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Production of Bioactive Peptides from Red Bean, Mung Bean, and Soy Bean using Bromelain Enzyme
Wilson Dwinanda Putra 2009-080-085
Thesis
Faculty of Biotechnology Atma Jaya Catholic University of Indonesia Jakarta 2013
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Production of Bioactive Peptides from Red Bean, Mung Bean, and Soy Bean using Bromelain Enzyme
Wilson Dwinanda Putra 2009-080-085
Thesis As partial fulfillment for the degree of Bachelor of Science in Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia
Faculty of Biotechnology Atma Jaya Catholic University of Indonesia Jakarta 2013
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INTELLECTUAL PROPERTY STATEMENT FORM I, Name : Wilson Dwinanda Putra NIM : 2009-080-085 Certify that my thesis is my own original work and no portion of my thesis has been copyrighted previously unless properly referenced. If there is a breach of items above, I will take full responsibility to Atma Jaya Catholic University of Indonesia for any legal action that might be caused.
Jakarta, January 31st 2013
Wilson Dwinanda Putra
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APPROVAL FORM
We hereby certify that: Name NIM Thesis title
: Wilson Dwinanda Putra : 2009-080-085 : Production of Bioactive Peptides from Red Bean, Mung Bean, and Soy Bean using Bromelain Enzyme Date of exam : January 31st, 2013 Has passed the thesis exam and confirmed that this thesis had been thoroughly examined, improved, and approved by advisors.
Approved by
Prof . Dr. Ir. Maggy T. Suhartono Advisor
Dr. Noryawati Mulyono, S.Si. Co-Advisor
Acknowledged
Dr. Diana Elizabeth Waturangi, M.Si. Dean
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PREFACE
This thesis is a final work as partial fulfillment for the degree of Bachelor of Science in Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia titled “Production of Bioactive Peptides from Red Bean, Mung Bean, and Soy Bean using Bromelain Enzyme” was conducted in Biochemistry and Enzyme Technology Research Laboratory, Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, from June 2012 until January 2013. Author would firstly like to thank God for His blessings until this thesis research is completed. Author also like to thank all people who were involved in this research, Prof. Dr. Ir. Maggy T. Suhartono and Dr. Noryawati Mulyono, S.Si. as advisors, Yanti, Ph.D. as examiner, technicians in Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia for their valuable comments, suggestions, and feedbacks in technical and data analysis. Furthermore, many people are appreciated for their assistance for data collection process. Author would like to thank partners, Maureen Novia and Yosep Sutanto, for their cooperation in this research, family for their love and supports, all of my friends for times we have spent together, and all other people that indirectly involved in the research. Lastly, may this thesis be useful for readers and for future research about similar topic or any other related field. Jakarta, January 31st 2013
Wilson Dwinanda Putra
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BIOGRAPHY Wilson Dwinanda Putra was born in Jakarta, November 14th, 1991 as the third son from Djap Darmanto and Tjong Lindawaty. He graduated from IPEKA Sunter 2 Senior High School, Jakarta in 2009 and obtained his degree in Bachelor of Science in January 2013 from Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia. During his course in Atma Jaya Catholic University of Indonesia, Author was involved in several commitees, including “BONSAI” charity event in 2009, “ELBOW” entrepreneurship seminar in 2010, Science Club Fieldtrip as public relation division in 2011, Science Club event division coordinator (2011 to 2012), and “MICROVONE” charity concert in 2012. Beside his organizational experiences, he have been worked several times in Atma Jaya Faculty of Biotechnology, as assistant in Basic Chemistry Laboratory in 2010, Biochemistry Laboratory in 2011, Biostatistics in 2011, and Food Technology Laboratory in 2012. He completed a short internship program at PT Sosro Tbk., Bekasi as a Chemical Analyst Staff from January until February 2012.
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CONTENTS Page List of Tables ............................................................................................
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List of Figures ...........................................................................................
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List of Appendices ....................................................................................
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PRODUCTION OF BIOACTIVE PEPTIDES FROM RED BEAN, MUNG BEAN, AND SOY BEAN USING BROMELAIN ENZYME Abstract ...........................................................................................
1
Introduction .....................................................................................
1
Literature Review ............................................................................
2
Materials & Methods .......................................................................
3
Results .............................................................................................
5
Discussions ......................................................................................
8
Conclusion .......................................................................................
11
References .......................................................................................
11
Appendices ......................................................................................
13
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LIST OF TABLES Page 1 BSA concentration for standard curve ..................................................
4
2 Tyrosin concentration for standard curve ..............................................
4
3 Composition of SDS-PAGE gel ...........................................................
5
4 Increment of DPPH scavenging activity (0.173 U/ml) ..........................
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5 Increment of ferric reducing activity ......................................................
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6 Increment of ACE inhibitory activity ...................................................
8
7 Bioactivity of many plant and animal samples in various studies ..........
10
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LIST OF FIGURES Page 1 The chemical structure of the compound DPPH free radical and nonradical ..................................................................................................
3
2 Renin angiotensin system (RAS) .........................................................
3
3 DPPH scavenging activity (%) of bean flour substrates hydrolyzed with bromelain enzyme ........................................................................
