FERMENTATION OF “KAMBAS” BITTER MELON (
Momordica
charantia
L.) JUICE USING
Lactobacilus fermentum
LLB3 FOR MAKING
PROBIOTIC BEVERAGES
FERMENTASI JUS PARE KAMBAS (Momordica charantia
L
.)
MENGGUNAKAN BAKTERI Lactobacillus fermentum LLB3 DALAM
UPAYA PEMBUATAN MINUMAN PROBIOTIK
BACHELOR THESIS
Submitted in partial fulfilment of the requirements for a Food Technology Bachelor’s degree in Faculty of Agricultural Technology
By:
EMILIA TRIVIANA SUSANTO 15.I1.0021
DEPARTMENT OF FOOD TECHNOLOGY
FACULTY OF AGRICULTURAL TECHNOLOGY
SOEGIJAPRANATA CATHOLIC UNIVERSITY
SEMARANG
i
STATEMENT OF THESIS AUTHENTICITY
I hereby declare that the thesis with the title “FERMENTATION OF “KAMBAS” BITTER MELON (Momordica charantia L.) JUICE USING Lactobacillus fermentum
LLB3 FOR MAKING PROBIOTIC BEVERAGES” contains no work that ever proposed
to gain a bachelorship title in a University and along with 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 partially or whole of the thesis is proven of plagiarism, I am willing to be revoked with all of the consequences in accordance with the law and regulations applied at Soegijapranata Catholic University and/or valid law and regulations.
Semarang, March 2019
Emilia Triviana Susanto 15.I1.0021
ii
FERMENTATION OF “KAMBAS” BITTER MELON (
Momordica
charantia
L.) JUICE USING
Lactobacillus fermentum
LLB3 FOR
MAKING PROBIOTIC BEVERAGES
FERMENTASI JUS PARE KAMBAS
(Momordica charantia
L.)
MENGGUNAKAN BAKTERI
Lactobacillus fermentum
LLB3 DALAM
UPAYA PEMBUATAN MINUMAN PROBIOTIK
BACHELOR THESISSubmitted in partial fulfilment of the requirements for a Food Technology Bachelor’s degree in Faculty of Agricultural Technology
By:
EMILIA TRIVIANA SUSANTO 15.I1.0021
This thesis has been approved and defended in front of the examination committees on January 30th, 2019
Semarang, March 4th, 2019
Faculty of Agricultural Technology Soegijapranata Catholic University
Supervisor Dean
Dra. Laksmi Hartayanie, MP Dr. R.Probo Y. Nugrahedi, S.TP., M.Sc.
Co-Supervisor
iii
SUMMARY
Nowadays, functional food becomes important for human life. Probiotic product is one of functional food that has been consumed by a lot of people around the world because of its benefits for human health. Commonly, probiotic product is presented as beverages such as yogurt, kefir, etc. Because of that, people who have high cholesterol, or vegetarian can not consume these kinds of product. As an alternative, probiotic beverages from vegetables based were made. Bitter melon is popular for its bitter taste. Because of that, bitter melon are not commonly consumed by people. However, despite its very bitter taste, it contains a lot of antioxidant and vitamin that gives a lot of benefits for human’s health. All of the beneficial components inside the bitter melon can improve the functional properties of probiotics beverages. The aim of this research is to examine the ability of “Kambas” bitter melon as media for lactic acid bacteria’s growth and also the effect of “Kambas” bitter melon fermentation on its component properties (antioxidant, bacteriocins and antimicrobial compound). The “Kambas” bitter melon juice were fermented by using Lactobacillus fermentum LLB3 and Instant lactic acid bacteria (as control) for 24 and 48 hours. The lactic acid bacteria content in “Kambas” Bitter Melon Juice was determined by total plate count and expressed as colony forming units (CFU/ml). The bacteriocin activity was determined using agar well-diffusion method and measuring the clear zone formed around the wells (expressed as Activity Unit / AU). The chemical analysis such as DPPH radical-scavenging activity, pH measurement, and determination of glucose content were measured at the different time of fermentation (24 and 48 hours). The result showed that optimum amount of lactic acid bacteria content in both fermented bitter melon were at 24 hours of fermentation with the amount of colony around 6.3 x 108 CFU/ml and 1.5 x 108 CFU/ml. Antioxidant activity also showed an increasing activity at 48 hours of fermentation with percentage of activity 84.58% for fermented bitter melon juice using Instant lactic acid bacteria and for fermented bitter melon juice using Lactobacillus fermentum LLB3 86.21%. Antimicrobial and bacteriocins inhibitory activity in all samples was decreased during fermentation. pH and glucose content of Lactobacillus fermentum LLB3 and Instant lactic acid bacteria also decreased along with the duration of the fermentation due to the formation of organic acid and the use of sugar for stimulating the growth of lactic acid bacteria. Among all of the samples, Lactobacillus fermentum LLB3 had better viability, antioxidant activity, antimicrobial and bacteriocins inhibitory activity than Instant lactic acid bacteria (as control).
