Journal of Biotechnology 347 (2022) 1–8

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Application of a recombinant GH10 endoxylanase from Thermoascus

aurantiacus for xylooligosaccharide production from sugarcane bagasse and
probiotic bacterial growth
Carlos Eduardo de Oliveira Nascimento a, Lorena Caixeta de Oliveira Simões a,
Josiani de Cassia Pereira a, Ronivaldo Rodrigues da Silva a, Evandro Antônio de Lima b,
Gabriel Cimonetti de Almeida a, Ana Lucia Barretto Penna a, Maurício Boscolo a, Eleni Gomes a,
Roberto da Silva a, *
a
Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, São José do Rio Preto, São Paulo, Brazil
b
Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, SP 1308-100, Brazil

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

Keywords: Xylooligosaccharides (XOs) are a promising class of prebiotics capable of selectively stimulating the growth of
Pichia pastoris the beneficial intestinal microbiota against intestinal pathogens. They can be obtained from xylan present in
Prebiotic residual lignocellulosic material from agriculture. Thus, in this study we produced XOs by extracting xylan from
Probiotic
sugarcane bagasse and hydrolyzing it using the GH10 xylanase from Thermoascus aurantiacus expressed by Pichia
Thermoascus aurantiacus
Xylanase
pastoris. An alkaline method to extract xylan is described, which resulted in 83.40% of xylan recovery and low
amounts of cellulose and lignin. The enzymatic hydrolysate exhibited a mixture of XOs containing mainly
xylobiose, xylotriose and xylotetraose. These oligosaccharides stimulated the growth of Lactobacillus casei,
L. rhamnosus, L. fermentum and L. bulgaricus strains, which were able to produce organic acids, especially acetic
acid. These findings demonstrate the possibility to redirect crop by-products to produce XOs and their use as a
supplement to stimulate the growth of probiotic strains.

1. Introduction linear (1 → 4)-linked β-D-xylopyranosyl backbone with branches at O-2

The huge amount of sugarcane (Saccharum officinarum) bagasse
derived as a by-product of the sugar and ethanol industries has mainly
attracted attention to the production of second generation (2 G) bio­
ethanol as an alternative to fossil fuels (Bezerra and Ragauskas, 2016;
Alokika et al., 2021). Sugarcane bagasse is a fibrous residue containing
about 32–45% cellulose, 20–32% hemicellulose and 17–32% lignin
Alokika et al., 2021). Due to its complex composition, it can also be used
to produce a multitude of other valuable products, like substrates for
fermentation processes, chemicals, animal feeds, oligosaccharides, etc.
Among them, because of the high xylan content, sugarcane bagasse is
also considered an excellent substrate for the low-cost production of
xylooligosaccharides (XOs) (Brienzo et al., 2010; Poletto et al., 2020;
Avila et al., 2020).
The molecular structure of xylans from sugarcane bagasse is typically
4-O-methyl-glucuronoarabinoxylans (4-O-MeGlcA-AX) consisting of a
and O-3 of 4-O-methylglucuronic acid and arabinofuranosyl units,
respectively (Bian et al., 2012; Carvalho et al., 2017). Hydrolyzing this
structure can produce free xylose and XOs - which are oligomers of β-1,
4-linked xylose that can vary from 2 to 10 xylose residues (Aachary and
Prapulla, 2011).
XOs emerge as an efficient prebiotic due to their chemical and bio­
logical characteristics such as stability with amylolytic enzymes, resis­
tance over a wide range of pH and the ability to promote the growth of
microorganisms of the genus Lactobacillus and Bifidobacterium known to
compose the beneficial intestinal microbiota (Poletto et al., 2020). In the
fermentation of these oligomers, short-chain organic acids are produced,
such as lactic and acetic acid, which act to control the pH of the intestine
(Ríos-Covián et al., 2016).
Enzymatic production of XOs is an ecologically safe process under
mild reaction conditions and with low generation of residual volume
(Poletto et al., 2020; Santibáñez et al., 2021). This process can include

* Corresponding author.
E-mail address: roberto.silva@unesp.br (R. da Silva).

