Investigating the effects of Bromelain and Papain on Pathogens and Probiotics

1. Group 01-14 Tevin Teo 3S1 Randall Choo 3S2 Nicholas Tan 3A1 Investigating the effects of Bromelain and Papain on Pathogens and Probiotics

2. Contents

3. Introduction

4. 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)

5. 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)

6. Introduction - Papain • Papain is a proteolyticenzyme which has medical uses • Papain possesses antibacterial activity against Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and antifungal activity against Candida albicans (Seenivasanet al., 2010).

7. Introduction to microbes used

8. 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 (Salyersand Whitt, 2002)

9. Microbes - Opportunistic pathogens 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

10. 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)

11. Beneficial Microbes Saccharomyces cerevisiae • Baker’s or brewer’s yeast • Used in bread making and ethanol fermentation

12. Objectives and Hypotheses

13. Objectives To investigate the effects of the crude enzyme extracts of bromelain and papain on the growth of microbes

14. Hypotheses Bromelainand papain extracts will • inhibit the growth of Escherichia coliand Staphylococcus epidermidis • enhance the growth of Lactobacillus casei and Saccharomyces cerevisiae

15. Experimental Methods

16. Apparatus • Autoclave • Incubator • UV-vis spectrophotometer • Biological safety cabinet • Incubator shaker • Centrifuge • Blender • Scalpel / knife

17. 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)

18. Variables

19. Experimental Outline General Procedure

20. Preparation of Agar

21. Preparation of Plant Extracts containing Enzymes

22. Growth of Microorganisms

23. Growth of Microorganisms E. coli Inoculation S. epidermidis

24. Growth of microorganisms L. casei Inoculation

25. Growth of Microorganisms S. cerevisiae Inoculation

26. Testing Effect of Plant Extracts on Growth of Microbes

27. Serial dilution and plating 10-1 dilution 10-3 dilution 10-5 dilution 1ml mixture + 9ml saline 1ml 10-2 dilution+ 9ml saline 1ml 10-4 dilution+ 9ml saline 10-4 dilution 10-2 dilution 1ml 10-3 dilution+ 9ml saline 1ml 10-1dilution+ 9ml saline

28. Serial Dilution and Plating

29. 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 Lactobacilluscaseiin the production of lactic acid and yoghurt

30. 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. PlantaMedica, 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.