iv
RINGKASAN
Pada era modern ini, pangan fungsional sudah menjadi bagian yang penting dalam kehidupan masyarakat. Salah satu jenis pangan fungsional yang banyak dikonsumsi karena manfaatnya yang baik untuk kesehatan tubuh adalah produk pangan yang mengandung probiotik. Umumnya, produk pangan probiotik ini disajikan dalam bentuk minuman probiotik seperti yogurt, kefir, dan lain lain. Oleh karena itu, beberapa konsumen yang mempunyai kolesterol yang tinggi, maupun vegetarian tidak bisa mengkonsumsi produk ini. Sebagai alternatif, sekarang mulai bermunculan produk minuman probiotik dengan bahan dasar jus buah. Pare umumnya dikenal dengan rasanya yang pahit. Rasa pahit tersebut menyebabkan pare tidak secara umum dikonsumsi oleh semua orang. Meskipun memiliki rasa yang pahit, didalam pare terkandung antioksidan yang tinggi dan beraneka macam vitamin yang mampu memberikan efek kesehatan bagi tubuh. Adanya kandungan yang bermanfaat di dalam pare ini mampu meningkatkan nilai fungsional dari minuman probiotik. Tujuan dari percobaan ini adalah untuk mengetahui kemampuan pare kambas sebagai media untuk pertumbuhan bakteri asam laktat dan untuk mengetahui bagaimana pengaruh fermentasi terhadap komponen yang terdapat di dalam pare (antioksidan , bakteriosin, dan senyawa antimikroba). Jus pare kambas difermentasi menggunakan bakteri Lactobacillus fermentum LLB3 dan bakteri asam laktat instan (sebagai kontrol) dengan tiga waktu fermentasi yang berbeda yaitu 24 jam dan 48 jam. Kandungan bakteri asam laktat dalam jus pare kambas yang telah difermentasi diuji dengan menggunakan uji angka lempeng total yang dinyatakan dalam satuan CFU/ml. Aktivitas penghambatan dari bakteriosin dan antimikroba diuji dengan metode agar well diffusion dan diukur zona bening yang terbentuk di sekitar sumuran. Uji kimia seperti uji antioksidan, pH, dan total gula juga dilakukan di setiap waktu fermentasi yang berbeda (24 dan 48 jam). Dari hasil yang diperoleh menunjukkan bahwa kandungan bakteri asam laktat dari jus pare baik yang difermentasi dengan menggunakan Lactobacillus fermentum LLB3 dan bakteri asam laktat keduanya memiliki pertumbuhan yang optimal pada fermentasi 24 jam dengan jumlah koloni 6,3 x 108 CFU/ml dan 1,5 x 108 CFU/ml. Aktivitas antioksidan dari kedua sampel juga menunjukkan peningkatan antioksidan pada fermentasi 48 jam dengan persentase antioksidan 84,58% untuk jus pare dengan bakteri asam laktat instan dan 86,21% untuk jus pare dengan Lactobacillus fermentum LLB3. Pada aktivitas bakteriosin dan antimikroba, pada semua sampel menunjukka penurunan aktivitas daya hambat bakteriosin dan antimikroba seiring dengan semakin lama waktu fermentasi. Hasil uji pH dan total gula juga menunjukkan penurunan seiring dengan semakin lamanya waktu fermetasi yang dikarenakan dihasilkannya asam laktat dan penggunaan gula sebagai komponen untuk menstimulasi pertumbuhan bakteri asam laktat. Secara keseluruhan, Lactobacillus fermentum LLB3 memiliki viabilitas, aktivitas antioksidan, dan daya hambat antimikroba dan bakteriosin yang lebih baik dibandingkan dengan bakteri asam laktat instan yang merupakan kontrol.
v
ACKNOWLEDGEMENT
Praise the Lord because of His grace and blessing, the author have an opportunity to complete the thesis entitled: “FERMENTATION OF “KAMBAS” BITTER MELON (Momordica charantia L.) JUICE USING Lactobacillus fermentum LLB3 FOR MAKING PROBIOTIC BEVERAGES”. This thesis was written to fulfill the requirement to acquire Bachelor Degree of Food Technology in Soegijapranata Catholic University, Semarang, Indonesia. This thesis is a part of the third year of Penelitian Unggulan Perguruan Tinggi (PUPT) 2017/2018.
The author would like to express sincere gratitude to all people who have given support and guidance. They are:
1. Almighty God who always bless the author from the beginning to the end.
2. Dra. Laksmi Hartajanie, MP as author’s supervisor and Dr. Ir. Lindayani, MP as author’s co-supervisor for giving the great opportunity to do this project and the excellent guidance during the proses.
