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Microwave-assisted extraction of carotenoids from carrot juice processing waste using flaxseed oil as a solvent
Journal Pre-proof Microwave-assisted extraction of carotenoids from carrot juice processing waste using flaxseed oil as a solvent Aysel Elik, Derya Koçak Yanik, Fahrettin Göğüş PII:
S0023-6438(20)30088-8
DOI:
https://doi.org/10.1016/j.lwt.2020.109100
Reference:
YFSTL 109100
To appear in:
LWT - Food Science and Technology
Received Date: 22 August 2019 Revised Date:
15 December 2019
Accepted Date: 25 January 2020
Please cite this article as: Elik, A., Yanik, Derya.Koç., Göğüş, F., Microwave-assisted extraction of carotenoids from carrot juice processing waste using flaxseed oil as a solvent, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2020.109100. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
CRediT author statement Aysel Elik: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization. Derya Koçak Yanık: Conceptualization, Methodology, Software, Data Curation, Writing Review & Editing, Visualization, Supervision, Project administration. Fahrettin Göğüş: Conceptualization, Methodology, Resources, Data Curation, Writing Review & Editing, Visualization, Supervision, Project administration, Funding acquisition.
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Microwave-assisted extraction of carotenoids from carrot juice processing waste using flaxseed
2
oil as a solvent
3
Aysel ELİK1*, Derya KOÇAK YANIK1, Fahrettin GÖĞÜŞ1
4
1
Gaziantep University, Engineering Faculty, Food Engineering Department, Gaziantep, Turkey
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*Address correspondence to: Gaziantep University, Engineering Faculty, Food Engineering
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Department, 27310 Gaziantep, Turkey. Tel: +90 (342) 317 23 36; Email: [email protected]
7
Abstract
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The present study was aimed to evaluate the effect of microwave technology on the extraction yield of
9
carotenoid from carrot juice processing waste using flaxseed oil as solvent. Response surface
10
methodology was employed to optimize the effect of microwave power, extraction time and oil to
11
waste ratio on percentage recovery of carotenoid. Optimum conditions obtained for microwave-assisted
12
carotenoid extraction were 165 W of microwave power, 9.39 min of extraction time and 8.06:1 g/g of
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oil to waste ratio which gave carotenoid recovery of 77.48 %. Selected physical and chemical
14
properties of flaxseed oil and carotenoid enriched flaxseed oil were investigated in terms of fatty acid
15
composition, acid value, peroxide value, p-anisidine value, colour parameters (L*, a* and b* values), β-
16
carotene content, total phenolic content and antioxidant activity. The results showed that the enriched
17
flaxseed oil was in good quality and had high phenolic content (214.05 ± 1.61µg GAE/ g oil) and
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antioxidant activity (inhibition % of DPPH = 70.67 ± 0.85).
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Key words: Waste utilization; Carrot juice; Flaxseed oil; Microwave; Carotenoids
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1. Introduction
1
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Carrot (Daucus carota L) is one of the most important root fruits grown worldwide and is an important
22
carotenoid source (Sharma, Karki, Thakur, & Attri, 2012). Besides the potential benefits to human
23
health due to biological activities such as antioxidant, anticarcinogenic activity (Kiss, Forgacs,
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Cserhati, Mota, Morais, & Ramos, 2000), carotenoids extracted from wastes has gained popularity as a
25
beneficial alternative to the synthetic carotenoids (Parjikolaei, El-Houri, Fretté, & Christensen, 2015).
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In the production of carrot juice, a pulp is wasted corresponding to 30-40% of the raw material. The
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process waste of carrot is rich in bioactive substances such as carotenoids, especially in β-carotene
28
(Oreopoulou & Tzia, 2007). Depending on the processing conditions, the total carotene content of this
29
waste can reach up to 2 g per kg dry matter (Sharma, Oberoi, & Dhillon, 2016).
30
Recovery of carotenoids in wastes is generally carried out using organic solvents such as hexane and
31
acetone which are environmentally harmful solvents. Carotenoids are oil-soluble pigments (Sachindra
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& Mahendrakar, 2005) and the use of oil as solvent can be a promising alternative method to organic
33
solvents. The use of oil as alternative solvent has the advantage of reducing the use of environmentally
34
harmful solvents. Furthermore, barrier properties of the oil against oxygen lead to retardation of the
35
oxidation time and degradation rate for the extract containing carotenoids (Parjikolaei, El-Houri, Fretté,
36
& Christensen, 2015). In the present study, flaxseed oil was selected as the solvent for the carotenoid
37
extraction as it is rich in polyunsaturated fatty acids (about 73% of total fatty acids), especially in
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omega-3 (Goyal, Sharma, Upadhyay, Gill, & Sihag, 2014). In this way, enriched oil to be obtained will
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have more beneficial effects in health due to the protection effect of omega-3 oils against
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cardiovascular diseases (Calder, & Yaqoob, 2009) and antioxidant effect of β-carotene (Tribble, 1999).
41
Moreover, direct use of oil for the extraction eliminates the extra cost of evaporation.
42
There are some studies in the literature, dealing with the carotenoid extraction using oil as a solvent.
43
However, most of these studies are based on the conventional solvent extraction process with a long 2
44
extraction time (1 to 48 hours) (Benakmoum, Abbeddou, Ammouche, Kefalas, & Gerasopoulos, 2008;
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Handayani, Indraswati, & Ismadji, 2008; Kang & Sim, 2008; Kessy, Zhang, & Zhang, 2011;
46
Parjikolaei, El-Houri, Fretté, & Christensen, 2015; Sachindra & Mahendrakar, 2005). In recent years,
47
ultrasound technology and vegetable oils have been used in a few studies in order to extract
48
carotenoids. They have been reported to provide better extraction yield (up to about 94 %) and shorter
49
time (max. 30 min) when compared to conventional processes (Li, Fabiano-Tixier, Tomao, Cravotto, &
50
Chemat, 2013; Goula, Ververi, Adamopoulou, & Kaderides, 2017).
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Microwave extraction can be regarded as one of the most appropriate method for β-carotene extraction
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from carrots (Kyriakopoulou, Papadaki, & Krokida, 2015) due to less time consumption and higher
53
extraction efficiency. When the carotenoid extraction studies used vegetable oils as a solvent are
54
examined, it is considered that the main drawback is long extraction time. The microwave causes rapid
55
disruption of the cell walls and shortens the duration of the extraction and increases the yield
56
(Milutinović et al., 2015). However, there is no exhaustive study available on carotenoid extraction
57
from carrot juice processing waste (CPW) using oil as a solvent under the microwave conditions. In
58
that respect, the main purpose of this study was to evaluate the effect of microwave technology on
59
extraction yield of carotenoids from CPW by using flaxseed oil as a solvent and to optimize the
60
extraction conditions (microwave power, extraction time and oil to waste ratio) to achieve optimum
61
recovery of carotenoids. Moreover, comparative studies between microwave-assisted carotenoids
62
extraction (MACE) and conventional carotenoid extraction (CCE) using flaxseed oil as a solvent was
63
carried out in terms of recovery of carotenoids and extraction time. Finally, fatty acid composition,
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colour parameters (L*, a*, b*), acid value, peroxide value, p-anisidine value, β-carotene content, total
65
phenolic content and antioxidant activity (inhibition % of DPPH) of carotenoid-enriched flaxseed oil
66
(CEFO) obtained under optimum conditions were compared to those of flaxseed oil (FO).
3
67
2. Materials and methods
68
2.1. Chemicals and reagents
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All chemicals and reagents used in this study were of analytical or HPLC grade. Standard of β-carotene
70
Type II (≥95% purity by HPLC assay), folin–ciocalteu’s phenol reagent, sodium carbonate (Na2CO3),
71
sodium chloride (NaCl), ascorbic acid, 1,1-diphenyl-2- picrylhydrazyl (DPPH), potassium hydroxide
72
(KOH) were purchased from Sigma-Aldrich. Gallic acid was provided by Merck.
73
2.2. Material
74
CPW used as a source of carotenoid in the experiments was supplied by a producer of carrot juice. The
75
waste was frozen at -40°C prior to freeze drying and then moisture content was reduced to below 1%
76
by using freeze dryer (CHRIST, model Alpha 1-4 LDplus, Martin Christ, Germany). The dried waste
77
was ground in a waring blender. The resulting powder (smaller than 0.85 mm) was stored at -20°C in
78
opaque containers until carotenoid extraction.
79
Flaxseed was supplied by the local market and pressed by using cold press oil machine (Karaerler, NF
80
100, Ankara, Turkey) to obtain the cold-pressed flaxseed oil in the laboratory.
81
2.3. Microwave assisted carotenoid extraction (MACE) using flaxseed oil
82
The certain quantity of the waste powder (the quantity varied as a function of the oil to waste ratio) was
83
weighed into the flask and mixed with flaxseed oil as solvent. Total amount (oil+ waste) was kept at 20
84
g. Thirty five ml extraction vessel (88 mm height and 25 mm diameter borosilicate glass with a silicone
85
cap) was placed in the microwave extraction device and the extraction process was operated after the
86
microwave power and extraction time were set up according to the experimental design (Table 1).
