Physicochemical properties and In Vitro starch digestibility of Thai tradisional rice cake and pie cake

PHYSICOCHEMICAL PROPERTIES AND

IN VITRO

_STARCH

DIGESTIBILITY OF THAI TRADITIONAL RICE CAKE AND PEA CAKE

BACHELOR THESIS

R. DHIMAS SATRIYO UTOMO

F24060455

FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

2011

i

PHYSICOCHEMICAL PROPERTIES AND

IN VITRO

_STARCH

DIGESTIBILITY OF THAI TRADITIONAL RICE CAKE AND PEA CAKE

BACHELOR THESIS

R. DHIMAS SATRIYO UTOMO

F24060455

FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

2011

ii

PHYSICOCHEMICAL PROPERTIES AND

IN VITRO

STARCH

DIGESTIBILITY OF THAI TRADITIONAL RICE CAKE AND PEA CAKE

R. Dhimas S. Utomo1, Yadi Haryadi1, Eko Hari Purnomo1, and Rungarun Sasanatayart2

1_{Department of Food Science and Technology, Faculty of Agricultural Engineering Technology,}

Bogor Agricultural University, IPB Darmaga Campus, PO. Box 220, Bogor, West Java, Indonesia 2_{School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand}

ABSTRACT

Physicochemical properties and in vitro starch digestibility of Thai traditional rice cake (kao ram phune) and pea cake were observed. Samples were prepared from flour slurries with 20% total solid, cooked at 95 o_{C for 25 min and cold set at room temperature for 6 hours to facilitate gel} forming. Proximate analysis, amylose content, thermal properties, pasting properties and granular morphology of flours were determined and related to the textural properties and resistant starch content of the corresponding cake. Results showed that pea flour had higher protein (21.84 %db) and lipid content (0.80 %db) compared to those of rice flour and sticky rice flour which were 7.21 %db and 0.41% db; 6.12 %db and 0.29 %db, respectively. Rice flour was mixed with sticky rice flour (in ratio of 100:0, 97.5:2.5, 95:5, 92.5:7.5, 90:10) to vary the amylose content (from 31.07 %db to 27.48 %db). Sticky rice flour was the lowest in amylose content (4.22 %db) whereas pea and rice flour 16.01 %db, 31.07 %db respectively. Conversely, pea cake was the highest in the amount of the resistant starch (9.62 % total starch), whereas cakes through ratios the values were (0.52 %, 0.45 %, 0.40 %, 0.34 %, respectively). Obtained cakes (pea cake, rice cake 100:0, rice cake 90:10) were cold set for 6 hours at room temperature, 6 hours at 4 oC, and 24 hours at 4 oC. Results showed that cold set increased hardness; 267.94 g - 521.71 g - 851.16 g (pea cake), 152.89 g - 197.78 g - 269.35 g (rice cake 100:0), and 69.73 g - 78.63 g - 121.90 g (rice cake 90:10). Resistant starch significantly increased after cold setting; 0.54 % - 0.73 % - 0.95 % (rice cake 100:0), 0.34 % - 0.45 % - 0.56 % (rice cake 90:10). However pea cake exhibited opposite trend as the RS content decreased during cold setting; 9.62 % - 7.46 % - 6.92 %. There was no clear trend for the color properties resulted from variation in amylose content and cold setting conditions.

iii R. DHIMAS SATRIYO UTOMO. F24060455. Physicochemical Properties and in Vitro Starch Digestibility of Thai Traditional Rice Cake and Pea Cake. Supervised by Yadi Haryadi, Eko Hari Purnomo, and Rungarun Sasanatayart. 2010

SUMMARY

This research was first aimed to investigate the effect of flour types (pea flour, rice flour, sticky rice flour) and mixed flour (rice flour and sticky rice flour) on physicochemical properties and starch digestibility of the corresponding pea cake and rice cake. Second, to investigate the effect of cold setting conditions on physicochemical properties and starch digestibility of pea cake and rice cake.

In the preliminary research, chemical composition of raw materials involved moisture, ash, protein, crude fat, and amylose content were determined as well as granular morphology, thermal and pasting properties. Experiment I was conducted in order to investigate the effect of mixing rice flour and sticky rice flour on granular morphology, thermal, and pasting properties. The resulted cakes were then analyzed in term of starch digestibiliity, textural and color properties (experiment II). At last, effect of cold setting condition on starch digestibility, textural, and color properties of pea cake and rice cake was investigated.

Results showed that pea flour, rice flour, and sticky rice flour were different significantly. Pea flour exhibited typically oval starch granules with average size of 11-44 μm, whereas rice flour and sticky rice flour were found to be polygonal shape sized 2.8-14 μm and 4.3-17.1 μm, respectively. Round shape granules were also found in the rice flour which supposed to be cassava. In the chemical composition, the highest ash (2.83 %db), protein (21.84 %db) and lipid content (0.80 %db) were obtained from pea flour. Rice flour possessed apparent amylose content of 31.07 %db, whereas pea flour and sticky rice flour were 16.01 %db and 4.22 %db, respectively. Rice flour and sticky rice flour were mixed at the ratios of 100:0, 97.5:2.5, 95:5, 92.5:7.5, 90:10 to vary the amylose content, and it was revealed that as the portion of sticky rice increased, the amylose content decreased. The ordered values through ratios were 31.07 %db, 29.62 %db, 29.24 %db, 28.12 %db, and 27.48 %db.

Pea flour and sticky rice flour possessed gelatinization temperature range of 69.19-77.11 o_C and 58.54-74.50 o_{C, with resulted endothermic enthalpy of 3.29 J/g and 10.43 J/g, respectively. Rice} flour behaved differently as two endothermic peaks were exhibited. The first peak corresponded to the melting of cassava starch which appeared at lower temperature (62.40-70.37 o_{C) with the endothermic} enthalpy of 1.56 J/g. The second peak at higher temperature (72.45-81.10 oC) with the endothermic enthalpy of 3.94 J/g corresponded to that of rice flour. Addition of sticky rice flour to the rice flour did not affect thermal properties significantly.

In term of pasting properties, pea flour, rice flour, and sticky rice flour exhibited their typical RVA curve. The highest peak viscosity (388.5 RVU) and breakdown viscosity (185.9 RVU) were obtained from sticky rice flour, whereas final viscosity (254.3 RVU) and setback viscosity (213.1 RVU) were owned by rice flour. There were no significant effects by adding sticky rice flour to the rice flour through all ratios.

The main textural properties (hardness and adhesiveness) of pea cake and rice cake were analyzed, both commercial and experimental one. Hardness and adhesiveness values of pea cake resulted from experiment were 267.94 g and -48.76 g.s, whereas commercial pea cake were 260.16 g and 66.39 g.s, respectively. Rice cake possesed hardness and adhesiveness values of 152.89 g and

-iv 53.85 g.s (experiment), 158.39 g and -42.81 g.s (commercial rice cake). Through ratios, hardness score was found to have positive correlation with apparent amylose content (R2 _{= 0.895). Conversely,} adhesiveness exhibited negative correlation with apparent amylose content (R2 _{= 0.899).}

Pea cake and rice cake were also different in the color properties. Pea cake was higher in L* value (64.41) and b* value (13.57) compared with that of rice cake (57.42; 2.68, respectively). Inversely, rice cake was higher in a* value (-2.98) than pea cake (-1.33). Through ratios, rice cake behaved disorderly in term of L*, a*, b* values.

Investigation on in vitro starch digestibility revealed that pea cake contained markedly higher amount of resistant starch (9.62 % total starch) compared with that of rice cake (0.54 % total starch). As the portion of added sticky rice flour increased, resistant starch content increased. The ordered values were 0.52 %, 0.45 %, 0.40 %, 0.34 %, respectively. However, only the extreme addition of sticky rice flour (ratio of 90:10) was statistically significant.

In the second experiment, cold setting conditions (6h, room temperature; 6h, 4 oC; 24h, 4oC) were conducted in which starch digestibility, textural, and color properties were influenced by. Hardness increased during cold setting. The increment in hardness values were; 267.94 g, 521.71 g, 851.16 g (for pea cake); 152.9.25 g, 197.78 g, 269.35 g (for rice cake 100:0); and 69.73 g.s, 78.63 g.s, 121.90 g.s (for rice cake 90:10). Adhesiveness increased as well as hardness with the values of; -48.76 g.s, -79.43 g.s, -96.68 g.s (for pea cake); -53.85 g.s, -82.92 g.s, -100.30 (for rice cake); and -85.83 g.s, -90.09 g.s, -114.20 g.s (for rice cake with 10 % sticky rice flour added). Longer storage (24 hours) in low temperature (4 oC) lowered the lightness of all samples that was mainly due to retrogradation. However, there was no exact trend for the a* and b* values through samples upon cold settings.

