International Journal of Biological Macromolecules 164 (2020) 1025–1036

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Co-optimization of pectin and polyphenols extraction from black

mulberry pomace using an eco-friendly technique: Simultaneous
recovery and characterization of products
Faramarz Khodaiyan a, Karim Parastouei b,c,⁎
a
Bioprocessing and Biodetection Laboratory, Department of Food Science and Engineering, University of Tehran, Karaj, Iran
b
Health Research Center, Life Style Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
c
Department of Nutrition and Food Hygiene, Faculty of Health, Baqiyatallah University of Medical Sciences, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The current work investigated a simultaneous recovery of pectin and polyphenols from black mulberry pomace
Received 29 April 2020 (BMP) using an eco-friendly extraction process. The microwave-assisted extraction variables were successfully
Received in revised form 7 June 2020 co-optimized using Box–Behnken design considering pectin and polyphenols yields as responses. The optimized
Accepted 10 July 2020
condition yielded about 10.95% pectin and 12.11% phenolics. The physicochemical analysis indicated a highly-
Available online 14 July 2020
esterified pectin (DE of 62.21%) with an average molecular weight of 620.489 kDa and galacturonic acid (GalA)
Keywords:
content of 70.15%. FTIR and NMR spectroscopies confirmed the predominant presence of highly esterified
Black mulberry pomace GalA-rich structure for BMP pectin. XRD analysis suggested an amorphous structure for the main part of BMP pec-
Pectin tin. Also, DSC results showed higher thermal stability for BMP pectin in comparison to commercial pectin. More-
Phenolic compounds over, the antiradical activity of BMP phenolic extract was very close to that of butylated hydroxyanisole (BHA)
and ascorbic acid. The results showed that the suggested procedure can be a promising solution for the manage-
ment of BMP waste generated in juice, syrup, or liquor processing plants. Also, the quality of the obtained prod-
ucts (BMP pectin and BMP phenolic extract) indicated that the products have the potential to be used as natural
ingredients in various food and pharmaceutical products.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction antioxidant, anti-cancer, and anti-inflammatory properties due to the

Daily, massive quantities of agro-food residues are generated in the
form of peel, husk, seed, pomace, etc. in food processing sectors. The dis-
posal of such a large volume of waste poses serious challenges such as
environmental pollution problems, and the loss of valuable compounds
[1]. A way to minimize these problems is to treat them as a potential
source of valuable compounds and reuse them in extraction and pro-
duction of marketable value-added products. This process can also pre-
vent the depletion of natural resources, enhance the agribusiness
opportunities, and support the rural livelihoods [2].
Mulberry is a member of Moraceae family and Morus genus originat-
ing from temperate and tropical regions of Asia, which is now widely
cultivated around the world [3]. Three of the most important mulberry
species are included white (Morus alba L.), red (Morus rubra L.), and
black (Morus nigra L.), which in the meantime, black mulberry has a
special place because of the extraordinary functional properties [4]. In
addition to its unique nutritional properties, black mulberry also has
⁎ Corresponding author at: Health Research Center, Life Style Institute, Baqiyatallah
University of Medical Sciences, Tehran, Iran.
E-mail address: Parastouei@gmail.com (K. Parastouei).
presence of phenolic compounds including phenolic acids, flavonols,
and anthocyanins [5]. This fruit is also rich in polysaccharides such as
pectin with good antioxidant, antitumor and antimicrobial properties
[6]. In general, black mulberry is consumed freshly, but a large quantity
of this fruit is used in food processing sectors to produce juice, syrup,
and liquor which in these cases, the generation of a large volume of
pomace is inevitable. Unfortunately, this valuable source of phenolics
[3–5] and pectin [6] is usually discarded as waste, while the extraction
of these compounds from black mulberry pomace (BMP), not only can
increase the financial benefits of production units but also can reduce
the volume of BMP waste and its subsequent environmental problems
[1]. Pectin is a plant polysaccharide with the main backbone of
(1 → 4) galacturonic acid partially esterified in some carboxylic groups.
This polysaccharide is widely used in the food and pharmaceutical in-
dustries as gelling, stabilizing, and thickening agents [1]. Phenolic com-
pounds, as powerful antioxidants, are other secondary metabolites of
plants widely applied in the food and drug industries [7]. The main phe-
nolic compounds found in black mulberry are phenolic acids (such as
chlorogenic, gallic, syringic, neochlorogenic, and caffeic acids), flavonols
(such as rutin and quercetin), and anthocyanins (such as cyanidin-3-O-
glucoside and cyanidin-3-O-rutinoside) [5].

https://doi.org/10.1016/j.ijbiomac.2020.07.107
0141-8130/© 2020 Elsevier B.V. All rights reserved.
1026 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036

