Improvement of Green Grass (Premna oblongifolia Merr.) Jelly’s Production Process and Its Effects on Physical and Functional Properties

IMPROVEME T

PRODUCTIO

FACULTY OF AG

BO

E T OF GREE GRASS (

TIO PROCESS A D ITS EFFECTS O PHY

FU CTIO AL PROPERTIES

BACHELOR THESIS

A DIKA BAGUS BA GU PRAKOSO

F24080065

OF AGRICULTURAL TECH OLOGY A D

BOGOR AGRICULTURAL U IVERSITY

BOGOR

2013

1

) JELLY’S

PHYSICAL A D

SO

D E GI EERI G

SITY

i

IMPROVEME T OF GREE GRASS (

) JELLY’S

PRODUCTIO PROCESS A D ITS EFFECTS O PHYSICAL A D

FU CTIO AL PROPERTIES

Andika Bagus Bangun Prakoso, Endang Prangdimurti, Faleh Setia Budi

Department of Food Science and Technology, Faculty of Agricultural Technology, Bogor Agricultural University, IPB Darmaga Campus, PO BOX 220, Bogor, West Java, Indonesia.

Phone: +6285780258659, e/mail: andika.prakoso@gmail.com

Green grass jelly, a traditional food from Indonesia, has good health effects. Unfortunately, green grass jelly is perishable since it shall have syneresis and contain high amount of microbes. The objective of this research was to find out the effects of steaming and addition of aHCO3 and

hydrocolloid on its physical, organoleptic, and health functional quality. Steaming could reduce the number of microbes contaminated green grass jelly. However, it could reduce the gel strength, increase syneresis rate, and change its colour to be brownish. The addition of aHCO3 was expected

to prevent the formation of brownish colour due to inhibition of chlorophyll degradation. The addition of carrageenan as hydrocolloid was also expected to reduce syneresis rate and improve gel strength due to steaming. aHCO3 concentrations used were 0%, 0.125%, and 0.583%. The chosen aHCO3

concentration was 0.125% based on the parameters of taste, colour, syneresis, and pH of green grass jelly. Carrageenan concentration of 2.00%, 2.25%, and 2.50% were selected based on the parameters of gel strength, colour, syneresis rate, and pH changes. Furthermore, concentration of 2.00% was selected as the best carrageenan concentration based on its sensory properties. Instead of the unsteamed selected formula, the steamed selected formula had lower total chlorophyll, higher total phenol, higher antioxidant capacity, and lower dietary fiber. However, this product was more safe from the microbiological aspects.

ii Andika Bagus Bangun Prakoso. F24080065. Improvement of Green Grass (

Merr.) Jelly’s Production Process and Its Effects on Physical and Functional Properties.

Supervised by Endang Prangdimurti and Faleh Setia Budi. 2013.

SUMMARY

Green grass jelly known to have functional efficacies of good health. Unfortunately, production process of green grass jelly is still relatively simple and it can be easily damaged if stored longer because it shall have syneresis and contaminated by high amounts of microbes. The objective of this study was to determine the effects of steaming and addition of NaHCO3 and hydrocolloid on

physical, organoleptic and health functional properties of green grass jelly. Steaming can reduce the amount of microbial contamination on green grass jelly. However, steaming also has negative effects, i.e. decrease gel strength, increase level of syneresis, and changing the color of green to be brownish. The addition of NaHCO3 was expected to prevent the formation of brownish color. Hydrocolloid was

also expected to reduce the level of syneresis and improve gel strength after steaming.

Productions of green grass jelly in the conventional way use the ratio of green grass jelly leaves with water at 1:15. NaHCO3 concentration was determined based on the pH need to reduce the

degradation of chlorophyll, i.e. pH of 7 and pH of 8. NaHCO3 concentrations used in the formulation

were 0% (as control), 0.125% (to reach pH about 7), and 0.583% (to reach pH about 8). Based on the analysis of taste, NaHCO3 concentration of 0.125% was chosen because it is tasteless. Based on the

analysis of color, NaHCO3 concentration of 0.125% has the most negative a value, meaning that the

gel has greenest color. Based on the rate of syneresis, NaHCO3 concentration of 0.125% has the lowest

rate of syneresis. In addition, the change in pH that occurred is not too volatile. NaHCO3 concentration

used was 0.125%. The optimum gelling time was 2.5 hours since it has the highest viscosity value. Alginate could not be used because it does not form a gel when mixed with the green grass extract, with or without CaCO3. LMP and mixture of alginate and LMP could not be used because it

could not form a gel when mixed with green grass extract without the use of CaCO3. The addition of

CaCO3 resulted in lighter green color. Hydrocolloid that can be used is the mixture of kappa and iota

carrageenan in the ratio of 1:1. By determination the variance of 0.25%, the concentrations of carrageenan which could form a good gel structure were 1.25% to 3.00%. Based on the analysis of texture, the carrageenan concentration which gave insignificantly different texture from commercial green grass jelly was 2.00%. Moreover, it has not too volatile pH changes. On the syneresis parameter, addition of 2.50% carrageenan was able to produce the lowest syneresis rate compared to other concentrations. Based on the analysis of color, carrageenan concentrations that produced insignificantly different color with the color of commercial green grass jelly was 2.00%. In addition, the concentration of 2.25% was also considered to be close to the color of green grass jelly commercial. It is also supported with a good rupture strength value of green grass jelly with the addition of 2.25% carrageenan.

Sensory analysis showed the best carrageenan concentration was 2.00%. Steaming could reduce the total chlorophyll contained in both of commercial green grass jelly and green grass jelly with the addition of 2.00% carrageenan by 70.81% and 22.45%. Steaming will lead to chlorophyll degradation reaction to form a brownish pheophytin. Steaming treatment on commercial green grass jelly could reduce 54.86% of the total phenol. On the other hand, the steamed green grass jelly with 2.00% carrageenan has increasing total phenol by 15.35%. The antioxidant capacity of green grass jelly with addition of 2.00% carrageenan and without steaming is higher than the commercial one without steaming treatment On the commercial green grass jelly, steaming treatment can reduce total dietary fiber of 9.30%, decline the soluble fiber of 1.61%, and decrease insoluble fiber of 27.80%. On the other hand, treatment of steaming green grass jelly with the addition of carrageenan 2% could reduce total dietary fiber of 11.06%, decrease 18.67% soluble fiber, and insoluble fiber increased by 8.00%. This steaming treatments could decrease S. aureus contained as much as 1.76 log CFU/ml.

iii

IMPROVEME T OF GREE GRASS (

) JELLY’S

PRODUCTIO PROCESS A D ITS EFFECTS O PHYSICAL A D

FU CTIO AL PROPERTIES

BACHELOR THESIS

On the partial fulfilment of the degree requirements of

BACHELOR OF AGRICULTURAL TECH OLOGY

At the Department of Food Science and Technology

Faculty of Agricultural Technology

Bogor Agricultural University

By

A DIKA BAGUS BA GU PRAKOSO

F24080065

FACULTY OF AGRICULTURAL TECH OLOGY A D E GI EERI G

BOGOR AGRICULTURAL U IVERSITY

BOGOR

2013

iv

Thesis Title : Improvement of Green Grass (Premna oblongifolia Merr.) Jelly’s Production Process and Its Effects on Physical and Functional Properties

ame : Andika Bagus Bangun Prakoso

Student o. : F24080065

Approved,

Acknowledged,

Head of Department of Food Science and Technology

(Dr. Ir. Feri Kusnandar, M.Sc) NIP 19680526 199303.1.004

Date of Comprehensive Test: February 12th 2013 Academic Supervisor I,

(Dr. Ir. Endang Prangdimurti, M.Si.)

NIP. 19680723 199203 2 001

Academic Supervisor II,

(Faleh Setia Budi, ST. MT.)

v

STATEME T OF THESIS A D I FORMATIO SOURCE

Hereby, I genuinely stated that the bachelor thesis entitled Improvement of Green Grass ( Merr.) Jelly’s Production Process and Its Effects on Physical and Functional Properties is an authentic work of mine under supervision of academic advisors and never being presented in any forms and university. All the informations taken and quoted from published or unpublished works of other writers had been mentioned in the texts and attached in the references at the end of the bachelor thesis.

Bogor, Februari 2013 The undersigned,

Andika Bagus Bangun Prakoso F24080065

vi © Created right owned by Andika Bagus Bangun Prakoso, 2013

All rights reserved

This thesis may not be translated or copied in whole or in part without written permission from Bogor Agricultural University in any forms or by any forms, print, photocopy, microfilm, etc.

ii

AUTHOR BIOGRAPHY

Andika Bagus Bangun Prakoso was born on Depok, June 22nd 1990 from the couple of Andarwanto and Diah Sulistiowati. He is the older brother with one younger sister. He studied in Public Elementary School of Anyelir I (1996/2002), Public Junior High School II Depok (2002/2005), Public Senior High School I Depok (2005/2008). In 2008, the author continued his education at Department of Food Science and Technology, Faculty of Agricultural Technology, Bogor Agricultural University with Admission Invitation of Bogor Agricultural University path. In 2011, the author joined Asean International Mobility for Students (AIMS) Program to University Putra Malaysia in order to take additional special courses. During his study in college, he joined organizations and extracurricular activities, i.e. Student Choir of Agria Swara; Campus Newspaper as reporter and editor; Department of Student Talent, Student Executive Board of Faculty of Agricultural Technology (BEM FATETA IPB); Red’s Voice as Announcer; Student Association of Food Science and Technology (HIMITEPA IPB); Theater Xantan of Faculty of Agricultural Technology; Telisik Pangan HIMITEPA IPB; and Indonesian Student Association of University Putra Malaysia (PPI UPM). He was active as committee of Graduation Ceremony of Faculty of Agricultural Technology (2009), OPEN HOUSE 46 (2009), MPKMB 46 (2009), Suksesi HIMITEPA IPB (2009), HACCP Seminar and Training (2010), RED’S CARPET (2010), Faculty Students Gathering (2010), FATETA Goes to OMI and IAC (2010), RED’S CUP (2010), SEREAL (2010), TECHNO F (2010), Inauguration Concert of Agria Swara (2010), LOFE (2010), FALCON (2010), Indonesian Students Gathering of PPI UPM (2011), Badminton Cup of PPI UPM (2011), Festival Corat/coret Batik (2012), and Year Book of FST 45 (2012). He has work experiences as private tutor (2008/2010), tutor of MSC College (2010), Lab Assistant of Food Analysis (2011), Internship Editor of GreenTV IPB (2012), and Lab Assistant of Sensory Evaluation (2012). The author obtained scholarships of Academic Achievements Improvement (PPA) and special scholarship to join AIMS Program 2011. He also joined International Conference “Future of Food Factors” and International Student Conference “The Power of Local Knowledge in Increasing Food Business Competitiveness” as presenter in 2012. Improving In March 2012, he started to conduct his final year project entitled “Improvement of Green Grass (Premna oblongifolia Merr.) Jelly’s Production Process and Its Effects on Physical and Functional Properties”.

