Revista de Ciencias Tecnológicas (RECIT). Volumen 3 (1): 10-22
Revista de Ciencias Tecnológicas (RECIT). Universidad Autónoma de Baja California ISSN 2594-1925
Volumen 8 (3): e405. Julio-Septiembre, 2025. https://doi.org/10.37636/recit.v8n3e405
1
ISSN: 2594-1925
Research article
Chemical and Thermal Characterization of an exopolysaccharide
from Lactiplantibacillus plantarum BAL-29-ITTG
Caracterización Química y Térmica de un exopolisacárido proveniente de
Lactiplantibacillus plantarum BAL-29-ITTG
Rony Obed Suchiapa Díaz1, Lucia María Cristina Ventura Canseco1, Alejandro Ramírez
Jiménez2*
1Tecnológico Nacional de México/Instituto Tecnológico de Tuxtla Gutiérrez, Carretera Panamericana, km.
1080. C.P. 29050, Tuxtla Gutiérrez, Chiapas, México.
2IIXM SECIHTI-Tecnológico Nacional de México/Instituto Tecnológico de Tijuana, Blvd. Industrial s/n, Cd
Industrial, 22430 Tijuana, Baja California, México.
Corresponding author: Alejandro Ramírez Jiménez, IIXM SECIHTI-Tecnológico Nacional de México/Instituto Tecnológico de Tijuana,
Blvd. Industrial s/n, Cd Industrial, 22430 Tijuana, Baja California, México. E-mail: aramirezj@secihti.mx; Tel.: +52 55-5197-0289. ORCID:
0000-0002-3011-1612.
Received: February 28, 2025 Accepted: September 3, 2025 Published: October 3, 2025
Abstract. - Exopolysaccarides (EPS) are biopolymers, which can be produced by lactic acid bacteria. In this work
an EPS from Lactiplantibacillus plantarum BAL-29-ITTG was characterized by 1H, 13C, COSY, TOCSY and HSQC
nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (FTIR), differential scanning calorimetry
(DSC), thermogravimetric analysis (TGA) and viscometry. Thermal analysis and viscometry results suggested that
ESP had a high molecular weight with a branched structure; to determine its main monosaccharides, the
experimental chemical shifts of hydrogens and carbons obtained by NMR were loaded and compared with the
database in the online software CASPER: http://www.casper.organ.su.se./casper/ Results showed that at least eight
monosaccharides are present as components of this EPS, the most likely monosaccharides identified were:
-D-
glucopyranose 1-4 and 1-6 linked: →4)-
-D-Glc-(1→; →6)-
-D-Glc-(1→ and
-D -manose 1-3, 1-4 and 1-6
linked: →3)-
-D-Man-(1→; →4)-
-D-Man-(1→; →6)-
-D-Man-
→, although, data from FTIR and NMR also
suggest N-acetylated residues.
Keywords: Exopolysaccharides; Monosaccharides determination; Nuclear magnetic resonance; Polysaccharides
composition; Polysaccharides characterization.
Resumen. – Los exopolisacáridos (EPS) son biopolímeros que pueden ser producidos por bacterias ácido lácticas,
En este trabajo, un EPS proveniente de Lactiplantibacillus plantarum BAL-29-ITTG fue caracterizado mediante
resonancia magnética nuclear (RMN) de 1H, 13C, COSY, TOCSY y HSQC, espectroscopía de infrarrojos (FTIR)
calorimetría diferencial de barrido (DSC), análisis termogravimétrico (TGA) y viscosimetría. Los resultados de
análisis térmicos y viscosimetría indican que este EPS tiene una estructura ramificada y una masa molar alta; para
determinar los monosacáridos principales, los desplazamientos químicos de carbono e hidrógeno obtenidos
mediante RMN fueron cargados y comparados con la base de datos del software en línea CASPER:
http://www.casper.organ.su.se/casper/ Los resultados mostraron que, al menos, ocho monosacáridos diferentes
componen este EPS, los más probables identificados fueron:
-D-glucosa con uniones 1-4 y 1-6: →4)-
-D-Glc-
(1→; →6)-
-D-Glc-(1→ y
-D-manosa con uniones 1-3, 1-4 y 1-6: →3)-
-D-Man-(1→; →4)-
-D-Man-(1→
y
→6)-
-D-Man-(1→, aunque los datos de FTIR y RMN sugieren también la presencia de residuos N-acetilados.
Palabras clave: Exopolisacáridos; Determinación de monosacáridos; Resonancia magnética nuclear; Composición
de polisacáridos; Caracterización de polisacáridos.
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1. Introduction
Carbohydrates are some of the main components
in living organisms. Monosaccharides can be in
lineal or in cyclic form, for the last one, anomers
or are possible. Polysaccharides are formed
through glycosidic linkage between many
monosaccharides, for this reason they also are
named glycans, because they have many
hydroxyl groups in their composition, the linkage
can be at different positions, more detailed
information on this can be found in the work of
Flitsch [1]. On the other hand, they generally are
attached to other biomolecules forming
glycoconjugates, for example, with proteins or
lipids form glycoproteins or glycolipids
respectively. For the mentioned above, their
complete chemical structure characterizations
can be highly complex.