87
Carotenoid extraction from the waste powder was carried out in a closed microwave system (CEM 4
88
Corporation, model Discover-SP, Matthews, NC, USA). The microwave chamber has been cooled by
89
air circulated in ice bath throughout the microwave application. The temperature did not exceed 110 oC
90
for even the harshest experimental runs (highest microwave power and longest time). Carotenoid-
91
enriched flaxseed oil was stored in dark glass bottles at -20°C until the analyses were performed.
92
2.4. Conventional carotenoid extraction (CCE) using flaxseed oil
93
Approximately, 0.95 g of the waste powder was mixed with 19.05 ml of oil (Oil to waste powder
94
ratio=20:1). Total amount (oil+ waste powder) was kept at 20 g as in MACE. The mixture was heated
95
at 65oC in a mixer incubator (Heidolph, Unimax 1010, Schwabach, Germany) for 270 min. The
96
samples were taken out at 12, 30, 60, 90, 120, 180, 240, 270 min and carotenoid contents of the
97
samples were determined.
98
2.5. Soxhlet extraction
99
Carotenoids in CPW were extracted by the soxhlet extraction method. Procedure with some
100
modifications described by Hiranvarachat, Devahastin, Chiewchan, and Raghavan (2013) was used for
101
the extraction of carotenoids. The waste powder (2 g) was placed in an extraction thimble, which was
102
then placed in an extraction chamber and extracted 6 h by using 150 mL of n-hexane, ethanol, acetone
103
(2:1:1 v/v/v) solvent mixture. Solvent mixture was evaporated under vacuum at 40oC using the rotary
104
vacuum evaporator (Heidolph, type basis Hei-VAP HL, Schwabach, Germany) in order to obtain
105
carotenoid extract. Extract was dissolved in n-hexane prior to analyses of total carotenoid and β-
106
carotene contents. Soxhlet extraction method was used to determine the total amount of carotenoid and
107
β-carotene content in CPW.
108
2.6. Total carotenoid content
5
109
The diluted extract obtained from soxhlet extraction was used to estimate the total carotenoids content
110
of CPW. Total carotenoid content of CPW was determined at 450 nm (absorbance maxima for β-
111
carotene) using a spectrophotometer (Optima, model SP-3000nano, Tokyo, Japan). The total carotenoid
112
content of CPW was expressed as µg β-carotene per g of CPW powder. The total carotenoid contents
113
of FO and CEFO were also determined spectrophotometrically, reading absorbance at 450 nm against
114
flaxseed oil as blank (Sachindra & Mahendrakar, 2005).
115
Percent recovery of carotenoid from CPW was calculated using Eq. (1) below.
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Recovery (%) = (CE / CW) x 100
117
where CE is amount of carotenoid extracted and CW is total carotenoid content of CPW.
118
2.7. Experimental design
119
Response surface methodology (RSM) was employed to determine the optimal conditions for MACE
120
from CPW using flaxseed oil as a solvent. RSM was performed using Design Expert, version 7 (Stat-
121
Ease, Design-Expert software, Minneapolis, USA). Microwave power (X1), extraction time (X2) and oil
122
to waste ratio (X3) were independent variables of the system and % recovery of carotenoid (Y1) was the
123
response of the system. The extraction parameters were optimized through a central composite
124
rotatable design (CCRD). The experimental design consists of 20 test points with 6 central points, 6
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star points (Table 1).
126
Significances of the model and all terms were determined by evaluating the Fisher test value at a
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probability of 0.05. CCRD uses least-squares regression to fit the experimental data to a second-order
128
polynomial model. The second-order polynomial equation expressed in Eq. (2) was used to calculate
129
predicted response.
(1)
6
=
130
+∑
İ
+ ∑
İ
+∑
∑
(2)
131
where Y are the dependent variable, β0, βi, βii and βij are the regression coefficients for the intercept,
132
linear, quadratic and interaction terms of variables i and j, respectively, and Xi and Xj are the
133
independent variables.
134
2.8. Quantification of β-carotene by HPLC
135
Rapid saponification was used to extract carotenoids from oil samples prior to the analysis of β-
136
carotene. The procedure for extraction with saponification was adapted from the method by Gimeno et
137
al. (2000).
138
The quantity of β-carotene using HPLC (Shimadzu, Prominence/LC-20AB, Kyoto, Japan) was
139
determined by the method proposed by Barba, Hurtado, Mata, Ruiz, and De Tejada (2006). Mobile
140
phase, mixture of methanol/ acetonitrile (90:10 v/v) + 9µM triethylamine, was eluted isocratically at a
141
flow rate of 0.9 ml/min. Supelcosil LC-18 5 µm (4.6 x 250 mm) HPLC column (Supelco, Bellefonte,
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PA, USA) was used for β-carotene analysis. Detection of β-carotene was made at a wavelength of 450
143
nm using UV–vis detector (Shimadzu, SPD-20A, Kyoto, Japan). Quantification of β-carotene was
144
performed through a calibration curve.
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2.9. Fatty acid composition
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The fatty acid compositions of FO and CEFO were identified according to procedure proposed by
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Yanık (2017), using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA,
148
USA).
149
2.10. Peroxide, acid and p-anisidine values
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150
The peroxide and acid values of oil samples were determined according to the method of AOCS Cd 8-
151
53 (AOCS, 1998) and AOCS Cd 3d-63 (AOCS, 1998), respectively. P-anisidine values of FO and
152
CEFO were determined according to AOCS Cd 18-90 (AOCS, 1998).
153
2.11. Total phenolic content (TPC)
154
Phenolics of FO and CEFO were extracted following the method described by Kıralan, Bayrak, and
155
Özkaya (2009).
156
TPC of FO and CEFO were determined with the Folin-ciocalteu colorimetric method presented by
157
Singleton, Orthofer, and Lamuela-Raventós (1999) with some modification. TPC values were
158
expressed as µg gallic acid equivalents (GAE)/ g oil.
159
2.12. DPPH-scavenging activity assay
160
Antioxidant activities of FO and CEFO were determined according to the procedure with some
161
modification reported by Kıralan, Bayrak, and Özkaya (2009). Inhibition (%) was calculated using the
162
following equation:
163
% Inhibition = (1- [Asample/Ablank]) x100
164
Where Asample is the absorbance of sample with DPPH solution, Ablank is the absorbance of DPPH
165
solution without sample solution at 517 nm.
166
2.13. Colour measurement
167
The colours of FO and CEFO were measured using a Hunter-Lab ColorFlex (HunterLab, model A-60-
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1010-615, Reston, VA). The colour value was expressed as L* (lightness; from 0 = black to 100 =
(3)
8
169
white), a* (negative values indicate greenness while positive values indicate redness) and b* (negative
170
values indicate blueness and positive values indicate yellowness).
171
2.14. Statistical analysis
172
The statistical comparisons of FO and CEFO in terms of some basic quality parameters have been done
173
using independent t-test. The data were statistically analyzed using the SPSS statistical software,
174
version 22.0 (SPSS Inc., Chicago, IL, USA). All analysis were performed three times and averaged.
175
Results expressed as means ± standard deviation (SD). Significant differences (p<0.05) between means
176
were determined.
177
3. Results and Discussion
178
3.1. RSM modeling
179
The most important factors influenced the recovery of bioactive compounds like carotenoids from
180
plants are microwave power, extraction time and as well as solvent to sample ratio when microwave-
181
assisted extraction (MAE) is concerned (Hiranvarachat, Devahastin, Chiewchan, & Raghavan, 2013;
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Mandal, Mohan, & Hemalatha, 2007). Therefore, microwave power, extraction time and oil to waste
183
ratio was studied as independent variables on extraction of carotenoid from carrot processing waste.
184
Percentage recovery of carotenoids was considered as the dependent variable. Three factors- five levels
185
CCRD was studied in a total of 20 experiments. Table 1 shows both the actual values of design
186
parameters and the experimental results obtained under the defined points.
187
The adequacy and fitness of the model and the relationship between variables were examined using
188
regression analysis and analysis of variance (ANOVA), respectively, and the results are given in Table
189
2. In this study, the model with high F-value (34.04) and a very small p-value (<0.0001) was highly 9
190
significant and the lack of fit test was found to be non-significant (p>0.05). Thus, the model builded is
191
sufficiently accurate for predicting the outcomes. The R2 value of 0.9521 implies that there was a good
192
correlation between experimental and predicted values of the response variable.
193
3.2. The effect of process variable on recovery of carotenoids
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According to the results of experiments carried out at the points designated by CCRD, the percentage
195
recovery of carotenoid (Y1) ranged from 45.81 to 77.68 % (Table 1). Highest Y1 value (77.68 %) was
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obtained in experimental run 4 under the following conditions: microwave power of 170 W of, an
197
extraction time of 9.46 min and oil to waste ratio of 8:1 g/g. While the microwave power (X1) and
198
extraction time (X2) have been found as highly significantly (p<0.001) effected on extraction of
199
carotenoids from carrot waste by flaxseed oil, the oil to waste ratio (X3) were found as non-significant
200
(p>0.05) (Table 2). However, the quadratic effect of X3, the interactive effect of X1*X3and X2*X3 were
201
found as statistically significant. The interactive effect of X1*X2 has also been found as non-significant.
