THE EFFECT OF DIFFERENT MEDIUM COMPOSITIONS ON PRODUCTION OF BACTERIOCIN OF LACTIC ACID BACTERIA ISOLATED FROM YELLOW BETUNG BAMBOO SHOOT (Dendrocalamus asper) PICKLE UNDER DIFFERENT FERMENTATION CONDITIONS PENGARUH PERBEDAAN KOMPOSISI MEDIUM TERHADAP PRODUKSI BAKTERIOSIN DARI BAKTERI ASAM LAKTAT YANG DIISOLASI DARI ACAR REBUNG BETUNG KUNING (Dendrocalamus asper) DALAM KONDISI FERMENTASI YANG BERBEDA BACHELOR THESIS Submitted in partial fulfilment of the requirements for a Food Technology Bachelor's degree in Faculty of Agricultural Technology By: LORENTIA SANTOSO 12.70.0078
DEPARTMENT OF FOOD TECHNOLOGY FACULTY OF AGRICULTURAL TECHNOLOGY SOEGIJAPRANATA CATHOLIC UNIVERSITY SEMARANG 2016
THE EFFECT OF DIFFERENT MEDIUM COMPOSITIONS ON PRODUCTION OF BACTERIOCIN OF LACTIC ACID BACTERIA ISOLATED FROM YELLOW BETUNG BAMBOO SHOOT (Dendrocalamus asper) PICKLE UNDER DIFFERENT FERMENTATION CONDITIONS PENGARUH PERBEDAAN KOMPOSISI MEDIUM TERHADAP PRODUKSI BAKTERIOSIN DARI BAKTERI ASAM LAKTAT YANG DIISOLASI DARI ACAR REBUNG BETUNG KUNING (Dendrocalamus asper) DALAM KONDISI FERMENTASI YANG BERBEDA BACHELOR THESIS Submitted in partial fulfilment of the requirements for a Food Technology Bachelor's degree in Faculty of Agricultural Technology By: LORENTIA SANTOSO 12.70.0078 This thesis has been approved and defended in front of the examination committees on 24th February 2016 Semarang, 2nd March 2016 Faculty of Agricultural Technology Soegijapranata Catholic University Supervisor
Dean,
Dra. Laksmi Hartayanie, MP
Dr. V. Kristina Ananingsih, ST, MSc
Co-Supervisor
Dr. Ir. Lindayani, MP
STATEMENT OF THESIS AUTHENTICITY I hereby declare that the thesis entitled “THE EFFECT OF DIFFERENT MEDIUM COMPOSITIONS ON PRODUCTION OF BACTERIOCIN OF LACTIC ACID BACTERIA ISOLATED FROM YELLOW BETUNG BAMBOO SHOOT (Dendrocalamus asper) PICKLE UNDER DIFFERENT FERMENTATION CONDITIONS” contains no work that ever proposed to acquire a bachelorship title in a University, and along to my knowledge, there is no work ever written or published by others, except the ones used as references in this thesis and mentioned in the list of references. If it is proven in the future that partially or whole thesis is the result of plagiarism, therefore I will be willing to be revoked with all the consequences in accordance with the law and regulations applied at Soegijapranata Catholic University and/or valid law and regulations.
