Industrial Crops and Products 77 (2015) 1028–1032

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Lignin antioxidants extracted from lignocellulosic biomasses by

treatment with ammonia water
Masahiro Kurakake a,b,∗ , Saya Hirotsu b , Miyuki Shibata b , Asami Kubota b ,
Atsushi Makino b
a
Department of Marine Biotechnology, Fukuyama University, 1-985 Sanzo, Higashimura-cho, Fukuyama, Hiroshima 729-0292, Japan
b
Department of Life and Nutritional Science, Fukuyama University, 1-985 Sanzo, Higashimura-cho, Fukuyama, Hiroshima 729-0292, Japan

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

Article history: In this study, it was found out that treatment with ammonia water efficiently produced thermostable
Received 7 May 2015 antioxidant from lignin of selected herbaceous and soft wood species. The SOD-like activity was enhanced
Received in revised form by ammonia water treatment and Madake (Phyllostachys bambusoides), Moso (Phyllostachys edulis) and
26 September 2015
Susuki (Miscanthus sinensis) showed high activity among the herbaceous species. The IC50 s for the SOD-
Accepted 28 September 2015
like activity of the lignin extract of Akamatsu (Pinus densiflora) and Moso were 0.0190 and 0.0097 mg/ml,
Available online 22 October 2015
respectively, where the IC50 value was represented using the total phenolic content. The lignin antiox-
idants extracted from Moso and Akamatsu had high thermostablility. This pretreatment also improved
Keywords:
Antioxidant
enzymatic hydrolysis of cellulose and hemicellulose for herbaceous species. After enzymatic hydrolysis
Lignin of the treated samples, the release of glucose from Moso was about 3.5 fold more than the untreated,
Lignocellulose achieving total sugars of 114.6 mg/g-sample. Pretreatment of Akamatsu with high lignin content did not
SOD provide any benefits.
DPPH © 2015 Elsevier B.V. All rights reserved.

1. Introduction
Lignocellulosic biomass, which originates from herbaceous or
woody species, consists of cellulose, hemicellulose, and lignin.
Cellulose is converted to bioethanol through saccharification and
fermentation. Various pretreatment procedures have been devel-
oped to enhance the affinity of cellulase for cellulose (Brodeur et al.,
2011). Hemicellulose can be converted to various kinds of sugar.
Especially, l-arabinose, xylose, xylooligosaccharides, and xylitol
are produced from arabinoxylan and xylan (Vázquez et al., 2000;
Kurakake et al., 2011; Albuquerque et al., 2014). These sugars are
then available as functional food ingredients.
The use of lignin is difficult because of its complicated struc-
ture where phenyl-propane units are bound together through
carbon–carbon and ether linkages. In Kraft pulping, lignin is
degraded and separated as black liquor, which is recycled as a fuel
for the pulping process. Many methods of delignification have been
studied for pretreating lignocellulose prior to enzymatic hydrolysis.
The structure of the degraded lignin varies depending on the type
∗ Corresponding author at: Department of Marine Biotechnology, Fukuyama Uni-
versity, 1-985 Sanzo, Higashimura-cho, Fukuyama, Hiroshima 729-0292, Japan.
Fax: +81 84 936 2023.
E-mail address: kurakake@fubac.fukuyama-u.ac.jp (M. Kurakake).
and intensity of the treatment. The degraded products may consist
of phenolic compounds and have antioxidative activities by radi-
cal scavenging (Catignani and Carter, 1982; Dizhbite et al., 2004).
Lignins from herb, wood, and crop residues were prepared by alka-
line, organosolv, and ball-milling treatments and the antioxidant
substances were investigated (Pan et al., 2006; Nadji et al., 2009;
Garcia et al., 2010; Lu et al., 2012; Ross et al., 2012; Hage et al., 2012;
Tangkhavanich et al., 2012).
Previously, we reported the use of pretreatment with ammo-
nia water for enhancing the enzymatic hydrolysis of cellulose and
hemicellulose (Kurakake et al., 2001). This treatment was effec-
tive for herb and crop residues with high arabinoxylan content,
likely because of the degradation of lignin with the alkaline ammo-
nia water. After the treatment, ammonia was easily removed by
vacuum-drying and the treated biomass can be used immediately
for the extraction of antioxidant.
In this study, Moso bamboo (Phyllostachys edulis), Madake
(Phyllostachys bambusoides), Susuki (Miscanthus sinensis),
Seitakaawadachisou (Solidago altissima), Kudzu (Pueraria lobata),
and Akamatsu (Pinus densiflora) were treated with ammonia
water, and then the antioxidant capacity of the degraded lignin
was investigated after pretreatment of cellulose and hemicellulose
for enzymatic hydrolysis.