202
Predicted model equation obtained from the experimental data was formulated by removing the non-
203
significant variables. However, some non-significant terms were not removed from the model in order
204
to support the model hierarchy. Second-order polynomial equation in terms of codded factors obtained
205
from CCRD model for recovery of carotenoid (%) was given as follows.
206
Recovery (%) = 62.49 + 5.77* X1 + 7.17* X2 – 1.44* X3- 1.80*X1*X2 + 1.97*X1*X3
207
-2.17*X2*X3 + 3.31*X32
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Based on the regression coefficient values (β), the highest influence on the recovery of carotenoids was
209
extraction time, followed by microwave power. However, it was observed that no significant effect of
210
oil to waste ratio was found on recovery of carotenoid. Extraction time has positive effect on the
211
response in linear term. It means that the increase in carotenoid content extracted corresponded to the
(4)
10
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rise in extraction time. This result was in good agreement with previous study about β-carotene
213
extraction from carrot using sunflower oil as a solvent under ultrasound treatment (Li, Fabiano-Tixier,
214
Tomao, Cravotto, & Chemat, 2013). It was found that β-carotene extraction yield could be increased by
215
increasing the time. In another study carried out by Goula, Ververi, Adamopoulou, & Kaderides
216
(2017), it was reported that the extraction yield was time dependent and increased with extended
217
ultrasonic times, especially from 10 to 30 min. Microwave power also showed a significant (p<0.0001)
218
positive linear effect on carotenoid extraction (Table 2). From Figure 1, it is understood that microwave
219
power had a strong sway on MACE. Microwave energy absorbed by materials depends on their
220
dielectric properties (Meda, Orsat, & Raghavan, 2017). Water, which has a large electric dipole
221
moment, is very efficient at absorbing the microwave energy. On the other hand, oils are esters of
222
long-chain fatty acids which have much less mobility compared to water molecules in response to
223
oscillating microwave irradiation. Therefore, they have rather small dipole moments and don't absorb
224
the radiation as much as water. However, the specific heat of oils is sufficiently smaller than the
225
specific heat of the water, and then oils tend to heating up much faster than water when exposed to the
226
same amount of microwaves (Hebbar, & Rastogi, 2012). The temperature rises of the mixture due to
227
the increase of microwave power reduce the viscosity of the solvent (flaxseed oil) and increase the
228
solubility of carotenoids (Hiranvarachat, Devahastin, Chiewchan, & Raghavan, 2013). This results in
229
an enhanced diffusion rate, thereby prominent accelerating extraction rate. Even though no significant
230
effect of oil to waste ratio was observed under microwave extraction using oil as solvent, the
231
interaction between extraction time and oil to waste ratio showed a synergistic effect on amount of the
232
carotenoid extracted (p<0.05). As shown in Figure 1B, extraction yield of the carotenoid increased with
233
longer extraction time with low ratio.
234
3.3. Experimental validation of optimized conditions
11
235
Microwave extraction system was optimized employing RSM. The optimum conditions were
236
determined by a quadratic model as 165 W of microwave power, 9.39 min of extraction time, 8.06:1
237
g/g of oil to waste ratio to obtain optimum value of recovery (77.87%). The carotenoid extractions
238
(three times) were performed at optimum conditions to validate the suggested model. The experimental
239
results gave 77.48 ± 0.16 % recovery of carotenoids which was close to the predicted value. The
240
experimental data confirmed the validity and the accuracy of predictive model.
241
3.4. Comparison of MACE and CCE
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The percent recovery of carotenoids was 77.48 % at optimum conditions (165W, 9.39 min and 8.06:1
243
g/g oil to waste ratio) using MACE. As it has been mentioned in introduction part, various conventional
244
extraction conditions were applied to extract carotenoids into oil systems. It was important to compare
245
the conventional extraction results with our finding to see how the recovery has been changed with
246
time and to what extent. As seen in the Figure 2, the extraction yield for conventional extraction
247
method increased with time in the first 30 min and almost 50 % of the carotenoids were extracted in
248
this time period. The extraction yield of carotenoids (about 87 %) reached to equilibrium at around 180
249
min and further increase in time did not affect the yield of carotenoid extraction up to 275 min. Percent
250
recovery of carotenoid under microwave was calculated for extraction times at 1, 3, 5, 7 and 9.39 min
251
on the basis of Eq. (4) while microwave power and oil to waste ratio were kept constant at optimal
252
values. While about 78 % of total carotenoids were extracted within the first 9.39 min of microwave
253
treatment, it was needed more than 120 min to achieve the same value for conventional treatment.
254
Although conventional extraction method had a higher recovery (about 87 %) of total carotenoids from
255
CPW, time required for extraction was quite long (around 180 min) resulting in consumption of
256
considerable energy.
257
3.5. Selected physical and chemical properties of FO and CEFO 12
258
Quality parameters of CEFO obtained under the conditions of optimum microwave treatment were
259
investigated and it was compared to those of FO. Physical and chemical quality parameters investigated
260
for both oils were reported in Tables 3 and 4. It is shown that the fatty acids of FO were mainly
261
constituted by linolenic (52.46 ± 0.02 %), oleic (19.77 ± 0.01 %), linoleic (15.35 ± 0.01 %), palmitic
262
(6.25 ± 0.03 %), and stearic (4.41 ± 0.01 %) acids (Table 3). Although percentages of some fatty acids
263
of CEFO were significantly (p<0.05) different from those of FO, fatty acid values found for CEFO
264
were still presenting a good quality flaxseed oil. The similar findings have been reported in the
265
previous study conducted by Yanık (2017).
266
The CIELAB L*, a* and b* parameters of FO and CEFO were compared. CEFO showed a higher a*
267
and b* values as well as lower L* value (Table 4). This means that the CEFO was darker and more red
268
and yellow in colour than FO. This is a clear indication of successful extraction of β-carotene into the
269
flaxseed oil during microwave treatment because β-carotene is known as the predominant
270
phytochemical component in orange carrot and it is a deep orange-yellow in colour (Poudyal, Panchal,
271
& Brown, 2010; Sun, Gantt, & Cunningham, 1996).
272
Acid, peroxide and p-anisidine values are important indicators of oil quality affected during processing
273
and storage stages of oils. Peroxide and p-anisidine are the primary and secondary products of oil
274
oxidation reactions, respectively; while acid value is a good indicator for hydrolytic reactions (Li, Cai,
275
Sun, & Liu, 2016). Acid values of FO and CEFO were found as 0.76 ± 0.08 and 1.16 ± 0.14 mg KOH/g
276
oil, respectively (Table 4). Significant (p<0.05) increase in acid value was observed after the
277
microwave treatment. This increase is probably due to the splitting of ester linkage of triglyceride
278
molecules during microwave treatment (Hassanein, El-Shami, & El-Mallah, 2003). In a study
279
conducted by Megahed (2011), acid value of flaxseed oil exposed to microwave irradiation showed a
280
similar trend. According to the Codex standard (Codex Alimentarius, 2017), acid value of cold-pressed 13
281
oil has to be less than 4.0 mg KOH/g oil. The results were lower than the limit established by Codex
282
Alimentarius.
283
The peroxide values of FO and CEFO were determined as 0.79 ± 0.03 and 3.95 ± 0.09 milliequivalents
284
(meq) peroxide/kg oil, respectively. Kreps, Vrbiková, Schmidt, Sekretár, & Híreš (2014) reported that
285
the peroxide values of sunflower and corn oils grew significantly after a 10 min of microwave
286
treatment. Even if the peroxide value showed a significant increase (p<0.05) during microwave
287
treatment, the peroxide values of FO and CEFO were still well within the limit of up to 15 meq
288
peroxide/ kg of oil (Codex Alimentarius, 2017).
289
P-anisidine values found for FO and CEFO were 0.018 ± 0.00 and 0.274 ± 0.00, respectively. A
290
significant increase in p-anisidine value of CEFO was observed after microwave treatment. Chiavaro,
291
Rodriguez-Estrada, Vittadini, & Pellegrini, (2010) reported that microwave treatment of canola oil,
292
high oleic sunflower oil and peanut oil for 15 min led significant increase of p-anisidine value over
293
fresh oils. However, p-anisidine value should be lower than 2 for good quality oil (Choo, Birch, &
294
Dufour, 2007). The microwave-treated oil obtained in the present study is still below this value and it
295
can be considered as a good quality oil after MAE.
296
Among the carotenoids, the β-carotene is the most abundant in the carrot processing waste (Sharma,
297
Karki, Thakur, & Attri, 2012). The percent β-carotene of the total carotenoid content was determined
298
for enriched flaxseed oil produced and CPW. Total carotenoid and β-carotene contents of CPW were
299
found as 4132.83 ± 1.05 and 2507.97 ± 0.49 µg/g CPW powder, respectively. β-carotene constituted
300
60.7 % of the total carotenoid content in CPW. Total carotenoid and β-carotene contents of CEFO
301
obtained under optimum conditions were 397.10 ± 0.8 and 236.35 ± 4.5 µg/g oil, respectively. It was
302
determined that β-carotene accounted for 59.5% of carotenoids in CEFO. It is comprehended from the
14
303
HPLC results that a large portion of carotenoid extracted from CPW was β-carotene (about 60%). β-
304
carotene content of FO after MACE was increased significantly (p<0.001).
