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

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 and Hypotheses

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

Experimental Methods

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

E. coli

Inoculation

Growth of Microorganisms

S. epidermidis

L. casei

Inoculation

Growth of microorganisms

S. cerevisiae

Inoculation

Growth of Microorganisms

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.