EFFECT OF EXTRACTION TIME AND RATIO OF SAMPLESOLVENT ON ANTIBACTERIAL ACTIVITY AND TOTAL PHENOLIC COMPOUND OF Caulerpa lentilifera PENGARUH WAKTU EKSTRAKSI DAN RATIO SAMPEL-PELARUT TERHADAP AKTIVITAS ANTIBAKTERIAL DAN TOTAL KOMPONEN FENOL PADA Caulerpa lentilifera BACHELOR THESIS
Submitted to the Faculty of Agricultural Technology in partial fulfillment of the requirements for obtaining Bachelor Degree
By: DEA NATHANIA HENDRYANTI 11.70.0097
DEPARTMENT OF FOOD TECHNOLOGY FACULTY OF AGRICULTURAL TECHNOLOGY SOEGIJAPRANATA CATHOLIC UNIVERSITY SEMARANG 2015
EFFECT OF EXTRACTION TIME AND RATIO OF SAMPLESOLVENT ON ANTIBACTERIAL ACTIVITY AND TOTAL PHENOLIC COMPOUND OF Caulerpa lentilifera PENGARUH WAKTU EKSTRAKSI DAN RATIO SAMPELPELARUT TERHADAP AKTIVITAS ANTIBAKTERIAL DAN TOTAL KOMPONEN FENOL PADA Caulerpa lentilifera
By : DEA NATHANIA HENDRYANTI NIM :11.70.0097 Department : Food Technology This thesis has been approved and defended in front of the examination committee at February 24th, 2015 Semarang, February 24th, 2015 Faculty of Agricultural Technology Soegijapranata Catholic University
Supervisor
Dean
Dr. Ir. Lindayani, MP.
Dr. Victoria Kristina Ananingsih, ST., MSc.
. Co-Supervisor
Kartika Puspa Dwiana, STP., MSi.
STATEMENT OF THESIS AUTHENTICITY
With this I state that Thesis with title “EFFECT OF EXTRACTION TIME AND RATIO OF SAMPLE-SOLVENT ON ANTIBACTERIAL ACTIVITY AND TOTAL PHENOLIC COMPOUND OF Caulerpa lentilifera” there is no work that has been proposed to get academic title on University, and as long as I know there is none work or opinion that had been wrote or published by another people, except that has been writing is referred in this manuscript and mentioned in references. If someday part or whole of this Thesis is proved and founded as plagiarism, then I deserved to be canceled with any risk of its punishment as the regulation that applicable in Soegijapranata Catholic University and/or the applicable of legislation.
Semarang, February 24th, 2015
Dea Nathania Hendryanti NIM : 11.70.0097
SUMMARY Seaweed are renewable living resource which has antibacterial potential. Nowdays, many researchers interested with Caulerpa lentilifera, a round seagrape which is also distributed in Indonesia (local name : Lato) but still poorly investigated in antibacterial activity. In fact, it has higher phenolic compound, which is act as an antibacterial agent in macroalgae, compared with almost all chlorophyta, phaeopyhta and rhodophyta species. The objectives of this research are to investigate the effect of extraction time (1, 2, 3 days) and ratio of sample-solvent (1:5, 1:10, 1:15 wv-1) in ethyl acetate extraction on antibacterial activity of dry C. lentilifera by using disk diffusion method followed by minimum inhibitory concentration (MIC) at 120, 60, 30, 15, and 7.5 µl and minimum bactericidal concentration (MBC) test against Gram-negative and Gram-positive bacteria. To determine the total phenolic compound to find out the relationship between antimicrobial activity and phenolic compound. The pathogenic bacteria used in this research are Staphylococcus aureus (FNCC 167), Escherichia coli (FNCC 194), Salmonella typhimurium (FNCC 0050) and Bacillus cereus (FNCC 0057). The antibacterial testing showed that inhibitory effect of C. lentilifera increased by increasing time of extraction and ratio of sample-solvent. The highest inhibitory action was achieved maximum level on 72 hours of extraction with ratio 1:15 wv-1. Caulerpa lentilifera has MIC value around 1.5 – 6 mg ml-1 which is classified as strong inhibitor and ratio of MBC : MIC value less than 4 which is considered as bactericidal agent against both Gram-negative and Gram-positive bacteria. Correlation analysis using SPSS showed very strong and very significant correlation between total phenolic compound and antibacterial activity with correlation coefficient is 0.594** and significant value is 0.001 at confident level of 95%. This correlation connection is directly proportional, means that the increasing amount of phenolic will followed by the increasing level of antibacterial activity of Caulerpa lentilifera. This research indicate that Caulerpa lentilifera possess a potential antibacterial activity against human pathogenic bacteria and one of its antibacterial agent is phenolic compound.
