Establishment of Axenic Culture and Callus Induction of Synsepalum Dulcificum Daniel (Miracle Berry)

ESTABLISHMENT OF AXENIC CULTURE AND CALLUS INDUCTION OF SYNSEPALUM DULCIFICUM DANIEL (MIRACLE BERRY)

TAN HONG WEE

Bachelor of Science with Honours (Plant Resource Science and Management) 2015

ESTABLISHMENT OF AXENIC CULTURE AND CALLUS INDUCTION OF SYNSEPALUM DULCIFICUM DANIEL (MIRACLE BERRY)

TAN HONG WEE

This project is submitted in partial fulfillment of the requirements for the Degree of Bachelor of Science with Honours

(Plant Resource Science and Management)

Faculty of Resource Science & Technology

UNIVERSITI MALAYSIA SARAWAK

2015

APPROVAL SHEET

Name of candidate : Tan Hong Wee

Title of dissertation: Establishment of Axenic Culture and Callus Induction of Synsepalum dulcificum Daniel (Miracle Berry)

(Prof Dr. Hamsawi Sani)

Supervisor

(Dr. Rebicca Edward)

Coordinator Plant Resource Science and Management Programme

Department of Plant Science and Environmental Ecology

Faculty of Resource Science and Technology

DECLARATION

I declare that this thesis entitle “Establishment of Axenic Culture and Callus Induction of

Synsepalum dulcifiucm Daniel (Miracle Berry) is the result of my own research except as cited in the references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.

Signature:

Name : TAN HONG WEE

Date : 15th June 2015

ACKNOWLEDGEMENT

First of all, I would like to express my thanks to Prof Dr. Hamsawi Sani for his comments, guidance and advice in the preparation of this Final Year Project (FYP) report. I would also like to express grateful to my family members especially my father, mother and my brother who had always encouraged and contributes plenty of information for the writing up of this thesis report. Thanks to my friends and colleagues for their patience and cooperation during the entire thesis writing process and during the whole experimentation process. Finally, I would also like to thank all lecturers in the Department of Plant Science

Resource and Management of Faculty Resource Science and Technology (FRST)

Universiti Malaysia Sarawak (UNIMAS) for the materials and facilities that had provided to me in fulfilment of this thesis report.

Establishment of Axenic Culture and Callus Induction of Synsepalum dulcificum Daniel

Tan Hong Wee

Plant Resource and Management Programme Faculty of Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Synsepalum dulcificum is a slow growing shrub from family Sapotaceae. The fruits contain high medicinal values which aid in chronic diseases due to the presence of active component Miraculin, a taste modifying properties. The current research emphasizes on the axenic cultures establishment and callus induction of Synsepalum dulcificum from leaf and seed explants. The sterilized leaf explants were obtained from both 25% Clorox® solution (Sodium hypochlorite) which produced 91.7% axenic cultures and 25% CaOCI solution (Calcium hypochlorite) that produced 94.4% axenic cultures with 20 minutes exposure time upon addition of 1% Benomyl solution respectively. The sterilized leaf explants were cultured onto Murashige & Skoog (MS) media supplemented with various concentrations of 2,4- Dichlorophenoxyacetic Acid (2,4-D), and Picloram. No callus was induced despite some of the leaf explants remained viable after certain period of time and most of the leaf explants results in browning conditions whereas callus was induced from seeds supplemented with 2.0 mg/L of 2,4-D after 3 weeks of culturing. The poor callus induction from leaves was highly due to the presence of high poly-phenolic compounds within the miracle berry plants. Future work should utilize different PGR‟s at different concentrations and also the use of different explants.

