Food Research International 148 (2021) 110627

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Production of a rich-carotenoid colorant from pumpkin peels using

oil-in-water emulsion followed by spray drying
Priscilla M. Lima *, Gustavo C. Dacanal , Lorena Silva Pinho , Luis Jaime Pérez-Córdoba ,
Marcelo Thomazini , Izabel Cristina Freitas Moraes , Carmen S. Favaro-Trindade
Universidade de São Paulo, Faculdade de Zootecnia e Engenharia de Alimentos, Av. Duque de Caxias Norte, 225, CEP 13635-900 Pirassununga, São Paulo, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Peels and seeds are byproducts generated during the processing of fruits and vegetables that have been cut off or
Byproduct rejected in the food industry. Pumpkin peels are an example of products that provide valuable nutritional aspects
Encapsulation but that have low commercial value. This work aimed at recovering carotenoids from pumpkin peels to produce
High-pressure
valuable powders. The pumpkin peel flour was obtained from convective drying and milling processes. Liquid-
Stability
Pigment
solid extraction produced the ethanol raw extract with a high carotenoid content. Carotenoid extract and Arabic
Dye gum suspensions were mixed in proportions of 1:2, 1:3, or 1:4 w/w. Emulsions produced via Ultra-Turrax (UT)
and Ultra-Turrax plus high pressure (UTHP) were evaluated and spray dried. The particles carotenoid concen­
trations varied from 159.1 to 304.6 µg/g and from 104.3 to 346.2 µg/g for samples primarily produced via UT
and UTHP, respectively. UTHP 1:3 particles showed the lowest degradation of carotenoids during 90 days of
storage, with a retention index of 79%. The homogenization and spray drying techniques were proven to be
suitable steps to preserve the carotenoids recovered from the byproduct studied. Microparticles can be used as a
natural dye with potential use in food, pharmaceuticals, and cosmetics.

1. Introduction emulsions, attributing greater protection to these compounds (McCle­

ments, 2010). Emulsions are obtained by homogenizing immiscible
The pumpkin (Cucurbita moschata) belongs to the Cucurbitaceae liquids, in which the dispersed phase is presented in the form of small
family. It is widely cultivated and consumed worldwide (Darrudi et al., droplets dispersed in the continuous phase (Mao, Roos, Biliaderis, &
2018), and it is a highly appreciated vegetable due to its functional Miao, 2017). β-carotene emulsions can be obtained by homogenization
properties, being a source of pro-vitamin A and carotenoids (Promsakha using ultrasonic treatment, high pressure, or agitation under high in­
na Sakon Nakhon et al., 2017). The pulp is usually used in the produc­ tensity, among other techniques (Hou et al., 2012).
tion of sweets, jams, and purees (Provesi et al., 2011). However, pro­ Homogenization under high pressure allows to control the size of the
cessing generates large amounts of peels and seeds, which are termed droplets in emulsions according to the number of cycles and pressure
byproducts. used in the process (Mao et al., 2018). High-pressure homogenization is
Like pulp, byproducts also have functional properties and can be known to induce greater stability of bioactive compounds such as
used as nutritional supplements or food ingredients (Massarolo et al., β-carotene compared with methods such as dispersing via Ultra-Turrax
2017). The peels are sources of carotenoids, that provide their orange (Sotomayor-Gerding et al., 2016). However, emulsions are thermody­
color (Martins & Ferreira, 2017). The carotenoids are recovered by namically unstable, and the droplet size distribution of emulsions
extraction, but they degrade when submitted to pH changes, heating, change when subjected to heating, freezing, or pH changes. As a
and light exposition. Alternatively, the literature has reported that it is consequence, the collapse of these droplets result in the exposure and
possible to obtain greater stability of these bioactives through different subsequent degradation of bioactive compounds (Liang et al., 2013).
encapsulation techniques (Janiszewska-Turak, 2017; Santos, Rubio, The literature reports that drying emulsions allows removing water
Balieiro, Thomazini, & Fávaro-Trindade, 2021). from the system and enables their application in foods. In this technique,
Lipophilic bioactive components may be incorporated into drying the emulsions enables the encapsulation of the oily phase within

* Corresponding author.
E-mail address: [email protected] (P.M. Lima).

https://doi.org/10.1016/j.foodres.2021.110627
Received 8 February 2021; Received in revised form 14 July 2021; Accepted 20 July 2021
Available online 22 July 2021
0963-9969/© 2021 Elsevier Ltd. All rights reserved.
P.M. Lima et al. Food Research International 148 (2021) 110627

