Industrial Crops and Products

Phytochemicals, nutritional and antioxidant properties of the miracle

fruit Synsepalum dulcificum

Zuxing He , Joo Shun Tan , Sahar Abbasiliasi , Oi Ming Lai , Yew Joon Tam Arbakariya B. Ariff

Synsepalum dulcificum, also called the miracle fruit, which has the sweet-inducing activity can be used as additives in food, medicine, and cosmetic industries. Some selected chemical properties of miracle fruit including percentage by weight, total anthocyanin, the phenolic and antioxidant content of different parts of miracle fruit as well as physicochemical analysis of seed oil, nutritional elements of fruit juice were determined in this study. The results showed that miracle fruit contains a large amount of vitamin C (40.1 mg/100 g fresh fruit weight (FW)), phenolic content (625.57 mg GAE/100 g FW), high antioxidant capacity (457.3 mol Trolox/100 g FW) and low total sugar content (5.6 g/100 g FW), suggesting that the fruit is healthy for human consumption. According to its fatty acid composition and Triacylglycerol (TAG) profile, miracle fruit seed oil is rich in oleic and palmitic acid.

Introduction

The miracle fruit, Synsepalum dulcificum, is an evergreen shrub belonging to the Sapotaceae family, Synsepalum genus. Miracle fruit is described as a berry that can change sour to sweet taste. The pulp of miracle fruit contains miraculin, a glycoprotein, which can cause sour food to taste sweet while it is tasteless. The sweet-inducing activity of miracle fruit could be exploited for use in food, medicine and cosmetic industries as sweeteners or additives.

The Miracle fruit plant is a shrub that grows up to 6.1 mhighinits native habitats but does not usually grow higher than 3 m in cultivation. The plant leaves are 5–10 cm long, 2–3.7 cm wide, and glabrous below while the flowers are brown in color. The ripened miracle fruits are red in color with about 2 cm long and they are clustered at the ends of the branches. Each fruit contains a thin layer of edible pulp surrounding an elongate-ovoid shape seed The plant can be harvested twice a year, so the yield is stable. The pulp of miracle fruit, the only part of the fruit that contains miraculin, takes only 4.44% weight of the fresh fruit.

Furthermore, antioxidants which are normally related to phenolic compounds, are considered to have the effect to improve the quality and nutritional value of food. At the same time, the search for antioxidants from natural sources has attracted increasing attention due to the widespread agreement of the potential health risks and toxicity of synthetic antioxidants such as BHA and BHT. The importance of antioxidant constituents of plant materials in the maintenance of health and protection from coronary heart disease and cancer is also raising interest among scientists, food manufacturers, and consumers as the trend of the future is moving toward the development of functional food with specific health effects.

Materials and Methods

2.1. Miracle fruit

Fresh ripened miracle fruits -  the skin and seed were separated from the pulp of the miracle fruits using a knife, and all of the skin, pulp, and seed were freeze-dried and ground into a fine powder. The powder was kept at − 30 ◦C prior to extraction.

2.2. Physicochemical analysis

Dry matter, crude protein, ash, total sugar and total dietary fiber, Weight percentages of different parts of miracle fruit, Vitamins A and C  were determined in this procedure.

2.3. Determination of anthocyanin, total phenolic, and antioxidant contents

2.3.1. Sample preparation

In the extraction method minor modifications were done. Ground samples were extracted with 100 mL of 80% aqueous methanol at room temperature using an orbital shaker for 120 min. The mixture was subsequently filtered and the filtrate was centrifuged for 10 min. The solvent supernatant was then transferred to tubes. All extracts were stored at - 30 °C prior to use in analyses.

2.3.2. Determination of total antioxidant

The total antioxidant in the extracts of skin, pulp, and the seed was determined according to the DPPH radical scavenging activity test where the absorbance was measured at 515 nm. The calibration curve was determined using Trolox 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid), as a standard with a range of concentration from 0.01 mM to 1.25 mM. The results were presented using Trolox equivalents as mmol/L. The results were then converted to Mtrolox/100 g of fresh fruit weight and M trolox/g of dry fruit weight.

2.3.3. Determination of phenolic content

The phenolic content in the extracts of skin, pulp, and seed were determined. The reaction mixture contained 50 L of extracts, 250 L of the Folin-Ciocalteu reagent (Sigma, America). The absorbance was measured at 765 nm. Gallic acid was used as standard and the results were presented as mg of gallic acid equivalents per 100 g fresh fruit weight (GAE/100 g fresh fruit weight (FW)).

