- Email: [email protected]
An improved microwave-assisted extraction of anthocyanins from purple sweet potato in favor of subsequent comprehensive utilization of pomace
Accepted Manuscript Title: An improved microwave-assisted extraction of anthocyanins from purple sweet potato in favor of subsequent comprehensive utilization of pomace Authors: Wei Liu, Chunv Yang, Chunjiao Zhou, Zhiyong Wen, Xinrong Dong PII: DOI: Reference:
S0960-3085(18)30788-0 https://doi.org/10.1016/j.fbp.2019.02.003 FBP 1042
To appear in:
Food and Bioproducts Processing
Received date: Revised date: Accepted date:
6 November 2018 26 January 2019 20 February 2019
Please cite this article as: Liu, Wei, Yang, Chunv, Zhou, Chunjiao, Wen, Zhiyong, Dong, Xinrong, An improved microwave-assisted extraction of anthocyanins from purple sweet potato in favor of subsequent comprehensive utilization of pomace.Food and Bioproducts Processing https://doi.org/10.1016/j.fbp.2019.02.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
An improved microwave-assisted extraction of anthocyanins from purple sweet potato in favor of subsequent comprehensive utilization of pomace
IP T
Wei Liu, Chunv Yang, Chunjiao Zhou, Zhiyong Wen, Xinrong Dong﹡
SC R
College of Science, Hunan Agricultural University, Changsha 410128, P. R. China
U
﹡Corresponding author: Xinrong Dong
A
N
E-mail: [email protected]
CC E
PT
ED
M
Graphical abstract
A
Highlights
An improved microwave-assisted extraction of anthocyanins from starch-rich material.
High efficiency as conventional microwave-assisted extraction. 1
Less solvent and clear extraction solution as sohx extraction.
Pomace in its original appearance without gelatinization of starch as USE.
High efficiency for extraction of anthocyanins in favor of utilization of pomace.
IP T
Abstract: In order to enhance the extraction efficiency of anthocyanins and
improved
microwave-assisted
extraction
(iMAE)
SC R
subsequent comprehensive utilization of pomace from purple sweet potato (PSP), of
anthocyanins
an
without
dextrinization of starch was developed in this work. Firstly, the extraction conditions of
N
U
PSP anthocyanins (PSPAs) were optimized by Response Surface Methodology (RSM)
A
and the optimal parameters were as follows: solid-to-liquid ratio of 1:3 (g / mL), ethanol
M
concentration of 30% with a small quantity of critic acid as regulating reagent of pH, microwave irradiation power of 320 w, extraction time of 500 s. Under the conditions,
ED
the yield of anthocyanins was 31.16 mg/100g PSP (RSD =1.45%, n=3), which was a
PT
relative error of -4.88% compared with the predicted value (32.76 mg/100g PSP) by model of Response Surface Methodology. By HPLC-ESI-MS/MS analysis, the
CC E
composition of anthocyanins obtained by iMAE is basically the same as that of ultrasound-assisted extraction with citric acid aqueous. And the residue obtained by
A
iMAE was in its original appearance and the total sugar content (TSC) in pomace was 88.2% of that in fresh PSP. The iMAE provided a method for the quick extraction of anthocyanins with some advantages of high efficiency as conventional MAE, but avoided gelatinization of starch which is useful for further utilization of starch in pomace. 2
Keywords: purple sweet potato; anthocyanins; microwave-assisted extraction; gelatinization of starch
IP T
1. Introduction Nowadays, purple sweet potato (PSP) has been cultivated widely in China as a
SC R
resource of food and industrial material (Liu, Mu, Sun, Zhang, & Chen, 2013).
PSP
contains a significant content of anthocyanins compared to white, yellow, and orange
U
ones. The purple sweet potato anthocyanins (PSPAs) were found to have remarkable
N
range of effective activities such as antioxidation (Teow et al., 2007), antimutation
A
(Yoshimoto et al., 1999), and hepato-protection (Hwang et al., 2011), memory
M
enhancing (Lu et al., 2012). Thus, there is a growing popularity in food, cosmetics, and
ED
pharmaceutical industries due to their non-toxicity, unique color, and nutritional benefits (Zhu et al., 2017).
PT
The most traditional industrial method for extraction of PSPAs from PSP is the solid-
CC E
liquid extraction by mechanical agitation. It is easy to cause starch gelatinization which is tedious for solid-liquid separation and unfavorable for the comprehensive utilization of starch in PSP residues. Some conventional solvent (such as methanol, acetone)
A
extraction (CSE) of anthocyanins have been described in the literatures (Kano, Takayanagi, Harada, Makino, & Ishikawa, 2005; Santos, Veggi, & Meireles, 2010; Burgos et al., 2013). While disadvantages including long time of extraction, solventconsuming were always found during the CSE (Sun, Liao, Wang, Hu, & Chen, 2007). 3
In order to improve the extraction efficiency, enzyme-assisted extraction has been currently widely used in extraction of value-added compounds from plant materials (Zhao & Li, 2015; Tanya et al., 2016; Zhu et al., 2018) with effective and nontoxic characteristic. However, it also has the disadvantages of strict pH value and long time.
IP T
Recently, ultrasonic-assisted extraction (UAE) were applied to extraction of anthocyanins (Chen, Zhao, & Yu, 2015; Bonfigli, Godoy, Reinheimer, & Scenna, 2017).
SC R
It has many advantages such as high efficiency, but there are still some disadvantages
such as a long time compared with microwave-assisted extraction (MAE). MAE is a
U
promising technology with a very short time for extraction of bioactive compounds
N
from natural resources (Yang, & Zhai, 2010; Bélafi-Bakó et al., 2012; Nora Pap et al.,
A
2013; Routary, & Orsat, 2014; Liazid, Guerrero, Cantos, Palma, & Barroso, 2011; Thais,
M
et al., 2018). However, the conventional MAE for extraction of PSPAs from PSP also
ED
has some disadvantages including using a large quantity of extraction solvent and bringing dextrinization of starch in PSP. In the conventional MAE process,
PT
dextrinization of starch will firstly bring difficulty of filtration. But more important, it
CC E
affects the subsequent utilization of starch. This not only causes waste of starch resource, but also brings about environmental problems. In this work, an improved microwaveassisted extraction (iMAE) of PSPAs from PSP was developed. It had some advantages
A
of high efficiency as conventional MAE, but used a small quantity of acidic ethanol aqueous as solvent and gave a clear extraction solution as conventional sohx extraction. More importantly, the PSP pomace extracted anthocyanins maintained in its original shape with high content of total sugar, which was conducive to its subsequent 4
comprehensive utilization.
2. Materials and methods 2.1. Materials
IP T
Fresh purple sweet potato (PSP) was obtained from a local market in Hunan Agriculture University. Prior to extraction, fresh PSP was washed and cut into about 2
SC R
mm granule in diameter. 2.2. Chemicals and reagents
U
Reagents of analytical grade (ethanol, citric acid, hydrochloric acid, potassium
N
chloride, sodium acetate) and reagents of HPLC grade (methanol and acetonitrile) were
A
purchased from sinopharm chemical Reagent Co., Ltd. (Shanghai, China).
M
2.3. The improved microwave-assisted extraction device and its work principle
ED
The improved microwave-assisted extraction (iMAE) has been performed in a Microwave Reactor of MCR-3 illustrated in Fig. 1. The improved apparatus for iMAE
PT
was composed of a 3-neck round-bottom flask, a soxh-like extractor improved from a
CC E
water segregator, a microwave reactor and a condenser.
A
Fig.1. Schematic diagram of improved microwave-assisted extraction
Compared the conventional MAE, the PSP granules (material) were put in soxh-like extractor but not in bottom of flask. In the process of microwave-assisted extraction, the sample (i.e. PSP granules) and solvent (i.e. ethanol aqueous) would be 5
simultaneously irradiated by microwave emitted from microwave device. The solvent was heated up, then evaporated and became into vapor. Then the solvent vapor entered into the condenser outside the microwave cavity and was condensed. The condensed solvent flowed into the extractor and soaked the sample irradiated simultaneously by
efficiently carried out.
SC R
2.4. Single factor conditions for iMAE of anthocyanins from PSP
IP T
microwave from the same microwave device. So, microwave-assisted extraction was
The iMAE was performed in a Microwave Reactor of MCR-3. Ten gram of PSP
U
granules were soaked into 2 mL 10% of citric acid aqueous solution for 30 min and then
N
placed into the extractor. Ethanol aqueous (adjusting pH = 2 with citric acid ) of 30 mL
A
was added into the 3-neck round bottom flask of 100 mL. Three single factors including
M
the concentration of ethanol aqueous (0, 10, 20, 30, 40, 50 and 60%, v/v), microwave
ED
irradiation power (80, 160, 240, 320 and 400 W) and extraction time (200, 300, 400, 500, 600 s) were investigated. Then, the mixture was centrifuged at 8000 rpm for 5 min
PT
and the supernatant was collected for analysis.
CC E
2.5. Optimization design by response surface methodology and statistical analysis In order to optimize the extraction conditions of anthocyanins from PSP, response
surface methodology (RSM) was applied to design of the experiment. Box-Behnken
A
Design (BBD) with three factors was used. The design consisted of 17 randomized runs with five replicates at the center of the domain. The independent variables and their levels were as follows: concentration of ethanol aqueous (20 to 40%, v/v), microwave irradiation power (160 to 320 w) and extraction time (300 to 500 s). The relation 6
between the coded values and actual values was obtained in the following equation:
X
xi xo Δxi
(1)
Where X is the coded value, xi is the actual value, x0 represents the actual value of xi
IP T
at the centre point, and Δxi corresponds to the step change value. The response function(Y) was partitioned into linear, quadratic, and interactive components. The
3
3
i 1
i 1
2
SC R
response variables were fitted to the following second order polynomial model (Eq. (2)). 3
Y β 0 βiXi βiiXi 2 βijXiXj i 1 ji 1
(2)
U
Where Y stands for the total anthocyanins content (TAC), β0 is the intercept, βi, βii,
N
and βij represent the coefficients of the linear, quadratic, and interactive effects,
ED
2.6.1 Validation experiment
M
2.6. Validate and Contrast experiment
A
respectively. Xi and Xj are coded values of the independent variables.
The optimal iMAE conditions were obtained by the single factor experiments and
PT
RSM, then the experiments under the optimal conditions were performed in triplicates.
CC E
2.6.2 Conventional ultrasound-assisted extraction (UAE) with citric acid aqueous as solvent
Ten gram of PSP granules and 100 mL of 3% (g/mL) critic acid aqueous were placed
A
into a conical flask of 250 mL and extracted by ultrasound wave for 10 min. The mixtures were filtrated with filter paper and the filtrate was collected. The residues of PSP were treated as above procedures until the anthocyanins in PSP were extracted completely. The filtrate was centrifuged at 8000 rpm for 5 min and the supernatant was 7
collected for analysis. 2.6.3 Conventional MAE under the optimal iMAE conditions Conventional MAE was carried out under the optimal iMAE conditions. And detailed description was as follows: Ten gram of PSP granules soaked with 2 mL of 10%
IP T
citric acid aqueous solution for 30 min and 30 mL of acidic ethanol aqueous ( pH = 2 adjusted by citric acid) were added into the 3-neck round-bottom flask of 100 mL and
SC R
extracted for 500s by MAE. Then, the mixture was further processed according to the Procedure 2.6.2.
U
2.6.4 Traditional hot reflux continuous extraction (HRCE) under the optimal iMAE
N
conditions
A
PSP granules were extracted for 60 min by HRCE using electric heating-jacket
M
instead of the microwave reactor and other optimal conditions of iMAE ( detailed
Procedure 2.6.2.
