ANALYSIS ON DEGREE OF HYDROLYSIS AND MOLECULAR WEIGHT OF LOTUS SEED PROTEIN ISOLATE BY ALCALASE ENZYME PRACTICAL TRAINING REPORT This practical training report is submitted for the partial requirement for Bachelor Degree
ANALYSIS ON DEGREE OF HYDROLYSIS AND
MOLECULAR WEIGHT OF LOTUS SEED PROTEIN
ISOLATE BY ALCALASE ENZYME
PRACTICAL TRAINING REPORT
This practical training report is submitted for the partial requirement
for Bachelor Degree
By :
Matius Inda Tatontos
12.70.0062
DEPARTMENT OF FOOD TECHNOLOGY
FACULTY OF AGRICULTURAL TECHNOLOGY
SOEGIJAPRANATA CATHOLIC UNIVERSITY
SEMARANG
2015
ANALYSIS ON DEGREE OF HYDROLYSIS AND
MOLECULAR WEIGHT OF LOTUS SEED PROTEIN
ISOLATE BY ALCALASE ENZYME
PRACTICAL TRAINING REPORT
This practical training report is submitted for the partial requirement
for Bachelor Degree
By :
Matius Inda Tatontos
12.70.0062
DEPARTMENT OF FOOD TECHNOLOGY
FACULTY OF AGRICULTURAL TECHNOLOGY
SOEGIJAPRANATA CATHOLIC UNIVERSITY
SEMARANG
2015
PREFACE
Gratitude to God The Almighty One, who has given His blessings so the writer can complete this practical training report entitled “Analysis on Degree of Hydrolysis and Molecular Weight of Lotus Seed Protein Isolate By Alcalase Enzyme
”. This practical training report is submitted as one of the requirements to gain bachelor degree of Agricultural Technology Faculty, Food Technology Department, Soegijapranata Catholic University.
In finishing this reports, the writer really gives thanks for people who has always support and help, they are :
1. Dr. Chun-Ping Lu, the advisor, who let the writer to join her laboratory, guide writer for this internship program, and practical training report.
2. Professor Bing-Huei Chen, Director College of Human Ecology, Fu Jen Catholic University, who has given and accepted writer to join the internship program in his college.
3. Dr. V. Kristina Ananingsih, ST., MSc., Dean of Faculty of Agricultural Technology, Soegijapranata Catholic University, who giving the information and chance about the internship program in Fu Jen Catholic University.
4. Ivone E. Fernandes, S.Si, M.Sc., the advisor, who help, give inputs, and ideas to the writer, so the writer can finish this practical training report well.
5. Chen Wen Chi, the mentor, who is in charge to take care and guide the writer to finish laboratory work for two months.
6. My parents, Djony Ignatius Tatontos and Agnes Lanny Santoso, and my brother, Andreas Aga Tatontos, for always supporting and pray for the writer.
7. Terry, Pito, Felly, Lisa, Stella, There, Lyra, who has been the best partners during internship program.
8. All people who has guide, accompany, and help the writer during internship program until finishing the report.
Finally, the writer realizes that this report is not perfect and there are some unintended errors. The writer will accept all suggestions from all the readers, so this report can be a good examples for the others. The writer hopes that this report can be useful for the others.
Semarang, 10 April 2015 Writer
CONTENTS
TITLE ........................................................................................................................... i APPROVAL PAGE ...................................................................................................... ii PREFACE ..................................................................................................................... iii CONTENTS ................................................................................................................. v LIST OF TABLES ....................................................................................................... vii LIST OF FIGURES ...................................................................................................... viii 1.
INTRODUCTION ................................................................................................. 1
1.1.Institution Profile .............................................................................................. 1 1.1.1.
Fu Jen Catholic University.................................................................... 1 1.1.2. College of Human Ecology/Department of Food Science .................... 2 1.1.3. Vision and Mission ............................................................................... 3 1.1.4. Faculty Members .................................................................................. 3
1.2.Purpose of Practical Training ........................................................................... 4
1.3.Time and Place of Practical Training ............................................................... 4 2. RESEARCH ........................................................................................................... 6
2.1.Overview ........................................................................................................... 6
2.2.Background of Research ................................................................................... 6
2.3.Literature Review ............................................................................................. 7 2.3.1.
Protein ................................................................................................... 7 2.3.2. Lotus Seed ............................................................................................. 7 2.3.3. Protein Hydrolysates ............................................................................. 9 2.3.4. Degree of Hydrolysis (DH) ................................................................... 9
2.4.Objectives ......................................................................................................... 11 3. RESEARCH METHODOLOGY ........................................................................... 12
3.1.Materials and Methods ..................................................................................... 12 3.1.1.
Tools ..................................................................................................... 12 3.1.2. Materials ............................................................................................... 12
3.2.Methods ............................................................................................................ 12 3.2.1.
