Group 01-14
Tevin Teo 3S1
Randall Choo 3S2
Nicholas Tan 3A1

Introduction
Objectives
Hypotheses
Enzymes used
Microbes used
Potential applications
Methodology
Materials
Apparatus
Variables
Procedures



 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)

Staphylococcus epidermidis
Opportunistic
Pathogens Escherichia coli
Microbes
Beneficial microbes
Lactobacillus casei
Saccharomyces cerevisiae

Staphylococcus epidermidis
 Gram-positive bacterium that is part of normal skin flora (Levinson, 2010)
 Forms biofilms on surgical implants, and creates mechanical barriers against antibiotics
 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)
 Main causative agent for urinary tract infections

 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
 Baker’s or brewer’s yeast
 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
Escherichia coli
Beneficial microbes
Lactobacillus casei
Staphylococcus epidermidis
Saccharomyces cerevisiae

Bromelain and papain extracts will

 inhibit the growth of Escherichia coli and Staphylococcus epidermidis enhance the growth of Lactobacillus casei and
Saccharomyces cerevisiae

 Autoclave
 Incubator
 UV-vis spectrophotometer
 Biological safety cabinet
 Incubator shaker
 Centrifuge
 Blender
 Scalpel / knife

 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 Independent
Temperature of growth of bacteria
(30

C) •
Species of bacteria
S. epidermidis
• E. coli
Concentration of
•
• L. casei
S. cerevisiae
(1 g in 10 ml saline)
Dependent
Growth rate of bacteria measured by:
• Absorbance at
600 nm
• Colony forming unit

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 Autoclaved at 10 psi
• MRS – L. casei for 10 min
• Potato dextrose –
S. cerevisiae
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 L. casei into 10 into 10 ml LB broth ml MRS broth
S. cerevisiae into 10 ml PD broth

E. coli
S. epidermidis
Inoculation

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
18 ml broth + 2 ml bromelain (test)
18 ml broth + 2 ml papain (test)
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
10 -5 dilution
1ml mixture +
9ml saline
10 -2 dilution
1ml 10 -1 dilution +
9ml saline
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
 Act as a disinfectant for hospitals to use to decontaminate instruments infected with Staphylococcus epidermidis
 Preserve food and reduce contamination of food with
Escherichia coli
 Increase the production of ethanol if the growth of
Saccharomyces cerevisiae is increased
 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.