ED
description as 2.6.3). Then, the mixture was further processed according to the
total
content
of
anthocyanins
was
determined
according
to
the
CC E
The
PT
2.7. Determination of total anthocyanins content (TAC)
spectrophotometric pH-differential method (Swer, Chauhan, Paul, & Mukhim, 2016). The sample extracted was mixed with hydrochloric acid - potassium chloride buffer
A
(pH=1.0) and sodium acetate buffer (pH=4.5), respectively. For each sample, the absorbance was measured at 520 and 700 nm. The TAC was calculated according to Eq. (3). TAC (mg 100g)
A520 A700pH1.0 A520 A700pH4.5 MW DF V 100 ε L mf 8
(3)
Where TAC is the total anthocyanins content expressed as cyanidin-3-glucoside equivalent (CGE) (mg/100g), MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol), DF is the dilution factor, V is the volumn of solution (mL), ε is the cyanindin-3-glucoside molar absorbance (26,900), L is the cell path length (1 cm), mf
2.8. Stability of anthocyanins under microwave irradiation
SC R
2.8.1 Comparative analysis of components in PSPAs extracts by HPLC
IP T
is the weight of the sample (g).
The HPLC analysis of PSPAs extracts was obtained using an Agilent 1200 series
U
high performance liquid chromatography. Chromatographic separations were carried
N
out using an Agilent C18 column (250 mm × 4.6 mm, 5 µm; Agilent Technologies Co.,
A
Ltd., Shanghai, China). And the detection wavelength was set at 520 nm, column
M
temperature was 25℃. And the mobile phase consisted of solvents A (acetic acid : water
ED
= 0.5 : 100, v/v) and B (acetonitrile), the gradient elution program was used as follows: 15% B between 0 and 30 min; 20% B between 30 and 80 min; 15% B between 80 and
PT
85 min. The flow rate was 0.50 mL/min and the injection volume was 20 µL. The
CC E
samples were dissolved in distilled water containing 50% methanol and were filtered through a 0.45 µm Millipore filter.
A
2.8.2 Comparative analysis of components in PSPAs extracts by HPLC-ESI-MS/MS Qualitative analysis of anthocyanins was conducted using the Agilent UHD Accurate
Mass Q-TOF LC-MS analysis. Chromatographic separation was carried out on Agilent Eclipse Plus-C18 column (250 mm × 4.6 mm, 5 µm). Flow rate was 1 mL/min, sample injection volume was 15 µL, the detection wavelength was set at 520 nm. Solvent A 9
was 0.1% formic acid in distilled water (v/v), and solvent B was purified acetonitrile. The following gradients were utilised: 0-20 min, 15%B; 20-40 min, 20-100%B; 40-50 min, 100%B. For the identification of anthocyanins, the ESI source was set in positive and negitive ionisation mode. The applied conditions were set as follows: capillary
IP T
temperature, 300℃; capillary voltage, 4000 V (positive ionisation mode)/3000 V (negitive ionisation mode); nebulizer pressure, 30 psi; dry gas flow at 10 L/min. Scan
SC R
range was measured from m/z 100 up to 1500.
2.8.3 Effect of microwave irradiation on total anthocyanins content (TAC)
U
The experiments were carried out as our previous method (Liu et al., 2018) and the
(critic acid used for regulating pH) were mixed and the
A
ethanol aqueous in pH = 2
N
detailed description were as follows. Eighty gram of PSP granules and 260 mL of 30%
M
mixures were extracted according to the Procedure 2.6.2. Then the filtrate was placed
ED
into a conical flask of 500 mL and then irradiated for 15 min by microwave of 320 W. In this process, appropriate amount of solution was taken out at intervals of 1 min and
PT
anthocyanins were analysized. The concentration of anthocyanins (Ca) was calculated
CC E
according to Eq. (4).
Ca ( mg/L ) = TAC*mf*10/V
(4)
Where Ca is the concentration of anthocyanins in solution. The meanings of other
A
symbols were the same as that of 2.7. 2.9 Analysis of pomace extracted anthocyanins The weight and the total sugar content (TSC) of pomace were checked according to the methods of literature (National Health and Family Planning Commission of the 10
People’s Republic of China, 2010).
3. Results and discussion 3.1 Determination of single factor experiment
IP T
3.1.1. Effect of adding acid on stability of anthocyanins PSPAs were more stable under the acid conditions than the neutral conditions and
SC R
alkaline conditions (Fan, Han, Gu, & Gu, 2008) and citric acid was used for adjusting the pH of extraction solvent in thi work. The experimental results indicated that the
U
good conditions were as follows: Ethanol aqueous as extraction solvent was adjusted
N
into pH = 2 by citric acid and the PSP granules (10 g) were simultaneously soaked by
A
2 mL of 10% (w/w) citric acid aqueous solution for 30 min before iMAE.
M
3.1.2. Effect of ethanol concentration on extraction yield of anthocyanins
ED
The effect of ethanol concentration varying from 0 to 60% on extraction yield of anthocyanins in PSP was investigated (Fig. 2(A)) while other extraction variables were
PT
fixed. The results indicated that the content of anthocyanins in extraction solution
CC E
increased as the ethanol concentration increased from 0 to 60%. It might be that more ethanol became gas when ethanol concentration increased. That is to say, the quantity of ethanol flowed into PSP granules increases with a higher concentration of ethanol.
A
So, more anthocyanins in PSP were extracted into solution with a higher concentration of ethanol aqueous. However, there was no significant differences in extraction yield from 30% to 60%. So 30% ethanol aqueous was used for the next experiment. 3.1.3. Effect of microwave irradiation power on extraction yield of anthocyanins 11
The effect of microwave irradiation power (MIP) on extraction yield of anthocyanins was shown in Fig. 2(B). The content of anthocyanins extracted from PSP was firstly increased with the increase of MIP from 80 to 240 w, and then there was a slow increase while the MIP changes from 240 to 400 w. Therefore, MIP of 240 w was chosen as a
IP T
compromise and fixed in the iMAE experiments.
SC R
Fig. 2. The effects of single factor on extraction yield of anthocyanins in PSP (A,
U
ethanol concentration ; B, microwave irradiation power; C, extraction time)
N
3.1.4. Effect of microwave irradiation time on extraction yield of anthocyanins
A
As we can seen in Fig. 2(C), the results shown that the content of anthocyanins
M
extracted from PSP increased rapidly when the extraction time varied from 200 to 500
ED
s, then it was increased smoothly until the time was 700 s. Actually, the anthocyanins in PSP were almost fully extracted out after 500 s.
PT
3.2 RSM statistical analysis
CC E
3.2.1 Analysis of the adequacy of the fitted model According to the results of above single factor experiments, ethanol concentration
from 20% to 40% (v/v), microwave irradiation power from 160 to 320 W and extraction
A
time from 300 to 500 s were chosen for RSM experiments. The optimization for extraction of anthocyanins from PSP was investigated using RSM, and three parameters for the following RSM design were X1 (ethanol concentration), X2 (microwave irradiation power), and X3 (microwave extraction time), 12
respectively. The response of each independent variable can be seen in Table 1.
Table 1 Total anthocyanins content of PSP used in the Box-Behnken design for response
IP T
surface methodology.
The regression equation in coded level neglecting insignificant terms was generated:
SC R
TAC mg 100g 25.75 3.71X1 5.91X 2 5.66X 3 1.50X1X 2 0.45X1X 3 1.40X 2 X 3 3.43X12 3.78X 2 2 0.78X 3 2
The analysis of variance for the fitted quadratic polynomial model for optimization
A
M
Table 2 ANOVA for quadratic model
N
U
of extraction parameters was presented in Table 2.
ED
The determination coefficient (R2) of the predicted model was 0.97, showing that only 0.03% of the variation can't be explained by the present model, and the regression
PT
model was highly significant (P<0.01) and could be used to monitor the optimization.
CC E
While the lack of fit was associated with a p-value of 0.7650 which is not significant. As can be observed in Table 2, the linear terms of ethanol concentration(X1, P<0.001), microwave irradiation power(X2, P<0.001), microwave extraction time(X3, P<0.001)
A
were highly significant; the quadratic terms of ethanol concentration(X12, P<0.01), microwave irradiation power(X22, P<0.01) reached also highly significant. The other terms were not significant (P>0.05). 3.2.2 Interactions of different experimental factors on the effects of response variables 13
RSM was also applied to understand the interaction relationship between the test variables affecting the selected process response (TAC). Three-dimensional response surface were illustrated in Fig. 3.
IP T
Fig. 3. Response surface plot for TAC contents showing the effect of independent variables (a, Ethanol concentration and microwave irradiation power; b, Ethanol
SC R
concentration and extraction time; c, microwave irradiation power and extraction time).
U
In Fig. 3, the effects of ethanol concentration, microwave irradiation power, and
N
microwave extraction time on TAC were shown. It was observed in Fig.3(a) that the
A
TAC was increased with an increase of ethanol concentration and microwave irradiation
M
power while the microwave extraction time was fixed. The higher TAC was obtained
ED
at ethanol concentration of 30 to 40% and the microwave irradiation power of 240 to 320 W. The effects of ethanol concentration and extraction time on the TAC of the
PT
extracts were reflected in Fig. 3(b), a similar linear increase in TAC with the increase
CC E
of extraction time and ethanol concentration at a fixed microwave irradiation power. Fig. 3(c) shown that the TAC was affected by microwave irradiation power and extraction time, exhibiting a clear increase in content with the raise of the two parameter.
A
Fom Fig. 3, it also clearly demonstrated that the effect of the microwave irradiation power and extraction time on the extraction efficiency of anthocyanins was greater than that of ethanol concentration, which was consistent with the results of Tab.2. 3.2.3. Optimization of technological parameters and the model validation 14
The optimal extraction conditions for TAC were determined by the canonical analysis of response surface methodology. It was found that the optimal conditions were ethanol concentration of 33.61%, microwave irradiation power of 281.98 W and extraction time of 500 s under a fixed ratio of solid to liquid of 1:3 (g/mL). Considering
IP T
the complexity of the actual operation, the confirmatory test was performed using the conditions as: ethanol concentration of 30% and extraction time of 500 s with a different
SC R
microwave irradiation power(MIP) of 240 W or 320 W due to the operational cause of microwave device (the microwave power could only be adjusted at intervals according
U
to its design). Compared with the predicted value of 30.63 mg/100g (for MIP of 240 W)
N
or 32.76 mg/100g(for MIP of 320 W), the TAC yield observed was 30.39
A
0.73(RSD=2.16%, n=3) or 31.160.44 mg/100g (RSD=1.45%, n=3) which gave a
M
relative difference of -0.78% or -4.88%, respectively. So, the optimized model was
ED
effective and feasible for the extraction of anthocyanins from PSP. 3.3 The advantages of iMAE for PSPAs
PT
Microwave-assisted extraction (MAE) has some advantages such as short time and
CC E
high efficiency for extraction of natural ingredients from botanic material. So it has been widely applied to extraction of bioactive compounds from natural resources (Yang, & Zhai, 2010; Routary, & Orsat, 2014; Liazid, Guerrero, Cantos, Palma, & Barroso,
A
2011; Thais, et al., 2018). However, conventional MAE for some starch-rich botanic resources such as PSP has some obvious disadvantages such as easy dextrinization of starch which brings difficulty of separation of extracting solution with the residues and more tanglesome constituents in extracting solution. On the other hand, Sohx extraction 15
has some specialty including using a little quantity of extraction solvent with high extraction efficiency and giving a clear extracting solution without multifarious tanglesome separation process. But a conventional Sohx extractor cannot be put into a small oven chamber of conventional microwave reactor. So microwave-assisted sohx-
IP T
extraction connot be carried out in ordinary laboratory. In this work, a device for microwave-assisted similar sohxlet extraction (MASSE) was developed and iMAE (i.e.
