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Microencapsulation of anthocyanin extract from purple rice bran using modified rice starch and its effect on rice dough rheology
Accepted Manuscript Microencapsulation of anthocyanin extract from purple rice bran using modified rice starch and its effect on rice dough rheology
Amit Baran Das, V.V. Goud, Chandan Das PII: DOI: Reference:
S0141-8130(18)35948-8 https://doi.org/10.1016/j.ijbiomac.2018.11.247 BIOMAC 11124
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
2 November 2018 22 November 2018 26 November 2018
Please cite this article as: Amit Baran Das, V.V. Goud, Chandan Das , Microencapsulation of anthocyanin extract from purple rice bran using modified rice starch and its effect on rice dough rheology. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.11.247
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ACCEPTED MANUSCRIPT Microencapsulation of anthocyanin extract from purple rice bran using modified rice starch and its effect on rice dough rheology
Amit Baran Dasa,b, V. V. Goud*b, Chandan Das*b Department of Food Engineering and Technology Tezpur University, Assam, India
Department of Chemical Engineering
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Indian Institute of Technology Guwahati, Assam, India
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___________________ *Corresponding author
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Tel: +913612582268/2272 Fax:+913612582291 E-mail: [email protected] (C. Das), [email protected] (V.V. Goud)
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ACCEPTED MANUSCRIPT Abstract The investigation aims to microencapsulate anthocyanin extract from purple rice bran with modified glutinous rice starch to provide stable anthocyanin powder. The modified glutinous rice starch was used as wall material where anthocyanin extract was a core material for
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microencapsulation. The microencapsulation was carried out in a spray dryer, and the process
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was optimized using a rotatable central composite design. The effect of independent parameters,
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viz., starch concentration, inlet air temperature, and atomizer pressure, on the dependent parameters, such as encapsulation efficiency, anthocyanin content, 2, 2-diphenyl-1-
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picrylhydrazyl (DPPH) scavenging activity, true density and water activity were investigated.
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The optimum values of the spray drying process parameters were 6.01% of starch concentration, 168.78°C inlet air temperature and 4.96 MPa atomizer pressure. The physical characteristics of
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spray-dried powders were investigated in term of water activity (0.51), solubility (51.83%), bulk
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density (1.404g/cm3), porosity (0.20), and diameter (6.44µm) and the Hausner ratio (1.020). The thermal, crystallinity and surface morphology of the microencapsulated particles were also
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comprehensively studied using DSC, XRD, FTIR, and SEM. The storage stability of
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microencapsulated anthocyanin showed more stability at 4°C than at 25°C for 90 days. The microencapsulated particles also have an effect on the steady-shear rheology of the rice dough.
properties.
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Keyword: Anthocyanin; microencapsulation; spray drying; thermal properties; rheological
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ACCEPTED MANUSCRIPT 1. Introduction Anthocyanin is the most widely used and recognized members of the bioflavonoid phytochemicals. The potential sources of anthocyanin are colored fruits and vegetables. Some pigmented rice varieties are also a good source of anthocyanin, especially purple rice bran [1].
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Besides the wide range of colors, the potential health benefits rendered anthocyanin as an
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attractive compound in the food system [2]. However, the stability of anthocyanin is a major
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challenge for the food industry. The stability of anthocyanin depends on various environmental factors, such as pH, light, temperature, oxygen, and enzymatic activities. For the low stability in
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various conditions during processing and storage, application of anthocyanin as an ingredient in
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foods is now quite challenging.
To obtain a stable and usable food ingredient from anthocyanin, several approaches are in
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consideration, such as encapsulation, emulsion, and freeze-drying. For anthocyanin,
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encapsulation method has been demonstrated and accepted by a few researchers [3-7]. The most commonly used encapsulation process is microencapsulation. Microencapsulation may be an
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efficient way to improve the bioavailability and stability of bioactive compounds and managing
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the release of active agent [2]. The main aim of microencapsulation is to protect the core material from environmental effects, thereby increases the shelf life of the product and promoting a
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controlled release of the core material [8]. There are various types of microencapsulation techniques, such as a vibrational nozzle, centrifugal extrusion, spray drying, and cocrystallization. The most commonly used technique is spray drying, due to its continuity in nature, low cost and less time which can produce particles with good quality [2]. For microencapsulation through spray drying process, the selection of wall material is the primary step. Most commonly used wall materials are maltodextrin, gum arabic, and emulsifying
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ACCEPTED MANUSCRIPT starches. The carbohydrates, such as, maltodextrins and starches have properties like good solubility and low viscosity at high solids contents which are highly desirable for an encapsulating agent. However, native starch is insoluble in cold water and, when heated with water it forms a paste-like texture with high viscosity, which decreases the spray drying
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efficiency and increases the surface crack, thereby limits its application for encapsulation using
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spray drying. These limitations can be overcome by the modification of native starch. Few
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numbers of researchers were used modified starch as a wall material in for encapsulation of various bioactive compounds [9]. However, the application of modified low amylose rice starch
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as wall material in microencapsulation of anthocyanin was scanty.
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The functional and physicochemical properties of a microencapsulated anthocyanin powder directly depend on the storage condition of the powder. The extrinsic factors like
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temperature, light, and oxygen, are the most determining factors of anthocyanin stability in
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powder [3]. Therefore, the effect of storage conditions on the stability of anthocyanin in microencapsulated powder needs to understand. On the other hand, the incorporation of
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microencapsulated powder significantly affect the rheological properties and textural properties
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of food product [10]. Therefore, a clear understanding of the effect of microencapsulated powder on the flow behavior of dough is required to design and develop a new food product. However,
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there is no information on the effect of microencapsulated anthocyanin powder incorporation on the rice dough rheology. Therefore, the present work is the first where anthocyanin from purple rice bran is microencapsulated with modified glutinous rice starch using a spray-drying technique. The objective of the present study was to microencapsulate anthocyanin extract from purple rice bran using modified starch through spray drying. The spray drying process for
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ACCEPTED MANUSCRIPT microencapsulation was optimized and validated. The physicochemical, thermal, and morphological properties of microencapsulated powder were also investigated extensively. The storage stability of a spray-dried powder in various temperatures was also carried out. In addition to that, the effect of microencapsulated anthocyanin powder on the rice dough rheology was also
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determined.
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2. Materials and Methods 2.1. Materials
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Purple rice (Oryza sativa L) from Manipur, India was used for this study. Bran was
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separated and stored in zip lock polyethylene bag at 28±2oC. Modified starch was prepared from rice starch according to the method described by Hu, Wei [11]. Glutenous rice starch (25 g) was
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dispersed in 100 mL anhydrous 1-butanol. The reaction was started by adding 1 mL of
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concentrated (36% by weight) HCl and proceeded at 30°C for 2 h, with constant stirring. The reaction was stopped by adding 14 mL of 1 M NaHCO3. The solution was cooled in an ice bath
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for 15 min and centrifuged at 5000 rpm (523.59 rad/s) for another 5 min. The precipitate was
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washed four times with 50% ethanol and dried in an air oven at 45°C. The anthocyanin was extracted from bran using ultrasound-assisted extraction process as follow by Das, Goud, and
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Das [1]. The extracted anthocyanin was separated from the extract through the adsorption/desorption process using amberlite XAD7 resin [12]. For adsorption, 1 g pre-treated XAD7 resin and 25 mL of purple rice bran extract was added to 250 mL flask. Then the flask was kept for shaking at 45 rpm (4.71 rad/s) in a shaker at room temperature (28±2°C) for 24 h. For desorption, the resins with anthocyanin were washed with 25 mL distilled water and then, 50
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ACCEPTED MANUSCRIPT mL 95% ethanol was added to the flask. The flask was kept in a shaker at 45 rpm (4.71 rad/s) for 24 h at room temperature (28±2°C).
2.2. Preparation of feed sample and encapsulation
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For microencapsulation of anthocyanin extract, modified starch was used as wall material
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and anthocyanin as a core material. The starch dispersion of various concentrations (5-10% w/v)
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was prepared in water with the combination of anthocyanin extract (40 mg cyanidin-3glucoside/L) (core) under magnetic agitation. The encapsulation of anthocyanin extract was done
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using spray dryer (Make: Labultima, Mumbai, India; Model: Lab plant LU 20 lab sprayer).
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During spray drying, the inlet air temperature was varied from 140 to 180°C and atomizer
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pressure ranged from 2.76 to 5.52 MPa as mentioned in Table 1.
