Carotenoid Extraction Using Edible Vegetable Oil: An Enriched Provitamin A Product
Carotenoid Extraction Using Edible Vegetable Oil: An Enriched Provitamin A Product
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The vegetable oils were obtained from a local Total carotenoid quantification
market in Rio de Janeiro, Brazil. The composition of The amount of total carotenoids from carrot was
the soy oil used, as declared by the manufacturer, was determined by spectrophotometry at 450 nm. Analyses
“100 % refined soy oil” and the olive oil was “extra virgin were performed in triplicate (Rodriguez-Amaya, 2001).
olive oil” (acidity ≤ 0.5 %).
According to the International Olive Council Carotenoid profile determination
classification (IOC, 2020), virgin olive oils are the oils The petroleum ether extract (2 mL) was dried under
obtained from the fruit of the olive tree (Olea europaea nitrogen flow and the residue solubilized in 200 µL of
L.) solely by mechanical or other physical means acetone. The carotenoid profile was determined by HPLC
under conditions, particularly thermal conditions, that (Pacheco et al., 2014), using an HPLC system, controlled
do not lead to alterations in the oil, and which have by the Empower software program with a column oven at
not undergone any treatment other than washing, 33 ºC and a photodiode array detector (PDA). Carotenoid
decantation, centrifugation and filtration. Extra virgin separation was obtained in a C30 column (S-3 Carotenoid,
olive oil is a virgin olive oil that has a free acidity no 4.6 mm × 250 mm; 3µ) by a gradient elution of methanol
higher than 0.8 %, expressed as oleic acid. and methyl tert-butyl ether. The elution started with a
mix of 80 % methanol and 20 % methyl tert-butyl ether.
Preparation of the edible oil enriched with At 0.5 min the ether concentration was increased to 25 %,
carotenoids at 15.00 min to 85 % and at 15.05 to 90 % ether. This
The process of preparing the edible oil enriched concentration was maintained at 90 % for 16.50 min
with carotenoids consisted of two steps: 1) extraction and then at 16.55 min returned to the initial condition
of the carotenoids with edible vegetable oils from the (20 %), remaining constant for up to 28 min. The flow
dehydrated matrix by agitation in a blender at 10,000 rate was 0.8 mL min–1 and the running time 28 min. The
rpm processed at room temperature; 2) removal of solid injection volume of the samples was 15 μL. Carotenoids
material dispersed in the oil after extraction by vacuum were identified based on their retention times and
filtration in a Buchner funnel (150 mm diameter) and UV/Vis absorption spectra, compared to the retention
quantitative filter paper (fast filtration). times of the carotenoid standards. The carotenoid
The extraction parameters were Ratio of Carrot quantification was carried out using analytical standards
Mass /Oil Volume (%M/V) that varied from 6.70 to (lutein, α-carotene, and β-carotene) previously isolated
63.33 and Time (min) that ranged from 5.0 to 13.5 in the laboratory according to Kimura and Rodriguez-
min. The final extraction volume was 100 mL. The Amaya (2001). After isolating the carotenoid of interest
enriched oil was kept in amber glass bottles at room and determining its chromatographic purity, the
temperature and protected from moisture for shelf concentration of the solution obtained was determined
life experiments. by spectrophotometry (Lambert-beer law). Using this
value, the required volume of the standard solution
Carotenoid analysis corresponding to 5 μg of the carotenoid was calculated.