6
4 Ferric reducing activity of bean flour substrates hydrolyzed with bromelain enzyme ................................................................................
7
5 ACE inhibitory activity (%) of bean flour hydrolyzed with bromelain enzyme .................................................................................................
7
6 SDS-PAGE result of hydrolysates of bean samples with different hydrolysis time .....................................................................................
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LIST OF APPENDICES Page 1 Procedure of ACE inhibitory activity .....................................................
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2 DPPH scavenging activity (%) of bean flour substrates (10 mg/ml) hydrolyzed with bromelain enzyme (0.272 U/ml) .................................
14
3 Tyrosin standard curve .........................................................................
15
4 Bradford standard curve .......................................................................
15
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Produksi Peptida Bioaktif dari Kacang Merah, Kacang Hijau, dan Kacang Kedelai menggunakan Enzim Bromelain Production of Bioactive Peptides from Red Bean, Mung Bean, and Soy Bean using Bromelain Enzyme WILSON DWINANDA PUTRA Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia Jalan Jenderal Sudirman 51, Jakarta 12930 Abstract. Food proteins have been known to contain peptide sequences capable of modulating health-related benefits in human. These proteins, termed bioactive peptides, can be used as safe, effective, natural antioxidant and antihypertension drug. The purposes of this study were to produce bioactive peptides derived from the various beans using bromelain enzyme and evaluate the effect of bioactive peptides as antioxidant and antihypertension agents. Bromelain enzyme extracted from pineapple and bean protein extracts were mixed to produce bioactive peptides at different hydrolysis time. 1,1-diphenyl 2-picrylhydrazyl (DPPH) scavenging activity and ferric reducing power were used to evaluate the effect of peptides as antioxidants. Antihypertensive capabilities were measured regarding inhibition of angiotensin-I converting enzyme (ACE) by sample. The highest activity for DPPH scavenging assay was mung bean (94.90 %) and for ferric reducing power assay was red bean (1.367) at 5 min hydrolysis time compared with ascorbic acid and α-tocopherol. It also showed that all samples have ACE inhibitory activity, while the highest activity was mung bean (96.67 %), which is higher than captopril as positive control. Enzyme activity, protein concentration and hydrolysis time variation seem to play role in bioactivity. This research showed the bioactive peptides produced from beans could act as potent radical scavengers and antihypertension drugs. Keywords: bioactive peptides; antioxidant; antihypertension Abstrak. Protein makanan telah diketahui memiliki sekuens peptida yang dapat memberikan efek yang menguntungkan bagi manusia, Protein ini, yang disebut peptida bioaktif, dapat digunakan sebagai antioksidan dan obat antihipertensi alami yang aman dan efektif. Tujuan dari penelitian kali ini adalah untuk memproduksi peptida bioaktif dari berbagai jenis kacangkacangan menggunakan enzim bromelain dan mengetahui efek peptida bioaktif sebagai antioksidan dan agen antihipertensi. Enzim bromelain diekstraksi dari nanas lalu dicampurkan dengan ekstrak protein kacang-kacangan dalam waktu hidrolisis berbeda untuk menghasilkan peptida bioaktif. Uji aktivitas penangkapan radikal 1,1-diphenyl 2-pikrilhidrazil (DPPH) dan kekuatan reduksi ferri digunakan untuk mengukur aktivitas antioksidan. Kemampuan antihipertensif diukur berdasarkan penghambatan enzim konversi angiotensin-I (ACE) oleh sampel. Aktivitas tertinggi untuk uji penangkapan DPPH adalah kacang hijau (94.90 %) dan untuk uji kekuatan reduksi ferri adalah kacang merah (1.367) dengan waktu hidrolisis 5 menit, dibandingkan dengan asam acrobat dan α-tokoferol. Hasil lain juga menunjukan semua sampel memiliki aktivitas penghambatan enzim ACE, dimana yang tertinggi adalah kacang hijau (96.67 %), yang lebih tinggi dibandingkan kaptopril sebagai kontrol positif. Aktivitas enzim, konsentrasi protein, dan waktu hidrolisis juga berperan dalam bioaktivitas. Penelitian ini menunjukkan peptida bioaktif yang dihasilkan dari kacang-kacangan dapat menjadi agen penangkap radikal bebas dan obat antihipertensi yang potensial. Kata kunci: peptida bioaktif; antioksidan; antihipertensi
INTRODUCTION Nowadays, consumer preferences for processed foods, concern in use of synthetic compounds and chemical additives, and
increased knowledge of the relationship between diet and health raise the demand for natural and functional food ingredients. Food proteins, besides their nutritional roles, contain peptide sequences encrypted in their
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primary structure that are capable of modulating specific physiological functions that are very useful to be used in medical field and any other fields (Correa et al. 2011). These protein fragments, termed bioactive peptides, are inactive within the sequence of the native protein, and could be released through enzymatic hydrolysis in vivo and in vitro (Correa et al. 2011). After release, bioactive peptides might exert antihypertensive, antibacterial, anticancer, and antioxidant activities, which have potential applications in food science, technology, and nutritional science (Shahidi & Zhong 2008). Bioactive peptides have been isolated from other food sources of protein, including egg, fish, oyster, cereals (rice, wheat, barley and maize) and soy bean (Wang & Gonzales 2005). The purposes of this study were to produce bioactive peptides derived from the various beans using bromelain enzyme extracted from pineapple and evaluate the effect of bioactive peptides as antioxidant and antihypertension agents.