Preparation of Lotus Seed Protein Isolate (LSPI) ................................. 12 3.2.2. Extraction of Lotus Seed Protein Hydrolysates .................................... 13
3.2.2.1.Effect of Different Substrate Concentration on DH ................. 13
3.2.2.2.Effect of Different Time of Hydrolysis on DH ......................... 15 3.2.3. SDS PAGE Analysis ............................................................................. 16
3.2.3.1.Gel Preparation.......................................................................... 16
3.2.3.2.Samples Preparation .................................................................. 17 4. RESULTS AND DISCUSSIONS .......................................................................... 18
4.1.Effect of Different Substrate Concentration on DH ......................................... 20
4.2.Effect of Different Hydrolysis Times on DH ................................................... 21
4.3.SDS PAGE Analysis ......................................................................................... 24 5. CONCLUSION ...................................................................................................... 27 6. REFERENCES ....................................................................................................... 28
LIST OF TABLES
Table 1. Composition of Lotus Seed ........................................................................... 8 Table 2. Effect of Different Substrate Concentration on Degree of Hydrolysis .......... 20 Table 3. Effect of Different Hydrolysis Times on Degree of Hydrolysis .................... 22
LIST OF FIGURES
Figure 1. Flag of Fu Jen Catholic University ............................................................... 2 Figure 2. Emblem of Fu Jen Catholic University ......................................................... 2 Figure 3. Organization Structure Department of Food Science .................................. 4 Figure 4. Map of Fu Jen Catholic University, Xinzhuang, New Taipei City ............... 5 Figure 5. Set Samples to Prepare LSPI ........................................................................ 13 Figure 6. Inactivation Process In Dry Bath Incubator .................................................. 14 Figure 7. Set Samples For Hydrolysis Process ............................................................. 15 Figure 8. SDS PAGE Analysis ..................................................................................... 17 Figure 9. Effect of Times of Hydrolysis on Degree of Hydrolysis .............................. 21 Figure 10. Effect of Substrate Concentration on Degree of Hydrolysis ....................... 23 Figure 11.Weight Molecular Distribution of Lotus Seed Protein Isolate ..................... 25
1. INTRODUCTION 1.1. Institution Profile 1.1.1. Fu Jen Catholic University
Fu Jen Catholic University is the first university in China established by the Catholic Church. Moved by the Christian understanding of love and inspired by the high ideals of Confucian education, it adopted the name "Fu Jen" to give expression to its universal vision and mission realized through holistic education in the Chinese cultural context. Fu Jen Catholic University was founded in Beijing in 1925 by the Benedictines of St. Vincent Archabbey in Latrobe, Pennsylvania, USA at the request of the Holy See. It was opened as a single college under the name of Fu Jen Academy. In 1929, the Ministry of Education officially recognized Fu Jen as a university. In 1959, the Chinese Regional Bishops' Conference, the Society of Jesus, and the Society of the Divine Word collaborated on the reestablishment of the University in Taiwan. In 1960, the Ministry of Education granted permission to restore Fu Jen in Taiwan. In 1961, the Graduate Institute of Philosophy admitted students. In 1963, the University was granted a share of the successful candidates of the University Entrance Examination and received the first freshmen of the College of Liberal Arts, Science and Engineering, and Law.
Currently, the University comprises 11 colleges, namely Liberal Arts, Arts, Foreign Languages, Science and Engineering, Human Ecology, Law, Social Sciences, Management, Medicine, Communication, Education, 48 departments, offering 47 master's programs, 22 in-service master's programs, 11 Ph.D. programs, and 16 departments in the School of Continuing Education. The land capacity of the university is about 35 hectares and current student enrollment is 26,000. The university has about 120 sister schools worldwide. The university strives to provide students with a diversified, holistic, interdisciplinary, and international learning environment.
The University’s flag color is yellow, which indicates the affinity of the University to the Holy See. The twelve stars in the middle symbolize the Virgin Mary.
Figure 1. Flag of Fu Jen Catholic University The University
’s emblem or the laurel wreath symbolizes peace, while the twelve stars in the middle are a symbol of the Virgin Mary. The Latin words at the bottom of the emblem signify the University's ideals —Truth, Goodness, Beauty, and Holiness.
Figure 2. Emblem of Fu Jen Catholic University 1.1.2.
College of Human Ecology / Department of Food Science
In 1963, the Department of Family Studies and Nutrition Sciences was established and grouped into the Family Studies section and the Nutrition Sciences section. Nutrition Sciences section was combined with the Food Sciences section as the Department of Nutrition and Food Sciences in 1971. The Graduate Institute of Nutrition and Food Scienc es was established and started to offer a master’s degree program in 1983. The doctoral program was joined to the Institute in 1995. Food Sciences section became an individual department in 2006. The Department of Food Science offers Bachelor’s degree pro gram and Master’s degree program.