So far, several studies have investigated the polysaccharides (such as Table 1

pectin and other cell wall polysaccharides) [6,8] and phenolics [3–5,8] Experimental and predicted results for the optimization of pectin and phenolic com-
pounds (TPC) extraction from black mulberry pomace.
of black mulberry or BMP which in all of them, the polysaccharides
and/or phenolics were separately extracted and characterized. How- Factors Unit Actual levels
ever, nowadays, with increasing the production of agricultural waste −1 0 +1
around the world, and developing the concept of sustainable waste val-
Microwave power (X1) W 200 500 800
orization and maximum utilization of food waste, the simultaneous pro- Irradiation time (X2) s 60 180 300
duction of several products from waste has received a lot of attention. pH (X3) – 1 2 3
Previously, several studies have shown the simultaneous extraction Liquid/solid ratio (X4) mL/g 20 30 40
process of pectin and phenolics from different agri-food waste such as
No. X1 X2 X3 X4 Pectin yield TPC yield (%)
eggplant peels [1], pomegranate peel [2], and mango peel [9] using a (%)
conventional extraction method with long processing time and high
Exp. Pred. Exp. Pred.
consumed energy. But no studies have been conducted on the co-
extraction of pectin and phenolics from BMP. 1 200 60 2 30 2.00 1.72 6.91 6.64
2 800 60 2 30 4.80 4.72 8.25 8.15
Therefore, for the first time, simultaneous recovery of pectin and
3 200 300 2 30 3.20 3.05 7.59 7.60
phenolics from BMP using microwave-assisted extraction (MAE), as 4 800 300 2 30 7.40 7.45 8.86 9.04
an eco-friendly extraction process, was studied to maximize the use of 5 500 180 1 20 9.80 9.55 11.05 11.20
waste for the production of the valuable compounds and minimize the 6 500 180 3 20 4.80 4.55 8.85 8.74
volume of the remained BMP waste. The co-extraction process was op- 7 500 180 1 40 2.80 2.82 5.20 5.22
8 500 180 3 40 5.60 5.62 5.26 5.08
timized using four MAE independent variables and two responses of
9 200 180 2 20 1.00 1.30 9.08 8.96
pectin and phenolics. Then, the compounds produced under optimum 10 800 180 2 20 8.80 9.00 11.87 11.63
conditions were characterized based on physicochemical, structural, 11 200 180 2 40 2.40 2.47 5.30 5.30
and functional properties. 12 800 180 2 40 2.20 2.17 5.70 5.58
13 500 60 1 30 5.40 5.37 8.07 7.96
14 500 300 1 30 8.00 8.00 8.65 8.43
2. Materials and methods 15 500 60 3 30 4.60 4.87 6.20 6.19
16 500 300 3 30 6.00 6.30 7.69 7.56
2.1. Materials 17 200 180 1 30 1.60 1.78 7.39 7.50
18 800 180 1 30 9.20 9.28 7.89 7.98
19 200 180 3 30 4.60 4.48 4.89 5.16
The BMP waste was collected from a black mulberry syrup-
20 800 180 3 30 4.60 4.38 7.43 7.65
manufacturing unit located in Karaj, Alborz, Iran. The pomace (with a 21 500 60 2 20 5.40 5.48 9.36 9.61
moisture content of 75–80% w/w) was washed using tap water twice, 22 500 300 2 20 7.20 7.12 11.83 11.91
cut into small pieces, then transferred into a stainless tray, and dried 23 500 60 2 40 2.20 2.25 5.88 6.14
24 500 300 2 40 4.80 4.68 5.60 5.69
in an air-circulating oven (55 °C, 48 h). The dried pomace was powdered
25 500 180 2 30 7.40 7.47 8.72 8.86
with a lab mill (IKA Universal M20) and then sieved using a sieve size of 26 500 180 2 30 7.60 7.47 8.90 8.86
400 μm. The obtained BMP powder (with a moisture content of 6–8% w/ 27 500 180 2 30 7.40 7.47 8.97 8.86
w) was stored in an airtight container at room temperature for the next
experiments.
Citric acid, hydrochloric acid, sodium hydroxide, gallic acid,
galacturonic acid, Folin-Ciocalteu reagent, DPPH• (2,2-Diphenyl-1- simultaneously using MAE process. After performing the extraction pro-
Picrylhydrazyl), ABTS•+ reagent were obtained from Merck Chemical cess and separating the residual BMP solids by centrifugation at
Co. (Darmstadt, Germany). The commercial citrus pectin (Galacturonic 10000 ×g for 20 min, pectin and phenolics samples were isolated as
acid ≥74.0%, P9561), meta-hydroxydiphenyl reagent, ascorbic acid, follows:
and butylated hydroxyanisole (BHA) were purchased from Sigma-
Aldrich Chemical Co. (St. Louis, MO, USA). Ethanol 96% and sulfuric 2.2.1. Pectin fraction (isolation and calculation)
acid were bought from Ghadir Co. (Tehran, Iran) and Dr. Mojallali chem- The liquid phase was precipitated by adding ethanol (96%) at a ratio
ical Co. (Tehran, Iran), respectively. The standard dextrans were pur- of 1:1 v/v, and stored at 4 °C for 24 h. Then, the precipitates were gath-
chased from Pharmacosmos (Holbaek, Denmark). All other chemicals ered by centrifugation at 10000 ×g for 20 min and was dried at 50 °C for
and reagents were of analytical grade. 12 h. To obtain a purified pectin sample, the dried pectin was dissolved
completely in distilled water and centrifuged (10,000 ×g, 20 min) to re-
2.2. Simultaneous extraction of pectin and phenolics move impurities. Then, ethanol (1:1 v/v) was added to it and stored
overnight (4 °C). The precipitates were gathered by centrifugation
MAE method, as an environmentally-friendly extraction process, (10,000 ×g, 20 min) and dried in an oven (50 °C, 12 h). The purification
was applied to the simultaneous extraction of pectin and phenolic com- process was repeated twice to reach a better quality for BMP pectin
pounds from BMP. The extraction process was performed on a domestic sample.
2450 MHz microwave oven (Butane Co., Iran) with an adjustable power The dried pectin was weighted and the extraction yield was calcu-
(100–1000 W) and time. Citric acid, as a natural organic acid, was used lated and reported as g of purified pectin per 100 g dry powder of
to adjust the pH of the solvent (distilled water). For this purpose, 5 g of BMP (based on dry matter).
dried BMP powder was mixed into the acidified distilled water at differ-
ent pH levels (including 1, 2, and 3) and different liquid/solid ratios 2.2.2. Total phenolic compounds (TPC) fraction (isolation and calculation)
(LSR; including 20, 30, and 40 mL/g). Then, the mixture was placed in The remained ethanolic solution after pectin removal was used for
the microwave oven and the irradiation time (at three levels: 60, 180, TPC measurement according to previous studies by Talekar et al. [2]
and 300 s) and microwave power (at three levels: 200, 500, and and Kazemi et al. [1].
800 W) were set up using a digital control system. The value of variables The TPC measurements were performed spectrophotometrically
for each run was according to Table 1. using the Folin-Ciocalteu method and a UV–Vis spectrophotometer as
The pectin and phenolics production processes were clearly illus- described by Kaderides et al. [10]. TPC of BMP phenolic extract samples
trated in Fig. 1. As it is obvious, both products were extracted was calculated according to the standard curve (R2 of 0.9913) of gallic
 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036 1027