Next, the solvent was removed and the standard
Extraction of carotenoids from carrot for analysis suspended in 1 mL of petroleum ether, producing a
The carotenoids were extracted according to solution with a concentration of 5 μg mL–1. This solution
Pacheco et al. (2014). The sample was homogenized and was used for the dilution of the analytical curve of seven
approximately 0.1 g of dried carrot powder was weighed points with the following final concentration 0.5; 1.5;
in a 2 mL microcentrifuge tube. One mL of acetone 2.5; 3.5; 5.0; 6.5 and 8.0 µg mL–1. The standards, with
was added, and the solid residue macerated using a purities in excess of 99 %, were used to make calibration
microturrax for 30 s. The mixture was centrifuged at curves, which were used to calculate the carotenoid
6,339 × g for 1 min and the supernatant transferred content in each sample.
to a 25 mL burette containing 5 mL of 5 % ethyl ether
solution in petroleum ether. Acetone (500 μL) was Determination of carotenoids in vegetable oil
added to the residue of the centrifuge tube and the Total carotenoid content and carotenoid profile
extraction procedure was repeated three or four times in vegetable oil (not enriched) and enriched vegetable
until the extract no longer exhibited the characteristic oils were evaluated by spectrophotometry and HPLC,
color of carotenoids. The resulting supernatant respectively. Approximately 0.5 g of each vegetable
(acetone extract) was transferred, quantitatively, to a oil (both not enriched and enriched with carotenoids)
burette. Next, the extract was washed three times using was weighed in 25 mL volumetric flasks and topped up
ultrapure water or until the wash water had turned with petroleum ether. The level of total carotenoids in
translucent. The extract was allowed to stand for 3 min the oil was determined by spectrophotometry at 450
before water decanting. The volume of ether extract nm. Carotenoid profiles were determined in a solution
was recorded, then 1 mL was transferred to a volume composed of 200 µL of oil and 800 µL of acetone by
cuvette and the total carotenoid contents determined HPLC, using the same method described for vegetable
by spectrophotometry. matrixes (Pacheco et al., 2014).
Recovery
80.00
85.00
62.00
64.00
88.00
65.00
81.00
81.00
78.00
80.00
80.00
Oil
olive oils
%
The quality of vegetable oils (soy and olive) was
evaluated both before and after the enrichment process
25.65 ± 0.16 d
29.07 ± 0.55 d
101.61 ± 0.07 b
103.16 ± 0.48 b
Retinol Equivalent
23.43 ± 1.33 d
74.3 ± 1.73 c
75.83 ± 2.76 c
135.7 ± 0.08 a
73.48 ± 8.33 c
70.64 ± 5.57 c
66.9 ± 0.7 c
with carotenoids. They were stored in amber glass bottles
µg RE mL–1
in oil *
at room temperature and protected from moisture. Total
Values represent the mean ± SD, different letters in the same column indicate statistical differences (p < 0.05); (C) = central point; *Retinol equivalent calculation: [(α-carotene/2) + β-carotene]/2.
carotenoids, acidity, peroxides and conjugated dienes
and trienes (CDT) were evaluated every 30 days for
150 days. The acidity and peroxide index analyses were
performed according to the official methods of AOCS
118 ± 13.41 c
45.85 ± 0.89 d
164.23 ± 0.27 b
168.25 ± 1.33 b
37.19 ± 1.91 d
106.65 ± 1.18 c
120.3 ± 2.72 c
123.63 ± 4.38 c
219.38 ± 0.21 a
113.03 ± 9.15 c
40.33 ± 0.3 d
(2009) Ca 5a-40 and Cd 8-53, respectively. Vegetable oils
Table 1 – Experimental design for the extraction parameters and predicted responses for the extraction of carotenoids from carrots using soy oil.
to the AOCS Ch 5-91 method.
A basic kinetic model was used to predict changes
21.92 ± 0.04 d
24.57 ± 0.42 d
77.97 ± 0.26 b
76.13 ± 0.75 b
19.32 ± 1.51 d
54.31 ± 0.44 c
56.61 ± 1.47 c
56.08 ± 2.27 c
104.06 ± 0.72 a
56.51 ± 3.97 c
57.94 ± 6.5 c
during storage according to Equation 1:
α-Carotene
dc
kC n (1)
Carotenoids in oil
dt
where the sign depends on the increment (+) or
degradation (-) of the compound of interest (total
1.58 ± 0.08 cd
2.81 ± 0.23 bc
4.07 ± 0.51 ab
2.88 ± 0.32 bc
1 ± 0.22 d
3.04 ± 0.28 b
1.2 ± 0.13 d
3.32 ± 0.14 b
3.05 ± 0.05 b
5.28 ± 0.59 a
carotenoids, acidity and CDT), C is the compound, t
3.04 ± 0.5 b
time, n the reaction order and k the rate constant.