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2.3. Encapsulation efficiency
The encapsulation efficiency of the microencapsulation process was measured using the
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method described by Akhavan Mahdavi, Jafari [4]. To estimate the efficiency, total anthocyanin
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content (TAC) and surface anthocyanin content (SAC) of the microcapsules were measured. For analysis, 1 mL of distilled water was added to 100 mg of sample and the microencapsulation was
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crushed in a mortar-pestle. Then, the samples were extracted using 10 mL ethanol. The surface anthocyanin from the microcapsules was extracted by quickly washing with ethanol (10 mL) in a vortex within 10s, and centrifuge at 3000 rpm (314.15 rad/s) for 3 min at 20°C. The clear supernatants were collected and filtered through 0.45µm-filter paper and used for anthocyanin determination. Encapsulation efficiency (%)
(TAC SAC ) 100 TAC 6
(1)
ACCEPTED MANUSCRIPT 2.4. Anthocyanin content and DPPH scavenging activity The anthocyanin contents of the microencapsulates were determined by the pHdifferential method as mentioned by Das, Goud, and Das [1]. The absorbance of the samples was measured at 515 and 700 nm using UV/VIS spectrophotometer (Make: Thermo Fisher Scientific,
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Waltham, Massachusetts, USA; Model: UV-2600).
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The DPPH scavenging activity of microencapsulates was measured according to Luo,
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Zhao [13] with some modifications. For analysis, microencapsulate (15 mg) was dissolved in a mixture (3 mL) of ethanol: acetic acid: water (50:8:42, v/v). The mixture was filtered through a
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filter paper (0.45µm) and diluted with methanol. The 100 µL filtrate was taken and added to 1.4
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mL DPPH radical methanolic solution (10-4 M). The absorbance was measured at 517 nm using a spectrophotometer (Make: Thermo Fisher Scientific, Waltham, Massachusetts, USA; Model:
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UV-2600). The DPPH radical scavenging activity was determined as follows
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Radical scavanging activity (%) =
Ao - As 100 Ao
(2)
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where Ao is absorbance of control blank, and As is absorbance of sample extract.
2.5. Physicochemical properties
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The physicochemical properties of the samples were investigated in terms of true density, water activity, Hausner ratio, bulk density, porosity, solubility, and diameter. The true density of the microencapsulated powders was measured using gas pycnometer (Make: Porous Materials Inc, Ithaca, New York, USA; Model: PYC-100A). The water activity of the microencapsulated samples was determined using a water activity meter (Make: Aqualab Pullman, USA; Model: 4TE). The Hausner ratio defining the flowability of powder, it is the ratio of tap volume to the bulk volume of powder. For bulk density, a 500mL measuring cylindrical was filled with the 7
ACCEPTED MANUSCRIPT sample from a height of 150mm at a constant rate and then weighing the contents. The porosity of the powder was calculated using the relationship between the bulk density and true density [4]. For solubility, the 2.5 g ground sample was mixed with 30 ml of water at 30°C, in a pre-
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weighed centrifuge tube, agitated for 30 min, and then centrifuged at 3000 rpm (314.15 rad/s) for
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10 min. The supernatant was carefully decanted into a pre-weighed evaporating dish. After
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evaporating the water from the supernatant, the mass of dried solids recovered was expressed as
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a percentage of dry solids in the sample and defined as the water solubility index (WSI).
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2.6. Gelatinization properties
A differential scanning calorimetry instrument (Make: NETZSCH, Selb, Germany;
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Model: DSC 214) was used to determine the gelatinization properties of microencapsulated
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powders. For the analysis, the initial temperature was 30°C and end at 250°C with 10°C/min rate. The onset, endset, and peak temperature of the powder were measured and analyzed using
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the DSC instrument’s software.
2.7. X-Ray Diffraction
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The crystallinity of the microencapsulated powder was studied at 35 kV and 20 mA CuKa radiation in an X-ray diffractometer (Make: Bruker Axs, Karlsruhe, Germany; Model: D8 FOCUS). Data were recorded over an angular range of 2° to 50° (2θ) with a scanning rate of 2°/min.
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2.8. FT-IR analysis The FT-IR spectra of microencapsulates were recorded using a spectrometer (Make: NICOLET, Wisconsin, USA; Model: IMPACT 410). The absorbance spectra of the
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microencapsulated powders were documented in the range of 4000 to 400 cm-1 having a
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resolution of 4 cm-1. All the experimental data were analyzed using OMNIC E.S.P.5.0 software.
2.9. Morphological properties
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The morphological properties of microencapsulated anthocyanin were carried out using a
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scanning electron microscope (Make JEOL Akishima, Tokyo, Japan; Model: JSM 6390 LV Singapore). The powdered samples were coated with gold after attaching with the tape stubs. The
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samples were analyzed under 20 kV energy with 5000X magnification.
2.10. Storage study
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The storage study of the microencapsulated anthocyanin powder was carried out for 90
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days in 4°C and 25°C [3]. An amount of 500 mg of microencapsulated powder was spread in closed petri dishes and stored at 4°C and 25°C in a temperature controlled cabinet without light
extract.
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exposure. Samples were taken after every 10 days and measured the anthocyanin content of
2.11. Steady-shear rheology The effect of incorporation of microencapsulates on rheological properties of rice dough was investigated in term of steady-shear rheology. The flow properties of dough were evaluated
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ACCEPTED MANUSCRIPT using a controlled shear stress rheometer (Make: Antron Paar, Graz, Austria; Model: Physical MCR 72,) with a plate and plate attachment having 50 mm diameter. For the analysis, various amount of microencapsulated powder (5, 10, 15, and 20% w/w) was incorporated in rice dough with a moisture content of 35% w/w. For the test, the shear rate varied from 0.1 to 100 s-1 and
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temperature was 25±1°C. The experimental data were fitted with Herschel–Bulkley, and
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Mizrahi-Berk model as follows,
kH O
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H
H
0.5 kM O
(4)
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Where σ shear stress,
and
are yield stress,
and
are consistency index,
and
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are flow behavior index, and γ is the shear rate.
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2.12. Experimental design
To study the effect of starch concentration (X1) (5-10%), atomizer pressure (X2) (2.76-
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5.52 MPa), and inlet air temperature (X3) (140-180°C) on microencapsulated anthocyanin during
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spray drying and to optimize the process parameter, three-factor rotatable center composite design (RCCD) was employed. RCCD is widely used in food processing to optimize the
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complex system [14]. The optimum spray drying condition for microencapsulated powder was obtained in terms of maximizing encapsulation efficiency (Y1), anthocyanin content (Y2), DPPH scavenging activity (Y3), true density (Y4) and minimum water activity (Y5). Twenty experiments were carried out to find out the optimum values of spray drying parameters. The real values of the independent variables are shown in Table 1.
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ACCEPTED MANUSCRIPT 2.13. Statistical analysis The experiment was designed using Design-Expert 7.0.0 software (State-Ease Inc. Minneapolis, MN, USA). For all the tests, the significant level was p≤0.05. 3. Results and discussion
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3.1. Analysis of variance
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For microencapsulation of anthocyanin extract, the effects of independent parameters on
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encapsulation efficiency, anthocyanin content, DPPH scavenging activity, water activity and true density of microencapsulated anthocyanin was investigated using RCCD. The results were
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investigated and fitted to the second-order polynomial equations (Eqs. 5 to 9) with a high
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coefficient of determination (R2) values (Table 2). ANOVA was carried out for the significance
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test of the model coefficients (Table 2).
Encapsulation Efficiency 90.21 4.17 x1 1.15 x2 0.41x3 1.14 x1 x2 2.47 x1 x3 0.60 x2 x3
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(5)
0.46 x12 1.34 x22 0.50 x32
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Anthocyanin content =2.81-0.30x1 +0.039x 2 -0.21x 3 -0.20x1x 2 +0.039x1 x3 +0.25x 2 x3 -0.28x12
(6)
-0.069x 22 -0.15x 32
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DPPH Scavenging Activity 52.02 4.35 x1 0.52 x2 2.09 x3 3.55 x1 x2 1.85 x1 x3 0.42 x2 x3
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1.70 x12 0.22 x22 2.06 x32
True density 1.83 0.045 x1 1.099 E 004 x2 0.062 x3 9.500 E 003x1 x2 0.022 x1 x3 0.060 x2 x3 0.028 x12 0.053x22 9.851E 003x32
Water Activity 0.52 0.011x1 3.566 E 003x2 0.013 x3 5.700E 003x1 x2 7.775E 003x1 x3 0.011x2 x3 5.032 E 003x12 7.201E 003x22 2.834 E 003x32
(7)
(8)
(9)
The lack of fit of five responses was insignificant (0.842, 0.269, 0.455, 0.312, and 0.730) (p≤0.05) which conferred that the developed relationships were suitable for the experimental
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ACCEPTED MANUSCRIPT data. From the ANOVA analysis, it can be inferred that the developed models can be efficiently used for the optimization of the microencapsulation process.
