Industrial Crops & Products 178 (2022) 114591

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Industrial Crops & Products

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Bioprocess development for production of xylooligosaccharides prebiotics

from sugarcane bagasse with high bioactivity potential
Mahak Gupta a, Ridhika Bangotra a, Surbhi Sharma a, Surbhi Vaid a, Nisha Kapoor a,
Harish Chander Dutt b, Bijender Kumar Bajaj a, *
a
School of Biotechnology, University of Jammu, Jammu, India
b
Department of Botany, University of Jammu, Jammu, India

A R T I C L E I N F O A B S T R A C T

Keywords: The xylooligosaccharides (XOS, prebiotics) production from xylan extracted from sugarcane bagasse (SB)
Aspergillus flavus MG-7 biomass was accomplished using an in-house produced xylosidase-free endoxylanase from an isolate Aspergillus
L. plantarum M-13 flavus MG-7. Delignification of SB by sodium hypochlorite, and xylan extraction by coupled sodium hydroxide
Prebiotics
and thermal treatment were optimized for various process variables based on Design of Experiments (DoE) to
Sugarcane bagasse
achieve enhanced xylan yield (96.11%, w/w). The extracted xylan was analysed by Fouriertransform infrared
Xylooligosaccharides
spectroscopy and thermogravimetric analysis to evaluate its structural morphology, purity and thermal stability.
Xylan hydrolysate obtained by endoxylanase was quantified for xylobiose (69.57%, w/w), xylotriose (13.17%)
and xylotetrose (8.377%), and XOS were examined by 1H NMR. Growth-promoting potential of XOS was
established for a probiotic Lactobacillus plantarum M-13 isolated from kalarei. XOS exhibited excellent bioactivity
potential in terms of high positive prebiotic activity score, significant antioxidant activity and antibacterial
activity. Efficient XOS production promises value-addition for biorefining of SB biomass.

1. Introduction 2021; Forsan et al., 2021).

Lignocellulosic biomass (LB) represented by agroindustrial/forestry
residues is an inexpensive, plentiful and renewable resource that can be
exploited for production of energy, biofuels, chemicals and materials of
high commercial value (Khaire et al., 2021; Meghana and Shastri, 2020).
However, hemicellulose, especially the xylan, major hemicellulosic
fraction of LB, is underutilized during bioconversion process, and thus,
undermines the overall process. Therefore, new/novel technologies are
being investigated for complete utilization of LB-polysaccharides for
successful biorefining (Alvarez et al., 2017). Sugarcane is one of the
largest conventional crops grown in various parts of the world including
India (Yang et al., 2021). The sugarcane bagasse (SB) left after the
extraction of sugarcane juice is a very rich source of carbohydrates
(40–45% of cellulose, 20–35% of hemicelluloses), and may be used for
the production of various commercial commodities (Meghana and
Shastri, 2020; Sharma et al., 2021a). Furthermore, extraction of xylan,
and its utilization for the production of various high value industrial
products such as xylitol, xylose and xylooligosaccharides (XOS) pre­
biotics promises value-addition in the biorefining of SB (de Freitas et al.,
Prebiotics are the non-digestible but fermentable oligosaccharides
which may enhance the growth, vigour, and metabolic potential of some
beneficial bacteria in the gastrointestinal tract (GIT) called as ‘pro­
biotics’ (Holscher, 2017). Prebiotics must be resistant to acidic gut
conditions and digestive enzymes so that these reach the colon, and get
fermented by the resident bacteria (Ghosh et al., 2021). Recently, there
has been tremendous research interest in gut microbiota composition
and health, and favourable interventions by probiotics and prebiotics
mainly due to their multifaceted health advantages in various systemic
disorders such as gastro-intestinal, cardiovascular, neurological, in­
flammatory, oncological and endocrine systems (Oniszczuk et al., 2021).
Beneficial gut microbiota (probiotics) in the host’s colon selectively
utilizes the prebiotics as substrates, and increases their numbers. Thus,
prebiotics enhance the growth and number of beneficial gut bacteria
which not only exclude various pathogenic bacteria but confer a variety
of other health benefits. Furthermore, the short chain fatty acids (SCFAs)
generated due to prebiotics fermentation possesses immunomodulatory
properties, and enhances the gut barrier integrity (Oniszczuk et al.,
2021). Various commonly used prebiotics include inulin,

* Corresponding author.
E-mail address: bajajbijenderk@gmail.com (B.K. Bajaj).