Lutein
Finally, the shelf life for total carotenoids was
calculated as the half shelf life (t1/2), which is the
time necessary to reach half the initial amount of the
compound of interest.
172.42 ± 12.81 cd
180.22 ± 4.04 cd
182.75 ± 6.13 cd
166.57 ± 2.31 cd
245.25 ± 0.27 b
248.26 ± 0.81 b
163.76 ± 1.85 d
63.24 ± 0.56 e
71.99 ± 1.23 e
57.70 ± 3.55 e
328.67 ± 0.06 a
Efficiency of extraction
Total
53.64 ± 0.74 bc
55.52 ± 4.13 bc
58.52 ± 0.01 b
58.85 ± 1.97 b
47.52 ± 0.42 c
97.07 ± 5.96 a
52.74 ± 0.6 bc
58.04 ± 1.3 b
TCO 100 100
Efficiency
E(%) (2)
TCM Ratio
%
–1
1
–1
1
0
0
0
0
0
–1
–1
1
1
0
0
0
0
0
6.50
13.50
6.50
13.50
10.00
10.00
5.00
15.00
10.00
10.00
10.00
Time
63.33
35.00
35.00
35.00
35.00
35.00
6.70
/Oil Volume (%M/V) and Time (min). The CCRD was 887.23 µg g–1, which was used to calculate the extraction
incorporated into the following responses: Efficiency, efficiency (Table 1).
Total Carotenoids, Lutein, α-carotene, β-carotene, 9-cis- The results obtained by the experimental design
β-carotene, 13-cis-β-carotene, retinol and oil recovery. (Table 1) show that the extraction efficiency (E) improved
The desirability function (ranged from 0 to 1) was with the lowest (%M/V) ratio, e.g. with a ratio of 6.7 %
carried out to optimize the efficiency, total carotenoids the highest E of 97 % (p < 0.05) was obtained, recalling
and recovery. All statistical analysis was performed at a the E express percentage of carotenoids from vegetable
significance level of 5 %, and was carried out using the that may be extracted by the oil. On the other hand, total
Statistica software program (Statsoft, Statistica 7.0). The carotenoids in oil increased with as the ratio increased.
response variables were modeled based on a polynomial Thus, the lowest and highest values were 57.7 µg mL –1
equation (Equation 3) as follows: and 328.67 µg mL–1 for the ratios 6.7 % and 63.3 %,
respectively (Table 1), wherever the ratio increase has
Yi 0 11 X1 12 X12 21 X 2 22 X 22 31 X1 X 2 (3) less E. Almost all the responses (lutein, α-carotene,
β-carotene, Retinol equivalent) presented the same
where X1 is the ratio of Carrot Mass/Oil Volume (%M/V); behavior, namely, that total carotenoid increased as
X2 time; βo the independent term; β11 the regression the ratio increased. An increase in the ratio resulted
coefficients for the linear response of the ratio; β12 the in a higher carotenoid content in the oil; however, the
regression coefficients for the quadratic response of E results decreased. This could be because the dry
the ratio; β21 the regression coefficients for the linear carrot amount rose, and made the availability of the
response of the time; β22 the regression coefficients for vegetable oil to interact with the carrot difficult. Thus,
the quadratic response of the time and β31 the regression the increment in the ratio increased results in higher
coefficients for the interaction of both factors. carotenoid content in the oil but with less E, which
To optimize the retinol equivalent (RE) content in turn could result in an economic loss since after
the desirability function was applied and optimization the extraction process, the dry carrot still retained the
was performed using three simultaneous responses. substance of interest.