LITERATURE REVIEW
may also be used in a variety of other conditions like hay fever, ulcerative colitis, removal of dead and damaged tissue after debridement in burns, preventing the collection of fluid in the lung pulmonary edema, relaxing muscles, stimulating muscle contractions, slowing clotting of blood, improving the absorption of antibiotics, preventing cancer, shortening delivery time, and helping the body to get rid of fat. Bromelain has even been widely studied as a complementary cancer treatment, as it contains powerful antioxidant components (Priya et al. 2011). Bromelain is a mixture of cysteine proteases and non proteases components, extracted from pineapple plant (Ananas comosus) and divided into two types, stem (E.C. 3.4.22.32) and fruit bromelain (E.C. 3.4.22.33) (Maurer 2001). Bromelain is stable at pH 3 - 8 (optimum pH 4.5 - 7.5) and at temperatures up to 60 °C (optimum temperature 35 - 45 °C). Bromelain powder can be stored originally packed and tightly closed up to two years at < 8 °C without reduction of enzyme activity (Poh & Majid 2011).
A. Pineapple & Bromelain Pineapple (Ananas comosus) is a plant originating from Brazil (South America) and one of Bromeliaceae family (another example; Tillandsia usneoides & Puya raimondii). Pineapple contains many essential minerals like manganese as a enzymatic reaction cofactor for energy production and antioxidant activities. One of the enzymes that use these minerals is superoxide dismutase (SOD), which also acts as an antioxidant (Subroto 2008). Pineapple plant has many functions especially for solving health problems, such as antiinflammatory, antimicrobial, therapeutic activity in blood clotting, also inhibition of tumor growth (Bartholomew et al. 2003). Besides the health related functions, the availability and cheap price of pineapple fruit are the good points that make this fruit is quite popular around communities (Sunarjono 2000). So many side effects found while using synthetic compounds, now the use of natural materials are important safety requirements before the products commercially applied. Pineapple fruit is one plant that is easily obtained in any season and already known has many benefits, like meat tenderizer beside papain enzyme (Sunarjono 2000). Bromelain
B. Bioactive Peptides Beans have long been recognized as a complementary source of protein with whole grains, like rice and wheat. Beans also contain nutrients other than protein, like minerals, vitamins, complex carbohydrates and dietary fiber (Astawan 2009). Red bean (Phaseolus vulgaris L.) contains 22.3 g protein per 100 g dry weight, which is similar to the mung bean (Vigna radiata) (20-25 g per 100 g dry weight), while soybean (Glycine max) contains highest protein, 35 g protein per 100 g dry weight (Astawan 2009). Bioactive peptides from enzymatic hydrolysis (treated with protease: alcalase,trypsin, etc) could posses antioxidative properties against free radicals. Free radicals are atoms or groups of atom that have one or more unpaired electrons. The existence of unpaired electrons causes the highly reactive compounds looking for a partner. This radical is often associated with cell damage, tissue damage, and the aging process (Fang et al. 2002). Antioxidants are chemical compounds that can contribute electron(s) or hydrogen atom (proton) to free radicals, so that free radicals may be quenched and turn into more stable form. According to sources there are two kinds of antioxidant, natural antioxidant and
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artificial antioxidant (synthetic, like butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), which prevent oxidative rancidity (lipid peroxidation) of foods). Synthetic antioxidants often show side effects such as induces cancer or tumour, so natural antioxidants could be answers for this problems (Race 2009). The human body has no supply of antioxidants within the muscle, so that if there are excess radicals in the body, the body needs extra antioxidants or exogenous antioxidants from outside the body (Aini 2007). There are so many antioxidant mechanisms, include radical scavenging (both hydrogen-donating capability (for example, 1,1-diphenyl-2-picryl hydrazyl) and free radical quenching activity), inhibition of lipid peroxidation, metal ion chelation, or a combination of these properties (Sarmadi & Ismail 2010).
Figure 1 The chemical structure of the compound DPPH free radical and non-radical (Molyneux 2004).
Antioxidants are naturally found in many types of plants, such as the berries (blueberry, acai berry, goji berry), pomegranate, peppers, beets (Carlsen et al. 2010). There is concern over the possibility of unknown side effects of synthetic antioxidants has become an alternative natural antioxidant that is needed. Antioxidants are expected to have features such as safe in use, do not give flavor, odor, and color of the product, effective at low concentrations, resistant to a wide range of processing products, and available at cheap price (Aini 2007). The most studied bioactive peptides are the antihypertensive peptides that modulate angiotensin-I converting enzyme (ACE, EC 3.4.15.1). Some of these peptides have also been used as functional foods for antihypertension problem (ACE inhibitor) such as „Ameals‟, „peptACE‟, and „Biozate‟, which contain highly purified peptides (small proteins) from milk, fish, and whey proteins, respectively. ACE is one of the key enzymes in the blood pressure regulation pathway (renin-angiotensin system). ACE acts by removing a dipeptide (two amino acids linked together) from the C-terminal of angiotensin I to produce angiotensin II, the latter being a very powerful vasoconstrictor (Aluko 2008).