a solid matrix, producing a dispersible powder (Yousefi et al., 2011). 2.4. Production of oil-in-water emulsions from raw extract
Spray drying is one of the most economical operational processes of
dehydration. In addition, the relatively short process time (5–100 s) The emulsions were prepared with different proportions of carot­
tends to preserve the most sensitive quality attributes such as nutrients, enoid raw extract from pumpkins peels and Arabic gum (Nexira Brazil
colors, and flavors (Shishir & Chen, 2017). The food products obtained Com. Ltd., Brazil) solution (20% w/w) (Table 1). Emulsions were pre­
from the spray drying current have a moisture content from 2% to 5% pared as 1:2, 1:3, and 1:4 (volume of concentrated extract per volume of
and water activity values varying from 0.2 to 0.6 (Shishir et al., 2016); Arabic gum solution). The production of emulsions followed two case
these features make them resistant to oxidative, microbiological, and studies:
enzymatic reactions (Tan et al., 2011). Therefore, spray drying has been
applied in food industries for decades (Shishir & Chen, 2017). i. Case study 1 (UT): The emulsions were produced by a homogeniza­
This work aimed to investigate the coupling of microencapsulation tion step in an Ultra-Turrax® IKA T25 (Labotechnic, Staufen, Ger­
processes by studying the stability of emulsions containing carotenoids many) at 10,000 rpm for 3 min (Xu et al., 2013).
from pumpkin peels and drying the emulsions by atomization in a spray ii. Case study 2 (UTHP): The emulsion was processed by homogenizing
dryer to produce particles that provide a better encapsulation efficiency in an Ultra-Turrax®, using the same conditions as case study 1, as
of the bioactive compound. The product obtained from these processes well as a high-pressure homogenization in which emulsions passed
was a colorant powder with a high carotenoid content and with the through a M− 110 Y microfluidizer (Microfluidics, MA, USA) at 100
potential to be applied as a natural dye in the food, pharmaceutical, and MPa and one processing cycle, to reduce the droplet size (Mutsokoti
cosmetic industries. et al., 2017).

2. Material and methods 2.5. Instability index of emulsion droplets by centrifugation

2.1. Production of pumpkin peel flour The instability index of the emulsion was measured in the LUMi­
Sizer® equipment (L.U.M. GmbH, Berlin, Germany) and was calculated
Pumpkin peels of C. moschata Duch variety were supplied by a local with the software package SepView 4.1 (L.U.M. GmbH). Subsequently,
agricultural holding (Doces Frutas de Minas, Caldas, MG, Brazil). A its stability was analyzed. The samples (0.4 mL aliquots) were placed in
domestic food processor (Walita Viva RI1364/06; Philips, Rio de polycarbonate cells (r = 130 mm) and immediately centrifuged (1163g),
Janeiro, RJ, Brasil) at maximum speed was used to crush the raw peels at 25 ◦ C, and measured at time intervals of 10 s, wavelength of 865 nm,
into small slices. The slices were placed in trays and dried in a convective and light factor of 1.0 for approximately 30 min, simulating accelerated
oven (Marconi, MA035/1152, Piracicaba, SP, Brazil) at 40 ◦ C in 18 h. A storage conditions of emulsions (Maher et al., 2011).
knife miller (Marconi, MA340) was used to mill the dried peels and
produce the coarse pumpkin peel flour (PPF). The flour samples were 2.6. Rheological measurements
sieved through 80 mesh.
The rheological behavior of all emulsions was determined by flow
curves using a rotational rheometer (TA Instruments 2000, New Castle,
2.2. Extraction of carotenoids from pumpkin peels flour
DE, USA), with parallel plate geometry (diameter of 60 mm and GAP of
1 mm). The flow curves at 25 ◦ C were generated over a logarithmic shear
First, the extraction of carotenoids from pumpkin peels flour was
rate range of 1–1000 s− 1. The emulsion viscosity was obtained by linear
conducted with ethanol 99.5% (Synth, Brazil) in a solvent–solid ratio
regression, considering the Newtonian fluid model by the Rheology
equal to 10% w/w (Ishida & Chapman, 2009). The suspension was
Advantage Data Analysis v.5.3.1 software.
shaken (280 rpm) (Orbital Shaker Marconi, MA420, Piracicaba, SP) at
40 ◦ C for 2 h (Das & Bera, 2013). In the second stage, the extract was
clarified by removing flour particles by filtration. The supernatant was 2.7. Production of solid microparticles by spray drying of emulsions
filtered through Whatman filter paper (0.45 μm) and then centrifuged at
7500 rpm for 4 min at 25 ◦ C in a centrifuge (Eppendorf 543 0R, The emulsions were dried using a spray dryer (model LM MSD 1.0,
Hamburg, Germany). Subsequently, 3% “w/w” of sunflower oil (pur­ LABMAQ, Ribeirão Preto, Brazil), with heated air flow of 2.50 m/s, inlet
chased from a local market) was added to the clarified extract. In the air temperature of 130 ◦ C, a feed flow rate of 0.6 L/h, a nozzle pressure
third stage, the initial volume of oil-extract was concentrated in a of 8.4 kgf/cm2, a nozzle diameter of 1.2 mm, and parameters deter­
TECNAL TE-211 rotary evaporator (Piracicaba, Brazil) at 48 ◦ C in 18 h, mined after numerous preliminary tests. The process yield, Y (%), was
until reaching the final volume equal to one third of the initial volume calculated as the ratio between total solid content (dry matter) in the
(D’Evoli et al., 2013). The extracts were manipulated and stored in the
absence of light to minimize the degradation of carotenoids. The Table 1
carotenoid raw extracts were composed of sunflower oil, ethanol, and Composition and type of homogenization process applied in the preparation of
extracted carotenoids. emulsions.
Homogenization case Trial* Vextract Vgum Proportion
study (mL) (mL) (Vextract /Vgum )
2.3. Chemical composition of the carotenoids-rich raw extract
Case study 1 UT 1:2 100 200 1:2
Case study 1 UT 1:3 75 225 1:3
The raw extract was characterized regarding the chemical compo­ Case study 1 UT 1:4 60 240 1:4
sition and the carotenoid content. The moisture content of the raw Case study 2 UTHP 100 200 1:2
extract was determined by Karl Fischer titration. The Kjeldahl method 1:2
Case study 2 UTHP 75 225 1:3
was used to quantify the raw protein level in FSA (Food Standards
1:3
Agency) (Association of Official Analytical Chemists, 1995), considering Case study 2 UTHP 60 240 1:4
the correction factor (NP = NT⋅4.27) for the conversion of total nitrogen 1:4
(NT) to protein nitrogen (NP) (Fujihara et al., 2001). The lipids content *
UT: Homogenization in ultra-turrax; UTHP: Homogenization in ultra-turrax
of the ether extract was determined according to the procedures plus high-pressure; Vextract : Volume of pumpkin peel extract; Vgum : Volume of
described by the Association of Official Analytical Chemists (1995). Arabic gum solution.