3.2. Response surface analysis
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Fig.1 (a and b) shows the relationship between encapsulation efficiency and the spray
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drying process parameters. As the inlet air temperature increased, the encapsulation efficiency
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increased significantly (p≤0.05). During spray drying, as the inlet air temperature increases the drying rate of droplets accelerated and hence there is a quick formation of particle crust which
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prevents leaching of anthocyanin and penetration heat in the droplet. Fig.1a and 1b show that for
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the increase of atomizer pressure the encapsulation efficiency increases significantly (P≤0.05), whereas, for the increase in starch concentration as wall material after a certain concentration
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(7%) there was drastically decreased in the encapsulation efficiency. In this regards, Jafari,
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Assadpoor [15] suggested that retention of core material significantly depends upon the viscosity of the initial emulsion. For the increase of solid concentration, there is an increase in viscosity
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which may be resulting eventual cracking on the particles which increase the diffusion of the
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anthocyanin from core to the wall, hence decrease the encapsulation efficiency [16]. Fig. 1(c, and e) illustrated that for the increase of inlet air temperature; anthocyanin
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content decreases and DPPH scavenging activity of microencapsulated powder, due to the thermal sensitivity of anthocyanin. For the increase in starch concentration up to 7% (w/v) the anthocyanin content and DPPH scavenging activity of powder was increased, beyond that both decreases. The increase in starch concentration increases the emulsion viscosity that suppresses the internal circulations and enhances the anthocyanin and DPPH scavenging activity of microencapsulate [16]. However at high starch concentration, due to the cracking on the particles
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ACCEPTED MANUSCRIPT wall, the core anthocyanin leached out to the surface and degraded [16]. Fig.1 (d and f) illustrated that higher atomizer pressure enhances the anthocyanin content and DPPH scavenging activity of particles. For the increase of atomizer pressure, the momentum of atomized droplets increases into the spray stream [15] and rapid development of the film around the droplet, which
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reduces the anthocyanin diffusion and enhances the anthocyanin retention and DPPH scavenging
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activity in microencapsulates.
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The water activity of microencapsulates was varied between 0.49 to 0.56 and showed a significant (p≤0.05) effect of independent variables during processing as shown in Fig.1 (g and
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h). At high inlet air temperatures, the atomized feed and drying air has a high temperature
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gradient thereby, the water activity of the microencapsulate was gradually decreased. It can be observed from Fig.1g, that water activity decreased with increases in starch concentration as wall
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material. The higher solids contents in feed reduce the total available moisture and decrease the
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water activity of powder [17]. The water activity of the powders was increased with increase in atomizer pressure (Fig.1h). At high atomization pressure the retention time of particles in the
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drying chamber reduces, and hence the water activity of finished particles increased.
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From the Fig. 1i, it was illustrated that for the increase in inlet air temperature, true density decreased. At very high temperatures, a high rate of drying implies a lower shrinkage of
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the droplets, and thereby a lower true density microencapsulated powder was obtained [16]. For the increase in starch concentration, a slight decrease in true density was noticed. For the increase in starch concentration the viscosity of the droplet increases and cause cracking on the particles surface which reduces the true density of particle [16]. It is observed that for the increase of atomization pressure up to 4.14 MPa there was an increase in true density beyond that there was a decrease in true density of microencapsulate.
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ACCEPTED MANUSCRIPT 3.3. Optimization and validation The section was aimed to find out the values of spray drying process parameters which would allow getting a microencapsulated powder with high encapsulation efficiency, anthocyanin content, DPPH scavenging activity, true density, and low water activity. Thereby
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during the optimization, the targets were set to achieve maximum encapsulation efficiency,
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maximum anthocyanin content, maximum DPPH scavenging activity, maximum true density,
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and minimum water activity. Optimum conditions were selected based on the highest desirability value. The developed models (Eqs. 5 to 9) were used for optimization of spray drying process
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parameters. The maximum desirability of spray drying conditions was 0.88.
The optimal
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condition of spray drying was 6.01 % modified starch, 4.96 MPa atomizer pressure, and 168.78°C inlet air temperature. In the optimum condition, the predicted values of the dependent
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variables were, encapsulation efficiency 94.22 %, DPPH scavenging activity 54.59 %,
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anthocyanin content 2.91 mg/L, true density 1.74 g/cm3, and water activity 0.51. For the validation of optimized condition, the experiment was conducted in optimum condition. At the
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optimized condition, encapsulation efficiency, anthocyanin content, DPPH activity, true density,
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and water activity of powder did not significantly (p≤0.05) varied from predicted values, hence
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the developed model was reliable.
3.4. Physico-chemical properties The physicochemical properties of microencapsulated anthocyanin powder were investigated in term of true density, Hausner ratio, bulk density, porosity, diameter, water activity, and solubility. It was observed that the true density was 1.74 (g/cm3), Hausner ratio
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ACCEPTED MANUSCRIPT (HR) 1.020, bulk density 1.404 (g/cm3), porosity 0.20, solubility 51.83 %, diameter 6.44 (µm), and water activity was 0.51 of the developed powder. 3.5. Gelatinization properties Gelatinization properties of the microencapsulated anthocyanin powder were analyzed
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through DSC and compared with both the starch samples. As shown in Fig 2, the
The onset, peak, and endset temperature signify the
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peak temperature than native starch.
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microencapsulated anthocyanin powder, as well as modified starch showed higher onset and
initiation of gelatinization, maximum gelatinization, and end of the gelatinization of the sample,
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respectively. There were distinct differences in the peak of melting temperatures of the native,
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modified starch and encapsulated powder. The onset, peak and endset temperature of native rice starch were 74.04, 73.21 and 77.21°C, respectively (Fig. 2).
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From the Fig. 2, it can be observed that the onset temperature of microencapsulated
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particles and modified starch were significantly changed. It may be due to the formation of various complexes with amylose for the hydrolysis of starch, especially amorphous regions and
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affect the thermal properties. A similar observation was reported by the previous researcher [18].
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The onset temperature of the samples was increased from 73.21ºC to 157ºC, due to the long chain complex formation of amylose-phenolic compounds, which needed a higher temperature to
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break. This increase could also be due to a high number of longer amylopectin double helices resulting from modification than in unhydrolyzed amylopectin molecule. Because branch points in unhydrolyzed amylopectin may reduce the length of helix-forming side chains. From the result, it can be inferred that the developed powder is thermally stable.
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ACCEPTED MANUSCRIPT 3.6. X-ray diffraction Fig.3 shows the X-ray diffraction pattern of microencapsulated anthocyanin powder, native and modified starch. As shown in Fig.3, a strong diffraction peak was observed around 15° which indicates A-type crystallinity of starch, a doublet at 17°, and 23° (2θ) [19]. After
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microencapsulation, the typical starch peaks disappeared and the loaded microencapsulated
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powders demonstrated an amorphous nature. The Fig. 3 illustrated that for microencapsulated
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anthocyanin powder, a sharp peak at 19.18° was observed which may be due to the formation of the biopolymer with an amorphous structure [20]. It may possible that there is a formation of V-
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amylose with other bioactive compounds present in anthocyanin extract, which is responsible for
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the distinct peak [21, 22]. In addition to that, the effect of the spray drying process may also be
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responsible for the distinct peak of microencapsulated anthocyanin powder.
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3.7. FT-IR analysis
The FT-IR spectrum of anthocyanin extract, starch, modified starch and encapsulated
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powder shown in Fig. 4. The infrared spectra of the samples were characterized with the help of
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previous investigations [18, 22]. The spectrum of native, modified starches and
groups.
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microencapsulated anthocyanin powder were having quite similar stretches and functional
The peaks were observed in the regions between 800 to 1,500 cm−1 (the fingerprint region), the region between 1,500 to 2,000 cm−1 (C-H stretch region), and finally the region between 2,500 and 3,500 cm−1 (O-H stretch region). The peaks between 1000 and 1250 cm-1 may be due to the C–O–C bond, which illustrated that the three samples were carbohydrate in nature. From the Fig.4, a band was observed at 1022 cm−1 which is responsible for more amorphous
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ACCEPTED MANUSCRIPT samples, while the bands at 1000 and 1047 cm−1 defined the more crystalline samples [23]. The predominant peaks located at 1636 cm−1 is also associated with ring stretching of C=C. From Fig. 4 it was observed that at 2950cm-1 there is a sharp peak which may be due to the C-H stretching vibration. The FT-IR spectrum for the anthocyanin extract shows a strong absorption
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band at 1032 cm-1 assigned to aromatic ring C-H deformation and 3489 cm-1 for OH. The
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absorption band between 1500 and 3000 cm-1 corresponding to the stretching of C–H, C=C
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asymmetric deformation in –OCH3, CH2 in pyran ring, typical of flavonoid compounds [18].