SC R
MASSE) of PSPAs was carried out with a small quantity of acidic ethanol aqueous as
solvent. The iMAE gave a clear extracting solution and maintained pomace in its
U
original appearance (Fig. 4) as Sohx extraction, but high efficiency as conventional
A
N
MAE.
ED
M
Fig. 4 The appearance of PSP residues extracted anthocyanins
For investigating the outstanding effect of iMAE on the extraction of PSPAs, several
PT
of conventional extraction methods including UAE, MAE and HRCE were designed
CC E
for comparing the extraction efficiency of PSPAs. The results were shown in table 3.
Table 3 The advantages of iMAE for PSPA compared with conventional extraction
A
methods
As shown in Table 3, iMAE showed a lot of advantages. First, in terms of the amount of anthocyanins extracted, iMAE was similar to HRCE. But, the former took much less 16
time. It might be that microwave irradiation not only heated the solvent in flask but also irradiated the PSP granules in extractor (Fig. 1) for iMAE. That is to say, microwave had an effective irradiation both on ethanol aqueous and PSP granules simultaneously. Meanwhile, conventional MAE with the same extraction conditions had a low
IP T
extraction efficiency. It was because the gelatinization of PSP starch leaded to the difficulty of separation of extraction solution from residues. Otherwise, the extraction
SC R
efficiency of PSPAs by iMAE (8.33 min) was close to the total efficiency of UAE for three times extraction (total in 30 min).
U
What must be emphasized is that plant residues usually is not considered for
N
comprehensive utilization in the traditional extraction process of natural active
A
ingredients. However, PSP contains a high content of starch which is an available
M
resource. So from the perspective of sustainable development, the weight and the total
ED
sugar content (TSC) of PSP pomace were further compared with those of fresh PSP. Ten gram of fresh PSP granules were dried to constant weight (National Health and
PT
Family Planning Commission of the People’s Republic of China, 2010) and 3.49 g of
CC E
dry matter was obtained. The total sugar content (TSC) of fresh PSP was also detected according to literature ( Chen, 2014 ). And it was 2.71 g in 10 g fresh PSP. The weight and the TSC of PSP pomace obtained by iMAE were 81.7% and 88.2% compared with
A
those of fresh PSP, both were similar to those of HRCE, but higher than those of UAE. Moreover, the pomace was still grainy and its appearance was close to that of fresh PSP granules, which is beneficial to the subsequent comprehensive utilization of residues. Last but the same important, a clear extraction solution was obtained in the process. 17
That is to say, it did not require tedious filtering. In conclusion, compared with the several conventional extraction methods, iMAE provided a new method with some significant advantages: small quantity of solvent, short time, high efficiency, easy to be separated and convenient utilization of PSP pomace.
IP T
3.4 Comparative analysis of stability of anthocyanins under microwave irradiation 3.4.1 HPLC Analysis of components in PSPAs extracts
SC R
HPLC analysis was performed for comparing constituents in extracts obtained by iMAE with UAE, MAE and HRCE. For determination of anthocyanins in extracts, the
U
detection wavelength was set at 520 nm according to the literature (Esatbeyoglu,
N
Rodriguez-Werner, Schlosser, Winterhalter, & Rimbach, 2017). Considering the
A
existence of chlorogenic acid in PSP, the HPLC also was carried out under detection
ED
M
wavelength of 325 nm. The results were shown in Fig. 5.
Fig. 5. HPLC analysis of extracts from PSP detected at 520 nm(A) and 325 nm(B)
( a,
CC E
PT
UAE; b, iMAE; c, HRCE; d, MAE).
HPLC analysis revealed that the constituents of anthocyanins and chlorogenic acid
in the extracts extracted by iMAE was basically same as that by UAE, MAE and HRCE.
A
3.4.2 HPLC-ESI-MS/MS Analysis of components in PSPAs extracts The chemical composition of PSPAs from iMAE was identified according to mass spectra data as compared with UAE. The total ion chromatograms for anthocyanins were shown in Fig. 6. 18
Fig. 6. HPLC-MS/MS analysis of anthocyanins from PSP on a positive ionisation mode (A, UAE; B, iMAE).
IP T
The major components identified by HPLC-ESI-MS/MS were shown in Table 4. Twelve anthocyanins were identified on a positive ionisation mode based on
SC R
fragmentation patterns of individual peaks and past studies (Zhu et al., 2017), the major
compounds of PSPAs were peonidin-3-sophoroside-5-glucoside, peonidin-3-caffeoyl-
U
p-hydroxy- benzoylsophoroside-5-glucoside, cyanidin-3-(6’’-
N
caffeoyl-6’’’-feruloylsophoroside)-5-glucoside, peonidin-3-(6’’-caffeoyl-6’’’-
A
feruloylsophoroside)-5-glucoside. And the chlorogenic acid was identified on a
M
negative ionization. So the results identified by HPLC-ESI-MS/MS indicated that the
ED
major components of anthocyanins and chlorogenic acid in both extracts were
PT
consistent.
CC E
Table 4 Molecular structure of PSPAs extracted by iMAE and UAE identified by HPLC-ESI-MS/MS
A
3.4.3 Effect of microwave irradiation on total anthocyanins content For testing the stability of PSPAs, the variation of concentration of anthocyanin (Ca)
in 30% ethanol aqueous solution ( pH = 2 ) during microwave irradiation (at 300 W) was further investigated according to the Procedure 2.8.3. And the detailed data of Ca after microwave treatment at 1 minute intervals were 21.12 (before processing), 21.63, 19
21.88, 24.96, 24.71, 24.71 24.63, 24.45, 24.46, 24.55, 24.88, 24.80, 24.55, 24.63, 24.63 and 24.80 mg/L (after treatment of 15 minutes), respectively. The results displayed that the Ca remained basically unchanged during microwave irradiation for 15 minutes,
IP T
which was bascally same as that in citric acid aqueous solution (Liu et al., 2018).
4. Conclusion
SC R
In this work, an improved method of microwave-assisted extraction (iMAE) without dextrinization of starch was developed. It has some advantages of high efficiency as
U
conventional MAE, but using a small quantity of solvent and giving a clear extraction
N
solution as sohx extraction, avoiding gelatinization of starch and giving PSP pomace in
A
its original appearance as UAE. The extraction conditions of PSPAs by iMAE with
M
acidic ethanol as solvent was optimized by response surface methodology with a Box-
ED
Behnken experimental design. The optimal conditions to obtain the highest TAC of PSPAs were as follows: ethanol concentration of 30% with a small quantity of critic
PT
acid as regulating reagent of pH, microwave irradiation power of 320 w, extraction time
CC E
of 500 s with a fixed solid-to-liquid ratio of 1:3 (g/mL). Under the optimal conditions, the yield of PSPAs was 31.16 0.44 mg/100g (n=3), which was 92.16% of TAC. Moreover, the results of analysis by HPLC-ESI-MS/MS demonstrated that the
A
constituents of PSPAs extracted by iMAE were consistent with those by UAE using citric acid aqueous as solvent. Compared with other conventional extraction method, the iMAE is very practical for preprocessing of analysis sample because of its advantages including less solvent, high efficiency, short time, simple post-treatment 20
process. Meanwhile, the most traditional industrial method for extraction of PSPAs from purple sweet potatoes is the solid-liquid extraction by mechanical agitation. It is easy to cause starch gelatinization and decay because of the long processing time. This is not
IP T
only unfavorable for the comprehensive utilization of starch in PSP residues, but also will cause environmental pollution. As is known to all, microwave-assisted extraction
SC R
is easy to industrialize. And the experimental results showed that the pomace extracted
anthocyanins by iMAE was in its original appearance and maintained a high TSC (total
U
sugar content, about 88.2% of that in fresh PSP). So, iMAE also provides a fast and
N
practical method for the industrial extraction of anthocyanins from purple sweet potato
M
A
and subsequent comprehensive utilization of starch in PSP pomace.
ED
Acknowledgments
This work was partially supported by scientific research fund of Hunan Provincial
A
CC E
interest.
PT
Education Department (grant No. 14c0563). The authors also declare no conflicts of
21
References Bélafi-Bakó, K., Petra, C., Sándor, B., Zsófia, C., & Cecília, H. (2012). Berry pectins: microwave-assisted extraction and rheological properties. Food and bioprocess technology, 5, 1100-1105. Bonfigli, M., Godoy, E., Reinheimer, M. A., & Scenna, N. J. (2017). Comparison
IP T
between conventional and ultrasound-assisted techniques for extraction of
SC R
anthocyanins from grape pomace. Experimental results and mathematical modeling. Journal of Food Engineering, 207, 56-72.
Burgos, G., Amoros, W., Munoa, L., Sosa, P., Cayhualla, E., & Sanchez, C., et al. (2013).
U
Total phenolic, total anthocyanin and phenolic acid concentrations and antioxidant
N
activity of purple-fleshed potatoes as affected by boiling. Journal of Food
M
A
Composition and Analysis, 30(1), 6-12.
Chen, D. J. (2014). Changes in active ingredients of American Ginseng during storage.
ED
Food Science, 2014, 35(14), 155-158.
PT
Chen, M. S., Zhao, Y., & Yu, S. J. (2015). Optimisation of ultrasonic-assisted extraction of phenolic compounds, antioxidants, and anthocyanins from sugar beet molasses.
CC E
Food chemistry, 172, 534-540. Esatbeyoglu, T., Rodriguez-Werner, M., Schlosser, A., Winterhalter, p., & Rimbach, G.
A
(2017). Fractionation, enzyme inhibitory and cellular antioxidant activity of bioactives from purple sweet potato (Ipomoea batatas). Food chemistry, 221, 447456.
Fan, G. J., Han, Y. B., Gu, Z. X., & Gu, F. R. (2008). Composition and colour stability of anthocyanins extracted from fermented purple sweet potato culture. LWT - Food 22
Science and Technology, 41(8), 1412-1416. Hwang, Y. P., Choi, J. H., Yun, H. J., Han, E. H., Kim, H. G., & Kim, J. Y., et al. (2011). Anthocyanins from purple sweet potato attenuate dimethylnitrosamine-induced liver injury in rats by inducing Nrf2-mediated antioxidant enzymes and reducing COX-2
IP T
and iNOS expression. Food and Chemical Toxicology, 49(1), 93-99. Kano, M., Takayanagi, T., Harada, K., Makino, K., & Ishikawa, F. (2005).
SC R
Antioxidative activity of anthocyanins from purple sweet potato, Ipomoera batatas
cultivar ayamurasaki. Bioscience, Biotechnology and Biochemistry, 69(5), 979-988.
U
Liazid, A., Guerrero, R. F., Cantos, E., Palma, M., & Barroso, C. G. (2011). Microwave
N
assisted extraction of anthocyanins from grape skins. Food Chemistry, 124(3), 1238-
A
1243.
M
Liu, W., Yang, C., Zhou, C., & Dong, X. (2018). Microwave assisted extraction of
ED
anthocyanins from purple sweet potato with phosphoric acid as solvent by response surface methodology. Chemistry ＆ bioengineering, 35(4), 35-40.
