Microencapsulation of vanilla (Vanilla planifolia Andrews) and powder
characterization

S.J. Calva-Estrada, M.R. Mendoza, O. Garcı́a, V.M. Jiménez-Fernández,

M. Jiménez

PII: S0032-5910(17)30831-8
DOI: doi:10.1016/j.powtec.2017.10.035
Reference: PTEC 12893

To appear in: Powder Technology

Received date: 20 September 2016

Revised date: 7 October 2017
Accepted date: 19 October 2017

Please cite this article as: S.J. Calva-Estrada, M.R. Mendoza, O. Garcı́a, V.M. Jiménez-
Fernández, M. Jiménez, Microencapsulation of vanilla (Vanilla planifolia Andrews) and
powder characterization, Powder Technology (2017), doi:10.1016/j.powtec.2017.10.035

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 ACCEPTED MANUSCRIPT

Microencapsulation of vanilla (Vanilla planifolia Andrews) and powder

characterization

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S.J. Calva-Estradaa, M.R. Mendozab, O. Garcíab, V.M. Jiménez-Fernándezc and M. Jiméneza*

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a
Instituto de Ciencias Básicas, Universidad Veracruzana, Xalapa, Veracruz, México. C.P. 91190.

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*Corresponding author: maribjimenez@uv.mx

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b
Unidad de Servicios de Apoyo en Resolución Analítica, Universidad Veracruzana, Xalapa,

Veracruz, México. MA
c
Facultad de Instrumentación Electrónica, Universidad Veracruzana, Xalapa, Veracruz, México.
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ABSTRACT
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Natural vanilla extract (NVE) and synthetic vanilla extract (SVE) were

microencapsulated by spray-drying, using whey protein concentrate (WPC) as wall material.

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Physicochemical properties, volatility profile, vanillin concentration and antioxidant activity

were evaluated by measuring total polyphenols, the inhibition of radical scavenging activity

by DPPH, chain-breaking activity and reducing power. Both microparticles exhibited high

vanillin retention and antioxidant activity during spray dried process, but microparticles of

SVE-WPC showed higher vanillin retention than microparticles of NVE-WPC during

storage. The micrographs of the microcapsules showed morphological characteristics that

were ideal for ensuring stability and integrity during storage to different water activities.

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WPC was found a suitable wall material to produce powder with vanilla extracts. Vanilla

powder represents an interesting food additive for incorporation into functional foods.

Keywords: Spray-drying, whey protein concentrate, volatile compounds, vanillin,

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antioxidant activity.

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1. Introduction

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Mexico is the centre of origin and domestication of vanilla (Vanilla planifolia
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Andrews). Vanilla is the only orchid with edible fruits, is commercially cultivated for its

pods, which contain vanillin considering the world's most popular flavouring substance [1].
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More than 95% of the vanilla consumed in the US is processed into extracts sold to
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manufacturers for flavouring [2], it has multiple uses in cottage industry, food, tobacco,

cosmetics and the pharmaceutical industry. The US is the largest importer of vanilla,
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importing as much as 40% of the world’s annual vanilla production. During this five-year

period, the US observed a 22% increase in consumption and in Germany, an astounding

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100% increase was recorded [2]. The antimicrobial, antimutagenic, anticarcinogenic and

antioxidant properties of vanillin have been studied [3]. Vanillin is widely used as a flavour

and fragrance and it is also used as a food preservative due to its antioxidant properties in

food industry [4]. Also it has been reported that vanillin is one of the most popular flavours

of the world, and it is extensively used in confectionary, food and beverage industries [5].

Due to the complexity and cost of cultivation of vanilla, the chemical industry has succeeded

in manufacturing than from some plants and trees and a solution smelling like natural vanilla

extract flavour. However, due to the number of aromatic compounds having the natural

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extract is nothing comparable substance synthetic vanilla. Although more than 12,000 tons

of vanillin is produced each year, less than 1% of this is natural vanillin from vanilla; the

remainder is synthesized much more cheaply via chemical processes [6]. However, vanillin

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has a short shelf-life, therefore, the stabilization of vanillin is very important for its prolonged

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functionality [7]. While technological innovations may reduce the complexity and cost of the

market for natural vanilla extracts.

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Microencapsulation is a technology used for the protection of active ingredients and

to reduce the degradation or decrease of aroma, flavour and physicochemical, nutritional and
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functional properties during processing and storage providing durability and protection

against external factors such as heat, light and moisture. Spray-drying is one of the methods
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is used for microencapsulation due to it is economic, flexible, and accessible and it is reported
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produce particles of good quality [8]. Numerous materials have been used as encapsulating
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agents, including proteins, gums and modified starches. Sometimes a blend of several
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ingredients can produce superior or lower performance compared to the use of one ingredient
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alone [9, 10]. However, in some works the use of alone material used as wall materials has

been efficient to protect the core material [11, 12]. Vanilla has been encapsulated in

polysaccharides, modified starches, and waxy amaranth starch by the spray-drying method