1.1.3. Vision and Mission
Uphold the spirit of pursuing truth, goodness, beauty and holiness, the Department of Food Science at the Fu Jen Catholic University integrates basic sciences with latest technology for excellence in education, research, and service. We are committed to promote the healthier, tastier and safer foods for improving eating quality, human health and wellness.
1.1.4. Faculty Members
There are 11 main instructors in Department of Food Science : 1.
Professor Chihwei P. Chiu 2. Professor John-Tung Chen 3. Professor Bing-Huei Chen 4. Associate Professor Rei-May Huang 5. Associate Professor Shau Chen 6. Associate Professor Meng-I, Marie, Kuo 7. Associate Professor Jung-Feng Hsieh 8. Assistant Professor Tsung-Yu Tsai 9. Assistant Professor Tsai-Hua Kao 10.
Assistant Professor Chun-Ping Lu 11. Assistant Professor Bang-Yuan Chen
All of those instructors have their own responsibility in their faculty. The organization structure Department of Food Science can be seen at Figure 3.
Director of Human Ecology
Professor Bing-Hui Chen
Director of Food Science
Assistant Professor Tsung-Yu Tsai Professor Chiwei P. Chiu Professor John Tung
Chien
Food Enzymology Lab. Food Physicochemistry Lab.
Associate Professor Jung-Feng Associate Professor Meng I-Marie Hsieh Kuo
Associate Professor Rey-May Associate Professor Shau-Chen
Huang
Food Microbiology Lab. Nutraceuticals & Food Processing Assistant Professor Bang-Yuan Lab.
Chen Assistant Professor Tsai-Hua Kao
Food Biochemistry Lab.
Assistant Professor Chun-Ping Lu Figure 3. Organization Structure Department of Food Science 1.2.
Purpose of Practical Training a.
Give the student an experience about food research in Taiwan.
b.
Give the student an opportunity to know and adapt with new culture and society in Taiwan.
c.
Give the student an experience to communicate in english.
1.3. Time and Place of Practical Training
The practical training is conducted in the College of Human Ecology, Departement of
rd nd
Food Science, Fu Jen Catholic University, Taipei, Taiwan, in Januari 13 to March 12 2015.
Figure 4. Map of Fu Jen Catholic University, Xinzhuang, New Taipei City The red indicator shows the location of Fu Jen Catholic University which is located at No. 510, Zhongzheng Rd., Xinzhuang Dist., New Taipei City 24205, Taiwan (R.O.C.) TEL +886-2-29052000.
2. RESEARCH 2.1. Overview
This research use lotus seed protein isolate (LSPI) as the main material. This research analyze degree of hydrolysis and molecular weight of LSPI. In the analysis on degree of hydrolysis (DH), factors that used were substrate concentration and hydrolysis time. Analysis on degree of hydrolysis used o-phthalaldehyde solution (OPA solution). While the determination of molecular weight using SDS PAGE analysis.
2.2. Background of Research
Lotus cultivated in China for more than 1000 years and served as an industrial crop grown over 40,000 ha. All parts of lotus can be used for humans needs, one of them are their seed. In China, lotus seed usually popped like popcorn, eat as a soup, and used as composite flour in bread making. Lotus seed also can be used as composite flour by blending it with other legumes like soybean or millets. This composite flour can be use as low cost proteinaceous and health food.
Lotus seed used in China folk medicines to treat tissue inflammation, cancer, skin diseases, leprosy, poison antidote, and generally prescribed to children as diuretic and refrigerant. Lotus seed can used to treat a lot of disease because it is a good source of bioactive peptides. Bioactive peptides obtained from hydrolysis of protein into protein hydrolysates. Protein hydrolysates widely used as nutritional supplements, functional ingredients, and flavor enhancers in many kind of foods. Previous studies have reported that food protein hydrolysates can scavenge free radicals for against aging, cardiovascular, and other diseases. Protein hydrolysates can be produced in vitro through enzymatic hydrolysis using commercial protease such as alcalase enzyme. The optimization of protein hydrolysates procedure will save cost and time to produce protein hydrolysates. So, with the optimization of protein hydrolysates procedure there will be a lot of protein hydrolysates can be produce and useful for food products.
2.3. Literature Review 2.3.1. Protein
Proteins are essential food components. Proteins are source of amino acids needed for growth and maintenance for human. Proteins also essential components of tissues in
organisms and have a large number of physiological processes within cells. Many of the physiological and functional properties of proteins are believed to attribute to biologically active peptides encrypted in the protein molecules. (Shahidi and Zhong, 2008).
