One-pot preparation of quercetin using natural deep eutectic solvents

Article abstract of DOI:10.1016/j.procbio.2019.10.019

In this study, we have established a green and efficient preparation method of quercetin. Rutin was first extracted from Sophora japonica using natural deep eutectic solvents (NADESs), then hydrolyzed into quercetin by rutin degrading enzyme (RDE) obtained from germinated tartary buckwheat in situ. Rutin solubility tests showed that most of the 11 NADESs increased the solubility of rutin by 67-3116 times compared to water. Thus, NADESs could be prior candidate to extract rutin. Extraction efficiency of rutin varied with different NADESs, and a maximum of 291.57 mg g^?1 was achieved in NADES ChGly, which was prepared by mixing choline chloride and glycerol at a molar ratio of 1:1. After that hydrolysis was performed directly in extraction system by adding RDE with degradation rate of up to 8.36 mg min^-1·L^-1. Our findings suggest that preparation of quercetin using NADESs was simple and feasible to operate, environmentally friendly, efficient, and inspired the preparation method of bioactive components from a new perspective.

Full text of DOI:10.1016/j.procbio.2019.10.019

ProcessBiochemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
One-pot preparation of quercetin using natural deep eutectic solvents
Yuan-Yuan Zanga,1, Xi Yanga,1, Zhi-Gang Chena,*, Tao Wub,*
a Glycomics and Glycan Bioengineering Research Center, College of Food Science &Technology, Nanjing Agricultural University, Nanjing, 210095, PR China
b The Department of Food Science, The University of Tennessee, 2510 River Drive, Knoxville, TN, 37996, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, we have established a green and efficient preparation method of quercetin. Rutin was first ex-
tracted from Sophora japonica using natural deep eutectic solvents (NADESs), then hydrolyzed into quercetin by
rutin degrading enzyme (RDE) obtained from germinated tartary buckwheat in situ. Rutin solubility tests
showed that most of the 11 NADESs increased the solubility of rutin by 67-3116 times compared to water. Thus,
NADESs could be prior candidate to extract rutin. Extraction efficiency of rutin varied with different NADESs,
and a maximum of 291.57 mg g−1 was achieved in NADES ChGly, which was prepared by mixing choline
chloride and glycerol at a molar ratio of 1:1. After that hydrolysis was performed directly in extraction system by
adding RDE with degradation rate of up to 8.36 mg min-1·L-1. Our findings suggest that preparation of quercetin
using NADESs was simple and feasible to operate, environmentally friendly, efficient, and inspired the pre-
paration method of bioactive components from a new perspective.
Natural deep eutectic solvents (NADESs)
Rutin
Sophora japonica
Rutin degrading enzyme (RDE)
Tartary buckwheat
1. Introduction
sulfuric acid or hydrochloric acid is often used for subsequent conver-
sion process [17], which results in an intense reaction with long time
Deep eutectic solvents (DESs), emerged recently as a novel class of
designer solvents, are attracting more and more attention [1]. They
overcome several limitations of traditional organic solvents and ionic
solvents that are high toxic, difficult to recycle, high cost, volatile and
so on. DESs are defined as eutectic mixtures composed of hydrogen
bond donors and acceptors. When the compositions are primary me-
tabolites, the formed solvents are also called natural deep eutectic
solvents (NADESs) [2]. NADESs have been widely studied in electro-
chemistry, separation processes and reaction processes due to their
unique physicochemical properties [3–7].
Quercetin (Scheme S1), 3,3′,4′,5,7-pentahydroxylflavone, is a fla-
vonoid with many biological activities, and it is widely distributed in
the flowers, leaves and fruits of many plants, mostly in the form of
glycosides and aglycones [8]. Studies have shown that quercetin has a
variety of pharmacological effects, e.g. anti-oxidation [9], anti-cancer,
anti-inflammation [10], and anti-virus [11].
and low yield. Biocatalyst was considered to address these challenges.
In recent study, it was reported that rutin degrading enzyme (RDE)
prepared from tartary buckwheat can specifically hydrolyze rutin to
quercetin. Its catalytic properties were then determined [18–20], but its
application in preparation of quercetin with NADESs has not been
discussed and known well.
An environmentally friendly preparation method of quercetin was
established in this study, in which NADESs were novelly applied to the
preparation of natural bioactive ingredients. The extraction of rutin and
its conversion to quercetin were completed in one solvent system.