[13]. However, whey proteins have been reported to be effective for microencapsulation by

the spray-drying of anhydrous milk fat or volatiles [8]. Its application in microencapsulation

vanilla (Vanilla planifolia Andrews) might be an effective process to the preservation of

antioxidant activity in the final product. Several chemical and kinetic methods have been

used to evaluate the antioxidant activity. The kinetic methods allow to measure the chain-

breaking activity of electron-transferring and hydrogen-donating antioxidant [14]. Phenolics

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are the main antioxidant components of vanilla and the chain breaking activity refers to the

ability of polyphenols to retard oxidation degradation associated with the elevated reactivity

of phenolics towards active free radicals as the most principle mechanism [15]. Therefore,

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the objective of this study was microencapsulated natural vanilla (NVE) extract and synthetic

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vanilla (SVE) extract by spray-drying using whey protein concentrate (WPC) as wall material

and evaluate the physical properties and antioxidant activity of microcapsules. In the first

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stage the physicochemical properties and the antioxidant activity of the vanilla extracts were

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evaluated. The second step was the microencapsulation the extracts by spray-drying and

finally, the physicochemical properties and the antioxidant activity of the microcapsules were
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evaluated. Finally, vanillin retention was evaluated under controlled conditions of storage.
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2. Material and methods

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2.1. Chemicals
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The 1,1-diphenyl-2-picryl-hydrazyl (DPPH.) (D9132), Folin–Ciocalteu reagent

(F9252), vanillin standard (1711009), sodium carbonate (S7795), trichloroacetic acid

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(T6399), gallic acid standard (27645), ascorbic acid (1043003), ferric chloride (157740),

potassium ferricyanide (P4066) were obtained from Sigma (Sigma-Aldrich GmbH,

Sternheim, Germany). All reagents were of analytical grade. Whey protein concentrate

(WPC) was purchased from Vilher (Guadalajara, México). Analytical grade solvents (ethanol

and toluene) were purchased from Sigma (Mexico D.F, Mexico).

2.2. Sample preparation

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The sample consisted of a synthetic ethanolic extract of vanilla (SVE), trademarked

as "PARDO", and a natural ethanolic extract of vanilla (NVE), trademarked as “GAYA”.

Both extracts were adjusted approximately to 20 % solids using a rotary evaporator at a

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temperature of 40°C and reduced pressure to reduce the ethanol content in the mixture. Then,

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the extracts adjusted extracts were analysed and used by microencapsulation.

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2.3. Physical properties of vanilla extracts

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Total solids, percentage of soluble solids (°Bx) and pH was described according to

the AOAC [16]. The colour was evaluated using a colorimeter (Hunter Lab, ColorFlex 45/0)
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through the parameters L*, a* and b* by CIE Lab scale and the Hue angle and Chroma were

calculated.
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2.4. Analysis of volatile compounds by GC-MS

The extraction of volatiles compounds was performed by SPME using a polyacrylate

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(PA) fibre 85μm, and the analysis was performed by gas chromatography (GC) (Agilent

Technologies model 6890N) coupled to a mass spectrometer (MS) (Agilent Technologies

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model 5975 inert XL). To solid phase micro-extraction by direct immersion (DI-SPME), the

sample (5 mL) was transferred to a head-space vial, which was sealed with PTFE cap. The

fibre was exposed and immersing in the sample into the vial and heated to 70°C for 30 min

with constant agitation, after this exposure time the fibre was retracted and inserted

immediately into the inlet of the GC. To chromatographic analysis, DB-5 column was used

5%-phenyl-methylpolysiloxane (Agilent Technologies) of 60 m x 10.25 mm and 0.25 μm

film thickness. The injector temperature was 280°C. Desorption time of the sample within

the nozzle was 5 min. The starting temperature was 40°C, which was maintained for 2 min,

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then increased to 200°C using a heating rate of 8°C/min, and finally increased to 250°C with

a heating rate of 50°C/min. Helium was used as the carrier gas with a flow rate of 1.0 mL/min.

Once obtained from the chromatogram, the identification of the chromatographic peaks was

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carried out by MS. Mass spectra were obtained by electron impact ionization at 70 eV, to

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identify the mass spectra obtained with a database (05 HP Chemstation, NIST Mass Spectral

search program, version 2.0d) and by comparison with a standard (vanillin, catalogue no.

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V1104, Sigma Aldrich) used under the same conditions.

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2.5. Vanillin estimation
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A vanillin stock solution in a 100 mL volumetric flask was prepared by dissolving

0.1 g of vanillin standard in 5 mL of ethanol. Aliquots of 1, 5, 10, 15 and 20 mL of the stock

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solution were taken and brought to a 250 mL volumetric flask separately dilute with distilled
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water. Two sets of solutions for each dilution prepared: a) neutral solution: were taken 10
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mL of each dilution and placed in a volumetric 100 mL flask separately dilute with distilled
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water, and b) alkali solution: were taken 10 mL dilution and were placed in a 100 mL

volumetric flask separately, and then were added 2 mL of 0.1M NaOH and dilute with
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distilled water. The absorbance of each of the alkaline solutions was measured using a UV-

Vis spectrophotometer at 348 nm using as a blank the neutral solutions [17].