2.3.2. Lotus Seed
Lotus (Nelumbo nucifera) is an aquatic plant, native to Asia from modern Afghanistan to Vietnam and to New Guinea and north Australia. It is extinct in the wild in Africa, but it is widely naturalized and commonly cultivated in water gardens around the world like China, Japan, Hawaii, India, and Korea. Nelumbo nucifera grows with roots in the muddy soil and leaves floating on top of the water surface. The flowers are found on thick stems. The plant grows up to 6 meters in height and spreads horizontally up to 3 meters. The leaves about 60 cm in diameter and the flowers about 20 cm in diameter. The Nelumbo nucifera is an integral part of tropical wetland ecosystems. Nelumbo
nucifera grows in shallow ponds, lagoons, marshes, flooded fields, and river. It is very
important for the ecosystems because many species depend on it for survival. The whole web species dependent on it for food, shelter, and other life requisites. Health condition of the lotus is also a good indicator of the health of the whole associated community (Murty, 2012). The taxonomy of Nelumbo nucifera is : Kingdom : Plantae Subkingdom : Viridiplantae Division : Tracheophyta Subdivision : Spermatophytina Class : Magnoliopsida Order : Proteanae Family : Nelumbonaceae Genus : Nelumbo Adans.
Species : Nelumbo nucifera Gaertn. Flowers, seeds, young leaves, and rhizomes are all edible. The hard seeds eaten like nuts, added as a thickening to soups, roasted like chestnuts, dried and ground into flour for making bread. As a food source, lotus seed consist of 10.5% moisture, 10.6-15.9% protein,1.93-2.8% crude fat, 70-72.17% carbohydrate, 2.7% crude fibre, 3.9-4.5% ash, and energy 348.45 cal/100 g. Lotus seed alson contains minerals like chromium (0.0042%), sodium (1%), potassium (28.5%), calcium (22.1%), magnesium (9.2%), copper (0.0463%), zinc (0.084%), manganese (0.356%) and iron (0.199%).
Table 1. Composition of Lotus Seed (100 g) Composition Lotus Seed Moisture (g)
10.5 Protein (g) 10.6-15.9 Crude Fat (g) 1.93-2.8 Carbohydrate (g) 70-72.17 Crude Fibre (g)
2.7 Ash (g) 3.9-4.5 Energy (cal) 348.45 (Sridhar and Bhat, 2007) Lotus seed is an important and famous as a traditional medicine in China. Lotus seeds used to treat tissue inflammation, cancer, diuretics, skin diseases and as poison antidote.
Lotus seeds are astringent and used to treat hyperdipsia, dermatopathy, halitosis, menorrhagia, leprocy, and fever. Seed powder mixed with honey can be use to treat cough. Lotus plants also provide several bioactive ingredients like alkaloids, flavonoids, antioxidants, antisteroids, antipyretic, anticancerous, antiviral and anti-obesity properties. Lotus seed can be use as an alternate protein source, supplement, and potential pharmaceutical source. As lotus seeds have potential nutririous and health advantage, blending its flour with other legumes or millets can develop low cost proteinacious and health food source (Sridhar and Bhat, 2007).
The bioactive peptides in lotus seed give a lot of functional properties of protein like antidote, antioxidant, and anticancer. The bioactive peptides are inactive within the
parent protein molecules. Bioactive peptides need certain processing approaches to release from lotus seed protein. Hydrolysis of lotus seed protein into lotus seed protein hydrolysates can release the peptides from lotus seed protein, so it can optimize the functional properties of its protein (Shahidi and Zhong, 2008).
2.3.3. Protein Hydrolysates
Protein hydrolysates are widely used in food systems as nutritional supplements, functional ingredients, and flavor enhancers in many kind of foods. Protein hydrolysates can be obtained from enzymatic hydrolysis of proteins. Enzymatic hydrolysis has been used for modification of functional and nutritional properties of food proteins (Liu and Chiang, 2008). Enzymatic hydrolysis using selective proteases will provides moderate conditions of the process, few or no undesirable side reactions or products, less salts, and the functionality of the final product can be controlled by selection of specific enzymes and reaction factors (Hrckova et.al., 2001). Enzymatic hydrolysis is generally used in laboratories and industries because more safe, cheaper, specific, and less destructive than chemical hydrolysis which can destroys all peptide bonds (Zhang et. al. ,2012).
In enzymatic hydrolysis, the type of enzyme is very important because it dictates the cleavage pattern of the peptide bonds. There are many enzyme that can be used like trypsin, subtilisin, chymotrypsin, thermolysin, pepsin, proteinase K, papain, and plasmin. Commercial protease such as Alcalase and Flavourzyme usually used to prepare peptides from protein. These enzymes are obtained from different sources, including plants, animals, and microorganisms, and each requires optimal conditions like temperature, pH, time course, enzyme/substrate ratio, etc. (Shahidi and Zhong, 2008) . Hydrolysate properties of protein can be measured by measuring the degree of
hydrolysis (DH).