https://doi.org/10.1016/j.indcrop.2022.114591
Received 30 October 2021; Received in revised form 1 January 2022; Accepted 18 January 2022
Available online 29 January 2022
0926-6690/© 2022 Elsevier B.V. All rights reserved.
M. Gupta et al.
fructooligosaccharides (FOS), galactooligosaccharides, lactulose, poly­
dextrose, isomaltooligosaccharides, xylooligosaccharides, lactitol, and
others (Guarino et al., 2020).
Xylooligosaccharides (XOS), the oligomers of β-1, 4-linked xylose
with numerous substitutions have recently emerged as a potential class
of prebiotics (Yang and Xu, 2018). XOS are produced by the hydrolysis
of xylan, and may differ with respect to degree of polymerization (DP) i.
e. the occurrence of monomeric units, degree of substitution (ratio of
arabinose to xylose), and the linkages between them (Khaleghipour
et al., 2021). Normally, DP of XOS in the range of 2–10 is preferable. Due
to the presence of β-1, 4-xylosidic bonds, the XOS are resistant to gastric
enzymes of the host’s gut, and are successfully fermented by beneficial
gut bacteria like Bifidobacterium species (B. adolescentis, B. infantis, B.
longum, B. bifidum, and others), and several Lactobacillus species which
result in increased growth and number of these bacteria (Holscher,
2017). XOS prebiotics have been reported to promote the selective
growth of a variety of other probiotics (Zhang et al., 2018). Further­
more, a wide bioactivity spectrum of XOS prebiotics viz. immunomod­
ulatory, anticancerous, antimicrobial, growth regulatory, antioxidant,
antimicrobial and other bioactive properties promote the host’s health
in multifaceted ways (Avila et al., 2020; Khaleghipour et al., 2021).
Additional health promoting benefits of XOS include gastrointestinal
function improvement, blood cholesterol lowering, reducing stomach
lesions, and others, thus, indicating a huge application potential of XOS
for food and pharmaceutical industries (Zhang et al., 2018a).
The enzymatic production of XOS is preferred due to several ad­
vantages such as no or low production of undesirable by-products, easy
recovery of pure XOS, and the environmental safety of the process (Avila
et al., 2020). Endoxylanases produce XOS from xylan, however,
β-xylosidase degrades the XOS to produce xylose. Therefore, endox­
ylanases intended for XOS production must be β-xylosidase-free (Surek
and Buyukkileci, 2017). Owing to numerous applications of XOS, in­
dustries are attempting to develop new technologies for production of
XOS with high purity, and with a desired degree of polymerization.
However, the production of XOS still remains an expensive and
complicated process despite high market value and demand (Forsan
et al., 2021).
Considering the importance of XOS for food and pharmaceutical
industries, the present study aimed at producing XOS from sugarcane
bagasse xylan by employing an in-house produced process-apt xylosi­
dase-free endoxylanase from an indigenously isolated Aspergillus flavus
MG-7. Design of experiments (DoE) based optimization of delignifica­
tion, and xylan extraction from sugarcane bagasse is being reported for
the first time to obtain high xylan yield. Furthermore, the enzymatic
hydrolysis of xylan using A. flavus MG-7 endoxylanase was optimized for
production of XOS. Also, the XOS were investigated for desired func­
tional attributes with respect to their growth promoting effect on a
probiotic L. plantarum M-13 (LPM-13), prebiotic activity score, antiox­
idant, and antibacterial activity. LPM-13 was previously isolated from
an indigenously fermented milk product kalarei, and characterized for
functional probiotic attributes, and used for development of fermented
foods (Gupta and Bajaj, 2018, 2017). Additionally, the extracted xylan,
and the XOS were studied by nuclear magnetic resonance (1H NMR),
Fourier transform infrared spectroscopy (FTIR) and thermogravimetric
analysis (TGA) to elucidate their structural and functional
characteristics.
2. Material and methods
2.1. Lignocellulosic biomass, process organisms, chemicals and reagents
Sugarcane bagasse (SB) was collected from a local sugarcane juice
vendor of Jammu, India. The biomass was finely chopped, washed with
hot distilled water and dried in oven at 60◦ C for 24 h. SB was grounded,
sieved to a uniform size (<4 mm) and stored in an air tight container at
room temperature.
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Industrial Crops & Products 178 (2022) 114591
A probiotic bacterial isolate L. plantarum M-13 (LPM-13) from kalarie
(Gupta and Bajaj, 2017) that possessed several probiotic attributes
(Gupta and Bajaj, 2018, 2017) was used for analysis of growth pro­
moting ability of XOS. LPM-13 was preserved in glycerol at –20 ◦ C,
revived in De Man, Rogosa and Sharpe (MRS) broth (at 37◦ C, 180 rpm,
18 h) and used for various experiments. The fungal isolate MG-7, an
isolate from Himalayan Shivalik foothills, was procured from Fermen­
tation Biotechnology Laboratory, School of Biotechnology, University of
Jammu, Jammu. The fungal strain MG-7 was capable of producing
xylosidase-free endoxylanase. The isolate MG-7 was identified as
Aspergillus flavus based on culture morphology, microscopic, and
sequence analysis of PCR-amplified internal transcribed spacer (ITS)
region (Sharma et al., 2021b). The ITS sequence showed high resem­
blance (99%) with that of Aspergillus flavus strains available at National
Centre for Biotechnology Information (NCBI) database, thus, the isolate
was identified and designated as A. flavus MG-7. The ITS sequence was
submitted to GenBank (https://www.ncbi.nlm.nih.gov/nuccor­
e/MG736928) with an accession number MG736928. Xylosidase-free
endoxylanase was in-house produced from A. flavus MG-7 under sub­
merged fermentation, and used for XOS production from xylan extracted
from SB.
The bacterial pathogens (Staphylococcus aureus, Escherichia coli,
Micrococcus luteus, Enterococcus faecalis, Bacillus cereus, Pseudomonas
aeruginosa, Streptococcus pneumoniae, Pseudomonas alcaligenes, Bacillus
subtilis and Klebsiella sp.) were procured from the culture repository of
the Fermentation Biotechnology Laboratory of the School of Biotech­
nology, University of Jammu. All the media, chemicals and reagents
used were of high analytical grade.
2.2. Polysaccharide composition of biomass
The polysaccharide composition (%, w/w) of raw and delignified SB
was determined (Sluiter et al., 2008) by HPLC (Shimadzu RID-20A)
using Aminex HPX 87 H column (temperature 80ºC) using water as
the mobile phase. The samples were run at an isocratic mode with a flow
rate of 0.6 ml/min and an end time of 20 min. The quantitative analysis
of various monosaccharides (glucose, xylose, mannose and arabinose)
was done using appropriate reference standards.
2.3. Pretreatment of sugarcane bagasse for delignification
Pretreatment of sugarcane bagasse was executed by four different
delignifying reagents i.e. sodium hypochlorite, sodium chlorite, aqueous
ammonia, and combination of hydrogen peroxide and acetic acid
(H2O2–Hac) at a biomass loading of 1:10 (%, w/v) under different set of
conditions (Reddy and Krishnan, 2016). Reaction conditions (reagent
concentration, temperature, time) for delignification were: sodium hy­
pochlorite (1% w/v, 40ºC, 1 h), sodium chlorite (1% w/v, 75ºC, 2 h),
aqueous ammonia (15% w/v, 40ºC, 16 h), H2O2–Hac (1:1 hydrogen
peroxide 21.6 M and glacial acetic acid 8.74 M, 60ºC, 7 h). After
delignification, the reaction contents were vacuum filtered and lignin
rich filtrate was examined for sugar loss (Dubios et al., 1956). The
sample was appropriately diluted with distilled water, and used for
sugar assay. To 1.0 ml of diluted sample, 1.0 ml of phenol (5% v/v) and
5.0 ml of sulphuric acid (96% v/v) was added, and reaction content was
incubated at room temperature for 20 min with intermittent shaking.
Sugar content was examined spectrophotometrically (A490) using xylose
standard and expressed as mg/g. The distilled water was used as blank.
The most efficient delignification reagent (sodium hypochlorite) which
incurred minimum sugar loss was selected, and the delignification
process was optimized for various process parameters.
2.4. Optimization of delignification of sugarcane bagasse by sodium
hypochlorite
Central composite design (CCD) of response surface methodology
M. Gupta et al.
(Design-Expert 6.0 software, Stat Ease, Inc., Minneapolis, Minnesota,
USA) was applied for optimization of sodium hypochlorite based
delignification of SB. The four process variables at minimum and
maximum level (indicated in parentheses) were: biomass loading
(5–10%, w/v), temperature (40–60◦ C), time (60–90 min) and sodium
hypochlorite concentration (0.50–1%, v/v). Thirty experiments devel­
oped by DoE were performed, and the results were examined by analysis
of variance (ANOVA). The significance of process variables, and the
model was determined (p < 0.05). Three dimensional response surface
plots generated by the model were used to determine the influence of
variables on the response (xylan yield %, w/w) individually and
interactively.
dry weight of extracted xylan (g)
Xylan (%, w/w) = × 100
weight of the sample (g)
The validation of the process model was done based on the point
prediction tool of the software. The experiments were performed at
optimal level of variables predicted by the software, and the experi­
mental and predicted responses were analysed.
2.5. Optimization of xylan extraction from delignified sugarcane bagasse
Three different approaches i.e. alkali hydrolysis, acid hydrolysis, and
coupled alkali (sodium hydroxide) and thermal treatment (121 ºC for
1 h), were examined for xylan extraction from delignified SB at biomass
loading of 1:10 (%, w/v) (Chapla et al., 2012). The coupled alkali (so­
dium hydroxide) and thermal treatment which yielded the maximum
xylan (%, w/w), was further optimized by DoE for four process variables
(minimum and maximum level in parentheses) i.e. extraction tempera­
ture (65–100ºC), biomass loading (5–10%, w/v), extraction time
(10–20 min) and sodium hydroxide concentration (10–14%, v/v).
Thirty experiments generated by the software were performed, and the
results were examined by ANOVA. The significance of the variables and
the model was ascertained. The individual and the interactive influences
of the process variables on xylan yield were studied by the 3-D response
surface plots and perturbation plot. The optimum level of the variables
predicted by the point prediction tool, was used to execute the experi­
ments, and the observed (experimental), and predicted responses were
analysed, to validate the process model.
2.6. Structural characterization of the xylan extracted from sugarcane
bagasse biomass
Xylan extracted from the delignified SB was characterized by Fourier
transform infrared (FTIR) spectroscopy, and thermogravimetric analysis
(TGA) (Yiin et al., 2017; Li et al., 2016). FTIR spectrum of the extracted
xylan was obtained using Thermo Nicolet, Avatar 370 spectrophotom­
eter ( 4000–400 cm− 1, resolution 4 cm− 1, KBr beam splitter). TGA of
xylan was done by heating the sample (20.0 mg) at 50–750 ◦ C with
10 ◦ C increase/min in continuous nitrogen gas flow (50 ml/min) (Perkin
Elmer, Diamond TG/DTA). Furthermore, the surface topography of the
residual SB biomass after delignification and xylan extraction, was
examined by scanning electron microscopy, SEM at 1000X to 3000X
(JEOL Model JSM - 6390LV instrument, accelerating voltage 30 kV)
(Sharma et al., 2021a). All the biomass samples were analysed at So­
phisticated Test and Instrumentation Centre (STIC), Cochin University
of Science and Technology (CUSAT), Kerala, India.
2.7. Xylosidase-free endoxylanase from fungal isolate Aspergillus flavus
MG-7
The xylanolytic activity of the A. flavus MG-7 was ascertained by
qualitative and quantitative assay (Bajaj et al., 2011). Xylanase pro­
duced under submerged fermentation was partially purified and
concentrated by ammonium sulphate fractionation (60–80%), followed
by dialysis for 8 h with intermittent change of buffer after every 4 h. The
3
Industrial Crops & Products 178 (2022) 114591
enzyme preparation so obtained was lyophilized. For ascertaining the
xylosidase-free mode of action of the xylanase, enzymatic hydrolysis of
the standard xylan (beech wood xylan, 0.5% w/v) was carried out by
using xylanase at different loadings (20 − 100 IU) at 50ºC under shaking
(150 rpm, 16 h). The endoxylanolytic nature of the xylanase was
examined (Bajaj et al., 2011) by thin layer chromatography (TLC) on
pre-coated silica gel plates (Macherey-Nagel, Alugram extra SIL
G/UV254).
2.8. Enzymatic production of xylooligosaccharides using A. flavus MG-7
endoxylanase
Enzymatic production of XOS from xylan extracted from SB was
accomplished using in-house generated xylosidase-free endoxylanase
from fungal isolate A. flavus MG-7. Optimization of various process
variables was done in a sequential manner using one variable at a time
(OVAT) approach. Enzymatic hydrolysis was carried out at 50ºC with
mild shaking (50 rpm). The reaction without enzyme was set as a con­
trol. Effect of enzyme dosage (xylanase loading 5 IU to 120 IU) on XOS
production was studied using xylan at 0.5% (w/v) as the substrate.
Similarly, the effect of xylan concentration (0.5–3.0%, w/v) at a selected
‘xylanase dosage’, was examined, and the effect of reaction time (16, 24
and 48 h) was determined using selected optimal values of ‘xylanase
loading’ and ‘xylan concentration’. The total reducing sugar content
(expressed as xylose equivalent mg/g) was examined for different ex­
periments, and the reaction conditions which yielded maximum xylose
equivalent were considered as the optimal ones for enzymatic hydrolysis
of xylan.
The qualitative analysis of XOS was executed by TLC (Bajaj et al.,
2011) while the quantitative analysis (XOS %, w/w) was done by HPLC
under operating conditions described in Section 2.2, at a column tem­
perature of 40ºC. Xylose and xylooligosaccharides (xylobiose X2, xylo­
triose X3 and xylotetrose X4) were used as the reference standards
(Megazyme, Prolab). The XOS hydrolysate was lyophilized and stored at
–20ºC for further use.
2.9. Nuclear magnetic resonance analysis of xylooligosaccharides
For obtaining the NMR spectrum (Bruker Avance III, 400 MHz), a
10 mg of lyophilized XOS was dissolved in 0.5 ml of D2O at the following
operating conditions i.e. 25ºC, 125 scans, proton 90 R at 10.65 ms, and
the recycle delay at 3 s. NMR analysis was done at Sophisticated Test
and Instrumentation Centre (STIC), Cochin University of Science and
Technology (CUSAT), Kerala, India.
2.10. Prebiotic potential of xylooligosaccharides for growth of L.
plantarum M-13
Effect of XOS on the selected probiotic bacterium LPM-13 was
determined by using modified MRS (% w/v, proteose peptone 1.0, beef
extract 1.0, yeast extract 0.5, polysorbate 0.1, ammonium citrate 0.2,
sodium acetate 0.5, magnesium sulphate 0.01, manganese 0.005,
dipotassium phosphate 0.2, pH 6.5 ± 0.2) in which the normal carbon
source (glucose) was replaced either with XOS or with any of the com­
mercial prebiotics (inulin, fructooligosaccharide FOS or lactulose), as
the sole carbon source. MRS with glucose was used as the standard for
the growth analysis of LPM-13 (Noori et al., 2017).
The filter sterilized prebiotic or glucose solution was added to the
pre-autoclaved medium broth (1%, w/v), and inoculated with the pro­
biotic LPM-13 (1% v/v, 108–109 cells/ml) and incubated (37◦ C,
180 rpm, 144 h). Samples withdrawn at different time intervals were
examined for growth of LPM-13 (log cfu/ml) based on viable plate count
on MRS agar.
M. Gupta et al.
2.11. Stability of xylooligosaccharides towards human gastric juice
The stability analysis of XOS in artificial human gastric juice was
evaluated using FOS as the control (Wang et al., 2015). Artificial human
gastric juice was prepared (% w/v, Na2HPO4⋅H2O 0.825; NaH2PO4
1.435; NaCl 0.8; KCl 0.02; CaCl2.2 H2O 0.01 and MgCl2.6 H2O 0.018, in
distilled water), and set at different pH (1− 5) with 1.0 M HCl. XOS or
FOS were suspended (1%, w/v) in artificial gastric juice, and incubated
at 37ºC for 6 h. Samples withdrawn at different time intervals were
examined for the total and reducing sugar content (Sharma et al., 2021a,
2021b). The stability of xylooligosaccharides (degree of hydrolysis) was
calculated according to the formula (Wang et al., 2015):
Hydrolysis degree (%) =
Reducing sugar released
× 100
Initial total sugar − initial reducing sugar
where reducing sugar released was the difference between the reducing
sugar content at specified time and the reducing sugar content present at
0 h.
2.12. Prebiotic activity score of xylooligosaccharides
Prebiotic activity score of XOS was examined (Zhang et al., 2018)
using probiotic LPM-13, and E. coli or S. aureus as pathogenic bacteria.
Activated culture of LPM-13 or bacterial pathogen was inoculated (1%
v/v, 108-109 cells/ml) into modified MRS broth containing XOS-SB or
glucose (1%, w/v), as the sole carbon source, and incubated at 37 ◦ C for
48 h under shaking (180 rpm). The growth of bacterial cultural was
examined by viable plate count, and expressed as log cfu/ml. The pre­
biotic activity score of XOS-SB was determined using the following
equation (Zhang et al., 2018):
ProbX48 − ProbX0 PathX48 − PathX0
Prebiotic activity score = −
ProbG48 − ProbG0 PathG48 − PathG0
where, ProbX48 and ProbX0 indicate the growth of probiotic LPM-13
in XOS medium at 48 h, and 0 h, respectively; PathX48 and PathX0
represent the growth of bacterial pathogen in XOS medium at 48 h, and
0 h, respectively; ProbG48 and ProbG0 indicate the growth of probiotic
LPM-13 in glucose medium at 48 h, and 0 h, respectively; PathG48 and
PathG0 indicate growth of bacterial pathogen in glucose medium at 48 h,
and 0 h, respectively.
2.13. Impact of xylooligosaccharides on antibacterial activity of
L. plantarum M-13
LPM-13 was inoculated (1% v/v, 108-109 cells/ml) into modified
MRS broth containing XOS (1%, w/v) as the sole carbon source, and
incubated at 37 ◦ C for 18 h under shaking (180 rpm). The cultural broth
was centrifuged (5000g for 20 min), and the supernatant was filter-
sterilized (0.22 µm filter), and used for antimicrobial activity assay.
The pathogenic bacteria used in the study were Staphylococcus aureus,
Escherichia coli, Micrococcus luteus, Enterococcus faecalis, Bacillus cereus,
Pseudomonas aeruginosa, Streptococcus pneumoniae, Pseudomonas alcali­
genes, Bacillus subtilis and Klebsiella sp. Either of the bacterial pathogen
was grown in nutrient broth (NB) under shaking (180 rpm) for 18 h at
37◦ C. The cell biomass was collected by centrifugation (5000g for
10 min), washed and resuspended in sterile saline (108-109 cells/ml)
(Millette et al., 2007).
For antibacterial activity, a mixture consisting of 100 µl of NB, 100 µl
of filter-sterile supernatant and 50 µl of either of the pathogen suspen­
sion was incubated at 37◦ C for 16 h under shaking (180 rpm). In the
control experiment C1, 100 µl of filter sterile supernatant was replaced
with 100 µl of NB, while control experiment C2 consisted of 100 µl of
NB, 100 µl of MRS and 50 µl of either of the pathogen suspension. The
microbial growth was monitored with a spectrophotometer (A600)
against a blank which consisted of 100 µl NB, 100 µl of filter sterile
4
Industrial Crops & Products 178 (2022) 114591
supernatant, and 50 µl of normal saline. The inhibition in the growth of
the pathogen by XOS was calculated using the following equation:
A1 – A0 = ACal
where A1 is the absorbance of sample; A0 is the absorbance of the
blank, and ACal is the absorbance calculated for each pathogen.
The inhibition of the pathogen by XOS was calculated as follows:
Inhibition(%) =
ACal
× 100
ANB
where ANB is absorbance of each pathogen suspension grown in NB.
2.14. Antioxidant activity of xylooligosaccharides
Antioxidant activity of XOS was analyzed spectrophotometrically
(A517) based on 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scav­
enging activity (Avila et al., 2020). XOS-SB was used at different con­
centrations (0.5–5.0 mg/ml in acetate buffer 50 mM, pH 6), and DPPH
radical scavenging activity was determined using the following
equation:
DPPH scavenging activity (%) = 1 − absorbance of sample
absorbance of control × 100
3. Results and discussion
3.1. Polysaccharide composition of sugarcane bagasse biomass
The holocellulose content of SB biomass was observed to be 57.87%
(w/w) that included cellulose (38.25%, w/w) and hemicelluloses
(19.62%, w/w), and three principle monomeric sugars i.e. glucose
(42.51% w/w), xylose (15.05% w/w) and arabinose (7.26% w/w).
Sharma et al. (2019) reported holocellulosic content of 58.6% in raw
sugarcane bagasse which represented cellulose and hemicellulose frac­
tions of 38.6% and 20.0%, respectively. Similarly, a detailed analysis of
sugarcane bagasse (Hesam et al., 2020) showed the following constitu­
ents (%, w/w): cellulose 33.36, hemicelluloses 25.01, lignin 23.27, ash
2.91, and crude protein 1.16. Composition of sugarcane bagasse biomass
may vary due to diversity of sugarcane varieties, climatic and cultivation
conditions, and other local factors (Sharma et al., 2019).
3.2. Delignification of sugarcane bagasse biomass
Delignification of SB aimed to remove lignin while maintaining the
integrity of carbohydrates in the substrate. Among the four pretreatment
methods, minimum sugar loss (0.75 ± 0.04 mg/g) was realized by so­
dium hypochlorite based pretreatment as compared to other pre­
treatments i.e. liquid ammonia (0.82 ± 0.02 mg/g), hydrogen peroxide-
acetic acid (H2O2–Hac) (0.95 ± 0.09 mg/g) and sodium chlorite
(1.18 ± 0.05 mg/g). Therefore, sodium hypochlorite pretreatment was
selected for further studies. Sodium hypochlorite is considered as a
potent delignifying agent as it creates adequate alkaline conditions, and
results in maximum lignin removal. Under alkaline pretreatment,
different chemical bonds in lignin may show different type of reactions.
For instance, the linkages between phenolic α-aryl ether bonds and
α-alkyl ether bonds are very weak, and get fractured in alkaline milieu
resulting in considerable depolymerisation of lignin. However, the non-
phenolic α-aryl ether linkages are very stable, and require much harsh
conditions for their breakage. Furthermore, after pretreatment of SB, a
yield of 91.55%, w/w was obtained, and weight loss of 8.45%, w/w was
accounted mainly due to removal of lignin, and some other extractives.
However, delignification also incurred a weight loss of cellulose (1.72%)
and hemicelluloses (3.55%) content.
Furthermore, after delignification, the holocellulose content of SB
was enriched by 12.93% (w/w), and resulted in increased cellulose
(41.49%, w/w) and hemicellulose content (29.31%, w/w). Also, the
glucose, xylose and arabinose content were increased to 46.11%,
M. Gupta et al. Industrial Crops & Products 178 (2022) 114591

