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Title: Microencapsulation<!–<query id="Q1">Please check

the presentation of the article title, and correct if necessary.
</query>–> of saffron anthocyanins using ␤ glucan and ␤
cyclodextrin: Nutraceutical, morphological, structural and the
release behavior of capsules during in-vitro digestion

Authors: Mudasir Ahmad, Bisma Ashraf, Asir Gani, Adil Gani

PII: S0141-8130(17)33376-7
DOI: https://doi.org/10.1016/j.ijbiomac.2017.11.122
Reference: BIOMAC 8602

To appear in: International Journal of Biological Macromolecules

Received date: 13-9-2017

Revised date: 3-11-2017
Accepted date: 19-11-2017

Please cite this article as: Mudasir Ahmad, Bisma Ashraf, Asir Gani, Adil
Gani, Microencapsulation of saffron anthocyanins using ␤ glucan and ␤
cyclodextrin: Nutraceutical, morphological, structural and the release behavior
of capsules during in-vitro digestion, International Journal of Biological
Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.11.122

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Microencapsulation of Saffron Anthocyanins using β glucan and β cyclodextrin:

Nutraceutical, Morphological, Structural and the release behavior of capsules during In-

vitro digestion.

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Mudasir Ahmad1, Bisma Ashraf1, Asir Gani2, Adil Gani1*

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Department of Food Science and Technology, University of Kashmir, Srinagar, India, 190006
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Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat

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Yai 90112, Songkhla, Thailand

*Address for correspondence

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Adil Gani (adil.gani@gmail.com)
Tel+ 8715022903
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*Note: The first and second author have contributed equally.
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Abstract
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In the present study, the saffron anthocyanins were encapsulated in β-glucan and β-cyclodextrin
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by spray drying technique to achieve their stability under adverse gastro-enviromental conditions.
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The microcapsules were subjected to simulated gastric conditions and release behavior of

monomeric anthocyanins, antioxidants and phenols were studied. The structural properties of
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microcapsules were analyzed by SEM and ATR-FTIR spectroscopy. The particle size distribution,

density, color, encapsulation efficiency and powder yield of samples were also evaluated. A
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characteristic band at 1700cm-1 by FTIR and specific enclosed particles in the cavities of wall

material were observed from the micrographs of SEM that confirmed the incorporation of
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anthocyanins in the microcapsules. The higher content of anthocyanins, antioxidants and phenols

in the intestinal conditions revealed the protection of core material from adverse conditions of

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stomach by encapsulation. Further studies are suggested to investigate the stability of encapsulated

anthocyanins in different environmental and processing conditions.

Keywords: Saffron; β-cyclodextrin; β-glucan; encapsulation; in-vitro digestion.

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1. Introduction

The saffron (Crocus sativus L.) belonging to the Iridaceae family is consider as the most

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expensive spice, derived from dried red colored stigma of the plant. It has been cultivated from

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about 3000 years ago[1]. Saffron is produced largely in Asia particularly in Iran [2] with more

than 90% of total annual saffron production in the world [2] . Saffron comprises of more than 150

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volatile compounds, including terpene, terpene alcohol, and terpene esters , together with aroma
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containing compounds and many non-volatile bioactive components many of which are
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carotenoids, including zeaxanthin, lycopene and various α- and β-carotenes. Saffron petals contain
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large amounts of anthocyanins, flavonoids & glycosides. Anthocyanins have been used in a wide
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variety of industries, including food, pharmaceutical & cosmetics due to its natural colorant,
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antioxidant & therapeutic properties (Like prevention of neuronal and cardiovascular, cancer and

diabetes illnesses). However the anthocyanins are rather unstable and influenced by the final
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processing temperature, storage, pH, light, oxygen, enzymes, proteins and metallic ions [3] .