2.3.4. Degree of Hydrolysis (DH)
Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate. Degree of hydrolysis also serves as a means of determining protein hydrolysate properties. The degree of hydrolysis (DH) is important variable affecting the attributes of the protein hydrolysates of a given enzyme/substrate system. It is generally agreed that with endopeptidases, lower DH produced hydrolysates with higher molecular weight fractions, which exhibited better emulsification and aeration properties but showed greater hydrophobicity. The relation between DH and bitterness, antioxidative or other peptide bioactivities is enzyme dependant (Himonides et al, 2011) There are several methods for determining DH; pH-stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), and formol titration methods. The pH-stat method is based on the number of protons released during hydrolysis. The pH-stat is simple and commonly used, but does not determine peptide bonds directly. The accuracy of the method also depends on the type of hydrolytic enzymes used, the size of the hydrolyzed peptides, and the reaction temperature. The SN-TCA method measures the amount of TCA-soluble nitrogen. The TNBS, OPA, and formol titration methods are based on the measurement of amino groups generated from hydrolysis. Generally, the TNBS and OPA methods are comparable and directly determine the DH. The TNBS method is laborious and use hazardous and unstable chemicals. The TNBS cannot be used to follow a hydrolysis reaction continuously. The OPA method is more accurate, easier, environmentally safer and faster, and has a broader application range as compared to the TNBS method (Zarei
et al , 2012).
OPA is a very high sensitivity detection reagent of amines contained in proteins, peptides, and amino acids. OPA is well soluble and stable in water solution at pH<11.5. It is sensitive to UV illumination and air oxidation. Absorbance at 340nm increase within 15seconds up to 1-3 minutes, then decreases more or less slowly. The reactivity of OPA to protein influenced by some factors. Buffer with a basic pH (pH 9.0 is optimal) results in greater fluorescence as primary amino groups are more likely to be protonated and thus more reactive. pH levels around the physiologic range (pH 6-9) provide quite acceptable results. Many buffer systems in the pH range of 6-9, such as PBS or sodium borate, are suitable for this reaction; however, they should not contain amines (e.g., Tris or glycine). o-Phthaldialdehyde offers several advantages for protein quantitation. OPA solution is stable for long periods while in solution and protein quantitation using OPA requires very little sample (5-
10 μl). (Held, 2006). In this research, degree of hydrolysis (DH) was taken as dependent variable. The independent variables are substrate concentration and time of hydrolysis. OPA solution is used to determine the degree of hydrolysis.
2.4. Objectives
The main objectives of this study are : 1. to determine which substrate concentration and hyrolysis time give the best degree of hydrolysis.
2. to determine the molecular weight of lotus seed protein isolate. 3. to optimize lotus seed protein hydrolysate procedure.
3. RESEARCH METHODOLOGY
This methods based on Frister H., et al (1988) using OPA modified method. This method use N,N-dimethyl-2-mercaptoethylammonium chloride as thiol component for determining peptides. This method give better result than method that using mercaptoethanol. OPA modified method give high accuracy, precision, and long-term stability solution.
3.1. Materials and Methods 3.1.1. Tools
Tools that used in this research were centrifuge, shaker, oven, dry bath incubator, freeze dryer, cap holding tabs, spectrophotometer, casting frames, casting stands, glass plates 1.5mm, well-forming comb, and anodes.
3.1.2. Materials
Materials that used in this research were defatted lotus seed powder, deoinized water,
0.5 N NaOH, 0.5 N HCl, phosphate buffer saline (PBS), alcalase enzyme pH 8.5, OPA solution (10mM sodium tetraborate, 20% sodium dodecyl sulfate, o-phthalaldehyde in methanol, β-mercaptoethanol), 12% separating gel solution 15 ml (40% acrylamide mix,
1.5M tris pH 8.8, 10% SDS, 10% APS, TEMED), 5% stacking gel solution 6 ml (40% acrylamide mix, 1.5M tris pH 8.8, 10% SDS, 10% APS, TEMED), isopropanol, sample buffer (1M tris-HCl pH 6.8, glycerol, SDS, bromphenol blue, dithiothreitol, water) 3.2.
Methods 3.2.1. Preparation of Lotus Seed Protein Isolate (LSPI)
Lotus seed contains moisture, protein, fat, and carbohydrate. In this research, only the protein from lotus seed that used. So, the preparation of lotus seed protein isolate (LSPI) was necessary to remove the unwanted substances. The main objective of this preparation is to get the lotus seed protein isolate (LSPI) so the determination degree of hydrolysis can be done accurately.