2.6. Quantification of Polyphenols

Samples were analysed for total polyphenol content according to the Folin-Ciocalteu

method measuring the absorbance at 750 nm using a spectrophotometer [18]. Calibration

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curve was performed using gallic acid as standard (R2=0.999). The results were expressed as

mg of gallic acid equivalents (GAE).

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2.7. Antioxidant activity of the extracts

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The antioxidant activity was determined using DPPH radical scavenging activity and

redox potential. Radical scavenging activity was measured in a sample solution of 500

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mg/mL by 1, 1-diphenyl-2-picrylhydrazil (DPPH) method, with absorbance measured at 517

nm. The median effective concentration (EC50) was calculated. Ascorbic acid was used as
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reference standard. The antioxidant activity was determined from the determination of k in

the eq. (1) proposed by Manzocco et al. [19].

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1 1
− 𝐴3 = −3𝑘𝑡 (1)
𝐴3 0
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Where, A0 is the initial optical density and A is the optical density at increasing time,
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t. The chain-breaking activity was expressed as (-D.O.-3/min/mgd.m.) assuming that all the dry
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matter (d.m.) of the extract possesses antioxidant properties. The data are expressed as the

mean of at least three repetitions. The reducing power was determined by measuring the

absorbance at 700 nm [20]. Ascorbic acid was used as reference standard.

2.8. Microencapsulation vanilla extracts

A solution consisting of NVE or SVE and whey protein concentrate (WPC) as wall

material was prepared at a ratio of 1:3 (w/w) in distilled water containing 30% solids. The

solution was homogenized using a homogenizer (Wiggen Hauser, model D500) at 15000 rpm

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for 5 min. The resultant solutions were fed to a Büchi Mini Spray Dryer B-290 with a nozzle

of 0.4 mm. Microencapsulation conditions were: inlet temperature of 150 ± 2°C, outlet

temperature 90 ± 5°C and a feed rate of 6% and a pressure of 50 mbar. The powders obtained

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were stored exclude light and were kept at -40°C until analysed.

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2.9. Physicochemical properties and antioxidant activity of the microcapsules

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Yield microencapsulation and antioxidant activity of the microcapsules was evaluated

in the microcapsules after removal of wall material, for that the core material was extracted
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placing 2 g of microcapsules in 15 mL of ethanol and 15 mL deionized water and after

homogenized by an ultrasonic homogenizer (Brason, model S-250D) of 200 watts using a

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pulse for 5 x 5 for 5 min with amplitude of 60%. The microencapsulation yield is defined as

the ratio of core material (vanillin) in the final dried microcapsules to that in the original
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solution. DPPH radical scavenging activity and redox potential in the microcapsules was
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determined with the same technique used by the extracts evaluation.

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The moisture content was determined gravimetrically by oven-drying at 60°C to

constant weight, and water activity of the powders after spray-drying was measured using a

water activity meter (AquaLab, 3TE, Decagon, USA). The solubility of the microcapsules

was determined by a gravimetric method, which consisted of adding 0.5 g of the sample to

an Erlenmeyer flask containing 50 mL of distilled water and homogenizing at 75 rpm for 30

min at room temperature. Then, the solution was centrifuged at 3000 g for 5 min and a 25

mL aliquot of the supernatant was transferred to a previously weighed Petri dish and

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maintained in an oven at 105°C until complete evaporation of the water. The dishes were

weighed and the solubility was calculated from the difference in weight.

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2.10. Flow properties of microcapsules

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Bulk density, tapped density, particle density, percentage compressibility, and the

angle of repose were determined. The particle density was measured by a pycnometric

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method using toluene. The particle density is the total mass of the particle divided by its total

volume. Bulk density was determined in ten grams of powder, which were loosely weighed
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into 25 mL graduate glass cylinder. The initial volume (V0) was recorded and bulk density

was calculated by dividing the sample weight by the volume. The compact density was
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determined by the method of "Tappin” [21]. The probe is hit on the flat surface to volume

constant. The compact density calculated by dividing the weight of the sample by the final
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volume (Vf) occupied by the sample. The percentage compressibility was determined as
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ration of compression to the initial and the Hausner ratio is calculated with the relation
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Hr=V0/Vf. For the angle of repose test, 5 g of sample microcapsule is weighed and added to

a dropping funnel which was placed at 10 cm of a flat surface, obstructing the departure

therefrom. Upon starting the assay was allowed to fall freely sample forming a cone on the

flat surface. Subsequently the height (h) of the cone formed and its radius (r) was measured.

The tangent of the angle of repose is given by the h/r ratio [22].

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2.11. Morphology of microcapsules

The microcapsules were equilibrated at different water activities (aw=0.0, 0.529, 0.753 and

0.902) for 25 days at 25°C. The outer structures of the microcapsules obtained were studied

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by Scanning Electronic Microscopy (SEM). The samples were coated with 60% gold and

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40% palladium using a Denton Vacuum DESK V and analysed using a JEOL JSM-5600 LV

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(Jeol, Tokyo, Japan) scanning electron microscope operated at 28 kV. Examinations were

made at different magnifications. ImageJ 7.50i software was used to analyse the obtained

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images for area, perimeter and Feret diameter.
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2.12. Vanillin retention during storage
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Twenty grams of microparticles were placed in containers stored at 25°C immediately

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after spray dried. The concentration of vanillin was determined each week in the both
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microparticles stored for one month. For the extraction of vanillin 1 g of microparticles were
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weighed and mixed with 10 mL of a 1:1 v:v water:ethanol solution. This operation was

repeated until the vanillin was completely extracted. The retention of vanillin as percentage
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was evaluated considering the ration with respect the initial vanillin concentration.