PT
Lu, J., Wu, D. M., Zheng, Y. L., Hu, B., Cheng, W., & Zhang, Z. F. (2012). Purple sweet
CC E
potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-ɑ-mediated mitochondrial biogenesis signaling in mice. Free Radical Biology and Medicine, 52(3), 646-659.
A
National Health and Family Planning Commission of the People’s Republic of China. 2010, National food safety standard, determination of moisture in foods. Standards Press of China. Nora, P., Sándor, B., Eva, P., Liisa, M., Miklòsnè, G., Ernő, G., Cecília, H., Riitta, L. K.(2013). Microwave-assisted extraction of anthocyanins from black currant Marc, 23
Food and Bioprocess Technology, 6:(10), 2666-2674. Routary, W., & Orsat, V. (2014). MAE of phenolic compounds from blueberry leaves and comparison with other extraction methods. Industrial Crops and Products, 58, 36-45.
IP T
Santos, D. T., Veggi, P. C., & Meireles, M. A. (2010). Extraction of antioxidant compounds from Jabuticaba (Myrciaria cauliflora) skins: Yield, composition and
SC R
economical evaluation. Journal of Food Engineering, 101(1), 23-31.
Sun, Y. Z., Liao, X. J., Wang, Z. F., Hu, X. S., & Chen, F. (2007). Optimization of
U
microwave-assisted extraction of anthocyanins in red raspberries and identification
N
of anthocyanin of extracts using high-performance liquid chromatography-mass
A
spectrometry. European Food Research Technology, 225, 511-523.
M
Swer, T. L., Chauhan, K., Paul, P. K., & Mukhim, C. (2016). Evaluation of enzyme
ED
treatment conditions on extraction of anthocyanins from Prunus nepalensis L. International Journal of Biological Macromolecules, 92, 867-871.
PT
Tany, L. S., Komal, C., Prodyut, K. P., Mukhim, C.(2016). Evaluation of enzyme
CC E
treatment conditions on extraction of anthocyanins from Prunus nepalensis L. International journal of biological macromolecules, 92, 867-871.
A
Teow, C. C., Truong, V. D., McFeeters, R. F., Thompson, R. L., Pecota, K. V., & Yencho, G. C. (2007). Antioxidant activities, phenolic and β-carotene contents of sweet potato genotypes with varying flesh colours. Food Chemistry, 103(3), 829-838.
Thais W. C., Karen E.L. M., Aline S.C. T., Gabriela N. M., Ana Iraidy S. B., Carlos A. C., Renata G. B., Ronoel L.O. G., Lourdes M.C. C., Renata V. T. (2018) . Phenolic
24
compounds recovery from grape skin using conventional and non-conventional extraction methods. Industrial Crops & Products, 111, 86-91. Yang, Z. D., & Zhai, W. W. (2010). Optimization of microwave-assisted extraction of anthocyanins from purple corn (Zea mays L.) cob and identification with HPLC-MS.
IP T
Innovative Food Science and Emerging Technologies, 11(3), 470-476. Yoshimoto, M., Okuno, S., Yoshinaga, M., Yamakawa, O., Yamaguchi, M., & Yamada,
SC R
J. (1999). Antimutagenicity of sweet potato (Ipomoea batatas) roots. Bioscience, Biotechnology, and Biochemistry, 63(3), 537-541.
U
Zhao, X., Li, J. (2015). Optimization of enzyme hydrolysis of purple sweet potato by
N
cellulase or pectinase for anthocyanin extraction. Food science and technology, 40(4),
A
277-281.
M
Zhu, Z. Z., Guan, Q. Y., Koubaa, M., Barba, F. J., Roohinejad, S., Cravotto, G., et al.
ED
(2017). HPLC-DAD-ESI-MS2 analytical profile of extracts obtained from purple
400.
PT
sweet potato after green ultrasound-assisted extraction. Food Chemistry, 215, 391-
Zhu, Z., Li, S., He, J., Rohit, T., Domenico, M., Francisco, J. B. (2018). Enzyme-
CC E
assisted extraction of polyphenol from edible lotus ( Nelumbo nucifera ) rhizome knot:
Ultra-filtration performance and HPLC-MS2 profile.
A
international, 111, 291-298.
25
Food
research
Figure captions
Fig.1. Schematic diagram of improved microwave-assisted extraction.
condenser PSP granule
SC R
microwave reactor
IP T
Figure 1
extractor
microwave device
A
CC E
PT
ED
M
A
N
U
ethanol aqueous
26
Fig. 2. The effects of single factor on extraction yield of anthocyanins (A, ethanol concentration ; B, microwave irradiation power; C, extraction time).
35
25
30
20
15
10
IP T
30
TAC (mg/100g)
TAC (mg/100g)
Figure 2
25
20
15
SC R
5 10
0
10
20
30
40
50
60
80
160
Acidic ethanol concentration (%)
(B)
U
(A) 30
N
25
20
A
TAC (mg/100g)
240
320
Microwave irradiation power (w)
M
15
200
300
400
500
Time (s)
A
CC E
PT
ED
(C)
27
600
700
400
Fig. 3. Response surface plot for TAC contents showing the effect of independent variables (a, Ethanol concentration and microwave irradiation power; b, Ethanol concentration and extraction time; c, microwave irradiation power and extraction
A
CC E
PT
ED
M
A
N
U
SC R
IP T
time).
28
SC R
IP T
Figure 3
(b)
A
CC E
PT
ED
M
A
N
U
(a)
(c)
29
Fig. 4 The appearance of residue of PSP after extracting anthocyanins
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Figure 4
30
Fig. 5. HPLC analysis of extracts from PSP detected at 520 nm(A) and 325 nm(B) (a, UAE; b, iMAE; c, HRCE; d, MAE).
(B)
A
CC E
PT
ED
M
A
(A)
N
U
SC R
IP T
Figure 5
31
Fig. 6. HPLC-MS/MS analysis of anthocyanins from PSP on a positive ionisation mode (A, UAE; B, iMAE).
6
2.5 2.0 3 4
1
1.5
9 6
2
1.0
8 5
0.5
7
0.0 0
2
4
6
8
10
12
14
16
18
20 22 24 26 Time (min）
32
34
36
38
40
42
44
36
38
40
42
44
N
6
4.0
1
2.0
3 2
4
A
3.0
9
6
1.0
5
0.0 2
4
6
8
10
12
14
16
18
8
7
20 22 24 26 Time (min）
(B)
A
CC E
PT
ED
0
M
Relative Abundance
×10
30
U
(A)
28
SC R
Relative Abundance
×10
IP T
Figure 6
32
28
30
32
34
Table 1 Total anthocyanins content of PSP used in the Box-Behnken design for response surface methodology. Run
Parameters
Response Factor 2 (X2)
Factor 3 (X3)
TAC (mg/100g)
1
30.00
160.00
300.00
8.42
2
40.00
240.00
500.00
31.56
3
30.00
240.00
400.00
29.06
4
30.00
320.00
500.00
31.16
5
20.00
160.00
400.00
8.32
6
40.00
320.00
400.00
25.75
7
30.00
240.00
400.00
26.45
8
20.00
240.00
500.00
9
30.00
160.00
500.00
10
20.00
240.00
300.00
11
30.00
320.00
300.00
12
40.00
240.00
300.00
13
20.00
320.00
400.00
14
30.00
240.00
400.00
24.95
15
40.00
160.00
400.00
17.23
16
30.00
240.00
400.00
25.15
17
30.00
240.00
400.00
23.14
SC R 23.54 21.84 10.62
U
23.34
N
A
M
ED PT CC E A
33
IP T
Factor1 (X1)
20.44 22.84
Table 2 ANOVA for quadratic model Sum of
Df
Squares
F
p-value
Square
Value
Prob > F
87.24
24.48
0.0002
785.13
X1
109.96
1
109.96
30.85
0.0009
X2
279.42
1
279.42
78.40
< 0.0001
X3
256.28
1
256.28
71.91
< 0.0001
X1X2
9.00
1
9.00
2.53
0.1561
X1X3
0.81
1
0.81
0.23
0.6481
X2X3
7.84
1
7.84
2.20
0.1816
X12
49.61
1
49.61
13.92
0.0074
2
60.24
1
60.24
16.90
0.0045
X3
2
2.55
1
2.55
0.71
0.4260
Residual
24.95
7
3.56
Lack of Fit
5.69
3
1.90
0.39
0.7650
Pure Error
19.26
4
4.81
Cor Total
810.08
16
U N A M
ED PT CC E A
34
significant
SC R
Model
X2
9
Mean
IP T
Source
Not significant
Table 3 The advantages of iMAE for PSPA compared with conventional extraction methods UAE*
MAE
HRCE
31.160.44
33.812.58
18.200.97
31.903.18
0.890.12
0.940.02
1.080.09
0.570.11
0.880.04
TAC of the PSP extracts (mg/g)
3.410.27
3.310.19
3.130.09
3.200.39
3.620.28
Extraction rate (%)*
89.88
92.16
100
53.83
94.35
The weight of residue (g)
2.910.22
2.850.06
2.590.15
/
2.820.03
Character of residue
granules
granules
mushy
Total sugar content of residue (g)
2.43
Solid to solvent (g/mL)
1:3
1:10
1:3
Critic acid for 1g PSP (g)
0.035
1.2
0.035
IP T
iMAE
Extraction time (min)
8.33
40
8.33
60
Character of extracting solution
clear and centrifuge easily
clear
gelatinization
clear and
and
centrifuge
centrifuge
easily
320 w
TAC (mg/100g )
30.390.73
Extracts of PSP (g)
2.39
and post-treatment process
2.34
U N
★
and
centrifuge easily
Microwave irradiation power.
granules 2.41
SC R
★
240 w
1:3
0.035
difficultly
A
CC E
PT
ED
M
A
In order to determine the total content of anthocyanins in PSP, four times of UAE (10 minutes each time) was performed. The relative yield of TAC was 40.91% (first time), 36.01% (second time), 14.08% (third time) and 9.00% (fourth time). So, the other extraction rate is a relative extraction rate compared with UAE.
35
Table 4 Molecular structure of PSPAs extracted by iMAE and UAE identified by HPLC-ESIMS/MS.
Peak
Retenti
[M+H]+/
Major
on
[M-H]-
fragment ion
time(m
(m/z)a
(m/z)
iMA
compounds
E
UAE
Reference
in) 2.6
773
611, 449, 287
Cyanidin-3-sophoroside-5-glucoside
√
√
2
2.6
787
625, 463, 301
Peonidin-3-sophoroside-5-glucoside
√
√
3
4.3
893
731, 449, 287
Cyanidin-3-p-hydroxybenzoylsophoroside-5-
√
glucoside 6.0
907
745, 463, 301
Peonidin-3-p-hydroxybenzoylsophoroside-5glucoside
5
7.3
949
787, 449, 287
Cyanidin-3-(6’’-feruloylsophoroside)-5glucoside
6
11.3
963
801, 463, 301
Peonidin-3-(6’’-feruloylsophoroside)-5-
25.2
1111
949, 449, 287
U
glucoside 7
√
SC R
4
IP T
1
√ √
√
√
√
√
2017)
√
√
√
√
√
√
Chlorogenic acid
√
√
Chlorogenic acid
√
√
Cyanidin-3-(6’’-caffeoyl-6’’’-
1069
907, 463, 301
Peonidin-3-caffeoyl-p-
A
25.8
N
feruloylsophoroside)-5-glucoside 8
hydroxybenzoylsophoroside-5-glucoside 26.1
1125
963, 463, 301
Peonidin-3-(6’’
M
9
-caffeoyl-6’’’-
feruloylsophoroside)-5-glucoside
4.2
353
191, 135, 85
11
5.8
353
191, 85
compounds 1-9 and [M-H]- for compound 10 and 11.