NADESs overcoming the limitations of organic extractions are safe and
non-toxic as extraction solvents. Bio-catalyst instead of hydrochloric
acid or sulfuric acid could get high yield of target product and low by-
products because of its specificity. In addition, the process was milder
and the time required was shortened.
In modern food and pharmaceutical industry, quercetin is usually
prepared by hydrolysis of rutin extracted from Sophora japonica because
of its high content [8]. Organic solvents or their combinations with
water are commonly used in the extraction due to the extremely low
2. Materials and methods
2.1. Chemical and biological materials
solubility of rutin in water, which leads to
a
complicated, en-
The tartary buckwheat was purchased from local market in Sichuan,
vironmentally harmful and low output process [12–16]. Concentrated
China. Sophora japonica was obtained from local pharmacy. Rutin
⁎ Corresponding authors.
E-mail addresses: zgchen@njau.edu.cn (Z.-G. Chen), twu2@utk.edu (T. Wu).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.procbio.2019.10.019
Received 29 April 2019; Received in revised form 16 October 2019; Accepted 17 October 2019
1359-5113/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Yuan-YuanZang,etal.,ProcessBiochemistry,https://doi.org/10.1016/j.procbio.2019.10.019

Y.-Y. Zang, et al.
ProcessBiochemistryxxx(xxxx)xxx–xxx
standard was obtained from Beijing Bailingwei Technology Co., Ltd.
NADESs component: alanine (99%), threonine (≧99%), lysine (≧98%),
proline (≧99%), and choline chloride (98%) were purchased from
Aladdin Chemical Reagent Co., Ltd.(Shanghai, China). 1,2-propanediol
(≧99%), xylitol (98%), triethylene glycol (98%), glycerol (≧99%), 1,2-
butanediol (98%), and ethylene glycol (≧99%) were purchased from
Nanjing Chemical Reagent Co., Ltd. (Nanjing, China).
stirred at 4 °C for 24 h. The supernatant was collected after cen-
trifugation at 8000 g for 20 min. Before storage at 4 °C, the volume was
dialysis with distilled water.
2.6.2. Enzyme activity determination of RDE from germinated tartary
buckwheat
RDE solution (100 μL) was added to 700 μL of 0.02 M Tris-HCl
buffer (pH = 7.0), followed by 200 μL of ethanol containing rutin
(0.75 mg mL−1). The mixture was incubated at 37 °C for 15 min. The
reaction was stopped by adding 1 mL of methanol. The degraded rutin
amount was determined by HPLC as described above (Figs. S2, S3). One
unit of RDE activity was defined as the amount of enzyme required to
catalyze 1 μg rutin per minute under the condition above.
2.2. Preparation and determination of physicochemical properties of
NADESs
Eleven NADESs were prepared by mixing hydrogen bond donors
and acceptors at a defined molar ratio as shown in Table S1. The
mixtures were heated at an elevated temperature (85–100 °C) under
constant stirring until a homogeneous liquid was formed. During pro-
cess the air was well insulated by adding laboratory film to the vessel to
prevent absorption of moisture from the environment.
2.7. Effect of germination condition, NADESs and water content on the
enzyme activity of RDE
The viscosity was determined by a rheometer at 40 °C; Nile red was
used to estimate the polarity, expressed as ENR (molar transition en-
ergy); the pH was measured using a pH meter; and rutin solubility was
determined by saturating NADESs with excess rutin followed by ultra-
sounding at 40 °C for 45 min [21].
2.7.1. Effect of germination condition on the enzyme activity of RDE
Tartary buckwheat seeds germinated at 15 °C, 20 °C, 25 °C, and
30 °C for different time were collected, RDE in each sample were ex-
tracted followed by enzyme activity determination.
2.7.2. Effect of NADESs and water content on the enzyme activity of RDE
Different kinds of NADESs were prepared, then diluted by adding
deionized water to final concentrations of 20%, 40%, 60%, and 80%,
(v/v). Enzyme activities of RDE in these systems were assayed by the
method in 2.6.2, respectively.