2.13. Statistical analysis

All analyses were performed in triplicate (n=3). Data analysis was performed using the

program Statistica 7.0 by StatSoft, Inc. (2004), the analysis of variance (ANOVA) and the

Tukey test (p < 0.05). The experimental data are presented as the mean and standard deviation

(SD).

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3. Results and discussion

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3.1. Physicochemical properties of natural and synthetic vanilla extracts

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Table 1 shows the physicochemical properties of natural vanilla (NVE) and synthetic

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(SVE) extracts. NVE extract showed a concentration of 19.8 % total solids, 14.6 % °Bx, and

a pH of 2.97. This extract showed a dark colour reflected by the L* (6.14), a* (1.89) and b*

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(0.76) colour parameters with Chroma value and hue angle of 2.03 and 21.90°, respectively.

Meanwhile, SVE extract showed a concentration of 20.22% of total solids, 12.66 °Bx, and a
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pH of 3.31. This showed a similar colour parameters to the natural extract (L*=5.76, a*=0.99

and b*=0.53) with Chroma of 1.12 and a hue angle of 28.16° located in the same plane of the
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colour chart CIE L*a*b* of the natural extract. Both samples presented similar parameters

colour possibly due to the extracts were adjusted to the similar total solids concentrations.
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3.2. Physicochemical properties of the microcapsules

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Physicochemical interactions that occur between vanilla and wall material help to

understand the behavior of microcapsules in food products. The binding that occurs at a

molecular level reflects changes, in the properties at a macroscopic level [23]. The

physicochemical properties of microcapsules with natural vanilla extract (NVE-WPC) and

microcapsules with synthetic vanilla extract (SVE-WPC) are shown in Table 2. No

significant differences (p >0.05) were found between the majority of physicochemical

properties made with vanilla natural (NVE) and synthetic vanilla (SVE) extracts. The

moisture content was 3.55 and 4.60 g /100 g for NVE-WPC and SVE-WPC, respectively.

While, water activity in both case was lower than 0.350. The microcapsules showed low

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moisture content and water activity reflecting adequate stability after spray-drying, and thus,

it could be considered biochemically and microbiologically stable. In general, biochemical

and microbiological reactions in a food system can be inhibited and the deterioration of the

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product prevented when the moisture content is less than 6% and the water activity is less

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than 0.6 [24]. These values were lower than those reported for CLA microcapsules using

whey protein concentrated and Arabic gum as wall material [25]. The low moisture content

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might due during atomization in high operation temperature, exposure of larger surface area

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enhances the heat and mass transfer rates therefore the moisture content in powders decrease

[26]. The microcapsules had a high solubility (around 92.67 - 95.30%) very close to having
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the WPC, indicating that in this case water solubility of the microcapsules only depended on

the wall material and which meant that it could also easily and effectively be reconstituted in
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water for use of the bioactive compounds of de vanilla extracts powders in aqueous systems,
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due to its higher solubility value [24]. Colour parameters were evaluated and no significant
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differences (p > 0.05) were found between the microcapsules prepared with NVE and SVE,
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this may be because these parameters correspond to the colour of whey protein concentrated.
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So, the microcapsules obtained showed a light brown colour. The colour parameter L* varied

from 68.25 to 65.26, a* varied from 3.04 to 2.89 and b* varied from 25.12 to 24.20 to NVE-

WPC and SVE-WPC, respectively. The Chroma of the microcapsules varied from 25.33 to

24.37 and hue angle was about 83.10° and 83.18° to NVE-WPC and SVE-WPC, respectively.

The same behaviour had flow properties, the microcapsules showed regular flow properties,

and no significant properties (p >0.05) were found in the flow properties of the NVE-WPC

and SVE-WPC microcapsules. Bulk and packet density from NVE-WPC and SVE-WPC

microcapsules presented especially desirable condition for the transport process and product

packaging. It is desirable for a powder to have a high bulk density as it will require less
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volume when packaged. In addition, a high bulk density usually means that there are less

empty spaces between particles available to be occupied by air, which can help to prevent

oxidation and increase the stability of the powder [24]. The particle density of the

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microcapsules varied of 740-750 kg/cm3, which indicates the presence of similar porosity in

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the microcapsules and/or the presence of microcapsules with a rough or toothed surface [24].