iii

PREFACE

Praise and gratitude to Allah, God Almighty, for all His grace this thesis was completed. The selected theme in this research conducted from April 2012 was green grass jelly, entitled “Improvement of Green Grass (Premna oblongifolia Merr.) Jelly’s Production Process and Its Effects on Physical and Functional Properties”. Acknowledgements go to all who have helped me in the completion of this thesis:

1. My beloved family: Dad Ir. Andarwanto, Mom Diah Sulistiowati, and friend of fighting at home Sis Anita Retno Rahmasari. Thanks for your love, patience, support, and pray.

2. Dr. Ir. Endang Prangdimurti, M.Si. and Faleh Setia Budi, ST. MT. as my beloved research supervisors. Thanks for the advice, guidance, attention, motivation, and evaluation have been given. May Allah repay your kindness.

3. Dian Herawati, S.TP. M.Si as my comprehensive examiner. Thanks for having the time, advice, and evaluations that have been given.

4. Assoc. Prof. Abdul Azis Arifin, Assoc. Prof. Yaya Sukardi, and Dr. Nordin Mohd. Som as lecturers who always guide and provide motivation during my study period at University Putra Malaysia.

5. Family in the one guidance, Sagita Nindyasari and Mohammad Iqbal Bijaksana.

6. My entire beloved friends in FST 45, especially Ranti, Bangun, Arum PP, Hilda, Hafiz, Fya, Yunita, and Sarinah, who have helped me to conduct this research.

7. My entire beloved junior in FST 46, especially Afi, Idong, Farah, Yanda, and Anan, who always supported me to finish this thesis.

8. My entire beloved friends in FST UPM, especially Wana, Nisa, Balqis, Wan, Uzai,. Harits, Erin, Faisal, Yana, and Izni. Thanks for happiness in Malaysia.

9. Big family of PPI UPM and MIT UPM 2011, especially Bu Imas, Teguh, Eldo, Vivi, Amitha, Ihsan, Chaeru, Yuan, Ami, Indah, Sari, and Maya.

10. Thanks for entire lab technicians and UPT administration staff.

Finally, the author hopes that this paper can be useful and contribute to the development of food science and technology. Thank you.

Bogor, January 2013

iv

TABLE OF CO TE TS

Page

PREFACE ... iii

TABLE OF CONTENTS ... iv

LIST OF TABLES ... vi

LIST OF FIGURES ... vii

LIST OF APPENDIX ... viii

I. INTRODUCTION ... 1

A. BACKGROUND ... 1

B. RESEARCH OBJECTIVE ... 2

II. LITERATURE REVIEW ... 3

A. GREEN GRASS PLANT ... 3

B. EFFICACIES OF GREEN GRASS PLANT ... 4

C. GREEN GRASS JELLY ... 5

D. HYDROCOLLOIDS ... 6

1. Alginate ... 6

2. Pectin ... 7

3. Carrageenan ... 8

E. ANTIOXIDANTS ... 10

F. CHLOROPHYLL ... 11

G. DIETARY FIBER ... 13

H. THERMAL PROCESS ... 14

III. RESEARCH METHODOLOGY ... 15

A. MATERIALS AND TOOLS ... 15

1. Production Materials ... 15

2. Chemicals ... 15

3. Tools ... 15

B. RESEARCH METHOD ... 15

1. DETERMINATION OF GELLING TIME ... 16

2. DETERMINATION OF NaHCO3 CONCENTRATION ... 17

3. DETERMINATION OF HYDROCOLLOID TYPE AND CaCO3 CONCENTRATION ... 19

v 4. DETERMINATION OF SELECTED HYDROCOLLOID CONCENTRATION WITH

STEAMING ... 20

5. SENSORY EVALUATION ... 22

6. FUNCTIONAL PROPERTIES ANALYSIS ... 22

7. MICROBIOLOGICAL ANALYSIS ... 25

8. DESIGN OF EXPERIMENT ... 26

III. RESULTS AND DISCUSSIONS ... 27

A. FIELD OBSERVATION OF COMMERCIAL GREEN GRASS JELLY PRODUCTION PROCESS ... 27

B. DETERMINATION OF GELLING TIME ... 27

C. DETERMINATION OF NaHCO3 CONCENTRATION ... 28

D. DETERMINATION OF HYDROCOLLOID TYPE AND CaCO3 CONCENTRATION ... 32

E. DETERMINATION OF SELECTED HYDROCOLLOID CONCENTRATION WITH STEAMING ... 35

F. SENSORY EVALUATION ... 40

H. FUNCTIONAL PROPERTIES ANALYSIS ... 42

I. MICROBIOLOGICAL ANALYSIS ... 48

IV. CONCLUSION AND RECOMMENDATION ... 49

A. CONCLUSION ... 49

B. RECOMMENDATION ... 49

REFERENCES ... 50

vi

LIST OF TABLES

Page

Table 1. Botanical classification of shrub green grass plant ... 3

Table 2. Green grass leaves’ composition... 4

Table 3. Studies of green grass leaves ... 5

Table 4. Results of green grass jelly’s color measurement ... 30

Table 5. Results of several hydrocolloid types and CaCO3 addition ... 33

Table 6. Texture analysis results of carrageenan addition treatments ... 35

Table 7. Results of color analysis for carrageenan addition ... 37

Table 8. Syneresis rate equation of green grass jelly with carrageenan addition ... 39

Table 9. Results of hedonic rating test ... 41

Table 10. The analysis results of total chlorophyll, total phenols, and antioxidant capacity ... 42

Table 11. The analysis result of dietary fiber and changes percentage in the treatments of without and with steaming ... 46

vii

LIST OF FIGURES

Page

Figure 1. General structure of pectin ... 6

Figure 2. General structure of alginate ... 6

Figure 3. Gelation mechanisms of alginate (a) Diffusion setting and (b) Internal setting ... 7

Figure 4. Schematic diagram of egg/box model ... 8

Figure 5. Molecule structure of kappa(a), iota(b), and lambda(c) carrageenans ... 9

Figure 6. Gelation mechanism of kappa and iota carrageenans ... 10

Figure 7. Chemical structure of chlorophyll ... 11

Figure 8. Chlorophyll degradation to be its derivatives ... 12

Figure 9. Research flow chart ... 16

Figure 10. Procedure of gelling time determination ... 17

Figure 11. Procedure of NaHCO3 concentration determination ... 18

Figure 12. Procedure of hydrocolloid type and CaCO3 concentration determination ... 20

Figure 13. Procedure of selected hydrocolloid concentration determination ... 21

Figure 14. The general curve obtained from the Stevens LFRA Texture Analyzer ... 21

Figure 15. Flow chart of conventional green grass jelly production process ... 27

Figure 16. Graph of viscosity of green grass jelly without addition of NaHCO3 ... 28

Figure 17. Syneresis graphs of NaHCO3 addition treatments ... 31

Figure 18. Graphs of pH changes by NaHCO3 addition treatments ... 32

Figure 19. Syneresis curve of green grass jelly with the addition of carrageenan ... 39

Figure 20. Graphs of pH changes by carrageenan addition treatments ... 40

Figure 21. Conversion of 10/hydroxychlorophyll to 10/methoxylactone of chlorophyll ... 43

viii

LIST OF APPE DIX

Page Appendix 1. Documentation of the conventional production process of commercial green grass

jelly ... 60

Appendix 2. Viscosity measurement result of green grass jelly without addition of NaHCO3 for determination of gelling time ... 61

Appendix 3a. The effects of NaHCO3 addition to the color of green grass jelly ... 63

Appendix 3b. Results of NaHCO3 addition effects on the color of green grass jelly before and after steaming ... 64

Appendix 4. Analysis of variance results of NaHCO3 addition to the color of green grass jelly before steaming ... 65

Appendix 5. Analysis of variance results of NaHCO3 addition to the color of green grass jelly after steaming ... 68

Appendix 6. T/test results of color measurement because of steaming treatment on each NaHCO3 concentration ... 71

Appendix 7. The syneresis measurement results of NaHCO3 addition treatments ... 74

Appendix 8. The results of pH measurement during 3 days by NaHCO3 additions ... 76

Appendix 9. The results of texture measurement by carrageenan additions ... 77

Appendix 10. Analysis of variance results of carrageenan additions to the texture of green grass jelly ... 79

Appendix 11. Results of color measurement by carrageenan additions ... 83

Appendix 12. Analysis of variance results of carrageenan additions to the color of green grass jelly ... 85

Appendix 13. Results of syneresis measurement of carrageenan additions ... 89

Appendix 14. pH changes measurement of green grass jelly by addition of carrageenan ... 91

Appendix 15. Results of sensory evaluation ... 93

Appendix 16. Analysis of variance results of carrageenan additions to the organoleptic preferences of green grass jelly ... 98

Appendix 17. Product of green grass jelly with addition of 2% carrageenan ... 101

Appendix 18. Chlorophyll analysis results ... 102

Appendix 19. Phenol analysis results ... 103

Appendix 20. Antioxidant capacity analysis results by DPPH method ... 105

Appendix 21. Dietary fiber analysis results ... 107

ix

Appendix 23. T/test results for total phenol analysis ... 112

Appendix 24. T/test results for antioxidant capacity analysis ... 113

Appendix 25. T/test results of dietary fiber measurement because of steaming treatment by carrageenan addition ... 114

Appendix 26. T/test results of dietary fiber measurement because of steaming treatment on commercial green grass jelly ... 115

Appendix 27. Results of moisture content analysis ... 116

Appendix 28. Results of ash content analysis ... 117

Appendix 29. Microbiological analysis result ... 118

1

I. I TRODUCTIO

A. BACKGROU D

Functional food is natural or processed food which contains one or more compounds based on scientific studies deemed to have specific physiological functions that are beneficial to health. One of the functional food that has been studied is green grass plant that contains both functional components and hydrocolloid gelling components. Based on the Food Category issued by Indonesian Drug and Food Control Agency (2006), green grass jelly is a product derived from the leaf extract of several types of plants, one of them is Premna oblongifolia Merr. This gel is formed in cool conditions and does not require another gelling agent. Green grass jelly consumed as drink or dessert.