http://dx.doi.org/10.1016/j.indcrop.2015.09.069
0926-6690/© 2015 Elsevier B.V. All rights reserved.
 M. Kurakake et al. / Industrial Crops and Products 77 (2015) 1028–1032
2. Materials and methods
2.1. Materials
DPPH (1,1-diphenyl-2-picrylhydrazyl) was purchased from
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Xanthine
and xanthine oxidase (buttermilk origin) were purchased from
Nacalai Tesque (Kyoto, Japan). WST-1 ((4-[3-(4-iodophenyl)-2-
(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium
salt was purchased from Wako Pure Chemical Industries Ltd.
(Osaka, Japan). All other chemicals were of reagent grade.
Moso bamboo (P. edulis), Madake (Giant timber bamboo; P. bam-
busoides, Susuki (M. sinensis), Seitakaawadachisou (S. altissima),
Kudzu (P. lobata), and Akamatsu (Japanese red pine; P. densiflora)
were sampled in Fukuyama, Japan. Cellulose and lignin contents (%)
in raw materials were 42.9, 24.3 (Moso); 50.1, 24.5 (Madake); 44.0,
18.8 (Susuki); 41.3, 17.3 (Seitakaawadachisou); 26.2, 16.2 (Kudzu);
and 51.8, 26.2 (Akamatsu) (Ooshima et al., 1992; Kurakake et al.,
2001; Okan et al., 2013; Ren et al., 2013; Nakhshiniev et al., 2014).
After washing with water and drying at 70 ◦ C for 1 day, the cutting
was ground to less than 1 mm by Cyclotec 1093 (Foss Ltd., Hillerod,
Denmark).
2.2. Treatment with ammonia water and extraction of lignin
The sample (2 g) and 25% NH3 (4 ml) were mixed in a 25-ml vial
and capped. The vessel was autoclaved at 120 ◦ C for 20 min accord-
ing to the previously published procedure (Kurakake et al., 2001).
The temperature was raised to 120 ◦ C from 100 ◦ C at 2.5 ◦ C/min.
After the incubation at 120 ◦ C for 20 min, it was cooled to 100 ◦ C
at a rate of 1.4 ◦ C/min. The heated mixture was vacuum-dried after
air-drying in a hood to remove ammonia. The treated (50 mg) was
mixed with 1 ml of an ethanol–water solution (0, 20, 40, 60, 80 and
100% ethanol) and incubated at ambient temperature for 1 h. At an
interval of 10 min, the mixture was stirred. After centrifugation at
1500 × g for 10 min, the supernatant was filtered through a 0.22 ␮m
membrane (hydrophilic PVDF) and used for the examination of the
antioxidative activities of the degraded lignin.
In the preparation of the degraded lignin from Moso and Aka-
matsu, 10 ml distilled water was added and mixed immediately
after the treatment with ammonia water. The black supernatant
containing the degraded lignin was separated by centrifugation at
1500 × g for 10 min. The lignin solution was neutralized with 1 M
sulfuric acid at a ratio 3:2 (lignin extract: sulfuric acid), and diluted
by 0.1 M Tri–HCl buffer (pH 7.4). The pH was adjusted to 7.2–7.5
and the solution examined for its antioxidative activity.
2.3. Determination of the radical-scavenging activity with DPPH
The diluted sample (0.2 ml) and 0.1 M Tris–HCl buffer (pH7.4,
0.8 ml) were mixed and 0.5 M DPPH (1 ml) dissolved in ethanol was
added. After mixing vigorously, the solution was stored for 20 min
at ambient temperature in the dark. The absorbance at 517 nm
was measured using a spectrophotometer U-2000 (Hitachi High-
Technologies Limited, Tokyo, Japan). A decrease in the absorbance
of DPPH indicates the presence of radical-scavenging activity. Two
Table 1
Summary of IC50 values of samples treated with ammonia water at high temperature.
Sample IC50 in DPPH radical-scavenging activity (mg/ml)
Madake (Phyllostachys bambusoides) 16.1 ± 2.5
Moso (Phyllostachys edulis) 20.5 ± 2.1
Susuki (Miscanthus sinensis) 14.6 ± 2.5
Seitaka (Solidago altissima) 44.0 ± 6.7
Kudzu (Pueraria lobata) 37.3 ± 4.2
1029
times of experiments were done and error values (standard devia-
tion) were calculated.