Key words: Synsepalum dulcificum, Miraculin, Axenic Culture, Callus Culture, Plant Growth Regulators (PGR)

ABSTRAK

Synsepalum dulcificum adalah sejenis pokok renek yang berasal dari keluarga Sapotaceae. Tumbuhan ini mengandungi nilai perubatan yang tinggi dengan adanya komponen aktif dalam Pokok Ajaib yakni Miraculin yang membantu dalam penyakit kronik dan turut berfungsi sebagai agen pengubahsuai rasa. Penyelidikan ini menekankan aspek penghasilan kultur arsenik dan pembentukan kalus dari bahagian daun dan biji Synsepalum dulcificum sebagai sumber eksplan. Daun eksplan yang steril terhasil dari penambahan 1% Benomyl dalam kedua-dua kepekatan larutan iaitu 25% Clorox® (Natrium hipoklorit) yang menghasilkan 91.7% aresenik dan 25% CaOCI (Kalsium hipoklorit) yang menhasilkan 94.4% arsenik dengan pendedahan masa selama 20 minit. Daun eksplan yang steril dikulturkan dalam media Murashige & Skoog (MS) yang ditambah dengan variasi kandungan kepekatan 2,4-Dichlorophenoxyacetic acid (2,4-D), dan Picloram. Walaubagaimanapun, tiada kalus terbentuk walaupun sebahagian daun eksplan masih dalam keadaan sihat tetapi kebanyakkan eksplan berubah warna menjadi perang manakala kalus terbentuk dari biji yang dikulturkan dalam 2.0 mg/L kepekatan 2,4-D. Kekangan pembentukan kalus daripada bahagian daun adalah disebabkan faktor utama iaitu kandungan kimia poli-phenolik yang tinggi dalam pokok ajaib. Kajian yang akan datang seharusnya menggunakan pelbagai PGR’s dalam kepekatan berbeza dan juga jenis eksplan yang lain.

Kata kunci: Synsepalum dulcificum, Miraculin, Kultur Arsenik, Kultur kalus, Hormon Pertumbuhan Tumbuhan

TABLE OF CONTENTS

TITLE PAGE I

APPROVAL SHEET II

DECLARATION III

ACKNOWLEDGEMENT IV

ABSTRACT/ABSTRAK V

TABLE OF CONTENTS VI

APPENDICES VII LIST OF FIGURES VIII

LIST OF TABLES IX

LIST OF ABBREVIATIONS X

1.0 INTRODUCTION 1

2.0 LITERATURE REVIEW

2.1 Botanical Description of Synsepalum dulcificum 4 2.2 History of Synsepalum dulcificum 6 2.3 Habitat and Distribution of Synsepalum dulcificum 7 2.4 Chemical Constituents of Miracle Berry 10 2.5 Economic Importance of Synsepalum dulcificum 12 2.5.1 Diabetic Health Benefits 12 2.5.2 Chemotherapy Health Benefits 13 2.5.3 Weight Loss Benefits 13 2.6 Growth of Synsepalum dulcificum 14 2.7 Plant Tissue Culture as an Alternative Propagation Method 15 2.8 Plant Tissue Culture and Sterilizing Agent 16 2.9 Plant Tissue Culture and Plant Growth Regulators 18

3.0 MATERIALS AND METHODS 3.1 Plant Tissue Culture Medium 20 3.1.1 Preparation of Murashige & Skoog (MS) Stock Solution 21 3.1.2 Preparation of Stock Packs for Used Medium 21

3.2 Establishment of Axenic Culture 22 3.2.1 Effect of Different Clorox ® (Sodium Hypochlorite) Concentrations 23 and Exposure Time 3.2.2 Effect of Different Calcium Hypochlorite (CaOCI) Concentrations 24 and Exposure Time 3.3 Induction of Callus Culture from Leaf Explants 25 3.3.1 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) on Callus Induction 26 3.3.2 Effect of Picloram on Callus Induction 27 3.3.3 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) in Activated Charcoal 28 on Callus Induction 3.4 Induction of Callus from Seed Culture 29 3.4.1 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) on Seed Culture 30

4.0 RESULTS AND DISCUSSIONS

4.1 Establishment of Axenic Culture 4.1.1 Effect of Different Clorox® Concentrations and Exposure Time 32 4.1.2 Effect of Different CaOCI Concentrations and Exposure Time 36 4.2 Induction of Callus Culture from Leaf Explants 4.2.1 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) on Callus Induction 39 4.2.2 Effect of Picloram on Callus Induction 44 4.2.3 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) in Activated Charcoal 50 on Callus Induction