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resulting powder and total solid content in the emulsion. a vortex mixer for 1 min. The suspensions were centrifuged in a 5430 R
centrifuge (Eppendorf AG, Hamburg, Germany) at 7000 rpm at 25 ◦ C for
2.8. Stability of carotenoids in microparticles during storage 3 min, then the hexane phase was removed with a pipette. The absor­
bance was measured with a Genesys 10S UV–Vis spectrophotometer at
The stability of free and encapsulated carotenoids in extracts was 450 nm.
analyzed according to the methodology described by Ostroschi et al.
(2018) with slight modifications. The samples were arranged in small 2.10. Color parameters
quantities in vials, aiming to expose the largest possible surface to air,
then stored in desiccators containing saturated magnesium chloride The color parameters of emulsions and microparticles was deter­
solution (MgCl2, 32.8% relative humidity [RH]). The desiccators were mined in a colorimeter (Mini Scan XE, HunterLab, Reston, VA, USA).
stored in greenhouses at 25 ◦ C. The samples were stored for 90 days Calibration was performed using black and white calibration plates
under the specified conditions and evaluated every 15 days in relation to provided with the instrument. The results are expressed according to the
the total carotenoid content and instrumental color measurements of CIELAB color system (L* = lightness, a* = redness, and b* = greenness).
CIELAB. All samples were evaluated for moisture, water activity, and The analyses of microparticles were carried out in triplicate 0, 15, 30,
average diameter at day 0 and at day 90. 60, and 90 days after the encapsulation. The samples were stored in glass
The profile of carotenoid stability was modeled by a zero-order re­ jars that were protected from light, in the presence of O2 (simple air) and
action equation (Eq. (1)). Researchers have reported the degradation of controlled humidity (33%, using a saturated solution of MgCl2), at 37 ◦ C.
carotenoids from several sources of food products, and zero-order ki­
netics described the decrease in the carotenoids during the storage (Liu 2.11. Mean diameter of pumpkin peel flour, emulsion droplets, and spray-
et al., 2019; Tan et al., 2021). dried microparticles
The reaction rate constant, k(μg⋅g− 1 ⋅s− 1 ), and the half-life time,
t1/2 (s), were evaluated from the analytical solution of the zero-order The mean size of flour particles, droplets in emulsions, and spray-
kinetic model, as described in Eqs. (2) and (3) (Idham et al., 2012). dried microparticles was evaluated with laser diffraction (Shimadzu
SALDI-201-V, Kyoto, Japan). Water was used as a dispersant medium for
∂c emulsion droplets, and ethanol was used as a dispersant medium for
− =k (1)
∂t spray-dried microparticles. The light scattering evaluated the measure­
ments of the particle size distribution, and the mean diameters were the
C = C0 − kt (2)
De Brouckere mean diameter (D4,3 ).
C0
t1/2 = (3) 2.12. Solubility
2k
In the above equations, c (μg/g d.b.) is the concentration of carot­
The solubility of spray-dried 1:2 UTHP sample was determined ac­
enoids in dry basis at a specific time (days). The retention of carotenoid,
cording to the methodology reported by Eastman and Moore (1984),
RC (%), was determined as the ratio between carotenoid concentration
with some modifications. The powder samples with 0.5 g were sus­
after 90 days of storage, C90 (μg/g d.b.), and the initial carotenoid
pended in 50 mL of distilled water, stirred at 110 rpm for 30 min using a
concentration, C0 (μg/g d.b.), as follows:
TE-420 shaker (Tecnal, Piracicaba, Brazil), and then centrifuged at
( )
C90 4000 rpm for 5 min. An aliquot (25 mL) of each supernatant was
RC = ⋅100 (4)
C0 removed, transferred to porcelain dishes, and dried to a constant weight
in an oven at 105 ◦ C. Solubility is expressed as a percentage (%). The
solubility was calculated using Eq. (6):
2.9. Measurement of total carotenoids in raw extracts, emulsions, and
microparticles Dried supernatant weight
Solubility (%) = × 100 (6)
Initial sample weight
An UV–Visible Ultraviolet spectroscopy measures the concentration
of total carotenoids in the hexane extract (Rodriguez-Amaya, 2001). A 2.13. Scanning electron microscopy (SEM) of microparticles
UV–Vis Genesys 10S spectrophotometer (Thermo Scientific, São Paulo,
SP, Brasil) was used and the concentration of total carotenoids in the The treatment 1: 2 UTHP was chosen for this analysis. The particles
pumpkin peel flour was computed using Eq. (5). were deposited on a double-sided carbon adhesive tape (Ted Pella Inc.,
Abs⋅V⋅104 Redding, CA, USA) in a vacuum chamber and observed using the
c= (5) Tabletop Microscope Hitachi TM 300 (Tokyo, Japan) scanning electron
Abs1%
1cm ⋅m
microscope. SEM images of microparticles and pumpkin flour were
In Eq. (5), c (µg/g) is the concentration of total carotenoids; Abs is the captured at a voltage of 15 kV from ×100 to ×1500 of magnification.
absorbance of the solution at wavelength of maximum absorption (λmax )
of 450 nm; V(mL) corresponds to the final volume of the solution; m (g) 2.14. Statistical analysis
is the mass of the sample; and Abs1% 1cm is the absorption coefficient of
β-carotene in hexane, or absorbance at the given wavelength of a 1% Statistical analyses were conducted using the JMP software (SAS
solution in spectrophotometer cuvette with a 1-cm light path, which is Analysis System) version 9.3, Analysis of variance (ANOVA) followed by
2710. the Tukey–Kramer post hoc test (α = 0.05) was used to compare the
The total carotenoid content was measured according to the meth­ means. Values with p < 0.05 were considered statistically significant.
odology proposed by (Yuan, Gao, Zhao, and Mao (2008) with some
modifications. The amount of sample used in the trials varied according 3. Results and discussion
to the source. The mass of the sample for the raw extract was 0.01 g,
while 0.1 g emulsion and 0.2 g microparticles were used. The sample 3.1. Characterization of pumpkin peel flour
was added to a solvent mixture containing 1 mL ethanol, 3 mL n-hexane,
and 1 mL deionized water. The drying and milling steps used for the production of pumpkin peel
The tubes containing the sample–solvent mixtures were stirred with flour generated a fine powder with a narrow particle size distribution.