2.4. Determination of SOD-like activity
SOD-like activity was determined using WST-1 as described
previously (Ueda et al., 1999). The diluted sample (0.1 ml), 2 mM
xanthine (0.1 ml), 3 mM EDTA (0.1 ml), 50 mM sodium phosphate
buffer (pH 8.0, 2.5 ml), and 1.5 mM WST-1 (0.1 ml) were mixed
in a test-tube. The mixture was incubated at 37 ◦ C for 20 min
after adding 60 mU/ml xanthine oxidase (0.1 ml). The absorbance
at 438 nm was measured by spectrophotometer. Superoxide pro-
duced by xanthine and xanthine oxidase converted WST-1 to
formazan dye. A decrease in the absorbance of the dye upon
addition of the sample indicates SOD-like activity. Two times of
experiments were done and error values (standard deviation) were
calculated.
2.5. Determination of total phenolic content
The diluted sample (1 ml) was mixed with Folin-Ciocalteu
reagent (1 ml). After 3 min, 10% sodium carbonate (1 ml) was added.
After the mixture was stored for 15 min at ambient temperature,
the absorbance at 700 nm was measured by spectrophotometer.
Gallic acid was used as a standard and the amount of total phenolic
content was expressed as mg of gallic acid per 1 g of the sample
used for the treatment. Two times of experiments were done and
error values (standard deviation) were calculated.
2.6. Enzymatic Hydrolysis and HPLC analyses of sugars
Fifty milligrams of sample and 1 ml of 10 mg/ml Meicelase (60 U
CMCase/ml and 47 U xylanase/ml) were incubated at pH 5.0 and
40 ◦ C for 24 h. After centrifugation at 1500 × g for 10 min, the super-
natant was filtered using a 0.22 ␮m membrane (hydrophilic PVDF),
prior to sugar analysis using HPLC under the following conditions:
column, 250 × 7 mm i.d. GL-C610 (Hitachi Kasei Limited, Tokyo,
Japan); mobile phase, water; column temperature, 60 ◦ C; flow rate,
1.0 ml/min; and detector, L-3300 differential refractive index mon-
itor (Hitachi High-Technologies Limited, Tokyo, Japan). Two times
of experiments were done and error values (standard deviation)
were calculated.
3. Results and discussion
The extract of antioxidants from Madake treated with ammo-
nia water was examined using an ethanol–water solution (0, 20,
40, 60, 80 and 100% ethanol) in which the treated Madake (50 mg)
was stored with the ethanol-water solution (1 ml) at ambient tem-
perature for 1 h. The radical-scavenging activities of the solution
was measured using DPPH. The extraction was improved by the
addition of ethanol where 60% ethanol was the most appropri-
ate for the extraction. The Moso, Susuki, Seitaka (abbreviation of
Seitakaawadachisou), and Kudzu samples were also treated with
ammonia water and their antioxidative activities were measured
from a 60% ethanol extract. Fig. 1 shows their IC50 s (50% inhibitory
IC50 in SOD-like activity (mg/ml)
11.6 ± 2.3
11.4 ± 1.0
11.4 ± 0.9
16.0 ± 3.8
14.6 ± 1.8
1030 M. Kurakake et al. / Industrial Crops and Products 77 (2015) 1028–1032
Fig. 1. IC50 for the DPPH radical-scavenging activity of each lignocellulose treated
with ammonia water. Bar; ( ) extracted with water alone and ( ) extracted with
60% ethanol.
concentration) for their radical-scavenging activity with DPPH. The
values were determined by the relationship between the activity
and sample concentration used for the extraction. A smaller IC50
value indicates a higher antioxidant capacity. Madake, Moso, and
Susuki had the high antioxidant capacities in the extraction with
60% ethanol in comparison with water alone. For Kudzu, the sam-
ple extracted with 60% ethanol had antioxidant activities similar to
that of water alone.
The SOD-like activities of the extracts were also measured.
Table 1 shows a summary of the antioxidative activities (IC50 ) for
samples treated with ammonia water at high temperature (120 ◦ C).
For each sample, the SOD-like activity was higher than the radical-
scavenging activity measured with DPPH. Although the DPPH
radical is stable and useful as a tool to measure antioxidant capac-
ity, its large structure makes it very different from active oxygen.
Instead, the measurement of SOD-like activity used superoxide,
a natural antioxidant. Madake, Moso and Susuki had high SOD-
like activities with IC50 values of 11.4–16.0 mg/ml, some 17–24
fold higher than Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid), a derivative of vitamin E. The extract did not
have as high an antioxidative activity as vitamin E. If the antioxi-
dants in the extract had been purified, a higher activity would be
expected. The antioxidants produced by ammonia water treatment
likely include phenolic compounds released from lignin because
the antioxidant capacity of lignin is known to be closely related to
Fig. 2. IC50 for the DPPH radical-scavenging activity and SOD-like activity of Moso
and Akamatsu treated with ammonia water. A; DPPH radical-scavenging activity
and B; SOD-like activity. Bar; ( ) untreated and ( ) treated with ammonia water.