2.3. Extraction of rutin from Sophora japonica
Sophora japonica powder (40 mg) was dissolved in 1.5 mL of desig-
nated NADES. The solution was sonicated in an ultrasonicator (Model:
NP-B-100-15; New Power Co., Ltd., Kunshan, China) at 20 Hz, 200 W,
for 45 min. Then the solutions were centrifuged at 7000 g for 30 min.
Rutin concentrations in the supernatant were determined by HPLC as
described below. The extraction efficiency was assessed using the fol-
lowing equation: extracted amount (mg·g−1)= mass of rutin (mg)/
mass of Sophora japonica powder (g).
2.8. Optimization of hydrolysis system of RDE
2.8.1. Single factor test
The single factor test was carried out with 5 factors and 5 levels,
namely temperature (25, 30, 37, 40, and 45 °C), reaction time (10, 15,
20, 25, and 30 min), pH (4, 5, 6, 7, and 8), substrate concentration
(0.25, 0.5, 0.75, 1, and 1.25 mg mL−1) and RDE amount (15, 20, 25, 30,
and 35 μL), each time only one factor was tested, calculated the rutin
degradation rate by the method described in 2.6.2.
2.4. Effect of water content in NADESs on the extraction efficiency of rutin
NADESs with high extraction efficiency were selected and water was
added by 0%, 10%, 20%, 30%, and 40% (v/v) to determine the effect of
water in NADESs on the rutin extraction efficiency.
2.8.2. Orthogonal analysis and hydrolysis optimization
An orthogonal L9 (3)3 test design was used to investigate the op-
timal hydrolysis condition of RDE. As seen from Table S2, the hydro-
lysis experiment was carried out with 3 factors and 3 levels, namely
temperature (30, 35, and 40 °C), substrate concentration (0.75, 1, and
1.25 mg mL−1) and RDE amounts (45, 50, and 55 μL). The rutin de-
gradation rate was the dependent variable.
2.5. HPLC analysis of rutin contents in extractions
The amounts of rutin in the extraction solutions were determined by
reversed phase high performance liquid chromatography (RP-HPLC) on
a 4.6 mm × 150 mm (5 μm) Zorbax Eclipse XDB-C18 column (Agilent
Technologies, Santa Clara, CA - USA). The HPLC was equipped with an
autosampler (Model: SIL-20A), a quaternary pump (Model: LC-20D)
and a diode array detector (Model: SPD-M20A) set at 360 nm (Fig. S1),
a degasser (Model: CBM-20A), and column thermostat (Model: CTO-
20A). The mobile phase was a mixture of methanol and water (50/50,
v/v) at flow rate of 0.6 mL·min−1 with isocratic system. The samples
were diluted 5 times with methanol and filtered before HPLC analysis.
3. Results and discussion
3.1. Preparation, physicochemical properties tests of NADESs
In recent years, NADESs have emerged as a new generation of de-
signer solvents. They can be simply prepared by mixing initial com-
ponents and stirring at a certain temperature; their properties could
also be tailored easily by changing their components [1].
2.6. Extraction and catalytic activity determination of RDE from
germinated tartary buckwheat
Physical properties of 11 NADESs such as viscosity, polarity, pH,
and solubility, were investigated systematically. As shown in Table 1,
most NADESs tested were relatively viscous. The highest viscosity
(3.18 Pa·s) was observed in GlyLys, 318 times as high as that of in
methanol. The polarity of the tested NADESs was similar to that of
methanol. This means NADESs could be classified as polar solvents. The
pH of NADESs ranged from 5.61 to 7.68, except for GlyLys (10.49),
indicating that they were in weak acid to weakly alkaline solvents.
The results of this study indicated that the solubility of rutin in 11
NADESs increased by 67-3116 times compared to that of in water
(Table 1), which varied significantly with the type of NADESs. The
2.6.1. Extraction of RDE from germinated tartary buckwheat
Ripened and uniform tartary buckwheat seeds were selected,
weighed to 5 g, and soaked with 1% sodium hypochlorite for 10 min.
The seeds were then washed with sterilized deionized water, soaked
with water at room temperature for 4 h, and then put in a plat with
sterilized gauze. The seeds were germinated in the dark at 15 °C, 20 °C,
25 °C, and 30 °C, respectively for 4 days with 100% humidity. The
samples were taken and the soaking water was changed every 24 h.