25.36% and 7.96%, respectively. This increase in sugar content was The different response in various experimental runs indicated the
mainly due to the removal of lignin and various other plant cell wall distinct impact of each of the process parameters on the response.
components. The lignin removal increases the porosity, and concentra­ Analysis of variance (ANOVA) showed that the model was significant
tion of carbohydrate polymers, and makes the biomass more amenable with F value of 2.84 and Prob>F value of 0.0269 (Table 2).
to bioconversion. Cellulose content of sugarcane bagasse biomass was In this case, A (temperature), D2 (sodium hypochlorite concentra­
reported to get increased from 42% to 55% after alkaline pretreatment tion), AB (interactive effect of temperature and biomass loading) and AC
(Nalawade et al., 2020). Similarly, cellulose content of sugarcane (interactive effect of temperature and time), were the significant model
bagasse biomass was increased considerably i.e. 52–79%, after auto­ terms. The lack of Fit F-value (0.87) implied its non-significance which
hydrolysis (hydothermal pretreatment) and alkaline delignification (de in turn substantiated the robustness of the model. The coefficient of
Oliveira Rodrigues et al., 2021). variation (CV) of 2.45 indicated the reliability and precision of the
The alkali (NaOH 1%, w/v) pretreatment of sugarcane bagasse experiment. Generally, a CV value of < 10% is desired to ensure credi­
resulted in increase of holocellulosic and cellulosic contents from 68.3% bility of the experimental data.
to 70.1% (w/w), and from 42.5% to 47.4% (w/w), respectively (Nath Coefficient of determination R2 (0.9261) explained the goodness of
et al., 2021). However, the original hemicellulosic (25.8%, w/w) con­ the model (value closer to 1.0 denotes better correlation between
tent was decreased slightly (22.1%, w/w), but acid-insoluble lignin observed and predicted responses), and indicated that 92.61% of the
(22.1% (w/w) content was decreased appreciably (12.3%, w/w) (Nath variability in the response could be explained by the model. Further­
et al., 2021). The raw SB biomass was reported to contain > 55% (w/w) more, the adjusted R2 (0.9705) and predicted R2 (0.9447) indicated
carbohydrate content (glucose 33.27%, xylose 21.44% and arabinose good agreement between experimental and predicted responses.
2.09%) of which 23% (w/w) constituted the hemicelluloses (Kundu Adequate precision i.e. signal to noise ratio of 26.204 (> 4.0) indicated
et al., 2021). Considerably high fraction of hemicelluloses (especially an adequate signal. All the statistical parameters were appropriate and
the xylan) in SB reflects its potential as an excellent substrate for XOS statistically significant. Thus, the model was used to navigate the design
production. Thus, loss of carbohydrate macromolecules during deligni­ space. A regression equation (I) obtained after multiple regression
fication is quite variable and depends on the type of delignifying analysis, for the response (Y, xylan, %, w/w) as a function of various
agents/methods used, and the type of biomass substrate being studied. variables is given below:
Response Y (Xylan, %, w/w) = +51.43 + 0.41 A− 3.80D2
+ 2.19AB− 8.48AC ………….(I).
3.3. Process optimization for delignification of sugarcane bagasse
The polynomial equation describes variations in the response Y
(xylan, % w/w) as a function of various variables i.e. A (delignification
For DoE-based optimization of process variables (biomass loading,
temperature), B (biomass loading), C (delignification time) and D (so­
temperature and time, and sodium hypochlorite concentration) for
dium hypochlorite concentration), and also provides detailed informa­
delignification of sugarcane bagasse, thirty designed experiments were
tion about the interactions within (i.e. A2, B2, C2 and D2), and between
performed, and the responses were fed to the response column (Table 1).
the independent variables (AB, AC, BC and BD). The interactive effect of
temperature (A) and biomass loading (B), and that of temperature (A)
Table 1
and time (C) on the response (xylan yield %, w/w) is presented in Fig. 1
Design of Experiment based optimization for delignification of sugarcane
(a) and (b), respectively. Three-dimensional (3-D) response surface plots
bagasse.
showed that the maximum response was obtained at moderate level of
Run Experimental variables* Xylan (%,w/w) temperature and maximum level of biomass loading and time. Pertur­
A B C D Experimental Predicted bation plot (Fig. 1c) showed an increase or decrease in the response
response response when each of the variable was changed keeping other variables constant
1 40.00 10.00 90.00 1.00 29.75 30.83 with respect to the chosen reference point. Sodium hypochlorite (D) had
2 50.00 7.50 75.00 0.75 41.53 40.30
3 50.00 7.50 45.00 0.75 30.66 43.38
4 60.00 5.00 90.00 1.00 31.00 61.61 Table 2
5 40.00 10.00 60.00 1.00 18.20 57.20 Analysis of variance for delignification of sugarcane bagasse based on Design of
6 50.00 7.50 75.00 0.75 43.93 32.76 Experiment.
7 40.00 10.00 90.00 0.50 66.50 59.29
Source* Sum of DF Mean F Prob
8 60.00 10.00 90.00 1.00 31.95 43.61
Squares Square Value >F
9 60.00 5.00 60.00 0.50 46.30 27.99
10 50.00 2.50 75.00 0.75 54.80 45.32 Model 3250.72 14 232.19 2.84 0.0269 Significant
11 40.00 5.00 90.00 1.00 65.80 21.95 A 4.11 1 4.11 0.050 0.0056 Significant
12 50.00 7.50 105.00 0.75 47.66 48.05 B 34.68 1 34.68 0.42 0.5246 –
13 40.00 5.00 60.00 0.50 33.10 55.85 C 145.78 1 145.78 1.78 0.2016 –
14 60.00 5.00 90.00 0.50 39.20 39.27 D 333.69 1 333.69 4.08 0.0616 –
15 60.00 5.00 60.00 1.00 40.80 39.35 A2 338.67 1 338.67 4.14 0.0599 –
16 50.00 7.50 75.00 0.75 47.40 31.53 B2 57.96 1 57.96 0.71 0.4130 –
17 40.00 5.00 60.00 1.00 27.40 36.55 C2 277.60 1 277.60 3.40 0.0852 –
18 50.00 7.50 75.00 1.25 32.26 38.20 D2 395.57 1 395.57 4.84 0.0039 Significant
19 50.00 12.50 75.00 0.75 60.60 54.84 AB 76.78 1 76.78 0.94 0.0078 Significant
20 50.00 7.50 75.00 0.25 41.13 59.65 AC 1150.06 1 1150.06 14.07 0.0019 Significant
21 30.00 7.50 75.00 0.75 37.06 33.78 AD 61.82 1 61.82 0.76 0.3982 –
22 60.00 10.00 60.00 0.50 54.35 43.64 BC 109.46 1 109.46 1.34 0.2653 –
23 50.00 7.50 75.00 0.75 55.60 43.70 BD 345.50 1 345.50 4.23 0.0576 –
24 50.00 7.50 75.00 0.75 67.53 28.78 CD 2.21 1 2.21 0.027 0.8715 –
25 40.00 5.00 90.00 0.50 45.10 51.43 Residual 1225.99 15 81.73 –
26 50.00 7.50 75.00 0.75 52.60 51.43 Lack of 777.52 10 77.75 0.87 0.6053 Not
27 60.00 10.00 90.00 0.50 40.60 51.43 Fit significant
28 70.00 7.50 75.00 0.75 38.60 51.43 Pure 448.47 5 89.69 –
29 60.00 10.00 60.00 1.00 56.55 51.43 Error
30 40.00 10.00 60.00 0.50 48.05 51.43 Cor Total 4476.71 29 –
*
(*A- Delignification temperature, ºC, B- biomass loading, %, w/v, C- delignifi­ (A- Delignification temperature, B- biomass loading, C- delignification time,
cation time, min and D- sodium hypochlorite, %, v/v) and D- sodium hypochlorite)