Fig. 1. Graphical scheme of the simultaneous extraction process of pectin and phenolic compounds from black mulberry pomace.

acid. The TPC measurements were used to calculate the TPC yields. The
yields were defined as g of gallic acid equivalents per 100 g dry powder
of BMP (based on dry matter).
2.3. Statistical analysis
In the current study, a Box–Behnken design (BBD) with four vari-
ables in three levels was considered to optimize the effect of indepen-
dent variables (microwave power (X1; W), irradiation time (X2; s), pH
(X3) and liquid/solid ratio (X4; mL/g)) on the extraction yield of pectin
and phenolic compounds as responses (dependent variables). The vari-
ables and their levels are shown in Table 1. Also, all computations and
graphics were made by Design-Expert software (version 7.0, Stat-Ease
Inc., USA) and Microsoft Excel software (version 2013, Microsoft, USA).
2.4. Physicochemical properties of BMP pectin
In this section, moisture, ash and protein content, total carbohy-
drates, galacturonic acid (GalA) content, degree of esterification (DE),
and weight-average molecular weight (Mw) of the BMP pectin ex-
tracted under optimum condition (microwave power of 700 W, irradia-
tion time of 300 s, pH of 1.42 and LSR of 20 mL/g) were determined.
Moisture and ash content was measured by the previously described
method [11]. The protein content was determined by the Kjeldahl
1028 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036
method (N × 6.25) [12]. The total carbohydrates were measured by the
phenol‑sulfuric acid photometric method as described by Dubois et al.
[13]. The galacturonic acid (GalA) content was determined colorimetri-
cally using 3,5-dimethylphenol reagent, and the standard curve of D-
galacturonic acid [14]. The degree of esterification (DE) was evaluated
using the titrimetric method as explained by Asgari et al. [15]. The Mw
was measured by a GPC device (Shimadzu LC-20A, Kyoto, Japan)
equipped with an Ultrahydrogel 250™ column and a refractive index
detector. The mobile phase of 0.1 M NaNO3 was used at a flow rate of
1 mL/min, a temperature of 35 °C and an injection volume of 20–50 μL.
2.5. Chemical structure of BMP pectin
The pectin obtained under optimum extraction condition (micro-
wave power of 700 W, irradiation time of 300 s, pH of 1.42 and LSR of
20 mL/g) was structurally compared with the commercial pectin by
Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR)
and X-ray diffraction (XRD) spectroscopies.
FTIR spectra were obtained by a Bruker FT-IR Tensor 27 spectrome-
ter (Billerica, Massachusetts, USA) using the KBr method at the wave-
number range and the resolution of 4000–500 and 4 cm−1,
respectively [15]. 1H NMR spectra were obtained by a Varian Unity
Inova 500 MHz spectrometer (Palo Alto, CA, United States) at the inter-
nal temperature, the relaxation delay, and the acquisition time of 24 °C,
1 s and 4 s, respectively [16]. XRD patterns were obtained by an X-ray
diffractometer (PHILIPS, Amsterdam, Netherland) at the internal tem-
perature, the relaxation delay, and the acquisition time of 24 °C, 1 s,
and 4 s, respectively [17].
2.6. Thermal analysis of BMP pectin
The BMP pectin isolated under optimum extraction condition (mi-
crowave power of 700 W, irradiation time of 300 s, pH of 1.42 and LSR
of 20 mL/g) and the commercial pectin were analyzed thermally by a
differential scanning calorimeter (DSC, NETZSCH 200 F3, Germany) at
the temperature range of 10 to 300 °C with a scanning rate of 10 °C/min.
2.7. Antioxidant analysis of BMP extract
The obtained BMP phenolic extract sample under optimum extrac-
tion condition (microwave power of 700 W, irradiation time of 300 s,
pH of 1.42 and LSR of 20 mL/g) at concentrations of 2–125 μg GAE/mL
were characterized using three methods including DPPH• scavenging
activity, ABTS•+ scavenging activity, and reducing power assay.
DPPH• scavenging activity was performed spectrophotometrically at
517 nm (SP-UV500DB model, Spectrum Instruments, Australia) accord-
ing to the method described previously by Kazemi et al. [1]. ABTS•+
scavenging activity, as one of the common tests for estimating the anti-
oxidant ability of natural materials, was performed using a UV–Vis spec-
trophotometer with sample detection at 734 nm as previously
described [18]. Reducing power assay was also done spectrophotomet-
