Accepted Manuscript Onion skin waste as a valorization resource for the by-products quercetin and biosugar In Seong Choi, Eun Jin Cho, Jae-Hak Moon, Hyeun-Jong Bae PII: DOI: Reference:
S0308-8146(15)00750-5 http://dx.doi.org/10.1016/j.foodchem.2015.05.028 FOCH 17574
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
22 January 2015 21 April 2015 6 May 2015
Please cite this article as: Choi, I.S., Cho, E.J., Moon, J-H., Bae, H-J., Onion skin waste as a valorization resource for the by-products quercetin and biosugar, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem. 2015.05.028
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1 2
Onion skin waste as a valorization resource for the byproducts quercetin and biosugar
3 4
In Seong Choi a,1, Eun Jin Cho a,1, Jae-Hak Moon b and Hyeun-Jong Bae a,c*
5 6 7 8 9 10 11
a
Bio-energy Research Center, Chonnam National University, Gwangju 500-757,
Republic of Korea b
Department of Food Science and Technology and Functional Food Research Center,
Chonnam National University, Gwangju 500-757, Republic of Korea c
Department of Bioenergy Science and Technology, Chonnam National University,
Gwangju 500-757, Republic of Korea
12 13 14 15 16 17 18 19 20
* Corresponding author at: Department of Bioenergy Science and Technology,
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Chonnam National University, Gwangju 500-757, Republic of Korea. Tel.: +82 62 530
22
2097; fax: +82 62 530 0029.
23
E-mail address:
[email protected] (H.-J. Bae)
24
1
25
These authors contributed equally to this work.
26
ABSTRACT
27
Onion skin waste (OSW), which is produced from processed onions, is a major
28
industrial waste. We evaluated the use of OSW for biosugar and quercetin production.
29
The carbohydrate content of OSW was analyzed, and the optimal conversion conditions
30
were evaluated by varying enzyme mixtures and loading volumes for biosugar
31
production and quercetin extraction. The enzymatic conversion rate of OSW to biosugar
32
was 98.5% at 0.72 mg of cellulase, 0.16 mg of pectinase, and 1.0 mg of xylanase per
33
gram of dry OSW. Quercetin extraction also increased by 1.61-fold after complete
34
enzymatic hydrolysis. In addition, the newly developed nano-matrix (terpyridine-
35
immobilized silica-coated magnetic naonparticles-zinc (TSMNP-Zn matrix) was
36
utilized to separate quercetin from OSW extracts. The nano-matrix facilitated easy
37
separation and purification of quercetin. Using the TSMNP-Zn matrix the quercetin was
38
approximately 90% absorbed. In addition, the recovery yield of quercetin was
39
approximately 75% after treatment with ethylenediaminetetraacetic aicd.
40 41 42 43
Keywords: Enzymatic hydrolysis, nano-matrix, onion skin waste, quercetin
44
1. Introduction
45
The importance of biomass as a renewable resource for biofuels and biochemicals has
46
increased considerably during recent decades. The use of biomass to produce energy has
47
economic and environmental benefits because it is highly productive, renewable, carbon
48
neutral, and easy to store and transport. However, the amount of biomass that can be
49
used for non-food purposes is limited and its use competes with food production and
50
supply (Cherubini, 2010; Mahro & Timm, 2007; Wi, Kim, Shobana, Yang, & Bae, 2009;
51
Wi, Choi, Kim, Kim, & Bae, 2013).
52
Bio-waste is defined as food and kitchen waste from households, caterers, and retail
53
premises, and comparable waste from food processing plants (EPC, 2008). Millions of
54
tons of bio-waste are produced every month. Part of the waste is allocated to landfills,
55
and part is exported to third world countries. This has a considerable impact on the
56
environment, particularly on wildlife, ecosystems, and human health. For this reason,
57
several new bio-waste treatment plants have been constructed and new processes for
58
generating energy from landfill waste have been developed (Galanakis, 2012;
59
Laufenberg, Kunz, & Nystroem, 2003; Lin et al, 2013). To minimize the environmental
60
hazards, it is necessary to integrate the various types of bio-waste into a biomass
61
economy. Bio-waste food processing residues, in particular, may be a good candidate
62
for assessment of economic potential, as they account for large quantities of biogenic
63
residues every year (Martin, 1998).