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RINGKASAN Rumput laut adalah sumber daya alam yang memiliki potensi sebagai agen antibakterial. Saat ini, banyak peneliti yang tertarik dengan Caulerpa lentilifera atau anggur laut bulat yang juga banyak terdapat di Indonesia (nama lokal : Lato) namun, masih sedikit penelitian terhadap aktivitas antibacterial dari rumput laut tersebut. Bukti menjelaskan bahwa rumput laut ini memiliki komponen fenol, yaitu berperan sebagai agen antibacterial pada rumput laut yang lebih tinggi bila dibandingkan dengan hamper selurud spesies chlorophyta, phaeopyhta dan rhodophyta. Tujuan penelitian ini adalah untuk menginvestigasi pengaruh waktu ekstraksi (1,2,3 hari) dan rasio sampelpelarut (1:5, 1:10, 1:15 wv-1) pada ekstrasi etil asetat Caulerpa lentilifera kering terhadap aktivitas antibakterial dengan menggunakan metode difusi kertas cakram dan dilanjutkan dengan uji konsentrasi minimal penghambatan (MIC) pada 120, 60, 30, 15, dan 7,5 µl dan konsentrasi minimal bakterisidal (MBC) melawan bakteri pathogen Gram-negatif dan Gram-positif. Untuk mengetahui total komponen fenol dan hubungannya dengan aktivitas antibakterial pada rumput laut tersebut. Bakteri patogen yang digunakan pada penelitian ini adalah Staphylococcus aureus (FNCC 167), Escherichia coli (FNCC 194), Bacillus cereus (FNCC 0057) dan Salmonella typhimurium (FNCC 0050). Uji antibakterial menunjukkan bahwa dengan meningkatnya waktu ekstraksi dan ratio sample-pelarut maka akan meningkatkan aktivitas penghambatannya. Aktivitas penghambatan terbesar tercapai pada waktu ekstraksi selama 72 jam dengan ratio 1:15 wv-1. Caulerpa lentilifera memiliki nilai MIC berkisar 1,5 – 6 mg ml-1 yang diklasifikasikan sebagai inhibitor kuat dan memiliki nilai ratio MBC:MIC kurang dari 4 sehingga dapat dikategorikan sebagai agen bakterisidal yang dapat melawan baik bakteri Gram-positif maupun Gram-negatif. Analisa korelasi dengan menggunakan SPSS menunjukkan korelasi yang sangat kuat dan sangat signifikan antara total komponen fenol dan aktivitas antibakterial dengan koefisien korelasi sebesar 0,594** dan nilai signifikansi sebesar 0,001 pada tingkat kepercayaan 95%. Hubungan ini saling berbanding lurus, artinya peningkatan jumlah fenol yang dapat terekstrak akan diikuti dengan peningkatan aktifitas antibakterial pada Caulerpa lentilifera. Penilitian ini mengindikasikan bahwa Caulerpa lentilifera memiliki aktivitas antibakterial yang potensial melawan bakteri patogen pada manusia dan salah satu agen antibakterialnya adalah komponen fenol.
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ACKNOWLEDMENT Praise in the name of Jesus Christ, because only His grace and favor, the author has finished the bachelor thesis entitle EFFECT OF EXTRACTION TIME AND RATIO OF SAMPLE-SOLVENT ON ANTIBACTERIAL ACTIVITY AND TOTAL PHENOLIC COMPOUND OF CAULERPA LENTILIFERA. This thesis can be done by prayer, supports, advice and encouragement from several individuals that the author very honored and grateful for. Therefore, the author would like to say special thanks to : 1. Dr. Victoria Kristina Ananingsih, ST., MSc. as the Dean of Faculty of Agricultural Technology who gives me a lot of opportunities that help me build and develop myself. 2. Dr. Ir. Lindayani, MP. as a supervisor and Kartika Puspa Dwiana, STP., MSi. as a Co-supervisor who are always give their time to help me with their great advice and motivation. Thank you for the great mentoring. 3. My parents and families who is always support and pray for every decision that the author made. 4. The laboratory assistants for all the help and support : Mbak Endah, Mas Soleh, Mas Supriyana and Mas Lylyx. 5. Balai Besar Perikanan Budidaya Air Payau (BBPBAP), Jepara and Pak Tri for providing me fresh Caulerpa lentilifera. 6. Yusup Nico Haryanto, who is support all my decisions and being a friend, brother, guardian and family. 7. Vonny Veronica, my partner who fight so hard to accomplished thesis research start from carrying boxes with weight up to 10 kg, or maybe more until support me on report writing days. Thanks a bunch for the help! 8. Dila, Fefe, Aline, Stella Meryl, Anggoro, Melnov, Meilsa, Jenny, Amel, Frisky, Mayang, Manda, Etha, Cik Ameju, and many other best friend in SCU, who has been support and help me during college days and thesis research. 9. All of the Food Technology Department, Van Deventer-Maas Stichting, GLORY 4 (Group of Leader on Research and Society), SALT (Soegijapranata Advance Leadership Training), Student Senate 2012/2013, Internship members with
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Assumption University, International Service Learning participants and committee with Fu Jen University of Taiwan, Generation of 2011 and all related parties who given the author such a great experiences and helped the author finishing this thesis. I realized that this report is still far from perfect and there are still many shortcomings due to the limitations of the author. However, the author hoped that this report can still be an inspiration and provide useful information for all the reader.