Anthocyanins can be directly absorbed by small intestinal epithelial cells [4], but it is difficult to
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transfer anthocyanins to the intestinal tract due to their instability in the adverse gastrointestinal
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environment [5]. Therefore, the challenge is to protect such promising molecules from

deterioration and increase their bioavailability. Currently, researchers are exploring ways to

stabilize the anthocyanins under adverse conditions and among them encapsulation technique is

considered as a promising protective method for active compounds which are senstive to moisture,

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heat, light or oxidation [6]. Encapsulation involves the packing of core material of smaller size

within hollow structures of wall matrix. Various wall materials like maltodextrin, pullulan,

curdlan, sodium alginate, pectin, β- Cyclodextrin and others have been used [7-9]. Barley β- glucan

is an unbranched polysaccharide consisting of β-d-glucopyranose units linked through (1 → 4) and

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(1 → 3) gly-cosidic bonds in cereals and (1 → 6) glycosidic bonds in fungal sources, respectively.

It has a unique inbuilt honey comb structure and its additional health benefiting properties like

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lowering of serum cholesterol level, reduces glycemic response, enhances immune system and

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growth of beneficial microflora in gut, makes it an interestingly more suitable for the use of wall

material [10]. The main objective of this study was to bring about the stabilization of saffron

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anthocyanins under adverse conditions of the stomach and increasing their bioavailability in the
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intestine by encapsulation in β-glucan. The β cyclodextrin was taken as comparison for evaluating
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the suitability and efficiency of β-glucan as wall material. The morphology, physical properties,
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encapsulation efficiency of wall materials and the release behavior of anthocyanins were
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investigated.
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2. Materials and Methods

2.1. Materials
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β-cyclodextrin and all other chemicals were purchased from Sigma-Aldrich (USA) and Hi - Media

laboratories (Mumbai, India).

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2.2. β-glucan

The encapsulating agent i.e., β-glucan was extracted from barley (Hordeum Vulgarea) according
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to our previously described method [16]. The percentage purity of β-glucan obtained from this

method was found to be 87% as already mentioned in the literature cited above.

2.2. Methods

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2.2.1. Preparation of concentrated anthocyanins

The 3 g of dried saffron petals were weighed and mixed with 60 ml of 50% acidified ethanol (it

was acidified with 2N HCl upto pH = 2) with continuous mixing on magnetic stirrer and kept in

a dark place at ambient temperature (25 °C). After 24 h, the extract was filtered by vacuum

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filtration. For complete extraction of anthocyanins, remaining petals on filter paper were washed

with the same volume of solvent three times. Acidified extract was concentrated by a rotary

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evaporator (Roteva Equitron) for 30 min at 40 °C to half of its volume and the total solid content

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of the extract obtained was 10° Brix.

2.2.2. Preparation of solutions

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The 5 g of each wall material was dissolved in 50 ml of water and the total solid concentration of

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solution obtained was 10%. Then, extract of anthocyanins from saffron petal and the wall materials
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were mixed in a weight ratio (w/w) of 1:5 (extract: wall material). The pH of mixtures were
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decreased to pH=2 with HCl (1.5 N) to stabilize anthocyanins [11] and homogenized for 10 min

using homogenizer (HG-15A, Witeg Germany).

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2.2.3. Encapsulation using spray drying

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The saffron extract and matrix mixtures were spray-dried using mini spray dryer (Buchi B-290).
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The air flow was ~ 140 L/h with an inlet and outlet temperatures of 130°C and 80°C, respectively.

2.2.4. Total anthocyanin content of the samples

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Encapsulated samples (0.1 g) were dissolved in 10 ml of distilled water in a volumetric flask far

from the light. The amount of anthocyanin was evaluated by pH differential method [12].
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Absorbance was measured at 510 nm and 700 nm by using spectrophotometer (Hitachi U2900).

Anthocyanins content was calculated as cyanindin-3-glucoside equivalents according to the

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following equation, using an extinction coefficient of 26,900 Lcm−1mg−1 and a molecular mass of

449.2 g mol−1. Triplet analysis was performed for each treatment.

Anthocyanin pigment (cyanidin-3-glucoside equivalents) = A×MW×DF×103

ε×l

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Where, A= (A520nm - A700nm) pH 1.0-(A520nm - A700nm) pH 4.5; MW= molecular weight for cyanidin-

3-glucoside; ε=molar extinction coefficient; DF=dilution factor; l= path length in cm; 103=

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conversion from gram to milligram.