The percentage compressibility of the microcapsules was near of 40% and angle of repose

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was approximately 40.52°. An angle of repose (40-45°) was indicative of powder with

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passable flow property and Hausner ratio (1.63-1.71) were indicative of powder viscosity

with some cohesiveness, suggestive of a very poor flow, with cohesive property [21, 27].
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Irregular surface morphology of spray dried powders might be attributed to the restricted and

very poor flowability and it is reported that large particles may tend to decrease the cohesive
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force, resulted in a reduced Hausner ratio [28]. The flow properties were similar to those
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reported microcapsule in which the WPC has been used [19], indicating that the flow
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properties of the microcapsules depends largely on the biopolymer used as wall material. The
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microparticles showed similar mean values of surface area around (270.80-320.60 μm2),
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perimeter (30.90-35.30 μm) and Feret diameter (9.50-10.25 μm), which coincides with the

behavior shown by the flow properties of both microparticles. The microcapsules developed

by the two extracts showed no significant (p > 0.05) yields of microencapsulation higher to

90%. Several authors have been found lower values of yield of encapsulated vanilla obtained

yields around 60-83.63% [13,29]. It is shown that the high retention of vanillin during

microencapsulation by spray-drying possible that WPC using as wall material is able to form

a reduced compact crust the time of formation of the shell of the microcapsule, the convection

heat transfer is limited in the interior of the capsule and therefore decreases the diffusion of

volatile compounds at reducing the surface loss and demonstrated the optimal spray-drying
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process without compromising the quality of the encapsulated extract. WPC is mainly

composed of β-lactoglobulin and its main gelling agent, whose hydrogel forms a three-

dimensional network that exhibits the ability to swell and retains a significant fraction of

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water within its structure. Possibly, WPC as an encapsulating agent forms a dense polymeric

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network in which vanillin molecule may be dispersed through the matrix [30]. Some authors

have suggested that there is no chemical interaction between vanillin and proteins used as

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wall material, since the aldehyde group and the ether vanilla did not show chemical alteration

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after encapsulation [31]. So that wall material could function as a container that retain and

protects encapsulated vanilla. Whey protein concentrate reduces the volatility of compounds
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it is found to be a suitable wall material for microencapsulation of volatile due that its exhibits

excellent film forming abilities [32].

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3.3. Antioxidant activity of natural and synthetic vanilla extracts

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Table 3 shows a concentration of 1.61 g of vanillin/100gd.m. for NVE and 20.3 g

vanillin/100gd.m. to SVE, resulting that the concentration of vanilla in synthetic extract was
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approximately 12 times higher than the natural extract which is possibly due to the extraction

process performed or the synthetic vanilla has fewer components because this is synthesized

from guaiacol and lignin [33]. Polyphenol concentration shows a similar behaviour, SVE

showed a concentration of polyphenols of 11.37 g of GAE/100gd.m., while NVE showed a

concentration of 0.89 g of GAE/100gd.m. Despite the difference in the concentrations of

vanillin, extracts NVE and SVE showed similar dose-dependent antioxidant activity

evaluated by the radical DPPH scavenging activity of 69.67% and 60.40%, respectively. The

SVE presented an EC50 of 17.1 mgd.m. (R2=0.957) for DPPH radical scavenging activity,

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while NVE showed a similar concentration (17.20 mgd.m.). These results indicate that a high

concentration of the extract is needed to achieve 50% inhibition of DPPH radicals as

compared to ascorbic acid employed as a control, which exhibited dose-dependent

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antioxidant activity with an EC50 of 0.30 ± 0.01 mgd.m. (R2=0.956). NVE showed a low

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concentration of vanillin but high chain breaking activity which possibly explains the

antioxidant activity similar to the SVE which showed a high concentration of vanillin but

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low chain breaking activity. Antioxidant activity by chain-breaking for SVE was of 0.01 -

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DO-3/min/mgd.m. (0.09 -DO-3/min/mgpolyphenol and 0.05 -DO-3/min/mgvanillin) and 0.15 -DO-
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/min/mgd.m. (5.20 -DO-3/min/mgpolyphenol and 25.00 -DO-3/min/mgvanillin) for NVE. Some
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synthetic polyphenol compounds possessing a rather high reactivity to active free radicals,

however show only a moderate chain-breaking antioxidant activity, because of the rather
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high chemical activity of the derived phenoxy radical and/or semiquinones. A real chaing
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breaking antioxidant activity is sometimes partly determined by the involvement of products

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of oxidative transformation of original antioxidant into inhibition [34]. Similarly, the chain-
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breaking activity of the reducing power of the natural extract (1.10 DO700nm/mgd.m.) was
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higher than that of the synthetic extract (0.91 DO700nm/mgd.m.), suggesting that the natural

extract contains compounds with greater capacity and higher reducing antioxidant activity.

It is reported that redox potential give indications of the overall reducing properties of all the

food antioxidants, including those which react slowly with radical species and it is an

indicator of the antioxidant efficiency of food products and the chain-breaking activity

measurements provide information on the capacity of antioxidant compounds to quench

radicals [14].