rically at 700 nm using the protocol described previously by
Mohammadian et al. [19].
In this stage, all experiments were triplicated for each sample and
the results were reported as mean value ± SD. To provide a better un-
derstanding, the experiments were carried out for two standard antiox-
idant samples (including ascorbic acid and BHA) at similar
concentrations and the results were compared to those of BMP phenolic
extract sample [1]. Also, the IC50 values (the concentration of sample
that can inhabit 50% of the free radicals) of DPPH• and ABTS•+ scaveng-
ing activity for each sample were calculated and compared with each
other.
3. Results and discussion
3.1. Co-optimization and statistical analysis
In this study, a Box–Behnken experimental design with four inputs
of microwave power, irradiation time, pH, LSR, and two outputs of pec-
tin extraction yield and TPC extraction yield was applied to optimize the
extraction conditions of pectin and TPC from BMP. The experimental
and predicted values are presented in Table 1. As can be seen, the ex-
traction yield of pectin and TPC ranged from 1.00 to 9.80% and 4.89 to
11.87%, respectively. Also, the optimum extraction condition for the re-
sponses according to the obtained statistical models (Eqs. (1) and (2))
were as follows: microwave power of 700 W, irradiation time of 300 s,
pH of 1.42, and LSR of 20 mL/g. Under this condition, the mathematical
model predicted a maximum extraction yield of 11.26% for pectin, and
12.49% for TPC, respectively. The obtained optimum yield was found
to be higher than the optimum yield for pectin extracted using MAE
process from several non-commercial sources such as pomelo peel
(2.93%) [20], pumpkin biomass (7.4%) [21], dragon fruit peel (7.42%)
[22], and banana peel (2.17%) [23]. However, it was lower than the op-
timum yield of commercial pectin sources such as sour orange peel
(29.1%) [24], sweet lemon peel (25.31%) [17], and orange peel
(18.59%) [25] extracted by MAE. In the case of TPC yield, the optimum
yield was found to be higher than the yield of the extract obtained
from pomegranate peels (10.6–11.8%) [2]; and lower than TPC yield
found for phenolic extract sample obtained from eggplant peel
(20.2%) [1], and sour cherry pomace (14.36%) [26].
For the validation and the predictability of the suggested models, the
optimized condition was repeated three times and the experimental
mean values of 10.95 ± 0.51 and 12.11 ± 0.27% were obtained for pec-
tin and TPC which were close to the predicted data and confirmed them.
The suggested statistical models for the extraction yield of pectin (Y1)
and TPC (Y2) were as follows:
Y1 ð%Þ ¼ 7:47 þ 1:85X1 þ 1:02X2 −0:55X3 −1:42X4 −2:19X21 −1:04X22
−0:29X23 −1:54X24 þ 0:35X1 X2 −1:90X1 X3 −2:00X1 X4
−0:30X2 X3 þ 0:20X2 X4 þ 1:95X3 X4 ð1Þ
Y2 ð%Þ ¼ 8:86 þ 0:74X1 þ 0:46X2 −0:66X3 −2:42X4 −0:74X21 −0:27X22
−1:06X23 −0:26X24 −0:20X1 X2 þ 0:51X1 X3 −0:60X1 X4
þ0:23X2 X3 −0:69X2 X4 þ 0:57X3 X4 ð2Þ
where X1-X4 are coded variables of microwave power, irradiation time,
pH, and LSR, respectively.
The regression coefficients of the suggested model based on BBD
were shown in Table 2. The results showed that the linear, quadratic
and interaction effects of the independent variables on the responses
were significant (p b 0.0001).
The results of analysis of variance (ANOVA) indicated that the ob-
tained regression models were significant (p b 0.05) while lack-of-fit
tests were insignificant (p N 0.05) for both responses, showing that
the fitness of the suggested models was significant [27]. Also, there is
an important correlation degree between inputs and outputs, because
the values of R2 for pectin yield and TPC were 0.9957 and 0.9930 show-
ing that only 0.43 and 0.7% of the total variation was unexplained
[1,15,17].
3.2. The role of extraction variables on the yield of pectin and TPC
The effect of extraction variables on the pectin and TPC yield was
shown in Figs. 2 and 3. The yields were microwave power-dependent
and by increasing the power up to 700 W both yields (pectin and poly-
phenols) were increased (Figs. 2A and 3A). This observation could be
due to the fact that with an increase in the microwave power and thus
the electromagnetic energy transferred on extraction solvent molecules,
 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036 1029