Semarang, March 2016
Lorentia Santoso 12.70.0078
SUMMARY Yellow Betung bamboo shoot is one of the bamboo shoot derived from an indigenous bamboo species (Dendrocalamus asper) in Indonesia. However, bamboo shoot as one of foods has a relatively short shelf life and easily damaged. One processing method to extend the shelf life of bamboo shoot is fermentation process. Bamboo shoot that are fermented into pickled bamboo shoot not only can have a longer shelf life, but also it can be one of the natural habitats for the growth of lactic acid bacteria (LAB). One of the capabilities of LAB is to produce antimicrobial compounds, such as bacteriocins. Bacteriocins of LAB were found to have antagonist activity against pathogenic bacteria. However, studies on the influence of fermentation conditions and the composition of the media to the bacteriocin production of LAB which isolated from Yellow Betung bamboo shoot pickle is still limited. Therefore, the aim of this study was to determine the antimicrobial activity of LAB bacteriocins isolated from Yellow Betung bamboo shoot pickle under different fermentation conditions and the medium compositions. A total of eleven isolates of LAB that have been previously isolated from pickled Yellow Betung bamboo shoot in four different fermentation conditions, were tested for their ability to produce bacteriocins. Isolates which isolated from the fermentation condition A (2.5% salt concentration at 15°C) were A20 and A43. While isolates obtained from the fermentation condition B (5% salt concentration at 15°C) were B1, B2, and B32. C18, C19, and C29 were isolated from the fermentation condition C (2.5% salt concentration at 30°C). A total of three isolates were also isolated from the fermentation condition D (5% salt concentration at 30°C), they were D11, D20, and D44. Isolate selection for the production of bacteriocins was based on the ability to against pathogenic bacteria S.aureus (FNCC 0047) and E.coli (FNCC 0091). Bacteriocin antimicrobial activity was tested by removing the effect of organic acids produced by LAB by adjusting the pH of the cell-free supernatant (bacteriocin) to 6.0. Testing the antimicrobial activity of bacteriocins was done by agar-well diffusion method. The inhibitory activity was observed by measuring the clear zone and calculate Activity Unit (AU in mm2 mL-1) of bacteriocins. All LAB isolates were grown in media without supplementation could not produce bacteriocins. Supplementation of carbon and nitrogen sources can induce the production of bacteriocins by LAB. However, only six of LAB isolates (Leuconostoc mesentroides ssp cremoris, Lactobacillus plantarum 1, Lactobacillus plantarum, Lactobacillus fermentum 1, Lactobacillus pentosus) which isolated from bamboo shoot pickle with fermentation condition C and D and also were supplemented with carbon and nitrogen source showed inhibitory activity against S.aureus (FNCC 0047), E.coli (FNCC 0091), and L.monocytogenes (FNCC 0156). Inhibitory activity magnitude of the neutralized cell-free supernatant (bacteriocin) ranged from 113-4663 mm2 mL-1. The highest bacteriocin inhibitory activity was found in bacteriocin D44 (produced by Lb.pentosus) which has been supplemented with 2% glucose and 2% yeast extract. The inhibitory activity of bacteriocins D44 (Lb. pentosus) against S.aureus (FNCC 0047) was equal to 4663 mm2 mL-1, E.coli (FNCC 0091) was 3109 mm2 mL-1, and L.monocytogenes (FNCC 0156) was 3179 mm2 mL-1. The results showed that the initial fermentation conditions (differences in salinity and temperature of fermentation) could determine different characteristics in each LAB produced mainly in terms of the ability to produce bacteriocins as an antimicrobial compound. In this study, was also found that supplementation of carbon and nitrogen sources were able to induce the production of bacteriocins which the result depended on each LAB isolate.