5
M. Gupta et al.
Fig. 1. Response surface plots for delignification of sugarcane bagasse showing (a) interaction between temperature and biomass loading; (b) different temperature
and time; (c) perturbation plot showing the effectiveness of the variables on the delignification of sugarcane bagasse.
the maximum effect on the response, and was followed by biomass
loading (B), delignification time (C) and temperature (A).
The experiments were performed at predicted optimal level of pro­
cess variables i.e. temperature 51.86ºC, biomass loading 12.91% w/v,
time 75.12 min, and sodium hypochlorite concentration 0.53% v/v), for
validating the model. The close proximity of experimental (75.05% w/
w, xylan) and predicted (72.65%, w/w, xylan) responses validated the
process model.
Presence of lignin and highly crystalline cellulose hinders the frac­
tionation of biomass, and necessitates a suitable pretreatment regime for
efficient delignification of biomass especially under mild process con­
ditions to avoid sugar degradation (Yiin et al., 2017). An efficacious
pretreatment promises effective and economic bioconversion at low
operating cost. Several factors influence the polysaccharide extraction
from LB in terms of yield and functional properties of extracted polymer,
therefore, for obtaining high quality and yield, the process variables
must be optimized (Wei et al., 2018). Optimization of delignification of
sugarcane bagasse (Valim et al., 2018) was achieved using alkaline
hydrogen peroxide with high efficiency (78.9%). Effectiveness of
delignification was indicated by the favourable morphological changes
in the biomass.
3.4. Process optimization for xylan extraction from delignified sugarcane
bagasse
Among various approaches examined for xylan extraction from the
delignified SB, sodium hydroxide coupled with thermal treatment yiel­
ded the maximum xylan (41.49%). Therefore, various process variables
such as temperature (A), biomass loading (B), extraction time (C) and
sodium hydroxide concentration (D), were optimized for xylan extrac­
tion using this approach. The responses obtained for DoE-generated
thirty experimental runs are presented in the Table 3.
6
Industrial Crops & Products 178 (2022) 114591
ANOVA-demonstrated model F-value of 1.30 implied the signifi­
cance of the model (Table 4). Based on Prob>F value, the model terms i.
e. B (biomass loading), D (sodium hydroxide concentration), C2
(extraction time), AB (interaction between temperature and biomass
loading), BC (interaction between biomass loading and extraction time)
and BD (interaction between biomass loading and sodium hydroxide
concentration), were significant. Insignificant lack of fit (P-value
0.1608), low standard deviation (3.04), and high R2 (0.9261), pointed
towards robustness and a reasonably good predictability of the model.
Adequate precision of 26.204 indicated a sufficient signal. The regres­
sion equation (II) obtained for the model is given below:
Response, Y (Xylan, %, w/w) = +51.43 + 1.20B− 3.73D− 3.18 C2
+ 2.19AB+ 2.62 BCE+ 4.65BD……………………(II).
The variables in coded form were: temperature (A), biomass loading
(B), extraction time (C) and sodium hydroxide concentration (D).
The 3-D response surface plots representing the interactive effect of
process parameters were studied. The interactions namely AB (temper­
ature and biomass loading), BC (biomass loading and time), and BD
(biomass loading and sodium hydroxide concentration) significantly
affected the response. The interactive influences of temperature (A) and
biomass loading (B) (Fig. 2a), biomass loading (B) and extraction time
(C) (Fig. 2b), and that of biomass loading (B) and sodium hydroxide
concentration (D) (Fig. 2c), showed a direct relationship with the
response. Thus, increasing the level of these process parameters led to
concomitant enhanced response (xylan yield). The perturbation plot
analysis illustrated that among all the variables, the variable B (biomass
loading) deviated maximally from the reference point, and was followed
by other variables i.e. A, C and D (Fig. 2d).
Experiments performed at predicted optimal level of variables
(temperature 89.03ºC, biomass loading 11.80% w/v, time 20.14 min
and sodium hydroxide concentration 14.19% v/v) showed a close pro­
pinquity between the experimental (96.11%, w/w) and the predicted
M. Gupta et al. Industrial Crops & Products 178 (2022) 114591