A
CC E
PT
a[M+H]+ for
ED
10
36
(Zhu et al.,
S0960-3085(18)30788-0 https://doi.org/10.1016/j.fbp.2019.02.003 FBP 1042
To appear in:
Food and Bioproducts Processing
Received date: Revised date: Accepted date:
6 November 2018 26 January 2019 20 February 2019
Please cite this article as: Liu, Wei, Yang, Chunv, Zhou, Chunjiao, Wen, Zhiyong, Dong, Xinrong, An improved microwave-assisted extraction of anthocyanins from purple sweet potato in favor of subsequent comprehensive utilization of pomace.Food and Bioproducts Processing https://doi.org/10.1016/j.fbp.2019.02.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
An improved microwave-assisted extraction of anthocyanins from purple sweet potato in favor of subsequent comprehensive utilization of pomace
IP T
Wei Liu, Chunv Yang, Chunjiao Zhou, Zhiyong Wen, Xinrong Dong﹡
SC R
College of Science, Hunan Agricultural University, Changsha 410128, P. R. China
U
﹡Corresponding author: Xinrong Dong
A
N
E-mail: [email protected]
CC E
PT
ED
M
Graphical abstract
A
Highlights
An improved microwave-assisted extraction of anthocyanins from starch-rich material.
High efficiency as conventional microwave-assisted extraction. 1
Less solvent and clear extraction solution as sohx extraction.
Pomace in its original appearance without gelatinization of starch as USE.
High efficiency for extraction of anthocyanins in favor of utilization of pomace.
IP T
Abstract: In order to enhance the extraction efficiency of anthocyanins and
improved
microwave-assisted
extraction
(iMAE)
SC R
subsequent comprehensive utilization of pomace from purple sweet potato (PSP), of
anthocyanins
an
without
dextrinization of starch was developed in this work. Firstly, the extraction conditions of
N
U
PSP anthocyanins (PSPAs) were optimized by Response Surface Methodology (RSM)
A
and the optimal parameters were as follows: solid-to-liquid ratio of 1:3 (g / mL), ethanol
M
concentration of 30% with a small quantity of critic acid as regulating reagent of pH, microwave irradiation power of 320 w, extraction time of 500 s. Under the conditions,
ED
the yield of anthocyanins was 31.16 mg/100g PSP (RSD =1.45%, n=3), which was a
PT
relative error of -4.88% compared with the predicted value (32.76 mg/100g PSP) by model of Response Surface Methodology. By HPLC-ESI-MS/MS analysis, the
CC E
composition of anthocyanins obtained by iMAE is basically the same as that of ultrasound-assisted extraction with citric acid aqueous. And the residue obtained by
A
iMAE was in its original appearance and the total sugar content (TSC) in pomace was 88.2% of that in fresh PSP. The iMAE provided a method for the quick extraction of anthocyanins with some advantages of high efficiency as conventional MAE, but avoided gelatinization of starch which is useful for further utilization of starch in pomace. 2
Keywords: purple sweet potato; anthocyanins; microwave-assisted extraction; gelatinization of starch
IP T
1. Introduction Nowadays, purple sweet potato (PSP) has been cultivated widely in China as a
SC R
resource of food and industrial material (Liu, Mu, Sun, Zhang, & Chen, 2013).
PSP
contains a significant content of anthocyanins compared to white, yellow, and orange
U
ones. The purple sweet potato anthocyanins (PSPAs) were found to have remarkable
N
range of effective activities such as antioxidation (Teow et al., 2007), antimutation
A
(Yoshimoto et al., 1999), and hepato-protection (Hwang et al., 2011), memory
M
enhancing (Lu et al., 2012). Thus, there is a growing popularity in food, cosmetics, and
ED
pharmaceutical industries due to their non-toxicity, unique color, and nutritional benefits (Zhu et al., 2017).
PT
The most traditional industrial method for extraction of PSPAs from PSP is the solid-
CC E
liquid extraction by mechanical agitation. It is easy to cause starch gelatinization which is tedious for solid-liquid separation and unfavorable for the comprehensive utilization of starch in PSP residues. Some conventional solvent (such as methanol, acetone)
A
extraction (CSE) of anthocyanins have been described in the literatures (Kano, Takayanagi, Harada, Makino, & Ishikawa, 2005; Santos, Veggi, & Meireles, 2010; Burgos et al., 2013). While disadvantages including long time of extraction, solventconsuming were always found during the CSE (Sun, Liao, Wang, Hu, & Chen, 2007). 3
In order to improve the extraction efficiency, enzyme-assisted extraction has been currently widely used in extraction of value-added compounds from plant materials (Zhao & Li, 2015; Tanya et al., 2016; Zhu et al., 2018) with effective and nontoxic characteristic. However, it also has the disadvantages of strict pH value and long time.
IP T
Recently, ultrasonic-assisted extraction (UAE) were applied to extraction of anthocyanins (Chen, Zhao, & Yu, 2015; Bonfigli, Godoy, Reinheimer, & Scenna, 2017).
SC R
It has many advantages such as high efficiency, but there are still some disadvantages
such as a long time compared with microwave-assisted extraction (MAE). MAE is a
U
promising technology with a very short time for extraction of bioactive compounds
N
from natural resources (Yang, & Zhai, 2010; Bélafi-Bakó et al., 2012; Nora Pap et al.,
A
2013; Routary, & Orsat, 2014; Liazid, Guerrero, Cantos, Palma, & Barroso, 2011; Thais,
M
et al., 2018). However, the conventional MAE for extraction of PSPAs from PSP also
ED
has some disadvantages including using a large quantity of extraction solvent and bringing dextrinization of starch in PSP. In the conventional MAE process,
PT
dextrinization of starch will firstly bring difficulty of filtration. But more important, it
CC E
affects the subsequent utilization of starch. This not only causes waste of starch resource, but also brings about environmental problems. In this work, an improved microwaveassisted extraction (iMAE) of PSPAs from PSP was developed. It had some advantages
A
of high efficiency as conventional MAE, but used a small quantity of acidic ethanol aqueous as solvent and gave a clear extraction solution as conventional sohx extraction. More importantly, the PSP pomace extracted anthocyanins maintained in its original shape with high content of total sugar, which was conducive to its subsequent 4
comprehensive utilization.
2. Materials and methods 2.1. Materials
IP T
Fresh purple sweet potato (PSP) was obtained from a local market in Hunan Agriculture University. Prior to extraction, fresh PSP was washed and cut into about 2
SC R
mm granule in diameter. 2.2. Chemicals and reagents
U
Reagents of analytical grade (ethanol, citric acid, hydrochloric acid, potassium
N
chloride, sodium acetate) and reagents of HPLC grade (methanol and acetonitrile) were
A
purchased from sinopharm chemical Reagent Co., Ltd. (Shanghai, China).
M
2.3. The improved microwave-assisted extraction device and its work principle
ED
The improved microwave-assisted extraction (iMAE) has been performed in a Microwave Reactor of MCR-3 illustrated in Fig. 1. The improved apparatus for iMAE
PT
was composed of a 3-neck round-bottom flask, a soxh-like extractor improved from a
CC E
water segregator, a microwave reactor and a condenser.
A
Fig.1. Schematic diagram of improved microwave-assisted extraction
Compared the conventional MAE, the PSP granules (material) were put in soxh-like extractor but not in bottom of flask. In the process of microwave-assisted extraction, the sample (i.e. PSP granules) and solvent (i.e. ethanol aqueous) would be 5
simultaneously irradiated by microwave emitted from microwave device. The solvent was heated up, then evaporated and became into vapor. Then the solvent vapor entered into the condenser outside the microwave cavity and was condensed. The condensed solvent flowed into the extractor and soaked the sample irradiated simultaneously by
efficiently carried out.
SC R
2.4. Single factor conditions for iMAE of anthocyanins from PSP
IP T
microwave from the same microwave device. So, microwave-assisted extraction was
The iMAE was performed in a Microwave Reactor of MCR-3. Ten gram of PSP
U
granules were soaked into 2 mL 10% of citric acid aqueous solution for 30 min and then
N
placed into the extractor. Ethanol aqueous (adjusting pH = 2 with citric acid ) of 30 mL
A
was added into the 3-neck round bottom flask of 100 mL. Three single factors including
M
the concentration of ethanol aqueous (0, 10, 20, 30, 40, 50 and 60%, v/v), microwave
ED
irradiation power (80, 160, 240, 320 and 400 W) and extraction time (200, 300, 400, 500, 600 s) were investigated. Then, the mixture was centrifuged at 8000 rpm for 5 min
PT
and the supernatant was collected for analysis.
CC E
2.5. Optimization design by response surface methodology and statistical analysis In order to optimize the extraction conditions of anthocyanins from PSP, response
surface methodology (RSM) was applied to design of the experiment. Box-Behnken
A
Design (BBD) with three factors was used. The design consisted of 17 randomized runs with five replicates at the center of the domain. The independent variables and their levels were as follows: concentration of ethanol aqueous (20 to 40%, v/v), microwave irradiation power (160 to 320 w) and extraction time (300 to 500 s). The relation 6
between the coded values and actual values was obtained in the following equation:
X
xi xo Δxi
(1)
Where X is the coded value, xi is the actual value, x0 represents the actual value of xi
IP T
at the centre point, and Δxi corresponds to the step change value. The response function(Y) was partitioned into linear, quadratic, and interactive components. The
3
3
i 1
i 1
2
SC R
response variables were fitted to the following second order polynomial model (Eq. (2)). 3
Y β 0 βiXi βiiXi 2 βijXiXj i 1 ji 1
(2)
U
Where Y stands for the total anthocyanins content (TAC), β0 is the intercept, βi, βii,
N
and βij represent the coefficients of the linear, quadratic, and interactive effects,
ED
2.6.1 Validation experiment
M
2.6. Validate and Contrast experiment
A
respectively. Xi and Xj are coded values of the independent variables.
The optimal iMAE conditions were obtained by the single factor experiments and
PT
RSM, then the experiments under the optimal conditions were performed in triplicates.
CC E
2.6.2 Conventional ultrasound-assisted extraction (UAE) with citric acid aqueous as solvent
Ten gram of PSP granules and 100 mL of 3% (g/mL) critic acid aqueous were placed
A
into a conical flask of 250 mL and extracted by ultrasound wave for 10 min. The mixtures were filtrated with filter paper and the filtrate was collected. The residues of PSP were treated as above procedures until the anthocyanins in PSP were extracted completely. The filtrate was centrifuged at 8000 rpm for 5 min and the supernatant was 7
collected for analysis. 2.6.3 Conventional MAE under the optimal iMAE conditions Conventional MAE was carried out under the optimal iMAE conditions. And detailed description was as follows: Ten gram of PSP granules soaked with 2 mL of 10%
IP T
citric acid aqueous solution for 30 min and 30 mL of acidic ethanol aqueous ( pH = 2 adjusted by citric acid) were added into the 3-neck round-bottom flask of 100 mL and
SC R
extracted for 500s by MAE. Then, the mixture was further processed according to the Procedure 2.6.2.
U
2.6.4 Traditional hot reflux continuous extraction (HRCE) under the optimal iMAE
N
conditions
A
PSP granules were extracted for 60 min by HRCE using electric heating-jacket
M
instead of the microwave reactor and other optimal conditions of iMAE ( detailed
Procedure 2.6.2.