4.3 Effect of 2,4-Dichlorophenoxyacetic (2,4-D) on Seed Culture 54

4.4 Factors Contributing to Inhibition of Callus Formation 57 4.4.1 Browning of Culture Materials 57 4.4.2 Age of Explants 59 4.4.3 Effect on the Presence of Contaminants on Explant Culture 61

5.0 CONCLUSIONS AND RECOMMENDATIONS 63

6.0 REFERENCES 65

APPENDICES 73

APPENDICES

Appendix Page

A Murashige and Skoog (1962), MS medium composition 73

B Results of Surface Sterilization from leaf explants Synsepalum 74 dulcificum Daniel (Miracle Berry)

C Statistical Analysis of Clorox® and CaOCI Solution 76

D Anova Analysis of Clorox® and CaOCI Solution 77

LIST OF FIGURES

Figure Page

1 Geographical range habitat of Synseplaum dulcificum in Tropical 7 West Africa Region

2 Map showing Benin within the Dahomey gap encircled 8

3 Map showing the current suitable habitat distribution of Synsepalum 8 dulcificum.

4 Seed Culture supplemented in 0.0 mg/L of 2,4-D for callus induction 31

Seed Culture supplemented in 2.0 mg/L of 2,4-D for callus induction 31

Seed Culture supplemented in 3.0 mg/L of 2,4-D for callus induction 31

Seed Culture supplemented in 5.0 mg/L of 2,4-D for callus induction 31

5 (a) Contamination occurred on Day 2 in 20% Clorox Concentration 34

(b) Fungi contamination in 25% Clorox Concentration on Day 3. 34

6 The effect of Clorox® solutions with 3 different treatments on the 35 percentage of axenic cultures

7 (a) Contamination in 20% CaOCI Concentration at Day 4. 37

(b) Contamination in 25% CaOCI Concentration at Day 5. 37

8 The effect of 3 different treatments concentrations of CaOCI 38 solutions on the percentage of axenic cultures.

9 (a) Leaf explants after 4 weeks culturing on 0.0 mg/L of 2,4-D. 40

(b) Leaf explants after 6 weeks culturing on 1.0 mg/L of 2,4-D. 40

(d) Leaf explants after 8 weeks culturing on 5.0 mg/L of 2,4-D. 40

(e) Leaf explants after 6 weeks culturing on 1.0 mg/L of 2,4-D under 43 light condition for 4 days.

(f) Leaf explants after 6 weeks culturing on 3.0 mg/L of 2,4-D under 43 light condition for 4 days.

(g) Leaf explants after 6 weeks culturing on 5.0 mg/L of 2,4-D under 43 light condition for 4 days.

10 (a) Leaf explants after 5 weeks culturing on 0.0 mg/L Picloram. 46

(b) Leaf explants after 7 weeks culturing on 1.0 mg/L of Picloram. 46

(d) Leaf explants after 6 weeks culturing on 5.0 mg/L Picloram. 46

11 (a) Leaf explants after 5 weeks supplemented with 0.0 mg/L Picloram 48 under light conditions for 4 days.

(b) Leaf explants after 2 weeks supplemented with 1.0 mg/L Picloram 48

(d) Leaf explants after 6 weeks supplemented with 5.0 mg/L Picloram 48 under light conditions for 4 days.

12 Contamination of contaminants after 10 weeks of culturing on 49 different concentration of Picloram media.

13 (a) Leaf explants after 5 weeks culturing on 0.1 mg/L 2,4-D 52 supplemented with activated charcoal.

(b) Leaf explants after 5 weeks culturing on 0.3 mg/L 2,4-D 52 supplemented with activated charcoal.

(d) Leaf explants after 5 weeks culturing on 0.8 mg/L 2,4-D 52 supplemented with activated charcoal.

14 (a) Seed culture on 2.0 mg/L of 2,4-D under dark condition. 54

(b) Induction of callus from seeds as indicated by the whitish 54 appearance on the top part of seeds.