3.8. Surface morphology
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The microencapsulated powder obtained under optimum spray drying conditions (6.01 % modified starch, 4.96 MPa atomizer pressure, and 168.78°C inlet air temperature) was
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characterized using SEM technique and compared with native starch and modified starch. As
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shown in Fig.5, the surface morphology of spray dried microcapsule was more uniform and smooth than native starch and modified starch surface. Moreover, no particle aggregation was
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observed in the microencapsulated anthocyanin powder [24]. Kanakdande, Bhosale [25]
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suggested that the microencapsulated powder should have a uniform and smooth surface having slightly spherical shape with minimum cracks and collapses on walls. Few broken microcapsules
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can be observed from Fig. 5, indicating quite inefficient encapsulating properties of the carrier materials. This result was in agreement with earlier reports [25].
3.9. Storage Study The storage stability of microencapsulated anthocyanin powder was studied for 90 days at 25 and 4°C. In both the temperatures, a decrease in anthocyanin content was observed as
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ACCEPTED MANUSCRIPT shown in Fig. 6. This phenomenon illustrated that after the microencapsulation, there can also be a significant decrease in the anthocyanin content. Hence, the degradation of anthocyanin in microencapsulated powder was higher at 25°C than 4°C. As similar types of observation were reported by the previous researcher [3]. Degradation of anthocyanin, during storage, may occur
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due to various reaction mechanisms, such as condensation reactions, oxidation, and cleavage of a
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covalent bond. During high-temperature storage, the reaction rate of these reactions is high,
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degradation during storage is difficult to establish [26].
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which enhance anthocyanin degradation. However, the exact mechanism for anthocyanin
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3.10. Steady shear rheology
The effect of microencapsulated powder on the steady-shear rheology of rice dough was
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determined and presented in Table 3. The Herschel–Bulkley, and Mizrahi-Berk model were and
) of the samples was less than
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applied for the investigation. The flow behavior index (
1, which depicted the shear thinning behavior. Moreover, as the proportion of microencapsulated
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powder increased in dough, the flow behavior index increased for both the model. This indicated
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that the microencapsulated powder decreases the pseudo-plasticity of the dough samples. It may be due to the formation of new networks in the rice dough in the presence of microencapsulated
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powder. For the increase of microencapsulated powder in rice dough, resulted in a very sticky and cohesive dough, which required a high force for shearing as compared to only rice dough and enhance the consistence of the dough. The Herschel–Bulkley model showed the higher coefficient of determination (R2) than Mizrahi-Berk model, hence it can be inferred that Herschel–Bulkley model can efficiently predict the effect of microencapsulated powder on the steady-shear rheology of rice dough.
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ACCEPTED MANUSCRIPT
4. Conclusions The microencapsulation of anthocyanin extract from purple rice bran with modified glutinous rice starch as wall material was successfully developed using the spray drying process.
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The process was optimized using RCCD followed by RSM and the optimum process condition
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for microencapsulation was 6.01% of starch concentration, 168.78°C drying temperature and
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4.96 MPa nozzle pressure. The physical characteristics of spray-dried powders were investigated in term of water activity, solubility, true density, bulk density, porosity, diameter, and Hausner
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ratio. The thermal properties and crystallinity of microencapsulate powder significantly differ
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from native and modified glutinous rice starch. The microencapsulate showed higher onset (157.0°C) and peak temperature (168.78°C). The morphological properties of the
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microencapsulate showed that the surface of microcapsules was more uniform and smooth
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surfaces than the native and modified starch. The storage study of microencapsulated powder illustrated that at low-temperature storage the anthocyanin degradation in particles was less than
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high-temperature storage. The microencapsulated particles also affect the steady-shear rheology
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of rice dough. The increase in microencapsulated powder concentration from 5 to 20% in rice
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dough, decrease the pseudo-plasticity of dough.
Acknowledgments
The authors are grateful to the Indian Institute of Technology Guwahati and Tezpur University for instrumental support.
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ACCEPTED MANUSCRIPT Figure Captions Fig. 1 Effect of temperature, starch concentration and atomizer pressure on microencapsulate. Fig. 2 Thermogram of a) microencapsulated powder, b) modified starch, and c) native starch Fig. 3 XRD of a) Microencapsulated powder b) Native, and c) Modified starch
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Fig. 4 FT-IR spectrum of native starch, modified starch and microencapsulated powder
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Fig. 5 Scanning electron microscope of a) and b) microencapsulated powder, c) native, and d)
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modified rice starch
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Fig. 6 Storage stability of anthocyanin in microencapsulated powder at 25°C and 4°C
Table Captions
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Table 1 Experimental design of spray drying of anthocyanin extract
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Table 2 Analysis of variance for the fitted quadratic polynomial model
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Table 3 Steady shear rheological parameters
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ACCEPTED MANUSCRIPT Table 1 Experimental design of spray drying of anthocyanin extract Independent Parameters
Dependent Parameters Anthocyanin
True
Pressure
Temperature
Encapsulation
Content
Scavenging
Density
Water
(%)
(MPa)
(°C)
Efficiency (%)
(mg/L)
Activity (%)
(g/cm3)
Activity
1.673
0.492
43.61
1.799
0.519
47.92
1.769
0.515
2.431
51.98
1.681
0.503
2.772
52.65
1.819
0.525
1.918
44.67
1.794
0.511
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Starch
4.14
160
80.97
1.54
2
8.99
4.96
150
84.94
1.803
3
8.99
3.32
170
88.75
1.619
4
7.5
5.52
160
95.38
5
6.01
3.32
150
96.74
6
6.01
3.32
170
88.53
7
7.5
4.14
180
87.91
1.969
42.63
1.715
0.501
8
6.01
4.96
170
95.32
2.932
52.71
1.692
0.512
9
7.5
4.14
160
92.23
2.912
54.86
1.865
0.529
10
8.99
4.96
170
89.02
2.087
41.32
1.62
0.490
11
8.99
3.32
150
85.10
2.554
48.07
1.698
0.495
12
6.01
4.96
150
99.17
3.040
61.94
1.949
0.569
13
7.5
4.14
160
90.83
2.892
52.01
1.795
0.514
14
7.5
4.14
140
88.66
2.721
47.92
1.989
0.558
15
7.5
4.14
160
90.83
2.892
52.01
1.865
0.529
16
5
4.14
160
95.83
2.43
54.73
1.817
0.523
17
7.5
4.14
160
87.33
2.642
50.71
1.832
0.529
18
7.5
4.14
160
89.35
2.592
49.92
1.775
0.519
19
7.5
4.14
160
90.83
2.922
52.9
1.865
0.529
20
7.5
2.76
160
91.58
2.708
51.45
1.67
0.500
25
ED
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CE
AC
41.84
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CR
IP
10
M
1
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Run
DPPH
ACCEPTED MANUSCRIPT Table 2 Analysis of variance for the fitted quadratic polynomial model Terms
Encapsulation
Anthocyanin
DPPH
True
Water
Efficiency
Content
Scavenging
Density
Activity
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.012
0.034
0.030
0.030
0.001
0.001
0.044
0.010
0.0006
0.548
0.254 0.243 0.004
0.0002
0.009
0.105
< 0.0001
< 0.0001
0.0002
0.461
0.054
0.014
0.109
0.014
0.002
0.515
0.0007
0.001
0.0001
0.004
0.012
0.027
0.167
0.654
0.0002
0.004
0.009
0.001
0.311
0.177
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CR
0.0008
0.842
0.269
0.455
0.730
0.312
R2
0.947
0.927
0.945
0.930
0.929
0.900
0.861
0.896
0.868
0.865
Adj-R2
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Lack of Fit
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0.209
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< 0.0001
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<0.0001
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< 0.0001
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Model
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Activity
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Table 3 Steady shear rheological parameters Herschel–Bulkley
Mizrahi-Berk
η
σ
R2
k (Pa sn)
η
σ
R2
Control
834
0.384
0.87
0.829
335
0.184
0.698
0.834
5
1096
0.458
156.9
0.891
524
0.630
12.31
0.888
10
1370
0.522
98.99
0.845
890
0.650
3.81
0.843
15
1469
0.687
40.94
0.876
681
0.715
5.572
0.873
20
1460
0.718
89.24
0.967
8.596
0.964
CR
US 724
AN M ED PT CE AC
27
IP
k (Pa sn)
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Sample
0.890
ACCEPTED MANUSCRIPT Highlights 1. Anthocyanin successfully microencapsulated using modified starch. 2. The storage stability of microencapsulated anthocyanin was high at low storage
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CE
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AN
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3. Microencapsulated powder affects the rice dough rheology.
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temperature.