ED
description as 2.6.3). Then, the mixture was further processed according to the
total
content
of
anthocyanins
was
determined
according
to
the
CC E
The
PT
2.7. Determination of total anthocyanins content (TAC)
spectrophotometric pH-differential method (Swer, Chauhan, Paul, & Mukhim, 2016). The sample extracted was mixed with hydrochloric acid - potassium chloride buffer
A
(pH=1.0) and sodium acetate buffer (pH=4.5), respectively. For each sample, the absorbance was measured at 520 and 700 nm. The TAC was calculated according to Eq. (3). TAC (mg 100g)
A520 A700pH1.0 A520 A700pH4.5 MW DF V 100 ε L mf 8
(3)
Where TAC is the total anthocyanins content expressed as cyanidin-3-glucoside equivalent (CGE) (mg/100g), MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol), DF is the dilution factor, V is the volumn of solution (mL), ε is the cyanindin-3-glucoside molar absorbance (26,900), L is the cell path length (1 cm), mf
2.8. Stability of anthocyanins under microwave irradiation
SC R
2.8.1 Comparative analysis of components in PSPAs extracts by HPLC
IP T
is the weight of the sample (g).
The HPLC analysis of PSPAs extracts was obtained using an Agilent 1200 series
U
high performance liquid chromatography. Chromatographic separations were carried
N
out using an Agilent C18 column (250 mm × 4.6 mm, 5 µm; Agilent Technologies Co.,
A
Ltd., Shanghai, China). And the detection wavelength was set at 520 nm, column
M
temperature was 25℃. And the mobile phase consisted of solvents A (acetic acid : water
ED
= 0.5 : 100, v/v) and B (acetonitrile), the gradient elution program was used as follows: 15% B between 0 and 30 min; 20% B between 30 and 80 min; 15% B between 80 and
PT
85 min. The flow rate was 0.50 mL/min and the injection volume was 20 µL. The
CC E
samples were dissolved in distilled water containing 50% methanol and were filtered through a 0.45 µm Millipore filter.
A
2.8.2 Comparative analysis of components in PSPAs extracts by HPLC-ESI-MS/MS Qualitative analysis of anthocyanins was conducted using the Agilent UHD Accurate
Mass Q-TOF LC-MS analysis. Chromatographic separation was carried out on Agilent Eclipse Plus-C18 column (250 mm × 4.6 mm, 5 µm). Flow rate was 1 mL/min, sample injection volume was 15 µL, the detection wavelength was set at 520 nm. Solvent A 9
was 0.1% formic acid in distilled water (v/v), and solvent B was purified acetonitrile. The following gradients were utilised: 0-20 min, 15%B; 20-40 min, 20-100%B; 40-50 min, 100%B. For the identification of anthocyanins, the ESI source was set in positive and negitive ionisation mode. The applied conditions were set as follows: capillary
IP T
temperature, 300℃; capillary voltage, 4000 V (positive ionisation mode)/3000 V (negitive ionisation mode); nebulizer pressure, 30 psi; dry gas flow at 10 L/min. Scan
SC R
range was measured from m/z 100 up to 1500.
2.8.3 Effect of microwave irradiation on total anthocyanins content (TAC)
U
The experiments were carried out as our previous method (Liu et al., 2018) and the
(critic acid used for regulating pH) were mixed and the
A
ethanol aqueous in pH = 2
N
detailed description were as follows. Eighty gram of PSP granules and 260 mL of 30%
M
mixures were extracted according to the Procedure 2.6.2. Then the filtrate was placed
ED
into a conical flask of 500 mL and then irradiated for 15 min by microwave of 320 W. In this process, appropriate amount of solution was taken out at intervals of 1 min and
PT
anthocyanins were analysized. The concentration of anthocyanins (Ca) was calculated
CC E
according to Eq. (4).
Ca ( mg/L ) = TAC*mf*10/V
(4)
Where Ca is the concentration of anthocyanins in solution. The meanings of other
A
symbols were the same as that of 2.7. 2.9 Analysis of pomace extracted anthocyanins The weight and the total sugar content (TSC) of pomace were checked according to the methods of literature (National Health and Family Planning Commission of the 10
People’s Republic of China, 2010).
3. Results and discussion 3.1 Determination of single factor experiment
IP T
3.1.1. Effect of adding acid on stability of anthocyanins PSPAs were more stable under the acid conditions than the neutral conditions and
SC R
alkaline conditions (Fan, Han, Gu, & Gu, 2008) and citric acid was used for adjusting the pH of extraction solvent in thi work. The experimental results indicated that the
U
good conditions were as follows: Ethanol aqueous as extraction solvent was adjusted
N
into pH = 2 by citric acid and the PSP granules (10 g) were simultaneously soaked by
A
2 mL of 10% (w/w) citric acid aqueous solution for 30 min before iMAE.
M
3.1.2. Effect of ethanol concentration on extraction yield of anthocyanins
ED
The effect of ethanol concentration varying from 0 to 60% on extraction yield of anthocyanins in PSP was investigated (Fig. 2(A)) while other extraction variables were
PT
fixed. The results indicated that the content of anthocyanins in extraction solution
CC E
increased as the ethanol concentration increased from 0 to 60%. It might be that more ethanol became gas when ethanol concentration increased. That is to say, the quantity of ethanol flowed into PSP granules increases with a higher concentration of ethanol.
A
So, more anthocyanins in PSP were extracted into solution with a higher concentration of ethanol aqueous. However, there was no significant differences in extraction yield from 30% to 60%. So 30% ethanol aqueous was used for the next experiment. 3.1.3. Effect of microwave irradiation power on extraction yield of anthocyanins 11
The effect of microwave irradiation power (MIP) on extraction yield of anthocyanins was shown in Fig. 2(B). The content of anthocyanins extracted from PSP was firstly increased with the increase of MIP from 80 to 240 w, and then there was a slow increase while the MIP changes from 240 to 400 w. Therefore, MIP of 240 w was chosen as a
IP T
compromise and fixed in the iMAE experiments.
SC R
Fig. 2. The effects of single factor on extraction yield of anthocyanins in PSP (A,
U
ethanol concentration ; B, microwave irradiation power; C, extraction time)
N
3.1.4. Effect of microwave irradiation time on extraction yield of anthocyanins
A
As we can seen in Fig. 2(C), the results shown that the content of anthocyanins
M
extracted from PSP increased rapidly when the extraction time varied from 200 to 500
ED
s, then it was increased smoothly until the time was 700 s. Actually, the anthocyanins in PSP were almost fully extracted out after 500 s.
PT
3.2 RSM statistical analysis
CC E
3.2.1 Analysis of the adequacy of the fitted model According to the results of above single factor experiments, ethanol concentration
from 20% to 40% (v/v), microwave irradiation power from 160 to 320 W and extraction
A
time from 300 to 500 s were chosen for RSM experiments. The optimization for extraction of anthocyanins from PSP was investigated using RSM, and three parameters for the following RSM design were X1 (ethanol concentration), X2 (microwave irradiation power), and X3 (microwave extraction time), 12
respectively. The response of each independent variable can be seen in Table 1.
Table 1 Total anthocyanins content of PSP used in the Box-Behnken design for response
IP T
surface methodology.
The regression equation in coded level neglecting insignificant terms was generated:
SC R
TAC mg 100g 25.75 3.71X1 5.91X 2 5.66X 3 1.50X1X 2 0.45X1X 3 1.40X 2 X 3 3.43X12 3.78X 2 2 0.78X 3 2
The analysis of variance for the fitted quadratic polynomial model for optimization
A
M
Table 2 ANOVA for quadratic model
N
U
of extraction parameters was presented in Table 2.
ED
The determination coefficient (R2) of the predicted model was 0.97, showing that only 0.03% of the variation can't be explained by the present model, and the regression
PT
model was highly significant (P<0.01) and could be used to monitor the optimization.
CC E
While the lack of fit was associated with a p-value of 0.7650 which is not significant. As can be observed in Table 2, the linear terms of ethanol concentration(X1, P<0.001), microwave irradiation power(X2, P<0.001), microwave extraction time(X3, P<0.001)
A
were highly significant; the quadratic terms of ethanol concentration(X12, P<0.01), microwave irradiation power(X22, P<0.01) reached also highly significant. The other terms were not significant (P>0.05). 3.2.2 Interactions of different experimental factors on the effects of response variables 13
RSM was also applied to understand the interaction relationship between the test variables affecting the selected process response (TAC). Three-dimensional response surface were illustrated in Fig. 3.
IP T
Fig. 3. Response surface plot for TAC contents showing the effect of independent variables (a, Ethanol concentration and microwave irradiation power; b, Ethanol
SC R
concentration and extraction time; c, microwave irradiation power and extraction time).
U
In Fig. 3, the effects of ethanol concentration, microwave irradiation power, and
N
microwave extraction time on TAC were shown. It was observed in Fig.3(a) that the
A
TAC was increased with an increase of ethanol concentration and microwave irradiation
M
power while the microwave extraction time was fixed. The higher TAC was obtained
ED
at ethanol concentration of 30 to 40% and the microwave irradiation power of 240 to 320 W. The effects of ethanol concentration and extraction time on the TAC of the
PT
extracts were reflected in Fig. 3(b), a similar linear increase in TAC with the increase
CC E
of extraction time and ethanol concentration at a fixed microwave irradiation power. Fig. 3(c) shown that the TAC was affected by microwave irradiation power and extraction time, exhibiting a clear increase in content with the raise of the two parameter.
A
Fom Fig. 3, it also clearly demonstrated that the effect of the microwave irradiation power and extraction time on the extraction efficiency of anthocyanins was greater than that of ethanol concentration, which was consistent with the results of Tab.2. 3.2.3. Optimization of technological parameters and the model validation 14
The optimal extraction conditions for TAC were determined by the canonical analysis of response surface methodology. It was found that the optimal conditions were ethanol concentration of 33.61%, microwave irradiation power of 281.98 W and extraction time of 500 s under a fixed ratio of solid to liquid of 1:3 (g/mL). Considering
IP T
the complexity of the actual operation, the confirmatory test was performed using the conditions as: ethanol concentration of 30% and extraction time of 500 s with a different
SC R
microwave irradiation power(MIP) of 240 W or 320 W due to the operational cause of microwave device (the microwave power could only be adjusted at intervals according
U
to its design). Compared with the predicted value of 30.63 mg/100g (for MIP of 240 W)
N
or 32.76 mg/100g(for MIP of 320 W), the TAC yield observed was 30.39
A
0.73(RSD=2.16%, n=3) or 31.160.44 mg/100g (RSD=1.45%, n=3) which gave a
M
relative difference of -0.78% or -4.88%, respectively. So, the optimized model was
ED
effective and feasible for the extraction of anthocyanins from PSP. 3.3 The advantages of iMAE for PSPAs
PT
Microwave-assisted extraction (MAE) has some advantages such as short time and
CC E
high efficiency for extraction of natural ingredients from botanic material. So it has been widely applied to extraction of bioactive compounds from natural resources (Yang, & Zhai, 2010; Routary, & Orsat, 2014; Liazid, Guerrero, Cantos, Palma, & Barroso,
A
2011; Thais, et al., 2018). However, conventional MAE for some starch-rich botanic resources such as PSP has some obvious disadvantages such as easy dextrinization of starch which brings difficulty of separation of extracting solution with the residues and more tanglesome constituents in extracting solution. On the other hand, Sohx extraction 15
has some specialty including using a little quantity of extraction solvent with high extraction efficiency and giving a clear extracting solution without multifarious tanglesome separation process. But a conventional Sohx extractor cannot be put into a small oven chamber of conventional microwave reactor. So microwave-assisted sohx-
IP T
extraction connot be carried out in ordinary laboratory. In this work, a device for microwave-assisted similar sohxlet extraction (MASSE) was developed and iMAE (i.e.
