Post on 19-Dec-2015
Investigating the effects of Bromelain
and Papain on Pathogens and
ProbioticsGroup 01-14Tevin Teo 3S1Randall Choo 3S2Nicholas Tan 3A1
ContentsIntroduction
Objectives
Hypotheses
Enzymes used
Microbes used
Potential applications
Methodology
Materials
Apparatus
Variables
Procedures
Introduction - Bromelain
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)
Introduction - Papain
• 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)
Introduction - Papain
• 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
Microbes
Opportunistic Pathogens
Staphylococcus
epidermidis
Escherichia coli
Beneficial microbes Lactobacillus
casei
Saccharomyces cerevisiae
Microbes - Opportunistic pathogens
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
Microbes - Opportunistic pathogens
Beneficial Microbes
Lactobacillus casei
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
Beneficial Microbes
Objectives
To investigate the effects of the crude enzyme extracts of bromelain and papain on the growth of microbes
Escherichia coli
Staphylococcus epidermidis
Harmful
microbes Lactobacillu
s casei
Saccharomyces cerevisiae
Beneficial
microbes
Bromelain and papain extracts will
inhibit the growth of Escherichia coli and Staphylococcus epidermidis
enhance the growth of Lactobacillus casei and Saccharomyces cerevisiae
Hypotheses
Apparatus
Autoclave Incubator UV-vis spectrophotometer Biological safety cabinet Incubator shaker Centrifuge Blender Scalpel / knife
Materials
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)
Variables
Controlled Independent Dependent
Temperature of growth of bacteria (30C)
Species of bacteria• S.
epidermidis• E. coli• L. casei• S. cerevisiae
Growth rate of bacteria measured by:• Absorbance
at 600 nm• Colony
forming unit
Concentration of extract (1 g in 10 ml saline)
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
General ProcedureExperimental Outline
Preparation of Agar
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
Preparation of Plant Extracts containing Enzymes
Skin of pineapple
and unripe papaya
are removed
Cut into small pieces
and blended
in normal saline (1g per 10ml)
Mixture is centrifug
ed at 7000 rpm
for 10 min
Supernatant
containing crude
enzyme extract is collected
Growth of Microorganisms
Inoculation of bacteria into broth and incubation at 30C 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
Testing Effect of Plant Extracts on Growth of Microbes
Incubated at 30C for 1 day with shaking
Absorbance is taken at 600 nm (correlated with growth)
5 replicates of each set-up are prepared0.1 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)
Cells are inoculated into their respective broths (preculture)
Incubated at 30C with shaking for 1 day
Serial dilution and plating
1ml mixture + 9ml saline
10-1 dilution
1ml 10-1 dilution + 9ml saline
10-2 dilution
1ml 10-2 dilution + 9ml saline
10-3
dilution
1ml 10-3 dilution + 9ml saline
10-4 dilution
1ml 10-4 dilution + 9ml saline
10-5 dilution
Serial Dilution and Plating
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
Applications
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
References
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.