64
Owing to its medicinal and nutritional value, world onion production has increased
65
by at least 25% over the past 10 years, with current production being around 83 million
66
tons (FAO 2013), making it the second most important horticultural crop after tomatoes
67
worldwide. Consequently, more than 500,000 tons of onion skin waste (OSW) are
68
discarded every year within the European Union, where it has become an environmental
69
problem (Benítez et al., 2011; Griffiths, Trueman, Crowther, Thomas, & Smith, 2002;
70
González-Sáiz, Esteban-Díez, Rodríguez-Tecedor, & Pizarro, 2008; Waldron, 2001).
71
The waste includes the brown skin, the outer layers, roots and stalks, as well as onions
72
that are not large enough for commercial use. This waste is not suitable as fodder for
73
animals, and hence is usually dumped. The brown skin and external layers are rich in
74
fiber and phenolic compounds, such as quercetin and other flavonoids (Downes, Chope,
75
& Terry, 2009; Jaime, Mollá, Fernández, Martín-Cabrejas, López-Andréu, & Esteban,
76
2002). Quercetin belongs to an important class of natural compounds, which are widely
77
used to treat several diseases, such as cancers of the prostate, breast, ovaries, colon,
78
rectum, and kidney (Hertog & Hollman, 1996; Jin et al., 2006). Thus, the extraction
79
methods of these valuable components from OSW should be worth investigating from
80
an economic point of view and environmental benefit. In recent years, several extraction
81
methods have been reported to obtain quercetin from onions, including conventional
82
solvent extraction (Wach, Pyrzyńska, & Biesaga, 2007), ultrasound-assisted extraction
83
(Jang, Asnin, Nile, Keum, Kim, & Park, 2013), and microwave assisted extraction
84
(Kumar, Smita, Kumar, Cumbal, & Rosero, 2014). Although many extraction methods
85
have been improved recently, developing more cost efficient methods and new
86
techniques remains a challenge.
87
The preparative purification of active constituents from plant extracts was also
88
considered in our study, since it represents an important step in the manufacture of
89
phytochemicals (Zhao, Dong, Wu, & Lin, 2011). Although several methods such as
90
liquid–liquid (Dinan, Harmatha, & Lafont, 2001) and ion exchange extractions (Skelly
91
& Crummett, 1971) have been employed for the separation of bioactive compounds
92
from natural resources, these methods are considered inefficient, since they take a long
93
time and consume a large amount of solvent. Biomagnetic separation techniques are
94
becoming increasingly important with a wide range of possible applications in
95
bioscience research. The magnetic nano matrix can be separated easily and quickly by
96
magnetic forces. The advantages of magnetic separation techniques include fast and
97
simple sample handling, and the opportunity to deal with large sample volumes without
98
the need for time-consuming centrifugation steps (Cho, Jung, Lee, Lee, Nam, & Bae,
99
2010; Safarikova & Safarik, 2001). Therefore, this report also describes a new magnetic
100
matrix that can separate phenolic compounds such as quercetin from OSW extracts. The
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newly developed magnetic matrix facilitated the separation and purification of quercetin.
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2. Materials and methods
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2.1. Sample and reagent preparation
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OSW was obtained from a local onion-skin processing factory (Damyang, Korea).
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Only waste with an onion outer covering was used. The OSW was lyophilized at -50 oC,
107
milled on an electrical grinder, and stored at -20 oC until further use. 4’-Chloro-
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2,2’:6’,2”-terpyridine and 3-(triethoxysilyl)propyl isocyanate were purchased from
109
Sigma-Aldrich (St. Louis, USA). All other materials were of analytical grade and
110
commercially available, including ferric chloride hexahydrate (FeCl3•6H2O), ferrous
111
chloride tetrahydrate (FeCl2•4H2O), ammonium hydroxide (25% [w/w]), and tetraethyl
112
orthosilicate (TEOS).