SC R
MASSE) of PSPAs was carried out with a small quantity of acidic ethanol aqueous as
solvent. The iMAE gave a clear extracting solution and maintained pomace in its
U
original appearance (Fig. 4) as Sohx extraction, but high efficiency as conventional
A
N
MAE.
ED
M
Fig. 4 The appearance of PSP residues extracted anthocyanins
For investigating the outstanding effect of iMAE on the extraction of PSPAs, several
PT
of conventional extraction methods including UAE, MAE and HRCE were designed
CC E
for comparing the extraction efficiency of PSPAs. The results were shown in table 3.
Table 3 The advantages of iMAE for PSPA compared with conventional extraction
A
methods
As shown in Table 3, iMAE showed a lot of advantages. First, in terms of the amount of anthocyanins extracted, iMAE was similar to HRCE. But, the former took much less 16
time. It might be that microwave irradiation not only heated the solvent in flask but also irradiated the PSP granules in extractor (Fig. 1) for iMAE. That is to say, microwave had an effective irradiation both on ethanol aqueous and PSP granules simultaneously. Meanwhile, conventional MAE with the same extraction conditions had a low
IP T
extraction efficiency. It was because the gelatinization of PSP starch leaded to the difficulty of separation of extraction solution from residues. Otherwise, the extraction
SC R
efficiency of PSPAs by iMAE (8.33 min) was close to the total efficiency of UAE for three times extraction (total in 30 min).
U
What must be emphasized is that plant residues usually is not considered for
N
comprehensive utilization in the traditional extraction process of natural active
A
ingredients. However, PSP contains a high content of starch which is an available
M
resource. So from the perspective of sustainable development, the weight and the total
ED
sugar content (TSC) of PSP pomace were further compared with those of fresh PSP. Ten gram of fresh PSP granules were dried to constant weight (National Health and
PT
Family Planning Commission of the People’s Republic of China, 2010) and 3.49 g of
CC E
dry matter was obtained. The total sugar content (TSC) of fresh PSP was also detected according to literature ( Chen, 2014 ). And it was 2.71 g in 10 g fresh PSP. The weight and the TSC of PSP pomace obtained by iMAE were 81.7% and 88.2% compared with
A
those of fresh PSP, both were similar to those of HRCE, but higher than those of UAE. Moreover, the pomace was still grainy and its appearance was close to that of fresh PSP granules, which is beneficial to the subsequent comprehensive utilization of residues. Last but the same important, a clear extraction solution was obtained in the process. 17
That is to say, it did not require tedious filtering. In conclusion, compared with the several conventional extraction methods, iMAE provided a new method with some significant advantages: small quantity of solvent, short time, high efficiency, easy to be separated and convenient utilization of PSP pomace.
IP T
3.4 Comparative analysis of stability of anthocyanins under microwave irradiation 3.4.1 HPLC Analysis of components in PSPAs extracts
SC R
HPLC analysis was performed for comparing constituents in extracts obtained by iMAE with UAE, MAE and HRCE. For determination of anthocyanins in extracts, the
U
detection wavelength was set at 520 nm according to the literature (Esatbeyoglu,
N
Rodriguez-Werner, Schlosser, Winterhalter, & Rimbach, 2017). Considering the
A
existence of chlorogenic acid in PSP, the HPLC also was carried out under detection
ED
M
wavelength of 325 nm. The results were shown in Fig. 5.
Fig. 5. HPLC analysis of extracts from PSP detected at 520 nm(A) and 325 nm(B)
( a,
CC E
PT
UAE; b, iMAE; c, HRCE; d, MAE).
HPLC analysis revealed that the constituents of anthocyanins and chlorogenic acid
in the extracts extracted by iMAE was basically same as that by UAE, MAE and HRCE.
A
3.4.2 HPLC-ESI-MS/MS Analysis of components in PSPAs extracts The chemical composition of PSPAs from iMAE was identified according to mass spectra data as compared with UAE. The total ion chromatograms for anthocyanins were shown in Fig. 6. 18
Fig. 6. HPLC-MS/MS analysis of anthocyanins from PSP on a positive ionisation mode (A, UAE; B, iMAE).
IP T
The major components identified by HPLC-ESI-MS/MS were shown in Table 4. Twelve anthocyanins were identified on a positive ionisation mode based on
SC R
fragmentation patterns of individual peaks and past studies (Zhu et al., 2017), the major
compounds of PSPAs were peonidin-3-sophoroside-5-glucoside, peonidin-3-caffeoyl-
U
p-hydroxy- benzoylsophoroside-5-glucoside, cyanidin-3-(6’’-
N
caffeoyl-6’’’-feruloylsophoroside)-5-glucoside, peonidin-3-(6’’-caffeoyl-6’’’-
A
feruloylsophoroside)-5-glucoside. And the chlorogenic acid was identified on a
M
negative ionization. So the results identified by HPLC-ESI-MS/MS indicated that the
ED
major components of anthocyanins and chlorogenic acid in both extracts were
PT
consistent.
CC E
Table 4 Molecular structure of PSPAs extracted by iMAE and UAE identified by HPLC-ESI-MS/MS
A
3.4.3 Effect of microwave irradiation on total anthocyanins content For testing the stability of PSPAs, the variation of concentration of anthocyanin (Ca)
in 30% ethanol aqueous solution ( pH = 2 ) during microwave irradiation (at 300 W) was further investigated according to the Procedure 2.8.3. And the detailed data of Ca after microwave treatment at 1 minute intervals were 21.12 (before processing), 21.63, 19
21.88, 24.96, 24.71, 24.71 24.63, 24.45, 24.46, 24.55, 24.88, 24.80, 24.55, 24.63, 24.63 and 24.80 mg/L (after treatment of 15 minutes), respectively. The results displayed that the Ca remained basically unchanged during microwave irradiation for 15 minutes,
IP T
which was bascally same as that in citric acid aqueous solution (Liu et al., 2018).
4. Conclusion
SC R
In this work, an improved method of microwave-assisted extraction (iMAE) without dextrinization of starch was developed. It has some advantages of high efficiency as
U
conventional MAE, but using a small quantity of solvent and giving a clear extraction
N
solution as sohx extraction, avoiding gelatinization of starch and giving PSP pomace in
A
its original appearance as UAE. The extraction conditions of PSPAs by iMAE with
M
acidic ethanol as solvent was optimized by response surface methodology with a Box-
ED
Behnken experimental design. The optimal conditions to obtain the highest TAC of PSPAs were as follows: ethanol concentration of 30% with a small quantity of critic
PT
acid as regulating reagent of pH, microwave irradiation power of 320 w, extraction time
CC E
of 500 s with a fixed solid-to-liquid ratio of 1:3 (g/mL). Under the optimal conditions, the yield of PSPAs was 31.16 0.44 mg/100g (n=3), which was 92.16% of TAC. Moreover, the results of analysis by HPLC-ESI-MS/MS demonstrated that the
A
constituents of PSPAs extracted by iMAE were consistent with those by UAE using citric acid aqueous as solvent. Compared with other conventional extraction method, the iMAE is very practical for preprocessing of analysis sample because of its advantages including less solvent, high efficiency, short time, simple post-treatment 20
process. Meanwhile, the most traditional industrial method for extraction of PSPAs from purple sweet potatoes is the solid-liquid extraction by mechanical agitation. It is easy to cause starch gelatinization and decay because of the long processing time. This is not
IP T
only unfavorable for the comprehensive utilization of starch in PSP residues, but also will cause environmental pollution. As is known to all, microwave-assisted extraction
SC R
is easy to industrialize. And the experimental results showed that the pomace extracted
anthocyanins by iMAE was in its original appearance and maintained a high TSC (total
U
sugar content, about 88.2% of that in fresh PSP). So, iMAE also provides a fast and
N
practical method for the industrial extraction of anthocyanins from purple sweet potato
M
A
and subsequent comprehensive utilization of starch in PSP pomace.
ED
Acknowledgments
This work was partially supported by scientific research fund of Hunan Provincial
A
CC E
interest.
PT
Education Department (grant No. 14c0563). The authors also declare no conflicts of
21
References Bélafi-Bakó, K., Petra, C., Sándor, B., Zsófia, C., & Cecília, H. (2012). Berry pectins: microwave-assisted extraction and rheological properties. Food and bioprocess technology, 5, 1100-1105. Bonfigli, M., Godoy, E., Reinheimer, M. A., & Scenna, N. J. (2017). Comparison
IP T
between conventional and ultrasound-assisted techniques for extraction of
SC R
anthocyanins from grape pomace. Experimental results and mathematical modeling. Journal of Food Engineering, 207, 56-72.
Burgos, G., Amoros, W., Munoa, L., Sosa, P., Cayhualla, E., & Sanchez, C., et al. (2013).
U
Total phenolic, total anthocyanin and phenolic acid concentrations and antioxidant
N
activity of purple-fleshed potatoes as affected by boiling. Journal of Food
M
A
Composition and Analysis, 30(1), 6-12.
Chen, D. J. (2014). Changes in active ingredients of American Ginseng during storage.
ED
Food Science, 2014, 35(14), 155-158.
PT
Chen, M. S., Zhao, Y., & Yu, S. J. (2015). Optimisation of ultrasonic-assisted extraction of phenolic compounds, antioxidants, and anthocyanins from sugar beet molasses.
CC E
Food chemistry, 172, 534-540. Esatbeyoglu, T., Rodriguez-Werner, M., Schlosser, A., Winterhalter, p., & Rimbach, G.
A
(2017). Fractionation, enzyme inhibitory and cellular antioxidant activity of bioactives from purple sweet potato (Ipomoea batatas). Food chemistry, 221, 447456.
Fan, G. J., Han, Y. B., Gu, Z. X., & Gu, F. R. (2008). Composition and colour stability of anthocyanins extracted from fermented purple sweet potato culture. LWT - Food 22
Science and Technology, 41(8), 1412-1416. Hwang, Y. P., Choi, J. H., Yun, H. J., Han, E. H., Kim, H. G., & Kim, J. Y., et al. (2011). Anthocyanins from purple sweet potato attenuate dimethylnitrosamine-induced liver injury in rats by inducing Nrf2-mediated antioxidant enzymes and reducing COX-2
IP T
and iNOS expression. Food and Chemical Toxicology, 49(1), 93-99. Kano, M., Takayanagi, T., Harada, K., Makino, K., & Ishikawa, F. (2005).
SC R
Antioxidative activity of anthocyanins from purple sweet potato, Ipomoera batatas
cultivar ayamurasaki. Bioscience, Biotechnology and Biochemistry, 69(5), 979-988.
U
Liazid, A., Guerrero, R. F., Cantos, E., Palma, M., & Barroso, C. G. (2011). Microwave
N
assisted extraction of anthocyanins from grape skins. Food Chemistry, 124(3), 1238-
A
1243.
M
Liu, W., Yang, C., Zhou, C., & Dong, X. (2018). Microwave assisted extraction of
ED
anthocyanins from purple sweet potato with phosphoric acid as solvent by response surface methodology. Chemistry ＆ bioengineering, 35(4), 35-40.
PT
Lu, J., Wu, D. M., Zheng, Y. L., Hu, B., Cheng, W., & Zhang, Z. F. (2012). Purple sweet
CC E
potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-ɑ-mediated mitochondrial biogenesis signaling in mice. Free Radical Biology and Medicine, 52(3), 646-659.