15 (a) Seed Culture in 0.0 mg/L of 2,4-D after several weeks of 55 culturing.

(b) Seed Culture in 3.0 mg/L of 2,4-D after several weeks of 56 culturing.

16 (a) Testfor the presence of contaminants from leaf explants obtained 62 from 2,4-D culture.

(b) Testfor the presence of contaminants from leaf explants obtained 62 from Pcloram culture.

17 (a) No presence of contaminants observed after 2 weeks of culturing 62 in broth culture of leaf explants from 2,4-D culture.

(b) No presence of contaminants observed after 2 weeks of culturing 62 in broth culture of leaf explants from Picloram culture.

LIST OF TABLES

Table Page

1 Treatments of different Clorox® Concentrations and Exposure Time on 23 Surface Sterilization.

2 Treatments of different CaOCI Concentrations and Exposure Time on Surface 24 Sterilization.

3 Callus Induction in different MS media supplemented with various 26 concentration of 2,4-D.

4 Callus Induction in different MS media supplemented with various 27 concentration of Picloram.

5 Callus Induction in different MS media supplemented with various 28 concentration of 2,4-D in activated charcoal.

6 Callus Induction from seed culture in different MS media supplemented with 30 various concentration of 2,4-D.

7 Percentage of axenic, contaminated and damaged leaf explants after different 34 Clorox® sterilization treatments.

8 Analysis of Variance (ANOVA) of 3 different treatments of Clorox® 77 Concentration on axenic cultures.

9 Percentage of axenic, contaminated and damaged leaf explants after different 37 CaOCI sterilization treatments.

10 Analysis of Variance (ANOVA) of 3 different treatments of CaOCI 77 Concentration on axenic cultures.

LIST OF ABBREVIATIONS

PGRs Plant Growth Regulators

2,4-D 2,4-Dichlorophenoxyacetic Acid

MS media Murashige and Skoog media

KOH Potassium hydroxide

HCl Hydrochloric Acid

ANOVA Analysis of variance

CaOCI Calcium hypochlorite

CRD Completely Randomized Design

Picloram 4-amino-3,5,6-trichloro Picolinic Acid

FDA Foods and Drugs Administration

1.0 INTRODUCTION

Miracle berry (Synsepalum dulcificum) or its vernacular name “Buah Ajaib” is an important source of medicinal plant which belongs to Sapotaceae family. This plant has its origin in Tropical West Africa which is a slow growing shrub that produced fruits occasionally throughout the year. As stated by Ogunsula and Ilori (2008), the fruits are small with approximately 2 to 3 cm long ellipsoid berries. The fruits are surrounded by edible pulp covering an elongate ovoid shape seed which changes colour into bright red upon ripens (Chen et al., 2012). The interesting facts about this fruit lie within the ability of its flesh pulp that is able to modify sour taste foods into sweet upon consumption (Yong,

2011). This unordinary effect was first documented in the scientific literature by Kurihara and Beidler (1968). The effect will usually last for about 30 minutes and beyond thus making it an extraordinary fruit that brings about all sour foods such as lemon to be tasted sweet when being consumed (Ogunsula and Ilori, 2008). This is due to the presence of

Miraculin compound which is a type of glycoprotein that is able to alter our sweet receptor buds by manipulating the configuration of proteins on our tongues.

At present, Synsepalum dulcificum has been treated as an important source of medicinal plant. It has been used to provide treatments for chronic disease patients who had typically developed their blunt taste sensation due to various treatments (Peregrin, 2009). Another issues concerning health benefits were an extravagantly consumption of sugar that may impose several negative effects which were hazardous to human health. Many artificial sweeteners such as aspartame had been used widely as it can alter our sweet desire without undesirable calories on blood sugar levels and health. Nevertheless, the circumstances after consumption of aspartame gave an unpleasant side effect. However, miracle berry eliminates the possibilities of having any side effects after consumption (Levin, 2012).