https://doi.org/10.1016/j.jbiotec.2022.02.003
Received 22 October 2021; Received in revised form 28 January 2022; Accepted 8 February 2022
Available online 10 February 2022
0168-1656/© 2022 Elsevier B.V. All rights reserved.
C.E.O. Nascimento et al.
enzymes like endo-1,4-β-xylanase, β-xylosidase, α-L-arabinofur­
anosidase and acetyl xylan esterase (Biely et al., 2016). GH10 and GH11
endoxylanases stand out for randomly hydrolyzing the β-1,4 glycosidic
bonds between xylose units, producing short-chain xylooligosaccharides
such as xylobiose (X2), xylotriose (X3) and xylotetraose (X4), which are
preferred for the metabolism of probiotic organisms (Poletto et al.,
2020).
The xylanases described in the literature belong to glycoside hy­
drolase (GH) families 5, 7, 8, 10, 11 and 43 (Lombard et al., 2014), of
which GH10 and GH11 are the most studied (Rashid and Sohail, 2021).
These enzymes differ in their physicochemical properties, primary
amino acid sequences, modes of action and substrate specificity (Gon­
çalves et al., 2012; Linares-Pasten et al., 2018). One of the most prom­
inent differences between them is the specificity of GH10 xylanases to
cleave glycosidic linkages closer to substituent residues and show a
higher affinity to shorter linear β-1,4-xylooligosaccharides than the
GH11 family of endoxylanases (Biely et al., 2016; Linares-Pasten et al.,
2018). In general, GH10 produces smaller xylooligosaccharides and
larger amounts of free xyloses while the GH11 enzyme produces higher
xylooligosaccharides and less free xylose.
Thus, in this study, we used the recombinant GH10 endo-1,4-
β-xylanase from Thermoascus aurantiacus (expressed in P. pastoris) to
produce XOs from xylan extracted from sugarcane bagasse. The result­
ing xylooligomers were used for the growth of probiotic bacteria:
L. casei, L. rhamnosus, L. fermentum and L. bulgaricus. The xylan extrac­
tion, the production and characterization of XOs, as well as the growth
of microorganisms and the production of organic acids have been
presented.
2. Materials and methods
2.1. Production of recombinant GH10 Xylanase
The xylanase was expressed by Pichia pastoris strain GS115 har­
bouring the plasmid pPIC9 with the xynA gene for GH10 xylanase
(AF127529.1) from Thermoascus aurantiacus CBMAI756. Induction and
enzyme production in shake flasks was performed as described in a
previous study (Franco, 2011).
The P. pastoris was maintained on slants of YPDA medium (1% yeast
extract, 2% peptone, 2% D-glucose, 2% agar). One transforming colony
was inoculated into a 250 mL flask containing 25 mL of BMGY medium
(buffered minimal glycerol complex) composed of 1% yeast extract, 2%
peptone, 100 mM phosphate buffer (pH 6.0), 1.34% yeast nitrogen base
without amino acids (YNB), 4 × 10− 5% biotin and 1% glycerol. The
culture was incubated at 30 ◦ C at 200 rpm for around 16–18 h until
reaching an OD600 nm of 2–6. Cells were collected by centrifugation at
1500 x g for 5 min at 4 ºC and transferred in 250 mL of BMMY medium
(BMGY exchanging 1% glycerol for 0.5% methanol) in a 1 L baffled
Erlenmeyer flasks, incubated under the same conditions for 96 h, with
the addition of 0.5% methanol (final concentration) every 24 h. After
that, supernatant was collected by centrifugation and used for enzyme
assays.
The presence of the endoxylanase was evaluated by sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-
PAGE was prepared at 10% concentration and Tris/Glycine/SDS was
used as running buffer. Next, the gel was stained using Coomassie blue
R-250.
2.2. Enzyme activity
The xylanolytic activity was evaluated using beechwood xylan 1%
(w/v) as a substrate and by quantifying the reducing sugar using the 3,5
dinitrosalicylic acid 10 g L− 1 (DNS) (Miller, 1959). The reactions were
carried out on a microplate containing 200 μL of reaction volume.
Initially, 20 μL of enzyme were added to 80 μL of 1% substrate dissolved
in 0.1 M acetate buffer, pH 5.0 and incubated at 60 ºC for 10 min. Then,