First, 4 g of defatted lotus seed powder was added by 40 ml of deionoized water. The solution was stirred for 30 minutes with shaker. The solution adjusted to pH 10 by 0.5N NaOH. The solution stirred again for 30 minutes. After 30 minutes, centrifuged it at 12,000 g x 30 minutes at 4°C. The supernatant (supernatant 1) was kept and the residue was added by 40 ml of deionized water. The solution was stirred for 30 minutes with shaker. The solution adjusted to pH 10 with 0.5N NaOH. The solution stirred again for 30 minutes. After 30 minutes, centrifuged it at 12,000 g x 30 minutes at 4°C. The supernatant (supernatant 2) was kept. Supernatant 1 was mixed with supernatant 2. The supernatant adjusted to pH 4 by 0.5N HCl. Centrifuged it at 12,000 g x 30 minutes at 4°C. The residue was kept. The residue washed with deoinized water two times. The residue made into smaller pieces. The residue neutralized with 0.5N NaOH and then stored in refrigerator. After 24 hours, the residue continued to lyophilisation process.
Figure 5. Set samples to prepare LSPI 3.2.2.
Extraction of Lotus Seed Protein Hydrolysates
3.2.2.1.Effect of Different Substrate Concentration on Degree of Hydrolysis
This method was using 3 substrate concentration; 2%, 4%, and 6%. Enzyme concentration that used was 5% of alcalase enzyme and 180 minutes for hydrolysis time. Each substrate concentration used 3 tubes. 20 mg (2%), 40 mg (4%), and 60 mg (6%) of lotus seed protein isolate (LSPI) was prepared. Each LSPI was added with 950µl phosphate buffer saline (PBS). The solution added with 50µl (5%) of alcalase enzyme pH 8.5. The tube was closed by cap holding tabs. Then, put it in the oven 50°C the dry bath incubator 110°C for inactivating process about 20 minutes. The samples was centrifuged for 3 minutes. The supernatant was kept in refrigerator and will be used as the samples for degree of hydrolysis analysis.
Figure 6. Inactivation Process In Dry Bath Incubator After kept for 24 hours, 10µl of supernatant (samples) was added by 490µl of deionized water (diluted 1/50). Blank solution was made by 950µl PBS and 50µl alcalase enzyme. The blank was put in the dry bath incubator 110°C for inactivating process about 20 minutes.
OPA solution prepared separately by adding 2500 μl of 100mM sodium tetraborate, 250 μl 20% sodium dodecyl sulfate (SDS), 4 mg o-phthalaldehyde in 100 μl methanol, 10 μl β-mercaptoethanol, and deionized water. Diluted samples was shaked. 20µl diluted samples and blank was added by 400µl OPA solution. The absorbance was determined by spectrophotometer with wavelenght 340 nm. Degree of hydrolysis determined by equation based on previous study : y = 0.2401x + 0.0118
L-Leucine mM
total
H (20 mg) 152.6551 H total (40 mg) 305.3103 H total (60 mg) 457.9654 H 4.9646
Ht−H0
DH% = x 100%
Htotal−H0
Remarks : t
H : hydrolysis for t minutes H : amount in original isolates H total : total hydrolysis with 6N HCl
3.2.2.2.Effect of Different Hydrolysis Times on Degree of Hydrolysis
This method was using 6% substrate concentration, 3% of alcalase enzyme, various hydrolysis times those are 0, 10, 20, 30, 60, 90, 120, and 180 minutes. Each hydrolisis time used 3 tubes. 24 tubes prepared and filled with 60 mg (6%) of lotus seed protein isolate (LSPI). Each LSPI was added with 970µl phosphate buffer saline (PBS). The solution added with 30µl (3%) of alcalase enzyme pH 8.5. The tube was closed by cap holding tabs. Then, put it in the oven 50°C for hydrolyzing process for 10, 20, 30, 60, 90, 120, and 180 minutes. The sample for 0 minutes hydrolysis time was put in the dry bath incubator 110°C for inactivating process about 20 minutes directly. After time of each hydrolysis time finished, the samples were put in the dry bath incubator 110°C for inactivating process about 20 minutes. The samples was centrifuged for 3 minutes. The supernatant was kept and used as the samples for degree of hydrolysis analysis.
Figure 7. Set Samples For Hydrolysis Process After kept for 24 hours, 10µl of supernatant (samples) was added by 490µl of deionized water (diluted 1/50). Blank solution was made by 970µl PBS and 30µl alcalase enzyme. The blank was put in the dry bath incubator 110°C for inactivating process about
20 minutes.
OPA solution prepared separately by adding 2500 μl of 100mM sodium tetraborate, 250 μl 20% sodium dodecyl sulfate (SDS), 4 mg o-phthalaldehyde in 100 μl methanol, 10 μl β-mercaptoethanol, and deionized water. Diluted samples was shaked. 20µl diluted samples and blank was added by 400µl OPA solution. The absorbance was determined by spectrophotometer with wavelenght 340 nm. Degree of hydrolysis determined by equation based on previous study : y = 0.2401x + 0.0118
L-Leucine mM H total (20 mg) 152.6551 H total (40 mg) 305.3103
total
H (60 mg) 457.9654 H 4.9646
Ht−H0
DH% = x 100%
Htotal−H0
Remarks : H t : hydrolysis for t minutes H : amount in original isolates H total : total hydrolysis with 6N HCl 3.2.3.