Table 3 process variables i.e. H2O2 5.63%, NaOH 12.91%, pretreatment time
Experimental and predicted response for xylan extraction (%, w/w) from 17.51 h (Hesam et al., 2020). A combination of alkalis (NaOH and
delignified sugarcane bagasse based on Design of Experiment. NH4OH) was reported to exert synergistic action on xylan extraction
Run Experimental variablesnew-a Xylan % (w/w) from sugarcane bagasse, and further the statistical optimization of alkali
A B C D Experimental Predicted
concentration and temperature increased the xylan recovery (Kundu
response response et al., 2021).
1 82.50 7.50 15.00 8.00 44.60 41.11
2 65.00 5.00 10.00 14.00 55.90 55.33 3.5. Physicochemical characterization of xylan extracted from sugarcane
3 100.00 5.00 20.00 10.00 51.00 51.92 bagasse
4 82.50 7.50 25.00 12.00 52.33 53.41
5 47.50 7.50 15.00 12.00 40.00 38.32
3.5.1. Thermogravimetric analysis of xylan
6 65.00 5.00 10.00 10.00 43.90 44.89
7 65.00 10.00 10.00 10.00 40.60 40.77 Thermogravimetric analysis of xylan was done to examine its ther­
8 82.50 7.50 15.00 12.00 43.86 41.63 mal stability and degradation pattern, to access its application potential
9 82.50 7.50 15.00 12.00 62.80 65.82 keeping in view that most of the industrial processes generally involve
10 100.00 5.00 20.00 14.00 31.50 31.79 high temperature milieu. TGA curve (Fig. S1a) showed that xylan
11 65.00 10.00 20.00 14.00 52.85 53.63
degradation occurred in three stages. The initial degradation at less than
12 65.00 5.00 20.00 10.00 36.80 38.87
13 100.00 10.00 10.00 14.00 66.70 67.80 150ºC could be attributed to the loss of water. The water content in the
14 65.00 10.00 20.00 10.00 53.60 46.12 sample is responsible for the low dip in the initiation of the thermal
15 100.00 10.00 20.00 10.00 49.75 48.26 degradation (Yiin et al., 2017). At around 80ºC, the degradation of xylan
16 117.50 7.50 15.00 12.00 75.95 71.85
led to a weight loss of 10%. After initial degradation phase, weight loss
17 82.50 7.50 15.00 12.00 61.2 62.77
18 82.50 12.50 15.00 12.00 84.16 86.58 occurred slowly which could be due to the existence of thermally stable
19 82.50 7.50 15.00 16.00 64.00 63.49 sugar moieties. In the second stage (150–450ºC), the decomposition and
20 100.00 5.00 10.00 14.00 31.60 31.02 elimination of carboxylic/hydroxyl groups resulted in a perceptible
21 100.00 10.00 20.00 14.00 85.75 84.26 weight loss (Fig. S1a) that was maximum (40%) around 200–300ºC
22 82.50 7.50 15.00 12.00 56.60 54.44
(Zhao et al., 2017c). The third stage of mass loss was observed around
23 65.00 10.00 10.00 14.00 56.90 56.87
24 82.50 7.50 15.00 12.00 58.33 57.81 500–600ºC (Fig. S1a) in which pyrolysis was completed and only char
25 82.50 7.50 15.00 12.00 39.66 59.74 residue was left (31–35%). At this point, all the elemental oxygen and
26 100.00 10.00 10.00 10.00 33.65 33.74 hydrogen had been liberated to gaseous compounds leaving only carbon
27 82.50 7.50 5.00 12.00 63.60 62.80
elements (Zhao et al., 2017c).
28 65.00 5.00 20.00 14.00 50.60 53.74
29 100.00 5.00 10.00 10.00 30.40 34.40
Since xylan is less thermally stable than cellulose and lignin, it de­
30 82.50 2.50 15.00 12.00 64.60 65.20 composes around 225–325ºC (Davila et al., 2016). Xylan degradation
a was reported to occur in two-step i.e. in first step, degradation of the
(A- Extraction temperature, ºC; B- biomass loading, %, w/v; C- extraction
glycosidic bonds along with the side-chain substituting groups occurs,
time, min; D-sodium hydroxide, %, w/v)
and in the second step breakage of the depolymerised fragments takes
place. The degradation and weight loss in this phase corresponds to
Table 4 random polymer backbone cleavages and depolymerization of the
Analysis of variance for xylan extraction (%, w/w) from delignified sugarcane remaining inorganic material from the xylan (Davila et al., 2016). Xylan
bagasse by employing Design of Experiment. from finger millet seed coat showed an initial degradation at 130 ◦ C
Source* Sum of DF Mean FValue Prob (Palaniappan et al., 2017), and significant decomposition at 200–400 ◦ C
Squares Square >F (weight loss 65–70%). Similarly, hemicelluloses from the apical, middle
Model 3392.86 14 242.35 1.30 0.0073 Significant
and basal segments of Neolamarckia cadamba exhibited an initial mass
A 713.95 1 713.95 3.84 0.0688 – loss due to moisture evaporation at 170 ◦ C, however, the maximum
B 903.07 1 903.07 4.86 0.0005 Significant weight loss (50%) was realized between 170 and 320 ◦ C (Zhao et al.,
C 155.55 1 155.55 0.84 0.3746 – 2017c). Thus, TGA analysis showed that the xylan extracted from the SB
D 36.65 1 36.65 0.20 0.0032 Significant
biomass was thermally stable and can be used for the production of
A2 111.53 1 111.53 0.60 0.4505 –
B2 247.44 1 247.44 1.33 0.2665 – xylooligosaccharides or other products.
C2 33.05 1 33.05 0.18 0.0091 Significant
D2 33.20 1 33.20 0.18 0.6785 – 3.5.2. Fourier transform infrared spectroscopy of xylan
AB 371.53 1 371.53 2.00 0.0007 Significant The FTIR analysis was carried out to ascertain the purity of the xylan
AC 5.52 1 5.52 0.030 0.8654
polymer for the presence of residual lignin components. FTIR spectral
–
AD 68.06 1 68.06 0.37 0.5540 –
BC 347.82 1 347.82 1.87 0.0093 Significant profile of xylan displayed typical signal patterns of xylan-rich hemicel­
BD 81.00 1 81.00 0.44 0.0090 Significant lulose (Li et al., 2016). The prominent absorption band at 3444.04 cm− 1
CD 218.30 1 218.30 1.18 0.2444 – was due to the stretching of the hydroxyl (OH) groups present in the
Residual 2786.30 15 185.75 –
xylan fractions as well as water involved in the hydrogen bonding
Lack of 2323.45 10 232.35 2.51 0.1608 Non
Fit significant (Fig. S1b) (Seesuriyachan et al., 2017). The fingerprint region
Pure 462.84 5 92.57 – (1800–600 cm− 1) allows the identification of major chemical groups in
Error the polysaccharides where as the position and intensity of the bands is
Cor Total 6179.16 29 – specific for each polysaccharide. A band observed at 1632 cm− 1 corre­
*
(A- extraction temperature, B- biomass loading, C- extraction time, D- so­ sponded to the mode of bending in water (Khaleghipour et al., 2021;
dium hydroxide) Zhang et al., 2016). The band at around 1400 cm− 1 and 1000 cm− 1
(Fig. S1b) were of typical xylan, and could be attributed to C-O, C-C
responses (92.75%, w/w), thus, validating the process model. The xylan stretching or C-OH bending in the hemicelluloses (Sukri and Sakinah,
yield was increased from initial 41.49% (w/w) to 96.11% (w/w) after 2018; Li et al., 2016).
optimization. Similar to current study, high yield of xylan (95.84%) was The band obtained at 883 cm− 1 was ascribed to the glycosidic link­
extracted from sugarcane bagasse after RSM based optimization of ages present between xylopyranose units in the xylan backbone (Kha­
leghipour et al., 2021). The band at 543.00 cm− 1 (Fig. 3b) resulted from