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Journal of Biotechnology 347 (2022) 1–8
100 μL of DNS was added to the reaction mixture and incubated at 95 ºC
for 10 min. For each reaction there was a corresponding blank where the
DNS was added before the substrate. Next, the reducing sugar was
measured in a spectrophotometer at 540 nm. All tests were performed in
triplicate. One unit of endoxylanase (U) was defined as the amount of
enzyme needed to produce 1 μmol of reducing sugar per min, using
xylose as a standard sugar.
The enzymatic activity on other substrates, such as carboxymethyl­
cellulose (CMC), microcrystalline cellulose (Avicel), 4-Nitrophenyl β-D-
glucopyranoside, 4-Nitrophenyl β-D-xylopyranoside and 4-Nitrophenyl
α-L-arabinofuranoside was also evaluated (Amo et al., 2019). For CMC
and Avicel, the DNS method similar to the xylanase assay was used. For
4-Nitrophenyl substrates, the following reaction conditions were used:
20 μL of enzyme was added to 80 μL of substrate (4 mM) dissolved in 0.1
M acetate buffer, pH 5.0 and incubated at 60 ºC for 10 min. Then, 100 μL
of 2 M sodium carbonate was used to quench the enzymatic reaction and
the nitrophenol was measured in a spectrophotometer at 410 nm.
2.3. Sugarcane bagasse
The sugarcane bagasse was donated by Itajobi mill located in the city
of Itajobi, São Paulo, Brazil. The bagasse was subjected to five water
washing cycles to remove particles such as gravel, soil and metals from
the crop. After washing, the material was dried in a drying oven at 40 ºC
for 48 h, reaching a humidity of ± 13%. Subsequently, the bagasse was
crushed using a Trapp 400 shredder (Trapp, Jaraguá do Sul, Brazil) with
an internal sieve of 1 mm and standardized with mesh 12 and mesh 16
sizes, where particulates sized < 1 mm, 1–1.41 mm and > 1.41 were
obtained.
2.4. Extraction and chemical composition analysis of xylan
Xylan extraction from sugarcane bagasse was carried out following
the method described by Kaur et al. (2019). Initially, the bagasse was
added to a NaOH 15% (w/v) solution, in a solid-liquid ratio 1:10, then
the mixture was autoclaved for 30 min at 121 ºC and filtered through
synthetic mousseline fabric. The filtrate was adjusted to pH 5 by the
addition of 85% phosphoric acid and the solution was concentrated 3
times using a rotary evaporator. Then, 96% ethanol was added to restore
the initial volume and the solution was centrifuged at 6000 x g for 10
min at 25 ◦ C. Afterwards, the precipitate was resuspended in 500 mL of
70% ethanol (this last step was repeated 3 times). Finally, the precipitate
was kept at 40 ºC for drying.
Chemical composition of the product, including cellulose, hemicel­
lulose and lignin was determined according to the NREL (National
Renewable Energy Laboratory – USA), Laboratory analytical procedures
NREL/TP 510–42618/42619 (Sluiter et al., 2008).
2.5. Production of xylooligosaccharides
Xylan hydrolysis was planned using experimental design, aiming at
the optimization of the process conditions, having the XOs concentration
as the response evaluated. Initially, the influence of the sugarcane xylan
(%) and enzyme (U g− 1 xylan) variables were evaluated using a face-
centered central composite design (CCD) (22, including 4 axial points
and 4 repetitions of central points, totaling 12 trials). Analysis of vari­
ance (ANOVA) was performed using Statistica software (Statsoft 7.0).
For the production of XOs, we used the optimal pH and temperature
for xylanase activity reported by Franco (2011). Thus, the enzymatic
hydrolysis of xylan was carried out in Erlenmeyer flasks (50 mL) sealed
with a rubber stopper containing sodium acetate buffer solution (0.05
mol L− 1, pH 5.0) in a final volume of 20 mL. The hydrolysis tests were
performed in duplicate, at 60 ºC, under agitation at 180 rpm, using xylan
at 20%, 25% and 30% (w/v), and enzyme at 140, 180 and 220 U per g
xylan. Samples were collected every 6 h (two Erlenmeyer flasks for each