Each sample was ground to a suspension and then dissolved in
20 mL of 0.02 M acetic acid buffer solution (pH = 5.0) which was
highest solubility was achieved in ChTEG (280.4
2.7 mg·mL−1),
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Table 1
water was added to lower its viscosity and increase rutin solubility.
As a promising green solvent, NADESs could be implemented in
various applications due to its unique properties, e.g. superior solubility
and adjustable physicochemical. The results of this study provide
foundation of using NADESs as solvents to extract various water-in-
soluble biologically active substances.
Viscosity and polarity of NADESs, and solubility of rutin in 80% NADESs.
b
NADESs
Abbreviation
Viscositya
(40 °C) (Pa·s)
ENR
pHc
Solubility of rutind
(mg·mL−1
(kcal·moL−1
)
)
ProAla
EGAla
GlyThr
GlyAla
GlyLys
ChGly
0.09
0.01
0.22
0.29
0.05
3.18
0.02
0.07
0.02
0.07
0.87
0.01
53.24-54.98
52.95-55.41
50.01
49.82
49.04-51.70
50.91
52.95-54.46
49.98-51.61
55.09-56.06
48.87-50.07
49.55
6.68
7.20
6.95
7.19
76.4
107.4
59
5.1e
4.7
3.9
165.0
5.0
4.1
3.3. Effect of water content in NADESs on extraction efficiency of rutin
10.49 79.2
5.67
6.92
5.61
6.95
7.45
7.68
6.8
6
2.3
Water is an important factor affecting the viscosity of NADESs. In
this study different water contents (0–40%) in NADESs system were
evaluated for the extraction of rutin. Based on the rutin yields (Fig. 1),
ChGly and EGAla were selected for this study. Fig. 2 shows that in-
creasing amounts of water in ChGly up to 20% significantly improved
the efficiency of the solvent in extraction of rutin. On the other hand,
addition of water into EGAla tended to decrease the efficiency of the
solvent. The maximum of ChGly appeared when the water content was
20% (291.57 mg g−1), and for EGAla, it is 10% (275.64 mg g−1).
The addition of appropriate amount of water could lower the visc-
osity and enhanced the extraction efficiency. Beyond the optimum level
(20% for ChGly, 10% for EGAla), the excess amounts of water might
interfere chemical interaction between the NADESs and their compo-
nents, resulting in decreased extraction efficiency [21].
ButThr
ChEG
25.0
4.5
5.2
2.7
4.3
146.1
280.4
31.6
178.2
97
ChTEG
GlyXyl
GlyPro
Methanol
Water
3.3
51.80
4
f
—
—
7.00
0.09
0.01
a
Detection at shear rate 0.268 [1·s−1].
Polarity: Nile red concentration, ∼ 0.1 mM; ENR=hcNA/λmax
Measured by pH meter.
b
c
.
d
The solubility of rutin in each solvents was determined by UV analysis of
saturated solutions at 40 °C.
e
Mean
Not detected.
SD (n = 3).
f
On the other hand, as it was reported in former study, the polarity of
water is 48.21 [23], which is similar to most of the NADESs
(48.87–56.06, Table 1), so the addition of water will not cause sig-
nificant changes in polarity.
which has the highest polarity. And the lowest was obtained in ChGly
(6 2.3), resulting from its high viscosity.
3.2. Research on extraction of rutin in NADESs
Traditionally organic solvents are used to prepare quercetin to solve
the problem of the poor solubility of rutin in water, which could pollute
the environment [22]. NADESs could be an alternative to organic sol-
vents for extraction due to high solubility of rutin in such solvents.
However, high viscosity of NADESs could be a barrier in applications.
Based on the previous study, the high viscosity of NADESs could be
reduced by adding a certain amount of water [19]. In this study, 80%
NADESs were used to extract rutin from Sophora japonica the traditional
extraction 80% methanol was selected as a reference solvent.
