Production of freeze-dried Mao juice powders and their functional properties

Wetanee Suravanichnirachorn1, Withida Chantrapornchai1*, Vichai Haruthaithanasan1, Suntaree Suwonsichon1, Udomlak Sukatta2 and Thanapoom Maneeboon3

ABSTRACT

One of the major problems in drying fruit juice is stickiness due to its high hygroscopicity. Thus, carrier agents were added into juice before drying for improving its properties. In this study, 35% w/w of maltodextrin, gum arabic and maltodextrin:gum arabic (1:1) were added into Mao fruit juices before passing the mixtures through freeze–dried process for 30 h. The Mao juice powders containing all carrier types had purple color and low water activity but moisture content and solubility time were significantly different (p 0.05). Mao juice powder with maltodextrin had the lowest moisture content (3.53 ± 0.62 %) and solubility time (131.78 ± 2.34 second). Mao juice powders had total anthocyanin content between 2.31 – 2.63 mg∙g fw and analysis using HPLC revealed that the major component was delphinidin. The antioxidant activities of Mao juice powder were investigated using DPPH, ABTS, FRAP and nitric oxide assays. The results showed that among all treatments, they have fairly similar antioxidant activity in FRAP assay (p > 0.05). However considering ABTS assay, the one with maltodextrin seemed to be a better antioxidant activities (p 0.05). Key Words: anthocyanin, freeze drying, Mao, maltodextrin, gum arabic

* Corresponding author; e-mail address: withida.c@ku.ac.th

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1Department of Product Development, Faculty of Agro-Industry, Kasetsart University, Bangkok, 10900 2Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, Bangkok, 10900, Thailand. 3Scientific Equipment and Research Division, Kasetsart University, Bangkok, 10900, Thailand.

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INTRODUCTION Anthocyanins are phytochemicals which widely used in functional foods due to their several

health benefits. They have been used for therapeutic treatments for diabetes, retinopathy, vision disorders and age-related diseases such as hypertension and heart failure. Moreover, they could enhance immune system, anti-inflammatory and anti-cancer activities (Motohashi and Sakagami, 2008).

Mao fruits are mostly found in the Northeast of Thailand such as Sakon Nakhon, Udon Thani, Loei, Nong Khai and Nakon Phanom (Butkhup and Samappito, 2008). They had good antioxidant capacity due to its high content of anthocyanin and phenolic compounds (Samappito and Butkhup, 2008; Musika and Sae-eaw, 2013). Freeze drying is a technique which favorable to a materials that easily deteriorated by heat. However, drying of fruit juices with high sugar content usually yields sticky and high agglomerates as sugar transforms into crystalline sugar at amorphous state through adsorption of small amounts of water resulting in high hygroscopic property. Hence, addition of some carriers in the mixtures could be useful (Cano-Chauca et al., 2005; Martinelli et al., 2007). Maltodextrin and gum arabic are carrier agents which mostly used in drying process for improving characteristic and extending shelf life of the powders, since they are highly water soluble and give low viscosity. Maltodextrins are oligosaccharides produced by hydrolysis of starch. Their properties depend on dextrose equivalent (DE) (Langrish et al, 2007). Gum arabic is a complex hetero-polysaccharide, it has good emulsifying capacity and low viscosity (Martinelli et al., 2007). Moreover, it is more expensive than maltodextrins.

In this study, we aimed to study physical properties, structure, anthocyanin content and their antioxidant activities (DPPH, ABTS, FRAP and Nitric oxide assay) of Mao juice powders were added with maltodextrin, gum arbic and maltodextrin:gum arabic (1:1)

MATERIALS AND METHODS 1. Preparation of Mao fruit powder with different carrier types. Fresh Mao fruits were purchased from Loei province, Thailand. The fruits with dark red and purple color were selected, sealed in plastic bags and kept in a freezer at -18C. Before investigation, Mao fruits were thawed overnight at 4C, washed with water to remove remaining sand, dirt and to reduce the initial microbial load, soaked with chlorine 50 ppm for 30 min and washed with water again. The Mao juices were squeezed from cleaned Mao fruits using juice extractor (GS 1000, Green Star, USA). Three different carrier types (maltodextrin DE 10 (Thai food and chemical, Thailand); gum arabic (Chemipan, Thailand); and the blend of maltodextrin and gum arabic (1:1)) were mixed into the juices at 35% w/w. The mixtures were freeze-dried using a freeze dryer (Supermodulyo-230 Thermo Scientific, USA) at -50C for 30 h. Mao juice powders were ground before transferring to aluminum foil bags. 2. Determination of Mao juice powder qualities (color, water activity, moisture content, solubility time and structure)