point), heated in a boiling water bath for 10 min to inactivate the
C.E.O. Nascimento et al.
enzyme, and centrifuged at 13000 x g. The supernatant was filtered
through a 0.22 µm filter and the liquid used for the qualitative and
quantitative analysis.
2.6. Quantification of Xylooligosaccharides
The hydrolysate resulting from the enzymatic hydrolysis of sugar­
cane xylan was analyzed using a Dionex ICS 5000 HPAEC-PAD system
(Thermo-Fisher Scientific, Sunnyvale, USA). All samples were filtered
through 0.22 µm filters and injected into the chromatograph (20 μL).
The flow rate was 1 mL min− 1 at 30 ºC. All eluents were prepared with
ultrapure water with N2 sparging. The xylooligosaccharides were
separated using a CarboPac™ PA-100 Column (4 mm × 50 mm) and a
CarboPac™ PA-100 Column (4 mm × 250 mm). Eluent A was 0.2 mol
L− 1 NaOH, eluent B was ultrapure water, and eluent C was 0.5 mol L− 1
sodium acetate in 0.15 mol L− 1 NaOH. The first phase, isocratic, was 5
min in 50% eluent A and 50% eluent B, followed by a linear gradient
targeting 44% eluent A and 16% eluent C, over 20 min. To identify the
products, Xylose (Sigma-Aldrich, St.Louis, USA), Xylobiose, Xylotriose,
Xylotetraose, Xylopentaose and Xylohexaose (Megazyme International
Ireland Ltd, Bray, Ireland) were used.
2.7. Capillary electrophoresis (CE) of oligosaccharides
Qualitative analysis of XOs that resulted from enzymatic hydrolysis
of sugarcane xylan, standard xylose and XOs (X2 to X6) were marked by
reductive amination reaction with the fluorophore 8-aminopyrene-
1,3,6-trisulfonic acid (APTS) (Sigma-Aldrich, St.Louis, USA). CE ana­
lyses were performed using a P/ACE™ MDQ system (Beckman Coulter,
Brea, USA) equipped with laser-induced fluorescence detection. Elec­
trophoresis conditions during separations were voltage 20 kV, electric
current 70–100 µA, temperature 20 ◦ C and running buffer 40 mM so­
dium phosphate, pH 2.5. To prevent the occurrence of cross contami­
nation, the capillary was washed between runs with water and then with
running buffer. The identification of XOs and xylose from samples was
done by comparing the retention times of standards. APTS-labeled XOs
were excited at 488 nm and emission was determined at 520 nm. This
analysis was performed in the Brazilian Biorenewables National Labo­
ratory (LNBR) at the Brazilian Center for Research in Energy and Ma­
terials (CNPEM), Campinas, SP, Brazil.
2.8. Probiotic bacteria growth
Five bacterial strains known to compose the intestinal microbiota
were used to evaluate the prebiotic potential of the XOs obtained. The
probiotic strains were Lactobacillus rhamnosus (commercial strain, pro­
vided by the company Chr. Hansen), and other species of Lactobacillus
isolated from Brazilian water Buffalo Mozzarella Cheese, including two
strains of Lactobacillus casei (I and II), Lactobacillus fermentum and
Lactobacillus bulgaricus (Silva et al., 2015).
Static bacterial growth was performed in tubes sealed with a rubber
stopper containing 10 mL of De Man, Rogosa and Sharpe (MRS), pH 5.0
at 30 ◦ C. First, the bacteria were cultivated in MRS supplemented with
1% glucose. After 12 h, the culture was centrifuged at 13000 x g and
serially diluted in sterile ultrapure water to standardize the inoculum.
Next, bacterial growth was prepared for comparative evaluation sup­
plementing the MRS medium with 1% glucose, glucose/XOs (0.5%/
0.5%) or 1% XOs. Bacterial growth was measured in a spectrophotom­
eter at 600 nm for 2, 4, 8, 12, 24 and 48 h.
2.9. Evaluation and quantification of organic acids produced in
fermentation
The amount of each organic acid (oxalic, formic, pyruvic, lactic,
acetic and propionic acid) released after probiotic bacterial growth was
determined using a 1220 Infinity Liquid Chromatograph (Agilent
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Journal of Biotechnology 347 (2022) 1–8
Technologies, Santa Clara, USA). All samples were filtered through 0.22