7
M. Gupta et al.
Fig. 2. Response surface plots for xylan extraction from delignified sugarcane bagasse showing (a) interaction between temperature and biomass loading; (b)
biomass loading and extraction time; (c) biomass loading and sodium hydroxide concentration; (d) perturbation plot showing the effectiveness of the variables on
xylan extraction from delignified sugarcane bagasse.
C–C–H or C–O–C stretching or bending (Seesuriyachan et al., 2017). The
absence of bands corresponding to lignin (1520 cm− 1) and pectin
(1570 cm− 1) residues substantiated the purity of the extracted xylan
(Davila et al., 2016). Furthermore, the removal of acetyl, uronic acid, or
ester groups by alkaline xylan extraction was confirmed by the absence
of bands at 1736 cm− 1 (Sukri and Sakinah, 2018; Wang et al., 2016).
Therefore, the FTIR analysis suggested that the extracted xylan was
significantly pure, and may potentially be used for XOS production or
other industrial applications.
3.5.3. Scanning electron microscopy of sugarcane bagasse after
delignification and xylan extraction
Scanning electron microscopy (SEM) was used to examine the
structural aberrations occurred in the biomass due to delignification and
xylan extraction. The structure of untreated material appeared strongly
bundled, rigid, highly ordered, compact, smooth and intact (Fig. S2a).
However, after delignification and xylan extraction, the biomass un­
derwent significant morphological changes, and appeared as disrupted,
shredded with increased surface area and porosity. The rough appear­
ance of delignified biomass surface was due to disrupted cell wall con­
tents, and/or re-deposition of pseudo-lignin on the surface (Zhao et al.,
2017a). The increased volume and surface area, and formation of
enlarged pores were attributed to the forced removal of lignin from the
biomass (Seesuriyachan et al., 2017). The effective removal of outer
lignin layer (peeling) decreased the cell wall thickness (Fig. S2b). Thus,
the biomass recalcitrance got decreased which made the hemicelluloses
more accessible by breaking the lignin–carbohydrate complex.
Xylan extraction made significant changes in the external as well as
8
Industrial Crops & Products 178 (2022) 114591
internal structure of the biomass (Fig. S2c), and it appeared heavily
damaged i.e. large scale deformations and shredded look, complete
surface exposure with drastic porosity, swelling and radically increased
volume (Kaweeai et al., 2016; Zhao et al., 2017b). Furthermore, the
swelling of the biomass due to increased hydration of fibrous material in
the middle lamella causes softening of cell walls which in turn eases the
breakage of structural tissue. The adjacent cells of the biomass get
separated resulting in the formation of pores of varying sizes. This may
be due to the removal of lignin, minerals, extractives and dissolution of
hemicelluloses from the biomass. In a similar study, SEM analysis of
ultra high pressure pretreated corncob biomass showed sieve-like
structure with pores of different diameters (Seesuriyachan et al.,
2017). Similarly, liquid ammonia pretreatment of giant reed biomass led
to increased porosity as shown by SEM analysis due to cellulose swelling
and lignin relocation, which resulted in easy extraction of xylan from
biomass (Zhao et al., 2017b). The SEM analysis of finger millet seed coat
showed irregular and rough surface morphology, and appeared largely
aggregated due to removal of lignin (Palaniappan et al., 2017). Thus,
delignification and/or xylan extraction results in enormous alterations
in the biomass structure and reduces its recalcitrance.
3.6. Xylosidase-free endoxylanase from fungal isolate A. flavus MG-7
The A. flavus MG-7 produced maximum xylanase (4.35 IU/ml) after
120 h of submerged fermentation. The concentrated xylanase obtained
after ammonium sulphate precipitation (activity 33.91 IU/ml), followed
by lyophilisation (activity 49.59 IU/mg), was used for hydrolysis of
xylan. TLC analysis of xylan hydrolysate demonstrated the presence of
M. Gupta et al.
Fig. 3. Thin layer chromatography analysis of xylan hydrolyzed products after
16 h where spot X: standard xylose; SX: standard beechwood xylan; HS1: xylan
hydrolyzed products with 20IU of enzyme; HS2: xylan hydrolyzed products
with 40IU of enzyme; HS3: xylan hydrolyzed products with 60IU of enzyme;
HS4: xylan hydrolyzed products with 80IU of enzyme and HS5: xylan hydro­
lyzed products with 100IU of enzyme.
various xylooligomers of varying degree of polymerization but complete
absence of xylose monomers thus, establishing the endoxylanolytic na­
ture of xylanase, and complete absence of xylosidase activity (Fig. 3).
TLC plate showed that standard xylan did not show any movement
because of its high molecular weight while standard xylose moved
maximally due to its low molecular weight, and xylanase-hydrolysed
xylan sample showed an intermediate movement between xylan and
xylose. Thus, it was established that the xylan has been cleaved endo­
lytically and resulted in the production of a mixture of oligosaccharides
but no xylose at all. Total absence of xylose signified the β-xylosidase-
free nature of endo-β-1,4-xylanase from A. flavus MG-7 (Khandeparker
et al., 2017).
Similar to current study, β-xylosidase-free endo-β-1, 4 xylanases were
produced from fungal species Neocallimastix patriciarum and Orpino­
myces sp., and used for XOS production from alkali-extracted eucalyptus
xylan (Puchart et al., 2018). A xylanase from Bacillus tequilensis BT21
had endoxylanolytic mode of action, and generated the xylobiose
products upon enzymatic hydrolysis of beechwood xylan (Khandeparker
et al., 2017). Thus, for production of XOS in high concentration,
β-xylosidase-free endoxylanases are highly preferred as the presence
β-xylosidases activity may hydrolyse XOS to produce xylose, and un­
dermine the overall process (Surek and Buyukkileci, 2017).
3.7. Production of xylooligosaccharides using A. flavus MG-7 xylanase
Xylan extracted from SB was used for the production of xylooligo­
saccharides (XOS) by using A. flavus MG-7 xylanase. XOS production by
enzymatic hydrolysis is generally preferred as it does not produce un­
wanted products like furfural, monosaccharides and other by-products
(Akpinar et al., 2009). For XOS production, xylanase must have
certain typical/ characteristic properties such as lack of β-xylosidase
activity, and ability to generate XOS with various degrees of polymeri­
zation (xylobiose X2, xylotriose X3, xylotetrose X4), efficient catalytic
9
Industrial Crops & Products 178 (2022) 114591
potential, higher specificity towards substrate, long shelf life, and
overall robust nature. Therefore, targeting process-apt xylanases from
the enormous microbial diversity has been a continuous practice (Kundu
et al., 2021; Khandeparker et al., 2017; Bajaj et al., 2011).
Commercial xylan from various biomass sources such as sugarcane
bagasse, rapeseed straw, corn cob and others have generally been used
for the XOS production. However, fewer studies have been undertaken
on XOS production using indigenously produced fungal xylanase (de
Freitas et al., 2021; Kundu et al., 2021; Khandeparker et al., 2017; Bajaj
et al., 2011). A. flavus MG-7 produced β-xylosidase-free endo-β-1,
4-xylanase which is suitable for the industrial production of xylooligo­
saccharides. Analysis of enzymatic hydrolysates via TLC and HPLC
revealed the presence of XOS with different degree of polymerization
(2− 4) i.e. xylobiose, xylotriose and xylotriose, but no xylose at all.
Furthermore, process for XOS production was optimized based on
‘one-variable-at-a-time’ (OVAT) approach to deduce the optimal level of
variables i.e. incubation period, xylan concentration and enzyme
loading. An enzyme dosage of 100 IU was found to be optimal as it
yielded maximum reducing sugar (RS) content (425 mg/g), and further
increase of dosage (120 IU) decreased the RS (378.42 mg/g) (Fig. 4a).
Since the endo-β-1,4-xylanase lacked β-xylosidase activity which is
required for complete hydrolysis of xylooligomers into xylose. There­
fore, accumulation of XOS might have caused end product inhibition
resulting in reduced RS yield at increased enzyme dose (Alvarez et al.,
2017).
Appropriate reaction time for enzymatic XOS production was 24 h as
it resulted in highest RS content (429.05 mg/g). However, upon longer
incubation (48 h), a decreased reducing sugar (RS) (Fig. 4b) yield was
obtained which may be attributed to inaccessibility of hydrolytic sites on
xylan substrate and/or restrained enzymatic hydrolysis due to end
product inhibition by the accumulated XOS (Sukri and Sakinah, 2018).
Similar to current study, an optimal incubation period of 24 h was re­
ported for XOS production from sugarcane bagasse (de Oliveira et al.,
2018), however, 72 h of incubation was documented to be optimal for
achieving a high concentration of XOS from brewer’s spent grain
(Amorim et al., 2019). The optimum substrate (xylan) concentration for
maximum XOS production was 2% (w/v) at enzyme loading of 100 IU,
and incubation time of 24 h as indicated by the maximum RS content
(947.49 mg/g). A further increase in xylan concentration, however, led
to a decrease in RS yield (Fig. 4c) which might be due to partial inhi­
bition of enzyme and/or increased viscosity of the reaction medium that
might have hindered the access of substrate by the enzyme.
HPLC analysis of enzymatic hydrolysate of xylan obtained under
optimal conditions showed a high XOS concentration of 91.12% (w/w)
that was comprised of xylotetrose (69.57%, w/w), xylobiose (13.17%,
w/v) and xylotriose (8.37%, w/w). Thus, XOS hydrolysate was rich in
xylooligomers exhibiting degree of polymerization of 2–4 oligomers.
This vindicated the effectiveness of the enzymatic regimen as the smaller
XOS (X2 to X4) are always preferred for food and pharmaceutical ap­
plications as prebiotic compounds (Reddy and Krishnan, 2016). Enzy­
matic hydrolysis of xylan (sugarcane bagasse) to XOS showed a
conversion yield of 42.3% (Khaleghipour et al., 2021) with xylobiose as
the predominant XOS followed by xylotriose. Contrary to current study,
xylan from areca nut husk upon enzymatic hydrolysis (Singh et al.,
2018) yielded a high amount of xylobiose (71%), moderate content of
xylotriose (26.2%), and a relatively low amount of xylotetrose (2%).
Thus, the endoxylanase produced from the indigenously isolated fungus
A. flavus MG-7 could be used for the efficient production of xylooligo­
saccharides from the sugarcane bagasse biomass for potential applica­
tions in food and pharmaceutical industries.
3.8. Nuclear magnetic resonance analysis of xylooligosaccharides
The ultrastructural characteristics of XOS produced from SB-
extracted xylan were studied by NMR. The peak signals in spectral re­
gion of 3.0–4.5 ppm (Fig. 5) were ascribed to the protons of xylose,
M. Gupta et al. Industrial Crops & Products 178 (2022) 114591

