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

i

ii

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

iii

good examples for the others. The writer hopes that this report can be useful for the others.

Semarang, 10 April 2015 Writer

iv

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

v

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

vi

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

vii

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

viii

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.

1

2

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 Sciences 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 program and Master’s degree program.

3

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.

4

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. Associate Professor Jung-Feng Hsieh

Food Physicochemistry Lab. Associate Professor Meng I-Marie Kuo

Associate Professor Rey-May Huang

Associate Professor Shau-Chen

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 Food Science, Fu Jen Catholic University, Taipei, Taiwan, in Januari 13 rd to March 12nd 2015.

5

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.

6

7

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.

8

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

9

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

10

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