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2.2.5. Physical properties of encapsulated anthocyanin powders

Bulk density measurement

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Bulk density of powders were measured by weighing the fixed amount of samples and pouring

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them into a 10 ml graduated cylinder. The bulk density was calculated through dividing the powder
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mass by the volume occupied in the cylinder (g/cm3) [13].
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Tapped density measurement

The known weight of sample was transferred to 10 ml graduated cylinder and the initial volume
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was assessed. Tapped density of the microparticles was calculated as the ratio of the sample weight
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(g) and the volume (mL) occupied after 10-20 tappings [14].
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2.2.6. Encapsulation efficiency and Powder yield determination

Powder yield was measured through determination of recovered product given by the ratio between
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the total recovered product mass and the mass of solid extract initially feed into the spray dryer

and it was expressed by the following equation [15]

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Y= (W2-W1) – Xwb (W2-W1) ×100

MvTs

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where y is the powder yield (%), Xwb is the moisture content in wet basis (wb), MV is the volume

of extract feed (L), Ts is the content of total solids (g dry matter/L), whileW1 andW2 are the weight

(g) of the powder receptacle before and after spray drying, respectively.

For encapsulation efficiency, 100 mg of each sample was dissolved in 10 ml of distilled water in

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a volumetric flask far from the light. This solution was then sonicated for 10 minutes to release the

anthocyanins from capsules. After centrifuging the solution for 10 minutes at 10,000 rpm, the

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supernatant was collected and the amount of anthocyanins released from capsule was evaluated by

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pH differential method [12].

The encapsulation efficiency (EE) was calculated using the following equation

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EE (%) = Anthocyanins in the capsules (μg/mg of matrix) × 100
Anthocyanins added to the solutions (μg/mg of matrix)

2.2.7. Color measurement

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The color of native compounds and encapsulated samples were measured by hunter lab digital
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colorimeter (Hunter Lab D25, Hunter associates Lab, Reston, USA). The samples were uniformly
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spreaded on white try of instrument. The hunter values L*, a*, and b* values were recorded, L*
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scale: light vs. dark, where a low number (0-50) indicates dark and a high number (51-100)

indicates light. a* scale: red vs. green, where a positive number indicates red and a
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negative number indicates green. b* scale: yellow vs. blue where a positive number indicates
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yellow and a negative number indicates blue [16].

2.2.8. Scanning Electron Microscopy (SEM)

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Particle structures of encapsulated powders were evaluated by SEM (Hitachi S-4100) after coated

with a gold- palladium mixture under vacuum.

2.2.9. Particle size analysis

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The size distribution and average diameter of the micro particles were determined by the light

scattering technique using laser diffraction particle size analyzer (Shimadzu SALD-2300).

2.2.10. Attenuated Total Reflectance-Fourier Transform Infrared (ATR–FTIR) Spectroscopy

ATR-FTIR (Agilent cary-630) analysis of microencapsulates were recorded at room temperature

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in the range of wave number from 4000 to 650 cm-1 to analyze the molecular organization in the

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samples.

2.2.11. Release of anthocyanin during simulated in-vitro digestion

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A static model that simulates digestion in the mouth, stomach and intestine was adapted. The

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simulated saliva solution was prepared by dissolving 0.2 % of α-amylase in phosphate buffered

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saline, to which pH was maintained at 6.8 ± 0.2. The simulated gastric juice (SGJ) was prepared
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by dissolving 3 g/L pepsin in sterile NaCl solution (9 g/L) and the pH was adjusted to 3.0 with 1.0
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mol/L HCl. The simulated intestinal juice (SIJ) was prepared by dissolving 3 gL-1 bile salts and

10 gL-1 pancreatin in phosphate-buffered saline, pH was maintained at 8 with 0.1M NaOH solution.
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The simulated gastric juice (SGJ) and simulated intestinal juice (SIJ) was prepared according to
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the method of Gani et al., [17]. Powder samples (100 mg) were placed in 125 ml flask and

incubated at 37°C under constant stirring. The samples were digested sequentially as follows:
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mouth – addition of 10 ml of salivary juice and mixing for 5 min and aliquot (1 ml) was collected;
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stomach – addition of 10 ml of SGJ and after constant stirring, aliquot (1 ml) was collected after

30 min & 1 hour of incubation; Intestines – addition of 10 ml of SIJ after constant stirring/mixing,
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aliquots (1 ml) were collected after 2 h and 4 h. All the aliquots were centrifuged at 6238 g for 5

min and supernatant liquid was filtered through a 0.22 μm membrane filter, the amount of

anthocyanin released was measured by pH differential method Lee et al., [12].