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RINGKASAN Rebung Betung Kuning merupakan salah satu jenis rebung yang berasal dari spesies bambu (Dendrocalamus asper) asli Indonesia. Namun, rebung sebagai salah satu bahan pangan memiliki umur simpan yang relatif singkat dan mudah mengalami kerusakan. Salah satu pengolahan untuk memperpanjang umur simpan rebung adalah melalui proses fermentasi. Rebung yang difermentasi menjadi acar rebung selain dapat memiliki umur simpan yang lebih panjang, juga dapat menjadi salah satu habitat alami untuk pertumbuhan bakteri asam laktat (BAL). Salah satu kemampuan BAL adalah menghasilkan beberapa senyawa antimikroba, diantaranya adalah bakteriosin. Bakteriosin dari BAL ditemukan memiliki aktivitas antagonis terhadap bakteri patogen. Namun, studi tentang pengaruh kondisi fermentasi dan komposisi media terhadap produksi bakteriosin BAL yang diisolasi dari acar rebung Betung Kuning masih terbatas. Oleh karena itu, tujuan penelitian adalah untuk mengetahui aktivitas penghambatan bakteriosin dari BAL yang diisolasi dari acar rebung Betung Kuning dalam kondisi fermentasi dan komposisi media yang berbeda. Sebanyak sebelas isolat BAL yang telah diisolasi sebelumnya dari acar rebung Betung Kuning dalam empat kondisi fermentasi yang berbeda, diuji kemampuannya dalam menghasilkan bakteriosin. Isolat yang diambil dari hasil fermentasi kode A (2,5% kadar garam pada suhu 15°C) adalah A20 dan A43. Sedangkan isolat yang diperoleh dari hasil fermentasi kode B (5% kadar garam pada suhu 15°C) adalah B1, B2, dan B32. Dari hasil fermentasi kode C (2,5% kadar garam pada suhu 30°C) diambil isolat C18, C19, dan C29. Sebanyak tiga isolat juga diambil dari hasil fermentasi kode D (5% kadar garam pada suhu 30°C), yaitu D11, D20, dan D44. Pemilihan isolat untuk produksi bakteriosin didasarkan pada kemampuannya dalam menghambat bakteri patogen S.aureus (FNCC 0047) dan E.coli (FNCC 0091). Daya hambat bakteriosin diuji dengan cara menghilangkan efek asam organik yang dihasilkan oleh BAL dengan menyesuaikan pH supernatan bebas sel (bakteriosin) menjadi 6,0. Pengujian daya antimikroba dari bakteriosin dilakukan dengan metode difusi sumuran agar. Aktivitas penghambatan diamati dengan mengukur zona bening dan menghitung Activity Unit (AU dalam mm2 mL-1) dari bakteriosin. Semua isolat BAL yang ditumbuhkan pada media tanpa suplementasi tidak dapat menghasilkan bakteriosin. Suplementasi sumber karbon dan nitrogen dapat menginduksi produksi bakteriosin oleh BAL. Namun, hanya enam isolat BAL (Leuconostoc mesentroides ssp cremoris, Lactobacillus plantarum 1, Lactobacillus plantarum, Lactobacillus fermentum 1, Lactobacillus pentosus) yang diperoleh dari acar rebung kondisi fermentasi C dan D yang telah disuplementasi dengan sumber karbon dan nitrogen yang mampu menunjukkan aktivitas antimikroba dalam menghambat indikator patogen (S.aureus (FNCC 0047), E.coli (FNCC 0091), dan L.monocytogenes (FNCC 0156)). Besarnya aktivitas penghambatan (AU/Activity Unit) dari supernatan yang telah dinetralkan (bakteriosin) berkisar antara 1134663 mm2 mL-1. Aktivitas penghambatan yang tertinggi ditemukan pada bakteriosin yang dihasilkan oleh isolat D44 (Lb. pentosus) yang telah disuplementasi dengan 2% glukosa dan 2% ekstrak yeast. Aktivitas penghambatan bakteriosin D44 (Lb. pentosus) dalam menghambat S.aureus (FNCC 0047) sebesar 4663 mm2 mL-1, E.coli (FNCC 0091) sebesar 3109 mm2 mL-1, dan L.monocytogenes (FNCC 0156) sebesar 3179 mm2 mL-1. Hasil penelitian menunjukkan bahwa kondisi fermentasi awal (perbedaan kadar garam dan suhu fermentasi) dapat menentukan karakteristik yang berbeda pada setiap BAL yang dihasilkan terutama dalam hal kemampuan menghasilkan senyawa antimikroba bakteriosin. Diketahui juga bahwa suplementasi sumber karbon dan nitrogen mampu menginduksi produksi bakteriosin yang hasilnya tergantung pada masing-masing isolat BAL.