According to Myers (2016) and de Paula et al. (2019), According to the chromatograms presented in
the first step consists in finding an individual desirability Figure 1, there was no change in the carotenoid profile for
level (di) varying between 0 and 1 for each response (yi). dried carrot, soy and olive oil enriched with carotenoids.
Each desirability level could have a different objective, Thus, it was not possible to observe carotenoid
e.g. desirability could be minimized, maximized or degradation or trans-cis conversion. All procedures were
just sufficient to reach a specific target. The following taken to prevent carotenoid degradation. In the case of
equation (Equation 4) explains how to calculate the carrots, processing and storage were as fast as possible
individual desirability level. in order to avoid prolonged contact with oxygen, which
is the main factor in the degradation of carotenoids. In
0 , yi Li the case of vegetable oils, transporting the carotenoids in
ri the oil can be considered a protective factor, since the oil
y Li
di i , Li yi Ti (4) itself protects the carotenoids from contact with oxygen.
Ti Li Oil recovery had an inverse relationship with
1 , yi Ti ratio, the higher the ratio the lower the oil recovery (p
< 0.05). With increases in the ratio there was greater
where ri is a weight assigned to each response according absorption of oil by the vegetal matrix, resulting in a
to the degree of importance of the desirability level as lower recovery of oil at the end of the extraction process.
follows: if ri = 1, the desirability became a priority to The ratio presented a linear relationship with the
reach its target value, if 0 ≤ ri ≤ 1 means less important. response variables as is observed in Table 2, where the p
yi is the value of the response for each treatment, Li value for the linear coefficient was significant in all cases
the minimum response, and Ti the maximum response (p < 0.05). On the other hand, time had no significant
(Derrien et al., 2017). effect and presented p values for linear coefficients in
The second step is to calculate the overall excess of 0.05. There were no quadratic effects (p >
desirability level (D) by applying Equation 5 as follow: 0.05) either for ratios or for time, in the same way as
there were no interactions (p > 0.05) between the two
D 3 d1 d2 d3 (5) factors.
The mathematical models (Figure 2) were adequate
where D is the overall desirability level and di the for almost all responses with the exception of Efficiency,
individual desirability level. which had a low R2Abj (0.338). For the other variables,
the R2Abj were 0.7818, 0.8760, 0.9452, 0.9588, 0.9653
Results and Discussion and 0.9659 for the following: oil recovery, lutein, total
carotenoids, α-carotene, retinol equivalent, β-carotene
The total carotenoid content in dried carrot was in that order. As was mentioned above, time was not
Figure 1 – HPLC chromatograms at 450 nm of dried carrot (A), enriched soy oil (B) and enriched olive oil (C). Peak identification: 1) lutein; 2)
α-carotene; 3) All-trans-β-carotene. Peak areas (UA): dried carrot (lutein 70828; α-carotene 1280403; β-carotene 2477785), enriched soy oil
(lutein 219304; α-carotene 3169265; β-carotene 8653354) and enriched olive oil (lutein 295806; α-carotene 2813841; β-carotene 8257522).
Table 2 – Summary of p values for the ANOVA factors (ratio and time) in carotenoid extraction.
Response variables Ratio (L ) (X1) Ratio (Q) (X12) Time (L) (X2) Time (Q) (X22) Interaction (X1.X2)
Efficiency (%) 0.01792 0.02479 0.187886 0.06106 0.375801
Total Carotenoids 0.000975 0.440916 0.269726 0.168092 0.759194
Lutein 0.000685 0.296159 0.113054 0.069905 0.142351
α-carotene 0.000146 0.518825 0.277433 0.120759 0.14765
β-carotene 0.000885 0.70367 0.194419 0.102397 0.9005
Retinol Equivalent 0.000566 0.654746 0.170564 0.070043 0.75335
Oil Recovery 0.002078 0.026651 0.165325 0.303689 0.323519
L and Q indicate the linear and quadratic effect, respectively; X1 and X2 are Ratio and Time, in this order.
