Transcript Investigating the effects of Bromelain and Papain

Group 01-14
Tevin Teo 3S1
Randall Choo 3S2
Nicholas Tan 3A1
Introduction
Methodology
Objectives
Materials
Hypotheses
Apparatus
Enzymes used
Variables
Microbes used
Procedures
Potential applications
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Bromelain is an enzyme found in pineapples, abundant in
the stem
It is a proteolytic enzyme that digests proteins
Has anti-inflammatory properties, as it directly degrades
fibrin and fibrinogen which are soluble proteins present in
blood plasma (Lotz-Winter, 1990)
• Papain is obtained from the skin of unripe papaya as
latex
• It acts as a debris-removing agent such as caries as this
involves the cleavage of polypeptide chains and
hydrolysis of collagen cross linkages (Amri and
Mamboya, 2012)
• Papain is a proteolytic enzyme which has medical
uses
• Papain possesses antibacterial activity against
Pseudomonas aeruginosa, Bacillus subtilis,
Staphylococcus aureus and antifungal activity
against Candida albicans (Seenivasan et al., 2010).
Introduction to microbes used
Staphylococcus
epidermidis
Opportunistic
Pathogens
Escherichia coli
Microbes
Beneficial
microbes
Lactobacillus
casei
Saccharomyces
cerevisiae
Staphylococcus epidermidis
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Gram-positive bacterium that is part
of normal skin flora (Levinson, 2010)
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Forms biofilms on surgical implants,
and creates mechanical barriers
against antibiotics
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Patients implanted with contaminated
devices contract infections (Salyers
and Whitt, 2002)
Escherichia coli

A food-borne pathogen that can
cause food poisoning

In the USA, hamburger meat
company Topps once recalled 21.7
million pounds of beef due to
potential E. coli contamination
(Dippold, 2005)
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Main causative agent for urinary
tract infections
Lactobacillus casei
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A probiotic found in yoghurt
and fermented milk

Effective in alleviation of
gastrointestinal pathogenic
bacterial diseases, especially
in children (World Health
Organization, 2002)
Saccharomyces cerevisiae
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Baker’s or brewer’s yeast
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Used in bread making and
ethanol fermentation
To investigate the effects of the crude enzyme extracts of
bromelain and papain on the growth of microbes
Harmful
microbes
Beneficial
microbes
Escherichia coli
Lactobacillus
casei
Staphylococcus
epidermidis
Saccharomyces
cerevisiae
Bromelain and papain extracts will
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inhibit the growth of Escherichia coli and Staphylococcus
epidermidis
enhance the growth of Lactobacillus casei and
Saccharomyces cerevisiae
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Autoclave
Incubator
UV-vis spectrophotometer
Biological safety cabinet
Incubator shaker
Centrifuge
Blender
Scalpel / knife
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Unripe papaya and pineapple
Escherichia coli ATCC 25922
Staphylococcus epidermidis ATCC 12228
Lactobacillus casei (from Yakult)
Saccharomyces cerevisiae (Carolina)
Luria-Bertani medium (for growth of E. coli)
Potato dextrose medium (for growth of yeast)
MRS medium (for growth of Lactobacillus)
Normal saline (0.85% sodium chloride)
Controlled
Temperature of
growth of bacteria
(30C)
Independent
Dependent
Growth rate of
Species of bacteria bacteria measured
• S. epidermidis
by:
• E. coli
• Absorbance at
Concentration of
• L. casei
600 nm
extract
• S. cerevisiae
• Colony forming
(1 g in 10 ml saline)
unit
General Procedure
Preparation of agar for microbe
growth
Preparation of plant extracts
containing enzymes
Growth of microorganisms
Testing effect of plant extracts on
growth of microorganisms
Serial dilution and plating
Types of agar
• LB – E. coli,
S. epidermidis
• MRS – L. casei
• Potato dextrose –
S. cerevisiae
Autoclaved at 10 psi
for 10 min
Poured onto Petri
dishes, solidified
and dried
Skin of
pineapple
and unripe
papaya are
removed
Cut into
small
pieces and
blended in
normal
saline (1g
per 10ml)
Mixture is
centrifuged
at 7000 rpm
for 10 min
Supernatant
containing
crude
enzyme
extract is
collected
Inoculation of bacteria into broth and incubation
at 30C for 1 day with shaking
E. coli and
S. epidermidis
into 10 ml LB
broth
L. casei into 10
ml MRS broth
S. cerevisiae
into 10 ml PD
broth
E. coli
Inoculation
S. epidermidis
L. casei
Inoculation
S. cerevisiae
Inoculation
Cells are inoculated into their respective broths (preculture)
Incubated at 30C with shaking for 1 day
5 replicates of each set-up are prepared
0.1 ml preculture +
18 ml broth + 2 ml
bromelain (test)
0.1 ml preculture +
18 ml broth + 2 ml
papain (test)
0.1 ml preculture +
18 ml broth + 2 ml
saline (control)
Incubated at 30C for 1 day with shaking
Absorbance is taken at 600 nm (correlated with growth)
10-1
dilution
10-3
dilution
1ml mixture +
9ml saline
10-2
dilution
1ml 10-1 dilution +
9ml saline
10-5
dilution
1ml 10-2 dilution +
9ml saline
10-4
dilution
1ml 10-3 dilution +
9ml saline
1ml 10-4 dilution +
9ml saline
Serial
dilution
until 10-5 of
original
0.1 ml of
10-4 and
10-5 diluted
cultures are
plated on
LB, MRS or
PD agar
Plates are
incubated
at 30
overnight
and
number of
colonies is
determined
To further
confirm
results,
compare
with
control set
ups
If our hypotheses are correct, bromelain and papain are able to
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Act as a disinfectant for hospitals to use to decontaminate
instruments infected with Staphylococcus epidermidis
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Preserve food and reduce contamination of food with
Escherichia coli
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Increase the production of ethanol if the growth of
Saccharomyces cerevisiae is increased
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Enhance growth of Lactobacillus casei in the production of
lactic acid and yoghurt
 Amri ,E. and Mamboya, F. (2012). Papain, a plant enzyme of biological importance: a
review. American Journal of Biochemistry and Biotechnology, 8(2), 99-104. Retrieved
March 22, 2014 from http://www.thescipub.com/ajbb.toc
 Fitzhugh, D.J., Shan, S. and Dewhirst, M.W. (2008). Bromelain treatment decreases
neutrophil migration to sites of inflammation. Clinical Immunology, 128(1), 66-74.
 Lotz-Winter, H. (1990). On the pharmacology of bromelain: an update with special
regard to animal studies on dose-dependent effects. Planta Medica, 56(3), 249-253.
 Maurer, H.R. (2001). Bromelain: biochemistry, pharmacology and medical use. Cellular
and Molecular Life Sciences, 58, 1234-1245. Retrieved March 22, 2014 from
http://www.volopharm.de/daten/Bromelain%20biochemistry,%20pharmacology%20and%20medical%20use.pdf
 Seenivasan, R., Roopa, L. and Geetha, S. (2010). Investigations on purification,
characterization and antimicrobial activity of enzyme papain from Carica papaya Linn.
Journal of Pharmacy Research, 3(5), 1092
 Salyers, A. and Whitt, D. (2002).Bacterial Pathogenesis: A Molecular Approach. 2nd ed.
Washington, D.C.: ASM Press.