2.2.12. Total phenolic content (TPC) & Antioxidant activity under simulated gastric conditions

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 The antioxidant activity and total phenolic content of samples were determined under

simulated gastro-intestinal conditions. The extracts obtained after saliva, gastric & intestinal stages

were first centrifuged at 6238 g for 5 min and then filtered through 0.22 μm membrane filter.

2.2.12.1. Total phenol content

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Total phenol content of extract was determined according to the method of Gani et al., [18],

with minor modifications. The 2400 μL of millipore water, 150 μL of 0.25 N folin ciocalteu`s

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reagent were added to the 150 μL of the extract and then mixed well by shaking. The mixture is

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allowed to react for 3 minutes then 300 μL of 1N Na2CO3 was added and mixed well again by

vortex mixer. The solution was incubated at room temperature in dark for 2h. The absorbance was

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measured at 725 nm using spectrophotometer. A calibration curve was prepared, using a standard

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solution of gallic acid (r2=0.993) and results were expressed as milligram gallic acid equivalents
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per gram of sample (mgGAE/g).
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2.2.12.2. Free radical scavenging activity by DPPH (1,1-dihpenyl-2-picrylhydrazyl)

Free radical scavenging activity of extract was determined according to the method of Khan et
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al.,[19] with modifications. The 80 μL of the sample were mixed with 200 μL of 0.05% DPPH
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solution, and total volume swas made to 4 ml with methanol and then allowed to react in the dark
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for 30 minutes. The absorbance was measured at 517 nm.

The scavenging percentage was calculated using following equation

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Scavenging activity (%) = A control - A sample / A control×100

Here, Acontrol is absorbance solution without sample and Asample is the absorbance solution
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containing sample

2.3 Statistical Analysis

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 Statistical analysis was performed using commercial statistical package SPSS.10.1 (USA)

and Primer of Biostatistics (version 4.0). The data were assessed by analysis of variance

(ANOVA), Duncan's Multiple Range Test and paired t- test at 5% significance level. The results

are presented as means ± standard deviation. All experiments were performed in triplicate.

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3. Result and discussion

3.1. Physical properties of encapsulated anthocyanin powders

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Bulk and Tapped density of the encapsulated saffron anthocyanin powders are depicted in

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Table 1. The results show that there is no difference observed in the bulk density when saffron

anthocyanins were encapsulated in β-Glucan and β-cyclodextrin (P>0.05). The values of bulk

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density ranges from 0.413 g/ml-0.419 g/ml, similar values were obtained for anthocyanins
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encapsulated in maltodextrin, gum Arabic and gelatin [20]. Bulk density is related to the molecular
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weight of material; material with higher molecular weight helps in better movement in empty
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areas, thus decreases the total volume and increases the bulk density. The bulk densities of powder
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was influenced by the size of the crushed particles, frangibility and flow properties [21]. The
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tapped density is another important physical character; it is related to the transport, packaging and

marketing of powders. Thus, this parameter is beneficial for determining the weight and quantity
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of material that will fit inside a container [21] The result shows low values for tapped density for

encapsulated saffron anthocyanins in β cyclodextrin and β glucan (SC and SG, respectively).
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Similar results were observed for the microencapsulation of rosemary essential oil with Gum

Arabic /starch/ maltodextrin /inulin. [22]

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3.2. Encapsulation efficiency and powder yield

Encapsulation efficiency is one of the most important quality parameter that determines the

potential of wall material to encapsulate or hold the core material inside the microcapsule. An

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efficient encapsulation method relies on achieving high retention of the core material inside the

wall material and minimum quantity on the surface of the powder particles. According to Jafari

et al. [23] the properties of wall and core materials as well as the emulsion characteristics and

drying parameters (especially conditions of the spray-drying such as inlet and outlet temperatures,