Fig. 3. IC50 for the DPPH radical-scavenging activity and SOD-like activity of lignin
extract from Moso and Akamatsu treated with ammonia water. Bar; ( ) DPPH
radical-scavenging activity and ( ) SOD-like activity.
its total phenolic content (Tangkhavanich et al., 2012). The treat-
ment of lignocellulosic wastes with ammonia water is an attractive
new method to produce antioxidants.
During the treatment of lignocellulosic wastes with ammonia
water at high temperature (120 ◦ C), antioxidants were formed from
herbaceous species. This treatment was also applied to Akamatsu,
a soft wood with high lignin content. Fig. 2 shows the antioxida-
tive activities of the extracts from Moso and Akamatsu treated
with ammonia water. The antioxidant activity of the extract from
Moso was improved by the treatment. On the other hand, the
antioxidant activity of the extract from Akamatsu was reduced by
the treatment, as reflected by the four-fold increase in the IC50 .
This indicates that the radical-scavenging activity of the Akamatsu
extract was less than that of the untreated extract. Similar trends
were observed for the SOD-like activities of the extracts. For the
measurement of the radical-scavenging activity with DPPH, the
IC50 values varied more between the treated and untreated extract
than the SOD-like activities. This suggests that the DPPH radical is
different from natural active oxides.
The degraded lignin was extracted immediately after the treat-
ment with ammonia water and the black solution containing
the soluble lignin in water was applied for the determination of
antioxidative activities. Fig. 3 shows the IC50 values for the DPPH-
scavenging and SOD-like activities of the lignin extract, which are
presented in terms of the sample concentration used in the treat-
ment. The results for the degraded lignin are similar to those of the
treated sample as shown in Fig. 2. The IC50 s for the SOD-like activ-
ity were about 13 mg/ml for both the Moso and Akamatsu lignin
extracts. When the IC50 for the Akamatsu extract was adjusted
based on its lignin content, the value decreased to 3.4 mg/ml (lignin
content; 26.2% (Ooshima et al., 1992)). This suggests that for Aka-
matsu, antioxidants with high activities are formed from the lignin
by the treatment with ammonia water. The total phenolic content
of the lignin extracts of Akamatsu and Moso were 0.683 ± 0.069
and 1.334 ± 0.044 mg-gallic acid/g-untreated sample, respectively.
When the IC50 values for SOD-like activity are adjusted for the total
phenolic content, they were 0.0190 and 0.0097 mg/ml, for Aka-
matsu and Moso, respectively. The phenolic hydroxyl groups are
formed in the depolymerization of lignin and have high antioxidant
capacity (Dizhbite et al., 2004; Pan et al., 2006). The substances with
phenolic hydroxyl groups include coniferyl alcohol, eugenol, propyl
Table 2
Thermostability of lignin extract from Moso and Akamatsu in SOD-like activity.
Sample Residual activity (%)
Moso (Phyllostachys edulis) 94.2 ± 2.5
Akamatsu (Pinus densiflora) 93.2 ± 8.3
Ferulic acid 56.7 ± 2.5
Ascorbic acid 0.6 ± 4.4
Samples of 65–69% in SOD-like activity were incubated at pH 7.2–7.5 and 100 ◦ C for
120 min.
 M. Kurakake et al. / Industrial Crops and Products 77 (2015) 1028–1032
Table 3
Sugars released from treated sample of Moso and Akamatsu by cellulase.
Sample Glucose Xylobiose
<Moso (Phyllostachys edulis)>
Untreated 17.6 ± 1.4 7.5 ± 0.6
Treated by ammonia 37.5 ± 1.5 24.2 ± 1.0
Removal of lignin 61.2 ± 2.9 32.8 ± 1.6
<Akamatsu (Pinus densiflora)>
Untreated 30.0 ± 0.8 0 4.8 ± 0.1
Treated by ammonia 22.4 ± 1.3 0 4.2 ± 0.2
Removal of lignin 37.3 ± 3.2 8.8 ± 0.7
Each sample (5%) and Meicelase (10 mg/ml) was incubated at pH 5 and 40 ◦ C for 28 h.
quaiacol, and syringol (Dizhbite et al., 2004). To act as antioxidants,
the phenolic hydroxyl group forms a phenoxyl radical and the
methoxy group in the ortho position stabilizes the radical (Barclay
et al., 1997; Hage et al., 2010). Although the phenolic content in
Moso was higher than that in Akamatsu, its SOD-like activity per
phenolic content was smaller. Herbaceous species such as Moso
have high hemicellulose content. During the ammonia treatment,
the arabinoxylan from the herbaceous species was efficiently dis-
solved in the water phase (Kurakake et al., 2001). Carbohydrates
such as hemicellulose are inhibitors of the antioxidant capacity, by
hydrogen bonding to the phenolic compounds from lignin (Dizhbite
et al., 2004).
