Evaluation of Spray-Dried and Freeze-Dried Red Pitaya Powder As a Functional Natural Colorant

Evaluation of spray-dried and freeze-dried red pitaya powder as a functional natural colorant in a model juice system

S.W. Chan1*,Y.J. Lee1,P. K. Lim1and C.P. Tan2

1School of Biosciences, Taylor’s University, No. 1, Jalan Taylor's, 47500 Subang Jaya Selangor, Malaysia

2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

*Corresponding author:

Dr. Chan Sook Wah

School of Biosciences,

Taylor’s University,

No. 1, Jalan Taylor's,

47500 Subang Jaya Selangor,

Malaysia.

Tel: +603- 5629 5553

Fax: +603- 5629 5311

E-mail address:

Abstract

Red pitaya (Hylocereuspolyrhizus) is an exotic fruit which belongs to the cactus family of Cactaceae. It is cultivated in many tropical countries such as Thailand, Cambodia, Vietnam, China, Australia and Malaysia. Pitaya fruit has recently drawn considerable attention due to its health beneficial claims such as antioxidant activity and the potential to reduce the risk of hypercholesterolemia and hypertension. Particularly, betacyanin in red pitaya is a potential source of natural colorant as an alternative to synthetic colorant. Consumption of synthetic colorants has been associated with many health problems such as liver and kidney damage. In this work, red pitaya powder was obtained via different processing methods (spray drying and freezing drying). Their physicohemical and antioxidant properties were compared and the stability of red pitaya betacyanin was analyzed by applying into a model juice system. As compared to spray-dried powder, freeze drying produced powder of higher yield with significantly (p < 0.05) lower moisture content, water activity; significantly (p < 0.05) higher bulk density, tapped density and particle size; better flowability and lower cohesiveness. However, betacyanin content and antioxidant activities of both spray-dried and freeze-dried red pitaya powder were comparable. Significant (p < 0.05) changes in betacyanin were observed during 4 weeks storage study. Betacyanin was best preserved in model juice at storage temperature of 4 °C, but completely degraded at room temperature and 40 °C after one week storage. With these findings, it can be concluded that betacyanin from red pitaya powder could be a potential natural colorant for chilled food products.

Keywords: red pitaya, spray drying, freeze drying, betacyanin, natural colorant

Introduction

Hylocereuspolyrhizus or better known as red pitaya fruit is originated from America and belongs to cactus family and plant order of Caryophyllales (Phebe & Chew 2009). This species have its peel and flesh in purple-red color. The flesh contains small black seeds which distributed evenly and its texture is delicate and juicy (Jamilah et al. 2011). Red pitaya fruit is cultivated worldwide in Malaysia, Bangladesh, China, Vietnam, Taiwan, Thailand, Australia, Israel and others (Ee et al. 2014a; Jamilah et al. 2011). In Malaysia, red pitaya fruit is consumed as fresh fruit as well as commercially manufactured to produce juices or jams products. The deep purple color of red pitaya flesh is contributed by the betacyanin pigment. It is reported that betacyanin has antioxidant properties which making it suitable to be used as food colorant and serves as functional ingredient in food product (Lee, Wu & Siow 2013).

One of the factors that affect the customers’ food choice is color. Synthetic colorant has been applied for ages in order to improve and enhance the food appearance. In Malaysia, 14 synthetic colorants have been permitted to be applied as coloring substance in food (Food Act 1983 (Act 281) & Regulations (as at 1st March 2014). However, due to the reported allergic and intolerance cases after consumption of synthetic colorant and the potential health hazard, natural colorant has recently gained market attention in order to replace synthetic colorant in the food application. According to Food Act 1983 (Act 281) & Regulations (2014), beet red is one of the permitted natural coloring substances to be used in food. Beet red, synonym to beetroot red is a coloring substances extracted from red beets roots. It is composed of mainly betalain pigments, purple-red betacyanins of which account for 75-95% of betanin (FAO 2012). Despite the rich betacyanin content in beet red, red pitaya betalain is more preferable due to the presence of geosmine and pyrazine in beet root which contribute to “earthy” taste (Chik et al. 2011).