2.3.4. Determination of total anthocyanin content

The total anthocyanin content was determined by the pH differential method.  Anthocyanin pigments transform reversible structures with a change in pH proven by strikingly different absorbance spectra. The colored oxonium form predominates at pH 1.0 and the colorless hemiketal form dominates at pH 4.5. The pH-differential method is based on this reaction and permits accurate and rapid measurement of the total anthocyanins.

2.4. Characterization of miracle fruit seed oil

The miracle fruit seed oil (MFSO) was characterized by Triacylglycerol (TAG) profile using high-performance liquid chromatography (HPLC), fatty acid composition using gas

chromatography (GC) and thermal behavior with differential scanning calorimetry.

2.4.1. Seed oil extraction

After the removal of the seed coating, the oil sample was extracted from the crushed seed kernels by Soxhlet extractor with petroleum ether (Sigma-Aldrich, America), following the method with some modifications. Leaching was carried out at the boiling point of the selected solvent for 8 h. The extracted phase (oil and solvent) was then distilled using a rotary evaporator with a vacuum pump to ensure complete removal of the solvent. The oil sample was stored at − 20 ◦C prior to using in the analysis.

2.4.2. GC analysis of seed oil of miracle fruit

The MFSO were analyzed as fatty acid methyl ester (FAMEs), prepared using the method with some modifications. After the esterification of the sample, hexane was added and vortexed for 5 min. The hexane layer containing FAME was placed in a chromatography vial to be analyzed using gas chromatography or kept at − 20 ◦C for further analysis. FAME was analyzed with Agilent 7890A (Santa Clara California, USA), GC equipped with Flame Ionization Detector (FID). The separation was carried out using capillary column BPX70 70% Cyanopropyl Polysilphenylene-siloxane (SGEAnalytical Science, Ridgewood Victoria, Australia), 30 m in length, with an internal diameter of 0.32 m and nitrogen as carrier gas with a flow rate fixed at 5.7 mL/min. The injector and detector temperatures were set at 250 and 280 ◦C, respectively. The split ratio used was 15:1. The oven temperature was programmed as follows: holding at 1 ◦C for 4.6 min, 100–170 ◦C at a rate of 10 ◦C/min, 170–230 ◦C at a rate of 1.5 ◦C/min, and hold at 230 ◦C for 7 min, 230–250 ◦C at a rate of 30 ◦C/min and hold at 250 ◦C for 1 min.

2.4.3. Determination of thermal behavior of seed oil of miracle fruit

The crystallization and melting thermograms of the MFSO were determined firstly, the sample was heated to 80 ◦C and held for 10 min to destroy any crystal memory contained in the sample. Thereafter, it was cooled to − 70 ◦C at a rate of 5 ◦C/min and held at − 70 ◦C for 10 min to obtain the crystallization thermogram. The sample was then heated from − 70 ◦C to 80 ◦C at a rate of 5 min/min and held at 80 ◦C for 10 min. An effort was made to ensure that all samples were of the same weight but not identical. All determinations were carried out with duplicate analysis.

2.4.4. Acylglycerol composition

Reacted samples were analyzed for their acylglycerol composition using Alliance e2695 Separation Modules High-performance liquid chromatography (HPLC) (Waters, USA) coupled with evaporative light scattering (ELS) 2424 detector (Waters, USA). The sample was prepared by adding a 5% w/v oil sample in acetone and 10 L of the sample was injected into Purospher®Star RP-18e column (5 m, 250 mm × 4 mm, Merck, Germany). Mobile phase set gradient of acetone (A) and acetonitrile (B) with a flow rate of 1 mL/min and programmed as follow; 0 min: 90% B, 8 min: 85% B, 40 min: 10% B, 50 min: 90% B, 52 min: 90% B. The drift tube temperature, nebulizer power, and gas pressure of the ELS detector were set at 45 ◦C, 60% (heating), and 35 psi, respectively.

2.4.5. Statistical analysis

All experiments and analyses were replicated three times for each sample. The results were presented as mean ± standard deviation(SD). The Differences of the physicochemical properties of fruits harvested from several plants at different times were statistically analyzed using one-way ANOVA in SPSS 17.0. The significance of differences between the means was determined using Tukey's test(p < 0.05). In most cases, the mean values for the different fruits harvested from different plants at different times were not significantly different (data are not shown).

