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

2. IntroductionObjectivesHypothesesEnzymes usedMicrobes usedPotential applicationsMethodologyMaterialsApparatusVariablesProcedures

4.  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. 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. 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).

7. Introduction to microbes used Microbes Opportunistic Pathogens Staphylococcus epidermidis Escherichia coli Beneficial microbes Lactobacillus casei Saccharomyces cerevisiae

8. 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)

9. 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. 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. Saccharomyces cerevisiae  Baker’s or brewer’s yeast  Used in bread making and ethanol fermentation

13. To investigate the effects of the crude enzyme extracts of bromelain and papain on the growth of microbes Escherichia coli Staphylococcus epidermidis Harmful microbes Lactobacillus casei Saccharomyces cerevisiae Beneficial microbes

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

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

17.  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. ControlledIndependentDependent Temperature of growth of bacteria (30  C) 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)

19. 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 Procedure

20. 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

21. 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

22. 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

23. E. coli Inoculation S. epidermidis

24. L. casei Inoculation

25. S. cerevisiae Inoculation

26. Incubated at 30  C for 1 day with shaking Absorbance is taken at 600 nm (correlated with growth) 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) Cells are inoculated into their respective broths (preculture) Incubated at 30  C with shaking for 1 day

27. 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

28. 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

29. 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

30.  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.