Semarang, February 2015
Dea Nathania Hendryanti Author
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CONTENTS Page SUMMARY…………………………………………………….…………………….. i RINGKASAN……………………………………………..……………..……………..…..….. ii ACKNOWLEDMENT….………………………………………...…………..…..… iii CONTENTS………………………………………………………..……………….… v LIST OF TABLES…….…………………………………………………..……….....vii LIST OF FIGURES……………………………………………………………........ viii LIST OF APPENDICES…………………………………………………….......…… ix 1.
INTRODUCTION……………………………………………………………... 1 1.1. Background…………………………………………………………………... 1 1.2. Literature review………………………………………..……………………. 2 1.2.1. Caulerpa lentilifera overview…………………………….………………. 2 1.2.2. Extraction process………………………………………………………… 4 1.2.3. Characteristic of phenolic compound…………………………………….. 4 1.2.4. Pathogenic bacteria………………………………………….…………… 6 1.2.5. Antibacterial testing………………………………………….…………... 7 1.3. Objectives……………………………………………………………………... 9
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MATERIALS AND METHODS……………………………………………….10 2.1. Material………………………………………………………………………. 10 2.2. Method……………………………………………………………………….. 10 2.2.1. Preliminary research……………………………………………………... 10 2.2.1.1. Preparation of sample........................................................................... 10 2.2.1.2. Well diffusion……...………………………………………………… 11 2.2.1.3. Disk diffusion……...………………………………………………… 11 2.2.2. Main research…………………………………………………………….11 2.2.2.1. Plant sample preparation and extraction……………………………... 11 2.2.2.2. Inoculum preparation…………………………………………………12 2.2.2.3. Antibacterial activity of seaweed extract using disk diffusion method………………………………………………………………...13 2.2.2.4. MIC and MBC determination………………………………………... 13 2.2.2.5. Total phenolic compound……………………………………………. 14 2.2.3. Data analysis……………………………………………………………... 14
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RESULT……………………………………………………………………….. 16 3.1. Preliminary result……………………………………………………………. 16 3.2. Main result…………………………………………………………………… 17 3.2.1. Antibacterial activity using disk diffusion method……………………….17 3.2.1.1. Comparison of antibacterial activity of C. lentilifera and commercial antibiotic....…………………………..……………..……19
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3.2.2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)…………………………………………………….. 20 3.2.3. Total phenolic compound………………………………………………... 22 3.2.3.1. Total phenolic compound of Caulerpa lentilifera............................ 22 3.2.3.2. Correlation…………………………...………………………………..23 4.