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The correlation between the results obtained in the different assays used to measure

the antioxidant activity of vanilla extract (Total Polyphenols, DPPH radical capture and

reducing power) were evaluated. A high linear correlation between the concentration of total

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polyphenols and antioxidant activity by the capture assay DPPH radical of the vanilla extract

was observed (R2=0.952). A high linear correlation was observed between the concentration

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of total polyphenols and reducing power of vanilla extract (R2=0.985). The antioxidant

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activity of some polar extracts might due to the presence of phenolic compounds, which exert

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their action by proton donation (free radical scavenging ability), or by interaction, radical

addition or combination or redox reactions (electron transfer). Phenolic compounds such as

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vanillin are excellent electron donors for hydrogen, reflecting electron transfer after the direct

reduction of salt oxidizing Fe[(CN)6]3 to Fe[(CN)6]2 in the reducing power assay. It is

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therefore possible to say that the reducing power of vanilla extract is closely related to the
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concentration of phenolic compounds [35]. A high linear correlation between the reducing
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power and antioxidant activity in the DPPH radical assay with an R2=0.969 was observed.
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This is possibly because of the radical DPPH test capability or easily an antioxidant or radical
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scavenger can transfer agent comprising an electron or hydrogen radical is evaluated for

stabilization, which determines the ability reducing (electron transfer capacity) of the extract

[36].

3.4. Antioxidant activity of natural and synthetic vanilla microcapsules

The SVE-WPC microcapsules showed a concentration of polyphenols of 0.82 g of

GAE/100gd.m., while NVE-WPC microcapsules showed a concentration of 0.19 g of

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GAE/100gd.m. The concentration of polyphenols was higher than that observed in the

microcapsules of other fruits [37]. In the capture assay on DPPH radicals, the antioxidant

activity of SVE-WPC was observed to be dose-dependent with R2=0.993 with a median

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effective concentration (EC50) of 29.50 mgd.m. NVE-WPC microcapsules showed dose-

dependent antioxidant activity (R2=0.998) with an EC50 of 36.2 mgd.m., indicating that very

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high concentrations of the extract are needed to achieve 45.70% of DPPH radical scavenging

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activity.

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Similarly to the extracts, the chain-breaking activity for polyphenols presents in SVE-
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WPC microcapsules (-0.008 -DO-3/min/mgpolyphenols) was lower than NVE-WPC

microcapsules dry matter (4.70 -DO-3/min/mgpolyphenols) (Table 3). The reducing power of the
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NVE-WPC microcapsules was 1.14 DO700nm/mgd.m.., suggesting that the compounds presents
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in NVE-WPC possessing higher ability to promote electron transfer [14]. It was observed

that the process of microencapsulation of vanilla extract favoured the retention of compounds
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with reducing power, compounds structurally capable of donating electrons and/or hydrogen

protons, such as phenolics compounds, which are closely related to the antioxidant activity
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of the vanilla extract [35].

3.5. Analysis of volatile compounds

Fifteen volatile compounds were identified in NV extract. The major compounds

identified included 4-hydroxy-3-methoxybenzaldehyde (86.30%), 3-aminobenzoic acid

(3.12%), linoleic acid ethyl ester (2.84%), hexadecanoic acid ethyl ester (2.55%), and 3-

phenyl-2-propenoic acid-1-methyl ester (1.34%) (Table 4). However, only two volatile

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compounds were identified in SV extract, 4-hydroxy-3-methoxybenzaldehyde (vanillin) with

an abundance of 58.10% and the second, 4-hydroxy-3-ethoxybenzaldehyde (ethyl vanillin),

with an abundance of 41.90%, which corroborates the replacement of natural extract for

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flavouring compounds of artificial origin. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is

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the main compound responsible for the characteristic aroma of vanilla extract (Vanilla

planifolia A.). Compounds such as p-hydroxybenzaldehyde, the vanillyl alcohol and esters

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of cinnamic acid are also involved in the aroma and taste of natural vanilla extract [1].

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Volatile analysis of the microcapsules was similar to the alone extracts, the presence
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of the two volatile compounds identified in the initial extract was observed, vanillin with an

abundance of 74.90% and 25.10% abundance ethylvanillin. Meanwhile, in case of

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microcapsules of natural extract were identified only 5 of the compounds of the fifteen initial
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extract: vanillin (83.00 %), hexadecanoic acid ethyl ester (2.00 %), ethyl linolenate (1.20 %),

(Z, Z, Z)-octadecatrienoic -9,12,15 acid ethyl ester (0.90 %) and octanoic acid ethyl ester
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(12.90%). Possibly the main reason for the high retention volatile of NVE within WPC after

spray-drying is because the phenolic compounds present in the NVE contain a hydroxyl
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group, which form bonds with relevant sites in the protein molecules of the WPC [38].

3.6. Micrographs of the microcapsules

The surface morphology of the microcapsules was observed by SEM. The microcapsules of

both extracts showed morphology, size, and very similar topography. Figure 1 show the

microcapsules prepared with natural extract equilibrated at four different water activities (aw)

after 25 days of storage at 25°C. The microcapsules exhibited in all aw a homogeneous

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spherical morphology with smooth surfaces, slightly toothed, without the presence of pores,

very heterogeneous in size and prone to agglomeration between smaller particles.