µm filters and injected into the chromatograph (20 μL). The flow rate
was 0.5 mL min− 1 at 20 ºC. All eluents were prepared with ultrapure
water. Organic acids were separated using Brownlee Choice Organic
Acids Column (5 µm x 250 mm × 4.6 mm – PerkinElmer, Waltham,
USA). The eluent used in the separation of organic acids was 100 mM
potassium phosphate buffer, pH 2.5 (isocratic gradient). Calibration
curves were made at the following concentrations: 25–100 mg L− 1 for
each organic acid.
3. Results and discussion
3.1. Hemicellulose extraction, chemical characterization and FTIR-ATR
Following the trend of sustainable technologies to take advantage of
residual biomass from industrial activity and agriculture, in this study,
xylan was extracted from sugarcane bagasse and was used for producing
XOs. The sugarcane bagasse, with the three different particulate sizes,
was treated to extract hemicellulose by an alkaline method.
The results show that the method was successful in extracting xylan
with a high yield and a low amount of cellulose and lignin. The highest
yield in the extraction of xylan was detected for the smallest particles,
with 83.40% yield for the particulate below 1 mm, 51.7% for the 1–1.41
mm particulate and 31.8% for the above 1.41 mm particulate. There­
fore, the particulate below 1 mm was chosen for continuing the work.
For xylan extraction, alkaline treatments are widely used in different
plant biomasses due to their reduced effect for damaging hemicellulose
and by breaking the ether and ester bonds between hemicellulose and
lignin (Tarasov et al., 2018; Schmitz et al., 2021). Additionally, the ef­
ficiency of NaOH for extracting hemicellulose is seen, which can vary
according to the particulate size of the substrate. The surface area is
enlarged with the reduction of granule size, leaving the hemicellulose
present in the biomass more exposed.
Jayapal et al. (2013) when using alkaline solutions associated with
steam explosion to extract hemicellulose from sugarcane bagasse ob­
tained a yield of 85% with sodium hydroxide (NaOH) and 53% when
using potassium hydroxide (KOH). Brienzo et al. (2010) also showed the
effectiveness of applying an alkaline pre-treatment to extract hemicel­
lulose from sugarcane bagasse. The amount of xylan present in the
hemicellulose obtained was 80.9%, a value close to that found in the
present study. Samanta et al. (2012) also reported a maximal xylan re­
covery of 83.5% from corn cobs in an alkaline method followed by steam
explosion. As well as the high yield and purity of xylan contribute to
greater accessibility of the enzyme to the substrate, the specificity of the
enzyme is also a property that interferes in the prebiotic function of the
xylan hydrolysate.
The analysis of the infrared spectra of xylan (< 1 mm) from beech­
wood (Sigma Aldrich, 99% purity) and sugarcane bagasse obtained is
presented in Fig. 1. The spectra are characteristic responses to xylan
extracted from different agro-industrial residues, such as sorghum
bagasse (Zhang et al., 2011), corn cobs (Samanta et al., 2012) and
sugarcane bagasse (Perrone et al., 2016; Kaur et al., 2019).
The vibration corresponding to the 897 cm− 1 is related to β (1→4)
glycosidic bonds, strongly present in the xylan main chain (Sporck et al.,
2017). The bands located in the 1158 cm− 1 refer to C-O-C stretch of β
(1→4) glycosidic bonds, and the band located in the 1320 cm− 1 ab­
sorption is associated with the –CH2 group present in hemicellulose and
cellulose. The bands found in the 1245 cm− 1 region are related to
infrared absorption by central hemicellulose chains. The bands located
in the 1035 cm− 1 absorption can be attributed to the C-O stretch present
in cellulose, lignin and hemicellulose (Gullón et al., 2011).
3.2. Enzyme specificity
The enzyme, whose molecular mass was estimated by SDS-PAGE to
be 32 kDa (Fig. 2), was not capable to hydrolyze
C.E.O. Nascimento et al. Journal of Biotechnology 347 (2022) 1–8

