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Primary recovery of miraculin from miracle fruit, Synsepalum
dulcificum by AOT reverse micellar system

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

LWT - Food Science and Technology


                 Miracle fruit, Synsepalum dulcificum, contains a glycoprotein known as miraculin. After consuming this glycoprotein, sour foods taste sweet and the effect lasts for up to 4 hours. With increasing demand for natural and “low-calorie” sweeteners, the use of miraculin as an additive is increasing enormously in the food, medicine and cosmetic industries. 



                 Synsepalum dulcificum, a shrub native to tropical West Africa, produces red berries that have the unusual ability to modify a sour taste into a sweet taste. The active ingredient in the berries, miraculin, is a taste-modifying protein that causes the sour taste components such as citric and ascorbic acids to be perceived as sweet after consumption in the mouth. 
                 The mechanisms behind the sweet inducing activity of miraculin have not yet been identified but histidine residues in miraculin have been linked to its taste modifying activity. Twenty micrograms of chromatographically purified miraculin produce a marked increase in the sweetness of lemon and concomitantly a marked diminution of sourness. 
                 However, the activity of miraculin is prone to be destroyed when the solution is boiled or exposed to a high concentration of organic solvents at room temperature. The activity was also decreased at high pH (pH > 12) and is greatly decreased (pH < 2.5). Although a lot of experiments have been carried out to explore the structure and mechanism of miraculin and to study the actual function of miraculin, the purification procedures for miraculin nowadays are thought to be labour-intensive, time-consuming and costly. 
                 The main objective of this study was to investigate the feasibility of using the reverse micelle extraction method to extract miraculin from S. dulcificum. The effects of various factors that might influence performance were evaluated, such as crude pH, surfactant concentration during forward extraction and pH, isopropanol concentration, and salt concentration in the aqueous phase during backward stripping. The significant factors were also optimized to enhance extraction yield and product purity. 


 Materials and methods

2.1. Miracle fruits

The skin and seeds of fresh miracle, S. dulcificum fruits were separated manually using a knife and the pulp was freeze-dried, then ground into a fine powder using a blender. The pulp powder was kept at 30 C prior to use in the extraction and purification procedures.


2.2. Chemicals

            Sodium di (2-ethylhexyl) sulfosuccinate (AOT) used without further purification, Isooctane, Bradford reagent, Sodium chloride, Isopropanol, Miraculin standard (~95% purity).


2.3. Preparation of miraculin extract

The extraction of miraculin was carried out with some modifications. In this method, 4 g of lyophilized pulp powder was suspended with 40 mL of water and homogenized for 2 min.  The homogenate was centrifuged at 12,000g for 30 min. After discarding the supernatant, the sediment was homogenized for 2 min in 30 mL of 0.5 M NaCl solution (pH 6.8). The homogenate was clarified by centrifugation at 12,000 g for 20 min and the colourless supernatant at pH 3 was stored at 30 C.


2.4 Forward extraction

We performed the forward extraction and backward stripping with some modifications. Briefly, the organic phase was prepared by dissolving various concentrations of AOT (0.03, 0.05, 0.1 and 0.2 mol/L) in isooctane. The pH of the crude was adjusted with either 1 mol/L NaOH or 1 mol/L HCl. The isoelectric point (pI) of miraculin is 9 and miraculin was reported to be stable during storage between pH 2.5 and 12. Based on this information, the pH of the crude in the aqueous phase was adjusted to various pH values ranging from 3 to 10 to avoid miraculin precipitation and loss of activity during the experiments. Equal volumes (0.5 mL each) of aqueous and organic solutions were mixed gently in a tube and the mixtures were then shaken mechanically for 10 min. The mixtures were then centrifuged at 4000 g for 5 min to reach a clear separation of the two phases.


2.5. Backward stripping

The reversed micellar solution with loaded protein was added to an equal volume of an aqueous solution which consisted of 0.02 mol/L phosphate buffer at the required pH (7, 8, 9, 10 and 11) in a tube. The required concentrations of NaCl (0, 0.5, 1, 1.5 and 2 mol/L) and isopropanol (0, 50, 100, 150 and 200 mL/L) were also added to the tube. The organic phase and fresh aqueous phase were shaken for 20 min. The mixtures were then centrifuged at 4000 g for 5 min to reach a clear separation of the two phases. The total protein in the stripping aqueous solution was determined.


2.6. Total protein assay

The total protein concentration in the crude sample was determined using the Bradford method using bovine serum albumin (BSA) as a standard. Ten mL of the sample was added to 200 mL of the diluted dye reagent (1 part dye reagent concentrate with four parts distilled, deionized water) in a microtiter plate and incubated at room temperature for at least 5 min. The absorbance was measured at 595 nm against a reagent blank.


2.7. Reverse-phase high-performance liquid chromatography analysis

The miraculin concentration in the sample was analyzed using reversed-phase high-performance liquid chromatography with some modifications. Sample (40 mL) was injected into the column and equilibrated with 1 mL/L trifluoroacetic acid (TFA) in water. The column was eluted using a linear gradient of acetonitrile with increasing concentration from 150 mL/L to 700 mL/L, and the flow rate was fixed at 1 mL/min. The absorbance of the sample was read at 280 nm. Miraculin standards were prepared at concentrations ranging from 100 to 1000 mg/L. The relationship between miraculin concentration (mg/L) and peak area (AU min) was observed as 0.00013 mg miraculin/L/peak area. The purity of the peak was analysed based on the percentage of total peak area using Empower software (System Software, Waters Co.) for data acquisition and analysis.


2.8. SDS-PAGE and silver staining

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a Bio-Rad electrophoresis unit as described by Laemmli (1970). The acrylamide gel was prepared as a 120 mL/L resolving gel and a 45 mL/L stacking gel. Protein samples recovered from the top phase were concentrated and precipitated using 100 mL/L trichloroacetic acid (TCA) solution, which removed the salts that affect the electrophoresis process. The pellets were resuspended in denaturing buffers (0.1 mol/L Tris HCl pH 6.8, 40 g/L SDS, 100 mL/L 2-mercaptoethanol, 200 mL/L glycerol and bromophenol blue). The electrophoresis was run at 110 V and 36 mA for 75 min. The gel was stained with a buffer solution consisting of 0.5 mL/L Coomassie Brilliant Blue G-250, 300 mL/L methanol and 100 mL/L acetic acid. After destaining, protein bands were visualized using the same buffer solution in the absence of Coomassie Brilliant Blue. The gel was then stained with a PageSilver™ silver staining kit (Fermentas, St. Leon-Rot, Germany).


2.9. Miraculin sensory analysis

The taste-modifying activities of miraculin were evaluated by five subjects by tasting 0.2 mL of partially purified miraculin solution and held in mouth (tongue) for 5 min. Subsequently, each subject expectorated out the partially purified miraculin solution, washed the mouth with distilled water and sipped 5 mL of 0.02 mol/L citric acid and finally evaluated the presence of taste modifying activities in the purified miraculin.


2.10. Definition

Specific miraculin in crude ¼ Miraculin in crude extract Total protein in crude extract Specific miraculin in back extraction aqueous phase ¼ Miraculin in back extraction aqueous phase Total protein in back extraction aqueous phase.


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