Fig. 1 shows a significant improvement of rutin extraction by using
NADESs as solvents compared to traditional methanol extraction. The
amounts of extracted rutin varied with NADES types. The highest yield
of rutin (284.81 mg g−1) was obtained in ChGly, followed by EGAla
(272.08 mg g−1). GlyXyl only yielded rutin 4.67 mg g−1. Based on rutin
yields, four NADESs solvents (PGAla, EGAla, ChGly, and ChTEG) were
clearly superior to methanol (149.75 mg g−1). NADESs have similar
polarity and pH to the methanol as seen in Table 1, but the rutin so-
lubility is higher (except for ChGly), it may because of the H-bound
between rutin and NADESs [2]. To improve the extraction efficiency,
3.4. Effect of germination condition, NADESs and water content on the
enzyme activity of RDE
3.4.1. Effect of germination condition on the enzyme activity of RDE
It has been studied that tartary buckwheat contains high active RDE,
which have strong pH and thermal stabilities [24]. Studies on its sub-
strate specificity indicate that the RDE has a strict substrate specificity
and only catalyzes rutin to quercetin [19]. RDE has not been available
commercially. Most of the researchers obtain RDE from plant materials
such as tartary buckwheat by ammonium sulfate precipitation or hy-
drophobic chromatography [19,24,25]. The catalytic activity of RDE
could be improved during the germination of tartary buckwheat.
However, some have reported that series of metabolic reactions oc-
curred during the germination process and RDE also underwent several
changes in this period [26]. Hence, germination temperature and time
were discussed to determine the optimum germination conditions.
Germination temperature and time have a great influence on the
catalytic activity of RDE. RDE extracted from the tartary buckwheat
which is germinated at 25 °C has the highest catalytic efficiency (Fig.
Fig. 1. Extraction yields (mg of rutin per g of Sophora japonica
powder) for different solvents. Extraction conditions: 40 mg
Sophora japonica powder, 1.5 mL 80% solvent, 50 °C, 1 h,
Ultrasound-assisted extraction (UAE) power 200 W. Extraction
efficiencies that were significantly higher in comparison with that
of 80% GlyXyl are indicated with *p < 0.05, **p < 0.01 and
***p < 0.001.
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Fig. 2. Effect of natural deep eutectic solvents (NADESs) water
content on the extraction efficiency of rutin. Extraction conditions:
40 mg Sophora japonica powder, 1.5 mL solvent, 50 °C, 1 h, UAE
power 200 W. Extraction efficiencies that were significantly
higher in comparison with that of 40% EGAla and 40% ChGly are
indicated with *p < 0.05, **p < 0.01 and ***p < 0.001 of
EGAla or #p < 0.05, ##p < 0.01 and ###p < 0.001 of ChGly.
S4). The degradation rate of RDE decreased with time. This result is in
line with the previous study. They theorized that the decreased activity
of RDE during germination might be due to the absorption and con-
version of nutrients [27]. Therefore tartary buckwheat was germinated
at 25 °C for 1d to extract RDE in the followed experiments.
maintain the active structure of the enzyme, improve the stability of the
enzymes [28], increase solubility of the substrate, inhibit side reactions,
and make it easier to recover the enzymes [29,30].
3.5. Optimization of hydrolysis system of RDE
3.4.2. Effect of NADESs and water content on the enzyme activity of RDE
In this study, NADESs provided a non-aqueous environment instead
of organic solvents, and the water content was optimized with rutin
degradation as an indicator.
The enzyme reaction system is characterized by mild reaction con-
ditions, specific substrate conversion and high conversion efficiency.
Generally, reaction time, pH, substrate concentration, amounts of en-
zyme, and temperature affect the catalytic efficiency of enzymes. In this
study, five factors were optimized by orthogonal test to improve the
enzymatic activity of RDE to obtain higher reaction efficiency.
The enzymatic activity of RDE increased with the reaction time and
it would level off after 15 min (Fig. 4A). This might be due to the
substrate that had been reacted or used up. The pH affects the dis-
sociation state of the active center of the enzyme. The optimum pH of
RDE suitable for the binding to the substrate was at pH 7.0 (Fig. 4B). At
too acidic or too alkaline pH, the enzyme structure would change, and
its activity would decrease. Reaction rate increased with substrate
concentrations up to 1.0 mg mL−1 (Fig. 4C). With the increase of the
substrate concentration, the reaction rate increased when the substrate
concentration was low and RDE would not be fully bounded, after the
enzyme binding reached saturation, the reaction rate would no longer
increase.
Fig. 3 shows that rutin degradation rate and the optimum water
contents varied with NADESs. In water/NADESs ratios of 20:80-80:20
(v/v), the catalytic activity of RDE in most NADESs systems increased
with the proportion of NADESs in the system. The maximum activity
occurred at 20% water, except for EGAla at 40% water. Pure NADESs
(0% water) with considerable high viscosity (Table 1) might cause
difficulties of substrate to bind to enzyme catalytic active center and
resulted in low degradation rate. The water activity of NADESs with
different water content was also determined, ChGly was taken as an
example (Fig. S5), which indicated that the Aw value has the similar
trend with water content.