305
The results (Table 4) showed that TPC and antioxidant activity of flaxseed oil increased after
306
carotenoid extraction under microwave. TPC of CEFO (214.05 ± 1.61 µg GAE/g oil) was about twelve
307
times higher than that of FO (17.10 ± 0.99 µg GAE/g oil). Furthermore, it was found that antioxidative
308
properties of FO after carotenoid extraction were also improved. While DPPH scavenging activity of
309
FO was 54.23 ± 1.37 %, this value reaches up 70.67 ± 0.85 % for CEFO. Presence of carotenoids such
310
as β-carotene which were reported to have stronger antioxidant capacity (Hiranvarachat & Devahastin,
311
2014) in FO after the microwave extraction may account for the better results obtained for its
312
antioxidant activity.
313
4. Conclusion
314
In the present study, the simultaneous optimization of MACE was established for extracting
315
carotenoids from CPW using flaxseed oil as a solvent. It was observed that microwave power and
316
extraction time are significantly effective in extracting carotenoids from the carrot waste to the flaxseed
317
oil. A 77.48 % recovery of carotenoid was achieved successfully at optimum conditions (165 W of
318
microwave power, 9.39 min of extraction time and 8.06:1 g/g of oil to waste ratio); hence the
319
carotenoid extraction by using oil under microwave irradiation is a promising and efficient process for
320
both waste uses and enrichment of oil. In this way, the enrichment of flaxseed oil with phenolics,
321
antioxidants and carotenoids has been developed by an alternative method based on innovative, fast
322
technology with the evaluation of CPW in food industry.
323
It was taken into account that peroxide, acid and p-anisidine values of enriched flaxseed oil were higher
324
than flaxseed oil when MACE applied, however, total phenolic and carotenoid contents, as well as 15
325
antioxidant activity (DPPH) of the enriched oil were simultaneously higher. It could be recommended a
326
study about the effect of different storage temperatures on quality parameters of untreated oil and
327
enriched oil produced under similar microwave technology for a future work. Additionally, this
328
carotenoid enriched oil could be directly used as carotenoids source in different products. In this
329
respect, it could be suggested to study the cost estimation for the production of carotenoid enriched oils
330
with the microwave technology as a future work.
331
Acknowledgements
332
Financial support for this study is provided by Scientific and Technological Research Council of
333
Turkey (TUBITAK) 1001 Project No: 217O125.
334
Declaration of conflicting interests
335
All authors declare that there is no conflict of interest.
336
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wet
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pomace.
LWT,
77,
45-51.
456
457
458
459
460
461
462
463
464
465
466
22
467
Figure Captions
468
Figure 1. 3D plots (A) extraction time (min) versus microwave power (W) (B) extraction time (min)
469
versus oil to waste ratio (g/g) (C) oil to waste ratio (g/g) versus microwave power (W)
470
Figure 2. The effect of extraction time on recovery (%) of carotenoids
471
472
473
474
475
476
477
478
479
480
481
482
483 23
484
Table Captions
485
Table 1
486
Experimental design for microwave extraction.
487
Table 2
488
Regression analysis and analysis of variance.
489
Table 3
490
Fatty acid composition (% fatty acid), %unsaturated fatty acid, and ratio of omega 3: omega 6 fatty
491
acids.
492
Table 4
493
Selected physical and chemical properties of FO and CEFO.
24
Table 1 Experimental design for microwave extraction. Run
Microwave power (W)
Extraction time (min)
Oil to waste ratio (g/g)
Recovery %
1
80.00
3.14
8:1
53.87 ±0.23
2
170.00
3.14
8:1
63.09±0.36
3
80.00
9.46
8:1
74.98±1.20
4
170.00
9.46
8:1
77.91±0.33
5
80.00
3.14
17:1
51.94±0.07
6
170.00
3.14
17:1
69.97±0.71
7
80.00
9.46
17:1
65.30±0.41
8
170.00
9.46
17:1
75.22±0.11
9
50.00
6.30
12.5:1
50.06±0.21
10
200.00
6.30
12.5:1
73.07±1.17
11
125.00
1.00
12.5:1
45.95±0.19
12
125.00
12.00
12.5:1
71.74±0.30
13
125.00
6.30
5:1
74.46±0.66
14
125.00
6.30
20:1
67.15±0.37
15
125.00
6.30
12.5:1
63.88±0.85
16
125.00
6.30
12.5:1
61.69±0.43
17
125.00
6.30
12.5:1
63.95±0.95
18
125.00
6.30
12.5:1
64.41±0.27
19
125.00
6.30
12.5:1
61.02±0.90
20
125.00
6.30
12.5:1
65.35±0.46
All the given values are means of two (n = 2) determinations ± SD
1
Table 2 Regression analysis and analysis of variance. Source
Model Linear Microwave Power (X1) Extraction Time (X2) Oil to waste ratio (X3) Interaction X1*X2 X1*X3 X2*X3 Quadratic X32 Residual Lack of Fit Pure Error Cor Total
SSa
DFb
Recovery (%) MSc F -Value
1440.35
7
205.76
34.04
p-value Prob > F < 0.0001d
454.61 702.14 28.48
1 1 1
454.61 702.14 28.48
75.21 116.16 4.71
< 0.0001d < 0.0001d 0.0507e
25.94 31.16 37.51
1 1 1
25.94 31.16 37.51
4.29 5.16 6.21
0.0605e 0.0424d 0.0284d
160.51 72.54 58.60 13.93 1512.89
1 12 7 5 19
160.51 6.04 8.37 2.79
26.55
0.0002d
3.00
0.1220e
R2 0.9521 0.9241 Adj R2 Pred R2 0.8516 a b Sum of Squares; Degree of freedom; cMean square d Significant at Prob > F less than 0.05 level; e Not significant at Prob > F higher than 0.05 level
2
Table 3
Fatty acid composition (% fatty acid), %unsaturated fatty acid, and ratio of omega 3: omega 6 fatty acids. Fatty acid
FO1
CEFO2
Palmitic acid (C16:0) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) Linolenic acid (C18:3) Eicosadienoic acid (C20:0) Other
6.25 ± 0.03a 0.08 ± 0.02a 4.41 ± 0.01a 19.77 ± 0.01b 15.35 ± 0.01b 52.46 ± 0.02a 0.22 ± 0.01a 1.46 ± 0.02a
6.17 ± 0.05a 0.08 ± 0.00a 4.55 ± 0.01b 19.73 ± 0.01a 15.33 ± 0.01a 52.47 ± 0.03a 0.21 ± 0.01a 1.46 ± 0.04a
% Unsaturated fatty acids
87.67 ± 0.01a
87.61 ± 0.03a
Omega 3: omega 6 fatty acids
3.42 ± 0.00a
3.42 ± 0.01a
All the given values are means of three (n = 3) determinations ± SD ab Means within a row with different letters are significantly different (p < 0.05) 1 Flaxseed oil 2 Carotenoid-enriched flaxseed oil
3
Table 4 Selected physical and chemical properties of FO and CEFO. Physical-chemical property
FO1
CEFO2
L*
72.77 ± 0.08b
58.69 ± 0.09a
a*
-2.53 ± 0.09a
28.35 ± 0.1b
b*
77.01± 0.18a
97.65 ± 0.21b
Acid value (mg KOH/g oil) Peroxide value (meq/kg oil) p-anisidine value (unit)
β-carotene content (µg/g oil)
0.76 ± 0.08a 0.79 ± 0.03a 0.018 ± 0.00a 1.85 ± 0.42a
1.16 ± 0.14b 3.95 ± 0.09b 0.274 ± 0.00b 236.35 ± 4.58b
Total phenolic content (µg GAE/ g oil)
17.10 ± 0.99a
214.05 ± 1.61b
DPPH (%inhibition)
54.23 ± 1.37a
70.67 ± 0.85b
All the given values are means of three (n = 3) determinations ± SD ab Means within a row with different letters are significantly different (p < 0.05) 1 Flaxseed oil 2 Carotenoid-enriched flaxseed oil
4
92
69
81.25
% Recovery
% Recovery
80
58
47
70.5
59.75
49
36
12.00
200.00 9.25
12.00
20.00
162.50 6.50
Extraction time (min)
9.25
125.00 3.75
6.50
87.50
Microwav e power (W)
1.00
16.25
Extraction time (min)
12.50 3.75
50.00
8.75 1.00
A
Oil to waste ratio (g/g)
5.00
B 85
% Recovery
76.5
68
59.5
51
20.00
200.00 16.25
162.50 12.50
125.00
Oil to waste ratio (g/g) 8.75
87.50
Microwav e power (W)
5.00
50.00
C Figure 1. 3D plots (A) extraction time (min) versus microwave power (W) (B) extraction time (min) versus oil to waste ratio (g/g) (C) oil to waste ratio (g/g) versus microwave power (W)
100
80
% Recovery
60
40 CCE MACE
20
0
0
50
100
150
200
250
300
Extraction time (min)
Figure 2. The effect of extraction time on recovery (%) of carotenoids (CCE: conventional carotenoid extraction; MACE: microwave-assisted carotenoid extraction)
Highlights • • • •
Microwave assisted extraction of carotenoids from carrot juice processing waste using flaxseed oil (solvent) was performed. A new process for carrot juice processing waste valorization in food industry was developed. Extraction conditions were optimized using RSM. Flaxseed oil with a high antioxidative capacity was obtained by enrichment with carotenoids and phenolics
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0023-6438(20)30088-8
DOI:
https://doi.org/10.1016/j.lwt.2020.109100
Reference:
YFSTL 109100
To appear in:
LWT - Food Science and Technology
Received Date: 22 August 2019 Revised Date:
15 December 2019
Accepted Date: 25 January 2020
Please cite this article as: Elik, A., Yanik, Derya.Koç., Göğüş, F., Microwave-assisted extraction of carotenoids from carrot juice processing waste using flaxseed oil as a solvent, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2020.109100. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
CRediT author statement Aysel Elik: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization. Derya Koçak Yanık: Conceptualization, Methodology, Software, Data Curation, Writing Review & Editing, Visualization, Supervision, Project administration. Fahrettin Göğüş: Conceptualization, Methodology, Resources, Data Curation, Writing Review & Editing, Visualization, Supervision, Project administration, Funding acquisition.