Fig. 4. Reducing sugars produced at different (a) enzyme loading; (b) incubation time; and (c) substrate concentration.

Fig. 5. Nuclear magnetic resonance spectra of xylooligosaccharides from sugarcane bagasse (SB) xylan.

arabinose, and glucuronic acid residues (Li et al., 2016). The charac­
teristic signals at 4.20–4.50 ppm (for anomeric protons of reducing end
of XOS) indicated the β-glycosidic linkages in the glycosyl residues,
while those at 3.8–3.0 ppm (Fig. 5) signified the non-substituted internal
xylose units (Zhang et al., 2016). The signal peaks at 3.8–3.2 ppm
showed the presence of β-xylopyranose residues at reducing and
non-reducing ends (Bowman et al., 2015). The characteristic signature
signals of XOS were similar to those reported previously, and some
overlaps were due to the mixed nature of XOS (Reddy and Krishnan,
2016).
The absence of signal at 5.00–5.40 ppm indicated that β-glycosidic
linked side-chains (glucuronic acid, α-L-arbinofuranosyl) have been

10
M. Gupta et al.
cleaved (Zhao et al., 2017c), and XOS were almost linear with few
attached branches. XOS derived from finger millet seed coat xylan
showed major signal at 3.0–5.0 ppm which corresponded to the β-linked
xylopyranoside residues, and the chemical shift signal at 5.10 ppm
indicated that 4-O-methyl α-D-glucuronic acid was attached to the XOS
backbone (Palaniappan et al., 2017). Similarly, XOS from Miscanthus
xylan showed signals in the range of 3.0–5.5 ppm in the 1H NMR which
was ascribed to xylose, arabinose, and glucuronic acid residues, thus,
displaying a typical pattern for xylooligosaccharides (Li et al., 2016).
3.9. Prebiotic potential of xylooligosaccharides generated from sugarcane
bagasse
The functional potential of XOS as prebiotics was ascertained based
on their growth promoting ability for a probiotic bacterium Lactobacillus
plantarum M-13 (LPM-13), isolated from an exotic source, and charac­
terized previously (Gupta and Bajaj, 2018, 2017). The results illustrated
that the XOS as a sole carbon source excellently supported the growth of
probiotic LPM-13 (18.77 log cfu/ml) that was comparable or even better
than that in standard MRS (with glucose as the carbon source).
Maximum growth of LPM-13 was observed at 72 h however, adequate
growth occurred even after 48 h (16.62 log cfu/ml). Furthermore, the
probiotic LPM-13 retained high viability (76%, 14.27 log cfu/ml) even
after prolonged incubation in MRS-XOS (144 h).
The growth of probiotic LPM-13 was comparable in MRS-XOS and
standard MRS up to 48 h, however, at 72 h growth was higher in MRS-
XOS than that in standard MRS. Furthermore, MRS-XOS supported
higher viability of probiotic LPM-13 (76%) as compared to standard
MRS (62%) after prolonged incubation (144 h). XOS due to their olig­
omeric structure are metabolized much slowly, and are capable of
supporting the growth/viability of probiotics for a much longer period.
In contrast, glucose being monomeric in nature is metabolized much
faster, and thus, do not support the sustained growth/viability of pro­
biotics over prolonged time periods. Furthermore, growth stimulatory
effect of XOS prebiotics on probiotic LPM-13 (18.77 log cfu/ml) was
comparable or even better than that of commercial prebiotics such as
FOS (18.78 log cfu/ml), lactulose (18.76 log cfu/ml) and inulin
(18.65 log cfu/ml). Thus, XOS-SB enriched with X2, X3, and X4 may be
an effective prebiotic for potential commercial applications.
Moreover, a concentration of XOS-SB or commercial prebiotics at 1%
(w/v) as the sole carbon source was observed to be adequate for sup­
porting growth of probiotic LPM-13. A higher prebiotic concentration
(as sole carbon source) may cause disproportion of carbon and the ni­
trogen source, and may be detrimental for growth as the nutrient
imbalance affects the enzymatic/metabolic/transport capabilities of
bacteria and leads to underutilization of nutrients, and hence impaired
growth/survival of probiotics (Noori et al., 2017). In a similar study,
XOS generated from xylan (extracted from banana pseudostem), using
an in-house produced endoxylanase (Aspergillus versicolour) exhibited
desirable prebiotic attributes, and adequately supported the growth of
probiotic bacteria L. plantarum and L. fermentum. Further, XOS with high
degree of polymerization supported growth of probiotic bacteria more
efficiently (de Freitas et al., 2021). Thus, XOS generated enzymatically
from LB-derived xylan may have excellent application potential for food
industries especially for production of probiotics-fortified functional
foods.
3.10. Stability of xylooligosaccharides under artificial gastric juice
Gastric-stability of oligosaccharide prebiotics is an imperative attri­
bute, and refers to their capability to withstand digestive processes,
gastric acidity, hydrolysis by enzymes and gastrointestinal absorption.
Prebiotics must resist the morbid gastrointestinal milieu, and reach the
large intestine to modulate the probiotic population (Ferreira-Lazarte
et al., 2017). XOS-SB exhibited moderate to mild hydrolysis in gastric
juice at different pH (1− 5) over an incubation time of 6 h. Maximum
11
Industrial Crops & Products 178 (2022) 114591
hydrolysis was observed at pH 1 (19.94%) followed by that at pH 2 and 3
(16.28 and 16.09, respectively), and at pH 4 (14.24%) and 5 (11.83%).
Similarly, FOS, a commercial prebiotic showed hydrolysis level of
11.16–17.67% in the gastric juice at different pH. An inverse relation­
ship between degree of XOS hydrolysis and pH of gastric juice (lower the
pH more is hydrolysis) may be due to higher vulnerability of glycosidic
bonds at low acidic pH (Azmi et al., 2012).
Additionally, the incubation time of probiotics in gastric juice also
contributes towards the degree of hydrolysis. Mild hydrolysis of XOS-SB
at high acidic pH (pH 1.0) over 6 h of incubation indicated their sub­
stantial resistance to gastric juice. Considering that food takes around
4 h to pass through the stomach, it may be inferred that XOS-SB could
reach the intestine in reasonably unaltered form, and be utilized by
probiotics present over there. In a similar study, oligosaccharides syn­
thesized from lactulose exhibited considerable resistance to gastroin­
testinal conditions, and showed a mild digestion (<15%)
(Ferreira-Lazarte et al., 2017). Similarly, XOS produced from sugarcane
straw and coffee husk (Avila et al., 2020), and from sugarcane bagasse
(Ghosh et al., 2021) were reported to have considerable stability to
digestive enzymes.
Various factors such as composition of sugar oligosaccahrides,
configuration of anomer, and linkages involved, may influence the de­
gradability of carbohydrates. Generally, β-linkages are more resistant
than α-ones, however, upon prolonged incubation at low pH (< 4.0)
even the former could undergo hydrolysis (Wang et al., 2015). Thus,
resistance of prebiotics to gastrointestinal milieu is absolutely essential
for getting them in large intestine in relatively unaltered form for suc­
cessfully delivering the health enhancing effects.
3.11. Prebiotic activity score of xylooligosaccharides
Prebiotic activity score (PAS) refers to comparative growth analysis
of a bacterial probiotic bacterium (Bifidobacterium spp. and Lactobacillus
spp.) and a potential bacterial pathogen (E. coli, S. aureus or Clostridium
spp. or others) using an oligosaccharide prebiotic or glucose as the sole
carbon source. Growth study of probiotic LPM-13 on MRS-XOS or
standard MRS (containing glucose) showed PAS of 1.5 and 1.46,
respectively, using E. coli and S. aureus as the reference pathogens. The
pectin-oligosaccharides produced from citrus peel pectin were reported
to have a PAC of 0.41 and 0.92 for L. paracasei LPC-37 and B. bifidum
ATCC 29521, respectively, with E. coli as the reference pathogen (Zhang
et al., 2018). Similarly, XOS from finger millet seed coat exhibited PAC
of 0.7–0.8 for several Lactobacillus spp. using E. coli as the reference
pathogen (Palaniappan et al., 2017).
A high and positive PAS score indicates relatively higher growth of
probiotics in medium containing prebiotic-oligosaccharides than that of
bacterial pathogens, and substantiates the premise that probiotics can
selectively and more efficiently utilize the oligosaccharide-prebiotics for
growth than the pathogenic bacteria. Thus, PAS analysis of XOS-SB
established its prospects for application as an ingredient for devel­
oping functional foods.
3.12. Impact of xylooligosaccharides on antibacterial activity of
L. plantarum M-13
Ever-rising incidences of antibiotic resistance among pathogens
necessitated the development of new/novel approaches for disease
control. Probiotics and/or prebiotics mediated antibacterial action may
be a potent approach for controlling/managing infectious diseases
caused by intestinal/food borne bacterial pathogens (Gupta and Bajaj,
2018; Palaniappan et al., 2017). Cell free supernatant of L. plantarum
M-13 grown in standard MRS has previously been shown to exhibit mild
antimicrobial activity against various bacterial pathogens (Gupta and
Bajaj, 2018). However, the cell free supernatant obtained after growing
L. plantarum M-13 in MRS with XOS as the sole carbon source, exhibited
a high inhibitory potential against a wide range of bacterial pathogens.
M. Gupta et al. Industrial Crops & Products 178 (2022) 114591