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feed flow rate, air flow and humidity, powder particle size, etc.) are the factors that can affect the

efficiency of encapsulation. Table 1 shows the average values of encapsulation efficiency and

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powder yield. Encapsulation efficiency of SG and SC was 45% and 63.25% respectively. Powder

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yield for SG was 45.33% while as 50% yield of powder for SC was found. Values close to these

are reported for encapsulation of crocin, picrocrocin and saffranal with maltodextrin/WPC matrix

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[24] . The larger internal cavity of β cyclodextrin may be responsible for its higher encapsulation

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efficiency; we have also achieved the similar type of results in our previous study on encapsulation
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of folic in β cyclodextrin [09]. The difference in the powder yield may be related to the different
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properties of the wall materials and their response during spray drying process. The other factors

like operating conditions, amount of total solid content in the feed were kept similar for both the
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samples. Since, the powder yield is influenced by total solid content in the feed solution, operating
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conditions of spray dryer and wall material to core material ratio [25].
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3.3. Color

The hunter values (L*, a* and b*) of β-glucan (G), β-cyclodextrin (B) and encapsulated
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anthocyanins in β-cyclodextrin and β-glucan (SC & SG) are shown in Table 2. The results showed

that β-cyclodextrin has highest L* value followed by β-Glucan, β-cyclodextrin microcapsules and
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β-glucan microcapsules. This reveals that microcapsules of β-cyclodextrin were whiter than

microcapsules of β-glucan. Encapsulated powders showed increase in a* value which may be

attributed due to high anthocyanin content [26]. The b* value of SG and SC were significantly

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different, whereas non-significant difference was observed in G & C. However, SG and SC showed

highest b* value than β-glucan and β-cyclodextrin, which clearly indicates that anthocyanin

imparts yellow-orange color to microcapsules. Similar results were reported by Jimenez et al. [27],

during encapsulation of Rubus sp. Juice through spray drying

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3.4. Morphology of the microcapsules containing saffron extract

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Scanning electron micrographs of anthocyanins and its encapsulated structures are depicted in

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Figure 1. The morphological characteristcs of encapsulated particles showed some particles

embedded in the hollow spheres of wall materials. Different type of wall material had a

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meaningful effect on the outward appearance of particles. There were differences found in

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configuration of native β-glucan and β-cyclodextrin which corresponds to difference in the
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configuration of encapsulated β-glucan and encapsulated β- cyclodextrin capsules. Similar reports
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were observed for the microencapsulation of rosemary essential oil with Gum arabic /starch/

maltodextrin /inulin [22]The compact cell wall structure of native β-glucan looks like hexagonal
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in shape with rough surface, whereas the encapsulate capsules were smooth, crack free surface
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[28]. The size of encapsulated β-cyclodextrin capsules was uneven and their shape and orientation
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was found to be irregular. However, the encapsulated β-glucan capsules had smaller size and more

granular shape than encapsulated β-cyclodextrin.

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3.5. Particle size analyzer

The distributions of particle size for encapsulated powders are shown in Figure 2a. The micro-

particles obtained after spray drying had different particle diameter. The 25%, 50%, 75% and 90%

sample had the particle size less than 47.20, 96.62, 260.22 and 391.31 µm for SG and 32.04, 62.40,

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101.50, 141.98 µm for SC. The particle size can be correlated to viscosities, particles having

greater molecular size possess more viscosity and vice versa [29]. The results showed that SG has

largest particle size compared to SC, which implies that particle size of capsules is influenced by

the type of the polymer as well. These particles sizes are best suited for oral deliveries as reported

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by Chen & Langer, [30].

