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

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

ii

iii

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

iv

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

others.

Semarang, 10 April 2015

Writer

v

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

vi

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

vii

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

viii

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

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.

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

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 13rd to March 12nd

2015.

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.

Assistant Professor Bang-Yuan

Chen

Nutraceuticals & Food Processing

Lab.

Assistant Professor Tsai-Hua Kao

Food Biochemistry Lab.

Assistant Professor Chun-Ping Lu

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.

6

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.

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<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).

11

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.

12

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.

13

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

for hydrolyzing process about 180 minutes. After 180 minutes, the samples were put in

14

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

Htotal (20 mg) 152.6551

Htotal (40 mg) 305.3103

Htotal (60 mg) 457.9654

H0 4.9646

DH% = Ht−H0

Htotal−H0 x 100%

Remarks :

15

Ht : hydrolysis for t minutes

H0 : amount in original isolates

Htotal : 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

16

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

Htotal (20 mg) 152.6551

Htotal (40 mg) 305.3103

Htotal (60 mg) 457.9654

H0 4.9646

DH% = Ht−H0

Htotal−H0 x 100%

Remarks :

Ht : hydrolysis for t minutes

H0 : amount in original isolates

Htotal : 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.

Isopropanol discarded and gel washed with water.

17

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

18

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.

19

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-

mercaptoethanol, 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.

20

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

Substrate (%) Absorbance (y) X x mean

Degree of

Hydrolysis

(%)

2 0.321 1.2878

61.96 38.5911 2 0.285 1.1379

2 0.322 1.2920

4 0.528 2.1499

104.305 33.0754 4 0.518 2.1083

4 0.492 2

6 0.582 2.3748

122.835 26.0199 6 0.585 2.3873

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.

21

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.

4.2. Effect of Different Hydrolysis Times on Degree of Hydrolysis

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.

0

5

10

15

20

25

30

35

40

45

2 4 6

De

gre

e o

f H

ydro

lysi

s (%

)

Substrate Concentration (%)

LSPI

22

Table 3. Effect of Different Hydrolysis Times on Degree of Hydrolysis

Time of Hydrolisis

(min) Absorbance (y) ӯ X

Degree of

Hydrolisis

(%)

180

0.536

0.532 108.35 22.822 0.528

0.532

120

0.506

0.499 101.45 21.299 0.508

0.482

90

0.438

0.450 91.25 19.048 0.435

0.477

60

0.387

0.417 84.4 17.535 0.450

0.414

30

0.391

0.394 79.6 16.476 0.411

0.380

20

0.373

0.354 71.25 14.633 0.342

0.346

10

0.302

0.317 63.55 12.933 0.328

0.320

0

0.217

0.223 44 8.617 0.223

0.229

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. From Table 3.

can be seen that on lotus seed protein isolate 180 minutes gave the highest degree of

hydrolisis and 0 minutes gave the lowest degree of hydrolysis. So, more longer the time

of hydrolysis on lotus seed protein isolate will give more high degree of hydrolysis, vice

versa. But in Hrckova et.al. (2001) with soy defatted flour and alcalase enzyme, 120

minutes gave the highest degree of hydrolysis. It happen because the sample was

different and the reaction factors could controlled by selection of specific enzymes

(Hrckova et.al., 2001). In this case, the specific enzymes was alcalase. Different

samples will give different reaction or final product with the use of an enzyme, because

enzyme split specific peptide bonds. This was happen because the sample was different.

Soybean contains about 40% of proteins, lotus seed about 10.6-15.9%. The amino acids

23

inside composition different and need different hydrolysis time. Figure 6 show the

effect of different hydrolysis times on degree of hydrolysis of lotus seed protein isolate.

Figure 10. Effect of Times of Hydrolysis on Degree of Hydrolysis

Based on Figure 10. can be seen that when time hydrolysis was 0 minutes, the degree of

hydrolysis is low. Then, the degree of hydrolysis increased by the increased of

hydrolysis times. After 90 minutes the degree of hydrolysis increased moderately.