Traditionally, this plant has been used as a febrifuge (antipyrectic), reducing the risk of gastroenteritis, nausea, and high blood pressure (Sunanto 1995). Some studies also mention the other benefits of green grass jelly, such as an anti/cancer (Ananta 2000), increasing the number of lymphocytes (Pandoyo 2000), decreasing the amount of free radicals (Handayani 2000), increasing the antioxidant capacity of lymphocytes (Koessitoresmi 2002), and not toxic for the body (Arisudana 2003; Nugrahenny 2003).

Consumption of green grass jelly is still limited because it is perishable. Green grass jelly stored too long will be contaminated by large amount of microbes. This is due to the lack of heating in the production process of green grass jelly since the gel will not be formed at the temperature of 80oC or more (Research Institute of Chemical Semarang 1976 in Ananta 2000). Pramitasari (2012) reported that steaming of green grass jelly at 100oC for 5 minutes could decline the total number of microbes on average of 1.07/1.10 log CFU/g, the amount of E. coli on average of 1.00/1.18 log CFU/g, and the number of Staphylococcus sp. on average of 0.29/0.71 log CFU/g. However, increasing temperature also incrases the syneresis and decreases its texture. This treatment could enhance the syneresis on average of 7.82/9.03% and reduce the gel strength on average of 1.97/2.99 g/cm2.

Addition of hydrocolloid could be a solution to solve this problem. Green grass jelly is expected to have a good texture despite having the heating process. Gelling hydrocolloid component of green grass jelly (Premna oblongifolia Merr.) itself has the same characteristics as other hydrocolloid, such as Low Methoxyl Pectin (LMP) and alginates. Calcium mineral is needed to form a gel, similar with alginates and iota/carrageenan. The gel mechanism of green grass jelly is an egg box model (Artha 2001). Source of calcium that can be used is CaCO3. As had been done previously by Camus

(2000), Setyaningtyas (2000), and Rustanti (2000) where the addition of hydrocolloid could reduce syneresis and improve gel strength of green grass jelly.

Heating will also lead to chlorophyll degradation reaction to form a brownish pheophytin (Gross 1991). Pramitasari (2012) reported that steaming at temperature of 100oC for 5 minutes could change the color of green green grass jelly to be brownish green because of heat and low pH. With the degradation of chlorophyll, the antioxidant capacity of green grass jelly is expected to decline due to the loss of metal at the center of the porphyrin ring on the chlorophyll structure (Ferruzzi et al. 2002). One solution that can be conducted to overcome the problem of chlorophyll degradation is addition of NaHCO3 that works to raise the pH so that the chlorophyll degradation reaction can not occur (Koca et

2

al. 2006). Functional properties of green grass jelly are also expected to decline due to steaming. Functional properties degradation of green grass jelly gel are not expected to occur significantly.

Thus, it is necessary to determine the formula of green grass jelly extract, NaHCO3,

hydrocolloid, and CaCO3 which are appropriate and able to be combined with the application of

steaming process that can provide the best effect in improving the quality and safety of the product microbiologically, giving a minimal effect on the physical and functional properties, and accepted by the panelists organoleptically.

B.

RESEARCH OBJECTIVE

The objective of this research was to find out the effects of steaming to decrease microbiological contamination, NaHCO3 addition to decline color changes because of steaming

treatment, hydrocolloids and CaCO3 addition to increase gel texture and decrease syneresis rate, and

observe its formulation on physical, organoleptic, and health functional quality of green grass jelly product.

3

II. LITERATURE REVIEW

A. GREE GRASS PLA T

Grass plant is a kind of plant originally from Indonesia and has other names in each region, such as Camcao and Kepleng (Java); Camcauh (Sunda); Daluman Kebo and Daluman Langis (Bali), Cao (Madura), and Cotok Balam (Bukit Tinggi). This plant spreads in West Java, Central Java, Sulawesi, Bali, Lombok, and Sumbawa. There are four types of grass plants known by Indonesian people, there are vines green grass, black grass, oil grass, and shrub or tree green grass. Koessitoresmi (2002) stated that there are two types of green grass jelly, vines green grass (Cyclea barbata L. Miers) and shrub or tree green grass (Premna oblongifolia Merr.). Botanical classification of shrub green grass plant (Premna oblingifolia Merr.) can be seen on Table 1.

Table 1. Botanical classification of shrub green grass plant (Premna oblongifolia Merr.) (Syamsulhidayat and Hutapea 1991)

Taxonomy Type

Kingdom Plantae

Division Spermatophyta

Subdivision Angiospermae

Class Dicotyledone

Ordo Lamiales

Family Verbenaceae

Genus Premna

Species Premna oblongifolia Merr.

Stem of this plant (Premna oblongifolia Merr.) does not spread or creep such Cyclea barbata

L. Miers, but shaped up. The leaves of this plant are oval (leaf length about 1.5 times width), oblong (long leaf support approximately 2.5 times the width), obovate (egg shape breech), leaf foot shaped round or heart (cordate), a bit rough, short taper leaf apex, not serrate leaf margins (flat) or slightly serrate (i.e. serrate the form of sharp gear and lead to the sharp gear of leaf end and dentate to the outside). Leaf with rather large bone has short fur and rare, some are not hairy. Leaf length is about 8.5 to 23 cm with a width about 3.5 to 10 cm. Petiole length is about 1.5 to 4 cm (Syamsuhidayat and Hutapea 1991).

4

B. EFFICACIES OF GREE GRASS PLA T

The main parts of the green grass plant regularly used are the leaves. The leaves are used as raw material in the production of traditional jelly that is custom to be mixed with fresh drinks. The chemical composition of green grass leaves reported by several researchers can be seen on Table 2.

Table 2. Green grass leaves’ composition (Premna oblongifolia Merr.)

Composition (% WB) Untoro (1985) Minawati (1985)

Protein 5.46 3.81

Water 81.00 82.62

Fat 0.94 1.11

Carbohydrate 11.94 10.48

Ash 0.66 1.98

Furthermore, green grass leaves (Premna oblongifolia Merr.) also contain 4.33% crude fiber WB (Untoro 1985) or 4.96% WB (Minawati 1985). Therefore, green grass leaves can be used as a source of food rich in fiber, but low in fat in the daily diet. Green grass leaves are used to make the gel that can also be used to treat a variety of diseases, i.e. gastric inflammation and high blood pressure (Sunanto 1995).

Several studies conducted on the efficacy, safety, and bioavailability of green grass leaves can be seen in Table 3. Several studies were also conducted in mice, such as lowering the amount of free radicals (Handayani 2000), not toxic for the body (Arisudana 2003), and anticancer activity (Pranoto 2003). Green grass leaves also contain β/carotene that can serve as a precursor of vitamin A and antioxidants (Jacobus 2003). Chalid (2003) tested the anti/cancer activity of green grass extracts (Premna oblongifolia Merr.) to C3H mice. The study showed that the addition of tumor volume of mice fed by green grass leaves extract (Premna oblongifoliaMerr.) is relatively lower than the comparator which was not fed by green grass leaves extract (Premna oblongifolia Merr.).

Pandoyo (2000) revealed that green grass is expected to have anticancer activity because of alkaloids composition. Kintzios and Barberaki (2004) stated that the alkaloid is a natural product derived from plants that have anticancer or antitumor properties. Most of the alkaloids are cytotoxic to inhibit the growth of some types of cancer and leukemia (Aryudhani 2011). In addition, the chlorophyll contained in the green grass leaves have antioxidant activity with protection mechanisms of linoleic acid oxidation and prevent the decomposition of hydroperoxide (Nurdin 2009; Marquez et al. 2005).

5 Table 3.Studies of green grass leaves

Aspect Research Result Reference

Safety

Increasing of lymphocyte cell proliferation on human peripheral blood cells in vitro

Pandoyo 2000

Reduction of Cytochrome P/420 content and increasing of Glutation S/Transferase content on mice Sprague Dawley

Nugrahenny 2003

Bioavailability Low bioavailability of carotenoid on mice Sprague Dawley

Wylma 2003

Low bioavailability of β/carotene content on mice Sprague Dawley

Jacobus 2003

Bioavailability of chlorophyll affected by vitamin A content on mice Sprague Dawley

Hendriyani 2003

Low bioavailability of flavonoid on mice Sprague Dawley

Raharjo 2004

Efficacy

Increasing of free radical production by macrofage on mice Balb/c

Handayani 2000

Reduction of allergy reaction on mice Balb/c Rachmini 2000 Increasing of human lymphocyte cell Koessitoresmi 2002 Antioxidant potention to increase life index of

lymphocyte cell on mice C3H

Setiawaty 2003

Reduction of proliferation on leukemia cancer K/ 562 and servix cancer Hela

Ananta 2000

Reduction of proliferation on breast cancer on mice C3H

Rochima 2012

Increasing of SOD content and reduction of catalase on mice C3H

Chalid 2003

Cancer Tissue Analysis

High HE score of liver tissue on mice C3H Widyanto 2010 Reduction of IHK (Caspace/3) and Increasing of

vascularization marker (CD31) tumor cell on mice C3H

Aryudhani 2011

C. GREE GRASS JELLY

Leaves of green grass plant used to make a kind of gel obtained from the crushed leaves mixed with water as solvent and would be thickened automatically (Sunanto 1995). Green grass jelly can be formed at room temperature, has green color because of its chlorophyll, opaque, and irreversible or the gel can not be form again after being destroyed.