Common methods suitable used to dry heat labile pigment are spray drying and freeze drying. Spray drying converts fluid materials into dry solid particles by atomizing the feed fluid into a hot gas medium to obtain powder instantaneously (Phisut 2012). Spray drying process is commercially used to produce fruit juice powders of good quality and facilitate easier transportation and storage. Freeze-drying refers to lyophilization is a drying process where the water is frozen at very low temperature and subsequently transformed from solid state into the vapor state through sublimation under low pressure. It became one of the best options for the production of heat-sensitive materials because this process does not involve heat (CiurzyńskaLenart 2011). Spray drying is more commonly used in the production of powder food colorant as it is more economical. However, it might increase loss of antioxidant content as this process involves high temperature. Freeze dried powder often give better properties but it involves high operating cost (LaokuldilokKanha 2015; Lee, Wu & Siow 2013). There are various studies reported on the physiochemical properties and antioxidant properties of red pitaya fruit as well as production of spray dried red pitaya powder (Ee et al. 2014a, 2014b; Lee, Wu & Siow 2013; Liaotrakoon et al. 2012; Woo et al. 2011). However, there is limited research on the production of freeze dried red pitaya powder and the comparison of physiochemical properties between spray dried and freeze dried red pitaya powder. Besides, study on the stability of red pitaya betacyanin in the food model system is very scarce. Hence, this research is aimed to compare the physiochemical and antioxidant properties of red pitaya powder produced by spray drying and freeze drying. The effects of temperature and light on the stability of betacyanin of spray dried and freeze dried red pitaya powder in a model juice system wereanalyzed.

Materials and Methods

Raw materials

All fresh red pitaya fruits used were purchased from fruits shop ‘Eats More Fruits’ located in Kota Kemuning, Shah Alam, Selangor.

Materials, chemicals, and reagents

Maltodextrin with dextrose equivalent (DE) 10% - 13% was obtained from V.I.S Foodtech Ingredient Supplies Sdn. Bhd., Kepong, Kuala Lumpur. Citric acid anhydrous was purchased from Bake with Yen Sdn. Bhd., Puchong, Selangor. Sugar (coarse sugar) was obtained from Central Sugars Refinery Sdn. Bhd., Shah Alam, Selangor. Methanol, trifluorocetic acid (TFA), acetonitrile, Folin-Ciocalteu’s (FC) reagent, sodium carbonate anhydrous, denatured ethanol, 1M sodium hydroxide, di-sodium hydrogen phosphate and iron (III) chloride were supplied by Merck, Germany. Sodium nitrite, aluminium chloride-6-hydrate, trichloroacetic acid and potassium ferricyanide were obtained from Bendosen Laboratory Chemicals, Norway. Gallic acid, catechin and betanin (red beet extract diluted with dextrin) were procured from Sigma-Aldrich, USA. Potassium di-hydrogen phosphate was obtained from Hamburg Chemical, Germany. All chemicals and reagents used were of analytical grade or high performance liquid chromatography (HPLC) grade. Distilled water was used throughout the analyses.

Preparation of red pitaya juice

Peel of red pitaya fruits was removed and the flesh was cut into smaller pieces. The flesh was then blended using blender to obtain the puree. The puree was filtered 3 times using muslin cloth to remove seeds before subjected to spray drying and freeze drying.

Spray drying of red pitaya juice

The filtered juice was mixed with distilled water at ratio of 1:2 (150 g of red pitaya juice mixed with 300 g of distilled water). 15% (w/w) of maltodextrin (67.5 g) was added to the red pitaya juice mixture and homogenized using blender. The red pitaya juice mixture was then spray dried using laboratory scale spray dryer (Lab-Plant SD-06, Labplant UK Ltd., UK). The spray drier was equipped with 0.5 mm spray nozzle (215 mm OD × 500 mm long). The pump speed of spray dryer was maintained at 11.58 mL/min, inlet temperature at 140 °C, fan setting at 4.3 m/s and pressure at 2 bars. The powder obtained was stored in Schott bottle at room temperature until further analysis.

Freeze drying of red pitaya juice

The filtered juice was mixed with same amount of maltodextrin as added into spray dried red pitaya juice (150 g of red pitaya juice mixed with 67.5 g of maltodextrin) and homogenized using blender. The red pitaya juice mixture was then poured into Schott bottle and frozen in ultra-low temperature freezer (Model: DW-86L388, Haier Group, China) at –80 °C for overnight. The red pitaya juice mixture was then freeze dried using benchtop freeze dryer (Model:FreeZone 4.5L, Labconco, USA) at –50 °C under pressure below 0.110 mBar for 55 hours. The dried red pitaya product was then ground using dry mill. The powder obtained was stored in Schott bottle at room temperature until further analysis.

Yield

The yield of red pitaya powder was calculated using equation below:

Yield (%) =

Solid content of juice is the dry weight of juice.