Table 2 therefore an increase in extraction yield [11,28,29]. As can be seen in

Analyze of variance for the fitted models of responses (pectin and TPC yield). Figs. 2 and 3, pH value had a reverse relationship with extraction yields
Source DF Some of squares Mean square F-value p-value and the responses were increased by decreasing this factor, which is
Pectin yield
probably due to the fact that the high concentration of acid in extraction
Model 14 160.421 11.459 198.32 b0.0001 medium can increase the damage to the plant tissue and thereby can
Linear 4 81.187 20.297 351.29 b0.0001 improve the release of the products [30,31]. The effect of LSR on the ex-
X1 1 41.070 41.070 710.83 b0.0001 traction yields was likewise indicated in Figs. 2 and 3. The extraction
X2 1 12.403 12.403 214.67 b0.0001
yield of pectin and TPC was decreased, when LSR was increased from
X3 1 3.630 3.630 62.83 b0.0001
X4 1 24.083 24.083 416.83 b0.0001 20 to 40 mL/g. One possible reason for the decline is that a high volume
Square 4 32.574 8.144 140.95 b0.0001 of extraction solvent can absorb the more microwave energy and thus
X21 1 25.618 25.618 443.39 b0.0001 can lead to a decrease in the energy available for cell wall destruction
X22 1 5.787 5.787 100.16 b0.0001 [25,32]. However, a key point in the mentioned figures is that when
X23 1 0.454 0.454 7.85 0.016
X24 1 12.676 12.676 219.39 b0.0001
using harsh conditions consisting of very high microwave power and ir-
2-Way interaction 6 46.660 7.777 134.60 b0.0001 radiation time and also very low pH and LSR, the extraction yield is de-
X1X2 1 0.490 0.490 8.48 0.013 creased which is probably related to the degradation of the target
X1X3 1 14.440 14.440 249.92 b0.0001 compounds by overheating [10,29].
X1X4 1 16.000 16.000 276.92 b0.0001
It should be noted that the MAE process showed great potential to
X2X3 1 0.360 0.360 6.23 0.028
X2X4 1 0.160 0.160 2.77 0.122 be considered as a green alternative method for cell disruption and
X3X4 1 15.210 15.210 263.25 b0.0001 extraction processes. In the current study, microwave factors had
Error 12 0.693 0.058 crucial effects in disrupting plant cells and increasing yields; for ex-
Lack-of-fit 10 0.667 0.067 5.00 0.178 ample, microwave power was the most effective factor in pectin
Pure error 2 0.027 0.013
yield. Moreover, the process was performed in a short processing
Total 26 161.114
R2 0.9957 time (300 s), low energy requirement, and free of hazardous chemi-
Adj. R2 0.9907 cal usage.
Pred. R2 0.9758