Starch digestibility was found to be influenced by cold setting conditions. The increments of resistant starch content were 0.54 % to 0.73 %, and finally 0.95 % for rice cake (100:0), whereas 0.34 % to 0.45 %, and finally 0.56 % for rice cake with 10 % sticky rice flour added (90:10). Pea cake behaved differently as the resistant starch amount in the cake decreased from 9.62 % to 7.46 %, and finally 6.92 % through cold setting conditions.

v

PHYSICOCHEMICAL PROPERTIES AND

IN VITRO

STARCH DIGESTIBILITY OF THAI TRADITIONAL

RICE CAKE AND PEA CAKE

BACHELOR THESIS

In the partial fulfillment of the requirement for degree of

SARJANA TEKNOLOGI PERTANIAN

at the Department of Food Science and Technology

Faculty of Agricultural Engineering and Technology

Bogor Agricultural University

By:

R. DHIMAS SATRIYO UTOMO

F24060455

FACULTY OF AGRICULTURAL ENGINEERING TECHNOLOGY

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

2011

vi

Title

: Physicochemical Properties and

in Vitro

Starch Digestibility of Thai

Traditional Rice Cake and Pea Cake

Nama

: R. Dhimas Satriyo Utomo

Student ID : F24060455

Approved by,

Advisor I,

Advisor II,

(Dr. Ir. Yadi Haryadi, M.Sc.)

NIP. 19490612.197603.1.003

(Dr. Eko Hari Purnomo, S.TP., M.Sc.)

NIP. 19760412.199903.1.004

Acknowledged by,

Head of Department of Food Science and Technology,

(Dr. Ir. Dahrul Syah, M.Sc.)

NIP. 19650814.199002.1.001

vii

STATEMENT LETTER OF THESIS

AND SOURCES OF INFORMATION

Hereby I genuinely stated that the bachelor thesis entitled

Physicochemical

Properties and

in Vitro

Starch Digestibility of Thai Traditional Rice Cake and

Pea Cake

is

an authentic work of mine under supervision of academic counselor and

never being presented in any forms and universities. All the information taken and

quoted from published or unpublished works of other writers had been mentioned in

the texts and attached in the bibliography at the end of this thesis.

Bogor, January 2011

The undersigned,

R. Dhimas Satriyo Utomo

F24060455

viii

AUTHOR BIOGRAPHY

The author was born in Karanganyar, Central Java, on August

28, 1988. He was graduated from SDN Kleco 1 Surakarta

elementary school in 2000, SMPN 1 Surakarta junior high

school in 2003, and SMAN 1 Surakarta senior high school in

2006. In the same year he joined Bogor Agricultural University

through Undangan Seleksi Masuk IPB (USMI). He was much

involved in student activities such as International Student

Association in Agricultural and related Sciences (IAAS), Himpunan Mahasiswa Ilmu

dan Teknologi Pangan (HIMITEPA), Komisi Pelayanan Siswa - Persekutuan

Mahasiswa Kristen IPB (KPS-PMK IPB), and Perhimpunan Mahasiswa Indonesia di

Thailand (PERMITHA).

The author did the academic things as well as extracurricular activities. In

2009, he won Silver Medal (oral presentation) and Bronze Medal (poster

presentation) in Pekan Ilmiah Mahasiswa Nasional (PIMNAS). He was also the best

poster presenter at Konferensi Nasional Mahasiswa Teknologi Pangan dan Ilmu Gizi,

held by Himpunan Mahasiswa Peduli Pangan Indonesia (HMPPI) in 2009. In the

same year he did a poster presen

tation entitled “

Microencapsulation Technique to

Maintain Antioxidant Capacity of Roselle (

Hibiscus sabdariffa

Lin.) in Roselle

Instant Drink Processing Using Spray Dryer

”

at the 3

rd

World Congress on Tea and

Health, which was held by International Society of Antioxidant in Nutrition and

Health (ISANH) in Dubai, United Arab Emirates. In the next year, he was selected

for a poster presentation

entitled “

An Overview of Functional Turmeric-Tamarind

Indonesian’s Traditional Drink”

at the USA/Ireland Functional Food Conference,

held by United States Department of Agriculture (USDA), University College Cork

(UCC), and TEAGASC in Cork City, Republic of Ireland. To improve his academic

skill, he attended Food Innovation Asia Conference 2010 held in Bangkok, Thailand.

Further, he did an oral presentation entitled “Comparative Study on Physicochemical

Properties and Resistant Starch Content of

Cake Prepared from Rice and Pea”

at the

International Conference on Agriculture and Agro-Industry, held in Chiang Rai,

Thailand. He joined also Distance Education Program, held by Southeast Asian Food

and Agricultural Science and Technology Center, and Texas A&M University, USA.

At last, the author was selected to be the Indonesian Delegates for the Credit

Transfer MIT (Malaysia-Indonesia-Thailand) Student Exchange Program in Mae Fah

Luang University, Thailand. Under such program, he did an undergraduate research

project entitled “Physicochemical Properties and in vitro Starch Digestibility of Thai

Tr

aditional Rice Cake and Pea Cake”

, supervised by Dr. Rungarun Sasanatayart,

Dr. Ir. Yadi Haryadi, M.Sc., and Dr. Eko Hari Purnomo, S.TP., M.Sc.

ix

FOREWORD

It was grace alone that this undergraduate research project entitled

”

Physicochemical Properties and

in Vitro

Starch Digestibility of Thai Traditional

Rice Cake and Pea Cake

”

had been completed accordingly. This research was

conducted at the School of Agro-Industry, Mae Fah Luang University (MFU,

Thailand) under Credit Transfer-MIT Program. The author would like to thank The

Almighty One, Jesus Christ, for the unconditional love, and is indebted to the large

number of people who have given freely of their time and experiences, provided him

with information, and outstanding efforts during the completion of this project. The

thanks and extensive gratitude of the author to:

1.

My beloved mother and brother, for the encouragement and forbearance at my

adventure of life. I do really love you both.

2.

My academic advisor Dr. Ir. Yadi Haryadi, M.Sc., for the considerable advice

and constructive thoughts.

3.

Dr. Eko Hari Purnomo, S.TP., M.Sc., as a co-advisor and was in charge of MIT

Program at once, for the ceaseless support and motivation.

4.

Dr. Rungarun Sasanatayart, my cheerful advisor in MFU. Thanks for the bright

ideas and technical support.

5.

All of faculty member of the Department of Food Science and Technology,

Bogor Agricultural University, for making the knowledge available.

6.

Directorate General of Higher Education - Ministry of National Education of the

Republic of Indonesia, which facilitated a pilot project on promoting student

mobility program between Bogor Agricultural University and Mae Fah Luang

University.

7.

Prof. Didik Sulistyanto as the Education Attache of the Embassy of Indonesia in

Bangkok, for the efforts to make this program successful.

8.

The big family of Perhimpunan Mahasiswa Indonesia di Thailand (PERMITHA),

for the brotherhood. Thanks for taking care of me during my stay in Thailand.

9.

All of my classmates, you guys are so worthwhile.

10.

My brothers and sisters in Komisi Pelayanan Siswa PMK IPB, thanks for the

Christ-like servanthood. Thanks for sharing life.

11.

The citizens of Pondok Syalom, thanks for the cheers and togetherness.

12.

Laboratory assistants in both MFU and IPB, for the technical assistance.

13.

I should also not forget individuals who deserve my gratitude for their silent

supports.

x

It is truly hoped that this manuscript will constitute a worthy addition to the

existing knowledge on food science and technology area, and that readers will

benefit useful information.

Bogor, January 2011

xi

TABLE OF CONTENT

Page

FOREWORD

………...

...

ix

TABLE OF CONTENT

………...

...

xi

LIST OF TABLES

………...

...

xiii

LIST OF FIGURES

……….

xiv

LIST OF APPENDICES

……….

xv

I.

INTRODUCTION

1.1 BACKGROUND

……….

1

1.2 OBJECTIVES

………..

2

II. LITERATURE REVIEW

2.1 THAI RICE CAKE AND PEA CAKE

………...

3

2.2 STARCH

……….

4

2.2.1 Rice Starch

………

5

2.2.2 Pea Starch

………..

5

2.3 ENZYMATIC DIGESTIBILITY OF STARCH

……….

5

2.3.1 Enzymatic Digestibility of Rice Starch

……….

6

2.3.2 Enzymatic Digestibility of Pea Starch

………..

7

2.4 PHYSICOCHEMICAL PROPERTIES OF STARCH

………

7

2.4.1 Gelatinization

………

7

2.4.2 Pasting Properties

………..

8

2.4.3 Instruments for Observing Gelatinization and Pasting

Behavior of Starch

……….

8

2.4.4 Syneresis and Retrogradation

………

8

2.5

APPLICATION OF DIFFERENTIAL SCANNING

CALORIMETRY (DSC) TO STARCH

………..

8

2.6

APPLICATION OF SCANNING ELECTRON MICROSCOPY

(SEM) TO STARCH

………...

10

III. RESEARCH METHODOLOGY

3.1 MATERIALS AND INSTRUMENTS

………

11

3.1.1 Materials

………

11

3.1.2 Instruments

………

11

3.2

EXPERIMENTAL DESIGN….

………..

11

3.3 METHOD OF ANALYSIS

……….

13

3.3.1 Moisture Content

………...

13

3.3.2 Ash Content

………...

13

3.3.3 Protein Content

………..

14

3.3.4 Crude Fat Content

……….

14

3.3.5 Apparent Amylose Content

………...

14

3.3.6

In Vitro

Starch Digestibility

………..

14

3.3.7 Granular Morphology

………

15

3.3.8 Thermal Properties

………

15

3.3.9 Pasting Properties

………..

15

3.3.10 Textural Properties

………

16

3.3.11 Color Properties

………

16

xii

IV. RESULT AND DISCUSSION

4.1

PRELIMINARY RESEARCH

……….………

...

17

4.1.1

Chemical Composition of Raw Materials

……….

17

4.1.2

Apparent Amylose Content

………...