113 114
2.2. Enzymatic hydrolysis and quercetin extraction/separation
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2.2.1. Enzymatic hydrolysis of OSW
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Cellulase (Cellulclast 1.5L) and pectinase (Pectinex SP-L) were purchased from
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Novozymes A/S (Bagsvaerd, Denmark). Xylanase (X2629 endo-1,4-β-D-xylanase) was
118
provided by Sigma-Aldrich. The cellulase activity was determined by NREL (1996),
119
and the pectinase and xylanase activities were measured according to Kittur, Kumar,
120
Gowda, and Tharanathan (2003) and Bae, Kim, and Kim (2008), respectively. The
121
cellulase, pectinase and xylanase activities were 0.122 filter paper units (FPU)/mg
122
protein, 240 international units (IU)/mg protein and 2.65 IU/mg protein, respectively.
123
Cellulase, pectinase and xylanase were added to OSW at concentrations of 0.16–0.79
124
mg, 0.06–0.16 mg, and 0.5–1.0 mg of protein/g OSW, respectively. Enzymatic
125
hydrolysis was performed on 1% substrate (w/v) with a citrate phosphate buffer (pH 4.8)
126
at 180 rpm for 48 h at 45°C. After enzymatic hydrolysis, the soluble sugars in the
127
enzymatic hydrolysis were measured using high-performance liquid chromatography
128
(HPLC) with a refractive index detector (2414; Waters, Milford, MA, USA). The
129
hydrolysate was loaded into a REZEX RPM (Phenomenex, Torrance, CA, USA)
130
column (300 × 7.8 mm) at 85 oC and eluted with deionized water at a flow rate of 0.6
131
ml/min.
132 133 134
2.2.2. Preparation of terpyridine-immobilized silica-coated magnetic nanoparticles (TSMNP) and TSMNP-zinc (Zn) for quercetin extraction and separation
135
The TSMNP were prepared as outline by Cho, Jung, Lee, Lee, Nam, and Bae (2010).
136
The silica-coated magnetic nanoparticles (SMNP, 1.0 g) were suspended in toluene (50
137
mL) under sonication for 30 min. Terpyridine derivative (0.5 g) was added and the
138
reaction mixture was refluxed for 24 h. After cooling to room temperature, the particles
139
were filtered, washed with toluene, and dried at 60 oC for 12 h in a vacuum oven. The
140
TSMNP (100 mg) were vortex-mixed in an aqueous zinc chloride solution (0.1 M, 5 ml)
141
for 1 h. Subsequently, the solution was removed by magnetic separation, and the
142
TSMNP-Zn conjugates were rinsed with deionized water before use. To estimate the
143
binding capacity of the zinc ions on the nanoparticles, the TSMNP-Zn in the suspension
144
were collected by magnetic separation. For the separation of quercetin from the OSW
145
hydrolysate, 5 ml of onion extracts (in methanol) was added to TSMNP-Zn (50 mg) and
146
stirred for 60 min. After removal of the methanol phase, these particles were washed
147
twice with methanol (10 ml) to remove the onion-skin residue and other non-specific
148
bound compounds. The particles were then treated with 100 mM of EDTA (in 1:1
149
MeOH : H2O, v/v) to recover the quercetin.
150 151
2.3. Biochemical analysis
152
2.3.1. Quantification of sugars
153
The neutral sugar composition of OSW was measured with alditol acetates containing
154
myo-inositol as an internal standard using gas chromatography (GC) (Choi, Wi, Kim, &
155
Bae, 2012; Wi, Chung, Lee, Yang, & Bae, 2011). Each sample was treated with 0.25 ml
156
72% sulfuric acid (H2SO4) for 45 min at room temperature and diluted with distilled
157
water to 4% H2SO4. The hydrolysis step was performed at 121°C for 1 h. A solution
158
containing a known amount of myo-inositol was used as an internal standard and
159
neutralized with ammonia solution. Sodium borohydride solution (1 ml) and 0.1 ml
160
glacial acetic acid (18 M) were added to degrade the sodium tetrahydroborate. Next, 0.2
161
ml methyl immidazole and 2 ml anhydrous acetic acid were sequentially added. Finally,
162
5 ml of deionized water were added and extracted with 2 ml dichloromethane. The
163
samples were analyzed by GC (GC-2010; Shimadzu, Otsu, Japan) using a DB-225
164
capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness, J&W; Agilent, Folsom,
165
CA, USA) and operated with helium. The operation conditions were as follows: an
166
injector temperature of 220 °C, a flame ionization detector at 250 °C, and an oven
167
temperature of 100 °C for 1.5 min with constant increases of 5 °C/min to 220 °C.