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3.6. Changes in molecular structure after encapsulation

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ATR-FTIR was performed to verify interaction of anthocyanins with polysaccharides and

to detect the structural changes in the microcapsules due to encapsulation of anthocyanin within

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the wall material (Figure.2b). Spectra of β-cyclodextrin depict the characteristic bands belonging

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to saccharides: 3268.86 cm−1 (O-H stretching vibration), 2924.566 cm−1 (C-H stretching
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vibration), 1637.826 cm−1 (O-H bending vibration), and 1155 cm−1 (C-O vibration) [31], Inclusive
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of these peaks β-glucan shows a characteristic peak at 890cm−1 (β-glycosidic anomeric bonds)

corresponds to the β-configuration [10]. The spectra displayed characteristic peaks at 1654.732cm-
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which refers to C = O stretching vibrations, peaks between 2923.47-2854.42cm-1 is related to CH
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stretching vibration band , 3339.67 cm-1 corresponds to stretching vibration of OH (which point
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out the presence of alcoholic grsoup ) [32]. The distinctive band in the region of 1700cm-1 from

SC & SG was seen which overlapped with the vibrational bands from anthocyanin, this region
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confirms the incorporation of anthocyanins in the microparticles capsules. It was observed that the

bands from the encapsulated anthocyanins are narrow when compared with the non-encapsulated
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anthocyanins, which suggested that the incorporation of anthocyanins changes the molecular order.

3.7. Release of anthocyanin during in-vitro digestion

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The in vitro release of monomeric anthocyanins from wall materials were studied under simulated

gastric and intestinal fluids. As shown in Table 3, the release of anthocyanins was effectively

controlled by protective coating of wall materials. The amount of anthocyanins released from SG

in simulated gastric fluid was less compared to SC microsphere. The results suggested that the

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microparticles coated by β-glucan and β-cyclodextrin were more resistant to gastric environment

of stomach & they protect the bioactive compounds during passage through human stomach. After

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that the microparticles of both SC & SG are transferred to simulated intestinal fluid separately to

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confirm the further release of anthocyanin. The results show that maximum amount of anthocyanin

monomer is released during 2hrs of simulated intestinal conditions. Results showed the control

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release of anthocyanins under these conditions, as there is increase in resident time which is

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beneficial for the absorption of bioactive substance by intestinal cells. It is perhaps due to neutral
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pH of intestine, the protective coatings are destroyed and thus the microparticles were degraded
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that resulted in the sustained release of monomeric anthocyanin from capsules in stimulated

intestinal fluids. The results of this study showed that β-glucan and β-cyclodextrin effectively
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prevent the release of anthocyanin in gastric conditions which increase their bioavailability by
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minimizing the chemical degradation of anthocyanin in gut environment. Our results are in
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accordance with those obtained for anthocyanins encapsulated in oxidized konjac glucomannan

microspheres stabilized by chitosan oligosaccharides [33].

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3.8. Total phenolic content (TPC) & Antioxidant activity under simulated gastric conditions
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In-vitro digestion was done to assess the potential of β-glucan & β-cyclodextrin for oral delivery

via a food or as a supplement for slow and targeted delivery of bioactive components in particular

section of human digestive system. The TPC and antioxidant activity of encapsulated saffron

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extract during simulated gastric digestive conditions are depicted in Table 4. The results showed

that phenolic contents and antioxidant activity showed an increasing trend from gastric to intestinal

digestion. The TPC of SG is higher than SC in simulated gastric fluid. Further, TPC of

microparticles was found to be higher in SIJ than SGJ. This showed that the process of

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microencapsulation provides protective effect in preventing loss of phenolic compounds during

harsh conditions of stomach. Similar results were reported during in-vitro release properties of

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encapsulated blueberry (Vaccinium ashei) extracts [34]. The higher levels of TPC in SIJ again

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confirms maximum release of core material in intestinal section. Since the polyphenols are mostly

absorbed in this section, therefore it is necessary that maximum of amount of bioactive compounds

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cmpounds reach in this section, which will goevern the health benefits thereof.

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4. Conclusion
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The findings of this study showed that β-glucan has potential to encapsulate saffron bioactives and

improves its stability during passage through simulated GI tract conditions. Encapsulation
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increased the availability of anthocyanins in intestinal section, which can lead to their maximium
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abosrbtion during oral digestion. The presence of anthocyanins in β-glucan matrix was clearly
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depicted through SEM and FT-IR spectroscopy. It can be concluded that encapsulation of saffron

extract (anthocyanins) in β-glucan could be very effective way for fortification of these bioactives
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in the different food systems. Moreover, using β-glucan as a wall material for encapsulation of

bioactives can provide additional health benefits arising from health promoting properties of β-
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glucan.