Gelling component in green grass jelly is a compound of hydrocolloid. Artha (2001) had succeeded to analyze that component and stated that major component of green grass jelly is a gelling

6 polymers with low methoxyl pectin with D/galacturonic acid as the main chain with β/(1,4)/glycosidic bond and galactose as side chains. It can form a gel chemically with the help of divalent cations minerals. The general structure of pectin molecules can be seen in Figure 1.

Figure 1. General structure of pectin (Shukla 2011)

The suitable degree of acidity (pH) to produce green grass jelly with the optimum texture is about pH of 4/7 (Garnawati 1978 in Ananta 2000). According to Wyanto (2000), the best concentration to produce green grass jelly is 1:15 or 6.67% (w/v). The main deterioration of green grass jelly is syneresis, the run out of water from green grass jelly. Syneresis can occur due to the termination of the bond on the fibriller or because fibriler, which is originally located rather far apart to each other, form bonds between fibriler to be close so that the liquid squeezed out (Setyaningtyas 2000).

D. HYDROCOLLOIDS

Hydrocolloid is a water/soluble polymer capable of forming a colloidal solution and able to thicken or form a gel from its solution. There are several kinds of hydrocolloids used in this research, i.e. alginate, pectin, and carrageenan.

1. Alginate

Alginate is an unbranched binary copolymer group consisting of D/mannuronic acid residues (M) and binds β (1,4) with L/guluronic (G) on some compositions that do not have a repetition (Draget 2009). Alginate showed characteristics on affinity for polyvalent cations, especially Ca2+ ions. This character is similar with pectin. This is influenced by the residual α/L/guluronic on alginate chain. General structure of the alginate molecule can be seen in Figure 2.

Figure 2. General structure of alginate (Draget 2009)

Alginate gel has a thermo/irreversible property, which stable to heat. This means that the alginate gel can be heated without being melted. However, the heating process must be conducted at neutral pH in order to avoid degradation of the alginate structure because it is too acidic or too alkaline. This degradation occurs due to the increased rate of polymer breakdown reaction (Draget et al. 1988).

7 Alginate gelation phenomena can be divided into two mechanisms, i.e. diffusion setting and internal setting (Draget 2009). Diffusion setting is characterized by gelation occurred because cross linking ion (Ca2+) diffuses from the media into the alginate solution (Figure 3(a)). Internal setting characterized by Ca2+ ion released in a controlled manner from a particular source of calcium into alginate solution (Figure 3(b)). Gelation mechanism used in this study was internal setting by using the internal sources of calcium in the form of CaCO3 which can be done on a wide range of pH (Draget et al. 1991). By using this gelation

system, the use of CaCO3 in the form of powder is very beneficial because it can increase the

surface area of the molecule that can reduce the transition time and gel can be formed more quickly and uniformly. Wyanto (2000) reported that formula with the mixture of 3% green grass extract and 1.75% alginate could provide good gel and preferable organoleptically.

(a) (b)

Figure 3. Gelation mechanisms of alginate (a) Diffusion setting and (b) Internal setting (Draget 2009)

2.

Pectin

Pectin is a polimer of galacturonic acid with the chain shape of 1/4/α/D/galacturonan with methyl ester bind partially. There are also branch of L/arabinose and 1/4/β/D/galactan inside (Glicksmann 1984). Degree of esterification (DE) is a ratio of esterified galacturonic acid units with total of galacturonic acid in the molecules. Low Methoxyl Pectin (LMP) is type of pectin with degree of esterification below 50%, while High Methoxyl Pectin (HMP) is type of pectin with degree of esterification above 50%.

To form a gel, LMP needs calcium ions because its molecule has high reactivity with calcium ions (May 2009). Calcium capability to form insoluble complex is associated with free carboxyl group in the pectin chain. The lower degree of esterification leads to the greater reactivity of pectin chain to the calcium ions. The gel conformation follows the egg/box model, the gel structures are formed due to calcium mineral induction in association with monosaccharide started from axial part of α (1,4)/O/glycosidic bond from six carbon monosaccharide structure that follow the formation of C1 and 1C, especially on the

8 galacturonic and guluronic acid (Walter 1991). The picture of egg/box model is presented on Figure 4.

Figure 4. Schematic diagram of egg/box model (Grant 1973 in Shukla et al. 2011) Artha (2001) reported that only LMP and alginate could be able to interact synergistically to form a gel with hydrocolloid of green grass jelly. Rustanti (2000) stated that gel product contains of 3% green grass extract and 1.75% LMP has high rupture strength, rupture point, and rigidity. Mixture of green grass extract with LMP (high or low DE) could better prevent the syneresis on the refrigeration temperature. Camus (2000) stated that mixture of 2% green grass extract and 1.75% hydrocolloid mixture of LMP and alginate (1:1) could provide the best formula on the hedonic and texture characteristics.

3. Carrageenan

Carrageenan is a high molecular weight linear polysaccharide comprising repeating galactose units and 3,6/anhydrogalactose (3,6 AG), both sulfated and non/sulfated, joined by alternating α/(1,3) and β/(1,4) glycosidic links (Imeson 2009). The distinct carrageenan structures differ in 3,6/anhydrogalactose and ester sulfate content. Variations in these components influence hydration, gel strength and texture, melting and setting temperatures, syneresis and synergism. These differences are controlled and created by seaweed selection, processing, and blending of different extracts.

The ester sulfate and 3,6/anhydrogalactose content of carrageenan are approximately 25% and 34% respectively for kappa carrageenan and 32% and 30% respectively for iota carrageenan. Lambda carrageenan contains 35% ester sulfate with little or no 3,6/ anhydrogalactose content. Even furcellaran, which in the past has been rather misleadingly called ‘Danish agar’, contains 16–20% sulfate content. These high sulfate levels contrast with agar/agar which has very low sulfate content, always below 4.5% and typically it is from 1.5– 2.5%. Structure of kappa, iota, and lambda carrageenan are shown in Figure 5.

Figure 5. Molecul 2009) If kappa requires heating to rather dark in colo provides elastic ge clear in color and water and solution when the hot solu This process is rev cooling.

Campo successive steps; c cause aggregation for gelation to proc are all capable of i more hydrophilic commercial carrag carrageenan is cha network of three adjacent spiral ch lambda carrageena this ‘‘crosslinking” 6.

(a)

(c)

olecule structure of kappa (a), iota (b), and lambda (c) c

appa carrageenan put in cold water, it will swell to for ting to 70°C to dissolve (Pebrianata 2005). Gel formed by

color and has easily cracked texture (Fardiaz 1989). On gel, syneresis free, and reversible (Pebrianata 2005). Th and soft in texture (Fardiaz 1989). Iota carrageenan poss lution of sodium salt. The ability of kappa and iota carrage t solution is allowed to cool, due to group/containing 3,6 is reversible, which means gel will melt when heated and

po et al. (2009) stated that gelation of carrageenans invo teps; coil/to/helix transition upon cooling and depend on

ation between helices. The presence of suitable cation, is an to proceed. For kappa carrageenans, the alkali metal ions (L le of inducing gelation. Iota types gel best with calcium ion philic and form fewer junction zones than do kappa

carrageenans, kappa and iota are gel/forming carrageen is characterized only as a thickener agent. Kappa and iota three/dimensional double helices, resulting from the ‘‘ ral chains that contain sulphate groups oriented towards t ageenan, the 2/sulphate group is oriented towards the intern nking”. Gelation mechanism of kappa and iota carrageenan

9 (b)

a (c) carrageenans (Imeson

to form rough spread that ed by kappa carrageenan is . On the other hand, iota The gel formed is more possesses soluble in cold arrageenans gelation occurs 3,6/anhidro/D/galactose. and form a gel again after

s involves two separate and nd on subsequent cation to , is an absolute requirement ons (Li+, Na+, K+, Rb+, Cs+) um ions. Iota carrageenan is appa carrageenan. Among ageenans, whereas lambda d iota carrageenans form a he ‘‘crosslinking” of the ards their external part. In internal part, thus avoiding eenan is presented in Figure

Figure 6. Gelation In a hot cooled, they inter double helices are Gelation occurs be due to the presenc form a rather firm Miller and Whistle Carrageen antihyperlipidemic potential. Sun polysaccharides is factors. Wijesekar structural features, glycosidic branchi antioxidant activit efficiently and don

E. A TIOXIDA TS

Antioxidants are c processes or reactions that food products, antioxidants foods, oxidation attack su intentionally added to a foo According to Ko antioxidants, especially phe oxygen and reacts with ox functions to break down antioxidants, works by elim ions such as copper and iron

elation mechanism of kappa and iota carrageenans (Be Mille a hot solution, the polymer molecules are in a coiled sta intertwine in double/helical structures. As the solution es are believed to nest together with the aid of potassi urs because the linear molecules are unable to form conti resence of structural irregularities. The linear helical porti r firm three/dimensional, stable gel in the presence of the a

histler 1996).

rageenan has biological activities of antitumor, idemic, and anticoagulant activities (Campo et al. 2009). I

un et al. (2009) pointed out that antioxidant activi des is usually not a function of one single factor, but rat esekara et al. (2011) stated that their antioxidant activ atures, such as degree of sulfating, molecular weight, type ranching. Low molecular weight molecules with high sulfat activity (Sun et al. 2009). Low molecules may incorpora

d donate proton effectively compared to the high ones (Ngo

TS

are compounds in biological systems that protect against s that lead to excessive oxidation (Aryudhani 2011). Mea idants are compounds that can protect materials or food pro such as oxidative rancidity. These compounds can be a food product (Fardiaz 1996).