Figure 2 Renin-Angiotensin System (RAS) (Belovic et al. 2012).
In disease conditions or as a result of genetic or environmental factors, the level of ACE is up-regulated and the resultant high levels of angiotensin II produces undesirable rates of blood vessel contraction that leads to the development of high blood pressure (hypertension). Though less active than commercial drugs, naturally occurring hypotensive peptides are believed to be safer than synthetic drugs as agents for the treatment of hypertension since synthetic drugs have some side effects. It is also important to note that food protein-derived ACE-inhibitory peptides can be consumed at higher levels since there is no risk associated with an overdose when compared to commercial drugs that need to be used at levels below the upper tolerable limit (Aluko 2008).
MATERIALS AND METHODS Materials. The materials used for this research were Palembang pineapple, red bean flour, mung bean flour, and soybean flour (both fluor samples from high standard commercial flours), ascorbic acid (a. acid) 320 mg, α-tocopherol (α-toco) 67 mg, captopril (12.5 mg), absolute ethanol, sodium carbonate, L-Tyrosine, Coomasie Brilliant Blue G-250 (CBB G-250), phosphoric acid, bovine serum albumin (BSA), NaOH, HCl, casein, sodium phosphate, acrylamide, NNmethylene-bis acrylamide, Tris, sodium dodecyl sulfate (SDS), bromphenol blue, potassium phosphate (KH2PO4 and K2HPO4), 1,1-diphenyl-2-picrylhydrazyl (DPPH), potassium ferricyanide, trichloroacetate (TCA), ferric chloride, Angiotensin Iconverting enzyme (ACE), ACE substrate (Hip-L-His-L-Leu), sodium borate, sodium chloride, and ethylacetate. In antioxidant assays, ascorbic acid and α-tocopherol were used as positive controls. Ascorbic acid was diluted in distilled water and α-tocopherol was diluted in absolute ethanol. In ACE inhibitory
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assay, captopril was diluted in distilled water. Other chemicals and reagents used were of the highest analytical grade commercially available. Protein Extraction. Flour mixed with water in the ratio of sample : distilled water = 1: 9. Next step, adjust the pH of solution to 8.6 by addition of 1 M NaOH solution and stirred for 30 min at a temperature of 50 – 55 °C. Then centrifuge at 3000×g for 30 min, which will form pellet and supernatant. pH of the supernatant was lowered by the addition of 2 M HCl solution to 4.5 for protein precipitation. Centrifugation perfomed again at 1500×g for 20 min. Pellet obtained was dried using incubator at 50 °C for 72 h. Then concentration of protein in the pellet was measured . Protein Concentration Assay (Bradford 1976). Bradford reagent was made by diluting 100 mg comasie brillant blue G-250 into 50 ml of absolute ethanol; 100 ml of H3PO4 85% (w/v), and then added by aquadest until 1000 ml. The reagent was incubated overnight and filtered to remove the insoluble matter. The obtained filtrate was kept as stock solution and diluted (10 times) when used. Bovine Serum Albumin (BSA) was used as standard analysis. A series of solution was made as standard to make the Lambert-Beer curve so the protein concentration could be calculated. The composition of standard solution was shown in Table 1. Table 1 BSA concentration for standard curve
Tube
[BSA] (mg/ml)
1 2 3 4 5 6
1.0 0.8 0.6 0.4 0.2 0.1
BSA volume 1 mg/ml (ml) 2.50 2.00 1.50 1.00 0.50 0.25
Aquadest volume (ml) 0.00 0.50 1.00 1.50 2.00 2.25
A 0.2 ml BSA for each concentration were added by 4 ml Bradford working solution and then incubated for 5 minutes before measured at 595 nm. Briefly, the sample analysis was done by adding 0.2 ml sample with 4 ml Bradford working solution. Blank used for this assay was 0.2 ml aquadest. Standard curve can be seen in Appendix 4. Extraction of Bromelain. Pineapple was peeled and separated from the flesh and crown of the fruit. Then pineapple core was blended
and 0.01 M potassium phosphate buffer pH 7 was added half of the total pineapple stem weight. The mixture was filtered using cheesecloth. Centrifugation was performed at 3000×g for 25 min. The filtrate is stored in dark bottles in the refrigerator (4 °C) for further use (Kosasih 2012). Enzyme Precipitation with Ethanol. A 50 ml cold absolute ethanol was slowly added to filtrate until ethanol level reach 60%. The mixture was incubated for 24 h 4 °C to precipitate bromelain, then centrifuge at 4000×g for 25 min to obtain pellet and filtrate. Pellets were weighed and dissolved using 0.02 M potassium phosphate buffer pH 7.0 as much as the pellet weight. A 0.5 ml of the pellet was dissolved in 0.02 M potassium phosphate buffer pH 7.0 (10 times dilution) (Kosasih 2012). Protease Activity Assay (Enyard 2008). Protease activity of bromelain enzyme was measured using casein 1% as enzyme substrate. A 800 µL casein was mixed with 200 µL enzyme then incubated at 37°C for 10 min. A 500 µL TCA was added to the mixture to stop the enzyme reaction. For blank solution, TCA was added before the addition of enzyme. Mixture was incubated 30 min in room temperature to let TCA works before centrifuged at 11000×g for 10 min. After that, 400 µL supernatant was pipetted to a new microcentrifuge tube and 1 ml Na2CO3 and 200 µL of Folin and Ciocalteu‟s phenol were added to the solution then incubated at 37oC for 30 min. Mixture was centrifuged 11000×g for 10 min. Supernatant was measured by spectrophotometer in 660 nm wavelength. A series of solution were made as standard to make the Lambert-Beer curve so that the activity of enzyme could be calculated. The composition of standard solution was shown in Table 2. Table 2 Tyrosin concentration for standard curve