Fig. 1. FTIR-ATR infrared spectrometry analysis.

Table 1
Statistical planning face centered 22 with the interaction of sugarcane xylan and
enzyme load as factors, with response to XOs production. Xylan hydrolysis was
performed at 60 ºC, pH 5 (acetate buffer solution, 0.05 mol L− 1), for 6 h at
180 rpm.
Assays U* Xylan (%) U* Xylan (%) Total XOs (g L¡1)

1 -1 -1 140 20 25.14
2 +1 -1 220 20 28.02
3 -1 +1 140 30 28.19
4 +1 +1 220 30 27.22
5 -1 0 140 25 32.06
6 +1 0 220 25 32.33
7 0 -1 180 20 26.01
8 0 +1 180 30 28.70
9 0 0 180 25 33.32
10 0 0 180 25 32.37
11 0 0 180 25 32.02
12 0 0 180 25 31.32

U: Enzyme unit.

and the results were confirmed in a validation experiment.

Fig. 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
of the recombinant GH 10 endoxylanase. Lane 1: Protein molecular weight
standards (Bio-Rad, USA). Lane 2: Endoxylanase.
Carboxymethylcellulose (CMC), Cellulose microcrystalline (Avicel), 4-
Nitrophenyl β-D-glucopyranoside, 4-Nitrophenyl β-D-xylopyranoside
and 4-Nitrophenyl α-L-arabinofuranoside. This favors the selective hy­
drolysis of xylan without hydrolyzing cellulose and releasing cello-
oligosaccharides (Simões et al., 2019). This stands out as another
advantage for the application of this xylanase in the production of XOs
because it avoids greater presence of glucose in the hydrolysate
obtained.
3.3. Optimization of xylooligosaccharide production
For the production of XOs, we used the experimental design (Table 1)
We observed that assays in which the xylose concentration was 25%
had better production of XOs for all enzyme concentrations evaluated.
The response surface (Supplementary material S1A) indicated that the
maximum production of XOs would occur with 220 U of xylanase g− 1
xylan and 25% xylan. The Pareto Diagram (Supplementary material
S1B) shows that the xylan variable and the interaction between factors
(xylan/enzyme) exhibited a 95% level of confidence on XOs production.
The linear and quadratic effects of the enzyme factor were not signifi­
cant, with p < 0.05.
Applying the desirability function (Software Statistica 10.0) with
interaction of factors (substrate/enzyme) in the conditions mentioned
above (220 U of xylanase/xylan (g), and 25% xylan), the XOs produc­
tion in the range of 25.140 at 33.320 g L− 1 can be predicted (Supple­
mentary material S1C). When validating this analysis, 28.62 g L− 1 of
XOs were obtained. In XOs production, a residual 3.04% glucose was
also detected. The tests were performed in triplicate.
Notably, we indicate the activity of this endoxylanase on xylan
extracted from sugarcane bagasse and the possibility of using the
resulting xylooligosaccharides as prebiotic agents. The production of
XOs via enzymatic hydrolysis is reported to be more advantageous than
acid hydrolysis of xylan, due to it occurring in milder reaction conditions
without using high molar acid solutions, such as sulfuric acid, and in­
cubation at high temperature. From the perspective of sustainable

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C.E.O. Nascimento et al. Journal of Biotechnology 347 (2022) 1–8

development, chemical hydrolysis may require higher energy con­

sumption and a residual volume of the solutions used, which needs
appropriate disposal in an ecologically safe way (Poletto et al., 2020;
Santibáñez et al., 2021).