Based on Hrckova et.al. (2001) soy defatted flour with alcalase showed the highest

increase in amino acids during the first 120 min of hydrolysis, but later the amount of

released amino acids increased moderately. So the graphic trend is the same Hrckova

et.al. (2001) that use soy defatted flour with alcalase. Based on Figure 10. can be seen

also that at first the increasing of DH is high, but then the increasing of DH is

decreasing slowly. Salwanee (2012) said this happened because 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. This happen because concentration of

peptide bonds available for hydrolysis is limited. At certain point, the DH will go down

or lower because all substrates has been produce to hydrolysates. The DH can also get

lower because enzyme inhibition and enzyme deactivation on the alcalase enzyme.

0

5

10

15

20

25

0 50 100 150 200

De

gre

e o

f H

ydro

lysi

s (%

)

Time (min)

LSPI

24

Enzymatic hydrolysis split specific peptide bonds. Based on Hrckova et.al. (2001)

alcalase could split soy defatted flour specific peptides bonds and mostly produced

histidine, leucine, and tyrosine. The total DH was about 35.1% for 480 minutes. In this

research data showed that the degree of hydrolysis (DH) was about 20% for 240

minutes. The difference happen because alcalase split specific peptide bonds (histidine,

leucine, and tyrosine). Based on Zeng, et. al. (2012) histidine content on lotus seed

protein is 23.66g/kg, leucine 64.04g/kg, and tyrosine 15.13g/kg so the cleaving or

splitting process is not well and made the DH low.

4.3. SDS PAGE Analysis

Polyacrylamide gel electrophoresis (PAGE) is one of the most famous techniques to

separate macromolecules such as DNA, RNA, and proteins. The separation in

electrophoresis based on electric charge of the molecules. When a charged molecule is

placed in an electric field, it will move toward the electrode with opposite charge. The

relative rate of movement depends on charge, molecular weight, and shape of the

protein. Proteins with greater negative charge are attracted toward the positive

electrode faster. The molecular weight and shape of the proteins are factors because of

the properties of the gel matrix. If the protein is large it will move slower, than small

globular protein will move faster (Spilatro, 2014). Gel electrophoresis can provide

information about molecular weights, charge of proteins, subunit structures of proteins,

and purity of a particular protein preparation.

Gel electrophoresis can be done by many techniques. Sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) is the most common method to used

for proteins. SDS-PAGE is useful for monitoring the fractions obtained during

chromatographic or othe purification content. SDS-PAGE also allows sample from

different sources to be compared for protein content. One of the important features of

SDS-PAGE is simple and reliable method to estimate molecular weight (MW) of

proteins. (AES, 2015). Protein molecular weight determination by SDS relatively

accurate because treatment with SDS creates a uniform charge to mass ratio between

different proteins. So the separation on polyacrylamide gel occurs mass only.

25

The system of SDS-PAGE consists of two gels ; separating (running) gel and stacking

gel. Separating gel is gel in which proteins are resolved on the basis of their molecular

weights. Stacking gel is gel in which proteins are concentrated prior to entering the

resolving gel. The differences in the compositions of the stacking gel, separating gel,

and electrophoresis buffer produce a system that is capable of finely resolving proteins

according to their MW. The sizes and molecular weight of protein sample can be

calculated by comparing their migration or distributin to a set of standard proteins run

on the same gel (Laemmli, 1970).

In this research, SDS PAGE method used to determine and give the distribution MW of

LSPI. Molecular weight can provide information about proteins and peptides from LSPI

with the use of alcalase enzyme. The weight molecular distribution of lotus seed protein

isolate can be seen at Figure 11.