All reagents were vortexed and 400 µL were pipetted into a new micro centrifuge tube. As much as 1 ml Na2CO3 and 200µL of Folin and Ciocalteu‟s phenol were added to the
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solution, then incubated at 37 oC for 30 min. Mixture was centrifuged 11000×g for 10 min. The obtained supernatant was measured at 660 nm wavelength. One unit of enzyme activity was defined as enzyme which produced 1µmol of tyrosin per minute. Standard curve can be seen in Appendix 3. Production of Bioactive Peptides. Protein extract was added with bromelain (protein sample : enzyme = 10 : 1), and was incubated at desired hydrolysis time (0, 5, 10, 20, 30, 60 min). The reaction was stopped by incubation at 90 °C for 10 min using water bath. DPPH Radical Scavenging Activity (Bersuder et al. 1998). This assay used to measure capability of samples as proton (hydrogen) donors. As much as 500 μl of bean sample solution was mixed with 500 μl of ethanol 99.5% and 125 μl ethanol 99.5% containing 0.02% (w/v) 2,2-diphenyl-1picrylhydrazyl (DPPH). The mixture was kept in the dark at room temperature for 60 min before the absorbance was measured spectrophotometrically at 517 nm. Radical scavenging activity (%) =
Control Blank Sample 100% Control
Where the control was the absorbance value of 500 μl of distilled water & 125 μl of ethanol 99.5% containing 0.02% DPPH & 500 μl ethanol 99.5%, and the sample containing 500 μl sample solution & 125 μl of ethanol 99.5% including 0.02% DPPH & 500 μl ethanol 99.5%, and the blank was 500 μlof sample solution & 125 μl ethanol 99.5% & 500 μl ethanol 99.5%. Ferric Reducing Power Activity (Oyaiza 1986). This assay used to measure capability of samples as electron donors. Sample solution (0.5 ml) was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% w/v potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. A total of 2.5 ml trichloroacetic acid (TCA) was added to the mixture, followed by centrifugation 700×g for 10 min. The upper layer (2.5 ml) of the solution was mixed with 2.5 ml of distilled water and 2.5 ml of 0.1% ferric chloride and the absorbance was read at 700 nm. Increasing absorbance of the reaction mixture indicates increasing reducing power.
ACE Inhibitory Activity. ACE inhibitory activity of the peptide sample was measured with a UV spectrophotometer based on the rate of formation of hippuryl hippurat acid-Lhistidyl-L-leusine (HHL) (Chusman & Cheung 1971). For each measurement, 10 μl peptide samples and 10 μl HHL 50 mM were incubated with 10 μl ACE 0.8 mU at 37 °C for 45 min. The reaction was stopped by adding 100 μl of 1 N HCl, then 600 μl ethyl acetate was added and homogenized using vortex for 30 seconds, and then centrifuged at 300×g for 10 min. A total of 100 μL supernatant (upper layer) was taken and dried at a temperature of 140 °C for 10 min. The residue obtained was dissolved with 600 μl 1 M NaCl. Absorbance was measured at wavelength of 228 nm. The complete procedure of measurement of ACE inhibitory activity can be seen in Appendix 1. ACE inhibitory activity (%) =
Ac As 100% ( Ac Ab )
Description: Ac = absorbance of control As = absorbance of the sample Ab = absorbance of blank
SDS-PAGE Analysis. To analyze and visualize protein pattern, SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) was done. As much as 10 μl sample were mixed with 40 μl sample buffer and were incubated 100 °C for 5 min before loading. Proteins were separated on a 6% (stacking gel) and 15% (separating gel) (Table 3) with 70 V for 3-4 h. After the run, the gels were stained with CBB-R 250 staining solution (15 min) and destained for 24 h (Laemmli 1970). Table 3 Composition of SDS-PAGE gel Composition 15% (µl)
6% (µl)
Solution A
2500
1000
Solution B
1250
-
Solution C
-
1250
MiliQ
1250
2750
APS 10%
100
50
TEMED
10
5
RESULTS In this research, DPPH radical scavenging activities of beans were analyzed with different hydrolysis time and protein concentration (Figure 3; Table 4). All samples showed antioxidant capacity in scavenging
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80 60
0 min 5 min 10 min
40 20 0
RB
MB
SB
Scavenging acticity(%)
(0.5 mg/ml)
A.acid α-toco (10mg/ml)(67mg/ml)
Scavenging activity(%)
100
thereafter. Both antioxidant assays (DPPH & Fe reducing) were compared to ascorbic acid and α-tocopherol as positive controls. For ACE inhibitory assay, all samples could act as antihypertension agents. Mung bean showed highest activity in different enzyme activity treatment in 5 min hydrolysis, 90.13 % and 96.67 % (0.173 U/ml and 0.272 U/ml, respectively) with 10 mg/ml protein concentration (Figure 5; Table 6). Antihypertensive activity of beans increased with hydrolysis treatment (in most samples) than without hydrolysis. Highest antihypertensive activity in all samples and different enzyme activity seen at 5 min hydrolysis, decreased thereafter. The SDS-PAGE results (Figure 6) showed that several different molecular weights of proteins were observed, which expected as bioactive peptides in bean samples. The breakdown of samples can be seen as protein bands disappear over increasing hydrolysis time (showed by arrows). Peptide concentrations are different depending on their size: in the first stages of storage peptides larger than 45 kDa were predominant, but as time increased, the concentration of lower MW peptides increased.