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The samples were sieved through size 80 mesh to remove the coarser Table 2
particles, resulting in a homogeneous powder with a mean diameter Chemical composition of pumpkin peel raw-extract.
(D4,3 ) of 240 μm. Fig. 1 shows the SEM images of the sieved particles. Parameters Mean ± SD
Peel flour particles have a non-spherical shape, showing a flattened
Moisture (g/100 g) 1.5 ± 0.0
surface, elongated, and with the presence of pores. According to Pietsch Protein (g/100 g) 0.4 ± 0.1
(2003) particles with an irregular surface have higher bulk porosity as Ethereal Extract (g/100 g) 3.6 ± 0.4
well as greater cohesiveness, because of mechanical interlocking and Total carotenoids (μg/g) 771.5 ± 3.7
abrasion between particles. α-carotene (µg/g) 196.0 ± 1.0
β-carotene (µg/g) 527.0 ± 6.0

Means and standard deviation (SD) were obtained from triplicates

3.2. Characterization of the raw extract
(n = 3).

3.2.1. Chemical composition of the raw extract

sufficient for the storage of the sample before the spray drying process.
The percentual composition and total carotenoids of the ethanol
In this test, LUMiSizer was utilized to evaluate physical stability of
extract are shown in Table 2. The moisture content of the extract was
emulsions by monitoring the transmission changes due to flocculation
1.5%. The lipid content was 3.63%, indicating a high extraction yield
and creaming, an approach that allowed checking the instability of the
when using ethanol as the solvent. The protein content was low, an
emulsions. When emulsions become unstable, the oil phase moves to the
expected finding because it is an alcoholic extract. Lima et al. (2019)
top, and the heavier aqueous phase moves to the bottom (Fig. 3). Then
found lower values of lipids (1.03 g/100 g extract) and higher values of
the change of transmission can reflect the instability of the emulsion.
proteins (1.53 g/100 g extract) in pumpkin peel flour.
Fig. 4, referring to emulsion produced by UTHP (1:2 ratio), clearly
The carotenoid content was high (771.5 µg/g), and α carotenes (196
reflects a separation of phases with the creaming phenomenon, identi­
µg/g) and β (527 µg/g) were identified in the extract, which has
fied by green profiles located at the top of the sample inside the bucket.
numerous beneficial properties to health because they act as antioxi­
At a low emulsifier concentration, the corresponding creaming rate is
dants and are precursors of vitamin A. Carvalho et al. (2012), when
the highest.
quantifying carotenoids in the pumpkin (Cucurbita moshata), found
All UT emulsions presented very close values, ranging from 0.60 to
234.2 μg/g to 405.0 µg/g of total carotenoids content, the α-carotene
0.68 (p > 0.05), without statistically significant differences between
content varied from 67.1 to 73.0 μg/g and total E-β-carotene content
treatments. On the other hand, the UTHP 1:2 emulsion was the least
varied from 142.0 μg/g to 244.2 μg/g.
stable among all treatments, probably due to the lower amount of Arabic
Considering that β-carotene has 100% pro-vitamin A activity, these
gum present within that formulation, which would provide greater
results are promising. No studies were found in the literature that re­
viscosity and, consequently, a lower tendency to cream. This view is
ported the centesimal composition of ethanol extract rich in carotenoids
consistent with the data because the samples with the highest Arabic
obtained from Cucurbita moschata pumpkin peels.
gum concentration were more stable for both treatments (p < 0.05) with
statistically significant difference. In the case of the UTHP treatment, the
3.2.2. Degradation of carotenoids during storage of the raw extract
high-pressure homogenization applied by the microfluidizer also helped
The concentration of total carotenoids in the raw extract was
to reduce the emulsion droplet size, making the droplets smaller than
determined after 0, 15, 30, 45, 60, 75, and 90 days of storage. Fig. 2
those obtained from the conventional process (UT). This phenomenon