Koschhar (1993), antioxidants can be classified into 5 phenolic compounds that can stop free radical chain ox ith oxygen to eliminate oxygen in a closed system, 3) se down fatty hydroperoxide into a final product that is

y eliminating solvent oxygen, and 5) chelating agent (seque nd iron that catalyzes the oxidation of fat.

10 Miller and Whistler 1996) ed state. As the solution is ution is cooled further, the otassium or calcium ions. continuous double helices l portions then associate to f the appropriate cation (Be

mor, immunomodulatory, 09). It also has antioxidant activity of marine algae ut rather a combination of activity depends on their t, type of major sugar, and sulfate content had the best orporate to the cells more s (Ngo et al. 2011).

ainst the harmful effects of Meanwhile, in relation to od products, especially fatty can be naturally present or

nto 5 types, ie: 1) primary oxidation of fat, 2) trap secondary antioxidants, at is stable, 4) enzymatic sequestrant), chelates metal

One type of antiox compounds that have a sin Wanasundara (1992) state antioxidants, free radicals compounds are flavonoid dihydrocalcone, calcone, flavones, flavonols, flavil antioxidant (Harbone 1987) as an antioxidant, phenoli protection from oxidation (Pratt and Hudson 1990). 0.17% phenol wb (solvent acid at 3.85% wb (solvent carotenoids, and chlorophyl

Beside the phenol antioxidant activity, namely type of carotenoid that has is also a potential compoun components, such as pheno Jacobus (2003) reported leaves as much as 1%, 3% carotene in the liver of mi suggests that the content o bioavailability.

F. CHLOROPHYLL

Chlorophyll is the chlorophyll derived from means leaf. The structure of

Figu

antioxidants in foods are phenolic compounds. Phenolic com single aromatic ring containing one or more hydroxyl states that the phenolic compounds proved to be an icals anchoring, and chelating metal ions. The most import onoid compounds. The main types of flavonoids foun cone, flavan, catechins (flavan/3/ol), leukoanthocyanid

flavilium salts, anthocyanidins, and auron. Flavonoids 1987). Flavonoids can reduce fat oxidation and reduce hype nolic compounds play a role in the process of lymph ation caused by free radicals and stimulates cell prolifer 990). Pandoyo (2000) revealed that the leaves of green g lvent water) and 0.36% wb (solvent hexane), and contain s olvent water) and 0.49% wb (solvent hexane). Many leave rophyll (Winarno 1995).

henolic compounds, there are other compounds in plant tis namely carotenoids. From all of the known types of carote at has the greatest vitamin A activity (Boileau et al. 1999). I mpound that binds singlet oxygen in order to function as a phenolic and carotenoid, are shown to have antioxidant a that the content of β/carotene in the ration of mice

3%, and 5% are at 2.022%, 2.126% and 2.451%. Wh of mice fed by these rations for 10 days was 2.81%, 2.4 tent of β/carotene in green grass jelly leaves is high, bu

YLL

is the green pigment found in most plants, algae, and cy from the Greek language “chloros” which means green ture of chlorophyll is presented in Figure 7.

Figure 7. Chemical structure of chlorophyll (Nollet 2000)

11 compounds are chemical droxyl groups. Shahidi and an effective source of important group of phenolic found in plants such as cyanidin (flavan/3,4/diol), onoids are very effective e hyperlipidemia. Functions lymphocyte cell membrane roliferation of lymphocytes reen grass jelly containing tain similar alcaloids tropic leaves contain flavonoids,

lant tissues which also have carotenoids, β/carotene is a 999). In addition, β/carotene n as an antioxidant. Several dant activity (Supari 1996). containing green grass While the content of β/

2.42% and 2.34%. This but do not have a high

nd cyanobacteria. Name of reen, and “phyllon” which

12 Structurally, chlorophyll is a porphyrin that contains basic ring tetrapirol, four rings bind to Mg and a fifth isocyclic ring closes to the third pyrrole ring. On the fourth ring, substituents of propionic acid are esterified by hydrophobic phytol groups. When the groups are removed from the structure of the core, then chlorophyll turned into a derivative that is hydrophilic (Gross 1991). During heating, there will be release of organic acids in the leaves tissue which in turn causes the chlorophyll to form brownish pheophytin (Gross 1991).

Chlorophyll is green, because it absorbs strongly in the red and blue of the visible spectrum. Chlorophyll a is less polar and blue/green, while chlorophyll b is more polar and yellow/green (Gross 1991). In solution, both chlorophyll a and b are fluorescent. The important characteristics of chlorophyll are extreme instability, sensitive to light, heat, oxygen, chemical degradation reactions include pheophytinisation, chlorophylide formation reaction, and the oxidation reaction. Changes in the structure of chlorophyll to its derivatives can be seen in Figure 8.

Figure 8. Chlorophyll degradation to be its derivatives (Van Boekel 1999)

Koca et al. (2006) suggested several ways that can be done to maintain the structure of chlorophyll during heating, i.e. the pH control applications, the High Temperature Short Time, and the combination of the High Temperature Short Time by adjusting the pH. Another way that can be taken is the use of an alkaline compound, such as NaHCO3, hexametaphospate, disodium glutamate, NaOH,

and Mg(OH)2. These compounds are used to raise the pH so that they can maintain the structure of

chlorophyll. Decrease of chlorophyll degradation caused by the effect of salt on electrostatic protection. The addition of cations can neutralize the negative charge on the surface of fatty acids and protein in the chloroplast membranes thereby reducing the attractiveness of hydrogen ions to the surface of the membrane (Nakatani et al. 1979 in Von Elbe and Schwartz 1996).

Chlorophyll in plant tissue may act as an antioxidant. Endo et al. (1985) showed the ability to degrade chlorophyll and pheophytin hydroperoxide, i.e. by incubation of linoleic acid methyl hydroperoxide substrate. The results showed that the two compounds do not have the ability to degrade hydroperoxide with parameters of peroxide numbers and carbonyl numbers. The ability to scavenge free radicals 2,2/diphenyl/1/picrylhydrazyl (DPPH), indicating that chlorophyll has the ability to capture (scavenger) lipid radicals produced during the process of oil autooxidation so it can break the chain of oxidation. Some researches indicate that chlorophyll and its derivatives have the ability as an antioxidant and antimutagenic (Marquez et al. 2005, Ferruzzi et al. 2002). The ability of chlorophyll and some of its compounds are widely used as a food dye, eliminating the less savory aroma, antioxidants, and cancer (Hasegawa et al. 1995; Keller et al. 1996 and Tassetti et al. 1997).

13 Kusharto et al. (2008) stated that the green leaves of grass jelly has the highest chlorophyll content (1709 ppm), compared to the three other leaves, mulberry respectively 844 ppm, katuk 1509 ppm, and gotu kola 832 ppm. Hendriyani (2003) revealed that in the powdered green grass leaves, there are total chlorophyll of 3950 ppm or 3.95 mg/g consisting of 3.45 mg/g of chlorophyll a and 0.50 mg/g of chlorophyll b. The amount of chlorophyll antioxidant activity using DPPH method reported by Kristopo et al. (2006) referred in Prangdimurti (2007), using chlorophyll green membranes isolated from mung bean sprouts. It was reported that at concentrations of 5x10/5 M, antioxidant activity of chlorophyll a is 10.857 + 0.277% and chlorophyll b is 8.937 + 0.454%.

G. DIETARY FIBER

Dietary fiber is a group of polysaccharides and lignin contained in the food that can not be hydrolyzed by human digestive enzymes (Trowell et al. 1976). Based on its origin, dietary fiber can be divided into two types, i.e. dietary fiber derived from carbohydrates, such as cellulose, hemicellulose, pectin, and gum, and dietary fiber derived from non/carbohydrate, such as phenolic compounds (lignin) (Gallaher 1996). Based on the solubility, dietary fiber can be divided into two types, namely water/soluble dietary fiber (Soluble Dietary Fiber, SDF), such as pectin, hemicellulose portion, and gum, and water insoluble dietary fiber (Insoluble Dietary Fiber, IDF), such as lignin, cellulose, and some hemicelluloses (Muchtadi et al. 2006). In addition, dietary fiber also contain sugar, such as glucose, galactose, xylose, mannose, arabinose, rhamnose, and fructose, and also acidic sugars, such as manuronic, galacturonic, glucuronide, guluronic, and 4/O/metil glucuronic acid.

Dietary fiber plays a role in prevention of various diseases. SDF can prevent coronary heart disease and diabetes, while the IDF can prevent constipation, diverticulosis, hemorrhoids, appendicitis, stomach pain, colon cancer, and obesity (Muchtadi et al. 2006). Dietary fiber also affects the bioavailability of fat soluble vitamins. This is presumably because there is influence of dietary fiber on bile acids (bile salts), where the acid plays an important role in the digestion and absorption of fats, including fatty acids (Leville 1977).

Dietary fiber can not be digested by humans, which is concluded that dietary fiber does not contain nutritional value (Desminarti 2001). But the colon microflora can ferment dietary fiber and form short/chain fatty acids which can then be absorbed by the intestine to be used by the body as an energy source (Dreher 1987). Dietary fiber has high water absorption, because the polymer size, complex structure, and contains many hydroxyl groups. It depends on the type of its polysaccharide.

William (1985) stated that dietary fiber has binding properties of organic materials, such as bile acids, and then wasted with feces. With the presence of dietary fiber that binds bile acids, the amount of free bile acids is reduced so it needs a new bile acid formed. This new bile acid formed from cholesterol present in the blood. Thus, the concentration of cholesterol in the blood decreases. According to Shinnik et al. (1990), soluble fiber such as pectin and gum can lower plasma cholesterol levels significantly. Green grass jelly (Premna oblongifolia Merr.) composed of low methoxyl pectin which also classified into water soluble dietary fiber (SDF). Pectin of green grass jelly consists of D/ galacturonic acid with side chains in the form of galactose.