significant (Table 2) for any of the variables (p > 0.05) an opposite trend. Left and center columns in the Figure
but it was retained in the models to improve the 3D 3 represent the ratio and time variables as well as making
model fit. it possible to observe the change in three responses in
Efficiency and oil recovery responses presented which the ratio and time changed.
a negative relationship with the ratio (negative sign in The right column indicates what was desirable
the X1 coefficient). On the other hand the rest of the in each response. Thus, the desirable amount of
responses presented a direct association with the ratio RE was fixed at 60 μg mL–1 (Figure 3C) based on the
(a positive sign in the X1 coefficient); thus the responses Recommended Daily Intake (RDI) for vitamin A which
increased as the ratio increased. is 600 μg RE for an adult (ANVISA, 2005a), that can be
As Efficiency did not have an adequate obtained from the ingestion of 10 mL (a spoonful) of
mathematical model, it was not considered in the provitamin A enriched oil. Thus, the desirability function
optimization with the desirability function (Figure 3); was used to achieve 60 μg of RE mL–1, and to maximize
thus, RE (Figures 3A, 3B and 3C for ratio, time and RE total carotenoids and recovery. On the other hand, it was
desirability, respectively) and total carotenoids (Figures desirable to maximize the recovery and total carotenoids
3D, 3E and 3F for ratio, time and total carotenoid as observed in Figures 3F and 3I respectively.
desirability, respectively) were chosen because they The results of the desirability function are in the last
presented the same behavior as the responses of lutein, row (Figures 3J and 3K for ratio and time respectively),
α-carotene and β-carotene. On the other hand, recovery thus, the optimum conditions were a ratio of 21 % and
(Figures 3G, 3H, and 3I for ratio, time and recovery 15 min to obtain an oil enriched with ~ 60 μg of RE mL–1
desirability respectively) was also chosen as it presented maximizing the total carotenoids and the recovery from
Figure 2 – Surface responses (mathematical models, their p value and the R2adj) of factors, Ratio (X1) and Time (X2) that enrich the oil with
carotenoids from carrots. Mathematical models present coefficients with significant effects (p < 0.05). Although the coefficients for time (X2)
were not significant (p > 0.05), they were maintained to improve the model fit. R2adj ≥ 0.70 were considered predictive (Toledo et al., 2019).
carrots, but when considering the lack of significant 6.9 % and 14.9 % respectively, these results validating
effect of time, it would be advisable to reduce the time the experiment. The same parameters (20 % ratio,
to a minimum (5 min) in order to minimize contact the extraction times of 5 and 10 min) were applied to
with oxygen. Contact with oxygen during blending in determine the amount of provitamin A carotenoids
extraction processes can trigger a deterioration in the from carrots in olive oil, and these parameters obtained
oil due to oxidative reactions and, therefore, reduce the similar RE contents (62.98 and 69.31 μg RE mL–1 for 5
shelf life of the product (McClements and Decker, 2007). and 10 min of extraction, respectively).
To validate the model and considering the results Even time was not significant for modeling RE,
of the desirability function, the parameters 20 % (M/V) and an increment in RE was observed. However, with
ratio and the extraction times of 5 and 10 min were an extraction time of 5 min, it was possible to reach
chosen to determine the amount of provitamin A in the Recommended Daily Intake for vitamin A content
terms of RE in the soy oil (not enriched and enriched). (600 μg retinol equivalent for an adult) using one dose of
The results confirmed an adequate RDI for the soy oil, the product (10 mL or a spoonful). In addition, 10 min
with values of 64.43 μg RE mL–1 for 20/5 (ratio/time) for carotenoid extraction promotes an increase of 100 %
and 70.53 μg RE mL–1 for 20/10 (ratio/time), varying by in the duration of the extraction step, which means
Figure 3 – Simultaneous optimization of process parameters (ratio and time) to achieve the Recommended Daily Intake (RDI) in retinol equivalent
to obtain enriched soy oil using carrots, by applying the desirability function in responses to the surface methodology.