1
Microwave-assisted extraction of carotenoids from carrot juice processing waste using flaxseed
2
oil as a solvent
3
Aysel ELİK1*, Derya KOÇAK YANIK1, Fahrettin GÖĞÜŞ1
4
1
Gaziantep University, Engineering Faculty, Food Engineering Department, Gaziantep, Turkey
5
*Address correspondence to: Gaziantep University, Engineering Faculty, Food Engineering
6
Department, 27310 Gaziantep, Turkey. Tel: +90 (342) 317 23 36; Email: [email protected]
7
Abstract
8
The present study was aimed to evaluate the effect of microwave technology on the extraction yield of
9
carotenoid from carrot juice processing waste using flaxseed oil as solvent. Response surface
10
methodology was employed to optimize the effect of microwave power, extraction time and oil to
11
waste ratio on percentage recovery of carotenoid. Optimum conditions obtained for microwave-assisted
12
carotenoid extraction were 165 W of microwave power, 9.39 min of extraction time and 8.06:1 g/g of
13
oil to waste ratio which gave carotenoid recovery of 77.48 %. Selected physical and chemical
14
properties of flaxseed oil and carotenoid enriched flaxseed oil were investigated in terms of fatty acid
15
composition, acid value, peroxide value, p-anisidine value, colour parameters (L*, a* and b* values), β-
16
carotene content, total phenolic content and antioxidant activity. The results showed that the enriched
17
flaxseed oil was in good quality and had high phenolic content (214.05 ± 1.61µg GAE/ g oil) and
18
antioxidant activity (inhibition % of DPPH = 70.67 ± 0.85).
19
Key words: Waste utilization; Carrot juice; Flaxseed oil; Microwave; Carotenoids
20
1. Introduction
1
21
Carrot (Daucus carota L) is one of the most important root fruits grown worldwide and is an important
22
carotenoid source (Sharma, Karki, Thakur, & Attri, 2012). Besides the potential benefits to human
23
health due to biological activities such as antioxidant, anticarcinogenic activity (Kiss, Forgacs,
24
Cserhati, Mota, Morais, & Ramos, 2000), carotenoids extracted from wastes has gained popularity as a
25
beneficial alternative to the synthetic carotenoids (Parjikolaei, El-Houri, Fretté, & Christensen, 2015).
26
In the production of carrot juice, a pulp is wasted corresponding to 30-40% of the raw material. The
27
process waste of carrot is rich in bioactive substances such as carotenoids, especially in β-carotene
28
(Oreopoulou & Tzia, 2007). Depending on the processing conditions, the total carotene content of this
29
waste can reach up to 2 g per kg dry matter (Sharma, Oberoi, & Dhillon, 2016).
30
Recovery of carotenoids in wastes is generally carried out using organic solvents such as hexane and
31
acetone which are environmentally harmful solvents. Carotenoids are oil-soluble pigments (Sachindra
32
& Mahendrakar, 2005) and the use of oil as solvent can be a promising alternative method to organic
33
solvents. The use of oil as alternative solvent has the advantage of reducing the use of environmentally
34
harmful solvents. Furthermore, barrier properties of the oil against oxygen lead to retardation of the
35
oxidation time and degradation rate for the extract containing carotenoids (Parjikolaei, El-Houri, Fretté,
36
& Christensen, 2015). In the present study, flaxseed oil was selected as the solvent for the carotenoid
37
extraction as it is rich in polyunsaturated fatty acids (about 73% of total fatty acids), especially in
38
omega-3 (Goyal, Sharma, Upadhyay, Gill, & Sihag, 2014). In this way, enriched oil to be obtained will
39
have more beneficial effects in health due to the protection effect of omega-3 oils against
40
cardiovascular diseases (Calder, & Yaqoob, 2009) and antioxidant effect of β-carotene (Tribble, 1999).
41
Moreover, direct use of oil for the extraction eliminates the extra cost of evaporation.
42
There are some studies in the literature, dealing with the carotenoid extraction using oil as a solvent.
43
However, most of these studies are based on the conventional solvent extraction process with a long 2
44
extraction time (1 to 48 hours) (Benakmoum, Abbeddou, Ammouche, Kefalas, & Gerasopoulos, 2008;
45
Handayani, Indraswati, & Ismadji, 2008; Kang & Sim, 2008; Kessy, Zhang, & Zhang, 2011;
46
Parjikolaei, El-Houri, Fretté, & Christensen, 2015; Sachindra & Mahendrakar, 2005). In recent years,
47
ultrasound technology and vegetable oils have been used in a few studies in order to extract
48
carotenoids. They have been reported to provide better extraction yield (up to about 94 %) and shorter
49
time (max. 30 min) when compared to conventional processes (Li, Fabiano-Tixier, Tomao, Cravotto, &
50
Chemat, 2013; Goula, Ververi, Adamopoulou, & Kaderides, 2017).
51
Microwave extraction can be regarded as one of the most appropriate method for β-carotene extraction
52
from carrots (Kyriakopoulou, Papadaki, & Krokida, 2015) due to less time consumption and higher
53
extraction efficiency. When the carotenoid extraction studies used vegetable oils as a solvent are
54
examined, it is considered that the main drawback is long extraction time. The microwave causes rapid
55
disruption of the cell walls and shortens the duration of the extraction and increases the yield
56
(Milutinović et al., 2015). However, there is no exhaustive study available on carotenoid extraction
57
from carrot juice processing waste (CPW) using oil as a solvent under the microwave conditions. In
58
that respect, the main purpose of this study was to evaluate the effect of microwave technology on
59
extraction yield of carotenoids from CPW by using flaxseed oil as a solvent and to optimize the
60
extraction conditions (microwave power, extraction time and oil to waste ratio) to achieve optimum
61
recovery of carotenoids. Moreover, comparative studies between microwave-assisted carotenoids
62
extraction (MACE) and conventional carotenoid extraction (CCE) using flaxseed oil as a solvent was
63
carried out in terms of recovery of carotenoids and extraction time. Finally, fatty acid composition,
64
colour parameters (L*, a*, b*), acid value, peroxide value, p-anisidine value, β-carotene content, total
65
phenolic content and antioxidant activity (inhibition % of DPPH) of carotenoid-enriched flaxseed oil
66
(CEFO) obtained under optimum conditions were compared to those of flaxseed oil (FO).
3
67
2. Materials and methods
68
2.1. Chemicals and reagents
69
All chemicals and reagents used in this study were of analytical or HPLC grade. Standard of β-carotene
70
Type II (≥95% purity by HPLC assay), folin–ciocalteu’s phenol reagent, sodium carbonate (Na2CO3),
71
sodium chloride (NaCl), ascorbic acid, 1,1-diphenyl-2- picrylhydrazyl (DPPH), potassium hydroxide
72
(KOH) were purchased from Sigma-Aldrich. Gallic acid was provided by Merck.
73
2.2. Material
74
CPW used as a source of carotenoid in the experiments was supplied by a producer of carrot juice. The
75
waste was frozen at -40°C prior to freeze drying and then moisture content was reduced to below 1%
76
by using freeze dryer (CHRIST, model Alpha 1-4 LDplus, Martin Christ, Germany). The dried waste
77
was ground in a waring blender. The resulting powder (smaller than 0.85 mm) was stored at -20°C in
78
opaque containers until carotenoid extraction.
79
Flaxseed was supplied by the local market and pressed by using cold press oil machine (Karaerler, NF
80
100, Ankara, Turkey) to obtain the cold-pressed flaxseed oil in the laboratory.
81
2.3. Microwave assisted carotenoid extraction (MACE) using flaxseed oil
82
The certain quantity of the waste powder (the quantity varied as a function of the oil to waste ratio) was
83
weighed into the flask and mixed with flaxseed oil as solvent. Total amount (oil+ waste) was kept at 20
84
g. Thirty five ml extraction vessel (88 mm height and 25 mm diameter borosilicate glass with a silicone
85
cap) was placed in the microwave extraction device and the extraction process was operated after the
86
microwave power and extraction time were set up according to the experimental design (Table 1).
87
Carotenoid extraction from the waste powder was carried out in a closed microwave system (CEM 4
88
Corporation, model Discover-SP, Matthews, NC, USA). The microwave chamber has been cooled by
89
air circulated in ice bath throughout the microwave application. The temperature did not exceed 110 oC
90
for even the harshest experimental runs (highest microwave power and longest time). Carotenoid-
91
enriched flaxseed oil was stored in dark glass bottles at -20°C until the analyses were performed.