Moisture content

The moisture content of red pitaya powder was determined using electronic moisture analyzer (Model: MOC63u UniBloc, Shimadzu, Japan).

Water activity

The water activity (aw) of red pitaya powder was determined using water activity meter (Model: Decagon AquaLab LITE, Decagon Devices, Inc., USA).

Bulk and tapped density

For bulk density determination, 5 g of powder was measured and gently poured into a 25 mL graduated measuring cylinder. The volume occupied by the powder was recorded and used to calculate the bulk density according to equation below(Jinapong, Suphantharika & Jamnong 2008; Saifullah et al. 2014; Victória et al. 2013):

Bulk density =

For tapped density, 5 g of powder was poured into a 25 mL graduated measuring cylinder. The samples were repeatedly dropped manually 100 times by lifting and dropping the cylinder under its own weight at a vertical distance of 10 cm. The volume occupied by the samples after tapped was recorded and calculated using equation below(Jinapong, Suphantharika & Jamnong 2008; Saifullah et al. 2014; Victória et al. 2013):

Tapped density =

Flowability and cohesiveness

Flowability and cohesiveness of the powders was estimated using Carr index (CI) and Hausner ratio (HR) (Jinapong, SuphantharikaJamnong 2008). CI and HR were calculated from the bulk and tapped densities of the powders using equation as follows:

CI (%) =

HR =

Flowability and cohesiveness of red pitaya powder classification are presented in Tables 1 and 2, respectively.

Particle size

Particle size of spray dried and freeze dried red pitaya powder was identified using laser diffraction method (Model: Mastersizer 2000, Malvern Instruments Ltd, UK). The condition set was air pressure at 1 bar, vibration rate of feed at 30% and refractive index at 1.44. The result was reported as surface-weighted mean diameter D [3, 2] (µm) and span for the measurement of particles size distribution.

Powder morphology

Morphology of spray dried and freeze dried red pitaya powder was observed using scanning electron microscope (SEM) (Model: JSM 6400, JEOL Ltd., Japan). SEM stubs were attached with double-sided adhesive tape and a thin layer of powder was stuck on the tape before subjected for drying. The samples were then coated with a thin layer of gold and the morphology was observed at 2000× magnification.

Color

The color of the powder and juice (L*, a*and b* values) was measured using colorimeter (Model:ColorFlex EZ, Hunter Associates Laboratory, Inc., USA).

Betacyanin content

The betacyanin content of the powder was identified using high performance liquid chromatography (HPLC) (Model: Prominence UFLC, Shimadzu, Japan). Betacyanin in sample was separated using Thermo Scientific Hypersil Gold column, (5 µm, 150 × 4.6 mm). The mobile phase used for elution is an isocratic solution contains a mixture of 0.5% trifluroacetic acid (TFA) (90%) and acetonitrile (10%). Isocratic solution contains a mixture of 0.5% trifluoracetic acid in water (90%) and acetonitrile (10%) was used as the mobile phase. The column oven condition was set at 25 °C, absorbance of 536 nm, flow rate at 1.00 mL/min for 15 minutes per each injection with 20 µL injection volume (Jamilah et al. 2011; Rizk, El-kady & El-bialy 2014).All samples were filtered using 0.45 µm nylon syringe filter prior to injection. The result was reported in mg/mL and the calibrated betaninstandard curve equation was y = 146234x + 2639.8 (R² = 0.9999).

Total phenolic content (TPC)

TPC was determined using Folin-Ciocalteu (FC) methodadopted from Lim, Lim & Tee (2006) with slight modification. Sample (0.3 mL) was mixed with 1.0 mL of FC reagent and stand for 3 mins. Then, 0.8 mL of 7.5% (w/v) sodium carbonate anhydrous solution was added into the mixture and vortex. The mixture was incubated in dark for 2 hours and the absorbance was recorded at 765nm using UV-Vis spectrophotometer (Model: GENESYS 10 UV, Thermo Scientific, USA). Denatured ethanol (70%) was used as blank. The polyphenol concentration was expressed as milligram of gallic acids equivalents (GAE) per 100 g of dry weight. The calibrated standard curve equation for gallic acid was y = 15.338x + 0.0801 (R² = 0.9945).