TPC yield 3.3. The physicochemical aspects of the BMP pectin

Model 14 97.970 6.998 122.03 b0.0001
Linear 4 84.892 21.223 370.08 b0.0001 The different physicochemical properties of BMP pectin extracted
X1 1 6.545 6.545 114.13 b0.0001
under optimum extraction condition (microwave power of 700 W,
X2 1 2.560 2.560 44.63 b0.0001
X3 1 5.252 5.252 91.59 b0.0001
irradiation time of 300 s, pH of 1.42, and LSR of 20 mL/g) were deter-
X4 1 70.535 70.535 1229.99 b0.0001 mined and the obtained results were shown in Table 3. As can be
Square 4 7.225 1.806 31.50 b0.0001 seen, moisture, ash and protein content of BMP pectin was found to
X21 1 2.891 2.891 50.41 b0.0001 be 7.64, 4.87, and 1.81%, respectively. Also, the total carbohydrates
X22 1 0.386 0.386 6.74 0.023
of pectin in the mentioned conditions were found to be 82.37%. Dif-
X23 1 5.975 5.975 104.20 b0.0001
X24 1 0.353 0.353 6.61 0.029 ferent values of the mentioned properties were reported for the pec-
2-Way interaction 6 5.854 0.976 17.01 b0.0001 tin of various sources, for example: the ash, protein, and total
X1X2 1 0.002 0.002 0.03 0.871 carbohydrates of the produced pectin from sour cherry pomace
X1X3 1 1.042 1.042 18.16 0.001
using MAE process were about 3.73, 1.41 and 26.43%, respectively
X1X4 1 1.428 1.428 24.90 b0.0001
X2X3 1 0.205 0.205 3.57 0.083
[26]. In another study, Kazemi et al. [33] reported a significantly dif-
X2X4 1 1.893 1.893 33.02 b0.0001 ferent values for the ash and protein content of pectin obtained from
X3X4 1 1.284 1.284 22.39 b0.0001 eggplant calyx (5.21 and 7.68%, respectively) and eggplant peel (9.03
Error 12 0.688 0.057 and 9.13%, respectively) using a similar MAE condition (microwave
Lack-of-fit 10 0.653 0.068 3.76 0.228
power: 700 W, irradiation time: 120 s, pH: 1.5, LSR: 20 mL/g). Differ-
Pure error 2 0.035 0.017
Total 26 98.658 ent values were also found for the carbohydrate and protein content
R2 0.9930 of pectin extracted from orange peel using the conventional method
Adj. R2 0.9849 (77.2% and 7.1% respectively), MAE method (80.2% and 7.0% respec-
Pred. R2 0.9611
tively), and combined surfactant and MAE method (84.2% and 5.8%
respectively) [34]. Probably, the used source, the extraction method,
and the process conditions are the main reasons for these differences
[11]. The determination of GalA content is an integral test of pectin
the temperature and thereby the mass transfer of the products were in- because this parameter is another definition for pectin purity.
creased. The acidified water, as a polar solvent, efficiently absorbs the Based on FAO recommendations, pectin should contain at least 65%
microwave energy, increases the temperature and leads to efficient (w/w) GalA to be used as an additive in the food industry [12]. The
heating. Then, the increase in temperature, caused by the thermal ef- BMP pectin extracted at optimum condition had a GalA content of
fects of microwave energy, improves the cell disruption in BMP powder 70.15%, meaning that it was higher than FAO limits. The DE value of
(which are in contact with solvent), then accelerates the release rate of BMP pectin found to be higher than 50% (DE = 62.21%), meaning
functional compounds (pectin and polyphenols) from the plant cells. that the BMP pectin belongs to the high methoxyl pectin (HMP)
However, beyond that power (700 W) the yields were decreased, prob- group. The HMP pectin can form gels at a low pH and in the presence
ably due to the excessive microwave power and energy, and the ther- of a high concentration of sugar. Therefore, it is suitable to be applied
mal degradation [10,27,28]. Irradiation time was another effective in the non-diet products such as all kinds of jams and jellies. Accord-
factor and the obtained results showed that this parameter also had a ing to Table 3, the BMP pectin extracted under optimum condition
direct relationship with the responses according to Figs. 2E and 3E. Sev- had an Mw of 620.489 kDa which was higher than those of pectin ex-
eral studies have been reported similar results and suggested that in- tracted by MAE process from various commercial and non-
creasing the extraction time during the microwave-assisted extraction commercial sources such as orange peel (Mw = 144.8 kDa) [34],
process leads to creating enough time for the extraction and the transfer sweet lemon peel (615.836 kDa) [17], and sour cherry extracted by
of compounds from plant fractions into the surrounding medium and MAE (Mw = 472.977 kDa) [26]. Previously, it has been
1030 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036