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Figure1. Scanning electron microscopy of different samples.
Here, NG= native β-glucan ; SG= encapsulated anthocyanins in β-glucan; NC= native β- cyclodextrin and SC=
encapsulated anthocyanins in β- cyclodextrin.

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SC
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Figure 2a: Particle size distribution of encapsulated powders. Here, SG and SC represents saffron anthocyanins
encapsulated in β- glucan and β- cyclodextrin, respectively.
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SC
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Figure 2a: Displays characteristic bands of different samples by ATR-FTIR.

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Here, S = Saffron anthocyanins; .NG= native β-glucan; SG= saffron anthocyanins encapsulated in β-
glucan; NC=native β-cyclodextrin and SC= saffron anthocyanin encapsulated in β- cyclodextrin,
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Table 1: Physical properties and the encapsulation efficiency of β-glucan and β-cyclodextrin.

Wall material Powder yield Encapsulation efficiency Bulk density Tapped density
(%) (%)

SG 45.33 45.00a±1.2 0.419Aa ±0.11 0.543Ba ±0.31

b Aa
SC 50.00 63.25 ±1.5 0.413 ±0.12 0.528Ba ±0.21
SC and SG represent saffron anthocyanins encapsulated in β-glucan and β-cyclodextrin respectively. Data with

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different superscript letters in the same column (small letters) and row (capital letters) are significantly different
(p˂0.05).

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Table 2 Color analysis of different samples.
SAMPLES L* a* b*

SC
SG 40.33±0.69a 27.32±1.155e 35.21±1.06b
SC 61.95±0.723b 13.45±0.352c 81.47±0.55d
S 61.45±0.88b 20.47 ±0.44d 52.57 ±1.07c
d
C 95.94±1.18 1.02±0.225a 11.51±0.66a

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G 79.40±1.84c 2.50±0.493b 11.88±3.89a
Values reported are mean ± standard deviation of the analysis performed in triplicates. Data with different superscript
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letters in the same column are significantly different (p˂0.05). (Here SG= saffron anthocyanins Encapsulated in β-
glucan, SC= Saffron anthocyanins encapsulated in β-cyclodextrin, S = saffron, C= cyclodextrin, G = β glucan).
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Table 3: Release percentage of anthocyanin from encapsulated powders during in vitro digestion.
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Digestion Duration of SC* SG*

digestion
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Mouth 5 min 15.83±0.20aB 11.50±0.45aA

bB
Gastric 30 min 38.63±0.55 22.93±1.00bA
conditions 60 min 41.26±1.10cA 39.83±0.76cA
dA
61.56±1.40dB
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Intestinal 2 hr 51.56±0.51
conditions 4 hr 71.76±1.66eA 80.33±1.52eB
SC and SG represent saffron anthocyanins encapsulated in β glucan and β-cyclodextrin, respectively.
Values reported are mean ± standard deviation of the analysis performed in triplicates. Data with
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Different superscripts in the same column with small letters and in the same row with capital letters are significantly
different (p˂0.05).
*Concentration of anthocyanins released µg/mg of encapsulated powder.
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Table 4 Total phenols and antioxidant activity of encapsulated samples during in vitro digestive
conditions.
Digestion Duration of Total phenols (mg GAE/ml) % Inhibition (DPPH)
digestion SG SC SG SC

Mouth 5 min 0.832±0.164b 0.987±0.157b 61.54±1.501ab 61.51±1.235a

Gastric 30 min 0.260±0.014a 0.334±0.074a 59.46 ±0.528a 62.89±0.303a
conditions 60 min 0.310±0.022a 0.318±0.081a 61.07±1.681ab 63.75±0.767a

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Intestinal 2 hr 1.317±0.034c 1.310±0.006c 62.65±2.448ab 63.55±2.594a
conditions 4 hr 1.341±0.057c 1.380±0.182c 64.06± 2.136b 70.44±7.154b
SC and SG represent saffron anthocyanin encapsulated in β-glucan and β-cyclodextrin respectively. Values reported

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are mean ± standard deviation of the analysis performed in triplicates. Data with different superscript letters in the
same column are significantly different (p˂0.05).

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