14

H. THERMAL PROCESS

Heating applied in food processing and preservation has the objective to eliminate or reduce microbes’ activity to grow, reproduce, and decompose nutritional components in food products. In addition, heating is also intended to obtain better aroma, texture, and appearance (Lewis 2006). There are several methods of food wet heating treatment, such as blanching, sterilization, and pasteurization.

Blanching is a cooking process wherein the food substance, usually a vegetable or fruit, is plunged into boiled water, removed after a brief, timed interval, and finally plunged into iced water or placed under cold running water (shocked) to halt the cooking process (Fellows 2000). Blanching is applied in the preparation process of commercial green grass jelly by its vendors. The objective of this process is to decrease initial amount of microbes, inactivate plant enzymes, and soften the tissue in order to ease hydrocolloid extraction.

Boiling is heating in boiling water at 100°C for a few minutes. In this method, heat/resistant bacterial spores can still be alive after boiling for several hours (Hariyadi 2000). Steaming is heating with hot steam at 100°C for a few minutes. Steaming works by boiling water continuously, causing it to vaporize into steam. The steam then carries heat to the nearby food, thus cooking the food. The food is kept separate from the boiling water but has direct contact with the steam, resulting in a moist texture to the food.

Sterilization is a condition that is obtained from food processing by using a high temperature in a period of time, so that no living microorganisms exist at normal storage temperatures. The products undergo sterilization may still contain spores of bacteria, but in dormant (not actively reproductive conditions), so its presence does not harm if the product is stored under normal conditions. Commercial sterilization standard used is 121°C for 15 minutes (Lewis 2006).

Pasteurization is a relatively mild heat treatment, in which food is heated to below 100oC. In low acid foods (pH > 4.5), it is used to minimize possible health hazards from pathogenic microorganisms and extend shelf life of foods for several days. In acidic foods (pH < 4.5), it is used to extend the shelf life for several months by destruction of spoilage microorganisms (yeast or mold) and enzyme inactivation. In both types of food, minimal changes are caused to the sensory characteristics or nutritive value (Fellows 2000).

Thermal treatment used in this research is pasteurization with steaming at 100oC for 5 minutes recommended by Pramitasari (2012). In previous research, boiling green grass jelly for 5 minutes resulted destruction of commercial green grass jelly structure and color change to be brownish green at the temperature which had not reached 100°C or about 70°C. Therefore, boiling is ineffective in improving the quality of green grass jelly. On the other hand, steaming of green grass jelly for 5 minutes resulted color change from green to be brownish green, but green grass jelly structure was not destroyed. Therefore, steaming is an effective thermal process.

Pramitasari (2012) reported that steaming green grass jelly at 100°C for 5 minutes could reduce the number of total microbes on average of 1.07 to 1.10 log CFU/g, the amount of E. coli on average of 1.00 to 1.18 log CFU/g, and the number of Staphylococcus sp. on average of 0.29 to 0.71 log CFU/g. Low decrease in microbial amounts was caused by lowest rate of heat penetration since the heating instrument was so simple, using conventional steamer pot. However, this steaming treatment can increase the gel syneresis on average of 7.82 – 9.03%. The gel strength of green grass jelly after steaming treatment decreased on average of 1.97 – 2.99 g/cm2.

15

II. RESEARCH METHODOLOGY

A. MATERIALS A D TOOLS

1. Production Materials

The materials used in the production of green grass jelly were green grass leaves (Premna oblongifolia Merr.) from Muara Empang Bogor, bottled water with the pH about 7.67, NaHCO3, sodium alginate, Low Methoxyl Pectin (LMP), and mixture of kappa and iota

carrageenan from Class Kimia Jakarta, and CaCO3 from Brataco.

2. Chemicals

The materials used in the analysis were phosphate buffer solution pH 4.00 and pH 7.00, methanol 85%, Folin/Ciocalteau reagent, sodium carbonate 5%, gallic acid, ascorbic acid, methanol 99.9%, DPPH reagent, acetone 99.8%, sodium phosphate, enzyme thermamyl Sigma A9972, HCl 4 N, enzyme pepsin 2844/01 (JT Baker), NaOH 4 N, pancreatin EC 232/ 468/9 (Merck), distillate water, ethanol 78 %, and ethanol 95%.

3. Tools

The tools used in this study were spatula, watch glass, analytical balance, basins, filter cloth, plastic stirrers, spoons, beaker glass, measuring cylinder, HDPE plastic cups, glass stirrers, HDPE plastic, conventional steamer pot, water bath, pH meter, LV Brookfield Viscometer, Stevens LFRA Texture Analyzer, Chromameter Minolta CR 200, vortex, test tube, cuvette, conical funnel, spectrophotometer, centrifuge, refrigerator, aluminum bowls, erlenmeyer flask, volumetric flask, volumetric pipette, micropipette, shaking incubator, drop pipette, agitators, filter paper Whatman No. 40, a filter with a vacuum pump, oven, electric furnaces, porcelain bowls, desiccator, small trays, small glasses, and small spoons.

B. RESEARCH METHOD

This study was conducted in several stages. The first study was conducted to determine the gelling time in order to standardize all of green grass jellies’ gelling time in this study by considering viscosity analysis. The next stage was determination of concentration NaHCO3

concentration used in the production of green grass jelly by considering physical properties. The next stage was determination of the hydrocolloid and CaCO3 concentration with steaming

treatment by considering physical and organoleptic properties. The last stage was the observation of green grass jelly’s chosen formula on the functional properties with the effects of steaming. The flow chart of this research can be seen on Figure 9.

16 Figure 9. Research flow chart

1. DETERMI ATIO OF GELLI G TIME

This stage was conducted to observe the optimum gelling time of green grass jelly by its viscosity. This was useful to standardize the gelling time for all treatments and formulations in this study, where the length of time would be determined during this stage took place. During the preparation, raw materials of green grass leaves selected based on the relatively similar size and rate of aging. First, leaf stems removed and cleaned by washing it with flow water. After washing, the leaves wiped with a tissue, wrapped in plastic bags, then stored in a refrigerator at a temperature of 10°C until use, not later than 6 days (Camus 2000)

To make green grass jelly, a comparison between green grass leaves and water used is at 1:15 or equal to 6.67% (w /v), in accordance with the best concentration reported by Wyanto (2000). Furthemore, this concentration is used by the commercial green grass jelly vendors. Green grass extract is made in accordance with the way taken from Rustanti (2000). First, leaves from preparation weighed, for instance to make 6.67% grass extract as much as 1000 ml, green grass leaves weighed as much as 66.7 grams. Volume of bottled drinking

Observation of chosen formula and commercial green grass jelly Determination of gelling time

Determination of NaHCO3 concentration

Determination of selected hydrocolloid concentration with steaming

Sensory evaluation

Physical analysis (color, texture, syneresis, and pH)

Formula choosing

Physical analysis (taste, color, pH, and

syneresis)

Determination of hydrocolloid type dan CaCO3

concentration with steaming

Physical analysis (color and gelling

capability)

Functional analysis (chlorophyll, phenol, antioxidant, and dietary

fiber) Physical analysis

17 water (mineral water) added to make the extract concentration is 1000 ml. The leaves then crushed by hand slowly in the water until all the leaves destroyed, become not smooth, and remain leaves bone. The results are filtered using two layers filter cloth while so that pulp and extract separated consistently.

Gelling time was determined by measure the viscosity of pure green grass extract. Viscosity was measured by using LV Brookfield Viscometer. As much as 150 ml sample was placed into a beaker glass 250 ml. By using spindle No. 2 and speed 30 rpm, the sample viscosity measurement was taken for 20 seconds until the needle on the reading obtained a stable position. The rotor spined and the needle would be moved to the viscosity of the sample obtained. Reading of viscosity grade performed after the needle was stable. The read viscosity sample tested showed in the cP (centipoise) unit. The flow diagram of this stage is shown in Figure 10.

Green grass leaves (Premna oblongifolia Merr.) and water 1:15

Crushed

Filtered using filter cloth

Green grass extract

Formed in gelling container

Gelling at room temperature

Observation of viscosity every 30 minutes for 5 hours Figure 10. Procedure of gelling time determination

2. DETERMI ATIO OF aHCO

3

CO CE TRATIO

This stage was conducted to determine the concentration of sodium bicarbonate to be used in the production of green grass jelly. Sodium bicarbonate itself was expected to reduce the degree of chlorophyll degradation by increase the pH. So, green grass jelly that had been treated by steaming does not undergo drastic color change.

Volume of the filtered extract was 750 ml and it was divided into three parts. The addition of NaHCO3 conducted with three types of treatments: the concentration of 0%

(without the addition of NaHCO3), 0.125% (NaHCO3 concentration used to raise the pH of

the green grass extract to be about 7.00), and 0.583% (NaHCO3 concentration used to raise

18 taste, gel color with Chromameter CR 200, syneresis rate, and pH changes. The flow diagram of this stage is shown in Figure 11.

Green grass leaves (Premna oblongifolia Merr.) and water 1:15

Crushed

Filtered using filter cloth

Green grass extract

Formed in gelling container

Gelling for ± 5 hours at room temperature

Green grass jelly

Steamed 100oC, 5 minutes

Observation of taste, color, syneresis, and pH changes Figure 11. Procedure of NaHCO3 concentration determination

2.1. Gel Color

Observation of gel color was conducted by using Minolta CR 200 Chromameter to assess changes in the gel color by the addition of NaHCO3 and also

selected hydrocolloid (in stage 4). In principle, this tool works by measuring the reflection of the color produced by the sample surface. Vibrating lights contained in the device will emit a beam of xenon and produce and spread the light evenly on the surface of the sample. Chromameter measurement results will be converted into the Hunter system with the symbol of L, a, and b. L value states brightness parameter that have a value of 0 (black) to 100 (white). L value indicates reflected light which produces achromatic colors of white, gray, and black. A value expressed mixed chromatic colors red and green, with a+ (positive) from 0 to +100 for the red color and the a/ (negative) from 0 to /80 for the green color. B value expresses mixed chromatic colors yellow and blue, with b+ (positive) from 0 to +70 for the yellow and b/ (negative) from 0 to /70 for blue.