more use of equipment, higher cost, as well as higher kinetics degradation, as well as acidity (Figure 4B) without
exposure of oils to oxidation. adjustment as per any kinetic order, due to the fact that it
According to the Brazilian Legislation for Enriched did not decrease sufficiently to describe a mathematical
or Fortified Foods (ANVISA, 1998), 100 mL or 100 g of model with significant parameters (p < 0.05). Thus,
the product, ready for consumption, must supply at acidity was stable for the 150 days of the experiment
least 15 % of the reference RDI for liquids and 30 % of (Table 3), and the peroxide index and CDT (Figures 4C
the reference case for solids. In this case, the vegetable and 4D) presented a first order kinetics degradation. For
oils with carotenoids obtained by the process presented 10 min extraction time the reaction order kinetics was:
can be considered fortified/enriched and should have 0, no model adjustment, 0 and 1 for total carotenoids, oil
the words “high content” or “rich” on the label. acidity, peroxide index and CDT respectively.
The reaction orders (n) of 0, 1 and 2 (Table 3 Based on the olive oil results, it was not possible
and Figure 4) were tested for the kinetic deterioration to adjust the data of total carotenoids and acidity (Figure
parameters (total carotenoids, acidity, peroxide index 4E and 4F) to any kinetic order. This is probably due to
and D as conjugates of dienes and trienes) of the the fact that the total carotenoids were stable through
enriched oil (soy and olive) with provitamin A from 150 days of storage time. Initially the acidity maintained
carrots. The criteria to choose the kinetic order were that stability (plateau), but after 90 days it presented a small
all the parameters have to be significant (p < 0.05) and increase and another plateau at the end of the experiment
at the same time the model must have the highest R2. (150 days). The peroxide index (Figure 4G) presented a
The second order was not adequate for any deterioration first order kinetics (n = 1) for not enriched, 5 min and
parameters (Table 3). 10 min of the blender in the extraction process; on the
In soy oil, regrading of 5 min extraction time, other hand the CDT (Figure 4H) presented a zero order
and total carotenoids (Figure 4A) followed a zero order kinetics (n = 0) for not enriched and 5 min of extraction
Table 3 – Basic kinetic parameter for zero, first and second order in stability study of vegetable oils enriched with provitamin A.
Deterioration parameters for soy oil
TO5 TO10 AOB AO5 AO10 POB PO5 PO10 DOB DO5 DO10
Zero order (n = 0), the equation is C = Co+kt
Co 174.83 184.36 0.14 0.26 0.25 –4.22 –3.62 2.02 0.38 0.36 0.40
k –0.277 –0.386 0.000 0.000 0.000 0.200 0.215 0.207 0.002 0.002 0.002
R2 0.88 0.96 0.11 0.56 0.49 0.78 0.78 0.90 0.71 0.74 0.89
First order (n = 1), the equation is Log(C) = Log(Co) + kt; or C = Co*Exp(Kt)
Co 175.56 185.62 0.15 0.26 0.251 1.07 1.30 6.71 0.38 0.36 0.413
k –0.002 –0.003 –0.001 –0.002 –0.002 0.023 0.022 0.011 0.004 0.005 0.004
R2 0.88 0.95 0.12 0.56 0.511 0.97 0.98 0.85 0.77 0.82 0.86
Second order (n = 2), the equation is (1/C) = (1/Co) + kt; or C = 1/((1/Co)-kt)
Co 170.06 185.29 0.15 0.26 –0.01 1.951 3.59 0.11 0.45 0.44 0.41
k 118.11 163.02 –0.010 –0.010 0.210 444.78 390.30 0.106 23662.48 20827.14 9621.43
R2 0.00 0.00 0.12 0.56 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Deterioration parameters for olive oil
TAB TA5 TA10 AAB AA5 AA10 PAB PA5 PA10 DAB DA5 DA10
Zero order (n = 0), the equation is C = Co+kt
Co 10.81 157.59 172.30 0.20 0.22 0.21 6.68 6.66 7.52 0.16 0.16 0.16