Moderate to high inhibition (30–80%) was observed against probiotics) in biorefining industries.
Gram-positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus,
Streptococcus pneumoniae, Enterococcus faecalis, Micrococcus luteus) and CRediT authorship contribution statement
Gram-negative pathogenic bacteria (Escherichia coli, Klebsiella sp.,
Pseudomonas aeruginosa, Pseudomonas alcaligenes). Maximum inhibition Mahak Gupta: Methodology, Experimentation, Software, Data
was observed against Klebsiella sp. (74.51%), and was followed by that curation. Ridhika Bangotra: Writing – original draft, Writing – review
against P. aeruginosa (72.96%), E. coli (71.94%), B. cereus (65.42%), S. & editing. Surbhi Sharma: Writing – review & editing. Surbhi Vaid:
aureus (64.92%), M. luteus (63.91%), P. alcaligenes (58.65%), Writing – review & editing. Nisha Kapoor: Writing – review & editing.
S. pneumonia (53.37%), E. faecalis (51.27%), and against B. subtilis Harish Chander Dutt: Writing– review & editing. Bijender Kumar
(49.42%). Bajaj: Conceptualization, Project Administration, Funding acquisition,
The potentiated antimicrobial activity of cell free supernatant of Resources, validation Supervision.
L. plantarum M-13 upon cultivation in MRS-XOS may be due to pro­
duction of short chain fatty acids as a result of metabolism of XOS by the
Declaration of Competing Interest
probiotic bacterium. Production of organic acids resulted in pH reduc­
tion of cultural broth (pH 2.8). Thus, it may be inferred that the path­
The authors declare that they have no known competing financial
ogenic bacteria were inhibited due to low acidic pH as well as due to
interests or personal relationships that could have appeared to influence
some other antimicrobials generated by L. plantarum M-13 (Gupta and
the work reported in this paper.
Bajaj, 2018). In a similar study, the cell free supernatant of L. plantarum
grown in medium containing XOS from finger millet seed coat displayed
Acknowledgements
strong antibacterial activity against several pathogens such as Shigella
flexneri, E. coli, Salmonella typhi, and S. aureus (Palaniappan et al., 2017).
Professor Bijender Kumar Bajaj (BKB) thankfully acknowledges the
Thus, metabolism of prebiotic XOS by probiotic bacteria generates
Department of Science and Technology ‘DST’-Govt. of India, for granting
metabolic products that exert antimicrobial activity against bacterial
financial aid in the form of Research Project (Ref. SR/SO/BB-66/2007),
pathogens.
and other funding agencies such as the Council of Scientific and Indus­
trial Research (CSIR-(Ref. No.: 37(1378)/09/EMR-II), Indian Council of
3.13. Antioxidant potential of xylooligosaccharides
Medical Research (ICMR Project Ref. no: 5/4/1/EXM/12-NCD-II), Uni­
versity Grants Commission (UGC Ref. F. Nos. F-32–550/2006/SR; 42-
Oxidative stress refers to excessive accumulation of free radicals
226/2013/SR) for financial support in the form of research projects.
which disturb the anti-oxidant defence system of the body. Free radicals
BKB gratefully acknowledges the Indo-US Science and Technology
may be implicated in conditions such as ageing and degenerative dis­
Forum for Research Fellowship at Ohio State University, USA,
eases viz. cancer, immune dysfunctioning, arthritis, cardiovascular
Commonwealth Scholarship Commission, for providing Commonwealth
complications, cataracts, liver damage, inflammation, renal failure,
Fellowship for research stay at Institute of Biological, Environmental
brain damage and others. Therefore, an effective antioxidant defence
and Rural Sciences, Aberystwyth University, Aberystwyth, UK, and
mechanism is vital for scavenging the free radicals (Zhang et al., 2018).
Institute of Advanced Study, Durham University, Durham, UK, for
XOS-SB showed good antioxidant potential as indicated by their ability
providing COFUND-International Senior Research Fellowship for
to scavenge DPPH radicals, and further the antioxidant activity
research at Department of Biosciences Durham University, UK. Mrs.
increased proportionally with the XOS-SB concentration. Maximum
Surbhi Vaid thankfully acknowledges Department of Biotechnology,
DPPH scavenging activity of 83.33% was observed at 5.0 mg/ml of
Govt. of India and British Council, UK, for providing Placement under
XOS-SB. In a similar study, xylooliogosaccharides produced from xylan
Newton Bhabha Ph.D. Placement Programme for Research Stay at
extracted from sugarcane straw and coffee husk exhibited antioxidant
Durham University, Durham, UK. Mrs. Mahak Gupta, Mrs. Surbhi Vaid
activity of 71% and 78%, respectively, at a concentration of 2 g/l (Avila
and Ms Surbhi Sharma acknowledge University of Jammu, Jammu, for
et al., 2020). Thus, antioxidant potential of XOS further adds to their
providing University Research Fellowship for research. Authors express
bioactivity potential, and hence augments their prospective for appli­
gratitude to the School of Biotechnology, University of Jammu for lab­
cations in food and pharmaceutical industries.
oratory facilities. SAIF, STIC, Cochin University of Science and Tech­
nology (CUSAT), Kerala, India, is thanked for physicochemical
4. Conclusions
characterization of samples.

Sugarcane bagasse (SB) extracted xylan could be a promising feed­

stock for the production of XOS, therefore leading to value-addition and
efficient valorization of sugarcane bagasse. DoE based optimization of
important variables effectively increased the yield of xylan from SB. The
FTIR and TGA analysis confirmed the purity and thermal stability of
xylan whereas SEM analysis of the biomass indicated that delignification
and xylan extraction adequately altered the complex structure of SB
biomass. Enzymatic hydrolysis of SB xylan by a β-xylosidase-free
endoxylanase indicated the high efficiency for XOS production. Struc­
tural elucidation of the xylooligosaccharides through NMR analysis
revealed that xylooligosaccharides were linear with no substitutions.
Furthermore, XOS prebiotics exhibited excellent functional attributes
and bioactivity spectrum viz. growth promoting potential of probiotic
L. plantarum M-13, high-positive prebiotic activity score, significant
antioxidant, and antimicrobial activity against bacterial pathogens. The
prebiotic potential of XOS promises their application prospective in food
industries for production of prebiotic-fortified functional foods.
Furthermore, the study emphasizes valorization of agricultural residues
(sugarcane bagasse) for production of value-added products (XOS
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.indcrop.2022.114591.
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