168
Uronic acids were measured colorimetrically according to the method adopted by
169
Blumenkrantz and Asboe-Hansen (1973) using
170
modification.
D-galacturonic
acid with slight
171 172
2.3.2. Quantification of quercetin
173
The quercetin concentration was measured according to Bordonaba and Terry (2008)
174
with slight modifications. Infra-red spectra were obtained in the range of 400-4000 cm-1
175
using a PerkinElmer FT-IR/ NIR 400 instrument. A field-emission scanning electron
176
microscope (FE-SEM) JEOL JSM-7500F was used to carry out scanning microscopy.
177
Thin-layer chromatography (TLC) was performed on a Merck Silica Gel 60 F 254
178
adsorbent, which comprised TLC plates with a fluorescent indicator with an excitation
179
wavelength of 254 nm. The compounds were visualized under UV light at 254 nm. The
180
UV absorption spectrum was measured on a Perkin Elemer Lambda 35 UV/Vis
181
spectrophotometer. HPLC was performed with a UV detector (Waters 2414, USA) using
182
a C18 column (250 × 10.0 mm; Varian Pursuit XRs, United States). HPLC-grade water
183
was supplied at a flow rate of 3.3 ml/min as a mobile phase, and maintained at room
184
temperature. The gradient program was as follows; linear gradient from solution A (25 %
185
acetonitrile) to solution B (65 % H2O, added formic acid 0.01 %) for 25 min, and finally
186
isocratic in solution B for 5 min.
187 188
3. Results and discussion
189
3.1. Carbohydrate composition of OSW
190
OSW comprises mainly, in order of concentration, carbohydrates, glucose, and uronic
191
acids, followed by xylose, with mannose, rhamnose, galactose, and arabinose present in
192
minor amounts (Table 1). The main polysaccharides of OSW are cellulose and
193
polyuronides. The Klason-lignin was obtained after acidic hydrolysis, and the amount
194
was higher than expected. This could be attributed to the presence of cell wall or co-
195
precipitated intracellular proteins (Jaime et al., 2002). The OSW contained virtually no
196
galactose as indicated by the high galacturonic acid/neutral sugar (sum of arabinose and
197
galactose) ratio (UA:NS). These results were consistent with previous studies on onion
198
tissues, in which a similar variation was observed in the cell wall composition among
199
tissues (Suutarinen, Mustranta, Autio, Salmenkallio-Marttila, Ahvenainen, & Buchert,
200
2003). However, the difference in the carbohydrate composition of OSW between
201
previous studies and the current study could be due to various factors, including the type
202
of cultivar, stage of maturation, environmental conditions, agronomic conditions,
203
storage time, and bulb section (Jaime et al., 2002; Ng, Parker, Parr, Saunders, Smith, &
204
Waldron, 2000).
205 206
3.2. Optimizing enzyme loading for saccharification
207
Several studies have been carried out on the carbohydrates and flavonoids, such as
208
flavonol (quercetin) and anthocyanins, in onions (Benítez et al., 2011; Bordonaba &
209
Terry, 2008; Downes et al., 2009). However, limited information is available regarding
210
the enzymatic hydrolysis used to enhance the quercetin extract from OSW. Quercetin is
211
the aglycone form of several other flavonoid glycosides, and exists as polyphenolic
212
compound with glucosides in OSW. With the aim of obtaining a more effective
213
quercetin extract, we conducted enzymatic hydrolysis before quercetin extraction. To
214
evaluate and compare the effects of different enzymes and their relative concentrations
215
in the hydrolysis yields from OSW, different volumes of cellulase, pectinase and
216
xylanase were added to 1% OSW (w/v) (Table 2). Our study focused mainly on the
217
glucose yield for biosugar production, which accounted for 40.6% of the OSW
218
carbohydrates. Although xylose, mannose and other monosugars were present, their
219
concentrations in the OSW were low. To identify the loading volumes that resulted from
220
synergistic interaction, the enzymes (cellulase, pectinase, and xylanase) were
221
individually loaded and the mixtures were added in different combinations to the OSW.
222
The degree of synergism was assessed by the amount of glucose released compared to
223
the amount released with hydrolytic enzyme alone.