The color values in CIE L*C*ho system were measured in reflectance mode using spectrophotometer Minolta CM-3500d (Konica Minolta Inc., Tokyo, Japan), under a standard

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illuminant D65, standard observer 10º. The water activity (aw) was determined by Aqua Lab (Series 3 TE, Decagon Devices, USA). The moisture content was analyzed according to a method in A.O.A.C. (2000). The powder structure were analyzed by scanning electron microscopy (JSM-5600LV, JEOL, Japan), operated at 10 kV with a working-distance of 10 mm. The solubility time was determined using a method modified from Goual et al. (2007). The powder 2 g and 50 mL distilled water were added to glass beaker 250 mL. The mixture was agitated with a magnetic stirrer (C-MAG HS 7, IKA, Germany) at position 2. The times for dissolving powder completely were obtained.

3. Determination of anthocyanin content Determination of total anthocyanin content (TAC)

The TAC was determined by the pH-differential method (Worsltad et al., 2005). Absorbance was measured by a spectrophotometer-UV/VIS UVmini-1240 (Shimadzu, Japan) at 510 and 700 nm. Distilled water was used as blank. The TAC was calculated using Eq. (1) and expressed as cyanidin-3-glucoside in mg∙g sample-1.

Total anthocyanins (mg/L) =

(1)

where A is absorbance = (A510– A700)pH 1.0- (A510– A700)pH 4.5, ε is cyanidin-3-glucoside molar absorbance (26900 L∙ (cm∙mg)-1), l is the cell path length (1 cm), MW is the molecular weight (449.2 g∙L-1) and DF is the dilution factor. Determination of individual anthocyanin by High-Performance Liquid Chromatography (HPLC)

Cyanidin-3-o-glucoside, Delphinidin, Pelargonidin and Malvidin were quantified by HPLC (Shimadzu, Japan) with an Inertsil ODS-3 C18 packed column (ID 5 µm, 250×4.6 mm). The HPLC analysis method used in this study was modified from the method of Salazar-González (2012). The acetonitrile: 4.5% formic acid were applied in a rate of 1.5 mL∙min-1 at proportions of 10:90, 13:87 and 100:0 during 0, 11, and 21 minutes, respectively, and measured at 520 nm. A standard solution of individual anthocyanins, suspended in ethanol:water (50:50), was injected to evaluate the area under the curve corresponding to 50-250 µg∙mL-1 of anthocyanin. This area was used to correlate the area observed on the chromatogram to calculate the amount of individual anthocyanins corresponding to individual anthocyanins. 4. Determination of antioxidant activity Many assays have been introduced to measure antioxidant capacity, each assay had different mechanisms. Antioxidant activities of Mao juice powders were determined using DPPH, ABTS, FRAP and Nitric oxide assays and compared with butylated hydroxytoluene (BHT), vitamin E and vitamin C.

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DPPH free radical scavenging activity The DPPH (1,1-diphenyl-2-picryl-hydrazyl) assay was slightly modified form Floegel et al.

(2011). A solution of 1 mM DPPH in methanol:water (80:20) was stirred for 40 min. The DPPH solution was diluted with 80% methanol to an absorbance of 0.700 ± 0.02 at 517 nm. Then, the DPPH solution 2.95 mL and diluted Mao juice powders solution (five different dilutions) 0.5 mL was added to a test tube. The mixture was shaken and kept in the dark for 30 min. The absorbance was measured at 517 nm using spectrophotometer. The results were calculated as percentage of free radical scavenging using Eq. (2).

% DPPH inhibition = - - -

(2)

where Ac, Abc, As and Abs are the absorbance of the control, the blank control, the sample and the blank sample, respectively.