This has been proven by Foods and Drugs Administration (FDA) in 1970‟s that miraculin compound were certified to be legally used as sugar substitute subsequently having examined for safety and efficacy. This had been further supported by Dr.Linda Barthoshuk in the 1960‟s in an experiment conducted for Smell and Taste in University of Florida

Center that no health risk were related to the consumption of miracle berry fruit. Hence, it has been commercialized as substitute for food additives as sweetener due to the facts that only little amount were utilised to make the food taste sweet as compared to the excessive used of food additives (Bartoshuk, 1974). In short, this fruit had been used as a medicinal plant which is believed to aid in diabetic patients and also as a source of daily products in food manufacturing as food sweeteners. Due to its increasing economically medicinal values and great commercial potential of miraculin, there has been a high growing demand for the production of a large scale yield of Synsepalum dulcificum.

However, raising the plants from seed posed several problems. As stated by Ogunsula and

Ilori (2008), the seed are recalcitrant and (Chen et al., 2012) this plants are “difficult to root” species thus production of large scale yield had been an obstacle. Following a great commercial potential of miraculin, there is a need to develop an effective way to yield more miracle berries. Therefore, there is a need to investigate other effective methods that can contribute to the regeneration of this species. Relatively little information were documented about the growth and development of Synsepalum dulcificum in tissue culture practices. Several successes have been reported in regenerating plants through propagation by stem cutting method by (Chen et al., 2012) and in vitro propagation through embryo and nodal cultures by (Ogunsula and Ilori, 2008). The recognition of the planting method had to be devised to search out an alternative approach in regenerating miracle berry plants other than conventional method of propagating by stem. Hence, an alternative approached

by using tissue culture method on leaves part as source of explants had been used in this study. It is worth seeking of developing this technology practices (tissue culture) to aid in future studies on this plant with potential application to its propagation for its recognition potential in nutritional and medicinal purposes.

According to Razdan (2003), tissue culture techniques have highly been used to assist the traditional techniques for plant improvement. Anchoring from this method, a number of plants can be regenerated or produced in vast amount in a short period of time. So far, there have been no report and research results documented regarding the regeneration of

Miracle berry plants through leaf explants by tissue culture methods. Hence, the need to investigate the regeneration of Miracle berry plant has been the centre of this research study. This research study emphasized on the callus induction of Miracle berry plant through leaf explants and seed culture by tissue culture methods. According to Ogunsula and Illori (2008), Miracle berry plant are recalcitrant and is rather difficult for this plant to be propagated by seeds or through cutting as the seeds desiccated in a rapid rate after being harvest. Therefore, it is essential to look for alternatives ways to regenerate this plant other than propagating methods. Hence, the present objectives of this study are:

1. To establish an axenic culture of Synsepalum dulcificum explants.

2. To determine the effect of different Plant Growth Regulators on callus formation

from leaf explants and seed culture.

2.0 LITERATURE REVIEW

2.1 The Botanical Description of Synsepalum dulcificum

The taxonomic classification and nomenclature of Synsepalum dulcificum are as follow:

Division: Magnoliophyta

Class : Magnoliopsida

Order : Ericales

Family : Sapotaceae

Genus : Synsepalum

Species : dulcificum

Scientific name: Synsepalum dulcificum

Synsepalum dulcificum is a small, evergreen shrub grows very slowly to a height of 4 to 6 feet in container, and 10 to 15 feet in natural habitat. The growing plant eventual size depends on where the plant is being grown. A 10 years old plant might be easily only 4 to

5 feet tall. It forms an oval to pyramidal shaped bush or small tree.

The leaves are dark green, glossy and glabrous. The foliage is elliptical in shape, with a cuneate base, entire leaf margin and an obtuse apice. The leaves are 5-10 cm long and 2-3 cm wide. The flowers are quite small and points downwards. It measures approximately 1 cm from the brown base to the creamy- white coloured pedal tips.

The flowers only partially open, revealing the stigma and pistil, a notable characteristic of the Sapotaceae family. As the flower ages and the ovary ripens, the petals turn a dark red and open up more, revealing the inside workings.

The fruit is the most miraculous part of this plant. Fruits are bright red, 2-3 cm long and ellipsoidal in shaped. The fruit itself is not particularly flavourful, however, once eaten; anything bitter or sour will become sweet taste and the effect may last for about thirty minutes to a few hours, depending on the conditions in which the plant itself was grown.

The colour of the miracle fruit plant does not vary much throughout its lifespan. Its bark is a light grey to medium brown. The foliage is a deep green like many tropical. Flowers are creamy-white with brown bases. The miracle fruit itself is bright red or scarlet in colour

(Pascoe, 2011).

It has an inconspicuous brown-and-white half inch flowers followed by bright scarlet, 1 inch football-shaped fruit, sweet and pleasant tasting. The plant starts fruiting when only 1 feet tall. It produces fruit practically year around. In native habitat, two large crops are available yearly, each after a rainy season. The mature bushes usually have a few fruits hanging around all years. It will usually takes about 2 to 3 years for seed to fruit whereas from flower to fruit usually results in 30 to 45 days (Anon, 2014). Synsepalum dulcificum or known as Miracle Berry is an important source of medicinal plants that is widely used worldwide. According to Ogunsula and Illori (2008), its habitat lies within evergreen tropical area which has a shrub plant habit. It has a distinctive characteristic that can altered or changed all sour foods into sweet due to the presence of Miraculin (a type of glycoprotein) that binds to our sweet receptors and changes its configuration on our tongues. Production of large scale yield of miracle berry has been hindrances since the plant are difficult to root species and the seeds are recalcitrant.

2.2 History of Synsepalum dulcificum Daniel

Miracle berry plants were originated in West Africa and it has been widely used as an important source of food to native African for centuries where they relies on this miracle berry as a source of foods which barely within few basic foods, mostly were of sour taste

(Anon, 2014). When these miracle berries were first tasted, the magnificent altering taste of sour foods into sweet had become a surprised. Anchoring from this finding, the West

African natives had utilised this miracle berry fruit to sweeten sour palm wine (beer) which were regarded as Pito, and fermented maize bread which were known as Kenkey. Their berries were used as food taste modifier and preservative where available food were preserved for a long periods of time. Due to this unique characteristics displayed by this berry, there is a tendency for the identification of this berry. As stated by Sabel (2012), in the early of the year 1700; these miracle berries were authenticated by a French explorer

Reynaud Des Marchais following the consumption by the native African. Subsequently during the mid-1880s, this berry fruit had been generalized and issued by a botanist after

Dr. W.F. Daniell. According to Anon (2014), this astonishment to West African was not botanically or taxonomically identified, described and named until the middle of the 19th century as Synsepalum dulcificum, which is a member of the Sapotaceae family, relative of the sapodilla (Manilkara zapota). Much research had been conducted on miracle berries as possible alternative for sugar substitute. As such, Synsepalum dulcificum plants were introduced to America by David Fairchild during the dawn of 20th century. Bill Whitman was the first to grow the plant successfully in the United States of America. He had a seedling all of 7" tall and a cutting of 4" bearing fruit upon the young twigs.

2.3 Habitat and Distribution of Synsepalum dulcificum

As stated by Akpla and Fandohan (2014), the current geographical range habitat of

Synsepalum dulcificum lies on southern Benin below 7˚ north as shown in Figure 1. The

Republic of Benin (6-12°N; 0.40-3°E) is a part of West African country located within the

Dahomey Gap as shown in Figure 2. Geographically, Benin had a coverage area of

112,622 km² of which 22.7 % of the area is legally protected. Much information regarding its distribution had been retrieved. Likewise, this species had been distributed to United

States of America (USA) in the year 1919 by Fairchild (1931). In fact, it has been successfully grown by Bill Whitman in the 19th century. This miracle berry had been widely produced and cultivated in Taiwan for large production (Fooladi, 2012).