It has been proved that several enzymes are more active in non-
aqueous or trace water-containing systems [28] RDE in NADESs with
up to 20% water of this study showed better catalytic activities. This
increased catalytic activity was due to several factors. The presence of
appropriate amounts of water in the non-aqueous system could
Rutin degradation shows a rapid rise from 15 μL to 25 μL, and a
slight rise from 25 μL to 55 μL, then keep in steady (Fig. 4D). It may
Fig. 3. Effect of NADESs species and water content on the degradation efficiency of rutin degrading enzyme (RDE). Degradation conditions: 100 μL crude RDE, 900
μL co-solvent with different water content (0%, 20%, 40%, 60%, and 80%, v/v) containing 150 μg rutin, 37 °C.
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Fig. 4. Single factor experiment on the effect of time (Fig. 4A), pH (Fig. 4B), substrate-concentration (Fig. 4C), RDE amount (Fig. 4D) and temperature (Fig. 4E) on
the degradation efficiency of RDE. Standard degradation conditions: 100 μL crude RDE 900 μL 80% ChGly containing 150 μg rutin, 37 °C.
because the substrate was fully bounded. Higher temperature promotes
mass transport, as it was reported in former study [31], the viscosity
decreases as the temperature rises, but after reaching the optimum
temperature of the enzyme, a further increase of temperature caused
destruction of molecular structure of the enzyme and irreversible de-
generation, resulting in a decrease in enzyme activity. As shown in
Fig. 4E, the maximum degradation rate was detected at 30 °C.
Orthogonal test table was designed based on single factor test re-
sults described above. The experimental results of orthogonal test are
presented in Table 2. Rj1 > Rj2 > Rj3 indicated that the factors af-
fecting hydrolysis from large to small are temperature > substrate
concentration > enzyme amounts. The optimum level of each factor
was determined by the value of K. The most optimum degradation
conditions were as follows: heating in a 35 °C water bath with a sub-
strate concentration of 1 mg mL−1 and an enzyme loading of 55 μL. This
optimum conditions resulted in a degradation rate of 8.07 mg min-1·L-1
(Table 2). Three parallel validation experiments were carried out ac-
cording to the hydrolysis conditions above, obtained a rutin degrada-
tion rate of 8.36 mg min-1·L-1, After optimization, the conversion rate
was increased to 12.5%. While traditional process could reach a yield of
50% [17]. However traditional ways pose a harsh reaction condition
Table 2
Design and results of L9 (34) orthogonal testa.
Exp.
number
T (°C) Substrate
concentrate
RDE
amount
(μL)
Degradation rate
(mg·min−1·L−1
)
(mg·mL−1
)
1
2
3
4
5
6
7
8
30
30
0.75
1
45
50
0.00
2.63
0.26
0.72
8.07
5.19
2.17
0.00
0.53
–
30
35
35
1.25
0.75
1
55
50
55
35
40
40
1.25
0.75
1
45
55
45
9
40
1.25
1.06
3.77
1.99
2.70
50
1.07
4.66
1.10
3.59
2.03
1.29
3.50
2.21
K1
K2
K3
Rj
–
–
–
a
Standard degradation conditions:100 μL crude RDE, 900 μL solvent (80%
ChGly) containing 150 μg rutin, 37 °C.
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with a lot by-product, and environmentally harmful reagents were
needed. In our study, the solvents were environmentally friendly and
the enzyme was extracted from plant.
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solvent, which is biodegradable, environmentally friendly and non-
polluting. The method described here can be applied to the extraction
and preparation of a variety of natural active substances, which have
broad application prospects in the pharmaceutical, food and related
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Declaration of Competing Interest
None.
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Acknowledgments
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This work was supported by Jiangsu Agricultural Industry
Technology System (JFRS-04) and the Priority Academic Program
Development of Jiangsu Higher Education Institutions (080-820830).
These founding sources had not involvement in study design; in the
collection, analysis or interpretation of data; in the writing of the re-
port; or in the decision to submit the article for publication.
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.procbio.2019.10.019.
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