shows the stability of the carotenoids of the extract obtained from the
provided greater stability to the emulsified system, reducing the
pumpkin peel flour. There was a decrease in the carotenoid content; this
creaming rate or a further separation phenomenon. Hosseini et al.
decline followed the zero-order rate equation for degradation kinetics.
(2015) reported similar results for emulsions prepared with Arabic gum
The carotenoid extracted from flour presented 53.17% retention at day
applying high-pressure homogenization. Those authors concluded that
90 of storage. The half-life of carotenoids in the raw extract was 96 ± 6
reducing the droplet size of emulsions containing high Arabic gum
days.
concentrations increased the viscosity of the emulsion, reducing the
speed of droplets and, consequently, the creaming rate.
3.3. Characterization of the emulsions As expected, the 1:4 emulsions prepared applying UTHP presented
the lowest rates of instability (p < 0.05), with statistically significant
3.3.1. Instability index of the emulsion droplets difference of the other trataments and, therefore, lower phase separation
The instability values of microemulsions prepared in three concen­ rates, and consequently, greater stability. By contrast, the emulsions
trations under two different treatments (UT and UTHP) are shown in prepared only with UT showed the highest instability rates (0.60–0.68).
Table 3. These data were obtained by simulating storage conditions at This outcome is due to the reduction in the droplet size of the emulsions
25 ◦ C for 24 days under Earth’s gravity. This time was considered

Fig. 1. (a) Particles of pumpkin peel flour obtained from SEM at magnification of x100, and (b) UTHP 1:2 spray-dried microparticles at magnification of x1500.

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Fig. 2. Stability profiles of carotenoids in the (a) free extract, and in the (b) spray-dried microparticles, evaluated during 90 days of storage.

the UT emulsification processes. On the other hand, the UT 1:3 and 1:4
Table 3
emulsions showed higher concentrations compared with the UTHP
Instability index (-) AND total carotenoids (μg/g) prepared under UT or UTHP
emulsions. This have occurred because producing emulsions in the
case studies.
microfluidizer at high pressure provoked a substantial increase in the
Trial Instability index Total carotenoids Droplet mean diameter temperature, reaching to 40 ◦ C, possibly degrading carotenoids.
(–) (μg/g) (μm)

UT 1:2 0.7 ± 0.01Aa 348.2 ± 31.4Ab 52.3 ± 4.6Aa

UT 1:3 0.6 ± 0.01Aa 229.6 ± 16.5Ba 41.8 ± 3.3Ba 3.5. Color parameters of emulsions
UT 1:4 0.6 ± 0.03Aa 189.9 ± 22.2Ca 40.9 ± 4.3Ba
UTHP 0.8 ± 0.02Aa 367.8 ± 23.0Aa 34.7 ± 1.7Ab Carotenoids are found in nature and play an important role in the
1:2
UTHP 0.5 ± 0.03 Ba
197.3 ± 6.2 Bb
18.4 ± 1.3 Bb coloration of food (Gordon et al., 1983). The addition of carotenoids to
1:3 emulsions leads to a yellow appearance that aids consumer acceptance
UTHP 0.3 ± 0.04Cb 105.9 ± 16.9Cb 2.6 ± 0.2Cb of food. The colorimetric results of L*, a*, and b* of carotene emulsions
1:4 produced using UT of UTHP with different extract/Arabic gum ratios are
Mean values, standard deviation, and coefficient of variation (n = 3). Capital shown in Table 4.
letters in the same column indicate a significant difference (p < 0.05) between The L* value was not different among the treatments. The a* value,
samples of the same treatment and lowercase letters in the same column indicate which is directly correlated with the carotenoid content (Desobry et al.,
a significant difference (p < 0.05) between treatments with the same ratio. UT: 1997), decreased as the amount of carrier agent increased (and thus the
Ultra-Turrax; UTHP: Ultra-turrax plus high-pressure. amount of extract decreased); this change was expected because the
emulsions had a reddish color. The a* value in UTHP emulsions was
when subjected to high homogenization pressure in the microfluidizer, lower than UT emulsions, probably due to the degradation of the
and a reduced tendency to cream also contributes to the stability of the
emulsions (Table 3).