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The absence of pores in the surface of the microcapsules is critical for stability, ensuring its

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integrity and resulting a good indicator of the efficiency of the microencapsulation process,

and that the pores facilitate the oxygen inlet and the exit of the encapsulated material by

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decreasing the efficiency microencapsulation in time and facilitating the oxidation of

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microencapsulated compound [39, 40]. The use of whey protein as a wall material has effect

on the morphology of the microcapsules making them more spherical and smooth surface
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[21]. Microcapsules shows smooth, skin forming behaviour which is a typical characteristic

of whey protein due to the principal component β-lactoglobulin, that possess interesting
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emulsifying and foaming properties [41]. The presence of smooth, spherical microcapsules
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is desirable for the stability of the encapsulated ingredient, because smooth, spherical
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microcapsules control release and facilitate solubility properties that improve the
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effectiveness of food additives to be incorporated in various complex matrices. The stability

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of the microcapsule was not affected by aw and storage temperature, and the microcapsules

not present caking and stickiness during the storage at different water activity. The

microcapsules remained unchanged during storage at 25°C in the ranges studied aw, all

replicas observed in the structure and shape of the microcapsules was not modified. This was

probably due to the fact that β-lactoglobulin present in the WPC is characterized by a high

content of charged amino acids that produce a hydrophilic surface [30], in addition to the

formation of a dense cross-linking matrix offers resistance against wall degradation in the

storage conditions studied.

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3.7. Vanillin retention during storage

Vanillin retention of the pure extracts was compared with the retention of the corresponding

microencapsulated extracts. Both microparticles showed slightly greater retention of vanillin

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compared to the non-microencapsulated extracts, demonstrating that microencapsulation

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process by spray-drying proves to be effective in the preservation of the vanillin during

storage (Figure 2). Vanillin retention of the microparticles was monitored for 30 days and

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presented two linear stages of degradation. In the first stage the vanillin retention was in the

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range of 55-70% in the first week of storage, with no significant differences for NVE-WPC

and SVE-WPC. Subsequently a second stage was presented with retention of 20% and 45%
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for NVE-WPC and SVE-WPC, respectively. The difference in the two stages could be due

to the fact that in the first stage the un-encapsulated vanillin, which is found on the surface
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of the particulates, is more exposed and degraded easily. In contrast, the second stage reflects
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the degradation of the vanillin inside the microparticle, indicating that the vanillin present in
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the SVE-WPC is more protected than in the NVE-WPC microparticles, which favor a greater
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retention of the compound during the storage. It has been reported that vanilla is distributed
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on the surface and it is easier to release in the primary release stage [42]. These results are

congruent with a higher concentration of vanillin in SVE-WPC found immediately after

spray drying. Similar results have been reported for the retention of carotenes encapsulated

during storage [43].

4. Conclusions

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Thus, the spray drying microencapsulation process used allowed for the production of

microcapsules with excellent yields of phenol compounds such as vanillin as well as

protection of the initial extract's antioxidant properties. The results showed that there was no

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difference in the yield encapsulation between the natural and synthetic vanilla extract, but

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there were differences in vanillin retention during the storage, being the microcapsules made

with synthetic vanilla extract which had a higher retention at the end of storage under

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controlled conditions. The microcapsules obtained showed morphological features ideal for

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ensuring stability and integrity, with desirable flow properties for industrial processes such

as transport and packaging. Thus, the results obtained in this work are promising to guarantee
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the encapsulation of compounds responsible for the antioxidant activity of vanilla extracts,

indicating the feasibility of these products for incorporation into functional foods.
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Acknowledgments
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The authors acknowledge the support to the L-IDEA, and the National Council of
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Science and Technology (CONACyT).

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FIGURE CAPTION

Figure 1. SEM micrographs of the NVE-WPC microparticles stored for 25 days at 15°C and

different water activities values. a). aw=0.0, b). aw=0.529, c). aw=0.753 and d). aw=0.902.

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Figure 2. Vanillin retention (%) of the extracts: NVE (○) and SVE (□), and the microparticles

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NVE-WPC (◊) and SVE-WPC (x) obtained by spray drying stored at 25°C during 30 days.

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Figure 1

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Figure 2

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Table 1. Physicochemical properties of natural vanilla extract (NVE) and synthetic vanilla

extract (SVE).

Physicochemical properties NVE SVE

19.8 ± 0.11a 20.22 ± 0.32a

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Solids total (%)
Brix (°) 14.6 ± 0. 81a 12.66 ± 0.23a
a
Color parameter L* 6.14 ± 0.48 5.76 ± 0.11a

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b* 0.76 ± 0.16a 0.53 ± 0.25a
b
a* 1.89 ± 0.25 0.99 ± 0.20a
Chroma 2.03 ± 0.67a 1.12 ± 0.50a

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b
Hue angle (°) 21.90 ± 0.61 28.16± 1.56a
a
pH 2.97 ± 0.11 3.31 ± 0.15a
Data are expressed as means ± SD. Different letters within the same row mean a significant

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difference (p < 0.05, n = 3). MA
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Table 2. Physicochemical and flow properties of microcapsules prepared with natural and

synthetic extracts of vanilla.