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Figure 1af
Figure 1gj
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Amit Baran Das, V.V. Goud, Chandan Das PII: DOI: Reference:
S0141-8130(18)35948-8 https://doi.org/10.1016/j.ijbiomac.2018.11.247 BIOMAC 11124
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
2 November 2018 22 November 2018 26 November 2018
Please cite this article as: Amit Baran Das, V.V. Goud, Chandan Das , Microencapsulation of anthocyanin extract from purple rice bran using modified rice starch and its effect on rice dough rheology. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.11.247
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ACCEPTED MANUSCRIPT Microencapsulation of anthocyanin extract from purple rice bran using modified rice starch and its effect on rice dough rheology
Amit Baran Dasa,b, V. V. Goud*b, Chandan Das*b Department of Food Engineering and Technology Tezpur University, Assam, India
Department of Chemical Engineering
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b
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a
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Indian Institute of Technology Guwahati, Assam, India
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___________________ *Corresponding author
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Tel: +913612582268/2272 Fax:+913612582291 E-mail: [email protected] (C. Das), [email protected] (V.V. Goud)
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ACCEPTED MANUSCRIPT Abstract The investigation aims to microencapsulate anthocyanin extract from purple rice bran with modified glutinous rice starch to provide stable anthocyanin powder. The modified glutinous rice starch was used as wall material where anthocyanin extract was a core material for
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microencapsulation. The microencapsulation was carried out in a spray dryer, and the process
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was optimized using a rotatable central composite design. The effect of independent parameters,
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viz., starch concentration, inlet air temperature, and atomizer pressure, on the dependent parameters, such as encapsulation efficiency, anthocyanin content, 2, 2-diphenyl-1-
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picrylhydrazyl (DPPH) scavenging activity, true density and water activity were investigated.
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The optimum values of the spray drying process parameters were 6.01% of starch concentration, 168.78°C inlet air temperature and 4.96 MPa atomizer pressure. The physical characteristics of
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spray-dried powders were investigated in term of water activity (0.51), solubility (51.83%), bulk
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density (1.404g/cm3), porosity (0.20), and diameter (6.44µm) and the Hausner ratio (1.020). The thermal, crystallinity and surface morphology of the microencapsulated particles were also
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comprehensively studied using DSC, XRD, FTIR, and SEM. The storage stability of
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microencapsulated anthocyanin showed more stability at 4°C than at 25°C for 90 days. The microencapsulated particles also have an effect on the steady-shear rheology of the rice dough.
properties.
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Keyword: Anthocyanin; microencapsulation; spray drying; thermal properties; rheological
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ACCEPTED MANUSCRIPT 1. Introduction Anthocyanin is the most widely used and recognized members of the bioflavonoid phytochemicals. The potential sources of anthocyanin are colored fruits and vegetables. Some pigmented rice varieties are also a good source of anthocyanin, especially purple rice bran [1].
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Besides the wide range of colors, the potential health benefits rendered anthocyanin as an
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attractive compound in the food system [2]. However, the stability of anthocyanin is a major
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challenge for the food industry. The stability of anthocyanin depends on various environmental factors, such as pH, light, temperature, oxygen, and enzymatic activities. For the low stability in
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various conditions during processing and storage, application of anthocyanin as an ingredient in
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foods is now quite challenging.
To obtain a stable and usable food ingredient from anthocyanin, several approaches are in
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consideration, such as encapsulation, emulsion, and freeze-drying. For anthocyanin,
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encapsulation method has been demonstrated and accepted by a few researchers [3-7]. The most commonly used encapsulation process is microencapsulation. Microencapsulation may be an
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efficient way to improve the bioavailability and stability of bioactive compounds and managing
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the release of active agent [2]. The main aim of microencapsulation is to protect the core material from environmental effects, thereby increases the shelf life of the product and promoting a
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controlled release of the core material [8]. There are various types of microencapsulation techniques, such as a vibrational nozzle, centrifugal extrusion, spray drying, and cocrystallization. The most commonly used technique is spray drying, due to its continuity in nature, low cost and less time which can produce particles with good quality [2]. For microencapsulation through spray drying process, the selection of wall material is the primary step. Most commonly used wall materials are maltodextrin, gum arabic, and emulsifying
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ACCEPTED MANUSCRIPT starches. The carbohydrates, such as, maltodextrins and starches have properties like good solubility and low viscosity at high solids contents which are highly desirable for an encapsulating agent. However, native starch is insoluble in cold water and, when heated with water it forms a paste-like texture with high viscosity, which decreases the spray drying
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efficiency and increases the surface crack, thereby limits its application for encapsulation using
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spray drying. These limitations can be overcome by the modification of native starch. Few
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numbers of researchers were used modified starch as a wall material in for encapsulation of various bioactive compounds [9]. However, the application of modified low amylose rice starch
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as wall material in microencapsulation of anthocyanin was scanty.
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The functional and physicochemical properties of a microencapsulated anthocyanin powder directly depend on the storage condition of the powder. The extrinsic factors like
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temperature, light, and oxygen, are the most determining factors of anthocyanin stability in
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powder [3]. Therefore, the effect of storage conditions on the stability of anthocyanin in microencapsulated powder needs to understand. On the other hand, the incorporation of
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microencapsulated powder significantly affect the rheological properties and textural properties
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of food product [10]. Therefore, a clear understanding of the effect of microencapsulated powder on the flow behavior of dough is required to design and develop a new food product. However,
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there is no information on the effect of microencapsulated anthocyanin powder incorporation on the rice dough rheology. Therefore, the present work is the first where anthocyanin from purple rice bran is microencapsulated with modified glutinous rice starch using a spray-drying technique. The objective of the present study was to microencapsulate anthocyanin extract from purple rice bran using modified starch through spray drying. The spray drying process for
4
ACCEPTED MANUSCRIPT microencapsulation was optimized and validated. The physicochemical, thermal, and morphological properties of microencapsulated powder were also investigated extensively. The storage stability of a spray-dried powder in various temperatures was also carried out. In addition to that, the effect of microencapsulated anthocyanin powder on the rice dough rheology was also
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determined.
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2. Materials and Methods 2.1. Materials
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Purple rice (Oryza sativa L) from Manipur, India was used for this study. Bran was
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separated and stored in zip lock polyethylene bag at 28±2oC. Modified starch was prepared from rice starch according to the method described by Hu, Wei [11]. Glutenous rice starch (25 g) was
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dispersed in 100 mL anhydrous 1-butanol. The reaction was started by adding 1 mL of
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concentrated (36% by weight) HCl and proceeded at 30°C for 2 h, with constant stirring. The reaction was stopped by adding 14 mL of 1 M NaHCO3. The solution was cooled in an ice bath
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for 15 min and centrifuged at 5000 rpm (523.59 rad/s) for another 5 min. The precipitate was
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washed four times with 50% ethanol and dried in an air oven at 45°C. The anthocyanin was extracted from bran using ultrasound-assisted extraction process as follow by Das, Goud, and
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Das [1]. The extracted anthocyanin was separated from the extract through the adsorption/desorption process using amberlite XAD7 resin [12]. For adsorption, 1 g pre-treated XAD7 resin and 25 mL of purple rice bran extract was added to 250 mL flask. Then the flask was kept for shaking at 45 rpm (4.71 rad/s) in a shaker at room temperature (28±2°C) for 24 h. For desorption, the resins with anthocyanin were washed with 25 mL distilled water and then, 50
5
ACCEPTED MANUSCRIPT mL 95% ethanol was added to the flask. The flask was kept in a shaker at 45 rpm (4.71 rad/s) for 24 h at room temperature (28±2°C).
2.2. Preparation of feed sample and encapsulation
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For microencapsulation of anthocyanin extract, modified starch was used as wall material
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and anthocyanin as a core material. The starch dispersion of various concentrations (5-10% w/v)
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was prepared in water with the combination of anthocyanin extract (40 mg cyanidin-3glucoside/L) (core) under magnetic agitation. The encapsulation of anthocyanin extract was done
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using spray dryer (Make: Labultima, Mumbai, India; Model: Lab plant LU 20 lab sprayer).
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During spray drying, the inlet air temperature was varied from 140 to 180°C and atomizer
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pressure ranged from 2.76 to 5.52 MPa as mentioned in Table 1.
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2.3. Encapsulation efficiency
The encapsulation efficiency of the microencapsulation process was measured using the
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method described by Akhavan Mahdavi, Jafari [4]. To estimate the efficiency, total anthocyanin
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content (TAC) and surface anthocyanin content (SAC) of the microcapsules were measured. For analysis, 1 mL of distilled water was added to 100 mg of sample and the microencapsulation was
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crushed in a mortar-pestle. Then, the samples were extracted using 10 mL ethanol. The surface anthocyanin from the microcapsules was extracted by quickly washing with ethanol (10 mL) in a vortex within 10s, and centrifuge at 3000 rpm (314.15 rad/s) for 3 min at 20°C. The clear supernatants were collected and filtered through 0.45µm-filter paper and used for anthocyanin determination. Encapsulation efficiency (%)
(TAC SAC ) 100 TAC 6
(1)
ACCEPTED MANUSCRIPT 2.4. Anthocyanin content and DPPH scavenging activity The anthocyanin contents of the microencapsulates were determined by the pHdifferential method as mentioned by Das, Goud, and Das [1]. The absorbance of the samples was measured at 515 and 700 nm using UV/VIS spectrophotometer (Make: Thermo Fisher Scientific,
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Waltham, Massachusetts, USA; Model: UV-2600).