SDS PAGE Analysis
Sodium dodecyl sulfate (SDS-PAGE) is widely used to analyze the proteins in complex extracts. SDS PAGE also can be used to determine the molecular weight (MW) of an
unknown protein. In this research, SDS PAGE method used to determine and give the distribution MW of protein and peptides from LSPI. Molecular weight can provide information about proteins and peptides from LSPI with the use of alcalase enzyme.
3.2.3.1.Gel Preparation
Casting frames prepared on the casting stands. The separating gel was prepared by mixing the 40% acrylamide mix, 1.5M tris pH 8.8, 10% SDS, 10% APS, and TEMED in small beaker. The solution swirled gently. Gap between the glass plates added by appropriate amount of separating gel solution. Made the top of separating gel horizontal by filling it with isopropanol until overflow. Let the solution gelated about 30 minutes.
The stacking gel was prepared by mixing the 40% acrylamide mix, 1.5M tris pH 8.8, 10% SDS, 10% APS, TEMED in small beaker. The stacking gel solution added to the top of separating gel until overflow. Well-forming comb inserted to the solution without trapping air under the teeth. Let it gelated about 30 minutes. Took out the comb. Took the glass plates out of the casting frame. The gel poured with some water if not used and set them in the buffer dam if used.
3.2.3.2.Samples Preparation
This method only want to show the molecular weight distribution of lotus seed protein isolate, but not show what kind of amino acids contain in the sample. 10 mg of LSPI mixed with 1 ml of deionized water. Then, sample diluted into 5 different concentration those were 5, 4, 3, 2, and 1 mg/ml. Each sample mixed with sample buffer with the ratio sample buffer : sample was 1:4. Samples were heated at 95°C for 5 minutes. Let the samples a little bit warm. Then put the 7µl of marker in the first lane and 20 µl samples into each wells, make sure not to overflow. The top was covered and connected to the anodes. The voltage set at 110 V for about 1 hour and 40 minutes.
Figure 8. SDS PAGE Analysis
4. RESULTS AND DISCUSSIONS
Lotus seed protein contain bioactive peptides. The bioactive peptides in lotus seed give a lot of functional properties of protein like antidote, antioxidant, and anticancer. The bioactive peptides are inactive within the parent protein molecules. Bioactive peptides
need certain processing approaches to release from lotus seed protein. The production of
protein hydrolysates can activate the bioactive peptides in lotus seed. Hydrolysis of
lotus seed protein into lotus seed protein hydrolysates can release the peptides from
lotus seed protein, so it can optimize the functional properties of its protein (Shahidi and
Zhong, 2008).Lotus seed protein hydrolysates can be produced in vitro through enzymatic hydrolysis
of proteins. Enzymatic hydrolysis has been used for modification of functional and
nutritional properties of food proteins (Liu and Chiang, 2008). Enzymatic hydrolysis using selective proteases will provides moderate conditions of the process, few or no undesirable side reactions or products, less salts, and the functionality of the final product can be controlled by selection of specific enzymes and reaction factors (Hrckova et.al., 2001).
The enzyme that used in this research is alcalase. Alcalase is famous as commercial
protease. Alcalase has been reported to be one of the most efficient protease to prepare
protein hydrolysates. Alcalase is an alkaline enzyme produced from Bacillus
licheniformis . Alcalase is liquid, brown, and has slight fermentation odor. Alcalase has
optimum temperature about 50 to 70°C and optimum pH about 8 to 10. Storage condition for alcalase is tightly closed in a dry and cool place about 0-10°C. (See et al 2011)
In this research, substrate concentration and hydrolysis time being tested in order to
optimize the lotus seed protein hydrolysates procedure. Optimization of lotus seed
protein hydrolysates procedure will make the production of lotus seed protein
hydrolysates more effective, efficient, and useful for industries in order to produce
nutritious food or pharmaceutical based on lotus seed protein.
In this research, the extraction of lotus seed protein hydrolysates done by some steps. First, the LSPI was added with 950µl phosphate buffer saline (PBS). Phosphate buffer saline used as a buffering agent to maintain the pH at certain level. The reactivity of OPA to proteins influenced by the pH. Based on Held (2006) buffer with a basic pH (pH 9.0 is optimal) results in more reactive primary amino groups. pH around 6-9 provide quite acceptable results. The solution added with 50µl (5%) of alcalase enzyme pH 8.5. Alcalase used as the enzyme because it has been reported to be one of the most efficient protease to prepare protein hydrolysates. The tube was closed by cap holding tabs. Then, put it in the oven 50°C for hydrolyzing process about 180 minutes. The temperature used is 50°C because alcalase has optimum temperature about 50 to 70°C.