92
2.4. Conventional carotenoid extraction (CCE) using flaxseed oil
93
Approximately, 0.95 g of the waste powder was mixed with 19.05 ml of oil (Oil to waste powder
94
ratio=20:1). Total amount (oil+ waste powder) was kept at 20 g as in MACE. The mixture was heated
95
at 65oC in a mixer incubator (Heidolph, Unimax 1010, Schwabach, Germany) for 270 min. The
96
samples were taken out at 12, 30, 60, 90, 120, 180, 240, 270 min and carotenoid contents of the
97
samples were determined.
98
2.5. Soxhlet extraction
99
Carotenoids in CPW were extracted by the soxhlet extraction method. Procedure with some
100
modifications described by Hiranvarachat, Devahastin, Chiewchan, and Raghavan (2013) was used for
101
the extraction of carotenoids. The waste powder (2 g) was placed in an extraction thimble, which was
102
then placed in an extraction chamber and extracted 6 h by using 150 mL of n-hexane, ethanol, acetone
103
(2:1:1 v/v/v) solvent mixture. Solvent mixture was evaporated under vacuum at 40oC using the rotary
104
vacuum evaporator (Heidolph, type basis Hei-VAP HL, Schwabach, Germany) in order to obtain
105
carotenoid extract. Extract was dissolved in n-hexane prior to analyses of total carotenoid and β-
106
carotene contents. Soxhlet extraction method was used to determine the total amount of carotenoid and
107
β-carotene content in CPW.
108
2.6. Total carotenoid content
5
109
The diluted extract obtained from soxhlet extraction was used to estimate the total carotenoids content
110
of CPW. Total carotenoid content of CPW was determined at 450 nm (absorbance maxima for β-
111
carotene) using a spectrophotometer (Optima, model SP-3000nano, Tokyo, Japan). The total carotenoid
112
content of CPW was expressed as µg β-carotene per g of CPW powder. The total carotenoid contents
113
of FO and CEFO were also determined spectrophotometrically, reading absorbance at 450 nm against
114
flaxseed oil as blank (Sachindra & Mahendrakar, 2005).
115
Percent recovery of carotenoid from CPW was calculated using Eq. (1) below.
116
Recovery (%) = (CE / CW) x 100
117
where CE is amount of carotenoid extracted and CW is total carotenoid content of CPW.
118
2.7. Experimental design
119
Response surface methodology (RSM) was employed to determine the optimal conditions for MACE
120
from CPW using flaxseed oil as a solvent. RSM was performed using Design Expert, version 7 (Stat-
121
Ease, Design-Expert software, Minneapolis, USA). Microwave power (X1), extraction time (X2) and oil
122
to waste ratio (X3) were independent variables of the system and % recovery of carotenoid (Y1) was the
123
response of the system. The extraction parameters were optimized through a central composite
124
rotatable design (CCRD). The experimental design consists of 20 test points with 6 central points, 6
125
star points (Table 1).
126
Significances of the model and all terms were determined by evaluating the Fisher test value at a
127
probability of 0.05. CCRD uses least-squares regression to fit the experimental data to a second-order
128
polynomial model. The second-order polynomial equation expressed in Eq. (2) was used to calculate
129
predicted response.
(1)
6
=
130
+∑
İ
+ ∑
İ
+∑
∑
(2)
131
where Y are the dependent variable, β0, βi, βii and βij are the regression coefficients for the intercept,
132
linear, quadratic and interaction terms of variables i and j, respectively, and Xi and Xj are the
133
independent variables.
134
2.8. Quantification of β-carotene by HPLC
135
Rapid saponification was used to extract carotenoids from oil samples prior to the analysis of β-
136
carotene. The procedure for extraction with saponification was adapted from the method by Gimeno et
137
al. (2000).
138
The quantity of β-carotene using HPLC (Shimadzu, Prominence/LC-20AB, Kyoto, Japan) was
139
determined by the method proposed by Barba, Hurtado, Mata, Ruiz, and De Tejada (2006). Mobile
140
phase, mixture of methanol/ acetonitrile (90:10 v/v) + 9µM triethylamine, was eluted isocratically at a
141
flow rate of 0.9 ml/min. Supelcosil LC-18 5 µm (4.6 x 250 mm) HPLC column (Supelco, Bellefonte,
142
PA, USA) was used for β-carotene analysis. Detection of β-carotene was made at a wavelength of 450
143
nm using UV–vis detector (Shimadzu, SPD-20A, Kyoto, Japan). Quantification of β-carotene was
144
performed through a calibration curve.
145
2.9. Fatty acid composition
146
The fatty acid compositions of FO and CEFO were identified according to procedure proposed by
147
Yanık (2017), using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA,
148
USA).
149
2.10. Peroxide, acid and p-anisidine values
7
150
The peroxide and acid values of oil samples were determined according to the method of AOCS Cd 8-
151
53 (AOCS, 1998) and AOCS Cd 3d-63 (AOCS, 1998), respectively. P-anisidine values of FO and
152
CEFO were determined according to AOCS Cd 18-90 (AOCS, 1998).
153
2.11. Total phenolic content (TPC)
154
Phenolics of FO and CEFO were extracted following the method described by Kıralan, Bayrak, and
155
Özkaya (2009).
156
TPC of FO and CEFO were determined with the Folin-ciocalteu colorimetric method presented by
157
Singleton, Orthofer, and Lamuela-Raventós (1999) with some modification. TPC values were
158
expressed as µg gallic acid equivalents (GAE)/ g oil.
159
2.12. DPPH-scavenging activity assay
160
Antioxidant activities of FO and CEFO were determined according to the procedure with some
161
modification reported by Kıralan, Bayrak, and Özkaya (2009). Inhibition (%) was calculated using the
162
following equation:
163
% Inhibition = (1- [Asample/Ablank]) x100
164
Where Asample is the absorbance of sample with DPPH solution, Ablank is the absorbance of DPPH
165
solution without sample solution at 517 nm.
166
2.13. Colour measurement
167
The colours of FO and CEFO were measured using a Hunter-Lab ColorFlex (HunterLab, model A-60-
168
1010-615, Reston, VA). The colour value was expressed as L* (lightness; from 0 = black to 100 =
(3)
8
169
white), a* (negative values indicate greenness while positive values indicate redness) and b* (negative
170
values indicate blueness and positive values indicate yellowness).
171
2.14. Statistical analysis
172
The statistical comparisons of FO and CEFO in terms of some basic quality parameters have been done
173
using independent t-test. The data were statistically analyzed using the SPSS statistical software,
174
version 22.0 (SPSS Inc., Chicago, IL, USA). All analysis were performed three times and averaged.
175
Results expressed as means ± standard deviation (SD). Significant differences (p<0.05) between means
176
were determined.
177
3. Results and Discussion
178
3.1. RSM modeling
179
The most important factors influenced the recovery of bioactive compounds like carotenoids from
180
plants are microwave power, extraction time and as well as solvent to sample ratio when microwave-
181
assisted extraction (MAE) is concerned (Hiranvarachat, Devahastin, Chiewchan, & Raghavan, 2013;
182
Mandal, Mohan, & Hemalatha, 2007). Therefore, microwave power, extraction time and oil to waste
183
ratio was studied as independent variables on extraction of carotenoid from carrot processing waste.
184
Percentage recovery of carotenoids was considered as the dependent variable. Three factors- five levels
185
CCRD was studied in a total of 20 experiments. Table 1 shows both the actual values of design
186
parameters and the experimental results obtained under the defined points.
187
The adequacy and fitness of the model and the relationship between variables were examined using
188
regression analysis and analysis of variance (ANOVA), respectively, and the results are given in Table
189
2. In this study, the model with high F-value (34.04) and a very small p-value (<0.0001) was highly 9
190
significant and the lack of fit test was found to be non-significant (p>0.05). Thus, the model builded is
191
sufficiently accurate for predicting the outcomes. The R2 value of 0.9521 implies that there was a good
192
correlation between experimental and predicted values of the response variable.
193
3.2. The effect of process variable on recovery of carotenoids
194
According to the results of experiments carried out at the points designated by CCRD, the percentage
195
recovery of carotenoid (Y1) ranged from 45.81 to 77.68 % (Table 1). Highest Y1 value (77.68 %) was
196
obtained in experimental run 4 under the following conditions: microwave power of 170 W of, an
197
extraction time of 9.46 min and oil to waste ratio of 8:1 g/g. While the microwave power (X1) and
198
extraction time (X2) have been found as highly significantly (p<0.001) effected on extraction of
199
carotenoids from carrot waste by flaxseed oil, the oil to waste ratio (X3) were found as non-significant
200
(p>0.05) (Table 2). However, the quadratic effect of X3, the interactive effect of X1*X3and X2*X3 were
201
found as statistically significant. The interactive effect of X1*X2 has also been found as non-significant.
202
Predicted model equation obtained from the experimental data was formulated by removing the non-
203
significant variables. However, some non-significant terms were not removed from the model in order
204
to support the model hierarchy. Second-order polynomial equation in terms of codded factors obtained
205
from CCRD model for recovery of carotenoid (%) was given as follows.