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The DPPH scavenging activity of microencapsulates was measured according to Luo,
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Zhao [13] with some modifications. For analysis, microencapsulate (15 mg) was dissolved in a mixture (3 mL) of ethanol: acetic acid: water (50:8:42, v/v). The mixture was filtered through a
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filter paper (0.45µm) and diluted with methanol. The 100 µL filtrate was taken and added to 1.4
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mL DPPH radical methanolic solution (10-4 M). The absorbance was measured at 517 nm using a spectrophotometer (Make: Thermo Fisher Scientific, Waltham, Massachusetts, USA; Model:
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UV-2600). The DPPH radical scavenging activity was determined as follows
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Radical scavanging activity (%) =
Ao - As 100 Ao
(2)
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where Ao is absorbance of control blank, and As is absorbance of sample extract.
2.5. Physicochemical properties
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The physicochemical properties of the samples were investigated in terms of true density, water activity, Hausner ratio, bulk density, porosity, solubility, and diameter. The true density of the microencapsulated powders was measured using gas pycnometer (Make: Porous Materials Inc, Ithaca, New York, USA; Model: PYC-100A). The water activity of the microencapsulated samples was determined using a water activity meter (Make: Aqualab Pullman, USA; Model: 4TE). The Hausner ratio defining the flowability of powder, it is the ratio of tap volume to the bulk volume of powder. For bulk density, a 500mL measuring cylindrical was filled with the 7
ACCEPTED MANUSCRIPT sample from a height of 150mm at a constant rate and then weighing the contents. The porosity of the powder was calculated using the relationship between the bulk density and true density [4]. For solubility, the 2.5 g ground sample was mixed with 30 ml of water at 30°C, in a pre-
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weighed centrifuge tube, agitated for 30 min, and then centrifuged at 3000 rpm (314.15 rad/s) for
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10 min. The supernatant was carefully decanted into a pre-weighed evaporating dish. After
CR
evaporating the water from the supernatant, the mass of dried solids recovered was expressed as
US
a percentage of dry solids in the sample and defined as the water solubility index (WSI).
AN
2.6. Gelatinization properties
A differential scanning calorimetry instrument (Make: NETZSCH, Selb, Germany;
M
Model: DSC 214) was used to determine the gelatinization properties of microencapsulated
ED
powders. For the analysis, the initial temperature was 30°C and end at 250°C with 10°C/min rate. The onset, endset, and peak temperature of the powder were measured and analyzed using
CE
PT
the DSC instrument’s software.
2.7. X-Ray Diffraction
AC
The crystallinity of the microencapsulated powder was studied at 35 kV and 20 mA CuKa radiation in an X-ray diffractometer (Make: Bruker Axs, Karlsruhe, Germany; Model: D8 FOCUS). Data were recorded over an angular range of 2° to 50° (2θ) with a scanning rate of 2°/min.
8
ACCEPTED MANUSCRIPT
2.8. FT-IR analysis The FT-IR spectra of microencapsulates were recorded using a spectrometer (Make: NICOLET, Wisconsin, USA; Model: IMPACT 410). The absorbance spectra of the
T
microencapsulated powders were documented in the range of 4000 to 400 cm-1 having a
CR
IP
resolution of 4 cm-1. All the experimental data were analyzed using OMNIC E.S.P.5.0 software.
2.9. Morphological properties
US
The morphological properties of microencapsulated anthocyanin were carried out using a
AN
scanning electron microscope (Make JEOL Akishima, Tokyo, Japan; Model: JSM 6390 LV Singapore). The powdered samples were coated with gold after attaching with the tape stubs. The
ED
M
samples were analyzed under 20 kV energy with 5000X magnification.
2.10. Storage study
PT
The storage study of the microencapsulated anthocyanin powder was carried out for 90
CE
days in 4°C and 25°C [3]. An amount of 500 mg of microencapsulated powder was spread in closed petri dishes and stored at 4°C and 25°C in a temperature controlled cabinet without light
extract.
AC
exposure. Samples were taken after every 10 days and measured the anthocyanin content of
2.11. Steady-shear rheology The effect of incorporation of microencapsulates on rheological properties of rice dough was investigated in term of steady-shear rheology. The flow properties of dough were evaluated
9
ACCEPTED MANUSCRIPT using a controlled shear stress rheometer (Make: Antron Paar, Graz, Austria; Model: Physical MCR 72,) with a plate and plate attachment having 50 mm diameter. For the analysis, various amount of microencapsulated powder (5, 10, 15, and 20% w/w) was incorporated in rice dough with a moisture content of 35% w/w. For the test, the shear rate varied from 0.1 to 100 s-1 and
T
temperature was 25±1°C. The experimental data were fitted with Herschel–Bulkley, and
IP
Mizrahi-Berk model as follows,
kH O
(3)
CR
H
H
0.5 kM O
(4)
M
Where σ shear stress,
and
are yield stress,
and
are consistency index,
and
M
AN
are flow behavior index, and γ is the shear rate.
US
M
ED
2.12. Experimental design
To study the effect of starch concentration (X1) (5-10%), atomizer pressure (X2) (2.76-
PT
5.52 MPa), and inlet air temperature (X3) (140-180°C) on microencapsulated anthocyanin during
CE
spray drying and to optimize the process parameter, three-factor rotatable center composite design (RCCD) was employed. RCCD is widely used in food processing to optimize the
AC
complex system [14]. The optimum spray drying condition for microencapsulated powder was obtained in terms of maximizing encapsulation efficiency (Y1), anthocyanin content (Y2), DPPH scavenging activity (Y3), true density (Y4) and minimum water activity (Y5). Twenty experiments were carried out to find out the optimum values of spray drying parameters. The real values of the independent variables are shown in Table 1.
10
ACCEPTED MANUSCRIPT 2.13. Statistical analysis The experiment was designed using Design-Expert 7.0.0 software (State-Ease Inc. Minneapolis, MN, USA). For all the tests, the significant level was p≤0.05. 3. Results and discussion
T
3.1. Analysis of variance
IP
For microencapsulation of anthocyanin extract, the effects of independent parameters on
CR
encapsulation efficiency, anthocyanin content, DPPH scavenging activity, water activity and true density of microencapsulated anthocyanin was investigated using RCCD. The results were
US
investigated and fitted to the second-order polynomial equations (Eqs. 5 to 9) with a high
AN
coefficient of determination (R2) values (Table 2). ANOVA was carried out for the significance
M
test of the model coefficients (Table 2).
Encapsulation Efficiency 90.21 4.17 x1 1.15 x2 0.41x3 1.14 x1 x2 2.47 x1 x3 0.60 x2 x3
ED
(5)
0.46 x12 1.34 x22 0.50 x32
PT
Anthocyanin content =2.81-0.30x1 +0.039x 2 -0.21x 3 -0.20x1x 2 +0.039x1 x3 +0.25x 2 x3 -0.28x12
(6)
-0.069x 22 -0.15x 32
CE
DPPH Scavenging Activity 52.02 4.35 x1 0.52 x2 2.09 x3 3.55 x1 x2 1.85 x1 x3 0.42 x2 x3
AC
1.70 x12 0.22 x22 2.06 x32
True density 1.83 0.045 x1 1.099 E 004 x2 0.062 x3 9.500 E 003x1 x2 0.022 x1 x3 0.060 x2 x3 0.028 x12 0.053x22 9.851E 003x32
Water Activity 0.52 0.011x1 3.566 E 003x2 0.013 x3 5.700E 003x1 x2 7.775E 003x1 x3 0.011x2 x3 5.032 E 003x12 7.201E 003x22 2.834 E 003x32
(7)
(8)
(9)
The lack of fit of five responses was insignificant (0.842, 0.269, 0.455, 0.312, and 0.730) (p≤0.05) which conferred that the developed relationships were suitable for the experimental
11
ACCEPTED MANUSCRIPT data. From the ANOVA analysis, it can be inferred that the developed models can be efficiently used for the optimization of the microencapsulation process.