120 100 80
60
0 min 5 min 10 min
40 20 0
RB
MB
(2 mg/ml)
SB
A.acid α-toco (10mg/ml)(67mg/ml)
100 80 60
0 min 5 min 10 min
40 20 0
RB
MB
SB
(1 mg/ml)
Scavenging activity (%)
Sscavenging activity(%)
free radicals. Red bean, mung bean, and soy bean hydrolysates exhibited the highest activity at 10 mg/ml protein concentration, both results (91.24 %, 94.90 %, 67.83 %, respectively) were carried out at 5 min hydrolysis time with 0.173 U/ml bromelain, and decreased thereafter. Results also showed that the increase of protein concentration gave better scavenging activity in each sample (from 0.5 mg/ml; 1 mg/ml; 2 mg/ml). However, in 10 mg/ml with the same enzyme activity, the scavenging activity was reduced. Higher enzyme activity (0.272 U/ml) which used at 10 mg/ml concentration showed higher scavenging activity (Appendix 2). It also peaked after 5 min hydrolysis, decreased thereafter. Due to this property, beans hydrolysates could scavenge by donating protons to free radicals and solution colour were changed to yellowish colour. Results of ferric reducing activity of hydrolysates obtained after various hydrolysis time treatments. It showed that all samples had antioxidant capacity in reducing ferric into ferrous form (Figure 4; Table 5). All samples showed highest activity at 2 mg/ml protein concentration with 5 min hydrolysis time (0.272 U/ml). Similarly, the results of DPPH scavenging activity, samples were peaked after 5 min hydrolysis and decreased
A.acid α-toco (10mg/ml)(67mg/ml)
100
80 60
0 min 5 min
40
10 min
20 min
20
30 min 0
60 min
RB
MB
(10 mg/ml)
SB
A.acid α-toco (10mg/ml)(67mg/ml)
Figure 3 DPPH scavenging activity (%) of bean flour substrates (RB: red bean; MB: mung bean; SB: soy bean) hydrolyzed with bromelain enzyme (0.173 U/ml).
2,5
0 min 2,0
5 min
1,5
10 min 20 min
1,0
30 min
0,5
60 min
0,0
Ferric reducing activity
Ferric reducing activity
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A.acid α-toco (1 mg/ml); 0.272 U/ml) (10mg/ml)(67mg/ml) MB
SB
Ferric reducing activity
RB
2,5 0 min 2,0
5 min 10 min
1,5
20 min 1,0
30 min 60 min
0,5 0,0 RB
MB
SB
(2 mg/ml; 0.272 U/ml)
A.acid α-toco (10mg/ml) (67mg/ml)
2,5 0 min 2,0
5 min 10 min
1,5
20 min 30 min
1,0
60 min 0,5
0,0 RB
MB
SB
A.acid
α-toco
(10 mg/ml; 0.173 U/ml) (10mg/ml)(67mg/ml) Figure 4 Ferric reducing activity of bean flour substrates (RB: red bean; MB: mung bean; SB: soy bean) hydrolyzed with bromelain enzyme.
ACE inhibitory activity(%)
100 80 0 min 5 min 10 min 20 min 30 min 60 min
60
40 20 0
ACE inhibitory activity (%)
RB MB SB C0.5 C1 (10 mg/ml; 0.173 U/ml) (Captopril; mg/ml) 100 80
0 min
60
5 min
40
10 min 20 min
20 0 RB MB SB C0.5 C1 (10 mg/ml; 0.272 U/ml) (Captopril; mg/ml)
Figure 5 ACE inhibitory activity (%) of bean flour substrates (RB: red bean; MB: mung bean; SB: soy bean hydrolyzed with different bromelain enzyme activity.
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Figure 6 SDS-PAGE result of hydrolysates (10 mg/ml; 0.272 U/ml) of bean samples with different hydrolysis time. Lane M: LMW Marker; Lane 1-3: Red bean (0, 5, 60 min, respectively); Lane 4-6: Mung bean (0, 5, 60 min, respectively); Lane 7-9: soy bean (0, 5, 60 min, respectively); Lane 10: Bromelain enzyme. Table 4 Increment of scavenging activity (0.173 U/ml) (RB: red bean; MB: mung bean; SB: soy bean)
Table 5 Increment of ferric reducing activity (RB: red bean; MB: mung bean; SB: soy bean)
Table 6 Increment of ACE inhibitory activity (RB: red bean; MB: mung bean; SB: soy bean)
DISCUSSION Free radicals with major species of ROS are unstable and react readily with other groups or substances in the body, resuting in
cell or DNA damage and, thus human disease. Therefore, removal of free radicals and ROS is probably one of the most effective defenses of a living body against various diseases. The beneficial effects of antioxidants are well known in scavenging free radicals and ROS or
9
in preventing oxidative damage by interrupting the radical chain reaction of lipid peroxidation (Kim et al. 2006). DPPH is a stable free radical that show maximum absorbance at 517 nm in ethanol. When DPPH encounters a proton-donating substance, such as antioxidant, the radical is scavenged. The color is changed from purple to yellow and the absorbance is reduced. The disappearance rate of DPPH free radicals was used to express the free radical scavenging activity of beans protein hydrolyzed with bromelain under optimized conditions (Shimada et al. 1992). Ferric reducing power activity also used to measure the antioxidant activity based on electron-donating activity. Compounds that have antioxidant activity reacts with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+) by reduction, which then forms a complex react with FeCl3 to form complex with FeCl2. The resultant solution will be measured by the spectrophotometric method at a wavelength of 700 nm (Hemalatha et al. 2010). The reducing ability of beans indicates that they could act as electron donors, reducing the oxidized intermediates of lipid peroxidation processes, and suggesting that the reducing power likely contributes to the antioxidant activity (Zhu et al. 2006). Results showed there were variation of scavenging activity and ferric reducing power between each sample and each treatment (hydrolysis time). It is also observed that protein concentration has effect on antioxidant activity, and most likely the higher sample concentration, the higher ability of samples to act as antioxidant. But this properties must be optimized with enzyme activity. Peptides and protein hydrolysates obtained from the proteolysis of various food proteins, are reported to posses antioxidant activities. This increasing antioxidant activity through hydrolysis suggests that this process contributed to antioxidant activity by releasing previously inactive peptides encrypted in the sequence of bean samples and then act as proton or electron donor (Correa et al. 2011). However, the longer hydrolysis with increasing antioxidant activity can not always be observed. In this study, mostly at 60 min hydrolysis time, samples showed reduction in activity compared to unhydrolyzed (0 min) samples. This possibly caused by shorter peptide chain length and also functionality of hydrolysis depended on enzyme characteristic (Klompong et al. 2007).
The distinctive behavior of protein hydrolysates in the antioxidant activity also could be explained by the different stereoselectivity of the radicals and different peptides present in samples capable of reacting and quenching different radicals. Also, the antioxidant effect of beans and beanderived peptides is not fully based on capability to donate hydrogen or electron, and differences is scavenging efficacy can be attributed to solubility and diffusivity of radicals. His, Phe, Tyr, Trp, among other aromatic and hydrophobic amino acids, seems to be involved in the antioxidant activity (Correa et al. 2011). The other method for measuring antioxidant activity is 2,2'-azinobis-3ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay. This assay is generated by oxidation of ABTS radical chromophore with potassium persulfate and is reduced in the presence of such hydrogendonating antioxidant. The solution are measured at 734 nm wavelength to find percentage of inhibition (Correa et al. 2011). Direct reaction of a substance is not the only mechanism by which the antioxidants may display their activity. Secondary antioxidants act through numerous possible mechanisms. One of the most important mechanisms of action of secondary antioxidants is chelation of prooxidant metals. Iron and other transition metals (copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel) promote oxidation by acting as catalysts of free radical reactions (Koncic et al. 2011). For example, the chelating properties of hydrolysates and peptide fractions can be evaluated by monitoring the formation of the complex between ferrozine and Fe2+ (ferrous) ion spectrophotometrically at 500 nm. When a compound has chelating properties, the formation of complex (Ferrozine/Fe2+) is disrupted resulting in a decrease of the red colour and this makes possible the estimation of the metal chelating activity of constituents (Yamaguchi et al. 2000). Many antihypertensive drugs have their primary action on systemic vascular resistance. Some of these drugs (captopril, lisinopril) produce vasodilation by interfering with vascular tone or by blocking the formation of angiotensin II or its vascular receptors. The other drugs have different medication mechanism, such as calcium channel blocker, beta blocker, and diuretic effect (Dilpardo 2010). Although some drugs
10
less commonly used because of a high incidence of side effects, like itching; rash; dry cough, therefore natural sources, are promising to be used as treatment for patient, including bioactive peptides. Related to that, results showed ACE inhibitory activity in all hydrolysates were high, and some of them had higher activity than captopril (Figure 5). This suggests that bean samples could be potent antihypertension
drugs with very minimum side effects. It also showed that different enzyme activity and hydrolysis time contribute to various result of ACE inhibitory activity. Red bean, mung bean, and soy bean have been known for its antioxidative characteristic, as mentioned in previous studies. This research also showed that proteins in each bean sample contributed to both antioxidant and antihypertensive capabilities (Table 7).