224
When cellulase, pectinase, and xylanase were added to the OSW, 0.6, 1.0, and 0.1
225
mg/ml glucose was released, respectively (data not shown). However, only a modest
226
increase in pectinase on the OSW was observed when a cellulase and xylanase mixture
227
was added at a fixed loading volume, despite its high pectin content (Table 1). Xylanase
228
increased from 0.1 to 1.0 mg/g OSW when the cellulase and pectinase loading volumes
229
were fixed, resulting in more than a 3- to 10-fold increase in the total released glucose.
230
Although OSW has a very low xylose content (Table 1), xylanase significantly
231
increased the enzymatic conversion rate. This could be attributed to the high pectin
232
content in OSW, which restricts the accessibility of cellulase to the glycosidic bonds
233
within the cellulose chain (Suutarinen, Mustranta, Autio, Salmenkallio-Marttila,
234
Ahvenainen, & Buchert, 2003). Although we used mandarin peel as biomass in a
235
previous study, our results showed a synergistic effect of pectinase and xylanase to
236
enhance enzymatic hydrolysis during mandarin peel bioconversion (Choi, Kim, Wi,
237
Kim, & Bae, 2013; Choi, Lee, Khanal, Park, & Bae, 2015). It is interesting to note that,
238
despite the high pectin content of mandarin peel waste, enhancement of enzymatic
239
hydrolysis was observed after xylanase addition. Although the amount of glucose
240
released increased after the pectinase treatment, the most efficient enzymatic hydrolysis
241
of OSW to glucose was achieved by addition of cellulase, pectinase and xylanase.
242
Cellulase increased from 0.16 to 0.72 mg/g OSW when xylanase and pectinase were
243
fixed at 1.0 and 0.16 mg/g OSW, respectively, causing a total released glucose increase
244
from 3.5 to 4.01 mg/mL (Fig. 1). The highest glucose concentration (4.01 mg/ml) was
245
obtained at 0.72 mg of cellulase, 0.16 mg of pectinase, and 1.0 mg of xylanase.
246
However, further improvement in glucose accumulation was not observed, even though
247
more enzymes were added. Figure S1A shows the HPLC peak before and after
248
enzymatic hydrolysis on the OSW. The conversion rates for OSW to glucose were 98.5%
249
(Fig. S1B). This is because xylanolytic and pectinolytic enzymes opened the surface
250
area of OSW and then, cellulose can subsequently access to the cellulose for hydrolysis
251
(Choi, Lee, Khanal, Park, & Bae, 2015). After complete enzymatic hydrolysis, the
252
remaining solid residues were used in further procedures for quercetin extraction and
253
separation.
254 255
3.3. Quercetin extraction and separation
256
3.3.1. TLC/HPLC
257
Quercetin was detected in the OSW extracts before and after enzymatic hydrolysis
258
using TLC (Fig. 2A). The percentage of quercetin in each OSW extract was quantified
259
using image analysis software. We observed that, after enzymatic hydrolysis, the
260
quercetin percentage in the OSW extract reached as high as 1.61-fold, which is much
261
higher than the value obtained by chemical extraction from an untreated sample (Fig.
262
2C). The quercetin content before and after enzymatic hydrolysis was determined using
263
HPLC (Fig. 2B). After treatment with enzyme, the yields of quercetin increased by
264
1.59-fold.
265 266
3.3.2. SEM /IR
267
Significant morphological changes were observed on the OSW surface before and after
268
enzymatic hydrolysis (Fig. S2). OSW surfaces treated with enzymes displayed a smaller
269
particle diameter than those on untreated surfaces. The morphological changes of the
270
OSW may facilitate the efficient extraction of quercetin. We also confirmed the
271
structural changes before and after enzymatic hydrolysis by FTIR spectroscopy (Fig.
272
S3). After enzymatic hydrolysis, the intensities of the absorption bands were decreased
273
in the carbohydrate region (O-H stretch : 3400 cm-1, C-H stretch : 2925 cm-1, C-O, C-C
274
stretch, C-OH, and C-O-C : 1200-950 cm-1).
275 276
3.3.3. Separation
277
Figure 3A illustrates the schematic process of quercetin purification using Zn-doped
278
TSMNP-Zn matrix. The TSMNP-Zn matrix is composed of Zn-charged terpyridine
279
chelate immobilized onto magnetic nanoparticles. The synthesis of the TSMNP as a
280
solid supporting material was performed following the procedure of Cho et al. (2010),
281
as detailed in the Material and Methods section. Subsequently, Zn ions were
282
immobilized onto the surface of TSMNP matrix.
