubated. Pure culture which was
obtained was stored in refrigerator for future use and it has
been re-cultured for every ten days.
Fig. 2: Microscopic view of White wild Yeast (10X).
Fig. 1: Betel leaves
The betel (Piper betel) is the leaf of a vine
belonging to the Piperaceae family (Fig.1), which includes
pepper and kava. It is valued both as a mild stimulant and
for its medicinal properties. Betel leaf contains moisture
85.4 per cent, protein 3.1 per cent, fat 0.8 per cent, minerals
2.3 per cent, fiber 2.3 per cent and carbohydrates 6.1 per
cent per 100 grams. Its minerals and vitamin contents are
calcium, carotene, thiamine, riboflavin, niacin and vitamin
C. Its calorific value is 44 [15].
Betel leaf is mostly consumed in Asia and
elsewhere in the world by some Asian emigrants, as betel
quid or paan, with or without tobacco, in an addictive
psycho-stimulating and euphoria-inducing formulation with
adverse health effects. The betel plant is an evergreen
and perennial creeper, with glossy heart-shaped leaves and
white catkin and it needs a compatible tree or a long pole
for support. Betel requires high land and especially fertile
soil.
2.1 Isolation, identification and pure culturing of wild yeast
One gram of spoilt grape was taken and serially diluted by
using sterilized saline solution in test tubes. 100µl of
inoculum were spread on YEPDA (yeast extract, peptone,
dextrose and agar) media [16] and incubated at 28°C for
three to four days. Yeast was identified based on colony
morphology and microscopic observations (Figure 2). A
21
2.2 Fermentation of betel leaves
2.2.1 Inoculum preparation
In a clean and dried 250 ml conical flask, 100 ml of YEPD
(yeast extract, peptone and dextrose) broth was taken,
plugged using cotton, sterilized and then cooled. Two loops
full of wild yeast was added to one conical flask and 0.05g
commercial yeast (Saccharomyces cerevisiae) was added to
another, incubated at 28°C on rotary shaker for 24 hrs.
Betel leaves were taken and sterilized using 1% sodium
hypochlorite and washed with distilled water. Betel leaves
were cut into small pieces.
In clean and dried eight (250 ml) conical flasks,
small pieces of betel leaf and distilled water were added
with different dilutions 1:10 (15 g leaf in 150 ml distilled
water), 1:15 (10 g leaf in 150 ml distilled water), 1: 20 (7.5
g leaf in 150 ml distilled water) and were labeled as given
below: Commercial yeast 01:10 (CY01:10); Commercial
yeast 01:15 (CY01:15); Commercial yeast 01:20
(CY01:20); White wild yeast 01:10 (WY01:10); White
wild yeast 01:15 (WY01:15); White wild yeast 01:20
(WY01:20); Control 01:10 (C01:10); Control 01:15
(C01:20). To each conical flask 20g of sugar was added
and heated at 60°C for 30 minutes and it was allowed to
cool at room temperature. 10% of commercial and white
wild yeast inocula were added to the respective conical
flasks and plugged using cotton in aseptic condition. The
contents were stirred till 2 days and then it was kept under
static condition at room temperature. After 21 days it was
filtered, fermented betel leaves were separated and wine
was pasteurized then stored for the future use.
Fermented betel leaves and nonfermented (fresh)
betel leaves were sterilized using 1% sodium hypochlorite
solution then washed by using distilled water.
Gold nanoparticles were synthesized using 1 mM
Chloroauric acid (HAuCl4) and Silver nanoparticles were
synthesized by using 1 mM silver nitrate [17]
3. Results and Discussion
3.1. Biosynthesis of gold nanoparticles:
Change in colour from light yellow to pink
in nonfermented betel leaf (Fig. 3) and from light yellow to
International Journal of Nanomaterials and Biostructures 2015; 5(1): 20-23
Fig. 3: Nonfermented betel leaf extract
Fig. 5: UV-Visible spectrum of gold nanoparticles
synthesized using fresh nonfermented leaf extract
Fig. 6: UV-Visible spectrum of gold nanoparticles
synthesized using fermented betel leaf extract due to
excitation of surface plasmon vibrations in gold
nanoparticles
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Fig. 4: Fermented betel leaf extract
wine red in fermented betel leaf (Fig. 4) after the addition
of gold chloride (HAuCl4) solution confirmed the presence
of gold nanoparticles.
Here, we can observe that fresh (nonfermented) betel leaf
extract contains less gold nanoparticles (Fig.5). Punuri et al
obtained similar results from nonfermented betel leaf which
displayed an intense peak at 547 nm for 0.5 mM HAuCl4
[18].
Fermented betel leaf extract contained more of
gold nanoparticles (Fig. 6). This is because of release of
secondary metabolites from the yeast during fermentation.
This aspect has to be further investigated. The peak formed
between 500-800 nm confirms the formation of gold
nanoparticles in the solution. The appearance of the peak is
due to the size dependant quantum mechanical
phenomenon called Surface Plasmon Resonance (SPR).
3.2 Biosynthesis of silver nanoparticles:
There was no color change from light yellow to wine red in
fresh, nonfermented as well as fermented betel leaf after the
addition of silver nitrate (AgNO3) solution which indicated
the absence of silver nanoparticles. Hence it is realized that
silver nanoparticles cannot be synthesized either from
fermented or nonfermented betel leaves in our laboratory
set up.
4. Conclusion:
The synthesis of gold nanoparticles from nonfermented and
fermented betel leaf extract was confirmed by UV visible
spectroscopy. Silver nanoparticles cannot be synthesized
either from fermented or nonfermented betel leaves.
Acknowledgements
We are thankful to the Department of PG studies &
Research in Biochemistry, P G Centre, Chikka Aluvara,
Mangalore University, Karnataka, for providing all the
necessary facilities required for the successful completion
of the project. Timely help of Mr. Rajkumar S. Meti,
Assistant Professor, Department of PG studies & Research
in Biochemistry, P G Centre, Chikka Aluvara, Mangalore
University by providing chemicals is gratefully
acknowledged.
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Source of support: Nil; Conflict of interest: None declared
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International Journal of Nanomaterials and Biostructures 2015; 5(1): 20-23