3.4. Total carotenoids in emulsions

Table 3 shows the total carotenoid content of the emulsions pro­

duced using UT or UTHP. The samples with higher extract content
within the formulations (1:2 ratio) presented the highest measured
carotenoid concentration for both treatments, 348.2 µg/g and 367.8 µg/
g, respectively. These concentrations were higher than even those found
in the fresh pulp of C. moschata (7.0-138.6 μg/g) (Assis et al., 2009;
Azizah et al., 2009) and Cucurbita maxima (2120 μg/100 g) (Kand­
lakunta et al., 2008). These findings indicate that the obtained pumpkin
Fig. 4. Transmission profile for UTHP emulsion (1:2) indicating the creaming
peel extract is rich in bioactive compounds (carotenoids) and thus justify
rate as a function of relative centrifugal force and transmission percentage.
its use. Besides, it can be observed that carotenoids are not degraded in

Fig. 3. Instability test and the appearance of the UT (a) UTHP (b) emulsions.

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Table 4
CIELAB color parameters of emulsions.
Trials L a b
Aa Aa
UT 1:2 26.4 ± 0.0 5.1 ± 0.3 36.3 ± 0.2Aa
UT 1:3 25.0 ± 0.1Ab 4.2 ± 0.1Aa 38.9 ± 0.7Aa
UT 1:4 27.5 ± 0.0Aa 1.0 ± 0.1Ba 34.9 ± 0.3Ba
UTHP 1:2 24.4 ± 0.1Ba 3.2 ± 0.2Ab 33.2 ± 0.5Bb
UTHP 1:3 30.4 ± 0.0Aa 1.0 ± 0.1Bb 33.0 ± 0.6Bb
UTHP 1:4 25.2 ± 0.0Ba − 2.2 ± 0.3Cb 35.2 ± 0.8Aa

Mean values, standard deviation, and coefficient of variation (n = 3). Capital

letters in the same column indicate a significant difference (p < 0.05) between
samples of the same treatment and lowercase letters in the same column indicate
a significant difference (p < 0.05) between treatments with the same ratio.

carotenoids in the high-pressure process due to release into the matrix,

leading to a loss of protection of the carotenoid–protein complex in the
chromoplasts. Zhang et al. (2019) reported similar results when they Fig. 5. Flow curves of emulsions obtained at 25 ◦ C and effects of proportions of
studied the effect of high-pressure homogenization on carotenoid bio­ Arabic gum (1:2; 1:3 e 1:4) and homogenization methods case study 1 (UT), and
accessibility. The UTHP 1:4 emulsion showed a negative a* value, case study 2 (UTHP) on emulsions viscosities.
approaching a green color trend. The b* values were positive for all
treatments, indicating that the samples are yellowish in color. agents, stabilizers, and wall materials for the production of spray-dried
oil-in-water emulsions (Choi et al., 2010). The encapsulation yield (YE)
3.6. Mean diameter of emulsion droplets of carotenoids was between 46.15% and 54.17% (Table 5); it was
affected by the total solid content. This finding is consistent with a
The mean droplet diameters of the UT emulsions were higher, previous report by Bustos-Garza et al. (2013), who reported a 58.65%
ranging from 40.9 to 52.3 μm, than those of the UTHP emulsions, which YE (astaxanthin, 100% Arabic gum) and a 50.95% YE (mixture of Arabic
were from 2.6 to 34.7 μm (Table 3). These values were higher than the gum and maltodextrin).
average diameters of the emulsions produced in the microfluidizer de­ The concentration of total carotenoids in microparticles was deter­
vice. Specifically, Mutsokoti et al. (2017) found lower values—from mined after 0, 15, 30, 45, 60, 75, and 90 days of storage (Fig. 2b). There
1.36 to 1.46 μm—from the present work when producing β-carotene- was a small decrease that followed the zero-order rate equation for
enriched microemulsions using the same conditions as the high-pressure degradation kinetics. Initially, the microparticles in the 1:2 emulsions
process. A reduction in droplet size was observed with increasing con­ showed the highest carotenoid concentration, an expected result due to