Table 7 Bioactivity of many plant and animal samples in various studies
The effect of bromelain on protein breakdown in beans at the different hydrolysis time was analyzed by SDS-PAGE (Figure 6). Bromelain catalyzes hydrolysis of protein and peptide amides with broad specificity for peptide bonds and is a preference for large uncharged residue. Molecular weight distribution of hydrolyzed protein is one of the most important properties in producing protein hydrolysates to be used as functional materials, which has a direct impact on their functional properties (Takano 2002). SDS-PAGE results showed that so many proteins detected with various molecular weight. Red bean sample showed quite many bands, mainly about 23-35 kDa (globulin types around 22 kDa-37 kDa), 40-50 kDa (amylase inhibitor types around 45-50 kDa) in unhydrolyzed condition (Meng & Ma 2002; Marambe & Wanasundara 2012). After hydrolysis, red bean sample showed reduction in bands, except protein below 30 kDa, which seem not being hydrolyzed. Protein with 4050 kDa (amylase inhibitor), showed reduction
of bands after 5 min and 60 min hydrolysis, suggest that it turned into small proteins or peptides that exert antioxidative capabilities. Mung bean showed most bands than any samples, which probably are legumin (16.5-56 kDa), vicillin (24-63.5 kDa), and globulin (2237 kDa) in unhydrolyzed condition (Marambe & Wanasundara 2012; Mendoza et al. 2001). Almost all bands in mung bean showed reduction in its thickness, started at 5 min hydrolysis, and vicillin was probably the main protein that exert bioactivity after being hydrolyzed. For soy bean, mostly below 45 kDa, which probably were subunits of glycinine or cystein protease types (20 kDa–40 kDa), and βconglycinin (63 kDa) (Wang & Gonzales 2005). All samples showed protein bands reduction afrer hydrolyzed with bromelain enzyme into small proteins or peptides. Cystein protease and glycinine seemed to play main roles in antioxidant and antihypertension capabilities. Different hydrolysis patterns related to different specificity of bromelain
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enzyme on each sample. This suggests that peptide production was probably a staged process in which low molecular weight peptides (below 30 kDa) were formed by the hydrolysis of midrange peptides (40 – 60 kDa) which were once originated by highmolecular-weight proteins or peptides.
CONCLUSION Herein we produce bioactive peptides by enzymatic hydrolysis using bromelain enzyme. The bioactive peptides produced were potent radical scavengers and antihypertensive activity by efficiently inhibited free radical chain reaction by scavenging DPPH free radical and reduce ferric into ferrous form. Results showed that each sample, especially in 5 min hydrolysis treatment, has maximum capability as well as positive controls. The inhibitory effect of peptides prepared from bean on ACE activity make these products, especially mung bean, a potential candidates to be developed and applied within the concept of functional foods. Further research is needed to know the biological effects in vivo and kinetic study.
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APPENDICES
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Appendix 1 Procedure of ACE inhibitory activity Volume (μl) Blank Control Distilled water 10 10 NaCl 4.0 M 10 10 Borate buffer 0.1 M pH 8.3 50 50 HHL 50 mM 10 10 HCl 1.0 N 100 Borate buffer 0.1 M pH 8.3 10 10 Sample solution ACE (0.8 mU) 10 10 Incubated at 370C for 45 min HCl 1.0 N 100 Ethylacetate 600 600 Homogenized using vortex for 30 seconds Centrifuged at 300×g for 10 min Filtrate
100 Dried in oven at 140 0C for 10 min 600
NaCl 1.0 M
Sample 10 10 50 10 10 10 100 600
100
100
600
600
Absorbance was measured at 228 nm
Appendix 2 DPPH scavenging activity (%) of bean flour substrates hydrolyzed with bromelain enzyme (0.272 U/ml) 100 Scavenging activity (%)
90 80 70 60
0 min
50
5 min
40
10 min
30
20 min
20
30 min
10
60 min
0 RB
MB
SB Samples
A.acid (10mg/ml)
α-toco (67mg/ml)
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Appendix 3 Tyrosin standard curve 1,2 y = 4,922x - 0,012 R² = 0,999
Absorbance
1 0,8 0,6 0,4 0,2 0 0
0,05
0,1
0,15
0,2
0,25
[Tyrosin] Appendix 4 Bradford standard curve 0,12 y = 0,094x + 0,005 R² = 0,991
Absorbance
0,1 0,08 0,06 0,04 0,02 0 0
0,2
0,4
0,6 [Protein]
0,8
1
1,2