IC50 value, a concentration of the sample required to inhibit 50% of radical, was calculated in mg∙mL-1 using an equation from a plot of %scavenging vs. concentration of Mao juice powders. ABTS free radical scavenging activity

The method for investigating ABTS [2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] radical cation scavenging activity was modified from Re et al. (1999). The ABTS radical cation was generated by mixing 7 mM ABTS:2.45 mM potassium persulfate solution at proportions of 2:1 and kept in the dark and refrigerated for 12–16 h. The ABTS solution was diluted with ethanol to an absorbance of 0.700 ± 0.02 at 734 nm. The ABTS solution 2 mL and diluted Mao juice powders solution (five different dilutions) 20 µL was added to a test tube. The mixture was shaken and kept for 6 min in the dark. The absorbance was measured at 734 nm using spectrophotometer. The results were calculated as the percentage of free radical scavenging using Eq. (2) and as IC50. Ferric reducing ability of plasma (FRAP)

The FRAP assay was modified from Benzie and Strain (1996) and Kubola and Siriamornpun (2011). The FRAP reagent was generated by mixing 300 mM acetate buffer: iron reagent solution: FeCl3·6H2O: deionized water at 100:10:10:12 (v/v). The FRAP reagent 1.8 mL was warmed to 37 °C, then deionized water 180 µL and diluted Mao juice powders solution 60 µL were added. The mixture was shaken and incubated at 37°C for 4 min. The absorbance was measured at 593 nm using spectrophotometer. The measurement was compared to a standard curve of FeSO4 concentrations and expressed in M FeSO4∙g sample-1. Nitric oxide radical scavenging activity

The Nitric oxide assay was done according to the method of Madan et al. (2010). Sodium nitroprusside in aqueous solution (10 mM) 1 mL, phosphate buffer saline (pH 7.4) 0.25 mL and diluted Mao juice powders solution 0.25 mL was mixed and incubated at 25°C for 2 h. Then, 0.5 mL

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of the mixture was removed and sulfanilic acid reagent (0.33% in 20% glacial acetic acid) 1 mL was added in the other. The mixture was shaken and kept for 5 min for completing diazotization. After that, naphthylethylene diamine dihydrochloride (0.1%) 1 mL was added, mixed and kept for 30 min in diffused light. The absorbance was measured at 540 nm using spectrophotometer. The results were calculated as the percentage of free radical scavenging using Eq. (2) and as IC50. 5. Statistical analysis All data were reported as mean ± standard deviation and statistically analyzed the regression and variance (ANOVA) using the software SPSS version 12. Duncan’s new multiple range test was used to determine the difference among means at 95% confidence level.

RESULTS AND DISCUSSION 1. Determination of Mao juice powder qualities

The color of Mao juice powders with all carrier types had purple color (ho were in a range of 331.66 - 333.78) and aw values were below 0.2, resulting in longer shelf life as less free water were available for microorganism growth and biochemical reactions (Ferrari et al., 2013). Among three carrier types, Mao juice powder with maltodextrin had lowest moisture content and solubility time values, because maltodextrin has better reconstitution properties and dispersibility (Cai and Corke, 2000). Mao juice powders had irregular structures with different sizes and the pore suffused in the powder (Fig. 1), because crystal structures were generated during freeze drying process (Franceschinis et al., 2014). Gurak et al. (2013) studied the structure of freeze-dried grape juice powders which encapsulated by gum arabic and maltodextrin, their results supported that the powder had deformed structure.

Table 1 The qualities of Mao juice powder with different carrier types

a-c mean within the same row followed by different letters were significantly different (p≤0.05). ns means within the same row were non significantly different (p 0.05). 2. Determination of total anthocyanin content

TAC of powders with different carrier types was in a range of 2.31 – 2.63 mg∙g fw which not significantly different (p 0.05). According to Tonon et al. (2008), TAC of açai juice powders with maltodextrins and gum arabic did not significantly different. HPLC determination indicated that the major anthocyanin in Mao juice powders was delphinidin, followed by cyanidin-3-o-glucoside, and pelargonidin. However, malvidin were not found in Mao juice powder (Table 2). Delphinidin amount

Quality values Maltodextrin Gum arabic Maltodextrin:gum arabic (1:1) L*ns 43.35 ± 1.50 42.18 ± 2.90 41.44 ± 2.23 C* ns 18.04 ± 0.70 17.37 ± 0.51 17.33 ± 0.16 ho ns 332.56 ± 2.26 331.66 ± 1.05 333.78 ± 0.01 aw

ns 0.149 ± 0.04 0.171 ± 0.05 0.148 ± 0.00 Moisture content (%) 3.53 ± 0.62b 5.41 ± 0.46a 4.20 ± 0.02b Solubility time (s) 131.78 ± 2.34c 631.27 ± 27.74a 431.33 ± 36.33b

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of Mao juice powder with different carrier types was not significantly different (p > 0.05), but cyanidin-3-o-glucoside content of Mao juice powders with maltodextrin and maltodextrin:gum arabic (1:1) were higher (p 0.05) than those with gum arabic.