18

4.1.3

Granular Morphology

………

18

4.1.4

Thermal Properties

………

19

4.1.5

Pasting Properties

………..

19

4.2

EXPERIMENT I

………...

23

4.2.1

Apparent Amylose Content

………...

23

4.2.2

Granular Morphology

………

23

4.2.3

Thermal Properties

………

25

4.2.4

Pasting Properties

………..

26

4.3

EXPERIMENT II ………

26

4.3.1

Textural Properties

………

26

4.3.2

Color Properties

……….

30

4.3.3

In Vitro

Starch Digestibility

………..

31

4.4

EXPERIMENT III

………...

33

4.4.1

In Vitro

Starch Digestibility

………..

33

4.4.2

Textural Properties

………

34

4.4.3

Color Properties

………

36

V.

CONCLUSIONS AND RECOMMENDATIONS

REFERENCES ………

...

40

xiii

LIST OF TABLES

Page

Table 1

Chemical composition of raw materials

………

17

Table 2

Apparent amylose content of raw materials

………

...

18

Table 3

Thermal properties of pea flour, sticky rice flour, rice flour

…….

21

Table 4

Thermal properties of mixture between rice flour and sticky rice

flour

………

25

Table 5

Textural properties of rice cake and pea cake

………

28

Table 6

L*,a*,b*

value of pea cake and rice cake

………..

31

xiv

LIST OF FIGURES

Page

Figure 1

Rice cake

………

3

Figure 2

Pea cake

………..

3

Figure 3

Flowchart of experiment I

……….…….

12

Figure 4

Flowchart of experiment II

……….

12

Figure 5

Flowchart of experiment

III …..……….

13

Figure 6

Granular morphology of pea flour, rice flour, sticky rice flour ..

19

Figure 7

DSC Thermogram of pea flour, rice flour, sticky rice flour

…..

.

20

Figure 8

RVA curve of

pea flour, rice flour, sticky rice flour

…………..

21

Figure 9

Apparent amylose content of mixture between rice flour and

sticky rice flour .………..

23

Figure 10 Granular morphology of mixture between rice flour and sticky

rice flour

……….

24

Figure 11 DSC thermogram of mixture between rice flour and sticky rice

flour ………

25

Figure 12 Pasting properties of mixture between rice flour and sticky rice

flour

………

26

Figure 13 Commercial and experimental pea cake

………

27

Figure 14 Commercial and experimental rice cake

………

27

Figure 15 Relationship between amylose content and hardness

(left),

adhesiveness (right)

………

30

Figure 16 Relationship between amylose content and digestible starch

(left), resistant starch (right)

………...

32

Figure 17 Resistant starch content of pea cake, rice cake, and rice cake

prepared from ratio of rice flour : sticky rice (90:10) flour at

various cold setting conditions

……….………

..

34

Figure 18 Textural properties (hardness, adhesiveness, springiness,

cohesiveness, gumminess, chewiness) of pea cake, rice cake,

and rice cake prepared from ratio of rice flour sticky rice flour

(90:10) at various cold setting conditions

………..

35

Figure 19

L*

value of pea cake, rice cake, and rice cake prepared from

ratio of rice flour : sticky rice flour (90:10) at various cold

setting conditions

………

37

xv

Figure 20

a*

value of pea cake, rice cake, and rice cake prepared from

ratio of rice flour : sticky rice flour (90:10) at various cold

setting conditions

………

37

Figure 21

b

* value of pea cake, rice cake, and rice cake prepared from

ratio of rice flour : sticky rice flour (90:10) at various cold

xvi

LIST OF APPENDICES

Page

Appendix 1

Physical and chemical properties of common starch

…………..

49

Appendix 2

Result of chemical composition analysis

……….

50

Appendix 3

ANOVA and Duncan`s test results for chemical composition

…

51

Appendix 4

Result of thermal properties analysis

………...

52

Appendix 5

ANOVA and Duncan`s test results for thermal properties

……..

54

Appendix 6

Result of pasting properties analysis

………...

58

Appendix 7

Result of textural properties analysis (experiment II)

………….

60

Appendix 8

Result of color properties analysis (experiment II)

……….

62

Appendix 9

Result of

in vitro

starch digestibility analysis (experiment II)

…

63

Appendix 10 ANOVA and Duncan`s test results for

in vitro

starch

digestibility (experiment II)

………

.

63

Appendix 11 Result of

in vitro

starch digestibility analysis (experiment III) ...

66

Appendix 12 ANOVA and Duncan`s test results for

in vitro

starch

digestibility

(experiment III) ...

………

66

Appendix 13 Result of textural properties analysis (experiment III)

…………

71

Appendix 14 Result of color properties analysis (experiment III)

………

74

1

I. INTRODUCTION

1.1

BACKGROUND

It has been established that carbohydrates are excellent source of human energy, providing 70-80 % of calories in the human diet worldwide (BeMiller and Whistler, 1996). It can be shown that grains as the source of carbohydrates have bigger proportion than that of vegetables, fruits, milk, meat and beans in the MyPyramid Food Intake Patterns (USDA, 2005). In cereal grains, carbohydrates are presented in the form of starch, playing role natively as energy storage (Collado & Corke, 2003). The amount of starch contained in a cereal grain varies but is generally between 60 and 75% of the weight of the grain, thus much of the food that humans consume is in the form of starch (Hoseney, 1998).

In addition to its nutritive value, starch is important because of its effect upon physical properties of many of our foods. Hence, it is not surprising that starches have an enormous number of food uses, including adhesive, binding, clouding, dusting, film forming, foam strengthening, anti-staling, gelling, glazing, moisture retaining, stabilizing, texturizing, and thickening applications (BeMiller and Whistler, 1996).

The commercial and technological uses of starch are extremely numerous, and as a result the academic aspects of the subject have received much attention. Starch has probably been investigated to a greater extent than any other biopolymer (Greenwood, 1976). Eyaru et al. (2009) reported the effect of various processing techniques on digestibility of starch in red kidney bean (Phaseolus vulgaris) and two varieties of peas (Pisum sativum). Chung et al. (2008) also reported the in vitro starch digestibility and some physicochemical properties of starch from common bean (Phaseolus vulgaris L.). Again, the influence of physicochemical properties to the in vitro digestibility of waxy rice starch gel was reported (Sasaki et al., 2009).

Physicochemical properties and enzymatic digestibility, and their correlation are likely to be the most important aspect at which many investigations have been conducted. Physicochemical properties are closely related to the processing condition, machinability, and quality of products, whereas starch digestion is a highly important metabolic response and the rate and extent of starch digestibility and absorption are nutritionally important as numerous studies have been carried out (Sasaki et al., 2009). The most desirable starch ingredient, from the nutritional point of view, is believed to be slowly digestible or even resistant. Many investigations concluded that generally, the amount of resistant starch (RS) increase on storage, especially low temperature storage (Sajilata et al., 2006).

Several studies of starch have concentrated on the raw materials. However, little emphasis has been given to the specific product. Therefore, investigating the physicochemical properties of starch based product is becoming important as well as its in vitro digestibility.

As widely known, rice is one of the leading food crops in Southeast Asia including China. China, India, Indonesia, Bangladesh, Vietnam, and Thailand are the top rice producing countries respectively as can be shown in the statistical report presented by FAOSTAT (2010). Rice can be ground into powder and utilized to produce many kinds of foods, including several types of cake. Rice cake is the most widespread of these types of cake. It is indigenous food of many countries in Asia and Southeast Asia.

In Thailand, especially in the North, rice cake (kao ram phune) is very popular. In certain parts, this type of cake is also made from pea (Pisum sativum). The processing aspect mainly comprises starch gelatinization (cooking) and retrogradation (cold setting). These processes may contribute to

2 the change in properties of starch, in particular digestibility. Until now, there is dearth information on the experimental data, how the process parameters contribute to the change of starch properties in the product of rice and pea cake.

Therefore this research was carried out to investigate how the starch properties in rice cake and pea cake comprising physicochemical aspects and enzymatic digestibility will be influenced by process parameters. The gained information would be very useful for developing these products.

1.2

OBJECTIVES

1. To investigate the effect of flour type and mixed flour on physicochemical properties and starch digestibility of the corresponding pea cake and rice cake.

2. To investigate the effect of cold setting conditions on physicochemical properties and starch digestibility of pea cake and rice cake.

3

II. LITERATURE REVIEW

2.1

THAI RICE CAKE AND PEA CAKE

Rice cake is indigenous food of many countries in Asia and Southeast Asia. Rice cake, in China, called migao, is famous for its soft-sticky texture and is usually served as a dessert. This cake is mainly prepared from rice flour and sticky rice flour (Ji et al., 2007). In Thailand, it is called kao ram phune (Figure 1) and is usually served with other ingredients such as vegetables, nut powder, spicy seasoning, etc. The vendors used to make it by soaking the rice grains overnight, wet milling, then boiling to gelatinize the starch.

In certain parts of Thailand, especially in the North, this type of cake is also made from pea (Pisum sativum) as presented in Figure 2. It is also usually served together with vegetables, nut powder, and spicy seasonings. The processing technique is a little bit different compared to rice cake. Pea grains are soaked overnight, then wet milled and filtered to separate coarse materials. The slurry then boiled to gelatinize the starch. After several minutes of cooling it forms a firm gel.