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ACKNOWLEDGMENT Firstly, I dedicated my greatest gratitude to Jesus Christ and The Virgin Mary, who have given their affection and blessing to the author for taking the time to complete this thesis entitled “THE EFFECT OF DIFFERENT MEDIUM COMPOSITIONS ON THE PRODUCTION BACTERIOCIN OF LACTIC ACID BACTERIA ISOLATED FROM YELLOW BETUNG BAMBOO SHOOT (Dendrocalamus asper) PICKLE UNDER DIFFERENT FERMENTATION CONDITIONS”. It has been written to fulfill the graduation requirements of the Food Technology Program at the Faculty of Agricultural Technology. I was engaged in researching and writing this thesis from September 2015 to February 2016. This thesis is a part of the second year of Penelitian Unggulan Perguruan Tinggi (PUPT) 2015/2016 entitled “Efek Probiotik dan Mikrostatik dari Bakteri Asam Laktat yang Berperan dalam Fermentasi Acar Rebung”.
The author really gives regards and thanks for people who has given guidance and help; they are: 1.
Dr. Victoria Kristina, ST, MSc, as the Dean of Faculty of Agricultural Technology Soegijapranata Catholic University Semarang, for giving the chance to finish this final research.
2.
Dra. Laksmi Hartayanie, MP as my supervisor and Dr. Ir. Lindayani, MP as my cosupervisor. I also wish to thank for their excellent guidance and support during this process. Thank you for always available and willing to answer my queries, without their cooperation I would not have been able to conduct this research.
3.
Elizabeth Ivone Fernandez, SSi, MSc as thesis coordinator for arranging the administration and schedule during this process.
4.
All lecturers in Food Technology Department for being a great teacher for giving the joyful of learning.
5.
The laboratory assistants for all their help and motivation throughout the unforgettable laboratory life in Food Technology Department.
6.
The administration staff for providing great service and information during the study activities.
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7.
My parents (Go Roy Santoso and Liliawati) and my beloved brothers (Yafet Haere, Danny Setiawan, and Surya Santoso) for always being number one supporter and saying my name in every prayer. If I ever lost interest, you kept me motivated.
8.
Ivana Aprilia Pratiwi and Veronica Dian Sari who had been perfect sisters during campus life.
9.
Anastasya Gumelar, Amelia Juwana, Melita Mulyani, Vivian, Michaela Jessica, Matius Inda, Lukas Terry, Anton Septian, Hendra Aditya, Michael Gurdamulya, Hengky Kurniawan, Michael Yefta, Asien, Toni Hartanto, Arnulf Budi, LIMUN Team, Student Organization Team, GLORY Team, Assistant Laboratory Team, AU Internship Team, all of student in Food Technology who had been a part of the author’s experiences during campus life.
10.
All people who has directly and indirectly helped during the experiment until finishing the thesis report.
Finally, the author realizes that there were unintended errors in writing this report. The author really allows all readers to give suggestions to improve its content in order to be made as one of the good examples for the next thesis. Big hope from the author that this report can be useful for others.