Figure 1 Scanning electron micrographs of the surface of Mao juice powders with maltodextrin (a), gum arabic (b) and maltodextrin:gum (c) at 500x and 2000x magnification.

Table 2 Total anthocyanin content and classification of individual anthocyanins by HPLC of Mao juice powder with different carrier types.

Carrier type TACns

(mg∙g fw)

Individual anthocyanin by HPLC Cyanidin-3-o-

glucoside (mg∙g fw) Delphinidinns

(mg∙g fw) Pelargonidin

(mg∙g fw) Malvidin (mg∙g fw)

Maltodextrin 2.63 ± 0.25 0.78 ± 0.05a 1.10 ± 0.12 nd. nd.

Gum arabic 2.34 ± 0.22 0.62 ± 0.02b 1.05 ± 0.14 nd. nd.

Maltodextrin:gum arabic 2.31 ± 0.06 0.73 ± 0.02a 1.16 ± 0.08 nd. nd. a-b mean within the same column followed by different letters were significantly different (p 0.05). ns means within the same column were non significantly different (p 0.05). nd. means not detected. 3. Determination of antioxidant activity

Antioxidant activities of Mao juice powders with different carrier types were investigated using DPPH, ABTS, FRAP and Nitric oxide assays and compared to a commercial antioxidant (BHT) and natural antioxidants (vitamin E and vitamin C). The results showed that among all treatments, Mao juice powders had the least antioxidant capacity (highest IC50 value and lowest FRAP values, which indicated that they have the least efficiency in reducing electrons). Considering among the three Mao juice powders with different carrier types, they have fairly similar antioxidant activity in FRAP assay (p > 0.05); but according to ABTS assay, the one with maltodextrin seemed to be a better antioxidant (p

b

500x

a

500x

c

500x

a

2000x

b

2000x

c

2000x

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0.05). However, Mao juice powders with maltodextrin and gum arabic had higher antioxidant capacity than those with maltodextrin:gum arabic (1:1) in DPPH assay.

Table 3 Antioxidant activities of Mao juice powder with different carrier types compared to BHT,

vitamin E and vitamin C Carrier types, commercial and natural antioxidants

DPPH IC50(mg∙mL-1)

ABTS IC50 (mg∙mL-1)

FRAP (mM FeSO4 g

-1 fw) Nitric oxide

IC50 (mg∙mL-1) Maltodextrin 17.71 ± 0.71bc 20.31 ± 1.45b 117.65 ± 3.04d 4.17 ± 0.40bc Gum arabic 17.48 ± 1.02b 25.15 ± 1.87c 119.32 ± 7.64d 3.71 ± 0.35b Maltodextrin:gum arabic 18.45 ± 0.08c 24.89 ± 0.89c 114.93 ± 2.61d 4.57 ± 0.09c BHT 0.33 ± 0.00a 0.53 ± 0.03a 1,251.60 ± 110.67c - Vitamin E 0.40 ± 0.00a 0.50 ± 0.02a 3,105.79 ± 133.16b - Vitamin C 0.15 ± 0.00a 0.22 ± 0.01a 12,296.85 ± 165.70a 0.47 ± 0.03a a-d mean within the same column followed by different letters were significantly different (p 0.05).

CONCLUSION Mao juice powders with maltodextrin, gum arabic and a combination of maltodextrin and gum arabic had slightly different in TAC and antioxidant capacities. However, the one with maltodextrin had a better potential to be used in the Mao juice powder production since it tends to have longer shelf life, lower solubility time, and lower price.

ACKNOWLEDGEMENTS The authors thank Kasetsart Agricultural and Agro-Industrial Product Improvement Institute,

Kasetsart University for tools and facilities support.

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