Figure 1. Rice cake

4

2.2

STARCH

Cereal grain stores energy in the form of starch. The amount starch contained in cereal grain varies but is generally between 60 and 75% of the weight of the grain. Starch is found in plants in the form of granules (Hoseney, 1998). The cold-water-insoluble starch granule forms the major constituent of all cereal crops. Being a macromolecule composed entirely of α-D-glucose, this polysaccharide is readily assimilated in the human diet; in fact, a very high proportion of the world`s food energy intake is starch (Greenwood, 1976).

Starch from different cereals vary widely in their characteristics involve size, shape, chemical composition, and physical properties. Types of granule shape are large lenticular or lens-shaped (25-40 μm), small spherical 5-10 μm), round, elliptical, polyhedral, and polygonal. In the chemical composition, starch is composed essentially of glucose. It may contain a number of minor constituents which can and do affect the starch properties. Cereal starches contain low levels of fats, which is generally polar lipids in amount of between 0.5 and 1 % (Hoseney, 1998). The presence of high amount of lipid in starch has unfavorable effects. The lipids repress the swelling and solubilization, also increase the pasting temperatures and reduce the water-binding ability. The formation of amylose-lipid complexes causes turbidity and precipitation in starch pastes and starch solutions. The oxidation of unsaturated lipids may cause the undesirable flavors in pregelatinized starch products. The other minor constituents are nitrogen substances (include proteins, peptides, amides, amino acids, nucleic acids, and enzymes), ash (corresponds partly with the amount of phospholipids in the starch granules), and phosphorus which occurs mainly as phospholipids (Collado and Corke, 2003).

One of the uniqueness of starch is that most starch granules are composed of a mixture of two polymers; an essentially linear polysaccharide called amylose, and a highly branched polysaccharide called amylopectin. Most of starches contain about 25 % amylose. While amylose is essentially a linear chain of (1→4)-linked α-D-glucopyranosyl units, many amylose molecules have a few α -D-(1→6) branches, perhaps 1 in 180-320 units, or 0.3-0.5 % of the linkages. The branches in branched amylose molecules are either very long or very short, and the branch points are separated by large distances so that the physical properties of amylose molecules are essentially those of linear molecules. Amylose molecules have molecular weights of about 106_{. The axial → equatorial position coupling of} the (1→4)-linked α-D-glucopyranosyl units in amylose chain gives the molecules a right-handed spiral or helical shape. The interior of the helix contains only hydrogen atoms and is lipophilic, while the hydroxyl groups are positioned on the exterior of the coil (BeMiller and Whistler, 1996). A helical conformation is common for amylose, and a double helix form when different helices pack together. An open channel in the center of a helix permits complexing with other molecular species, such as iodine, organic alcohols, or acids. Methods based on the iodine reaction remain a convenient means for estimating amylose content, giving accurate and reproducible results (Collado and Corke, 2003). The second polymer is amylopectin, constituting about 75% of most common starches. It is a very large, very highly branched molecule, with branch-point linkages constituting 4-5% of the total linkages (BeMiller and Whistler, 1996). Like amylose, amylopectin is composed of α-D-glucose linked primarily by α-(1→4) bonds. Amylopectin is branched to a much greater extent than is amylose, with 4-5% of the glycosidic bonds being α-(1→6) bonds (Hoseney, 1998). Amylopectin consists of a chain containing the only reducing end-group, called a C-chain, which has numerous branches, termed B-chains, to which one to several third-layer A-chains are attached. A-chains are unbranched. B-chains are branched with A-chains or other B-chains. The branches of amylopectin molecule are clustered and occur as double helices. Molecular weights of from 107 _{to 5x10}8 _{make amylopectin molecule among} the largest molecules found in nature (BeMiller and Whistler, 1996).

5 2.2.1 Rice Starch

Rice starch exhibits typical characteristics compared to other starches (Appendix 1). Starch granules in rice endosperm grow as a single entity inside the cellular amyloplasts. Rice only contains compound granules in which many granules have developed within a single amyloplast. Compound granules having diameters up to 150 μm form as clusters containing between 20 and 60 individual granules (Champagne, 1996) which are the smallest known to exist in cereal grains (3 to 8 μm) as reported by Bao and Bergman (2004). There are some variations in starch granule size between different rice genotypes. Sodhi and Singh (2003) reported that a group of rice varieties grown in India had starch granules from 2.4-5.4 μm. The average starch size from some waxy rice ranged from 4.9 to 5.7 μm (Qi et al., 2003). Rice starch granules have a smooth surface but angular and polygonal shapes. The polygonal starch granules may be formed by compression of the starch granules during grain development (Hoseney, 1998).

2.2.2 Pea Starch

Being a legume starch, yellow pea (Pisum sativum) which is also known as field pea, garden pea, or smooth pea, exhibits a definitely different characteristic from rice which is grain starch, both of in physical and chemical properties. It has a relatively large average granular size (28.82 μm) compared to other starches, with oval, round, spherical, elliptical, or irregular shapes. However, yellow pea starch has a closely range of gelatinization temperature (61.7-75.1 o_{C) compared to rice starch} (61-80 o_{C), as reported by Collado and Corke (2003). Pea starch also contains higher amount of amylose} (34.2-40.8 %) compared to any other type of starches. This typical characteristic of legume starch leads to less enzyme susceptibility (Singh et al., 2010).

2.3

ENZYMATIC DIGESTIBILITY OF STARCH

Such enzymes can hydrolyze the starch with the presence of water. The hydrolases are a large group of enzymes which have in common the involvement of water in formation of product. One of the important subgroups is carbohydrases. Amylases are carbohydrases that catalyse the hydrolysis of α-D-1,4-glycosidic linkages of starch and related oligo- and polysaccharides by the transfer of glycosyl residue (donor) to H2O as the acceptor (Naz, 2002).

α-Amylase (EC. 3.2.1.1, 1,4-α-D-glucan glucanohydrolase) is an endo-enzyme that cleaves both amylose and amylopectin molecules internally, producing oligosaccharides. The larger oligosaccharides may be singly, doubly, or triply branched via (1→6) linkages, since α-amylase acts only on the (1→4) linkages of starch (BeMiller and Whistler, 1996). An α-1,4 linkage neighbouring an α-1,6 branching point in the substrate is resistant to attack by the enzyme (Naz, 2002). α-Amylase also does not attack double-helical starch polymer segments or polymer segments complexed with a polar lipid which is stabilized single helical segments (BeMiller and Whistler, 1996).

Glucoamylase (amyloglucosidase) is used commercially, in combination with an α-amylase, for producing D-glucose (dextrose) syrups and crystalline D-glucose. The enzyme acts upon fully gelatinized starch as an exo-enzyme, sequentially releasing single D-glucosyl units from the nonreducing ends of amylose and amylopectin molecules, even those joined through (1→6) bonds. Consequently, the enzyme can completely hydrolyze starch to D-glucose, but is used on starch that has been previously depolymerized with α-amylase to generate small fragments and more reducing ends (BeMiller and Whistler, 1996).

6 Various kinds of enzymes that catalyze hydrolysis of amylose and amylopectin molecules are obtained from a variety of sources, such as fungi, bacteria, cereals, and other plants, and in the case of α-amylase even from animal sources. Their efficiency, specificity, and optimum conditions of activity depend on the source (Collado and Corke, 2003). The node of enzymatic degradation of the granule also depends both on the enzyme source and the type of starch (Greenwood, 1976). Although the endo-enzyme implies random cleavage, numerous experiments have suggested that the endo-enzyme action follows a definite pattern depending on the source of the enzyme (Naz, 2002).

Depending on their rate and extent of digestion, starch can be classified into three categories (Englyst et al., 1992); these include rapidly digested starch (RDS), slowly digested starch (SDS), and resistant starch (RS). The fraction of starch that is said to be RDS in vitro is defined as the amount of starch digested in the first 20 min of enzyme digestion, whereas SDS is defined as the starch that is digested after the RDS but in no longer than 120 min under standard conditions of substrate and enzyme concentration (Englyst et al., 1992). RDS and SDS are digestible starch, or that we call non-resistant starch (NRS). Total starch is the sum of non-non-resistant starch and non-resistant starch.

In the last 25 years the digestibility of foods has been classified by a number of metrics, the most popular of which is the glycemic index (Dona et al., 2010). However,aside from glycemic index (GI), restistant starch (RS) content has been established as an important measure to characterize starch digestibility (Frei et al., 2003). Resistant starch (RS) itself has been defined as the portion of starch that is not hydrolyzed by the enzymes in the small intestine and passes to the large intestine, or to be the total starch minus amount of glucose released within 120 min of in vitro digestion (Singh et al., 2010a).

The beneficial effects of resistant starch have received much attention. Resistant starch acts as a fermentation substrate in the colon, similar to non-starch carbohydrates, with positive implications for the prevention of food-borne diseases, such as colon cancer and hypolipidemia (Frei et al., 2003). According to Hu et al. (2004), RS is slowly absorbed in the small intestine resulting in decreased postprandial glucose and insulin responses. This behavior has significant implications for the use of RS in food formulations for persons with certain forms of diabetes. The slowed starch absorption also implies long-term benefits in controlling hyperlipidaemia. In the colon, RS increases fecal bulk, lowers colonic pH and the portion fermented by the intestinal microflora produces a range of short-chain fatty acids (SCFA), primarily acetate, propionate and butyrate. SCFA production has a positive impact on bowel health, including increased absorption of magnesium and calcium, epithelial proliferation, the balance of bacterial species, and bacterial metabolism of bile salts. Whether through its indirect effect on bile salts or through dilution effects, RS is thought to provide a degree of protection against bowel cancer.