3.2. Response surface analysis
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Fig.1 (a and b) shows the relationship between encapsulation efficiency and the spray
IP
drying process parameters. As the inlet air temperature increased, the encapsulation efficiency
CR
increased significantly (p≤0.05). During spray drying, as the inlet air temperature increases the drying rate of droplets accelerated and hence there is a quick formation of particle crust which
US
prevents leaching of anthocyanin and penetration heat in the droplet. Fig.1a and 1b show that for
AN
the increase of atomizer pressure the encapsulation efficiency increases significantly (P≤0.05), whereas, for the increase in starch concentration as wall material after a certain concentration
M
(7%) there was drastically decreased in the encapsulation efficiency. In this regards, Jafari,
ED
Assadpoor [15] suggested that retention of core material significantly depends upon the viscosity of the initial emulsion. For the increase of solid concentration, there is an increase in viscosity
PT
which may be resulting eventual cracking on the particles which increase the diffusion of the
CE
anthocyanin from core to the wall, hence decrease the encapsulation efficiency [16]. Fig. 1(c, and e) illustrated that for the increase of inlet air temperature; anthocyanin
AC
content decreases and DPPH scavenging activity of microencapsulated powder, due to the thermal sensitivity of anthocyanin. For the increase in starch concentration up to 7% (w/v) the anthocyanin content and DPPH scavenging activity of powder was increased, beyond that both decreases. The increase in starch concentration increases the emulsion viscosity that suppresses the internal circulations and enhances the anthocyanin and DPPH scavenging activity of microencapsulate [16]. However at high starch concentration, due to the cracking on the particles
12
ACCEPTED MANUSCRIPT wall, the core anthocyanin leached out to the surface and degraded [16]. Fig.1 (d and f) illustrated that higher atomizer pressure enhances the anthocyanin content and DPPH scavenging activity of particles. For the increase of atomizer pressure, the momentum of atomized droplets increases into the spray stream [15] and rapid development of the film around the droplet, which
T
reduces the anthocyanin diffusion and enhances the anthocyanin retention and DPPH scavenging
IP
activity in microencapsulates.
CR
The water activity of microencapsulates was varied between 0.49 to 0.56 and showed a significant (p≤0.05) effect of independent variables during processing as shown in Fig.1 (g and
US
h). At high inlet air temperatures, the atomized feed and drying air has a high temperature
AN
gradient thereby, the water activity of the microencapsulate was gradually decreased. It can be observed from Fig.1g, that water activity decreased with increases in starch concentration as wall
M
material. The higher solids contents in feed reduce the total available moisture and decrease the
ED
water activity of powder [17]. The water activity of the powders was increased with increase in atomizer pressure (Fig.1h). At high atomization pressure the retention time of particles in the
PT
drying chamber reduces, and hence the water activity of finished particles increased.
CE
From the Fig. 1i, it was illustrated that for the increase in inlet air temperature, true density decreased. At very high temperatures, a high rate of drying implies a lower shrinkage of
AC
the droplets, and thereby a lower true density microencapsulated powder was obtained [16]. For the increase in starch concentration, a slight decrease in true density was noticed. For the increase in starch concentration the viscosity of the droplet increases and cause cracking on the particles surface which reduces the true density of particle [16]. It is observed that for the increase of atomization pressure up to 4.14 MPa there was an increase in true density beyond that there was a decrease in true density of microencapsulate.
13
ACCEPTED MANUSCRIPT 3.3. Optimization and validation The section was aimed to find out the values of spray drying process parameters which would allow getting a microencapsulated powder with high encapsulation efficiency, anthocyanin content, DPPH scavenging activity, true density, and low water activity. Thereby
T
during the optimization, the targets were set to achieve maximum encapsulation efficiency,
IP
maximum anthocyanin content, maximum DPPH scavenging activity, maximum true density,
CR
and minimum water activity. Optimum conditions were selected based on the highest desirability value. The developed models (Eqs. 5 to 9) were used for optimization of spray drying process
US
parameters. The maximum desirability of spray drying conditions was 0.88.
The optimal
AN
condition of spray drying was 6.01 % modified starch, 4.96 MPa atomizer pressure, and 168.78°C inlet air temperature. In the optimum condition, the predicted values of the dependent
M
variables were, encapsulation efficiency 94.22 %, DPPH scavenging activity 54.59 %,
ED
anthocyanin content 2.91 mg/L, true density 1.74 g/cm3, and water activity 0.51. For the validation of optimized condition, the experiment was conducted in optimum condition. At the
PT
optimized condition, encapsulation efficiency, anthocyanin content, DPPH activity, true density,
CE
and water activity of powder did not significantly (p≤0.05) varied from predicted values, hence
AC
the developed model was reliable.
3.4. Physico-chemical properties The physicochemical properties of microencapsulated anthocyanin powder were investigated in term of true density, Hausner ratio, bulk density, porosity, diameter, water activity, and solubility. It was observed that the true density was 1.74 (g/cm3), Hausner ratio
14
ACCEPTED MANUSCRIPT (HR) 1.020, bulk density 1.404 (g/cm3), porosity 0.20, solubility 51.83 %, diameter 6.44 (µm), and water activity was 0.51 of the developed powder. 3.5. Gelatinization properties Gelatinization properties of the microencapsulated anthocyanin powder were analyzed
T
through DSC and compared with both the starch samples. As shown in Fig 2, the
The onset, peak, and endset temperature signify the
CR
peak temperature than native starch.
IP
microencapsulated anthocyanin powder, as well as modified starch showed higher onset and
initiation of gelatinization, maximum gelatinization, and end of the gelatinization of the sample,
US
respectively. There were distinct differences in the peak of melting temperatures of the native,
AN
modified starch and encapsulated powder. The onset, peak and endset temperature of native rice starch were 74.04, 73.21 and 77.21°C, respectively (Fig. 2).
M
From the Fig. 2, it can be observed that the onset temperature of microencapsulated
ED
particles and modified starch were significantly changed. It may be due to the formation of various complexes with amylose for the hydrolysis of starch, especially amorphous regions and
PT
affect the thermal properties. A similar observation was reported by the previous researcher [18].
CE
The onset temperature of the samples was increased from 73.21ºC to 157ºC, due to the long chain complex formation of amylose-phenolic compounds, which needed a higher temperature to
AC
break. This increase could also be due to a high number of longer amylopectin double helices resulting from modification than in unhydrolyzed amylopectin molecule. Because branch points in unhydrolyzed amylopectin may reduce the length of helix-forming side chains. From the result, it can be inferred that the developed powder is thermally stable.
15
ACCEPTED MANUSCRIPT 3.6. X-ray diffraction Fig.3 shows the X-ray diffraction pattern of microencapsulated anthocyanin powder, native and modified starch. As shown in Fig.3, a strong diffraction peak was observed around 15° which indicates A-type crystallinity of starch, a doublet at 17°, and 23° (2θ) [19]. After
T
microencapsulation, the typical starch peaks disappeared and the loaded microencapsulated
IP
powders demonstrated an amorphous nature. The Fig. 3 illustrated that for microencapsulated
CR
anthocyanin powder, a sharp peak at 19.18° was observed which may be due to the formation of the biopolymer with an amorphous structure [20]. It may possible that there is a formation of V-
US
amylose with other bioactive compounds present in anthocyanin extract, which is responsible for
AN
the distinct peak [21, 22]. In addition to that, the effect of the spray drying process may also be
M
responsible for the distinct peak of microencapsulated anthocyanin powder.
ED
3.7. FT-IR analysis
The FT-IR spectrum of anthocyanin extract, starch, modified starch and encapsulated
PT
powder shown in Fig. 4. The infrared spectra of the samples were characterized with the help of
CE
previous investigations [18, 22]. The spectrum of native, modified starches and
groups.
AC
microencapsulated anthocyanin powder were having quite similar stretches and functional
The peaks were observed in the regions between 800 to 1,500 cm−1 (the fingerprint region), the region between 1,500 to 2,000 cm−1 (C-H stretch region), and finally the region between 2,500 and 3,500 cm−1 (O-H stretch region). The peaks between 1000 and 1250 cm-1 may be due to the C–O–C bond, which illustrated that the three samples were carbohydrate in nature. From the Fig.4, a band was observed at 1022 cm−1 which is responsible for more amorphous
16
ACCEPTED MANUSCRIPT samples, while the bands at 1000 and 1047 cm−1 defined the more crystalline samples [23]. The predominant peaks located at 1636 cm−1 is also associated with ring stretching of C=C. From Fig. 4 it was observed that at 2950cm-1 there is a sharp peak which may be due to the C-H stretching vibration. The FT-IR spectrum for the anthocyanin extract shows a strong absorption
T
band at 1032 cm-1 assigned to aromatic ring C-H deformation and 3489 cm-1 for OH. The
IP
absorption band between 1500 and 3000 cm-1 corresponding to the stretching of C–H, C=C
US
CR
asymmetric deformation in –OCH3, CH2 in pyran ring, typical of flavonoid compounds [18].