Semarang, March 2016 Author,
Lorentia Santoso
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LIST OF CONTENTS Page SUMMARY .................................................................................................................... i RINGKASAN................................................................................................................... ii ACKNOWLEDGMENT ............................................................................................... iii LIST OF CONTENTS .................................................................................................... v LIST OF TABLES ........................................................................................................ vi LIST OF FIGURES ...................................................................................................... vii LIST OF APPENDICES ............................................................................................. viii 1. INTRODUCTION ..................................................................................................... 1 1.1. Background of Research ..................................................................................... 1 1.2. Literature............................................................................................................. 2 1.2.1. Lactic Acid Bacteria (LAB) ..................................................................... 2 1.2.2. Bamboo Shoot Pickle ............................................................................... 3 1.2.3. Importance of Lactic Acid Bacteria in Vegetable Fermentation .............. 4 1.2.4. Bacteriocins .............................................................................................. 5 1.2.5. The Effect of Medium Composition on Bacteriocin Production .............. 8 1.3. Objectives ........................................................................................................... 9 2. MATERIALS AND METHODS............................................................................. 10 2.1. Materials ........................................................................................................... 10 2.2. Methods ............................................................................................................ 11 2.2.1. Experimental Design .............................................................................. 11 2.2.2. Production of Crude Bacteriocins........................................................... 12 2.2.3. Bacteriocin Inhibitory Activity Test ....................................................... 12 3. RESULT .................................................................................................................. 15 4. DISCUSSION .......................................................................................................... 28 5. CONCLUSIONS AND SUGGESTIONS ............................................................... 35 5.1. Conclusions....................................................................................................... 35 5.2. Suggestions ....................................................................................................... 35 6. REFERENCES ........................................................................................................ 36 7. APPENDICES ......................................................................................................... 42
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LIST OF TABLES Page Table 1. Result of Antimicrobial Activity .................................................................... 15 Table 2. Result of API Software Identification (24 hours Incubation) ........................ 16 Table 3. Detection of Bacteriocin Inhibitory Activity Against Pathogenic Bacteria ... 17 Table 4. Effect of Adding Some Nutrient Components in MRS-B Medium on Bacteriocin Inhibitory Activity (AU mm2 mL-1) by LAB Isolates which Isolated from Yellow Betung bamboo shoot pickle (C and D) ...................... 18
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LIST OF FIGURES Page Figure 1. Flowchart of Experimental Design ............................................................... 11 Figure 2. Flowchart of Bacteriocin Production under Different Medium Compositions .................................................................................................. 12 Figure 3. Flowchart of In Vitro Detection of Bacteriocin Inhibitory Activity using Agar-Well Diffusion Method ................................................. 13 Figure 4. Flowchart of Selection and Activation of Bacteriocin Producing LAB in Bacteriocin Production ...................................................................... 14 Figure 5. Effect of Medium Compositions on Bacteriocin Inhibitory Activity (AU mm2 mL-1) of C and D Isolates Against L. monocytogenes (FNCC 0156), S. aureus (FNCC 0047), and E. coli (FNCC 0091) ............... 20 Figure 6. Effect of Medium Compositions on Bacteriocin C18 Inhibitory Activity Test using Agar-Well Diffusion Method) ........................................ 22 Figure 7. Effect of Medium Compositions on Bacteriocin C19 Inhibitory Activity Test using Agar-Well Diffusion Method ......................................... 23 Figure 8. Effect of Medium Compositions on Bacteriocin C29 Inhibitory Activity Test using Agar-Well Diffusion Method ......................................... 24 Figure 9. Effect of Medium Compositions on Bacteriocin D11 Inhibitory Activity Test using Agar-Well Diffusion Method ......................................... 25 Figure 10. Effect of Medium Compositions on Bacteriocin D20 Inhibitory Activity Test using Agar-Well Diffusion Method ......................................... 26 Figure 11. Effect of Medium Compositions on Bacteriocin D44 Inhibitory Activity Test using Agar-Well Diffusion Method ......................................... 27 Figure 12. Identification of Isolate A43 using API 20 strep ........................................ 42 Figure 13. Identification of Isolate D44 using API 50 CH ........................................... 43
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LIST OF APPENDICES Page Appendix 1. Species Identification of LAB Isolates using API 20 strep ..................... 42 Appendix 2. Species Identification of LAB Isolates using API 50 CH ....................... 43 Appendix 3. Media Used for Growth and Bacteriocin Inhibitory Activity Test of LAB Isolated from Yellow Betung Bamboo Shoot Pickle ......... 44 Appendix 4. Composition of Standard Solution McFarland No. 2 and 3 .................... 45
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