3.4. Types of XOs produced in the xylanase reaction

In native xylan, xylose residues are partially acetylated while some

arabinose residues are esterified with ferulic acid. However, as alkaline
extraction was carried out, it is assumed that there has been a saponi­
fication of ester bonds which eliminates acid substituents, resulting in a
neutral arabinoglucuronoxylan, which was further submitted to enzyme
hydrolysis (Biely et al., 2016).
Xylanases described in the literature belong to glycoside hydrolase
(GH) families 5, 7, 8, 10, 11 and 43 (Lombard et al., 2014), of which
GH10 and GH11 are the most studied xylanases (Rashid and Sohail,
2021). These enzymes differ in their physicochemical properties, pri­
mary amino acid sequences, modes of action and substrate specificity
(Gonçalves et al., 2012; Karlsson et al., 2018).
Considering the enzyme specificity, GH10 xylanases require two
consecutive unsubstituted xylopyranosyl to attack the xylan substrate,
and are able to cleave xylopyranosyl linkages closer to side-chain resi­
dues, whereas GH11 xylanases cleave xylopyranosyl linkages one link­
age before the xylopyranosyl substituted with methyl-glucuronic acid,
and hydrolyze the larger unsubstituted regions of glucuronoxylan (Biely
et al., 2016). Therefore, it is assumed that GH10 produces smaller
xylooligosaccharides and larger amounts of free xyloses, while the GH11
enzyme produces higher xylooligosaccharides and less free xylose. Since
the recombinant xylanase used is this work is a GH10 enzyme, it was
expected to produce a certain amount of free xylose and short-chain
xylooligosaccharides. In our study, this was confirmed as the chro­
matographic profiles of XOs composition (Fig. 3). The main products
released were XOs with 2–4 xylopyranosyl residues (xylobiose to xylo­
tetraose) and low xylose production.
Many studies have reported that XOs chains from X2 up to X4 are
preferred types of oligomers for food industry applications (Gullón et al.,
2011). The XOs have the ability to stimulate the growth of probiotic
microorganisms present in the intestinal microbiota, such as those of the
genera Lactobacillus and Bifidobacterium, thus inhibiting the growth of
pathogenic microorganisms and can also improve calcium absorption.
They also have cytotoxic effects on leukemic cells, a positive effect on Fig. 3. Types of xylooligosacharides obtained by the action of recombinant
type II diabetes mellitus and still exhibit antioxidant activity (Jain et al., endoxylanase on xylan from sugarcane bagasse. The cleavage pattern was
2015; Mussatto and Mancilha, 2007). analyzed by capillary electrophoresis. The products released were xylose (X1)
A study by Okazaki et al. (1990) showed that probiotic microor­ and mainly xylobiose (X2), xylotriose (X3) and xylotetraose (X4).
ganisms can have their growth stimulated by XOs of different degrees of
polymerization. When evaluating the XOs assimilation by Bifidobacte­ 1% XOs or media with a combination of glucose and XOs (0.5% XOs +
rium adolescetis, a preference for X2 and X3 was seen. In another study 0.5% glucose), diauxic growth (growth in two phases) occurred that
with commercial XOs containing a higher concentration of X2 and X3, it might indicate the possible sugar consumption of microorganisms,
was also demonstrated that B. adolescencis and B. longum grow better in glucose first and then the xylose oligomers. Even so, in all cultures there
these oligomers (Carvalho et al., 2020). was high growth in the media containing XOs. Notably, the culture in
In this work, the XOs produced according to the experimental design, the medium containing 1% XOs showed better growth than the culture
with a predominance of xylobiose, xylotriose and xylotetraose, were with glucose and XOs (0.5% XOs + 0.5% glucose) for the bacteria L. casei
used to evaluate bacterial growth. I (Fig. 4D) and L. bulgaricus (Fig. 4E), and there was a similar response
for both media in the cultivation of L. casei II (Fig. 4B) and L. fermentum
3.5. Bacterial growth and organic acid production on XO-containing (Fig. 4C).
media The presence of glucose in the xylan hydrolysate was ~3% glucose
(for 1 g xylan hydrolysate, there was around 0.03 g glucose, i.e. for 1 g
Evaluating the bacterial growth, reduced growth was noted for all XOs per 100 mL MRS, there was around 0.03% glucose) which is much
Lactobacillus strains in the absence of sugars as a carbon source and there lower than for the tests supplemented with 0.5%. glucose. Because of
was a rapid growth in the medium with 1% glucose (Fig. 4). All strains of this, the lag phase of diauxic growth in the medium containing 1% XOs
Lactobacillus used in the present work were able to grow in the presence was reached at a lower optical density in comparison to the media with
of XOs. glucose additives. This indicates that the low glucose concentration
Despite the specificity of the enzyme for hydrolyzing xylan, a low present in 1% XOs was consumed more quickly compared to other media
concentration of glucose in the hydrolysates was seen, which may be due and the second phase of bacterial growth corresponds to the consump­
to the xylan extraction process, as glucose is a minor component of the tion of xylooligomers.
fraction of this heteropolysaccharide. Because of this, in a medium with