206
Recovery (%) = 62.49 + 5.77* X1 + 7.17* X2 – 1.44* X3- 1.80*X1*X2 + 1.97*X1*X3
207
-2.17*X2*X3 + 3.31*X32
208
Based on the regression coefficient values (β), the highest influence on the recovery of carotenoids was
209
extraction time, followed by microwave power. However, it was observed that no significant effect of
210
oil to waste ratio was found on recovery of carotenoid. Extraction time has positive effect on the
211
response in linear term. It means that the increase in carotenoid content extracted corresponded to the
(4)
10
212
rise in extraction time. This result was in good agreement with previous study about β-carotene
213
extraction from carrot using sunflower oil as a solvent under ultrasound treatment (Li, Fabiano-Tixier,
214
Tomao, Cravotto, & Chemat, 2013). It was found that β-carotene extraction yield could be increased by
215
increasing the time. In another study carried out by Goula, Ververi, Adamopoulou, & Kaderides
216
(2017), it was reported that the extraction yield was time dependent and increased with extended
217
ultrasonic times, especially from 10 to 30 min. Microwave power also showed a significant (p<0.0001)
218
positive linear effect on carotenoid extraction (Table 2). From Figure 1, it is understood that microwave
219
power had a strong sway on MACE. Microwave energy absorbed by materials depends on their
220
dielectric properties (Meda, Orsat, & Raghavan, 2017). Water, which has a large electric dipole
221
moment, is very efficient at absorbing the microwave energy. On the other hand, oils are esters of
222
long-chain fatty acids which have much less mobility compared to water molecules in response to
223
oscillating microwave irradiation. Therefore, they have rather small dipole moments and don't absorb
224
the radiation as much as water. However, the specific heat of oils is sufficiently smaller than the
225
specific heat of the water, and then oils tend to heating up much faster than water when exposed to the
226
same amount of microwaves (Hebbar, & Rastogi, 2012). The temperature rises of the mixture due to
227
the increase of microwave power reduce the viscosity of the solvent (flaxseed oil) and increase the
228
solubility of carotenoids (Hiranvarachat, Devahastin, Chiewchan, & Raghavan, 2013). This results in
229
an enhanced diffusion rate, thereby prominent accelerating extraction rate. Even though no significant
230
effect of oil to waste ratio was observed under microwave extraction using oil as solvent, the
231
interaction between extraction time and oil to waste ratio showed a synergistic effect on amount of the
232
carotenoid extracted (p<0.05). As shown in Figure 1B, extraction yield of the carotenoid increased with
233
longer extraction time with low ratio.
234
3.3. Experimental validation of optimized conditions
11
235
Microwave extraction system was optimized employing RSM. The optimum conditions were
236
determined by a quadratic model as 165 W of microwave power, 9.39 min of extraction time, 8.06:1
237
g/g of oil to waste ratio to obtain optimum value of recovery (77.87%). The carotenoid extractions
238
(three times) were performed at optimum conditions to validate the suggested model. The experimental
239
results gave 77.48 ± 0.16 % recovery of carotenoids which was close to the predicted value. The
240
experimental data confirmed the validity and the accuracy of predictive model.
241
3.4. Comparison of MACE and CCE
242
The percent recovery of carotenoids was 77.48 % at optimum conditions (165W, 9.39 min and 8.06:1
243
g/g oil to waste ratio) using MACE. As it has been mentioned in introduction part, various conventional
244
extraction conditions were applied to extract carotenoids into oil systems. It was important to compare
245
the conventional extraction results with our finding to see how the recovery has been changed with
246
time and to what extent. As seen in the Figure 2, the extraction yield for conventional extraction
247
method increased with time in the first 30 min and almost 50 % of the carotenoids were extracted in
248
this time period. The extraction yield of carotenoids (about 87 %) reached to equilibrium at around 180
249
min and further increase in time did not affect the yield of carotenoid extraction up to 275 min. Percent
250
recovery of carotenoid under microwave was calculated for extraction times at 1, 3, 5, 7 and 9.39 min
251
on the basis of Eq. (4) while microwave power and oil to waste ratio were kept constant at optimal
252
values. While about 78 % of total carotenoids were extracted within the first 9.39 min of microwave
253
treatment, it was needed more than 120 min to achieve the same value for conventional treatment.
254
Although conventional extraction method had a higher recovery (about 87 %) of total carotenoids from
255
CPW, time required for extraction was quite long (around 180 min) resulting in consumption of
256
considerable energy.
257
3.5. Selected physical and chemical properties of FO and CEFO 12
258
Quality parameters of CEFO obtained under the conditions of optimum microwave treatment were
259
investigated and it was compared to those of FO. Physical and chemical quality parameters investigated
260
for both oils were reported in Tables 3 and 4. It is shown that the fatty acids of FO were mainly
261
constituted by linolenic (52.46 ± 0.02 %), oleic (19.77 ± 0.01 %), linoleic (15.35 ± 0.01 %), palmitic
262
(6.25 ± 0.03 %), and stearic (4.41 ± 0.01 %) acids (Table 3). Although percentages of some fatty acids
263
of CEFO were significantly (p<0.05) different from those of FO, fatty acid values found for CEFO
264
were still presenting a good quality flaxseed oil. The similar findings have been reported in the
265
previous study conducted by Yanık (2017).
266
The CIELAB L*, a* and b* parameters of FO and CEFO were compared. CEFO showed a higher a*
267
and b* values as well as lower L* value (Table 4). This means that the CEFO was darker and more red
268
and yellow in colour than FO. This is a clear indication of successful extraction of β-carotene into the
269
flaxseed oil during microwave treatment because β-carotene is known as the predominant
270
phytochemical component in orange carrot and it is a deep orange-yellow in colour (Poudyal, Panchal,
271
& Brown, 2010; Sun, Gantt, & Cunningham, 1996).
272
Acid, peroxide and p-anisidine values are important indicators of oil quality affected during processing
273
and storage stages of oils. Peroxide and p-anisidine are the primary and secondary products of oil
274
oxidation reactions, respectively; while acid value is a good indicator for hydrolytic reactions (Li, Cai,
275
Sun, & Liu, 2016). Acid values of FO and CEFO were found as 0.76 ± 0.08 and 1.16 ± 0.14 mg KOH/g
276
oil, respectively (Table 4). Significant (p<0.05) increase in acid value was observed after the
277
microwave treatment. This increase is probably due to the splitting of ester linkage of triglyceride
278
molecules during microwave treatment (Hassanein, El-Shami, & El-Mallah, 2003). In a study
279
conducted by Megahed (2011), acid value of flaxseed oil exposed to microwave irradiation showed a
280
similar trend. According to the Codex standard (Codex Alimentarius, 2017), acid value of cold-pressed 13
281
oil has to be less than 4.0 mg KOH/g oil. The results were lower than the limit established by Codex
282
Alimentarius.
283
The peroxide values of FO and CEFO were determined as 0.79 ± 0.03 and 3.95 ± 0.09 milliequivalents
284
(meq) peroxide/kg oil, respectively. Kreps, Vrbiková, Schmidt, Sekretár, & Híreš (2014) reported that
285
the peroxide values of sunflower and corn oils grew significantly after a 10 min of microwave
286
treatment. Even if the peroxide value showed a significant increase (p<0.05) during microwave
287
treatment, the peroxide values of FO and CEFO were still well within the limit of up to 15 meq
288
peroxide/ kg of oil (Codex Alimentarius, 2017).
289
P-anisidine values found for FO and CEFO were 0.018 ± 0.00 and 0.274 ± 0.00, respectively. A
290
significant increase in p-anisidine value of CEFO was observed after microwave treatment. Chiavaro,
291
Rodriguez-Estrada, Vittadini, & Pellegrini, (2010) reported that microwave treatment of canola oil,
292
high oleic sunflower oil and peanut oil for 15 min led significant increase of p-anisidine value over
293
fresh oils. However, p-anisidine value should be lower than 2 for good quality oil (Choo, Birch, &
294
Dufour, 2007). The microwave-treated oil obtained in the present study is still below this value and it
295
can be considered as a good quality oil after MAE.
296
Among the carotenoids, the β-carotene is the most abundant in the carrot processing waste (Sharma,
297
Karki, Thakur, & Attri, 2012). The percent β-carotene of the total carotenoid content was determined
298
for enriched flaxseed oil produced and CPW. Total carotenoid and β-carotene contents of CPW were
299
found as 4132.83 ± 1.05 and 2507.97 ± 0.49 µg/g CPW powder, respectively. β-carotene constituted
300
60.7 % of the total carotenoid content in CPW. Total carotenoid and β-carotene contents of CEFO
301
obtained under optimum conditions were 397.10 ± 0.8 and 236.35 ± 4.5 µg/g oil, respectively. It was
302
determined that β-carotene accounted for 59.5% of carotenoids in CEFO. It is comprehended from the
14
303
HPLC results that a large portion of carotenoid extracted from CPW was β-carotene (about 60%). β-
304
carotene content of FO after MACE was increased significantly (p<0.001).