Furthermore, Kurihara and Beidler (1968) stated that these miracle berries were also encountered in Oban Hills in Cross River National Park and were also available in Japan

(Madison, 2008). Therefore, this remarkably proven that their distributions in Asia region were significant. Nonetheless, the present study of this species did not yield any documentation on how it has been distributed to Malaysia. In brief, this species were believed to have distributed across a number of regions.

Figure 1: Geogrphical range habitat of Synseplaum dulcificum in Tropical West Africa Region (Adapted from Fleurs, 1997).

Figure 2: Map showing Benin within the Dahomey gap encircled (Adapted from Olson et al., 2001).

Figure 3: Map showing the current suitable habitat distribution of Synsepalum dulcificum (Adapted from Akpla et al., 2014).

Climatically, Benin is subdivided in three main regions: the semi-arid, the sub-humid dry and the sub-humid humid. According to Inglett and May (1968), miracle berry were farms growing based and around dwellings occurs in surrounding forests, and is common on the coast in Ghana. The predicted range under the current climate conditions shows that the suitable areas for Synsepalum dulcificum were located in the south of Benin as shown in

Figure 3, which was characterized by wet subequatorial climate. The highly current

suitable areas for the species cultivation (6,510 km²) were imbedded in the West Plateau and Ouémé-valley phytodistricts. The moderately suitable habitats (3,671 km²) were mainly in the east Plateau phytodistrict while the poorly suitable areas correspond to the

Coastal phytodistrict and the southern part of Pobe and Zou phytodistrict. However, globally apart from the south–eastern part of Zou phytodistrict, there were only some spots of poorly suitable habitat in the remaining parts of this phytodistrict up to the centre of

Benin. Moreover, no information and records were obtained in the centre and north of the country.

According to Sabel (2012), Synsepalum dulcificum originates from Tropical West Africa, which had been highly commercial farmed by farmers of rare fruit in Taiwan, Jamaica,

Puerto Rico, Guam, and South Florida. Its habitat lies within and grows ideally in a humid, warm environment and while resistant to varying water levels; it cannot withstand any sort of freezing. This plant was well adapted to a slightly acidic soil environment (pH 4.5-5.8) and produces edible fruits about 3 years after planting. The fruit grows in a single season ranges throughout from May to September, though berries are generated year-round. When grown from seedlings, it takes three to four years before fruiting occurs; the bush grows slowly and eventually reaches six to fifteen feet in height when fully mature (Adansi,

1970).

2.4 Chemical Constituents of Miracle Berry

The active component constituent in this miracle berry is miraculin compound (Gollner,

2008). Miraculin (MCL) is a glycoprotein molecule extracted from fruit Synsepalum dulcificum which is native to the African. According to Sabel (2012), miraculin is a single polypeptide chain which consists of 191 amino acids with the presence of two glycosylated polypeptides which were Asn-42 and Asn-186 linked by disulfide bond. As stated by

Temussi (2006), miraculin is a macromolecule compound which has a molecular mass of

24,600 and it has been verified to be approximately 400,000 times sweeter than sucrose on same molecular mass basis. Its properties as food modifier had been remarkably recognised which were able to convert taste bud stimuli from sour taste into sweet.

According to Gimbson (n.d.), this glycoprotein molecule adheres to the sweet receptors of the tongue and it triggers the inactivation of the sour and bitter taste bud which leads the sour taste food into sweet. Upon consumption, this miraculin compound will retained on tongue bud and this configuration will triggers sweetness after tasting sour foods in tandem this effect usually lasts for one to two hours (Koizumi et al., 2011). The sweet receptors will recognized the sour food as sugar which taste sweet and this effect will last for at most an hour right until the saliva washes off the protein molecule. This effect is apparent when it comes in contact upon acidic pH. Miraculin compound has the ability to evoke the sweetness taste from several acid formulations which primarily includes hydrochloric acid, oxalic acid, lactic acid, formic acid, acetic acid and citric acid. The actual sweetness effect is dependent upon the rate of acidity (Kurihara and Beidler, 1968).

In addition to miraculin, other secondary metabolites chemical constituents have also been reported to be isolated from miracle berries. These include tannins and other poly-phenolic