DISCUSSION………………………………………………………………….. 24 4.1. Preliminary result……………………………………………………………..24 4.1.1. Comparison of agar disk diffusion and agar well diffusion…………….. 24 4.1.2. Choice of solvent………………………………………………………...24 4.1.3. Preparation of sample……………………………………………………25 4.1.4. Effect of concentration of sample………………………………………. 26 4.2. Main result…………………………………………………………………… 27 4.2.1. Effect of ratio sample : solvent (wv-1) on antibacterial activity………... 27 4.2.2. Effect of extraction time on antibacterial activity………………………. 27 4.2.3. Comparison of Caulerpa lentilifera and Amoxicillin as antibacterial Agent………………………………………….....……………...………...28 4.2.4. Determination of MIC and MBC……………………………………….. 30 4.2.5. Correlation between total phenolic compound (TPC) and antibacterial activity…………………………………………………………………... 32
5. CONCLUSIONS AND SUGGENSTIONS………………………………………..34 5.1. Conclusions………………………………………………………….………....34 5.2. Suggestions………..…………………………………………………………... 34 6. REFERENCES…………………………………………………………………..... 35 7. APPENDICES…………………………………………………………………...... 41
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LIST OF TABLE
Table 1. Proximate Composition (g/100g sample dry basis) of Caulerpa lentilifera... 2 Table 2. Inhibition Zone (mm) of C. lentilifera against Staphylococcus aureus……. 16 Table 3. Inhibition zone of C. lentilifera dry extract ……….................…...……...... 17 Table 4. Comparison of inhibition zone (mm) between sample and commercial antibiotic…………………………………..…………………….…...….…. 19 Table 5. MIC and MBC value of C. lentilifera dry extract............................………...20 Table 6. Total Phenolic Content (mg GAE g-1 of extract) of C. lentilifera………..... 22 Table 7. Inhibition zone of C. lentilifera against B. cereus FNCC 0057......................52 Table 8. Inhibition zone of C. lentilifera against E. coli FNCC 194............................ 53 Table 9. Inhibition zone of C. lentilifera against S. thypimurium FNCC 0050........... 54 Table 10. Inhibition zone of C. lentilifera against Staph. aureus FNCC 167.............. 55 Table 11. Similarity of turbidity between sample and one of the control.....................56
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LIST OF FIGURE Figure 1. General morphology (a) and detail of branchelets (b) of Caulerpa lentillifera……………………………………………………...……………. 3 Figure 2. Chemical structure of phenolic compound………………………..……….. 5 Figure 3.General mechanisms of bioactive compound in killing bacteria (a); Cytoplasmic membrane as a place where phenolic compounds are active (b)…...................................................…………………………….… 6 Figure 4. Single colony of B. cereus for preparing working culture……………..….. 12 Figure 5. Comparison between 18 hours of intermediate culture (a) and control (b)...13 Figure 6. Diagram of experimental design………………………………………..…. 15 Figure 7. Inhibition zone of ethyl acetate dry extract at concentration of 100 mg ml-1 using disk diffusion method.……………………………… 17 Figure 8. Inhibition zone of C. lentilifera against S. thypimurium (a), Staph. aureus (b), E.coli (c) and B. cereus (d)…………………………..… 18 Figure 9. Comparison of inhibition zone of Amoxicillin and ethyl acetate extract of C. lentilifera………………………………..…………….....................…20 Figure 10. Negative tubes considered as MIC value (a); Positive tubes (b); pattern background could shows clearer difference of turbidity (c). Red circle marked the similar turbidity between sample and one of the control......... 21 Figure 11. Negative result on minimum bactericidal concentration (MBC) testing using 60 µl of sample extract (100 mg ml-1)............….............………….. 22 Figure 12. Figure of total phenolic compound determination by using Folin-Ciocalteau method............................................................................. 23 Figure 13. Effect of ratio and time of extraction on total phenolic compound............ 23 Figure 14. Standard cureve of Gallic Acid................................................................... 41 Figure 15. Picture of Gallic Acid standard with concentrations 0-100 ppm................ 41 Figure 16. Sample preparation from fresh C. lentilifera to crude extract.................... 51 Figure 17. Picture of 15 µl (a), 30 µl (b), 60 µl (c) and 120 µl (d) of sample against B. cereus (code number 1).............................................................. 57 Figure 18. Picture of 60 µl (a) and 120 µl (d) of sample against E. coli (code number 2)............................................................................................57 Figure 19. Picture of 60 µl (a) and 120 µl (d) of sample against S. thypimurium (code number 3)............................................................................................58 Figure 20. Picture of 15 µl (a), 30 µl (b), 60 µl (c) and 120 µl (d) of sample against Staph. aureus (code number 4).....................................................................58
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LIST OF APPENDICES Appendix 1. Gallic acid standard...........…………………………………………… 41 Appendix 2. Output of One-Way Anova on preliminary result of ethyl acetate dry extract (confidence level of 95%) against Staph. aureus………….. 42 Appendix 3. Output of Two-Way Anova between ratio of solid-liquid and extraction time on inhibition zone of ethyl acetate dry extract of C. lentilifera………………………………………………….....…….. 42 Appendix 4. Output of Independent Two Sample T-Test between inhibition zone of sample (1:15 wv-1 for 72 hours) and amoxicillin…………..…. 46 Appendix 5. Output of Two Way Anova between ratio solid-liquid and extraction time on total phenolic content..…………………………….. 47 Appendix 6. Output Bivariate Correlation between inhibition zone and total phenolic content on C. lentilifera……………………………………… 48 Appendix 7. Mathematics calculation for equalization ratio of sample:amoxicillin... 49 Appendix 8. Mathematics calculation to determine MIC value………................….. 50 Appendix 9. Sample preparation……….………………………...............…….……. 51 Appendix 10. Inhibition zone of ethyl acetate dry C. lentilifera extract….................. 52 Appendix 11. Minimum inhibitory concentration (MIC) of C. lentilifera ...……..… 56 Appendix 12. Minimum bacterial concentration (MBC) of C. lentilifera ...........….. 57
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