centration of carrier material (Arabic gum), which can be attributed to the greater amount of extract present (Table 5).
several factors: the faster adsorption of the emulsifier to the surfaces of The free extract showed a significant decrease in the carotenoid
the oil droplets during homogenization leading to lower interfacial content after 45 days of storage compared with the carotenoid concen­
stresses, thus facilitating droplet breakage and more emulsifier available tration in microparticles (Fig. 2b), due to the absence of microencap­
to cover the surface of droplets formed during homogenization (Ziani sulation that offers good protection to the bioactive components. This
et al., 2011). degradation occurs due to the high degree of unsaturation, which favors
oxidation and isomerization reactions (Chen et al., 2014).
3.7. Rheological characterization of emulsions The emulsions were spray dried and, according to Table 5, carot­
enoid retention values after the atomization process were high for the
Rheological measurements provide valuable information on the two processes used for emulsion production, using the different pro­
properties and performance of food emulsions (Chung et al., 2018; portions of Arabic gum. When considering the 1:2 and 1:4 ratios, the
Paglarini et al., 2018). The experimental data were adjusted by the UTHP emulsion had lower retention values compared with the UT
Newtonian model (R2 > 0.998), according to which the viscosity is
constant as the shear rate increases. Tonon et al. (2011) observed similar
Table 5
rates in emulsions containing flaxseed oil and Arabic gum and by Bae
Process yieldY, Initial concentration of carotenoids C0 , retention of carotenoids
and Lee (2008) in emulsions with avocado oil containing maltodextrins
at day 90 of storage RC, half-life time t1/2 , and mean diameter of solid micro­
and soy protein isolate.
particles measured at 0-day and at day 90 of storage.
The viscosity of emulsions ranged from 13.1 to 19.1 mPa⋅s, with
increasing values as the emulsifier concentration increased (Fig. 5). Trial Y(%) C0 (μg/g RC(%) t1/2 (days) D4,3 (μm) D4,3 (μm)
d.b.) at 0-day at day 90
These results might be ascribed to the increase in the proportion of the
emulsifier and the reduction in the average diameter of the droplets UT 1:2 46.1 304.6 ± 59.1 ± 160.5 ± 17.1 ± 18.9 ±
(Table 3). Emulsion viscosity increased as the total solid content ± 2.4 20.8 10.2Bb 30.8 1.2Aa 1.8
UT 1:3 50.2 231.7 70.8 ± 220.7 ± 18.9 ± 19.9 ±
increased because Arabic gum is characterized by chains with a partially ±
± 1.2 22.6 10.6Aa 21.9 2.6Aa 3.4
ramified structure, and is generally used as a thickening agent in food­ UT 1:4 54.2 159.1 ± 54.3 ± 176 ± 19.4 ± 19.7 ±
stuffs, making them more viscous (Aniesrani Delfiya et al., 2015). ± 1,8 18.4 7.5Bb 16.5 2.7Aa 3.1
Emulsions produced with the highest solid content were the most UTHP 47.5 346.2 ± 75.0 ± 301 ± 10.8 ± 13.5 ±
viscous and had the smallest average drop diameter. Carmona et al. 1:2 ± 2,2 32.6 5.1Aa 18.3 0.6Bb 1.2
UTHP 50.9 201.2 79.0 ± 249.3 ± 13.1 ± 13.7 ±
(2018) reported similar results when studying emulsions rich in carot­
±
1:3 ± 1,7 17.6 7.0Aa 14.5 1.2Ab 0.6
enoids extracted from palm oil produced by using Arabic gum. UTHP 53.5 104.3 ± 70.7 ± 142.9 ± 14.7 ± 14.9 ±
1:4 ± 3,3 12.1 6.6Aa 16.4 1.2Ab 1.4
3.8. Characterization of spray-dried microparticles Mean values, standard deviation, and coefficient of variation (n = 3). Capital
letters in the same column indicate a significant difference (p < 0.05) between
Encapsulation yield (YE) and stability of carotenoids during storage samples of the same treatment and lowercase letters in the same column indicate
Biopolymers, such Arabic gum, have been used as emulsifying a significant difference (p < 0.05) between treatments with the same ratio.