283
First, we measured the fluorescence spectra of terpyridine derivatives after the
284
addition of zinc ions and quercetin to understand the interaction behavior between the
285
TSMNP-Zn matrix and quercetin (Fig. 3B). In the absence of Zn ions, the terpyridine
286
moiety exhibited a strong fluorescence emission band (λmax = 530 nm) when excited at
287
270 nm. It is interesting to note that the largest decrease in fluorescence intensity
288
resulted from the addition of Zn ions to a solution of terpyridine derivatives. The
289
fluorescence quenching effect can be explained as a reverse photo-induced electron
290
transfer (PET) when the Zn ions are bound to the nitrogen atoms in the terpyridine,
291
behaving as a PET donor (Shinkai & Takeuchi, 2004). Furthermore, the fluorescence
292
emission of the terpyridine-Zn (Ter-Zn) decreased slightly after the addition of quercetin.
293
The hallmark of TSMNP-Zn binding to quercetin is its reversibility in the presence of
294
EDTA. We therefore confirmed that the fluorescence emission of Ter-Zn binds to
295
quercetin after addition of excess EDTA. As expected, the fluorescence intensity of Ter-
296
Zn-quercetin gradually decreased with increasing EDTA, indicating the quantitative
297
binding of EDTA to the Zn ion attached to the terpyridine moiety.
298
The changes in UV visible light absorption of quercetin in the presence of the
299
TSMNP-Zn matrix were examined in a methanol solution. The UV visible light
300
spectrum of quercetin showed an intense absorbance at 210 and 297 nm. When
301
quercetin was added to the TSMNP-Zn matrix, a decrease in absorption was observed.
302
These results indicated the formation of a complex between quercetin and the TSMNP-
303
Zn matrix (Fig. 3C). We also calculated the percentage of binding of the TSMNP-Zn
304
matrix to quercetin and the recovery yield from the change in intensity at 297 nm.
305
Quercetin showed almost 90% binding to the TSMNP-Zn matrix. In addition, the
306
recovery yield of quercetin was ~75% after treatment with EDTA. Thus, the TSMNP-Zn
307
matrix could facilitate quercetin separation from OSW extracts.
308 309
4. Conclusions
310
This study aimed to evaluate the potential of enzymatic hydrolysis for glucose
311
production and quercetin extraction from OSW. The amount of glucose produced from
312
the OSW accounted for ~98.5% of the total glucose in the original material.
313
Furthermore, the quercetin yield increased by 1.61-fold after complete enzymatic
314
hydrolysis. Quercetin was ~90% absorbed using the TSMNP-Zn matrix. These results
315
indicate that enzymatic hydrolysis and TSMNP-Zn matrix are highly efficient for
316
recovering valuable biosugar and quercetin from OSW. This new approach for the
317
extraction of flavonoids and other biosugars is potentially useful for obtaining value-
318
added products from agricultural waste.
319 320
Acknowledgements
321
This work was supported by Priority Research Centers Program (2010-0020141)
322
through the National Research Foundation of Korea (NRF) funded by the Ministry of
323
Education, Science and Technology, Republic of Korea.
324
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Figure Legends
431 432
Fig. 1. Glucose accumulation after enzymatic hydrolysis.
433
Xylanase and pectinase were fixed as 1.0 and 0.16 mg/g OSW, respectively. The
434
released glucose was accumulated from 3.5 to 4.01 mg/ml with increased cellulase from
435
0.16 to 0.72 mg/g OSW.
436 437
Fig. 2. (A) Thin-layer chromatography (TLC) plates showing the
presence of
438
quercetin in isolated onion skin wastes (OSW) samples before and after enzymatic
439
hydrolysis. (B) High-performance liquid chromatography (HPLC) chromatograms of
440
OSW before and after enzymatic hydrolysis. (C) Quantification of quercetin in OSW.
441
The intensity of the quercetin band was quantified using image analysis software. To
442
obtain a linear correlation between the intensity of the band and the amount of quercetin,
443
the extract was exposed at several different exposure times.