Fig. 2. Independent factors effects of microwave-assisted extraction on pectin of black mulberry pomace.

recommended that pectin with high average molecular weight is
more suitable to be used as a gelling agent, while pectin with lower
Mw can offer higher antioxidant properties [35]. Probably, the plant
source is the main reason for the differences found in Mw of pectin
samples. In addition to the source, the MAE extraction factors such
as microwave power, irradiation time, and pH have also been re-
ported to alter the Mw of pectin [36]. The high value of Mw/Mn was
attributed to the presence of smaller chains in the pectin fraction
due to the high microwave power and low pH used in the extraction
process [17].
 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036 1031

3.4. The structural properties of BMP pectin
The structural properties (FTIR, 1 H NMR, and XRD spectros-
copies) of BMP pectin extracted under optimum extraction condi-
tions were evaluated and compared with the commercial citrus
pectin.
FTIR spectra (Fig. 4) showed several characteristic peaks at
about 3365, 2904, 1704, 1607 and 900–1250 cm −1 related to
O\\H stretching vibration, C\\H of CH, CH2, and CH3, ester carbonyl
stretching bands, free carboxyl stretching vibrations, and C-O-C
and O\\H of pyranose rings, respectively [30,37,38]. These peaks
confirmed the presence of pectin in the isolated sample. However,