2.3.1 Enzymatic Digestibility of Rice Starch

Many of researches have been carried out to investigate the digestibility of rice starch. The glycemic response of rice is known to be relatively high compared to other starchy foods (Frei et al., 2003). The same authors also reported the effect of cooking and storage to the in vitro starch digestibility and the glycemic index of six different rice cultivars from the Philippines. The results indicate substantial differences in the estimated glycemic index between rice cultivars. Values ranged between 68 and 109 for cooked rice and between 64 and 87 for stored rice containing retrograded starch. Storage under refrigeration also has been reported to slow the rate of rice starch digestion. The other result was that starch hydrolysis tended to be more rapid and more complete for waxy cultivars than for high amylose cultivars. Sasaki et al. (2009) reported the effect of physicochemical characteristics on the in vitro digestibility among waxy rice cultivars in the form of starch gels. The

7 authors suggested that the differences in amylopectin chain distribution reflect the stability, perfection, and extent of recrystallinity in the starch gel, inducing a difference in digestibility of the starch gel. Perez et al. (1991) reported that rice cultivars with similar amylose content varied in starch digestibility, the difference being associated with other properties, such as gelatinization temperature, cooking time, amylograph consistency, and volume expansion upon cooking. Processing techniques are also reported to impact the rate of rice starch digestion. Parboiling reportedly decreases rice starch rate of digestion (Tetens et al., 1997). Rashmi and Urooj (2003) found that the steaming of rice created more resistant starch than boiling or pressure cooking.

2.3.2 Enzymatic Digestibility of Pea Starch

Legume starch is generally less in digestibility compared to grain starch. It is mainly due to the composition between amylose and amylopectin. RS contents increased with increasing amylose content which means pea starch will be less in digestibility (Perera et al., 2010). This is also affected by morphological characteristics of pea starch which has a large granular average size. Parera et al. (2010) reported an apparent direct negative relationship between large size granules and starch digestibility. In other case, Lindeboom et al. (2004) reported that the small barley and wheat starch granules hydrolyze faster than the large granules. The presence of some non-starchy substances such as proteins over the granule surface may also limit the rate of enzymatic hydrolysis. Granule surface proteins and lipids can reduce surface accessibility by blocking the adsorption sites and therefore influences enzyme binding (Oates, 1997).

2.4.

PHYSICOCHEMICAL PROPERTIES OF STARCH

2.4.1 Gelatinization

Starch granules are insoluble in cold water. They swell slightly but shrink back to their original size and consistency on drying. When heated in a water suspension to progressively higher temperature, very little happens until a certain critical temperature is reached. At that point starch granules begin to swell, simultaneously losing polarization crosses. This is termed gelatinization (Pomeranz, 1991). Other definition is the collapse (disruption) of the starch granule manifested in irreversible changes in properties such as granular swelling, native crystallite melting, loss of birefringence, and starch solubilization. The point of initial gelatinization and the range over which it occurs is governed by starch concentration, method of observation, granular type, and heterogeneity within the granule population under observation (Collado and Corke, 2003).

The gelatinization temperature (GT) is always a temperature range. For a single starch granule in excess water, this temperature range might be 1 to 2 oC, whereas for the entire population the range might be 10 to 15 o_{C (Eliasson and Gudmundssond, 2006). Rice starch (20 %) has GT ranged from 60} to 77 o_{C while waxy rice starch at the same concentration ranged from 60 to 78} o_{C (Eliasson and} Gudmundssond, 2006). After than a decade of study most agree that variation in rice GT is a result of differences in the proportion of amylopection that is short versus long chains, thus degree of cristallinity is what is being measured as gelatinization temperature (Bao and Bergman, 2004).

Investigating gelatinization characteristics of starches is very important. Gelatinization is a physical process that is unique to starches and is responsible for its change properties during the preparation and processing of food (McWilliams, 2005; Collado and Corke, 2003). The heat energy required to completely gelatinize starch in rice is critical to the rice processor, who must optimize heat

8 input, cooking time, and temperature and, at the same time, minimize the cost of entire process (Bao and Bergman, 2004).

2.4.2 Pasting Properties

The terms gelatinization and pasting have often been applied to all changes that occur when starch is heated in water. However, gelatinization includes the early changes and pasting includes later changes. Pasting is the phenomenon following gelatinization in the dissolution of starch. It involves granular swelling, exudation of molecular components from the granule, and eventually total disruption of the granules (Collado and Corke, 2003). Simply stated, starch paste can be described as a two phase system composed of a dispersed phase of swollen granules and a continuous phase of leached amylose (Collado and Corke, 2003).

2.4.3 Instruments for Observing Gelatinization and Pasting Behavior of Starch

The gelatinization and pasting behavior can be recorded by using a Brabender Visco Amylograph, Rapid Visco Analyzer (RVA), or other viscometers which record the viscosity continuously as the temperature is increased, held constant for a time, and then decreased. However, RVA is likely to be preferred as it gives results in much shorter time (Bao and Bergman, 2004; Hoseney, 1998). At the initial step, the viscosity increases rapidly with the increase of temperature as the granule swells. The peak viscosity is reached when granules swelling have been balanced with the granules broken by stirring. With continued stirring, more granules rupture and fragment, causing a further decrease in viscosity. On cooling, some starch molecules partially re-associate to form a precipitate or gel, in which amylose molecules aggregate into a network, embedding remnants of starch granules (Bao and Bergman, 2004). Rice starch pasting parameters have been reported to be correlated with amylose content (Keeratipibul et al., 2008) but different in waxy rice compared to nonwaxy rice (Bao and Bergman, 2004).

2.4.4 Syneresis and Retrogradation

As the starch paste is cooled, the starch chains become less energetic and the hydrogen bonds become stronger, giving a firmer gel. As a gel ages or if it is frozen and thawed, the starch chains have a tendency to interact strongly with each other and thereby force water out of the system. The squeezing of water out of the gel is called syneresis (Hoseney, 1998). Longer storage gives rise to more interaction between the starch chains and eventually the formation of crystals. This process, called retrogradation is the crystallization of starch chains in the gel. The gel becomes more opaque as retrogradation progress. In addition, it becomes more rigid or rubbery, perhaps partially as a result of crystallization and partially just from interaction of the starch (Hoseney, 1998).

2.5

APPLICATION OF DIFFERENTIAL SCANNING CALORIMETRY

(DSC) TO STARCH

Thermal analysis is broad term that encompasses numerous techniques that measure chemical or physical changes of a substance as a sample is subjected to a controlled temperature program over time. The most popular modern thermal analysis techniques are those that dynamically follow (a sequence

9 of) physicochemical changes that a substance undergoes during heating or cooling. One of them is differential scanning calorimetry or DSC (Schenz and Davis, 1998).

Differential scanning calorimetry (DSC) has been the most commonly used methods of thermal analysis in food science. This technique has come to dominate the studies of starch-related transitions (Eliasson, 2003). It measures the differential temperature or heat flow to or from a sample versus a reference material, and this is displayed as a function of temperature or time. This technique can differentiate between two types of thermal events: endothermic and exothermic (Schenz and Davis, 1998). Endotherms typically associated with the melting of mono-, di-, oligo-, and polysaccharides, denaturation of proteins (Kalentunc and Breslauer, 2003). Starch gelatinization is also an endothermic process with enthalpy values in the range of 10 to 20 J/g (Eliasson and Gudmundssond, 2006).

To gather, interpret, and calculate the proper onset (To), peak (Tp), conclusion temperatures (Tc) and heat transition, as well as the heat capacity of the sample, the instrument must be calibrated with well characterized standards, such as inidium. Inidium has a ΔH of fusion of 28.4 J/g, and melting point of 156.64 oC (Schenz and Davis, 1998).

Regarding to the sample size, it must be small to obtain a near-instantaneous response to the heat transfer and resulting high precision in the determination (Blond and Simatos, 1996). The usual sample size (6-12 mg) can be placed in either small (up to 20 mg) volatile or nonvolatile sample pans (usually sealed volatile sample pans or often called hermetic pans are used most commonly in food science work) or stainless steel capsule that can withstand high-pressure buildup inside them, such as that caused by the volatilization of water (Schenz and Davis, 1998).

Regarding to the heating rate, it should be slow enough to obtain distinct and reproducible peaks for each transition. For most applications, a rate of 10 o_{C/min suits well. However, in cases where} precise temperature determinations are desired, slower rates (1-2 oC/min) must be used (Blond and Simatos, 1996).

Since DSC measures heat flow, larger sample and faster heating rates will give larger signal. However, too large sample and too fast heating rates also broaden transitions. In general, it is best to use the minimum sample size and slowest heating rate that is practicable to give the desired resolution of transitions and thermal data (Schenz and Davis, 1998).

This instrument will display the result of observation in the DSC thermogram. The start of the peak (where it deviates from the base line) corresponds to the start of birefringence loss. The area under curve is a measure of the energy (enthalpy, ΔH) required for the transition from an ordered to a disordered state (i.e., for the crystalline area to melt). The end point of the lost of birefringence and the end of the peak are not quite the same, as there is a considerable lag in the DSC. However, in general, the two correlate well (Hoseney, 1998). One major controversy in the interpretation of DSC curves relates to the onset temperature, the meaning of the peak, and the determination of the baseline. Most researchers agree onset temperature is more significant than peak temperature, since peak temperature is greatly influenced by scan rate and sample size and does not always relate to a specific physical change. Although one may report peak temperatures to compare to other reports, onset temperatures should be used to interpret data (Schenz & Davis, 1998).