0%, 0.125%, and 0.583% NaHCO3

19

2.2. Level of Acidity (pH) (AOAC 2000)

Measurement of pH was conducted on the acidity of green grass jelly’s syneresis water. Before used, the pH meter was calibrated with buffer solution of pH 4.00 and pH 7.00. Furthermore, because the green grass jelly is semi solid product, it is necessary to prepare the sample before pH measurements performed. Green grass jelly homogenized by shredding into small parts, then chopped or crushed until smooth. Then, 20 ml of syneresis water was placed in a glass beaker, its pH value then measured.

2.3. Gel Syneresis (AOAC 1995)

Gel syneresis observed according to AOAC (1995) method by storing gel at refrigerator temperature (10°C) for 24 hours, 48 hours, and 72 hours. Each gel was put into the cup to hold the water released from the gel during storage. Gel syneresis was calculated by measuring the weight loss during storage and compared with the initial weight of the gel.

Gel Syneresis = A/B A ×100% Where:

A = initial weight of the gel before storing (gram) B = weight of the gel after storing (gram)

3. DETERMI ATIO OF HYDROCOLLOID TYPE A D CaCO

3

CO CE TRATIO

This stage used various hydrocolloids, such as 2% alginate, 2% LMP, 2% mixture of alginate and LMP (1:1), and 2% mixture of kappa and iota carrageenan (1:1), combined with green grass jelly extract 6.67%. In determination of CaCO3 concentration, the concentrations

used were 0% and 1%. Moreover, there was only one NaHCO3 concentration added, the

chosen concentration from the previous stage. This treated green grass jelly would be steamed at 100°C for 5 minutes. Determination of hydrocolloid type and CaCO3 concentration with

steaming treatment would be observed based on the subjective color and gelling capability. The flow diagram of this stage is shown in Figure 12.

20 Green grass leaves (Premna oblongifolia Merr.)

and water 1:15

Crushed

Filtered using filter cloth

Green grass extract

Formed in gelling container

Gelling for ± 5 hours at room temperature

Green grass jelly

Steamed 100oC, 5 minutes

Observation of color and gelling capability

Figure 12. Procedure of hydrocolloid type and CaCO3 concentration determination

4. DETERMI ATIO

OF

SELECTED

HYDROCOLLOID

CO CE TRATIO WITH STEAMI G

This stage used the selected hydrocolloid, mixture of kappa and iota carrageenan (1:1). The concentrations used were determined by variance of 0.25% from 1.25% to 3.00%. This treated green grass jelly would be steamed at 100°C for 5 minutes. Determination of selected hydrocolloid concentration with steaming treatment would be observed based on the parameters of color with Chromameter CR 200, gel texture with Stevens LFRA Texture Analyzer, syneresis rate, and pH changes. Parameters of texture observed were rupture strength (g/cm2), rupture point (cm), and rigidity (g/cm). From these results, three best formulas would be selected. The flow diagram of this stage is shown in Figure 13.

0% and 1% CaCO3

NaHCO3

2% Hydrocolloid

21 Green grass leaves (Premna oblongifolia Merr.)

and water 1:15

Crushed

Filtered using filter cloth

Green grass extract

Formed in gelling container

Gelling for ± 5 hours at room temperature

Green grass jelly

Steamed 100oC, 5 minutes

Observation of color, texture, syneresis rate, and pH changes Figure 13. Procedure of selected hydrocolloid concentration determination

4.1. Gel Texture

Gel texture was measured by using Stevens LFRA Texture Analyzer. Measurement conditions were used according to the research that has been conducted by Camus (2000). The distance between the probes and the gel of 55 mm, probe speed of 2 mm/sec, chart paper speed of 30 cm/min, the probe diameter of 0.5 inches, sensitivity of 100 mV, and strain of 50%. The general curve obtained from the Stevens LFRA Texture Analyzer can be seen in Figure 14.

Figure 14.The general curve obtained from the Stevens LFRA Texture Analyzer NaHCO3

1.25% / 3.00% Selected Hydrocolloid

Rupture Penetration (cm)

R

u

p

tu

re

W

ei

g

h

t

(g

22 Parameters observed in this measurement were gel rupture strength, rupture point, and rigidity. Calculation for rupture strength, rupture point, and rigidity was done by using the formulas from Angalet (1986) and Fry and Hudson (1983) as follow:

Rupture strength (g/ cm2) = Load of rupture Surface area of probe base =

AB 1.27 cm2

Rupture Point = Rupture penetration = AC Rigidity (g/ cm) = Load of rupture

Rupture penetration×

probe speed paper speed =

AB AC

5. SE SORY EVALUATIO

This stage was a continuation of the previous stage, where three green grass jelly formula that showed the best value on the parameters of color, texture, syneresis, and pH changes would be selected as the one chosen formula by consumers’ organoleptic acceptance. Type of test used was hedonic rating test (Meilgaard et al. 1999). In hedonic rating test, panelists asked to reveal personal perceptions about preferences of like or dislike the green grass jelly products. Three chosen formula and one formula without the addition of hydrocolloid as control would be judged based on color, aroma, texture, flavor, and overall parameters. Panelists needed at least 70 untrained panelists. The scale used is the category scale with seven values.

6. FU CTIO AL PROPERTIES A ALYSIS

The chosen formula of green grass jelly would be analyzed on the health functional properties of total chlorophyll, total phenol, and antioxidant capacity.

6.1. Total Chlorophyll ( ollet 2000)

A total of + 2.5 green grass jelly was extracted with 10 ml of 99.8% acetone solution, then mixed well, and stored for 1 night in refrigerator. Sample centrifuged at 3000 rpm for 15 minutes, then filtered. The absorbance of the filtrate then measured at 645 nm and 663 nm using a spectrophotometer to measure the contents of total chlorophyll, chlorophyll a, and chlorophyll b. Calculation was done by the following formulas:

Total chlorophyll (mg/L) = 20.2 A645 nm + 8.02 A663 nm

Chlorophyll a (mg/L) = 12.7 A663 nm – 2.69 A645 nm

Chlorophyll b (mg/L) = 22.9 A645 nm – 4.68 A663 nm

6.2. Total phenol (Sakanaka

2005 in Yoga 2008)

In brief, 1.8 grams of green grass jelly was extracted with 10 ml of 99.9% methanol solution, mixed well, and sentrifuged at 3000 rpm for 15 minutes. The supernatans were filtered through a filter paper. Filtrates were adjusted to be 10 ml in the volumetric flask. Volume of 0.5 ml of deionized water and 0.125 ml of a known dilution of the extract were added to a test tube, followed by addition of 0.125 ml of

23 Folin–Ciocalteu reagent. They were mixed well and then allowed to stand 6 min before 1.25 ml of a 7% sodium carbonate solution was added. The mixture was diluted to 3 ml with deionized water. The color was developed for 90 minutes at room temperature and the absorbance was measured at 760 nm using a spectrophotometer. The measurement was compared to a standard curve of prepared gallic acid solutions and expressed as mg of gallic acid equivalents per litre extract.

6.3. Antioxidant capacity (Sharma and Bhat 2009)

A total of + 1 gram green grass jelly was extracted with 7 ml of methanol, homogenized, and centrifuged at 3000 rpm for 15 minutes, until the supernatants were obtained. Supernatants were filtered. Then, 2 ml solution of DPPH 0.25 mM added, homogenized, and incubated at the dark room temperature for 30 minutes. The absorbance then measured at 517 nm using a spectrophotometer. Ascorbic acid standard curve made with the preparation of such samples.

6.4. Dietary Fiber (Asp.

1983)

Sample preparation

Samples were freeze dried. A total of 1.0 g of sample was added into the erlenmeyer, 25 ml of sodium phosphate buffer pH 6.00 added and made into a suspension. Then 100 µL enzyme termamyl added, closed, and incubated at 80oC for 15 minutes with shaking incubator, then it was removed and cooled. Then conducted adjusting the pH to 1.5 by addition of 4 N HCl. Samples then added with 1 ml enzyme pepsin (0.1 g/ml), incubated at 37°C, while agitated for 120 minutes.

pH adjustion was conducted to reach pH of 6.8 by addition of 4 N NaOH, then added 1 ml enzyme pancreatin (0.1 g/ml), closed, and incubated at 37°C for 120 minutes while agitated. pH was then adjusted to 4.5 by adding 4 N HCl, then filtered using Whatman filter paper No. 40 which has been dried in oven 105oC for 3 hours and obtained constant weight (B). Filtering was conducted by using a vacuum pump and flushing water destilata as much as 2 x 10 ml, so that residue and filtrate obtained.

Determination of Insoluble Dietary Fibre

The residue obtained was washed with 2 x 10 ml of 78% ethanol and 2 x 10 ml of acetone pro analysis. The mixture solution of the residue dried at 105oC, until a constant weight for 3 hours (C = constant weight after analysis and dried). Porcelain bowl was heated in an oven at 105oC for 1 hour, cooled, and weighed (D = weight of porcelain bowls). Filter paper and residue in the furnace incinerated at 500oC for 5 hours, cooled, and weighed (E = weight after incineration).

Determination of Soluble Dietary Fibre

The filtrate was added by 500 ml of 95% ethanol warm (60°C) and precipitated for 24 hours. The precipitate was filtered with filter paper which has dried in oven 105oC for 3 hours and has constant weight (F = weight of filter paper), washed

24 with 2 x 10 ml of 78% ethanol and 2 x 10 ml of acetone. The precipitate was dried at a temperature of 105°C for 3 hours, cooled in a desiccator, and weighed (G = weight after analyzed and dried). Porcelain bowl was heated in an oven 105oC for 1/3 hours, cooled in a desiccator, and weighed (H = weight of porcelain bowls), then filter paper and residue incinerated at temperatures about 550°C for 5 hours, cooled in a desiccator, and weighed (I = weight after incinerated).