Fig. 3. Independent factors effects of microwave-assisted extraction on the phenolic compounds of black mulberry pomace.
1032 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036
Table 3
Physicochemical analysis of pectin from black mulberry pomace.
Parameters Value
Moisture content (%) 7.64 ± 0.85
Ash content (%) 4.87 ± 0.92
Protein content (%) 1.81 ± 0.43
Total carbohydrates (%) 82.37 ± 1.12
GalA content (%) 70.15 ± 2.12
DE (%) 62.21 ± 3.54
Mw (kDa) 620.489
Mn (kDa) 169.455
Mw/Mn 3.66
two characteristic signals are very important and represent the DE
value of pectin sample: the signals at 1704.84 and 1607.63 cm −1 .
According to these signals, the DE of BMP pectin was higher than
50% [39], and confirmed the result obtained by the titrimetric
method as presented in Table 3.
The 1 H NMR spectra of the BMP and commercial pectin were
presented in Fig. 5. The chemical shifts observed at about 5.3, 5.1,
4.2, 3.9, 3.7, and 2.3 ppm were related to H-1, H-5, H-4, H-3,
-OCH3, H-2, and -COCH3, respectively [39,40]. The comparison be-
tween spectra obtained for pectin samples suggested that the ap-
peared signals for BMP pectin were almost identical to those of
commercial one, meaning that the spectra showed the predomi-
nant presence of pectin structure in the isolated sample. It should
also be stated that the signals of methyl and acetyl groups are at-
tributed to the DE of pectin and their sharpness are directly related
to this parameter [1]. Therefore, it can be concluded that the 1 H
NMR spectrum also confirmed the reported data for the titrimetric
determination of DE (Table 3).
The XRD patterns of the BMP pectin and the commercial sample
were presented in Fig. 6. In these patterns, the crystallinity of a
polymer is showed by a series of sharp peaks while the absence of
these peaks indicates the amorphous nature of the polymer [41].
Therefore, both the BMP and commercial pectin has an amorphous
nature. However, because of the presence of a few sharp signals in
the pattern of BMP pectin, it seems that there are also some crystal-
line regions in the isolated pectin. A slight difference between the
two patterns is probably due to the difference in the source and
the extraction conditions of pectin [11,15,17].
Fig. 4. FTIR spectra of pectin of black mulberry pomace and commercial pectin.
3.5. The thermal analysis of the BMP pectin
The DSC was used to determine the thermal properties of the BMP
pectin obtained under optimum condition (microwave power of
700 W, irradiation time of 300 s, pH of 1.42, and LSR of 20 mL/g) and
the commercial pectin. According to Fig. 7, the endothermic peak for
BMP pectin and the commercial pectin was found at 115.82 and
111.36 °C, respectively. The endothermic peak can be depicted due to
the existence of moisture and hydrogen bonding among GalA units. It
also represents the ability of pectin sample to hold water and is related
to the hydrophilic groups of pectin structure [42]. The exothermic transi-
tions at 251.82 °C for BMP pectin and 246.33 °C for commercial citrus pec-
tin are due to the degradation of pectin samples at those temperatures. As
can be seen, the exothermic peak of BMP pectin appeared at a slightly
higher temperature than the commercial pectin. This observation reveals
that the thermal stability of BMP pectin was higher than commercial pec-
tin, meaning that BMP pectin sample might be preferred during thermal
processing. Pectin obtained from various sources can show various ther-
mal properties. However, the extraction technique and condition are
also effective in the thermal profile of this polymer. Previously, Dranca
et al. [43] reported that pectin extracted from Malus domestica ‘Fălticeni’
apple pomace using various non-conventional techniques (such as
ultrasound-, enzyme-, and microwave-assisted techniques) showed dif-
ferent thermal properties. The authors also reported that the obtained
pectin using MAE process had a higher thermal stability than commercial
citrus and apple pectin [43].
3.6. Antioxidant potential of BMP extract
The antioxidant potential of the BMP phenolic extract obtained under
optimum extraction condition and the standard antioxidants were mea-
sured by three widely used methods including DPPH•, ABTS•+, and reduc-
ing power assay, since choosing only one method for estimation may
compromise the effectiveness of obtained results [1,9].
Fig. 8 revealed that DPPH• scavenging activity of samples had a
concentration-dependent behavior, thus, the IC50 value was established
for samples to provide a reliable comparison with each other [1]. The
IC50 value for ascorbic acid, BHA, and BMP phenolic extract were
30.96, 41.42, and 63.76 μg/mL, respectively. Therefore, it could be con-
cluded that the strongest sample was ascorbic acid, followed by BHA,
and then BMP phenolic extract. However, as can be seen in Fig. 8, the
 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036
Fig. 5. 1H-NMP spectra of pectin of black mulberry pomace and commercial pectin.
inhibition percentage of BMP phenolic extract was very close to the
standards at the highest concentration.
In addition, the obtained results of ABTS•+ scavenging activity for
samples also suggested similar results. As it is obvious in Fig. 9, the
trends of curves were very similar to those of DPPH•, were in a dose-
dependent manner. As it was expected, the IC50 values indicated that
ascorbic acid (IC50 value: 29.66 μg/mL) was stronger than BHA (IC50
value: 32.91 μg/mL) and BMP extract (IC50 value: 46.61 μg/mL). But at
the highest concentration (125 μg/mL), all of them were very close to-
gether in their highest inhabitation level. Likewise, similar results
were also observed for reducing power of samples. Fig. 10 suggested
that the trends of the curves were similar to the previously mentioned
results, and all samples increased with an increase in their
concentration.
Based on the earlier results, the BMP phenolic extract presented a
great antioxidant potential and bioactivity, which was very close to
those of commercial standards. Therefore, it should be considered as a
potential natural antioxidant with high bioactivity, to be used as an
Fig. 6. XRD patterns of pectin of black mulberry pomace and commercial pectin.
1033
ingredient in the pharmaceutical or food industry in formulating vari-
ous health-promoting products.
4. Conclusion
In the current research, BBD was employed to co-optimize the si-
multaneous extraction of pectin and phenolics from BMP using MAE
process. The co-optimization results revealed that the highest pectin
(10.95%) and TPC (12.11%) yields were achieved under MAE optimum
condition (microwave power of 700 W, irradiation time of 300 s, pH
of 1.42 and LSR of 20 mL/g). Pectin produced under optimum condition
was rich in high-methylated GalA (70.15%) and had an Mw value of
620.489 kDa with a wide molecular weight distribution (Mw/Mn =
3.66). The structural properties of BMP pectin was analyzed using vari-
ous methods and the obtained results confirmed the predominant pres-
ence of GalA units with high DE value, and an amorphous structure with
a good thermal stability (degradation temperature = 251.82 °C). The
evaluation of antioxidant capacity suggested a dose-dependent behav-
ior for BMP extract and standard samples (ascorbic acid and BHA). At
a concentration of 125 μg/mL, the antioxidant capacity of BMP extract
sample was close to those of standard samples. The results showed
that the simultaneous production of pectin and polyphenol from BMP
waste not only produces two marketable products but also offers
lower overall production cost and allows the sustainable valorization
of BMP. Therefore, it should be concluded that the proposed process
for the valorization of BMP was fully in line with circular economy con-
cepts; and the employing of green-chemistry extraction process can in-
crease its benefits.
Author statement
Karim Parastouei: Data curation, Investigation, Methodology, Writ-
ing – original draft, Conceptualization, Formal analysis, Project adminis-
tration, Resources, Supervision.
Faramarz Khodaiyan: Software, Validation, Visualization, Writing –
review & editing.
1034 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036

Fig. 7. DSC thermograms of pectin of black mulberry pomace and commercial pectin.

Fig. 8. DPPH radical scavenging activity of black mulberry pomace BMP phenolic extract compared with BHA and ascorbic acid.

Fig. 9. ABTS radical scavenging activity of black mulberry pomace BMP phenolic extract compared with BHA and ascorbic acid.
 F. Khodaiyan, K. Parastouei / International Journal of Biological Macromolecules 164 (2020) 1025–1036
Fig. 10. Reducing power of black mulberry pomace BMP phenolic extract compared with BHA and ascorbic acid.
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