k 0.016 –0.038 –0.078 0.001 0.001 0.001 0.067 0.055 0.044 0.001 0.001 0.001
R2 0.134 0.18 0.44 0.67 0.73 0.80 0.84 0.90 0.89 0.96 0.96 0.88
First order (n = 1), the equation is Log(C) = Log(Co) + kt; or C = Co*Exp(Kt)
Co 10.90 157.62 172.36 0.20 0.23 0.21 6.98 6.94 7.68 0.16 0.16 0.16
k 0.001 –2e-4 –0.0005 0.0026 0.003 0.004 0.006 0.005 0.004 0.003 0.003 0.003
R2 0.13 0.18 0.44 0.66 0.71 0.80 0.90 0.95 0.93 0.96 0.95 0.90
Second order (n = 2), the equation is (1/C) = (1/Co) + kt; or C = 1/((1/Co)-kt)
Co 0.11 159.75 175.88 0.20 0.24 0.22 0.11 0.05 0.11 0.17 0.17 0.17
k 0.105 171.08 83.43 0.010 39063.07 0.012 0.105 0.317 0.105 0.014 0.015 0.0116
R2 0.00 0.00 0.00 0.65 0.78 0.78 0.00 0.00 0.00 0.96 0.93 0.9
Bold numbers indicate statistical significance (p < 0.05) for parameters. The model with all significant parameters was chosen and the highest determination
coefficient (in gray background) being the best to describe each response. In the heading of the deterioration compound TO and TA indicates total carotenoids for soy
and olive oil, AO and AA means acidity for soy and olive oil, PO and PA represents the peroxide index for soy and olive oil and DO and DA is the conjugated dienes and
trienes for soy and olive oil, the letter O means oil, B indicates time zero, 5 and 10 are min of the extraction time. C is the compound of interest, Co is the intercept
or the initial amount of the compound C, k is the rate constant and n is the order of the reaction. R2 is the determination coefficient.
time, and a first-order kinetics for 10 min of the blender The shelf life for total carotenoids, calculated as the
for the extraction of the carotenoids. half shelf life (t1/2), for soy oil was 323 days (10.8 months)
The stability of the carotenoids in the soybean for not enriched and 237 days (7.9 months) for 5 min of
oil and olive oil was evaluated over 150 days of storage the extraction process. It was not possible to calculate the
(Figures 4A and 4B). The results of the study showed shelf life of soy and olive oils in terms of acidity as they
80 % and 97 % of carotenoid retention in soybean oil did not present a kinetic trend in any order (0, 1 or 2).
and olive oil, respectively. Carotenoids showed greater The time to achieve maximum concentration
stability in olive oil than in soybean oil (Figure 4A and according to government laws (ANVISA, 2005b)
4B respectively). According to Velasco and Dobarganes pertaining to the peroxide index for soy oil (maximum
(2002), virgin olive oil has high resistance to oxidative concentration allowed is 10 meq kg–1) was 98 days for
deterioration due to the low content of polyunsaturated soy oil with no carotenoids, followed by soy oil with
fatty acids and the high amounts of phenolic compounds. carotenoid extracted for 5 min (91 days) and soy oil with
There is a greater amount of phenolic compounds in carotenoid extracted for 10 min (39 days). On the other
virgin olive oil compared to other refined oils, as they hand, olive oil presented greater shelf life as a function
are eliminated or drastically reduced during the refining of the peroxide index (maximum concentration allowed
process. These phenolic compounds in virgin olive oil is 20 meq kg–1). Thus, the treatments of olive oil with
probably improve the stability of the carotenoids. no carotenoids, olive oil with carotenoid extracted after
Figure 4 – Experimental data and models for total carotenoids. Soy oil on the left, Olive oil on the right. Total carotenoids (A, E), acidity (B, F),
peroxide index (C, G) and conjugated dienes and trienes (D, H) by kinetic modeling for oil enriched with provitamin A extracted from carrots.