Ji et al. (2007) reported that two endotherms were observed by DSC studies of cake prepared from rice flour and sticky rice flour. The first transition with a peak temperature (Tp) at approximately 58.1 o_{C is characteristic of melting of retrograded amylopectin. The second endotherm observed by} DSC above 100 o_{C is characteristic of melting of the amylose-lipid complex of starch.}

10

2.6

APPLICATION OF SCANNING ELECTRON MICROSCOPY (SEM) TO

STARCH

Scanning electron microscope (SEM) has been widely used in the area of research which is focused on starches, especially in examining granular morphology of starches. It gives satisfying information of starches granule size and shape among botanical sources which are necessary to be understood. In the best conditions, and with the most advanced equipment, the resolving power of the scanning electron microscope can reach 5 nm for biological matter (Blond and Simatos, 1996).

The morphological changes of starch during heating in excess water is also can be studied by SEM (Eliasson and Gudmundssond, 2006). Collado and Corke (2003) suggested that functional properties of the starches are related not only to their structure as polymers but also to the packing of polymers within the granules. Bao and Bergman (2004) also reported that the clarity of starch suspensions which is very important for many food applications varies among different rice cultivars and may be attributed to amylose content and granular size.

The mechanism of SEM in generating image reveals quite a bit of complexity, but it can be simplified (Hafner, 2007). Principally, the sample is bombarded by electrons energy. Under this bombardment, each point of the object spontaneously emits varied radiations, depending on its chemical nature, surface state and the conditions of the electron probe emission. This radiation is captured by appropriate detectors (Blond and Simatos, 1996). This instrument is composed by several supporting equipments such as a column containing a thermoelectronic emission system (electron gun), an electron probe focusing system (condenser and objective), and a scanning system (electromagnetic deflection) controlling the scan amplitude (Blond and Simatos, 1996).

One thing that must be concerned in SEM is sample preparation. Sample preparation for scanning electron microscopy depends on their hydration. The samples must be dehydrated before being placed under vacuum in the microscope column. If the moisture content does not exceed 16 % (e.g., cereal grains or dry products) they can be observed as they are. Contrarily, if their moisture content is higher, it is essential to dehydrate them either by lyophilization or critical point drying (Blond and Simatos, 1996). If it is preferred to avoid the inconvenience of dehydration, it is possible to use a cryotransfer stage. This special device allows the sample to be observed directly, after very fast freezing on a liquid nitrogen cooled stage (-170 o_{C). Since the sample is preliminary fractured and} coated with metal under vacuum at -170 o_{C, the temperature is never interrupted. The results with this} technique are outstanding (Blond and Simatos, 1996).

The remarkable advantages of SEM are apparently admitted. However, this technique also presents certain drawbacks, like the inability to study hydrated media without resorting to complex techniques, and multitude of problems related to the preparatory techniques, which do not occur without fairly significant disruption in the sample material or the production of foreign elements that must be eliminated. These factors must be taken into account when interpreting the photographic documentation (Blond and Simatos, 1996). As stated above that samples must be observed in dehydrated state, biological samples exhibit at three major drawbacks: 1) they are poor conductor, and thus poor emitters, causing strong discharges during observation, which disrupts the electromagnetic detection and consequently, the image; 2) the low energy electrons penetrate easily, so that the image is formed from both true secondary electrons and second generation electrons, which weakens image clarity, but accentuates its relief; 3) some substances, such as starch, are very sensitive to the action of high energy electrons, and break up when the intensity is too high (Blond and Simatos, 1996).

11

III. RESEARCH METHODOLOGY

3.1

MATERIALS & INSTRUMENTS

3.1.1

Materials

Pea flour (Yunnan Xing Yi Foodstuff Co. Ltd., China), rice flour (Thai Better Foods Co. Ltd., Thailand), and sticky rice flour (Thai Better Foods Co. Ltd., Thailand), were obtained from local market. Potato amylose standard (Sigma Chemical Co., St. Louis) was used for apparent amylose content analysis, while for resistant starch analysis, Resistant Starch Assay Kits Megazyme International Ireland, Ltd. (pancreatic α-amylase, amyloglucosidase, GOPOD reagent buffer and enzymes, D-Glucose standard solution, resistant starch control) were used.

3.1.2

Instruments

.

The main instruments were a scanning electron microscope (SEM, LeO 1450 VP, England), a differential scanning calorimeter (DSC, Mettler Toledo, TGA/SDTA 851e, Switzerland) a rapid visco analyzer (RVA, Model 4D, Newport Scientific, Australia), a texture analyzer (TA-XT2, Stable Micro System, Texture Technologies Corp., USA), a spectrophotometer (UV Vis. Biochrom/Libra S22, England), and color analyzer (ColorQuest XE HunterLab, Hunter Associates Laboratory Inc., Virginia-USA).

3.2.

EXPERIMENTAL DESIGN

This research was divided into three parts. The preliminary research was investigation on the properties of raw materials (rice flour, sticky rice flour, and pea flour) involved chemical composition, apparent amylose content, granular morphology, thermal and pasting properties. In experiment I (Figure 3), the effect of mixing rice flour with sticky rice flour at various ratios (100:0, 97.5:2.5, 95:5, 92.5:7.5, 90:10) on apparent amylose content, granular morphology, thermal and pasting properties was also investigated. In experiment II (Figure 4), trial was conducted to determine the solid content, proper cooking time and temperature to obtain rice cake. Similar trial was conducted for pea cake. Cakes resulted from various ratios of rice flour and sticky rice flour were then analyzed in terms of starch digestibility, textural and color properties. Commercially prepared pea cake and rice cake were also analyzed for comparison.

In experiment III (Figure 5) various cold setting conditions were conducted on pea cake and the extreme ratios of rice cake (100:0 and 90:10). First condition was 6 hours at room temperature / ± 25 o_{C which represented vendors that make cakes in the morning, place them in room temperature} then sell them in the afternoon or evening. Second condition was 6 hours at refrigeration temperature (4 o_{C) which represented some vendors that place the cakes in iced box during time between after} cooking to selling (approximately 6 hours). Third condition was 24 hours at 4 o_{C which came from} theoretical point of view that RS content will increase after storage for 24 hours or longer at refrigeration temperature for some starches. Experiment for 12 or 24 hours at room temperature was impossible to be conducted as spoilage occurred on the cakes. Clearer experimental steps can be seen on the following figures.

12

Figure 3. Flowchart of experiment I

Figure 4. Flowchart of experiment II RF:SRF (100:0) RF:SRF (97.5:2.5) RF:SRF (95:5) RF:SRF (92.5:7.5) RF:SRF (90:10) Add water to obtain 20% solid content

Gel forming (6 h, room T) Cooking

95 o_{C for 25 min with continue} stirring

Analysis: 1. Textural peoperties 2. Color properties

3. In vitro starch digestibility Rice cake RF:SRF (100:0) Rice cake RF:SRF (97.5:2.5) Rice cake RF:SRF (95:5) Rice cake RF:SRF (92.5:7.5) Rice cake RF:SRF (90:10) Sticky Rice Flour (SRF) Mixing Rice Flour (RF) RF:SRF (100:0) RF:SRF (97.5:2.5) RF:SRF (95:5) RF:SRF (92.5:7.5) RF:SRF (90:10) Analysis: 1. Amylose content

2. Granular morphology (SEM) 3. Thermal properties (DSC) 4. Pasting properties (RVA)

13 Figure 5. Flowchart of experiment III

3.3

METHOD OF ANALYSIS

3.3.1

Moisture Content (AOAC, 1995)

Empty moisture cans were pre-dried in 105 o_{C oven for 15 min and cooled in desiccator. Then} cans and 1-2 g of sample were weighed and dried in 105 o_{C oven for 3 hrs. After cooling in} desiccators, and cans containing sample were weighed until constant weight is obtained (∆ weight less than 0.0005 g).

% misture (wb) = w - (w1-w2) x 100 w

w = original sample weight

w1 = sample weight + can after drying w2 = weight of dried empty can

3.3.2

Ash Content (AOAC, 1995)

Sample of 5-10 g was weighed into a tared crucible. Very moist sample should be pre-dried first. Crucibles were placed in cool muffle furnace. Tongs, gloves, and protective eyeware should be used if the muffle furnace was warm. Sample was then ignited 12-18 hrs (or overnight) at about 550 o_{C. Muffle furnace was turned off and wait to open it until the temperature has dropped to at least 250} o_{C, preferably lower. Door must be opened carefully to avoid losing ash that may be fluffy. Crucibles} were transferred to a desiccator with a porcelain plate and desicant then weighed after cooling.