444
S: standard; BH: before enzymatic hydrolysis; AH: after enzymatic hydrolysis
445 446
Fig. 3. (A) Schematic representation of the method of quercetin purification using
447
TSMNP. (B) Changes in the fluorescence emission of terpyridine when added to Zn2+,
448
quercetin, and EDTA in succession. Cter, 6.0µM; CZn2+, 1.0 mM; Cque, 1.0 mM; CEDTA,
449
1.0 mM; λex, 270 nm. Solvent; CH3CN:H2O = 1: 1. (C) Changes of the absorption
450
spectra of quercetin when added to TSMNP-Zn and EDTA in succession.
451
Appendix A. Supplementary data
452 453
Fig. S1. Enzymatic hydrolysis of onion skin waste (OSW). (A) High-performance
454
liquid chromatography (HPLC) peak before and after enzymatic hydrolysis of OSW. (B)
455
Enzymatic conversion rates of OSW to glucose.
456 457
Fig. S2. Micromorphological characteristics of onion skin before and after enzymatic
458
hydrolysis observed under an SEM (A) before EH (× 100), (B) before EH (× 5000), (C)
459
after EH (× 100), and (D) after EH (× 5000).
460 461 462
Fig. S3. FTIR analysis of an onion extract before and after enzymatic hydrolysis.
463
464 465
466 467
468 469
470 471 472 473
Table 1. Chemical composition (% dry weight) of onion skin waste. Carbohydrate Arabinose Onion skin waste (This study) Onion peela Papery scales c
Brown skin 474 475 476 477 478 479
a
b
Xylose
Rhamnose
Mannose
Galactose
Glucose
Total
Klason lignin
Ash
UA:NS
0.4±0.1
4.2±0.6
2.5±0.4
3.7±1.0
1.8±0.4
40.6±1.7
22.9±1.0
76.1±1.7
9.4±0.1
5.7±0.3
10.1
0.4
0.8
0.8
1.6
0.8
17.9
17.1
38.9
4.2
nd
12.1
0.8
3.2
0.8
1.6
1.6
35.5
37.9
80.6
nd
nd
16.0
0.4
2.2
1.2
1.1
1.2
32.3
17.0
55.5
1.5
5.7
10.2
From Jaime et al. and for Allium cepa L. cv Sturon from British Onion Producers Association. From Ng et al. for Allium cepa L. cv Sturon from British Onion Producers Association. c From Suutarinen et al. for Allium cepa L. from local farm in Finland. Values present means of triplications ± standard deviations. nd: No data b
Uronic acid
480 481
482 483
Table 2. Glucose concentration of OSW hydrolysate under different enzyme loading conditions. Cellulase (mg/g OSW) 0.16
Pectinase (mg/g OSW) 0.06
Xylanase (mg/g OSW) 0.1
Glucose (mg/mL) 0.4
Conversion rate (%) 9.9
0.16
0.06
0.5
1.65
40.6
0.16
0.06
0.75
2.53
62.3
0.16
0.06
1.0
3.3
81.3
0.16
0.16
0.1
0.55
13.5
0.16
0.16
0.5
1.76
43.3
0.16
0.16
0.75
2.66
65.5
0.16
0.16
1.0
3.5
86.2
0.36
0.06
0.1
0.67
16.5
0.36
0.06
0.5
2.19
53.9
0.36
0.06
0.75
2.88
70.9
0.36
0.06
1.0
3.72
91.6
0.36
0.16
0.1
0.86
21.2
0.36
0.16
0.5
2.37
58.4
0.36
0.16
0.75
3.16
77.8
0.36
0.16
1.0
3.83
94.3
0.72
0.06
0.1
1.46
36.0
0.72
0.06
0.5
2.87
70.7
0.72
0.06
0.75
3.63
89.4
0.72
0.06
1.0
3.92
96.6
0.72
0.16
0.1
1.60
39.4
0.72
0.16
0.5
2.94
72.4
0.72
0.16
0.75
3.85
94.8
0.72
0.16
1.0
4.01
98.5
484 485 486
Highlights
487 488 489
• Onion skin waste (OSW) is an attractive source for value-added byproducts production
490
• 98.5% of glucose was produced from OSW with optimal enzyme dosage
491
• Quercetin yield increased by 1.61-fold from OSW enzymatic hydrolysis
492
• High separation (90%) and purification of quercetin was obtained using TSMNP-Zn
493