Determination of Total Dietary Fibre

Level of total dietary fiber was obtained by sum the value insoluble dietary fiber with soluble dietary fiber. Blank was done without samples, the calculations are:

1. % Insoluble dietary fiber (IDF) in dry base

IDF = {(C/B) / (E/D)} x 100 A

2. % Soluble dietary fiber (SDF) in dry base

SDF = {(G/F) / (I/H)} x 100 A

3. % Total dietary fiber = % IDF + % SDF Where:

A = dry sample weight (g) B, F = filter paper weight (g)

C, G = filter paper and residue weight after dried (g) D, H = porcelain bowl weight (g)

E, I = porcelain bowl and ash weight after incinerated (g)

6.5. Moisture Content (AOAC 2000)

The empty aluminum bowl was dried in an oven for 15 minutes, then cooled for 10 minutes in a desiccator and then weighed.The empty bowl that have been dried weighed. 1/2 grams of homogenized sample was put into the bowl. The bowl containing sample was put into the oven for minimum 6 hours. The contact between the bowl with oven wall avoided. The cup was put into desiccator and cooled. Once cool, the bowl is weighed again until obtain the relatively constant weight.

Moisture content (g/100 g wet basis) = W/(W1/W2) W ×100 Where: W = weight of sample before dried (g)

W1 = weight of sample and bowl after dried (g) W2 = weight of empty bowl (g)

6.6. Ash Content (AOAC 2000)

The empty porcelain bowl was put into the oven for 3 hours, then put into a desiccator for 15 minutes and weighed. The samples were pulverized then weighed 1/2 grams and put into the porcelain bowl of known weight constant. Sample was loaded

25 into the furnace at a temperature of 500oC for minimum 6 hours. The sample was cooled in a desiccator for 15 minutes, then weighed. Ash content of the sample can be calculated using the following formula:

Ash content (g/100 g wet basis) = (W1/W2) W ×100 Ash content (g/100 g dry basis) = ash content (WB)

(100/moisture content WB )×100%

Where: W = weight of sample before incinerated (g) W1 = weight of sample and bowl after incinerated (g) W2 = weight of empty bowl (g)

7. MICROBIOLOGICAL A ALYSIS (BAM 2001)

Test bacteria that will be added to the product in fresh condition in which bacteria have not entered a phase of death (age ± 24 hours), Staphylococcus aureus. Fresh culture in slant the TSA as much as 1 loop was scratched upward selective medium, BPA + EYT. Then, it was incubated for 24 hours at 35°C. After the incubation process was done, transfer of the colony was conducted on BHIB liquid media. Then incubated for 18/20 hours at temperature of 35°C and obtained fresh culture containing 109/1010 cell S. aureus.

A solid test sample weighed of 10 grams then placed in a sterile plastic and added a solution of 0.85% NaCl physiological as much as 90 ml. This solution was then homogenized in a stomacher apparatus for a minute. This solution is the solution concentration of 10/1. Inoculation is done up to 10/5. The method used was spread plate, sterile agar poured and allowed to freeze then followed by sample poured and razed with a hockey stick.

From each dilution rate, 1 ml taken and inoculated on three plates containing BPA medium supplemented with EYT for each 0.4 ml, 0.3 ml, and 0.3 ml. The suspension was then razed using a sterile glass hockey stick. After the suspension was not visible on the surface of media, plate incubated in the inverted position on the temperature of 35oC for 45/ 48 hours. After incubation, the number of colonies of S. aureus was observed and counted that have a characteristic round, slick and smooth, convex, 2/3 mm of diameters, gray to black, and surrounded by an opaque zone outside the clear zone. Plates were selected based on the containing colonies of 20/200.

8. DESIG OF EXPERIME T

A common experimental design used in this study was completely randomized design (CRD). The design of the experiment was generally prepared with 2 replications. General model completely randomized design for stage 2, stage 4, and stage 5 with 1 factor is as follows:

114 Appendix 25. T/test results of dietary fiber measurement because of steaming treatment on chosen formula

t/Test: Paired Two Sample for Means IDF t/Test: Paired Two Sample for Means SDF t/Test: Paired Two Sample for Means TDF

Steamed Fresh Steamed Fresh Steamed Fresh

Mean 0.545423 0.495543 Mean 1.224394 1.496118 Mean 1.769817 1.991661

Variance 0.00783 0.003289 Variance 0.005139 0.007648 Variance 0.001957 0.012398

Observations 4 4 Observations 4 4 Observations 4 4

Pearson Correlation 0.111497 Pearson Correlation /0.39517 Pearson Correlation 0.950873

Hypothesized Mean Difference 0 Hypothesized Mean Difference 0 Hypothesized Mean Difference 0

df 3 df 3 df 3

t Stat 0.998248 t Stat /4.07994 t Stat /6.28217

P(T<=t) one/tail 0.195864 P(T<=t) one/tail 0.013296 P(T<=t) one/tail 0.004072

t Critical one/tail 2.353363 t Critical one/tail 2.353363 t Critical one/tail 2.353363

P(T<=t) two/tail 0.391727 P(T<=t) two/tail 0.026591 P(T<=t) two/tail 0.008145

115 Appendix 26. T/test results of dietary fiber measurement because of steaming treatment on commercial green grass jelly

t/Test: Paired Two Sample for Means IDF t/Test: Paired Two Sample for Means SDF t/Test: Paired Two Sample for Means TDF

Steamed Fresh Steamed Fresh Steamed Fresh

Mean 0.346994 0.475512 Mean 1.21855 1.241221 Mean 1.565544 1.716733

Variance 0.00047 0.009441 Variance 0.002765 0.002151 Variance 0.005498 0.003613

Observations 4 4 Observations 4 4 Observations 4 4

Pearson Correlation 0.521152 Pearson Correlation /0.8087 Pearson Correlation 0.342241

Hypothesized Mean Difference 0 Hypothesized Mean Difference 0 Hypothesized Mean Difference 0

df 3 df 3 df 3

t Stat /2.92618 t Stat /0.48172 t Stat /3.88419

P(T<=t) one/tail 0.030595 P(T<=t) one/tail 0.331481 P(T<=t) one/tail 0.015119

t Critical one/tail 2.353363 t Critical one/tail 2.353363 t Critical one/tail 2.353363

P(T<=t) two/tail 0.061189 P(T<=t) two/tail 0.662961 P(T<=t) two/tail 0.030239

116 Appendix 27. Results of moisture content analysis

Sample R W W1 W2

Moisture Content % WB Average ±

SD Steamed chosen

formula green grass jelly

1 2.0283 4.3653 4.3206 97.80 97.68 ± 2.0378 4.4234 4.3774 97.74 0.12 2 2.0161 4.5884 4.5414 97.67

2.1305 4.4534 4.4007 97.53 Unsteamed chosen

formula green grass jelly

1 2.0670 4.6321 4.5897 97.95 97.68 ± 2.0086 4.8056 4.7549 97.48 0.23 2 2.0562 4.7065 4.6551 97.50

2.0507 4.5733 4.5283 97.80 Steamed

commercial green grass jelly

1 2.4123 4.2722 4.2565 99.35 99.43 ± 2.4267 4.1427 4.1311 99.52 0.10 2 2.4678 4.2849 4.2689 99.35

2.4218 4.5441 4.5322 99.51 Unsteamed

commercial green grass jelly

1 1.9999 5.4490 5.4356 99.33 99.43 ± 2.0000 5.0485 5.0388 99.52 0.09 2 2.0159 5.1744 5.1618 99.37

117 Appendix 28. Results of ash content analysis

Sample R W W1 W2

Ash Content % Wet

basis

Average ± SD

% Dry basis

Average ± SD Steamed chosen formula

green grass jelly

1 1.1424 30.0694 30.0682 0.11 0.10 ± 5.99 5.78 ±

1.1703 25.7245 25.7234 0.09 0.01 5.36 0.58

2 1.1904 20.5645 20.5634 0.09 5.27

1.1385 20.9845 20.9832 0.11 6.51

Unsteamed chosen formula green grass jelly

1 1.1043 25.5627 25.5614 0.12 0.10 ± 6.71 5.81 ±

1.1037 20.7846 20.7834 0.11 0.01 6.20 0.84

2 1.1987 20.4330 20.4320 0.08 4.76

1.1267 27.8965 27.8954 0.10 5.56

Steamed commercial green grass jelly

1 1.1890 20.4220 20.4212 0.07 0.07 ± 3.84 3.96 ±

1.1638 25.5627 25.5618 0.08 0.01 4.41 0.64

2 1.1037 24.4870 24.4864 0.05 3.10

1.1423 24.2799 24.2790 0.08 4.49

Unsteamed commercial green grass jelly

1 1.1199 20.0525 20.0518 0.06 0.07 ± 3.56 3.93 ±

1.1868 30.3284 30.3277 0.06 0.02 3.36 0.96

2 1.1683 20.8465 20.8454 0.09 5.37

118 Appendix 29. Microbiological analysis result

Treatments Dillution Inoculum

Total

Colony cfu/ml

log cfu/ml Average log cfu/ml Decrease of log cfu/ml Unsteamed Chosen Formula Green Grass Jelly

10/5 0.3 230

5.5E+07 7.74

7.52

1.76

0.3 166

0.4 156

10/6 0.3 12

3.2E+07 7.51

0.3 2

0.4 18

10/7 0.3 1

2.0E+07 7.30

0.3 1

0.4 0

10/8 0.3 0

0.3 0

0.4 0

Steamed Chosen Formula

Green Grass Jelly

10/3 0.3 19

1.4E+05 5.14

5.76

0.3 31

0.4 87

10/4 0.3 1

2.2E+05 5.34

0.3 9

0.4 12

10/5 0.3 1

1.2E+06 6.08

0.3 7

0.4 4

10/6 0.3 1

3.0E+06 6.48

0.3 0

0.4 2

Appendix 30. T/test result of microbiological analysis

Unsteamed Steamed

Mean 7.5175 5.76

Variance 0.032292 0.393867

Observations 4 4

Pearson Correlation /0.59882 Hypothesized Mean Difference 0

df 3

t Stat 4.691987

P(T<=t) one/tail 0.009153 t Critical one/tail 2.353363 P(T<=t) two/tail 0.018305 t Critical two/tail 3.182446

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