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C.E.O. Nascimento et al.
Fig. 4. Growth of probiotic strains on MRS media under the influence of different carbon sources. Bacterial growth was assessed by measuring optical density at
600 nm after 2, 4, 8, 12, 24 and 48 h.
After 48 h of cultivation on MRS containing 1% XOs, organic acids
such as oxalic, formic, pyruvic, lactic, acetic and propionic acid were
produced according to analyses performed by chromatography. All
strains had a similar profile for metabolite production (Fig. 5). The re­
sults show a higher production of acetic acid in all fermentations,
reaching 11.17 g L− 1 for the L. fermentum.
Studies have shown that the inhibitory effect on the proliferation of
pathogenic bacteria are related to the competition between pathogenic
and probiotic strains for binding sites that are present in the villi of the
gastrointestinal region. The production of acids such as acetic and lactic
acid promotes a decrease in pH, reducing or even inhibiting pathogenic
bacterial growth (Crittenden and Playne, 1996; Mcnamara et al., 2015;
Schrezenmeir and De Vrese, 2001).
Mathew et al. (2018), when evaluating the metabolites resulting
from the fermentation of (A)XOs by L. brevis, found predominantly lactic
6
Journal of Biotechnology 347 (2022) 1–8
and acetic acid after 48 h of fermentation, 1.76 ± 0.02 and
1.14 ± 0.03 g L− 1 respectively.
Kaur et al. (2019) also reported the effect of XOs on growth of
Lactobacillus strains. XOs from sugarcane bagasse were more effective
than fructooligosaccharides (FOS) for stimulating of growth of
L. acidophilus, L. brevis and L. viridescens.
Food components that are not degraded by human digestive enzymes
and that can be fermented by the intestinal microbiota are attractive for
prebiotic function (Karlsson et al., 2018). Glucose, the preferred sugar
for the energy metabolism of living organisms, does not favor the se­
lective growth of the beneficial microbiota. On the contrary, XOs stand
out for being metabolically restricted to xylose fermenting organisms
which, when fermented, can generate organic acids that act to control
intestinal pH, favoring probiotic organisms in competition between
members of the intestinal microbiota.
C.E.O. Nascimento et al.
Fig. 5. Organic acid production in 48 h of bacterial growth on MRS with 1% XOs. The acids were detected and quantified by HPLC UV/VIS using a Brownlee Choice
Organic Acids column (250 mm × 4.6 mm×5 µm – PerkinElmer, Waltham, USA).
4. Conclusion
This study showed a successful and high yield alkaline xylan
extraction from sugarcane bagasse, and the potential use of this xylanase
to obtain XOs capable of stimulating the growth of Lactobacillus species.
Fermenting XOs, these bacteria produced organic acids that may
potentiate their antagonism against pathogenic microbiota in the in­
testine. In future studies, we can investigate the combination of this
xylanase with other xylanases and auxiliary enzymes, such as α-L-ara­
binofuranosidases, and evaluate the resulting xylan hydrolysate from
sugarcane bagasse and other plant substrates. The validation of XOs as
prebiotics reinforces the importance of using agro-industrial by-prod­
ucts for this biotechnological proposal.
CRediT authorship contribution statement
Carlos Eduardo de Oliveira Nascimento, Lorena Caixeta de Oli­
veira Simões, Josiani de Cassia Pereira, Ronivaldo Rodrigues da
Silva, Evandro Antônio de Lima, Gabriel Cimonetti de Almeida:
Investigation, Methodology, Formal analysis, Visualization. Carlos
Eduardo de Oliveira Nascimento, Ronivaldo Rodrigues da Silva:
Writing – original draft. Ronivaldo Rodrigues da Silva, Roberto da
Silva: Writing – review & editing. Ana Lucia Barretto Penna, Maur­
ício Boscolo, Eleni Gomes, Roberto da Silva: Funding acquisition.
Roberto da Silva: Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors would like to acknowledge the financial support pro­
vided by the São Paulo Research Foundation - FAPESP (Fundação de
Amparo à Pesquisa do Estado de São Paulo) process 2017/16482–5.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jbiotec.2022.02.003.
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