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P.M. Lima et al. Food Research International 148 (2021) 110627

emulsion. In this study, the proportion of wall material (Arabic gum) did carotenoids during storage (Table 6).
not influence the retention of carotenoids in microparticles. Carotenoid In samples produced at high pressure, the ΔE ranged from 3.4 to 4.5,
retention was influenced only by the homogenization process. Many indicating a smaller variation in color during the storage period. A
factors can contribute to this outcome, for example, the increased sur­ higher value of ΔE was observed for the 1:2 sample, which probably
face area resulted in a high degradation by oxidation. These reactions occurred due to the lower amount of wall material present, offering less
are related to hydroxylation, oxidation, and epoxidation, with conse­ protection to the bioactive component.
quent formation of volatile compounds, such as apocarotenoids (Limbo
et al., 2007). 3.10. Size distribution during storage of spray-dried microparticles
However, on day 90 of storage, the microparticles produced from the
UTHP emulsions presented high carotenoid retention compared with Initially, the microparticles produced in case study 2 (UTHP) showed
those produced from the UT emulsions (Table 5). The retention of ca­ mean diameters ranging from 10.81 to 14.77 µm, while the size of the
rotenoids in the 1:3 sample did not differ for either process. Our results microparticles produced in case study 1 (UT) ranged from 17.14 to
are higher than those reported by Matioli and Rodriguez-Amaya (2003), 19.45 µm. After the storage period, the powders showed no significant
who when encapsulating lycopene using Arabic gum as a card reader difference.
material verified a loss of 50% on day 18 of storage at room temperature According to the Tables 3 and 5, there was a reduction in the size of
and in the absence of light. particles when compared to the size of the droplets of the emulsions. The
reduction in the size of the particles may have occurred due to stress
3.9. Color parameters of microparticles during the atomization process of the emulsions. Because emulsions are
in a liquid state, the spray atomization combined with the drying of
Color is another parameter related to the stability of carotenoids, droplets may provoke the rupture of emulsion particles and some
because a color change can indicate possible degradation. The color encapsulated compounds may have been released and exposed to
change during storage was measured by the coordinates L*, a*, b*, and degradation. Consequently, the solid microparticles showed a minor size
ΔE (total color difference). compared with the liquid emulsion droplets.
The L* coordinate indicates the luminosity of the sample; in both Authors have reported similar values concerning the reduced size
processes, the 1:4 sample presented higher L values due to the greater distribution of spray-dried particles in relation to the initial oil droplet
presence of Arabic gum and lower concentration of extract and, there­ size. One example is the study of spray drying of emulsions of purple
fore, lower concentration of pigment. The L* value remained more sta­ cactus pear fruit, which resulted in the mean diameter of 47.7 µm for oil
ble for UTHP compared with UT emulsions, probably because emulsions droplets and spray-dried particles ranging from 2 to 58 µm (Toledo-­
with smaller particle sizes present greater compound retention and, Madrid, Gallardo-Velázquez, & Osorio-Revilla, 2018). Another example
therefore, greater color stability. Sheu and Rosenberg (1995) reported is the drying of emulsions containing echium oil and sinapic acid and
similar results when encapsulating ethyl caprylate in different wall Arabic gum, by spray drying and freeze drying, which resulted in
materials. The b* oordinate varies from blue to yellow; in this study emulsions with a mean size of 14.31 and 10.03 µm, respectively, and
there was no change in the b* values during storage. The a* coordinate smaller spray-dried particles, with size ranging from 2.3 to 5.14 µm
varies from green to red and is related to the carotenoid concentration (Comunian et al., 2019).
(Desobry et al., 1997). In this study, samples with a higher concentration The 1:2 samples had the lowest mean particle diameter (Table 5);
of extract showed higher values of a* during storage. There was no these emulsions contain the lowest concentration of the carrier agent
significant decrease (p < 0.5) in the a* values (Table 6). and have decreased viscosity.
An ΔE value of 0–0.5 implies an imperceptible difference in color The particles produced from the UTHP emulsions showed lower
between two samples, 0.5–1.5 indicates a small difference, 1.5–3.0 mean diameters relative to the UT emulsions. This was probably due to
specifies a significant difference, 3.0–6.0 represents a notable difference, the smaller droplet size of these emulsions, which presented higher
6.0–12.0 implies an extremely notable difference, and >12.0 denotes solvent evaporation rates and shorter film formation times, factors that
color with a different hue (Kim et al., 2002). According to Table 6, in the are related to greater encapsulation efficiency and retention of carot­
UT samples, the ΔE ranged from 6.75 to 8.84, indicating a great varia­ enoids during the storage period (Table 5). Shen and Quek (2014) made
tion in color during storage at 25 ◦ C. The 1:4 sample showed greater a similar finding when they used high pressure emulsions as a system to
variation, which may be related to the degradation of carotenoids during encapsulate astaxanthin. Moreover, Shu et al. (2006) reported similar
the period, as it was the treatment that showed the least retention of results when they encapsulated lycopene using conditions similar to the
present work.
There was no significant difference between the treatments
Table 6
CIELAB color indexes (L, a, b) and total color difference (ΔE) of microparticles regarding the mean diameters after storage except for the 1:2 samples.
measured at production (0-day) and at day 90 of storage, at 25 ◦ C. Specifically, there was a small variation, possibly linked to increased
moisture during the storage, promoting particle agglomeration.
Trial L0 a0 b0 L90 a90 b90 ΔE

UT 1:2 79.9 ± 6.5 ± 52.1 ± 75.1 ± 5.2 ± 47.6 ± 6.8 ± 3.11. Solubility
0.3 0.6 2.6 0.5 0.4 2.2 0.5Ba
UT 1:3 80.1 5.3 48.6 ± 74.2 ± 4.5 44.3 ± 7.4 ±
The powders produced in this study had high water solubility
± ± ±
0.4 0.3 0.9 0.7 0.5 2.0 1.2Ba
UT 1:4 81.2 ± 4.1 ± 43.5 ± 76.7 ± 2.9 ± 36.1 ± 8.8 ± (87.54–92.62%), which can be attributed to the amphiphilic property of
1.0 0.1 1.3 0.4 0.4 2.1 1.0Aa Arabic gum. This finding is important considering that carotenoid-rich
UTHP 77.9 ± 6.9 ± 51.1 ± 74.7 ± 6.3 ± 48.0 ± 4.5 ± materials have poor water solubility and that the aim of this study
1:2 1.3 0.8 2.2 0.2 0.5 2.0 1.2Ab
was to produce a colorant for food application. There was no difference
UTHP 79.2 ± 4.8 ± 43.0 ± 75.4 ± 4.1 ± 41.5 ± 4.2 ±
1:3 1.1 0.5 1.1 1.3 0.6 1.4 0.4Ab regarding the water solubility of the powders. The proportion of extract
UTHP 80.0 ± 3.3 ± 36.6 ± 77.8 ± 2.6 ± 34.1 ± 3.4 ± did not affect the solubility, as stated by the particles with the highest
1:4 0.9 0.5 1.9 0.6 0.2 0.9 1.1Bb extract content (1:2), which showed a higher solubility in water.
Averages followed by the same capital letter in the same column and lowercase Solubility is an important feature that will indicate the quality of
in the same row do not differ statistically from each other, by Tukey testing at these products (Jayasundera et al., 2011) in addition to its applicability.
5% probability. L0 , a0 and b0 were CIELAB indexes measured at 0-day, and L90 , The obtained powders had high solubility due to the Arabic gum that
a90 and b90 were CIELAB indexes measured at day 90 of storage. was used as an encapsulating material. In fact, Arabic gum is a vehicle

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