Physicochemical properties NVE-WPC SVE-WPC

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Moisture (%) 3.55 ± 0.30a 4.60 ± 0.20a

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aw 0.350 ± 0.01a 0.335 ± 0.02a
Color
L 68.25 ± 1.55b 65.26 ± 1.03a

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a 3.04 ± 0.05a 2.89 ± 0.06a
b 25.15 ± 0.50a 24.20 ± 0.60a
Hue angle (°) 83.10 ± 0.40a 83.18 ± 0.50a

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Chroma 25.33± 0.60a 24.37± 0.70a
Solubility (%) 95.30 ± 3.20a 92.67 ± 4.50a
Bulk density (kg/m3) 130.20 ± 5.25a 137.60 ± 3.70a
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Compact density (kg/m3) 225 ± 3.15a 227 ± 5.20a
Particle density (kg/m3) 750 ± 4.25a 740 ± 3.10a
Angle of repose (°) 44 ± 2.30a 40.52 ± 3.50a
Compressibility (%) 41.77 ± 1.25a 38.76 ± 1.50a
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Hausner ratio 1.71 ± 0.10a 1.63 ± 0.20a

Surface area (μm2)* 150.90 ± 50.50a 120.80 ± 60.10a
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Perimeter (μm)* 50.30 ± 20.90a 40.90 ± 15.10a

Feret diameter (μm)* 16.25 ± 12.15a 15.30 ± 10.10a
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Yield Microencapsulation (%) 94.4 ± 1.50a 95.7 ± 2.20a

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Data are expressed as means ± SD. Different letters within the same row mean a significant

difference (p < 0.05, n = 3). *Parameters determined by ImageJ 7.50i software.

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Table 3. Antioxidant properties and chain-breaking activity of the microcapsules and

vanilla extracts.

Properties NVE SVE NVE-WPC SVE-WPC

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Vanillin 1.61 ± 0.02b 20.30 ± 0.01d 0.15 ± 0.01a 1.60 ± 0.13c
(g/100gd.m.)

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DPPH radical scavenging 69.67 ± 5.25 60.40 ±7.80 45.70 ± 4.60 39.60 ± 3.50
activity (%)

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DPPH EC50 17.20 ± 0.60a 17.10 ± 0.7a 36.20 ± 3.50b 29.50 ± 0.09b
(mgd.m.)
Total Phenols 0.89 ± 0.01b 11.37 ± 0.01c 0.19 ± 0.01a 0.82 ± 0.01b

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(g GAE/100gd.m.)
Reducing power 1.10 ± 0.06a 0.91 ± 0.03a 1.14 ± 0.07a 0.454 ± 0.07a
(D.O.700 nm /mgd.m.) MA
k 0.15 ± 0.03b 0.01 ± 0.01a 0.01 ± 0.00a 0.008 ± 0.00a
(-D.O.-3/min/mgd.m.)
k 5.20 ± 0.70b 0.09 ± 0.01a 4.7 ± 0.5b 0.008 ± 0.00a
(-D.O.-3/min/mgpolyphenol)
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k 25.00 ± 3.00d 0.05 ± 0.01a 6.16 ± 0.5c 0.50 ± 0.00b

(-D.O.-3/min/mgvanillin)
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NVE= natural vanilla extract; SVE= artificial vanilla extract; NVE-WPC= microcapsules of
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natural vanilla extract and whey protein concentrate (WPC); SVE-WPC microcapsules of

artificial vanilla extract and WPC; d.m.= dry mass; D.O.=Optical density; nm= nanometers;
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g=grams; mg= milligrams; n.d.= not determined. The inhibition of DPPH radical is expressed

in median effective concentration (EC50); Total phenols are expressed as gallic acid

equivalents (GAE). k= reaction rate. Data are expressed as means ± SD. Different letters

within the same row mean a significant difference (p < 0.05, n = 3).

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Table 4. Volatile compounds in area percentage presents in extracts and microcapsules of

natural and synthetic vanilla.

Rt
Compound name Percentage (%)a
(min)

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SVE NVE- SVE-
NVE
WPC WPC

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12.110 propylbenzene 0.22 - - -
13.209 2-methoxyphenol 0.49 - - -
14.187 butylbenzene 0.84 - - -

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15.114 4-methoxyphenol 0.44 - - -
16.173 pentylbenzene 0.16 - - -
17.460 trans-1-phenyl-1-pentene 0.11 - - -

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18.055 hexyl-benzene 0.12 - - -
19.863 3-phenyl-2-propenoic acid-1-methyl ester 1.34 - - -
20.395 4-hydroxy-3-methoxybenzaldehyde 86.30 58.10 83.00 74.90
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21.626 3-aminobenzoic acid 3.12 - - -
21.900 4-hydroxy-3-ethoxybenzaldehyde - 41.90 - 25.10
25.917 pentadecanoic acid ethyl ester 0.31 - - -
26.884 hexadecanoic acid ethyl ester 2.55 - 2.00 -
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28.858 linoleic acid ethyl ester 2.84 - 1.20 -

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28.961 9,12,15-octadecatrienoic acid ethyl ester 0.87 - 0.90 -

29.196 octanoic acid ethyl ester 0.22 - 12.90 -
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The components of honey sample were identified by their mass spectra and retention times
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with that of the Wiley and NIST mass spectral databases. a Percentage of component based
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on the area normalization. RT: Retention time. –Not detected.

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Graphical Abstract

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Highlights

 WPC is an alternative to produce powder with vanilla extract.

 Vanillin was successfully microencapsulated using spray drying.
 Microcapsules presented good physicochemical and antioxidant properties.

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 Microcapsules were not altered during storage period.
 Natural and synthetic vanilla extracts produced powder with similar properties.

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