Untreated Akamatsu has high antioxidative activity. The original
antioxidants were degraded by the severe treatment with ammonia
water at high temperature, and new antioxidants were produced
from the lignin degraded by the treatment. This suggests that the
antioxidants of untreated Akamatsu have poorer thermostability
than that of the treated. As the treatment with ammonia water
was done at high temperature, it is understood that the antioxi-
dants produced from the lignin have high thermostability. Lignin
extracts from Moso and Akamatsu were diluted to 65–69% based on
their SOD-like activity and incubated at pH 7.2–7.5 and 100 ◦ C for
120 min in a block heater. As shown in Table 2, the residual SOD-
like activities were more than 93% in the lignin extracts of Moso
and Akamatsu, although the activities of the reference substances
(ferulic acid and ascorbic acid) were significantly decreased. It is
possible that the thermostable antioxidants isolated from lignin
would be useful as food ingredients.
The sugar recovery from cellulose and hemicellulose after cel-
lulase treatment was examined for each sample (Moso, Madake,
Susuki, Seitaka, and Kudzu) treated with ammonia water at high
temperature. The amounts of glucose released from the treated
samples by cellulase (Meicelase) were measured. The enzyme
treatment improved the release of glucose from cellulose, in par-
ticular, for Susuki and Seitaka, 38.3 and 44.1 mg-glucose/g in the
untreated were efficiently increased to 166 and 121 mg-glucose/g
in the treated, respectively. This treatment provides the benefit on
both the production of antioxidants from lignin and glucose from
cellulose.
After extraction of lignin from Moso and Akamatsu (see Fig. 3),
the residues were washed with water and acetone, dried and sub-
jected to enzymatic hydrolysis by cellulase. Table 3 shows the sugar
composition released from the residues. In both samples, removal
of lignin after treatment with ammonia water enhanced the enzy-
matic hydrolysis. For Moso, the release of glucose was about 3.5 fold
larger than that from the untreated sample. The total sugar released
from cellulose and arabinoxylan was increased to 114.6 mg/g-
sample. For Akamatsu, a soft wood with high lignin content, a total
sugar content of only 52.5 mg/g-sample was achieved. A higher
treatment temperature is necessary to efficiently degrade and
extract the rigid lignin of Akamatsu for improvement of the enzy-
matic hydrolysis. The phenolic content extracted from lignin was
1031
Xylose l-Arabinose Total (mg/g-sample)
8.2 ± 0.7 0 33.3 ± 1.7
11.9 ± 0.5 4.7 ± 0.2 78.3 ± 1.9
15.7 ± 0.8 4.9 ± 0.2 114.6 ± 3.4
0 34.8 ± 0.8
4.7 ± 0.3 31.3 ± 1.4
3.4 ± 0.3 3.0 ± 0.3 52.5 ± 3.3
also small. Organic solvent like ethanol or dioxane is used to extract
rapidly degraded lignin, which remains undissolved into the water
phase (Nadji et al., 2009; Hage et al., 2012; Li et al., 2012; Lu et al.,
2012). An organosolv treatment using an organic solvent plus an
ammonia water treatment seems to be effective to extract a large
amount of lignin from Akamatsu.
For herbaceous species in particular, ammonia water treat-
ment not only functions as a pretreatment prior to the enzymatic
hydrolysis of cellulose and hemicellulose, but also improves the
production of antioxidants from lignin. Additionally, the used
ammonia can be easily separated. This method should prove useful
for ensuring separate applications of each component of lignocel-
lulose: cellulose, hemicellulose and lignin.
4. Conclusion
This treatment with ammonia water is an attractive new method
to produce efficiently antioxidants from lignocellulosic wastes. The
thermostable antioxidants obtained from lignin by the treatment
would be useful as food ingredients, rubbers, latex, plastics etc. This
treatment also improves sugar recovery from herbaceous species
by enzymatic hydrolysis. It was seemed that an organosolv treat-
ment with an ammonia water was effective to extraction of lignin
in Akamatsu.
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