5 min and olive oil with carotenoids after 10 min of de Orçamentos Familiares (POF, Family Budget Research)
blender reached 169, 197 and 98 days respectively. This 2008-2009 carried out by the Instituto Brasileiro de
greater shelf life of olive oil against soy oil is because the Geografia e Estatística (IBGE). Thus, it can be used for
maximum allowed concentration is two times that of soy carotenoid fortification and to increase provitamin A
oil and the carotenoids presented a protective effect in intake for needy populations. The differential in the
the olive oil. proposed process used in this study is that carotenoids
The use of oil for the extraction of provitamin were extracted from natural sources. Thus, there were
A carotenoid from carrots was shown to be a superior no additions of synthetic substances; furthermore, the
strategy for enriching the oil. Soybean oil is the technology used in the extraction process was simple and
vegetable oil most commonly consumed by the Brazilian low-cost.
population, and olive oil is in second place, according to Vegetable oils composed of unsaturated fatty
the most recent survey on consumption called Pesquisa acids are highly recommended for their health benefits.
These oils are at the base of the food pyramid and are dispersion into liquids). In this step, the proportion of
recommended as the primary sources of fat. Foods high the oil/carrot powder as well as shear rate and time
in good fats include vegetable oils (such as olive, canola, must be optimized; 2) the second step would consist
sunflower, soy, and corn), nuts, seeds, and fish (Willett of the mixing of the product previously obtained with
and Skerrett, 2017). an increased quantity of oil (hide mixture tank). In this
Red palm oil is a source of provitamin A stage the proportion, time, temperature, and rotation
carotenoids. However, it is also rich in saturated fat, must be optimized, and 3) the purification of the
which could contribute to an increase in cholesterol product could be done by filtration by, for example, a
and the development of atherosclerotic complications plate filter followed by a filter press.
(Willett and Skerrett, 2017). In addition to its health-
jeopardizing aspects, it has a strong flavor, which could Conclusion
prompt low acceptance by consumers.
The enriched oil can be used directly for The process developed to extract carotenoids
consumption or as a replacement for the oil used in showed that it was possible to enrich edible vegetable
the preparation of food. The enrichment of foods with oils with carotenoids from natural sources, using only
provitamin A aims to prevent and / or mitigate the VAD the vegetable oil as the vehicle of extraction. Small
problem and increase the antioxidant potential by the quantities of enriched oils, 10 mL for soybean oil or
action of β-carotene. According to the International olive oil, can supply the Recommended Daily Intake for
Vitamin A Consultative Group (IVACG, 2017), vitamin A for an adult.
carotenoids immersed in oil have a higher conversion Lastly, some final considerations: the proposed
efficiency to equivalent retinol (6 times higher) when process consists of simple and low-cost technology that
compared to carotenoids in vegetable matrices (eg: 1 can be used to enrich edible oils (soybean and olive oil)
µg RE = 2 µg β-carotene in oil or 12 µg β-carotene in with provitamin A. The ingestion of small quantities
mixed foods). of the enriched oils can contribute to preventing and/
The choice of dietary lipid and its fatty acid or mitigating vitamin A deficiency, which is considered
profile is vital to the enhancement of the bioavailability a public health issue in a number of countries and by
of carotenoids. Lakshminarayana and Baskaran (2013) certain populations worldwide.
showed that dietary unsaturated fat appears to be a
suitable carrier for carotenoids when oxidative stress Authors’ Contributions
is a critical issue in nutrition–related degenerative
disorders. Specific fatty acids or vegetable lipids play Conceptualization: Borguini, R.G.; Godoy,
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are also involved in the regulation of the oxidative Nascimento, L.S.M.; Jesus, M.S.C.; Couto, G.A. Data
mechanism. analysis: Chávez, D.W.H. Design of methodology:
The present study is in accordance with the Borguini, R.G.; Pacheco, S.; Chávez, D.W.H. Writing
concept of food–to–food fortification (Chadare et al., and editing: Santiago, M.C.P.A.; Borguini, R.G.;
2019), that often uses foods that are available in the Pacheco, S.; Chávez, D.W.H.
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