After 180 minutes, the samples were put in the dry bath incubator 110°C for inactivating process about 20 minutes. This process used to deactivated the alcalase enzyme, so the hydrolysis process stopped. The samples was centrifuged for 3 minutes. The supernatant was kept in refrigerator and will be used as the samples for degree of hydrolysis analysis. The same steps done to made the blank solution.
This research use OPA solution to determine the DH of lotus seed protein isolate. Based on Zarei et al (2012) the TNBS and OPA methods are comparable and directly determine the DH. OPA also very high sensitive to detect reagent of amines contained in proteins, peptides, and amino acids. o-Phthaldialdehyde offers several advantages for protein quantitation. OPA solution is stable for long periods while in solution and protein quantitation using OPA requires very little sample (5-
10 μl). OPA solution prepared separately by adding 2500 μl of 100mM sodium tetraborate, 250 μl 20% sodium dodecyl sulfate (SDS), 4 mg o- phthalaldehyde in 100 μl methanol, 10 μl β- mercap toethanol, and deionized water. The SDS and β-mercaptoethanol used to solubilizes most proteins effectively. Samples that are resistant can be solubilized by boiling in SDS and
β-mercaptoethanol prior to addition of the reagent. (Held, 2006). Diluted samples was shaked. 20µl diluted samples and blank was added by 400µl OPA solution. The absorbance was determined by spectrophotometer with wavelenght 340 nm.
4.1. Effect of Different Substrate Concentration on Degree of Hydrolysis
Enzymatic hydrolysis influenced by some factors such as pH, time, enzyme concentration, and substrate concentration. Substrate concentration become important because it can influence the degree of hydrolysis. In this research effect of different substrate concentration on degree of hydrolysis of lotus seed protein isolate can be seen at Table 2. Table 2. Effect of Different Substrate Concentration on Degree of Hydrolysis
Degree of Substrate (%) Absorbance (y) X x mean Hydrolysis
(%) 2 0.321 1.2878 2 0.285 1.1379 61.96 38.5911 2 0.322 1.2920 4 0.528 2.1499 4 0.518 2.1083 104.305 33.0754 4 0.492
2 6 0.582 2.3748 6 0.585 2.3873 122.835 26.0199 6 0.638 2.6081
From Table 2. can be seen that different substrate concentration gave different degree of hydrolysis. 2% substrate concentration of lotus seed protein isolate gave the highest degree of hydrolysis and 6% substrate concentration gave the lowest degree of hydrolysis. So, more low the substrate concentration of lotus seed protein isolate give more high degree of hydrolysis, vice versa. Based on Zhang et. al. (2012) said that if substrate concentration is high, it will reduce the availability of water in the reaction system and the diffusion motions, so the substrate becomes aggregated. Hence, the hydrolysis was inhibited. When the hydrolysis is inhibited, means that the cleaving process is inhibited, so the protein is still in the form of complex protein. So with low substrate concentration of lotus seed protein isolate, more peptides will produce because the degree of hydrolysis is high. Figure 5 show the effect of different substrate concentration on degree of hydrolysis.
Figure 9. Effect of Substrate Concentration on Degree of Hydrolysis On Figure 9 can be seen clearly that low substrate concentration give high DH and high substrate concentration give low DH. Himonides et al (2011) said that degree of hydrolysis (DH) is the proportion of cleaved peptide bonds. So, high DH means the cleaving process run well and produce more protein hydrolysate. If the production of protein hydrolysate is high means that 2% substrate concentration optimize the procedure. Based on Himonides et al (2011) lower DH produced hydrolysates with higher molecular weight fractions, which exhibited better emulsification and aeration properties but showed greater hydrophobicity. The relation between DH and bitterness, antioxidative or other peptide bioactivities is enzyme dependant.
Enzymatic hydrolysis also influenced by hyrolysis time. When enzyme is added into a substrate, enzyme will be absorbed into the suspended particles. Then, the hydrolysis will run simultaneously. After an initial rapid phase of hydrolysis, the rate of hydrolysis will entering a stationary phase. At certain hydrolysis time, the DH will much lower than before because the substrate is limited. Effect of different hydrolysis times on degree of hydrolysis of lotus seed protein isolate can be seen on Table 3. 5 10 15 20 25 30 35 40 45 2 4 6 D
e g re e o f H y d ro ly si s (% ) Substrate Concentration (%) LSPI
4.2. Effect of Different Hydrolysis Times on Degree of Hydrolysis
Table 3. Effect of Different Hydrolysis Times on Degree of Hydrolysis Degree of
Time of Hydrolisis Absorbance (y)
X Hydrolisis ӯ
(min) (%)