A
National Health and Family Planning Commission of the People’s Republic of China. 2010, National food safety standard, determination of moisture in foods. Standards Press of China. Nora, P., Sándor, B., Eva, P., Liisa, M., Miklòsnè, G., Ernő, G., Cecília, H., Riitta, L. K.(2013). Microwave-assisted extraction of anthocyanins from black currant Marc, 23
Food and Bioprocess Technology, 6:(10), 2666-2674. Routary, W., & Orsat, V. (2014). MAE of phenolic compounds from blueberry leaves and comparison with other extraction methods. Industrial Crops and Products, 58, 36-45.
IP T
Santos, D. T., Veggi, P. C., & Meireles, M. A. (2010). Extraction of antioxidant compounds from Jabuticaba (Myrciaria cauliflora) skins: Yield, composition and
SC R
economical evaluation. Journal of Food Engineering, 101(1), 23-31.
Sun, Y. Z., Liao, X. J., Wang, Z. F., Hu, X. S., & Chen, F. (2007). Optimization of
U
microwave-assisted extraction of anthocyanins in red raspberries and identification
N
of anthocyanin of extracts using high-performance liquid chromatography-mass
A
spectrometry. European Food Research Technology, 225, 511-523.
M
Swer, T. L., Chauhan, K., Paul, P. K., & Mukhim, C. (2016). Evaluation of enzyme
ED
treatment conditions on extraction of anthocyanins from Prunus nepalensis L. International Journal of Biological Macromolecules, 92, 867-871.
PT
Tany, L. S., Komal, C., Prodyut, K. P., Mukhim, C.(2016). Evaluation of enzyme
CC E
treatment conditions on extraction of anthocyanins from Prunus nepalensis L. International journal of biological macromolecules, 92, 867-871.
A
Teow, C. C., Truong, V. D., McFeeters, R. F., Thompson, R. L., Pecota, K. V., & Yencho, G. C. (2007). Antioxidant activities, phenolic and β-carotene contents of sweet potato genotypes with varying flesh colours. Food Chemistry, 103(3), 829-838.
Thais W. C., Karen E.L. M., Aline S.C. T., Gabriela N. M., Ana Iraidy S. B., Carlos A. C., Renata G. B., Ronoel L.O. G., Lourdes M.C. C., Renata V. T. (2018) . Phenolic
24
compounds recovery from grape skin using conventional and non-conventional extraction methods. Industrial Crops & Products, 111, 86-91. Yang, Z. D., & Zhai, W. W. (2010). Optimization of microwave-assisted extraction of anthocyanins from purple corn (Zea mays L.) cob and identification with HPLC-MS.
IP T
Innovative Food Science and Emerging Technologies, 11(3), 470-476. Yoshimoto, M., Okuno, S., Yoshinaga, M., Yamakawa, O., Yamaguchi, M., & Yamada,
SC R
J. (1999). Antimutagenicity of sweet potato (Ipomoea batatas) roots. Bioscience, Biotechnology, and Biochemistry, 63(3), 537-541.
U
Zhao, X., Li, J. (2015). Optimization of enzyme hydrolysis of purple sweet potato by
N
cellulase or pectinase for anthocyanin extraction. Food science and technology, 40(4),
A
277-281.
M
Zhu, Z. Z., Guan, Q. Y., Koubaa, M., Barba, F. J., Roohinejad, S., Cravotto, G., et al.
ED
(2017). HPLC-DAD-ESI-MS2 analytical profile of extracts obtained from purple
400.
PT
sweet potato after green ultrasound-assisted extraction. Food Chemistry, 215, 391-
Zhu, Z., Li, S., He, J., Rohit, T., Domenico, M., Francisco, J. B. (2018). Enzyme-
CC E
assisted extraction of polyphenol from edible lotus ( Nelumbo nucifera ) rhizome knot:
Ultra-filtration performance and HPLC-MS2 profile.
A
international, 111, 291-298.
25
Food
research
Figure captions
Fig.1. Schematic diagram of improved microwave-assisted extraction.
condenser PSP granule
SC R
microwave reactor
IP T
Figure 1
extractor
microwave device
A
CC E
PT
ED
M
A
N
U
ethanol aqueous
26
Fig. 2. The effects of single factor on extraction yield of anthocyanins (A, ethanol concentration ; B, microwave irradiation power; C, extraction time).
35
25
30
20
15
10
IP T
30
TAC (mg/100g)
TAC (mg/100g)
Figure 2
25
20
15
SC R
5 10
0
10
20
30
40
50
60
80
160
Acidic ethanol concentration (%)
(B)
U
(A) 30
N
25
20
A
TAC (mg/100g)
240
320
Microwave irradiation power (w)
M
15
200
300
400
500
Time (s)
A
CC E
PT
ED
(C)
27
600
700
400
Fig. 3. Response surface plot for TAC contents showing the effect of independent variables (a, Ethanol concentration and microwave irradiation power; b, Ethanol concentration and extraction time; c, microwave irradiation power and extraction
A
CC E
PT
ED
M
A
N
U
SC R
IP T
time).
28
SC R
IP T
Figure 3
(b)
A
CC E
PT
ED
M
A
N
U
(a)
(c)
29
Fig. 4 The appearance of residue of PSP after extracting anthocyanins
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Figure 4
30
Fig. 5. HPLC analysis of extracts from PSP detected at 520 nm(A) and 325 nm(B) (a, UAE; b, iMAE; c, HRCE; d, MAE).
(B)
A
CC E
PT
ED
M
A
(A)
N
U
SC R
IP T
Figure 5
31
Fig. 6. HPLC-MS/MS analysis of anthocyanins from PSP on a positive ionisation mode (A, UAE; B, iMAE).
6
2.5 2.0 3 4
1
1.5
9 6
2
1.0
8 5
0.5
7
0.0 0
2
4
6
8
10
12
14
16
18
20 22 24 26 Time (min）
32
34
36
38
40
42
44
36
38
40
42
44
N
6
4.0
1
2.0
3 2
4
A
3.0
9
6
1.0
5
0.0 2
4
6
8
10
12
14
16
18
8
7
20 22 24 26 Time (min）
(B)
A
CC E
PT
ED
0
M
Relative Abundance
×10
30
U
(A)
28
SC R
Relative Abundance
×10
IP T
Figure 6
32
28
30
32
34
Table 1 Total anthocyanins content of PSP used in the Box-Behnken design for response surface methodology. Run
Parameters
Response Factor 2 (X2)
Factor 3 (X3)
TAC (mg/100g)
1
30.00
160.00
300.00
8.42
2
40.00
240.00
500.00
31.56
3
30.00
240.00
400.00
29.06
4
30.00
320.00
500.00
31.16
5
20.00
160.00
400.00
8.32
6
40.00
320.00
400.00
25.75
7
30.00
240.00
400.00
26.45
8
20.00
240.00
500.00
9
30.00
160.00
500.00
10
20.00
240.00
300.00
11
30.00
320.00
300.00
12
40.00
240.00
300.00
13
20.00
320.00
400.00
14
30.00
240.00
400.00
24.95
15
40.00
160.00
400.00
17.23
16
30.00
240.00
400.00
25.15
17
30.00
240.00
400.00
23.14
SC R 23.54 21.84 10.62
U
23.34
N
A
M
ED PT CC E A
33
IP T
Factor1 (X1)
20.44 22.84
Table 2 ANOVA for quadratic model Sum of
Df
Squares
F
p-value
Square
Value
Prob > F
87.24
24.48
0.0002
785.13
X1
109.96
1
109.96
30.85
0.0009
X2
279.42
1
279.42
78.40
< 0.0001
X3
256.28
1
256.28
71.91
< 0.0001
X1X2
9.00
1
9.00
2.53
0.1561
X1X3
0.81
1
0.81
0.23
0.6481
X2X3
7.84
1
7.84
2.20
0.1816
X12
49.61
1
49.61
13.92
0.0074
2
60.24
1
60.24
16.90
0.0045
X3
2
2.55
1
2.55
0.71
0.4260
Residual
24.95
7
3.56
Lack of Fit
5.69
3
1.90
0.39
0.7650
Pure Error
19.26
4
4.81
Cor Total
810.08
16
U N A M
ED PT CC E A
34
significant
SC R
Model
X2
9
Mean
IP T
Source
Not significant
Table 3 The advantages of iMAE for PSPA compared with conventional extraction methods UAE*
MAE
HRCE
31.160.44
33.812.58
18.200.97
31.903.18
0.890.12
0.940.02
1.080.09
0.570.11
0.880.04
TAC of the PSP extracts (mg/g)
3.410.27
3.310.19
3.130.09
3.200.39
3.620.28
Extraction rate (%)*
89.88
92.16
100
53.83
94.35
The weight of residue (g)
2.910.22
2.850.06
2.590.15
/
2.820.03
Character of residue
granules
granules
mushy
Total sugar content of residue (g)
2.43
Solid to solvent (g/mL)
1:3
1:10
1:3
Critic acid for 1g PSP (g)
0.035
1.2
0.035
IP T
iMAE
Extraction time (min)
8.33
40
8.33
60
Character of extracting solution
clear and centrifuge easily
clear
gelatinization
clear and
and
centrifuge
centrifuge
easily
320 w
TAC (mg/100g )
30.390.73
Extracts of PSP (g)
2.39
and post-treatment process
2.34
U N
★
and
centrifuge easily
Microwave irradiation power.
granules 2.41
SC R
★
240 w
1:3
0.035
difficultly
A
CC E
PT
ED
M
A
In order to determine the total content of anthocyanins in PSP, four times of UAE (10 minutes each time) was performed. The relative yield of TAC was 40.91% (first time), 36.01% (second time), 14.08% (third time) and 9.00% (fourth time). So, the other extraction rate is a relative extraction rate compared with UAE.
35
Table 4 Molecular structure of PSPAs extracted by iMAE and UAE identified by HPLC-ESIMS/MS.
Peak
Retenti
[M+H]+/
Major
on
[M-H]-
fragment ion
time(m
(m/z)a
(m/z)
iMA
compounds
E
UAE
Reference
in) 2.6
773
611, 449, 287
Cyanidin-3-sophoroside-5-glucoside
√
√
2
2.6
787
625, 463, 301
Peonidin-3-sophoroside-5-glucoside
√
√
3
4.3
893
731, 449, 287
Cyanidin-3-p-hydroxybenzoylsophoroside-5-
√
glucoside 6.0
907
745, 463, 301
Peonidin-3-p-hydroxybenzoylsophoroside-5glucoside
5
7.3
949
787, 449, 287
Cyanidin-3-(6’’-feruloylsophoroside)-5glucoside
6
11.3
963
801, 463, 301
Peonidin-3-(6’’-feruloylsophoroside)-5-
25.2
1111
949, 449, 287
U
glucoside 7
√
SC R
4
IP T
1
√ √
√
√
√
√
2017)
√
√
√
√
√
√
Chlorogenic acid
√
√
Chlorogenic acid
√
√
Cyanidin-3-(6’’-caffeoyl-6’’’-
1069
907, 463, 301
Peonidin-3-caffeoyl-p-
A
25.8
N
feruloylsophoroside)-5-glucoside 8
hydroxybenzoylsophoroside-5-glucoside 26.1
1125
963, 463, 301
Peonidin-3-(6’’
M
9
-caffeoyl-6’’’-
feruloylsophoroside)-5-glucoside
4.2
353
191, 135, 85
11
5.8
353
191, 85
compounds 1-9 and [M-H]- for compound 10 and 11.
A
CC E
PT
a[M+H]+ for
ED
10
36
(Zhu et al.,