Results and Discussion

3.1. Proximate chemical analysis

The weight percentage of pulp, seeds, and skin in the miracle fruit is summarized. The seeds occupied 64.11% of lyophilized fruit, while skin and pulp take 15.47% and 20.42%, respectively. The data of a dry basis. However, the water content in the fresh fruit (65.33%) obtained in this study was lower than that reported by the same researchers (75.22%). The edible part of miracle fruit is low because of the high proportion of seed. The proximate chemical composition of miracle fruit flashes. The total sugar, total dietary fiber, ash, carbohydrate, vitamin A and vitamin C were found to be 5.6 g/100 g FW, 12.5 g/100 g FW, 1.0 g/100 g FW, 22.5 g/100 g FW, 37.3 g/100 g FW, and 40.1 mg/100 g FW, respectively. It is interesting to note that the FL flashes of miraculous fruit did not contain any fat. It is well known that fruits are the main sources of vitamin C.

The vitamin C content in miracle fruit is close to that in Citrus fruits which are commonly thought to be excellent sources of vitamin C. Moreover, as the potential functional food in diet for the patients of diabetes and obesity, total sugar content in miracle fruit is low, which is lower than berries. The total sugar content of miracle fruit is similar to raspberry (5.58 g/100 g FW), which is considered as fruit with low sugar content.

3.2. Determination of antioxidant phenolic, and anthocyanin content

The total content of antioxidant, phenolic, and anthocyanin in different parts of miracle fruit determined where phenolic content was found in the skin, pulp and seeds. The study confirmed that phenolic substances are concentrated in the skin. Because of the different weight ratios, skin contribution. In comparison to berries, which are usually considered as a fruit rich in phenolic content, the total phenolic content in the flesh of miracle fruit is much higher. Considering the edible part in miracle fruit is quite little, the phenolic content in flesh is very high.

The high antioxidant capacity of miracle fruit indicates that it can be used as a supplement to improve human health. It is well known that dietary antioxidants can stimulate cellular defenses and help to prevent cellular components against oxidative damage. The total anthocyanin content of miracle fruits may be possibly responsible for the red color of the fruit.

Thus, antioxidant capacity may significantly affect the fruit itself. Moreover, evaluation of the total antioxidant capacity of fruits, vegetables, and other plant products cannot be performed accurately by any single method due to the complex nature of phytochemicals and different detection methods may deliver variation. High vitamin C and phenolic content may together contribute to the high performance of antioxidant capacity of miracle fruit.

3.3. Proximate analysis of seed oil of miracle fruit

The weight of seed oil from miracle fruit was 6% of the total weight of the freeze-dried seeds. It was light green in color and viscous at room temperature.

Miracle fruit seed oil(MFSO) may have the same potential application as palm oil-producing edible oil due to its similar fatty acid composition. Palmitic acid, stearic acid, oleic acid, and linoleic acid made up 91.8% of MFSO. Oleic acid is the most common monounsaturated acid and also the most common acid produced in nature. Stearic acid was reported to be as effective as oleic acid in the role of lowering the plasma cholesterol level.

Triacylglycerol (TAG) composition analysis

HPLC chromatogram shows the triacylglycerol composition in the seed oil. The MFSO contained 77% triacylglycerol. The results indicated that the most prominent monounsaturated TAG was palmitic acid.

All the triacylglycerol species identified in MFSO were either monounsaturated or polyunsaturated forms with a total degree of unsaturation of 74.3%. What was notable, the graph also demonstrates there was a large amount of diacylglycerol(DAG) in MFSO, which was around 22.4%. Comparison of TAG profile of MFSO and some other oils. The TAG profile of miracle fruit seed oil is extremely similar to the one of palm oil. Unlike sunflower oil, soybean oil, and sesame oil, which were dominated by oleic acid: linoleic acid: oleic acid (OLO), and OLL in TAG profile, MFSO contained a lot of POO and POP.

Conclusion

For a full understanding of potential utilization of miracle fruit, some Physico-chemical properties of this fruit including percentage weight and nutritional elements (ash, crude protein, fat, total dietary fiber, carbohydrate, and energy), total anthocyanin, phenolic, and antioxidant content of different parts of miracle fruit were demonstrated.

The melting and crystallization behavior, fatty acid composition, and triacylglycerol composition of miracle fruit seed oil were also presented. Although the edible part of miracle fruit is not very much, the flesh of miracle fruit contains a large amount of vitamin C, phenolic content which gives high antioxidant capacity.

The seed of miracle fruit is also a good source of antioxidants because of a large weight ratio. Moreover, the total sugar content in the flesh of miracle fruit is low, indicating that it may be healthy for human consumption, especially as sweeteners for patients suffering from diabetes and obesity. The yield of oil from seeds of miracle fruit is 6%, the oil is green, viscous, and fluid at room temperature. Fatty acid composition analysis found 10 fatty acids in the seed oil, the major components are oleic acid, palmitic acid, and linoleic acid.

Reference:

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