Legend :

I : 7 µl marker lane

II : 20 µl sample, concentration 5 mg/ml

III : 20 µl sample, concentration 4 mg/ml

IV : 20 µl sample, concentration 3 mg/ml V : 20 µl sample, concentration 2 mg/ml

VI : 20 µl sample, concentration 1 mg/ml

Figure 11.Weight Molecular Distribution of Lotus Seed Protein Isolate

Based on Figure 11. can be seen that protein from lotus seed protein isolate separate

based on its molecular weight. The thick marker sign that a lot of peptides in that

marker. Based on figure 11, molecular weight of lotus seed protein isolate using

180 130 100

75

63

48

35

28

17

10 I II III IV V VI

26

alcalase are mostly at 10, 17, 35, and 48 KDa. This can happen because the protein split

into some amino acids due to reaction with alcalase enzyme. Based on Hrckova et.al.

(2001) alcalase and novozym could split soy defatted flour mostly into histidine,

leucine, and tyrosine. But with the use of flavourzyme, the soy defatted flour mostly

split into arginine, leucine, phenylalanine. This shows that different enzyme can be used

to produce different amino acids. This result just show the molecular weight of lotus

seed protein isolate. Identification about the amino acids in lotus seed protein isolate

need more further research.

27

5. CONCLUSION

Practical training (internship program) in Fu Jen Catholic University give a knowledge

about food research in Taiwan. Food sciences research in Taiwan especially Fu Jen

Catholic University generally leads to biotechnology and functional properties of food.

It can be seen at this research about protein hydrolysate. Protein hydrolysate widely

used as nutritional supplements, functional ingredients, and can scavenge free radicals

for against aging, cardiovascular, and other diseases. The optimization of protein

hydrolysate procedure give the effective and efficient materials and method to produce

lotus seed protein hydrolysate.

This internship program also give knowledge about culture and society in Taiwan.

Tourism sites in Taiwan offer historical stories (museum), scenery, and the most famous

one is local food. Local food in Taiwan is delicious, cheap, famous around the world,

and some food can be found in Indonesia. Taiwan is one of world tour destination, so

there are a lot of foreigner or tourist in Taiwan. It makes the society very nice to

foreigner. Some foreigner also live in Taiwan for study, this make a lot of cross culture

mix in Taiwan. Internship program also make the writer more fluent to communicate

and write in english.

Based on the research can be conclude that on lotus seed protein isolate with 5%

alcalase enzyme and 180 minutes hydrolysis time, 2% substrate concentration give the

best degree of hydrolysis. While, the best hydrolysis time on lotus seed protein isolate

with 6% substrate concentration and 3% alcalase enzyme, is 180 minutes. Analysis on

molecular weight results that mostly the molecular weight of LSPI are at 10 – 17 KDa

and 35 – 48 KDa.

28

6. REFERENCES

AES Electrophoresis Society. 2015. Gel Electrophoresis of Proteins.

www.aesociety.org/areas/pdfs/Garfin_1DE_WebArticle9-07.pdf. (accessed on

2015-05-15)

Anonyme. OPA, Amine Detection Reagent. Interchim. FT-02727A.

Anonyme. http://www.ag.auburn.edu/hort/landscape/LOTUS_LIT.html#PlantUses

(accessed 2015-03-03)

Frister, H., H. Meisel, E. Schlimme. (1988). OPA Method Modified by Use of N,N-

dimethyl-2-mercaptoethylammonium chloride as thiol component. Fresenius Z.

Anal Chem (1988) 330 : 631-633.

Held, P.G. (2006). Quantitation of Total Protein Using OPA. BioTek Instrument, Inc.

Rev. 2-20-01.

Himonides, A.T., A.K.D. Taylor, and A.J. Morris. (2011). A Study of the Enzymatic

Hydrolysis of Fish Frames Using Model Systems. Food and Nutrition Sciences,

2011, 2, 575-585.

Hrckova, M., M. Rusnakova, and J. Zemanovic. (2001). Enzymatic Hydrolysis of

Defatted Soy Flour by Three Different Proteases and their Effect on the

Functional Properties of Resulting Protein Hydrolysates. Czech J. Food Sci. Vol.