3. All lectures in Food Technology Department for giving all great lessons during the learning activities.
4. All laboratory assistants and administration staffs in Food Technology Department for the help and motivation during the laboratory and learning activities.
5. Author’s parents (Adi Susanto and Lina Sinatra Wijaya; Budi Nur and Fanny Agustin), sisters (Elaine Septiana Susanto, Evelyn Octaviana Susanto, and Eidina Budi Theomarga), brother (Yuwono Gusman, Shun Watanabe, Radithya Budi Theomarga), niece (Alisha Mikaela Gustiana), Wanna One, Cookie von channel, Chestnut von channel, Cotton and Candy for always supporting and cheering the author everyday. 6. Angela Novita and Elizabet Vivin as author’s partners for always listening, supporting,
and encouraging the author during the process also Mbak Agata and Cik Donna Larissa as PUPT Team for always supporting, giving advices, and new knowledge during process.
7. Katherine Kristalia, Sarah Florentia, Francy Francesca, Josephine Permatasari, Elisabeth Merline, Jeanny Kurniawan, Viola Aulia, Fanny Margaretha, and all my friends that cannot be mentioned one by one, who always support and accompany author during campus and boarding house life.
The author realizes that there were unintended errors in writing this report. The author really allows all readers to give suggestions to improve the content. However, the author hopes this report can be an inspiration and provide useful information for others.
Semarang, March 2019 Author,
vi
LIST OF CONTENTS
STATEMENT OF THESIS AUTETICITY……….. i
APPROVAL PAGE………...………... ii
SUMMARY ... iii
RINGKASAN ... iv
ACKNOWLEDGEMENT ... v
LIST OF CONTENTS ... vi
LIST OF TABLES ... viii
LIST OF FIGURES ... ix
LIST OF APPENDICES ... xi
1 INTRODUCTION ... 1
1.1 Background of Research ... 1
1.2 Literature Review ... 3
1.2.1 “Kambas” Bitter Melon (Momordica charantia L.) ... 3
1.2.2 Lactic Acid Bacteria (LAB) ... 3
1.2.3 Fermentation of Lactic Acid Bacteria ... 4
1.2.4 Free Radical and Antioxidant for Human Health ... 5
1.2.5 Bacteriocins ... 6
1.2.6 Probiotic Beverages ... 7
1.3 Objectives ... 8
2 MATERIALS AND METHODS... 9
2.1 Materials ... 9
2.2 Equipment ... 9
2.3 Methods ... 10
2.3.1 “Kambas” Bitter Melon Juice Preparation ... 10
2.3.2 Inoculum of Lactic Acid Bacteria (Lactobacillus fermentum and Instant LAB) Preparation ... 10
2.3.3 Fermentation of “Kambas” Bitter Melon using Lactobacillus fermentum and Instant LAB ... 11
2.3.4 Probiotic Bacteria Content (Total Plate Count) ... 12
2.3.5 Free Radical Scavenging Activity Using 2.2-Diphenyl-1-Picryl Hydrazyl (DPPH) ... 12
2.3.6 Bacteriocins and Antimicrobial Inhibitory Activity Test ... 13
2.3.7 pH Measurement ... 14
2.3.8 Glucose Content Analysis (Phenol Method) ... 14
2.3.9 Data Analysis ... 14
2.3.10 Experimental Design ... 15
3 RESULTS ... 16
3.1 Fermentation of “Kambas” Bitter Melon Juice using Lactobacillus fermentum and Instant LAB ... 16
3.2 LAB Content in Fermented Bitter Melon Juice ... 16
3.3 Free Radical Scavenging Activity Using 2.2-Diphenyl-1-Picryl Hydrazyl (DPPH) 19 3.4 Bacteriocins and Antimicrobial Inhibitory Activity Test ... 20
vii
3.4.1 Antimicrobial Inhibitory Activity Against Pathogenic Bacteria (Escherichia
coli, Staphylococcus aureus, and Salmonella typhimurium) ... 23
3.4.2 Bacteriocins Inhibitory Activity Pathogenic Bacteria (Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium) ... 24
3.5 pH Measurement ... 26
3.6 Glucose content Analysis (Phenol Method) ... 27
4 DISCUSSIONS ... 29
4.1 Characteristics of Fermented Bitter Melon Juice by Lactobacillus fermentum LLB329 4.2 Antimicrobial and Bacteriocins Inhibitory Activity Test ... 32
4.2.1 Antimicrobial Inhibitory Activity Test ... 32
4.2.2 Bacteriocins Inhibitory Activity Test ... 34
5 CONCLUSIONS AND SUGGESTIONS ... 36
5.1 Conclusions... 36
5.2 Suggestions ... 36
6 REFERENCES ... 37
viii
LIST OF TABLES