3.8. Surface morphology
AN
The microencapsulated powder obtained under optimum spray drying conditions (6.01 % modified starch, 4.96 MPa atomizer pressure, and 168.78°C inlet air temperature) was
M
characterized using SEM technique and compared with native starch and modified starch. As
ED
shown in Fig.5, the surface morphology of spray dried microcapsule was more uniform and smooth than native starch and modified starch surface. Moreover, no particle aggregation was
PT
observed in the microencapsulated anthocyanin powder [24]. Kanakdande, Bhosale [25]
CE
suggested that the microencapsulated powder should have a uniform and smooth surface having slightly spherical shape with minimum cracks and collapses on walls. Few broken microcapsules
AC
can be observed from Fig. 5, indicating quite inefficient encapsulating properties of the carrier materials. This result was in agreement with earlier reports [25].
3.9. Storage Study The storage stability of microencapsulated anthocyanin powder was studied for 90 days at 25 and 4°C. In both the temperatures, a decrease in anthocyanin content was observed as
17
ACCEPTED MANUSCRIPT shown in Fig. 6. This phenomenon illustrated that after the microencapsulation, there can also be a significant decrease in the anthocyanin content. Hence, the degradation of anthocyanin in microencapsulated powder was higher at 25°C than 4°C. As similar types of observation were reported by the previous researcher [3]. Degradation of anthocyanin, during storage, may occur
T
due to various reaction mechanisms, such as condensation reactions, oxidation, and cleavage of a
IP
covalent bond. During high-temperature storage, the reaction rate of these reactions is high,
US
degradation during storage is difficult to establish [26].
CR
which enhance anthocyanin degradation. However, the exact mechanism for anthocyanin
AN
3.10. Steady shear rheology
The effect of microencapsulated powder on the steady-shear rheology of rice dough was
M
determined and presented in Table 3. The Herschel–Bulkley, and Mizrahi-Berk model were and
) of the samples was less than
ED
applied for the investigation. The flow behavior index (
1, which depicted the shear thinning behavior. Moreover, as the proportion of microencapsulated
PT
powder increased in dough, the flow behavior index increased for both the model. This indicated
CE
that the microencapsulated powder decreases the pseudo-plasticity of the dough samples. It may be due to the formation of new networks in the rice dough in the presence of microencapsulated
AC
powder. For the increase of microencapsulated powder in rice dough, resulted in a very sticky and cohesive dough, which required a high force for shearing as compared to only rice dough and enhance the consistence of the dough. The Herschel–Bulkley model showed the higher coefficient of determination (R2) than Mizrahi-Berk model, hence it can be inferred that Herschel–Bulkley model can efficiently predict the effect of microencapsulated powder on the steady-shear rheology of rice dough.
18
ACCEPTED MANUSCRIPT
4. Conclusions The microencapsulation of anthocyanin extract from purple rice bran with modified glutinous rice starch as wall material was successfully developed using the spray drying process.
T
The process was optimized using RCCD followed by RSM and the optimum process condition
IP
for microencapsulation was 6.01% of starch concentration, 168.78°C drying temperature and
CR
4.96 MPa nozzle pressure. The physical characteristics of spray-dried powders were investigated in term of water activity, solubility, true density, bulk density, porosity, diameter, and Hausner
US
ratio. The thermal properties and crystallinity of microencapsulate powder significantly differ
AN
from native and modified glutinous rice starch. The microencapsulate showed higher onset (157.0°C) and peak temperature (168.78°C). The morphological properties of the
M
microencapsulate showed that the surface of microcapsules was more uniform and smooth
ED
surfaces than the native and modified starch. The storage study of microencapsulated powder illustrated that at low-temperature storage the anthocyanin degradation in particles was less than
PT
high-temperature storage. The microencapsulated particles also affect the steady-shear rheology
CE
of rice dough. The increase in microencapsulated powder concentration from 5 to 20% in rice
AC
dough, decrease the pseudo-plasticity of dough.
Acknowledgments
The authors are grateful to the Indian Institute of Technology Guwahati and Tezpur University for instrumental support.
19
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ACCEPTED MANUSCRIPT Figure Captions Fig. 1 Effect of temperature, starch concentration and atomizer pressure on microencapsulate. Fig. 2 Thermogram of a) microencapsulated powder, b) modified starch, and c) native starch Fig. 3 XRD of a) Microencapsulated powder b) Native, and c) Modified starch
T
Fig. 4 FT-IR spectrum of native starch, modified starch and microencapsulated powder
IP
Fig. 5 Scanning electron microscope of a) and b) microencapsulated powder, c) native, and d)
CR
modified rice starch
AN
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Fig. 6 Storage stability of anthocyanin in microencapsulated powder at 25°C and 4°C
Table Captions
M
Table 1 Experimental design of spray drying of anthocyanin extract
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Table 2 Analysis of variance for the fitted quadratic polynomial model
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Table 3 Steady shear rheological parameters
24
ACCEPTED MANUSCRIPT Table 1 Experimental design of spray drying of anthocyanin extract Independent Parameters
Dependent Parameters Anthocyanin
True
Pressure
Temperature
Encapsulation
Content
Scavenging
Density
Water
(%)
(MPa)
(°C)
Efficiency (%)
(mg/L)
Activity (%)
(g/cm3)
Activity
1.673
0.492
43.61
1.799
0.519
47.92
1.769
0.515
2.431
51.98
1.681
0.503
2.772
52.65
1.819
0.525
1.918
44.67
1.794
0.511
T
Starch
4.14
160
80.97
1.54
2
8.99
4.96
150
84.94
1.803
3
8.99
3.32
170
88.75
1.619
4
7.5
5.52
160
95.38
5
6.01
3.32
150
96.74
6
6.01
3.32
170
88.53
7
7.5
4.14
180
87.91
1.969
42.63
1.715
0.501
8
6.01
4.96
170
95.32
2.932
52.71
1.692
0.512
9
7.5
4.14
160
92.23
2.912
54.86
1.865
0.529
10
8.99
4.96
170
89.02
2.087
41.32
1.62
0.490
11
8.99
3.32
150
85.10
2.554
48.07
1.698
0.495
12
6.01
4.96
150
99.17
3.040
61.94
1.949
0.569
13
7.5
4.14
160
90.83
2.892
52.01
1.795
0.514
14
7.5
4.14
140
88.66
2.721
47.92
1.989
0.558
15
7.5
4.14
160
90.83
2.892
52.01
1.865
0.529
16
5
4.14
160
95.83
2.43
54.73
1.817
0.523
17
7.5
4.14
160
87.33
2.642
50.71
1.832
0.529
18
7.5
4.14
160
89.35
2.592
49.92
1.775
0.519
19
7.5
4.14
160
90.83
2.922
52.9
1.865
0.529
20
7.5
2.76
160
91.58
2.708
51.45
1.67
0.500
25
ED
PT
CE
AC
41.84
US
CR
IP
10
M
1
AN
Run
DPPH
ACCEPTED MANUSCRIPT Table 2 Analysis of variance for the fitted quadratic polynomial model Terms
Encapsulation
Anthocyanin
DPPH
True
Water
Efficiency
Content
Scavenging
Density
Activity
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.012
0.034
0.030
0.030
0.001
0.001
0.044
0.010
0.0006
0.548
0.254 0.243 0.004
0.0002
0.009
0.105
< 0.0001
< 0.0001
0.0002
0.461
0.054
0.014
0.109
0.014
0.002
0.515
0.0007
0.001
0.0001
0.004
0.012
0.027
0.167
0.654
0.0002
0.004
0.009
0.001
0.311
0.177
AN
CR
0.0008
0.842
0.269
0.455
0.730
0.312
R2
0.947
0.927
0.945
0.930
0.929
0.900
0.861
0.896
0.868
0.865
Adj-R2
AC
Lack of Fit
CE
PT
0.209
T
< 0.0001
IP
<0.0001
M
< 0.0001
ED
Model
US
Activity
26
ACCEPTED MANUSCRIPT
Table 3 Steady shear rheological parameters Herschel–Bulkley
Mizrahi-Berk
η
σ
R2
k (Pa sn)
η
σ
R2
Control
834
0.384
0.87
0.829
335
0.184
0.698
0.834
5
1096
0.458
156.9
0.891
524
0.630
12.31
0.888
10
1370
0.522
98.99
0.845
890
0.650
3.81
0.843
15
1469
0.687
40.94
0.876
681
0.715
5.572
0.873
20
1460
0.718
89.24
0.967
8.596
0.964
CR
US 724
AN M ED PT CE AC
27
IP
k (Pa sn)
T
Sample
0.890
ACCEPTED MANUSCRIPT Highlights 1. Anthocyanin successfully microencapsulated using modified starch. 2. The storage stability of microencapsulated anthocyanin was high at low storage
AC
CE
PT
ED
M
AN
US
CR
IP
3. Microencapsulated powder affects the rice dough rheology.
T
temperature.
28
Figure 1af
Figure 1gj
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6