20, No. 1: 7–14.

Laemmli, U.K. (1970). Cleavege of Structural Proteins During The Assembly of The

Head of Bacteriophage T4. Nature 227 : 680-685. doi : 10.1038/227680a0.

Liu, B.L. and Pei, S.C. (2008). Production of Hydrolysate with Antioxidative Activity

and Functional Properties by Enzymatic Hydrolysis of Defatted Sesame

(Sesamum indicum L.). International Journal of Applied Science and Engineering

2008. 6, 2: 73-83.

Murty, D. (2012). Story of The Sacred Lotus. Buddhist Council of NSW.

Salwanee, S., W.M.W. Aida, S. Mamot, M.Y. Maskat, and S. Ibrahim. (2012). Effects

of Enzyme Concentration, Temperature, pH and Time on the Degree of

Hydrolysis of Protein Extract from Viscera of Tuna (Euthynnus affinis) by Using

Alcalase. Sains Malaysiana 42(3)(2013): 279–287.

See, S.F., L.L. Hoo, and A.S. Babji. (2011). Optimization of Enzymatic Hydrolysis of

Salmon (Salmo salar) skin by Alcalase. International Food Research Journal

18(4): 1359-1365 (2011).

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Shahidi, F. and Zhong, Y. (2008). Bioactive Peptides. Journal of AOAC International

Vol. 91, No. 4, 2008.

Spilatro, S.R. (2014). Electrophoresis and Electroblotting of Proteins.

www.marietta.edu/~spilatrs/biol309/labexercises/ (accessed 2015-05-19)

Sridhar, K.R. and Bhat, R. (2007). Lotus – A Potential nutraceutical source. Journal of

Agricultural Technology 3(1): 143-155.

Wu, J.Z., Y.B. Zheng, T.Q. Chen, J. Yi, L.P. Qin, K. Rahman, W.X. Lin. (2007).

Evaluation of the quality of lotus seed of Nelumbo nucifera Gaertn from outer

space mutation. Food Chemistry 105 (2007) 540–547.

Zarei, M., Afshin, E., Azizah, A.H., Farooq A., and Nazamid S. (2012). Production of

Defatted Palm Kernel Cake Protein Hydrolysate as a Valuable Source of Natural

Antioxidants. Int. J. Mol. Sci. 2012, 13, 8097-8111; doi:10.3390/ijms13078097.

Zeng, H.Y., L.H. Cai, X.L. Cai, Y.J. Wang, and Y.Q. Li. (2012). Amino acid profiles

and quality from lotus seed proteins. J Sci Food Agric 2013; 93; 1070-1075.

Zhang, H., L. Yu, Q. Yang, J. Sun, J. Bi, S. Liu, C. Zhang and L. Tang. (2012).

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Peanut Peptides. Molecules 2012, 17, 5661-5674;

doi:10.3390/molecules17055661.