% ash (wb) = weight after ashing – tare weight of crucible x 100 original sample weight

Rice cake (100:0)

Rice cake

(90:10) Pea cake

Analysis: 1. Textural peoperties 2. Color properties

3. In vitro starch digestibility Cold

setting 6h, 25o_C

Cold setting 6h, 4o_C

Cold setting 24h, 4o_C

Cold setting 6h, 25o_C

Cold setting 6h, 4o_C

Cold setting 24h, 4o_C

Cold setting 6h, 25o_C

Cold setting 6h, 4o_C

Cold setting 24h, 4o_C

14

3.3.3

Protein Content (Kjeldahl Method, AOAC 1995)

Sample of 100-250 mg was put into Kjeldahl flask. Then 1±0.1 g of K2SO4, 40±10 mg of HgO, and 2±0.1 ml of H2SO4 were added. Boiling stones around 2-3 items were then put and boil the solution for 1-1.5 hrs. At distillation stage, a little amount of water is transferred step by step through flask wall and shaken carefully to reform the crystal. Solution was then transferred to the distillation ware, rinsed 5-6 times with 1-2 distilled water, followed by adding rinsing water to distillation ware and 8-10 ml of 60% NaOH-5% Na2S2O3. Erlenmeyer was placed under the condenser with 5 ml H3BO3 and 2-4 drops red-methylene blue added. End of condenser must be soaked in H3BO3 solution. At titration stage, sample solution was diluted into 50 ml, and then titrated with 0.02 N standardized HCl until grey color appears. Volume of HCl for titration was then reported.

% N = N HCl x corrected acid volume x 14 g/mole N x 100 g of sample

% N x 6.25 = % protein

3.3.4

Crude Fat (AOAC, 1995)

Total fat content was analyzed by soxhlet method. All the glassware (round bottom flask) was rinsed with hexane and dried in an oven at 102 oC for 30 min and cooled in a desiccator. Accurately 1-2 g of sample was weighed and covered with filtered paper. Sample was put into soxhlet extractor and condenser was set above the round bottom flask. Hexane solvent was poured into round bottom flask adequately. Sample and solvent was heated or refluxed above 5 hours or until solvent have dropped clearly to the flask. Sample was taken out of extractor then the solvent was distilled until there was almost no solvent in flask. The round flask with extracted oil was stored in oven at 105 o_{C, cooled to} desiccator and weighed until it has constant weight. Total fat content (%) = (X-Y) / W x 100%, where X = weight of empty flask and extracted fat, Y = weight of empty flask, and W = weight of sample.

3.3.5

Apparent Amylose Content (Juliano, 1971)

Sample (12% MC) of 0.1 g was weighed into 100 ml volumetric flask in duplicate. Then 1 ml of 95% ethanol and 9 ml of 1 M NaOH were added. The flasks were then boiled in waterbath for 10 min to gelatinize the sample. After cooling, distilled water was added to make the volume exactly 100 ml. After mixing, sample were kept standing overnight at room temperature. Blank solution was prepared following the previous steps except taking sample to the volumetric flask. After 24 hrs, flask was thoroughly mixed and 5 ml of sample solution was then transferred to an empty 100 ml volumetric flask. About 70 ml distilled water then added, followed by 1 ml of glacial acetic acid and 2 ml of iodine solution. The volume was adjusted to exactly 100 ml with distilled water. After mixing, let it be kept standing for 20 min to develop dark purple color. The absorbance was measured at 620 nm after setting zero with the blank solution. The value of the absorbance was calculated into the apparent amylose content using standard calibration curve developed from potato amylose standard.

3.3.6

In Vitro

Starch Digestibility (Megazyme International Ireland Inc., 2008)

The samples were incubated in a shaking water bath with pancreatic α-amylase and amyloglucosidase for 16 h at 37 o_{C to hydrolyzed digestible starch to glucose. The reaction was} terminated with 4 ml ethanol and the indigested resistant starch (RS) was recovered by centrifugation

15 (5000g, 10 min). The supernatant was then decanted and washed with 50% ethanol twice to remove the digested starch. The sediment was solubillized in 2 ml of 2 M KOH in an ice bath, neutralized with 8 ml sodium acetate (1.2 M) and the RS was hydrolyzed to glucose with of amyloglucosidase (0.1 ml, 3300 U/ml) for 20 min. The glucose oxidase / peroxidase reaction was used to measure glucose released from the digested and resistant starches. Absorbance was read at 510 nm after a 20 min incubation period at 50 oC. Starch fractions were calculated as follows:

Resistant starch = ∆E x F x 10.3/0.1 x 1/1000 x 100/W x 162/180

= ∆E x F/W x 9.27

Non-resistant starch = ∆E x F x 100/0.1 x 1/1000 x 100/W x 162/180 = ∆E x F/W x 90

Total starch = Resistant starch + Non-resistant starch Where,

∆E = absorbance (reaction) read against the reagent blank

F = conversion from absorbance to microgram (the absorbance obtained for 100 μg of D-glucose in the GOPOD reaction is determined, and F = 100 (μg of D-glucose) divided by the GOPOD absorbance for this 100 μg of D-glucose.

100/0.1 = volume correction (0.1 ml taken from 100 ml) 1/1000 = conversion from micrograms to milligrams W = dry weight of sample analyzed

= “as is” weight x [(100-moisture content)/100]

100/W = factor to present RS as a percentage of sample weight

162/180 = factor to convert from free D-glucose, as determined, to anhydro-D-glucose as occurs in starch

10.3/0.1 = volume correction (0.1 ml taken from 10.3 ml) for samples containing 0-10 % RS where the incubation solution is not diluted and the final volume is ~ 10.3 ml

3.3.7

Granular Morphology

Samples were magnified from 500 to 5000 X under SEM. Detector SE1 was used with acceleration potential of 5.00 kV during micrography using SEM.

3.3.8

Thermal Properties

Samples of approximately 3 gram were weighed into aluminium pans followed by addition of 6 μl of water. The pans containing mixtures were then hermetically sealed to prevent moisture loss and equilibrated at ambient temperature for 2 h. Samples which have been equilibrated were heated from 10 o_{C to 120} o_{C at a rate of 10} o_{C/min using differential scaning calorimeter (DSC). The onset} temperature (To), peak temperature (Tp), conclusion temperature (Tc), the gelatinization enthalpy

change (ΔH) were also calculated and expressed as joule per gram sample (J/g).

3.3.9

Pasting Properties

Paste viscosity was determined by using rapid visco analyzer (RVA). A programmed heating and cooling cycle was used where the samples were held at 50 o_{C for 1 min, heated to 95} o_{C at 6} o_{C/min, and held at 95} o_{C for 2.7 min, prior to cooling from 95 to 50} o_{C at 6} o_{C/min and holding at 50}

16 o_{C for 2 min. Parameters recorded were pasting temperature, peak viscosity, final viscosity (viscosity} at 50 o_{C), breakdown viscosity (peak-trough viscosity), and setback viscosity (final-trough viscosity).}

3.3.10

Textural Properties

Textural properties were analyzed by using texture analyzer (TA). Sample was sliced to smaller size (6x6x1) cm. Texture profile analysis was conducted under condition of 100 mm/min pretest speed, 50 mm/min test speed, 100 mm/min post test speed, 20% strain, P/36R probe, with weight calibration as follows; return distance of 30 mm and return speed of 10 mm/sec.

3.3.11

Color Properties

The ColorQuest (XE HunterLab, Hunter Associates Laboratory Inc., Virginia-USA) based on CIE system (L*, a*, b*) was used to investigate the color of rice cake and pea cake. The instrument was calibrated each time before its use using area view of 0.375 RSIN/RSEX, light tab reflectant and white tile reflectant.

3.3.12

Statistical Analysis

Results were expressed as mean of values ± standard deviation of independent determinations. Analysis of variance and comparison of means using Duncan`s test (p≤0.05) were performed using the statistical software SPSS 16.0 for windows, SPSS Inc., Chicago, USA.

17

IV. RESULT AND DISCUSSION

4.1

PRELIMINARY RESEARCH

4.1.1

Chemical Composition

Table 1 summarizes the analytical results for the chemical composition of pea flour, rice flour, and sticky rice flour involving moisture content, ash content, protein, and crude fat content. Pea flour, rice flour, and sticky rice flour possessed moisture content of 7.73 %, 11.30 %, and 9.77 %, respectively. The moisture content of pea flour was in range (7.7-9.1 %) with those reported by other authors (Naguleswaran and Vasanthan, 2010; Chung et al., 2008), whereas for rice flours ranged from 6.10-12.52 % (Dias et al., 2010; Tavares, 2010; Liu et al., 2006; Chun and Yoo, 2004) and moisture content of 10.05 % for sticky rice flour was reported by Zhu et al., 2010. The difference in moisture content among them was mainly due to the difference in manufacturing.

In term of ash content, pea flour (2.86 %) was the highest followed by rice flour (0.38 %) and sticky rice flour (0.17 %), respectively. Range of ash content for pea flours (2.24-3.73 %), rice flours (0.40-0.72 %), and sticky rice flour (0.16-0.29 %) were reported by Dias et al., (2010); Naguleswaran and Vasanthan (2010); Petitot et al. (2010); Singh et al. (2010); Sung et al. (2008); Maninder et al. (2007); Latha et al. (2002); Lumdubwong and Seib (2000).

Table 1. Chemical composition of raw materials

Sample Moisture (%) Ash (%) Protein (%) Crude fat (%)

Pea flour 7.68 2.83 21.84 0.80

Rice flour (RF) 11.21 0.39 7.21 0.41

Sticky rice flour (SRF) 10.37 0.18 6.12 0.29

The reported crude fat content of pea flours were 0.80-0.99 % (Naguleswaran and Vasanthan, 2010), which were near to the crude fat content determined in this study. However the crude fat content of rice flour (0.41 %) and sticky rice flour (0.29 %) were markedly lower than those reported by Dias et al. (2010) and Latha et al. (2002), for rice flour (0.87-0.90 %) and Sung et al. (2008), for sticky rice flour (0.45-0.94 %). The flours might be previously defatted (Maninder et al., 2007).