Total flavonoid content (TFC)

Estimation of the TFC in crude extracts was performed according to the procedures described byThoo et al. (2010) with slightmodifications. Sample (0.25 mL) was added into test tube followed by 1.25 mL distilled water and 75 µL of 5% sodium nitrite. The mixture was incubated for 6 mins and 150 µL of 10% aluminium chloride-6-hydrate solution was added into the mixture. The mixture was incubated for 5 mins and 0.5 mL of 1M sodium hydroxide and 250 µL of distilled water were added into the mixture. The absorbance was then recorded using UV-Vis spectrophotometer at wavelength of 510 nm and distilled water was used as blank. The flavonoid concentration was expressed as milligram of catechin equivalents (CE) per 100 g of dry weight. The calibrated standard curve equation for catechin was y = 0.0024x + 0.0112 (R² = 0.9996).

Ferric reducing antioxidant power (FRAP)

The ferric reducing power of the fruit extracts was determined by using the potassium ferricyanide–ferric chloride method (Lim, Lim & Tee 2006). Sample (1 mL) was mixed with 2.5 mL of 0.2M phosphate buffer (pH 6.6) and 2.5 mL 1% potassium ferricyanide. After that, it was incubated at 50 °C for 20 minutes. After that, 2.5 mL of 10% trichloroacetic acid was added to the incubated mixture. The incubated mixture (2.5 mL) was transferred into new test tube. 2.5 mL of distilled water and 0.5 mL 1% iron (III) chloride were added into the mixture and the sample was incubated for 30 minutes. The absorbance of the mixtures was then recorded using UV-Vis spectrophotometer at wavelength of 700 nm and 70% denatured ethanol was used as blank. The polyphenol concentration was expressed as milligram of gallic acids equivalents (GAE) per 100 g of dry weight. The calibrated standard curve equation for gallic acid was y = 21.581x + 0.1902 (R² = 0.9935).

Stability studies: temperature and light

Stability studies were conducted by incorporating spray dried and freeze dried red pitaya powder into model juice. 600 mL of model juice was prepared with powder concentration of 5 % (30 g) , brix value around 9 – 10% (addition of sugar around 32 g) and pH value between 3.1 – 3.2 by using citric acid (approximately 0.71 g). Model juice was pasteurized at 98 °C for 5 sec before hot filled into universal bottle and held for 5 minutes before it was cooled under running tap water. Model juices were stored in five different conditions: 4 °C with light and without light, room temperature with light and without light and 40 °C. Betacyanin content of juices incorporated with spray dried and freeze dried red pitaya powder was determined and sampled on weekly basis until week 5. The results were expressed in betacyanin retention (%) based on the following formula:

Betacyanin retention (%) =

Statistical analysis

Results were analyses using Statistical Package for Social Sciences (SPSS) Version 21 software (IBM, SPSS Inc.). All results are reported in mean ± SD of triplicate determinations. Independent t-test was used to determine significant differences between two samples and One-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, was used to conclude significant differences between three or more samples at level of p < 0.05. Repeated measures was used to determine significant changes over 5 weeks of storage stability study of model juice at level of p < 0.05.

Results and Discussions

Preliminary study

It was the first attempt for this research team to produce red pitaya powder using freeze drying. Therefore, a preliminary study was done in order to identify the suitable method to obtain an ideal sample. Pure filtered red pitaya juice was freeze dried at the initial step. However, the end product turned out to be unsatisfied due to high stickiness of the ground powder. This might be due to the incomplete removal of moisture from the red pitaya juice as it contained high moisture and sugar content. High sugar or soluble solid will decrease the drying rate hence it might require longer time to obtain a powder of better quality (Heldman & Lund 2007). Therefore, based on several other studies done by Estupiñan, Schwartz & Garzón (2011), Mehrnoush, Mustafa & Yazid (2012), and Murali et al. (2014), maltodextrin was added into the red pitaya juice before subjected to freeze drying.

Physiochemical properties of red pitaya powder

Yield

In food industry, yield is an important economic consideration. The higher the yield is, the more efficient the process is. It was observed that the yield of freeze-dried red pitaya powder (96.05%) was around three times higher compared to spray-dried powder (33.25%)(Table 3).As red pitaya juices contain high sugar naturally, it might create stickiness problem during spray drying (Lee, Wu & Siow 2013). Although maltodextrin was added to increase the glass transition temperature of the powder, however the end result showed that the yield of spray drying is still not satisfied. Product loss during spray drying process was largely due to the sticking of product on the wall of drying chamber and cyclone hence led to decrease in yield. During freeze drying, the red pitaya juices were frozen in the bottle and the end product was all collected within the same bottle. Therefore, there is no or insignificant product loss.