30

APPENDIX

Effect of Different Substrate Concentration on Degree of Hydrolysis

y = 0.2401x + 0.0118

2%

0.321

0.321 = 0.2401x + 0.0118

0.3092 = 0.2401x

x = 1.2878

0.285

0.285 = 0.2401x + 0.0118

0.2732 = 0.2401x

x = 1.1379

0.322

0.322 = 0.2401x + 0.0118

0.3102 = 0.2401x

x = 1.2920

X mean = 1.2878+1.1379+1.2920

3= 1.2392

1.2392 x 50

= 61.96

4%

0.528

0.528 = 0.2401x + 0.0118

0.5162 = 0.2401x

x = 2.1499

0.518

31

0.518 = 0.2401x + 0.0118

0.5062 = 0.2401x

x = 2.1083

0.492

0.492 = 0.2401x + 0.0118

0.4802 = 0.2401x

x = 2

X mean = 2.1499+2.1083+2

3= 2.0861

2.0861 x 50

= 104.305

6%

0.582

0.582 = 0.2401x + 0.0118

0.5702 = 0.2401x

x = 2.3748

0.585

0.585 = 0.2401x + 0.0118

0.5732 = 0.2401x

x = 2.3873

0.638

0.638 = 0.2401x + 0.0118

0.6262 = 0.2401x

x = 2.6081

X mean = 2.3748+2.3873+2.6081

3= 2.4567

2.4567 x 50

32

= 122.835

DH% = Ht−H0

Htotal−H0 x 100%

2%

DH% = Ht−H0

Htotal−H0 x 100%

= 61.96−4.9646

152.6551−4.9646 x 100%

= 38.5911%

4%

DH% = Ht−H0

Htotal−H0 x 100%

= 104.305−4.9646

305.3103−4.9646 x 100%

= 33.0754%

6%

DH% = Ht−H0

Htotal−H0 x 100%

= 122.835−4.9646

457.9654−4.9646 x 100%

= 26.0199%

Effect of Different Hydrolysis Times on Degree of Hydrolysis

180

y1 = 0.536, y2 = 0.528, y3 =

y mean = 0.532

0.532 = 0.2401x + 0.0118

0.5202 = 0.2401x

x = 2.167

2.167 x 50

= 108.35

DH% = Ht−H0

Htotal−H0 x 100%

33

= 108.35−4.9646

457.9654−4.9646 x 100%

= 22.822%

120

y1 = 0.506, y2 = 0.508, y3 = 482

y mean = 0.499

0.499 = 0.2401x + 0.0118

0.4872 = 0.2401x

x = 2.029

2.029 x 50

= 101.45

DH% = Ht−H0

Htotal−H0 x 100%

= 101.45−4.9646

457.9654−4.9646 x 100%

= 21.299%

90

Y1 = 0.438, y2 = 0.435, y3 = 0.477

y mean = 0.450

0.450 = 0.2401x + 0.0118

0.4382 = 0.2401x

x = 1.825

1.825 x 50

= 91.25

DH% = Ht−H0

Htotal−H0 x 100%

34

= 91.25−4.9646

457.9654−4.9646 x 100%

= 19.048%

60

y1 = 0.387, y2 = 0.450, y3 =0.414

y mean = 0.417

0.417 = 0.2401x + 0.0118

0.4052 = 0.2401x

x = 1.688

1.688 x 50

= 84.4

DH% = Ht−H0

Htotal−H0 x 100%

= 84.4−4.9646

457.9654−4.9646 x 100%

= 17.535%

30

y1 = 0.391, y2 = 0.411, y3 = 0.380

y mean = 0.394

0.394 = 0.2401x + 0.0118

0.3822 = 0.2401x

x = 1.592

1.592 x 50

= 79.6

DH% = Ht−H0

Htotal−H0 x 100%

35

= 79.6−4.9646

457.9654−4.9646 x 100%

= 16.476%

20

y1 = 0.373, y2 = 0.342, y3 =0.346

y mean = 0.354

0.354 = 0.2401x + 0.0118

0.3422 = 0.2401x

x = 1.425

1.425 x 50

= 71.25

DH% = Ht−H0

Htotal−H0 x 100%

= 71.25−4.9646

457.9654−4.9646 x 100%

= 14.633%

10

y1 = 0.302, y2 = 0.328, y3 =0.320

y mean = 0.317

0.317 = 0.2401x + 0.0118

0.3052 = 0.2401x

x = 1.271

1.271 x 50

= 63.55

36

DH% = Ht−H0

Htotal−H0 x 100%

= 63.55−4.9646

457.9654−4.9646 x 100%

= 12.933%

0

y1 = 0.217, y2 = 0.223, y3 = 229

y mean = 0.223

0.223 = 0.2401x + 0.0118

0.2112 = 0.2401x

x = 0.880

0.880 x 50

= 44

DH% = Ht−H0

Htotal−H0 x 100%

= 44−4.9646

457.9654−4.9646 x 100%

= 8.617%