Protein content of 21.84 %, 7.20 %, and 6.12 % were observed during analysis for pea flour, rice flour, and sticky rice flour, respectively. Other results in protein content analysis by another authors were 21.4-26.8 % for pea flour, 6.93-8.11 % for rice flour, and 6.35-8.85 % for sticky rice flour (Naguleswaran and Vasanthan, 2010; Petitot et al., 2010; Tavares et al., 2010; Chung et al., 2008; Sung et al., 2008; Maninder et al., 2007; Chun and Yoo, 2004). Protein contents in legume grains range from 17% to 40 %, contrasting with 7-13 % of cereals, and being equal to the proteins contents of meats (18-25 %) (Genovese and Lajolo, 2001 in Costa et al., 2006). Hence, legume seeds including pea, are of prime importance in human nutrition due to their high protein content and are better known as a rich source of protein rather than rice (Singh, et al., 2004).

69 12.6 Non-resistant starch content of rice cake (90:10)

Univariate Analysis of Variance

Tests of Between-Subjects Effects

Dependent Variable:VAR00008

Source

Type III Sum of

Squares df Mean Square F Sig.

Corrected Model .049a 2 .025 43.603 .006

Intercept 59464.520 1 59464.520 1.056E8 .000

ricecake_90_non_resistant_s

tarch .049 2 .025 43.603 .006

Error .002 3 .001

Total 59464.571 6

Corrected Total _{.051 5}

a. R Squared = .967 (Adjusted R Squared = .945)

Post Hoc Tests

ricecake_90_non_resistant_starch

Homogeneous Subsets

VAR00008

Duncan

Sample N

Subset

1 2 3

24h, 4C 2 99.4418

6h, 4C _{2 99.5531}

6h, 25C _{2 99.6634}

Sig. _{1.000 1.000 1.000}

Means for groups in homogeneous subsets are displayed. Based on observed means.

70

Appendix 13. Result of textural properties analysis (experiment III)

Sample Cold setting Measurement Hardness (g) Adhesiveness (g.sec) Springiness Cohesiveness Gumminess Chewiness

Pea cake

6h, 25C

1 269.43 -49.08 0.97 0.90 242.48 234.22

2 283.23 -52.08 0.97 0.90 255.42 246.96

3 262.65 -47.32 0.96 0.89 234.94 225.25

4 265.48 -53.27 0.96 0.90 237.72 228.67

5 258.92 -42.07 0.96 0.90 233.53 224.77

Mean 267.94 -48.76 0.96 0.90 240.82 231.97

SD 9.37 4.42 0.00 0.00 8.85 9.19

6h, 4C

1 510.61 -88.54 0.96 0.97 495.54 477.77

2 492.40 -84.28 0.96 0.93 456.10 437.58

3 572.68 -76.97 0.97 0.91 521.83 507.76

4 534.21 -71.62 0.94 0.92 490.17 460.28

5 498.65 -75.74 0.96 0.91 455.47 438.63

Mean 521.71 -79.43 0.96 0.93 483.82 464.40

SD 32.66 6.84 0.01 0.02 28.26 29.41

24h, 4C

1 893.65 -95.96 0.96 0.86 765.18 733.95

2 850.56 -87.12 0.96 0.88 744.55 715.41

3 911.11 -100.26 0.95 0.88 801.79 764.79

4 870.27 -96.66 0.96 0.88 769.35 739.76

5 730.20 -103.42 0.96 0.88 641.72 614.93

Mean 851.16 -96.68 0.96 0.87 744.52 713.77

71 Sample Cold setting Measurement Hardness (g) Adhesiveness (g.sec) Springiness Cohesiveness Gumminess Chewiness

Rice cake

6h, 25C

1 151.66 -49.33 0.94 0.84 127.68 120.37

2 148.48 -38.05 0.95 0.87 128.46 122.13

3 169.16 -51.73 0.94 0.89 149.92 141.32

4 148.03 -72.54 0.92 0.80 118.98 108.88

5 147.12 -57.62 0.90 0.78 114.81 103.22

Mean 152.89 -53.85 0.93 0.84 127.97 119.18

SD 9.25 12.63 0.02 0.04 13.57 14.67

6h, 4C

1 _{207.74 -80.29 0.94 0.92 190.98 178.72}

2 183.49 -74.91 0.94 0.88 161.11 151.17

3 179.18 -90.77 0.88 0.88 158.12 138.43

4 _{208.81 -76.36 0.92 0.90 188.84 173.11}

5 _{209.68 -92.29 0.93 0.91 191.26 177.05}

Mean 197.78 -82.92 0.92 0.90 178.06 163.70

SD 15.10 8.12 0.03 0.02 16.90 17.94

24h, 4C

1 268.08 -97.24 0.94 0.93 249.10 234.12

2 265.17 -103.54 0.94 0.93 247.63 231.52

3 277.88 -96.62 0.94 0.93 257.72 241.44

4 284.88 -111.69 0.92 0.94 267.46 245.08

5 250.73 -92.72 0.91 0.93 232.34 211.02

Mean 269.35 -100.36 0.93 0.93 250.85 232.64

72 Sample Cold setting Measurement Hardness (g) Adhesiveness (g.sec) Springiness Cohesiveness Gumminess Chewiness

*Rice cake (90:10)

6h, 25C

1 70.63 -99.58 0.82 0.89 62.59 51.15

2 75.06 -74.93 0.82 0.86 64.24 52.94

3 62.33 -86.70 0.80 0.85 53.10 42.63

4 70.75 -96.38 0.83 0.89 62.86 51.94

5 69.86 -71.55 0.84 0.86 59.73 50.26

Mean 69.73 -85.83 0.82 0.87 60.50 49.79

SD 4.61 12.49 0.01 0.02 4.45 4.12

6h, 4C

1 87.49 -95.64 0.84 0.85 74.35 62.54

2 67.23 -106.87 0.77 0.80 53.77 41.29

3 90.07 -92.66 0.89 0.53 47.97 42.80

4 72.52 -78.75 0.88 0.56 40.87 35.75

5 75.83 -76.54 0.88 0.54 40.54 35.52

Mean 78.63 -90.09 0.85 0.66 51.50 43.58

SD 9.80 12.56 0.05 0.16 13.90 11.09

24h, 4C

1 129.94 -110.72 0.88 0.87 113.38 99.51

2 109.58 -98.09 0.89 0.83 90.63 80.95

3 120.14 -106.48 0.89 0.84 100.92 89.34

4 134.04 -112.31 0.89 0.85 114.11 101.51

5 115.83 -143.78 0.84 0.88 101.34 84.79

Mean 121.91 -114.28 0.88 0.85 104.08 91.22

SD 10.05 17.39 0.02 0.02 9.82 9.02

73

Appendix 14. Result of color properties analysis (experiment III)

Sample Cold setting Measurement L a b

Pea cake

6h 25C

1 64.26 -1.45 13.36

2 64.81 -1.45 13.40

3 64.37 -1.23 13.54

4 64.21 -1.27 13.46

5 64.39 -1.25 14.10

Mean 64.41 -1.33 13.57

SD 0.24 0.11 0.30

6h 4C

1 65.57 -1.49 14.91

2 65.67 -1.12 15.59

3 65.35 -1.50 14.43

4 65.68 -1.53 14.57

5 65.39 -1.25 15.73

Mean 65.53 -1.38 15.05

SD 0.15 0.18 0.59

24h 4C

1 61.19 -1.08 10.29

2 61.20 -1.11 10.29

3 61.24 -1.08 10.24

4 61.28 -1.09 10.21

5 61.20 -1.09 10.34

Mean 61.22 -1.09 10.27

SD 0.04 0.01 0.05

Sample Cold setting Measurement L a b

Rice cake

6h 25C

1 57.43 -2.97 2.69

2 57.39 -2.96 2.57

3 57.44 -3.00 2.65

4 57.44 -2.99 2.72

5 57.42 -2.99 2.77

Mean 57.42 -2.98 2.68

SD 0.02 0.02 0.08

6h 4C

1 58.94 -3.25 4.20

2 58.56 -3.24 4.21

3 59.14 -3.29 4.21

4 59.54 -3.43 4.45

5 57.98 -3.33 4.16

Mean 58.83 -3.31 4.25

SD 0.59 0.08 0.12

24h 4C

1 57.68 -3.42 3.83

2 56.88 -3.35 3.90

3 57.46 -3.27 4.00

4 56.69 -3.22 3.99

5 57.64 -3.27 3.89

Mean 57.27 -3.31 3.92

74

Sample Cold setting Measurement L a b

*Rice cake (90:10)

6h 25C

1 56.29 -3.08 2.20

2 56.18 -3.04 2.15

3 56.24 -3.02 2.08

4 56.14 -3.05 2.42

5 55.68 -3.02 2.36

Mean 56.11 -3.04 2.24

SD 0.24 0.02 0.14

6h 4C

1 56.78 -3.22 3.46

2 56.91 -3.15 3.59

3 56.77 -3.24 3.61

4 56.25 -3.22 3.43

5 56.33 -3.17 3.43

Mean 56.61 -3.20 3.50

SD 0.30 0.04 0.09

24h 4C

1 55.61 -3.22 3.56

2 58.10 -3.07 3.93

3 56.56 -3.08 3.63

4 54.74 -3.06 3.45

5 58.31 -3.11 3.59

Mean 56.66 -3.11 3.63

SD 1.55 0.07 0.18