Table 1. LAB Content of Fresh and Fermented “Kambas” Bitter Melon Juice (CFU/ml) ... 18
Table 2. DPPH Scavenging Activity of Fresh and Fermented “Kambas” Bitter Melon Juice (%) ... 19
Table 3. Antimicrobial Activity Against Pathogenic Bacteria (mm2 ml-1) ... 23
Table 4. Bacteriocins Inhibitory Activity Against Pathogenic Bacteria (mm2 ml-1) ... 24
Table 5. pH Measurement of Fresh, Pasteurized, and Fermented “Kambas” Bitter Melon Juice ... 26
ix
LIST OF FIGURES
Figure 1. Whole fruit of “Kambas” Bitter Melon (a); “Kambas” Bitter Melon that had been chopped and without seeds (b); and Fresh Bitter Melon Juice that had been filtered (c) ... 10
Figure 2. Sterilized tomato extract (a); Two milliliters inoculum of Lactobacillus fermentum (left) and Instant Lactic Acid Bacteria (right) + 18 ml of pasteurized “Kambas” Bitter Melon Juice (b) ... 11
Figure 3. Flowchart of Research Design... 15
Figure 4. Fresh “Kambas” Bitter Melon Juice (a); “Kambas” Bitter Melon Without Fermentation (b); Fermented “Kambas” Bitter Melon Juice (24 hours) (c); Fermented “Kambas” Bitter Melon Juice (48 hours) (d) ... 16 Figure 5. Total plate count of fresh “Kambas” bitter melon juice ... 17
Figure 6. Total plate count of “Kambas” bitter melon fermentation using Lactobacillus fermentum (LLB3) 0 hour fermentation (a); 24 hours fermentation (b); and 48 hours fermentation (c) ... 17 Figure 7. Total plate count of “Kambas” bitter melon fermentation using Instant LAB
0 hour fermentation (a); 24 hours fermentation (b); and 48 hours fermentation (c) ... 17
Figure 8. Clear zone that indicate the present of Lactic Acid Bacteria (LAB) in sample ... 18
Figure 9. LAB content (CFU/ml) of fresh and fermented “Kambas” bitter melon using Lactobacillus fermentum (B3) and Instant LAB in different time of fermentation (0 hour, 24 hours, and 48 hours). ... 18 Figure 10. DPPH scavenging activity (%) of fresh and fermented “Kambas” bitter
melon using Lactobacillus fermentum LLB3 and Instant LAB in different time of fermentation (0 hour, 24 hours, and 48 hours)... 20
Figure 11. Bacteriocins and Antimicrobial Activity of fresh bitter gourd juice (a); fermented “Kambas” bitter melon using Lactobacillus fermentum LLB3 for 0 hour fermentation (b); 24 hours fermentation (c); and 48 hours fermentation (d) ... 21
Figure 12. Bacteriocins and Antimicrobial Activity of fresh bitter gourd juice (a); fermented “Kambas” bitter melon using Instant LAB for 0 hour fermentation (b); 24 hours fermentation (c); and 48 hours fermentation (d) ... 22
Figure 13. Antimicrobial Activity (mm2 ml-1) of Fresh “Kambas” Bitter Melon, Fermented “Kambas” bitter melon using Lactobacillus fermentum LLB3 and Instant LAB against Escherichia coli FNCC 0091, Staphylococcus aureus FNCC 0047, and Salmonella typhimurium FNCC 0187 ... 23
x
Figure 14. Bacteriocin Inhibitory Activity of Fresh “Kambas” Bitter Melon, Fermented “Kambas” bitter melon using Lactobacillus fermentum LLB3 and Instant LAB against Escherichia coli FNCC 0091, Staphylococcus aureus FNCC 0047, and Salmonella typhyimurium FNCC 0187 (mm2 ml-1).... 25
Figure 15. pH measurement of fresh, pasteurized, and fermented “Kambas” bitter melon using Lactobacillus fermentum LLB3 and Instant LAB with different fermentation time (0 hour, 24 hours, 48 hours). ... 26
Figure 16. Glucose content analysis of fresh and fermented “Kambas” bitter melon using Lactobacillus fermentum LLB3 and Instant LAB with different fermentation time (24 hours and 48 hours). ... 27
xi
LIST OF APPENDICES
Appendix 1. Media Used for Total Plate Count and Bacteriocins and Antimicrobial
Inhibitory Activity Test ... 43
Appendix 2. Composition of Standard Solution McFarland 3 ... 45
Appendix 3. Standard Curve of Glucose for Glucose Content Analysis ... 46
Appendix 4. Statistical Analysis of DPPH Scavenging Activity ... 47
Appendix 5. Statistical Analysis of pH Measurement ... 49