305
The results (Table 4) showed that TPC and antioxidant activity of flaxseed oil increased after
306
carotenoid extraction under microwave. TPC of CEFO (214.05 ± 1.61 µg GAE/g oil) was about twelve
307
times higher than that of FO (17.10 ± 0.99 µg GAE/g oil). Furthermore, it was found that antioxidative
308
properties of FO after carotenoid extraction were also improved. While DPPH scavenging activity of
309
FO was 54.23 ± 1.37 %, this value reaches up 70.67 ± 0.85 % for CEFO. Presence of carotenoids such
310
as β-carotene which were reported to have stronger antioxidant capacity (Hiranvarachat & Devahastin,
311
2014) in FO after the microwave extraction may account for the better results obtained for its
312
antioxidant activity.
313
4. Conclusion
314
In the present study, the simultaneous optimization of MACE was established for extracting
315
carotenoids from CPW using flaxseed oil as a solvent. It was observed that microwave power and
316
extraction time are significantly effective in extracting carotenoids from the carrot waste to the flaxseed
317
oil. A 77.48 % recovery of carotenoid was achieved successfully at optimum conditions (165 W of
318
microwave power, 9.39 min of extraction time and 8.06:1 g/g of oil to waste ratio); hence the
319
carotenoid extraction by using oil under microwave irradiation is a promising and efficient process for
320
both waste uses and enrichment of oil. In this way, the enrichment of flaxseed oil with phenolics,
321
antioxidants and carotenoids has been developed by an alternative method based on innovative, fast
322
technology with the evaluation of CPW in food industry.
323
It was taken into account that peroxide, acid and p-anisidine values of enriched flaxseed oil were higher
324
than flaxseed oil when MACE applied, however, total phenolic and carotenoid contents, as well as 15
325
antioxidant activity (DPPH) of the enriched oil were simultaneously higher. It could be recommended a
326
study about the effect of different storage temperatures on quality parameters of untreated oil and
327
enriched oil produced under similar microwave technology for a future work. Additionally, this
328
carotenoid enriched oil could be directly used as carotenoids source in different products. In this
329
respect, it could be suggested to study the cost estimation for the production of carotenoid enriched oils
330
with the microwave technology as a future work.
331
Acknowledgements
332
Financial support for this study is provided by Scientific and Technological Research Council of
333
Turkey (TUBITAK) 1001 Project No: 217O125.
334
Declaration of conflicting interests
335
All authors declare that there is no conflict of interest.
336
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wet
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pomace.
LWT,
77,
45-51.
456
457
458
459
460
461
462
463
464
465
466
22
467
Figure Captions
468
Figure 1. 3D plots (A) extraction time (min) versus microwave power (W) (B) extraction time (min)
469
versus oil to waste ratio (g/g) (C) oil to waste ratio (g/g) versus microwave power (W)
470
Figure 2. The effect of extraction time on recovery (%) of carotenoids
471
472
473
474
475
476
477
478
479
480
481
482
483 23
484
Table Captions
485
Table 1
486
Experimental design for microwave extraction.
487
Table 2
488
Regression analysis and analysis of variance.
489
Table 3
490
Fatty acid composition (% fatty acid), %unsaturated fatty acid, and ratio of omega 3: omega 6 fatty
491
acids.
492
Table 4
493
Selected physical and chemical properties of FO and CEFO.
24
Table 1 Experimental design for microwave extraction. Run
Microwave power (W)
Extraction time (min)
Oil to waste ratio (g/g)
Recovery %
1
80.00
3.14
8:1
53.87 ±0.23
2
170.00
3.14
8:1
63.09±0.36
3
80.00
9.46
8:1
74.98±1.20
4
170.00
9.46
8:1
77.91±0.33
5
80.00
3.14
17:1
51.94±0.07
6
170.00
3.14
17:1
69.97±0.71
7
80.00
9.46
17:1
65.30±0.41
8
170.00
9.46
17:1
75.22±0.11
9
50.00
6.30
12.5:1
50.06±0.21
10
200.00
6.30
12.5:1
73.07±1.17
11
125.00
1.00
12.5:1
45.95±0.19
12
125.00
12.00
12.5:1
71.74±0.30
13
125.00
6.30
5:1
74.46±0.66
14
125.00
6.30
20:1
67.15±0.37
15
125.00
6.30
12.5:1
63.88±0.85
16
125.00
6.30
12.5:1
61.69±0.43
17
125.00
6.30
12.5:1
63.95±0.95
18
125.00
6.30
12.5:1
64.41±0.27
19
125.00
6.30
12.5:1
61.02±0.90
20
125.00
6.30
12.5:1
65.35±0.46
All the given values are means of two (n = 2) determinations ± SD
1
Table 2 Regression analysis and analysis of variance. Source
Model Linear Microwave Power (X1) Extraction Time (X2) Oil to waste ratio (X3) Interaction X1*X2 X1*X3 X2*X3 Quadratic X32 Residual Lack of Fit Pure Error Cor Total
SSa
DFb
Recovery (%) MSc F -Value
1440.35
7
205.76
34.04
p-value Prob > F < 0.0001d
454.61 702.14 28.48
1 1 1
454.61 702.14 28.48
75.21 116.16 4.71
< 0.0001d < 0.0001d 0.0507e
25.94 31.16 37.51
1 1 1
25.94 31.16 37.51
4.29 5.16 6.21
0.0605e 0.0424d 0.0284d
160.51 72.54 58.60 13.93 1512.89
1 12 7 5 19
160.51 6.04 8.37 2.79
26.55
0.0002d
3.00
0.1220e
R2 0.9521 0.9241 Adj R2 Pred R2 0.8516 a b Sum of Squares; Degree of freedom; cMean square d Significant at Prob > F less than 0.05 level; e Not significant at Prob > F higher than 0.05 level
2
Table 3
Fatty acid composition (% fatty acid), %unsaturated fatty acid, and ratio of omega 3: omega 6 fatty acids. Fatty acid
FO1
CEFO2
Palmitic acid (C16:0) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) Linolenic acid (C18:3) Eicosadienoic acid (C20:0) Other
6.25 ± 0.03a 0.08 ± 0.02a 4.41 ± 0.01a 19.77 ± 0.01b 15.35 ± 0.01b 52.46 ± 0.02a 0.22 ± 0.01a 1.46 ± 0.02a
6.17 ± 0.05a 0.08 ± 0.00a 4.55 ± 0.01b 19.73 ± 0.01a 15.33 ± 0.01a 52.47 ± 0.03a 0.21 ± 0.01a 1.46 ± 0.04a
% Unsaturated fatty acids
87.67 ± 0.01a
87.61 ± 0.03a
Omega 3: omega 6 fatty acids
3.42 ± 0.00a
3.42 ± 0.01a
All the given values are means of three (n = 3) determinations ± SD ab Means within a row with different letters are significantly different (p < 0.05) 1 Flaxseed oil 2 Carotenoid-enriched flaxseed oil
3
Table 4 Selected physical and chemical properties of FO and CEFO. Physical-chemical property
FO1
CEFO2
L*
72.77 ± 0.08b
58.69 ± 0.09a
a*
-2.53 ± 0.09a
28.35 ± 0.1b
b*
77.01± 0.18a
97.65 ± 0.21b
Acid value (mg KOH/g oil) Peroxide value (meq/kg oil) p-anisidine value (unit)
β-carotene content (µg/g oil)
0.76 ± 0.08a 0.79 ± 0.03a 0.018 ± 0.00a 1.85 ± 0.42a
1.16 ± 0.14b 3.95 ± 0.09b 0.274 ± 0.00b 236.35 ± 4.58b
Total phenolic content (µg GAE/ g oil)
17.10 ± 0.99a
214.05 ± 1.61b
DPPH (%inhibition)
54.23 ± 1.37a
70.67 ± 0.85b
All the given values are means of three (n = 3) determinations ± SD ab Means within a row with different letters are significantly different (p < 0.05) 1 Flaxseed oil 2 Carotenoid-enriched flaxseed oil
4
92
69
81.25
% Recovery
% Recovery
80
58
47
70.5
59.75
49
36
12.00
200.00 9.25
12.00
20.00
162.50 6.50
Extraction time (min)
9.25
125.00 3.75
6.50
87.50
Microwav e power (W)
1.00
16.25
Extraction time (min)
12.50 3.75
50.00
8.75 1.00
A
Oil to waste ratio (g/g)
5.00
B 85
% Recovery
76.5
68
59.5
51
20.00
200.00 16.25
162.50 12.50
125.00
Oil to waste ratio (g/g) 8.75
87.50
Microwav e power (W)
5.00
50.00
C Figure 1. 3D plots (A) extraction time (min) versus microwave power (W) (B) extraction time (min) versus oil to waste ratio (g/g) (C) oil to waste ratio (g/g) versus microwave power (W)
100
80
% Recovery
60
40 CCE MACE
20
0
0
50
100
150
200
250
300
Extraction time (min)
Figure 2. The effect of extraction time on recovery (%) of carotenoids (CCE: conventional carotenoid extraction; MACE: microwave-assisted carotenoid extraction)
Highlights • • • •
Microwave assisted extraction of carotenoids from carrot juice processing waste using flaxseed oil (solvent) was performed. A new process for carrot juice processing waste valorization in food industry was developed. Extraction conditions were optimized using RSM. Flaxseed oil with a high antioxidative capacity was obtained by enrichment with carotenoids and phenolics
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: