Synopsis Final Project Mikhael

8/2/2019 Synopsis Final Project Mikhael

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UTILIZATION OF ROSELLE (Hibiscus sabdariffa L.)

CALYCES EXTRACT AS THE SOURCE OF ANTIOXIDANT

AND COLORANT IN MODIFIED JAMMikhael Dominico

1), Herry Cahyana

2), Julia Ratna Wijaya

2)

ABSTRACT(15 pages : 2 tables, 13figures)

Exercises and healthy diet has become more important in human life, yet

their schedule has turned tighter and leaving them with limited time to prepare

their food. Functional food becomes one interest and potential needs to answer

this challange. Roselle (Hibiscus sabdariffa L.) calyces has been used as herbs,food, and additives for its anthocyanins content. In this project, modified jam

product containing roselle extract was developed in respond to this condition.

Formulation using 2% low methoxyl pectin (LMP) with 30% addition of calcium

ions was found to be the most suitable to form intact jam that was targeted so that

the jam can be directly placed on top of the bread without the need of spreading

it. Analysis showed that different concentration of extract and pH affect the

functional properties of anthocyanins as antioxidant. Higher concentation showed

higher anthocaynins and phenolic content. Higher pH produced showed

antioxidant activity which was supported by lower anthocyanins monomers and

phenolic content. Between product made from red and purple calyces, product

made from 15% dried red roselle calyces extract at pH 4.3 was found to be theone with the highest acceptance level based on sensory evaluation while the one

with the highest antioxidant activity was obtained from the 20% dried purple

roselle calyces at pH 4.6.

Keywords: modified, jam, roselle, anthocyanins, antioxidant

References: 8 (2002-2009)

1) Student of Food Technology Department Universitas Pelita Harapan

2) Full time lecturer at Food Technology Department, Universitas Pelita Harapan


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INTRODUCTION

People start to consider that exercises and healthy diet are important. A

survey has stated that most used reason was 34% to lose weight and 30% to stay

healthy (Anonym1, 2006). Consumption of functional food increases because

people start to realize its potential. Unfortunately, high living cost, tight schedule,

and daily routine only allow these people with limited time. Modification of food

through genetic, processing, and composition has also been conducted. The

purpose is to provide better options and answers of the current issues.

Roselle calyces (Hibiscus sabdariffa L.) have been known for its potential

as antioxidant, anti-carcinogenic, and colorant source due to its active

components, specifically anthocyanins. It has been studied and used to make jam,

jellies, and beverages since it has appealing red color and unique taste. Jam is gel-

based product which is viscous and made using mixture of acid (commonly from

fruit), sugar, and pectin. In general, jam is consumed together with bread as

topping for breakfast. However, its application still needs tools such as knife to

apply it on the bread. The possibility of developing an innovative jam giving a

simpler method of application during the consumption is required. In this

research, the jam will be made as intact product rather than common jam which is

viscous and slurry so that it can be directly placed on top of the bread. Processing

steps in producing jam can contribute to changes both in physical and chemical of

the final product (modified roselle jam). Physical change can be color alteration,

while changes in functional property such as antioxidant activity are the example

for chemical change.

RESEARCH METHODOLOGY

Materials

The samples used in this research are dried red and purple calyces of

roselle flower (Hibiscus sabdariffa L.), sugar, LMP (Low Methoxyl Pectin),

calcium citrate, water, and sodium citrate. Materials for analysis are HCl 37%,

DPPH (2,2-diphenyl-1-picrylhydrazyl ) solution, methanol, Folin Ciocalteau,


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Na2CO3 2%, galic acid, buffer solution KCl (pH 1), and buffer solution Na-acetate

(pH 4.5).

Procedure

Preliminary Research

Preliminary research was carried out to find the maximum concentration

of extract that can be obtained from roselle calyces extraction process and also

appropriate formulation for the modified jam. Determining the maximum

concentration of extract was meant to prepare the stock extract (Figure 1). This

stock extract was used to prepare the samples and produce the modified jams for

analysis. The stock extract was made as concentrated as possible thus calyces

water ratio used for the research was conducted in ascending starting from 5%

with an increase of 5% each (10%, 15%, and so on) until the maximum possible

extraction (the one level below where the product cannot be extracted again).

Figure 1. Extraction procedure of roselle calyces

Table 1. Ingredients for one formulation

Ingredients Amount (g)

Dried roselle extract 60

Sugar 40

Pectin (LMP/HMP) 2

Calcium citrate 0.6

Water

Heat up to 95C (Boiling)

Add roselle calyces

Boil for 4 minutes

Filter

Roselle stock extract


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The parameters observed for appropriate formulation were the type of

pectin (High Methoxyl Pectin/HMP or Low Methoxyl Pectin/LMP) and pH of

extract. The pH range used was 2.8, 3.0, 3.2, 3.4, 3.7, 4.0, 4.3, 4.6, 4.9, 5.2. The

formula used was shown in the table 1 with the procedure as in Figure 2. The final

formulation was used for making modified jam for analysis.

Figure 2. Processing step of modified jam

Sample Preparation for Analysis

Active components (mainly anthocyanins) from roselle modified jam are

extracted using acidified methanol for 2 hours, filtered, and kept under cool

condition in covered container (Adrian, 2008). The analyses are conducted within

a week after the sample was made.

Sample Analysis

Both red and purple calyces are analyzed for total monomericanthocyanins, total phenolic content, and antioxidant activity. Additional test of

color, sensory evaluation, and LC-MS was conducted as supporting information.

Result of Preliminary Research

In this research, ratio of 1:5 (20%) was selected for making the initial

extract from which further dilution would be done for 10% and 15%. It is because

extraction through decoction method is done under continues heating thus

Mix roselle extract andsugar

Heat up to 60C

Add ca citrate (for LMP)

Heat up to 7275C

Add LMP

Heat up to 90C

Shaped and cooled down


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controlling water evaporation rate is difficult. The other reason for choosing 20%

concentration is due to the condition that at higher concentration, the extract yield

was almost impossible to be obtained since there was no water left in the extract.

Most of the water was absorbed by the flower and the rest was evaporated.

Table 2 The result of dried roselle extraction at different concentration

Concentration (%) Yield (g) pH Brix

5 228.28 2.97 2.9

10 183.29 2.81 4.9

15 171.68 2.83 6.9

20 105.51 2.72 9.0

25 - - -

Some trials of jam production were conducted by using different pectin

types (HMP / High Methoxyl Pectin and LMP / Low Methoxyl Pectin) (Table 3).

It was found that the modified jam was formed (intact) at pH 4.3 to 5.2 with 2%

LMP and 0.3% calcium ions (out of total pectin used), but at 5.2 the jam produce

sandy texture and excessive syneresis, thus pH 5.2 was excluded.

Table 3 Product texture of modified jam using different pectin source

Texture of Modified jam

LMP HMP

2.8 Watery Slurry

3.0 Watery Slurry3.2 Watery Slurry

3.4 Watery Slurry

3.7 Watery Slurry

4.0 Slurry Slurry

4.3 Intact Slurry

4.6 Intact Slurry

4.9 Intact Slurry

5.2 Intact Slurry

Total Monomeric Anthocyanins

Figure 3. showed that increase in concentration will increase the amount of

total monomeric anthocyanins from the products made from dried purple calyces

roselle. Effect of pH cannot be seen clearly. The highest level of total monomer

anthocyanins were achieved in product at pH 4.6 with 20% roselle extract for both

red (0.645mg/10g product) and purple (6.169mg/10g product). However, the

amount of anthocyanins from original extract of each type was much higher

Pectin type

pH


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1.080mg/10g (red calyces) and 11.360mg/10g (purple calyces) than the one

contained in the products. It indicated the loss or degradation of anthocyanins

occurred during process of making products. Product using extract from purple

calyces was found to have higher total monomers compare to the red calyces.

Figure 3. Comparison of total monomeric anthocyanins from different roselle calyces in products

Concentration of anthocyanins and pH have been known as factors that

affect activity and stability of anthocyanins. Different source of anthocyaninsmight have different stability toward pH, but most anthocyanins are stable at

acidic pH. Rein (2005) stated that acylation at B ring of the anthocyanins can

create a stronger and more stable bond at pH around 4 to 5.5. Increase in

anthocyanins concentration will create better stability through self-association.

Rise in temperature at pH 2 to 4 leads to the further loss in anthocyanins through

the hydrolysis of glycosidic bond of the anthocyanins which can lead to higher

loss of the anthocyanins monomers. Reduction of anthocyanins monomers from

extract to product could be derived from losses of both anthocyanins and other

compound inside the extract during processing which might aid the stability of

anthocyanins.

0.264a

0.280a

0.2

48a

0.409cd

0.381c

0.347b

0.439d

0.617e

0.412cd

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

pH 4.3 pH 4.6 pH 4.9

TotalMonomericAnthocyanins

(mg/gproduct)

Purple Calyces 10% Purple Calyces 15%

Purple Calyces 20%

0.02

3ab

0.0

38cd

0.01

4a

0.0

32bc

0.032bc

0.0

21ab

0.0

46d

0

.065e

0.0

49d

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

pH 4.3 pH 4.6 pH 4.9

TotalMonomericAnthocyanins

(mg/gproduct)

Red Calyces 10% Red Calyces 15%

Red Calyces 20%


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Total Phenolic Content

Figure 4 below shows the comparison of total phenolic content. Purple

roselle showed higher phenolic content compare to red roselle. The effect of

concentration was clearly shown; the higher concentration resulted in the higher

the phenolic compounds, while the higher pH showed the significant reduction of

phenolic for the product with purple roselle (at pH 4.3 to 4.9), but did not

significant in the product with red roselle. The result indicated that phenolic

compound was more stable at the lower pH.

Figure 4. Comparison of total phenolic content from different roselle calyces in products

Roselle calyces do not only contain anthocyanins but also some other

phenolic compounds in small amount. The extractable polyphenol (EPP) in roselle

calyces such as hydroxybenzoic acid, hydroxycinnamic acid, anthocyanidins, and

flavonols can be easily absorbed and contribute to functional properties of roselle

calyces as antioxidant (Sayago-Ayerdi, et al., 2007).

Antioxidant Activity (DPPH Method)

The statistical analysis showed that the increase in concentration improve

the antioxidant activity of the product. Interaction between pH and concentration

on the products and extract were found either on the dried purple or red calyces.

The highest antioxidant activity was found in the product made by using the pH

4.6 at extract concentration of 20% for purple calyces and 15% for red calyces,

respectively. The result from product made using red roselle calyces showed

1.167b,1

1.678b,1

2.097b,1

1.042ab,2

1.654ab,2

1.894ab,3

1.110a,4

1.490a,4

1.839a,4

0.000

0.500

1.000

1.500

2.000

2.500

pH4.310%

pH4.315%

pH4.320%

pH4.610%

pH4.615%

pH4.620%

pH4.910%

pH4.915%

pH4.920%

TotalPhenolicContent

(mg/gprodu

ct)

Purple Roselle Modified Jam

0.502ab

0.611c

0.738d

0.514b

0.581c

0.734d

0.453a

0.594c

0.694d

0.000

0.500

1.000

1.500

2.000

2.500

pH4.310%

pH4.315%

pH4.320%

pH4.610%

pH4.615%

pH4.620%

pH4.910%

pH4.915%

pH4.920%

TotalPhenolicContent

(mg/gprodu

ct)

Red Roselle Modified Jam


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lower antioxidant activity compare to the purple one which is showed by the

higher value of IC50.

Figure 5. Comparison of antioxidant activity from different roselle calyces in products

The small value of IC50 obtained from the results showed that both roselle

calyces have high antioxidant activity, thus only small amount or small

concentration required to reduce the 50% of the DPPH radicals. The activity of

antioxidant in product made using purple calyces extract appeared to be stronger

at the higher pH, while the one made of red roselle calyces showed the higher

activity at a more acidic condition. It could be resulted from the nature of the

extract composition that makes different activity and antioxidant stability.

Color Evaluation

Hue showed the intensity of red color, when Hue angle decreased, the

intensity of red color is increased (Brewer, et al., 2005). This theory support the

result of the experiment; the modified jam using red roselle definitely showed

more intense red color as the concentration increase because the amount of total

pigment inside the product was increased.

Referring to Figure 6, both product made with purple and red calyces

extract showed red purple color based on the Hue but different visually (purple

calyces showed a more deep purple color while the red one was more translucent).

Red roselle calyces tend to show the decrease of Hue as the concentration

increase, while the purple calyces showed that the higher concentration has the

0.965c

0.752b

0.577a

0.958c

0.716b

0.549a

1.123d

0.689b

0.532a

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

pH4

.310%

pH4

.315%

pH4

.320%

pH4

.610%

pH4

.615%

pH4

.620%

pH4

.910%

pH4

.915%

pH4

.920%

IC50(g)

Purple Roselle Modified Jam

2.222b

2.009ab

1.953ab

3.02

5cd

1.725a

2.939c

2.021ab

3.106cd

3.4

40d

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

pH4

.310%

pH4

.315%

pH4

.320%

pH4

.610%

pH4

.615%

pH4

.620%

pH4

.910%

pH4

.915%

pH4

.920%

IC50(g)

Red Roselle Modified Jam


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higher Hue. However, the highest Hue in product made using purple calyces

(20% concentration; pH 4.9) still have lower Hue compare to the red one.

Figure 6. Comparison of color evaluation of purple and red roselle calyces in products

Sensory Evaluation

The reason of conducting sensory evaluation for this product was to find

out the profile of the product. According to SNI 3746-2008 (Anonym3, 2008),

standard requirement of jam is total soluble solid (TSS) at least 65 Brix. One

parameter which commonly used to determine the quality of jam is itsspreadability. Since jam usually consumed as bread topping, it needs to be spread

on top of it. However, modified jam product which is discussed in this research is

a new type of product which is hope can substitute the function of jam as bread

topping but with easier application (only need to be placed on top of the bread)

which is why it is called as modified jam. To allow the product easily placed, it

needs to be intact and not friable.

Figure 7 and 8 showed the result for color and intactness of the test. It can

be seen that in general, increase of concentration resulting in the increase of color

in red roselle but not that significant in purple roselle. It is because visually, the

color of modified jam with purple roselle is dark (almost navy blue) and the

method for scoring the color intensity is by observing the sample in the table

(white table), thus it is possible that the color intensity could not be seen clearly.

Result of statistical analysis for intactness showed interaction present. Based on

the graphs, there cannot be seen any trend whether concentration or pH cause

0.685

0.712

0.779

0.634

0.597

0.758

0.490

0.449

0.628

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.8000.900

pH 4.3 pH 4.6 pH 4.9

Hue

Extract Concentration 10% 15% 20%

0.021

0.059

0.144

0.067

0.027

0.022

0.183

0.187

0.379

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.8000.900

pH 4.3 pH 4.6 pH 4.9

Hue

Extract Concentration 10% 15% 20%


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decrease or increase in intactness. Intactness of the product can be related with the

type of gelling agent used, how it interacted with other ingredients in the formula,

and the gel characteristic of it.

Figure 7. Sensory evaluation result for color and intactness of red roselle calyces in

products

Figure 8. Sensory evaluation result for color and intactness of purple roselle calyces inproducts

In term of application, the average score for all the product was around 3

(average; normal) (Figure 9 and 10). The method of application which was asked

from the panelists were how difficult the effort that they have to give to transfer

the samples from the wrapper (PE plastic) to the bread. Syneresis present in low

amount in all formulation. It is the nature of gel product to undergo syneresis.

Presence of leech out liquid syneresis) could cause the product became sticky and

made it more difficult to be moved from plastic to bread. Sandy texture also found

1

2

3

4

5

pH

4.3

10%

pH

4.3

15%

pH

4.3

20%

pH

4.6

10%

pH

4.6

15%

pH

4.6

20%

pH

4.9

10%

pH

4.9

15%

pH

4.9

20%

Score

Product

Color

Intactness

1

2

3

4

5

pH

4.3

10%

pH

4.3

15%

pH

4.3

20%

pH

4.6

10%

pH

4.6

15%

pH

4.6

20%

pH

4.9

10%

pH

4.9

15%

pH

4.9

20%

Score

Product

Color

Intactness


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in several formulation possibly due to un-homogenize stirring or unbalance

proportion of pectin : calcium ratio. The ratio of pectin : calcium used in this

research was based on previous research by Marcia (2009) through trial and error.

Hui (2006) stated that LMP can still form gel up to neutral pH, however it is still

possible that pH can still give effect towards the gel formation of the product.

Figure 9. Sensory evaluation result for syneresis, application, and sandiness of red roselle

calyces in products

Figure 10. Sensory evaluation result for syneresis, application, and sandiness of purple

roselle calyces in products

The overall acceptance of the product evaluated by 30 panelists is shown

in figure 4.10. It can be seen that in general the acceptance is above average (3.0).

The best result obtained by formulation made using 15% red roselle calyces

extract at pH 4.3 (average score 3.6). It is possible that product with 20% extract

concentration tasted to sour for most panelists thus it is not preferable. Product

with purple roselle was lack of roselle specific taste which might affect the overall

1

2

3

4

5

pH

4.3

10%

pH

4.3

15%

pH

4.3

20%

pH

4.6

10%

pH

4.6

15%

pH

4.6

20%

pH

4.9

10%

pH

4.9

15%

pH

4.9

20%

Score

Product

Syneresis

Application

Sandiness

1

2

3

4

5

pH

4.3

10%

pH

4.3

15%

pH

4.3

20%

pH

4.6

10%

pH

4.6

15%

pH

4.6

20%

pH

4.9

10%

pH

4.9

15%

pH

4.9

20%

Sco

re

Product

Syneresis

Application

Sandiness


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taste (acid and sweetness). Figure 11 provide the result of sensory evaluation for

each parameter from the best formulation.

Figure 11. Sensory evaluation result of product (red roselle, pH 4.3, 15% extract)

Liquid ChromatographyMass Spectroscopy (LC-MS)

Additional test to support results from analysis was conducted using LS-MS

equipment. The result can be seen from Figure 12 and 13.

3.63.7

3.2

3.4

3.02.7

3.43.2

1.61.5

2.32.0

Color

Smoothness

Intactness

Application

Sweetness

Acidity

Roselle Flavor

Roselle Taste

Off Flavor

Off Taste

Sandiness

Syneresis

pH 4.3 15%

Red Roselle


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Figure 12 LC-MS result of crude roselle extract from red calyces

Figure 13 LC-MS result of crude roselle extract from purple calyces

99.0 319.2 539.4 759.6 979.8 1200.0

Mass (m/z)

0

115.4

0

10

20

30

40

50

60

70

80

90

100

%I

ntensity

Mariner Spec /91:93 (T /4.25:4.35) -83:85 (T -4.25:4 .35) ASC=>NR(2.00) [BP= 219.2, 115]

219.18

191.22

159.22

279.10155.31

237.14318.11118.19 401.15 508.59 1045.60839.02756.82678.72

0 4 8 12 16 20

Retention Time (Min)

197.4

20

30

40

50

60

70

80

90

100

%I

ntensity

BPI=>NR(2.00)

T3.2

T3.7

T1.7 T11.6

99.0 319.2 539.4 759.6 979.8 1200.0

Mass (m/z)

0

165.3

0

10

20

30

40

50

60

70

80

90

100

%I

ntensity

Mariner Spec /69:71 (T /3.20:3.30) -50: 54 (T -3.20:3.30) ASC=>NR(2 .00)[BP = 175.2,165]

175.25

203.21

235.20132.35

207.24

219.17

236.18156.28428.96318.06 544.88 842.87759.86620.47 1101.74

126 156 186 216 246 276

Mass (m/z)

0

12.0

10

20

30

40

50

60

70

80

90

100

%I

ntensity

Mariner Spec /80:81 (T /3.72:3.77) -77: 78 (T -3.72:3.77) ASC=>NR(4.00) [BP= 219.1, 12]

219.15

205.18

219.68

175.07

205.63156.04 219.92183.27 234.75203.53 254.22154.04 170.82140.14


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Both figures showed that there are two compounds which present in high

amount at both calyces. Purple calyces roselle appeared to have two more detected

compounds, which is seen by the amount of detected peaks from the sample

reading. From the two results, both purple and red roselle were presumed to have

similar compound T 3.4 (red) and T 3.2 (purple). The other compounds T 4.3

(red) and T 3.7 (purple) might have similar basic structure but different form. It

was due to the fact that at red roselle, heavier compound was detected while it was

not appeared in the purple one.

CONCLUSION AND SUGGESTION

Result of initial extraction of roselle calyces for stock extract was the 1:5

ratio (roselle:water) using boiling method for 4 minutes. The final formulation

was the one using 2% Low Methoxyl Pectin (LMP) with added calcium 30% out

of the total pectin used. The different pH (4.3, 4.6, and 4.9) and extract

concentration (10%, 15%, and 20%) significantly affect the anthocyanins

monomers, phenolic content, and antioxidant activity. Higher pH caused decrease

in the three aspects, while higher concentration increased them. Purple calyces

contained higher extracted components (pigments; anthocyanins and other

phenolic compound) compare to the red calyces showed by the antioxidant

activity and the Hue. Based on sensory evaluation, the best accepted product by

panelists was modified jam made by using 15% extract of red roselle calyces, at

pH 4.3. Product made with purple calyces has higher functional values compare to

the red one and its acceptance was not different much from the product made

using red roselle calyces.

Research regarding anthocyanins of roselle calyces can still be required,

especially for extract purification, research towards other roselle varieties, and

stability test. Study about the antioxidant mechanism of anthocyanins and the

correlation between pigmen discolorations with its activity would help further

application and storage method of anthocyanins. Development in modified jam

product is needed to obtain the most suitable formulation through the utilization of

other gelling agents, different formulation, characterization of product, packaging


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and storage. The relation between LMP and calcium ions is also interesting to be

studied to ensure the proper proportion in the future studies.

REFERENCES

Adrian, Charles. Studi Kestabilan Pigmen Antosianin Kulit Buah Anggur (Vitis

vinifer L.) pada ProdukHard Candy. Skripsi Sarjana Teknologi Pertanian

Fakultas Teknologi Industri. Universitas Pelita Harapan. Karawaci, 2008.

Anonym1. Health and Food. Special Eurobarometer 246/ Wave 64.3 (2006).

Anonym2. Selai Buah. Standar Nasional Indonesia (SNI) 3746 - 2008. Jakarta:

BSN, 2008.

Hui, Yiu H. Handbook of Food Science, Technology, and Engineering. USA:

CRC Press, 2006.

Marcia, Cynthia. Pemanfaatan dan Uji Kestabilan Antosianin dari Rosella

(Hibiscus sabdariffa L.) pada ProdukJelly. Skripsi Sarjana Teknologi

Pertanian Fakultas Teknologi Industri. Universitas Pelita Harapan.

Karawaci, 2009.

Rein, Maarit. Copigmentation reactions and color stability of berryanthocyanins. Academic Dissertation Faculty of Agriculture and Forestry.

University of Helsinki. Helsinki, 2005.

Sayago-Ayerdi, Sonia G, Sara Arranz, Jose Serrano, and Isabel Goni. Dietary

Fiber Content and Associated Antioxidant Compounds in Roselle Flower

(Hibiscus sabdariffa L.) Beverage. Journal Agricultural and Food

Chemistry 55 (2007).

Tsai, Pi-Jen, John McIntosh, Philip Pearce, Blake Camden, and Brian R. Jordan.

Anthocyanin and Antioxidant Capacity in Roselle (Hibiscus sabdariffa

L.) Extract. Food Research International 35 (2002): 351-356.

Synopsis Final Project Mikhael

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