Exopolysaccharides (EPS) are extracellular
biopolymers that can be produced by plants,
fungi or bacteria [2, 3]. Bacterial polysaccharides
also included lipopolysaccharides (LPS),
peptidoglycans, teichoic acids (TA), capsular
polysaccharides (CPS) [4]. Although EPS can
also be extracted from plants, bacterial EPS have
more diverse structures, therefore more diverse
properties and bioactivities, hence, they have
recently received significant attention in different
fields of science and technology for different
applications, for example in medicine or food
package [2, 5]. These biopolymers are mainly
composed of repetitive sugar units and,
depending on their location, could be found in
capsular form, they are closely associated with
the cell surface and in free form, weakly bound
or even totally secreted into the extracellular
environment. According to their chemical
composition, EPS are classified into
homopolysaccharides, which are made up of
units of a single type of monosaccharide such as
glucose or fructose and heteropolysaccharides,
which are made of two or more different
monosaccharide units (glucose, xylose, fructose,
mannose, galactose, rhamnose, N-
acetylglucosamine, gluronic acids, etc.) having
different chemical structures and linkages,
[2, 5, 6].
The number of investigations dedicated to the
applications of bacterial EPS has increased in
recent decades. Due to bacterial EPS possess
various unique beneficial properties such as, high
adhesive capacity, biocompatibility,
biodegradability, gelation ability, non-toxicity,
pseudoplasticity, viscoelasticity and thixotropic
nature [2, 7]. EPS have also been reported to
withstand various environmental stresses, such
as high temperature, high pH, freezing, thawing,
or high salt concentrations [8], therefore, they
have wide commercial applications, for example,
in the food packages, pharmaceutical and
cosmetic industries, among others [2].
Furthermore, some bacterial EPS also possess
antitumor, antioxidant, antibiofilm, anti-
inflammatory, antibacterial, antiviral,
cholesterol-lowering, prebiotic, wound healing
or immunomodulatory activities [9, 10, 11].
As mentioned before, EPS can be extracted from
different sources but, particularly lactic acid
bacteria (LAB) are microorganisms present in
food production and in turn these are beneficial
to human health. [12, 13]. In general, LAB are
widely recognized as safe, due to this they are
mainly used in fermented foods [14]. LAB
exhibits a variety of probiotic effects, which are
closely related to their metabolites, including
organic acids, bacteriocin, exopolysaccharides
(EPS) and others [15, 16, 17]. However, the
development and application of EPS from BAL
are relatively limited compared to
polysaccharides derived from animals and plants
[18].
The bioactivities presented by the EPS produced
by the BAL are related to their structure. Mainly,
the monomeric composition, molecular weight
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and charge of EPS depend on the bacteria,
likewise, the biological functions such as
antioxidant, antitumor and antibiofilm activity
depend on the composition and structure of EPS
[19, 20]. Certain structural features may be
associated with specific bioactivities. For
example, the presence of α-(1 → 3) and α-(1 →
6) linkages in the main chain and α-(1 → 3)
linkages in the branched chains of the EPS
produced by L. plantarum (LAB) has been
associated with immunomodulatory activity [19].
Jiang and Yang, reported that, although EPS
produced by lactic acid bacteria (LAB) contain
similar monosaccharide components (galactose,
glucose, rhamnose, etc.), these had different
properties owing their structural difference [21].
They also reported that, these properties were
affected by their molecular weight distribution,
the type of glycosidic bond, their charge, side
chains, the rigidity of the molecules that form the
EPS, among others; regarding this, it was
mentioned that the structural characteristics, uses
and bioactivities of EPS depend on the type of
microorganism and the medium in which they
were grown up [2, 5]. Because bacterial EPS
have many functional groups, for example,
hydroxyl, carboxyl, carbonyl or acetyl, which
allow their modification to obtain new properties
[8].
Ramírez-Pérez et al. reported that LAB isolated
from a fermented beverage, known as “taberna”
(Lactiplantibacillus plantarum BAL-03-ITTG,
Lactiplantibacillus plantarum BAL-05-ITTG,
Limosilactobacillus fermentum BAL-21-ITTG,
Lactiplantibacillus pentosus BAL-22-ITTG,
Lactiplantibacillus fabifermentans BAL-27-
ITTG, Lactiplantibacillus paraplantarum BAL-
28-ITTG, Lactiplantibacillus plantarum BAL-
29-ITTG), can use different carbon sources and,
when they are cultured in MRS broth (de Man,
Rogosa y Sharpe), these can be produced as free
and capsular EPS. The highest production of
ERPS from Lactiplantibacillus plantarum BAL-
29-ITTG, was 478.0 ± 16.97 mg L-1) [22]. On the
other hand, the EPS production from
Lactiplantibacillus plantarum BAL-29-ITTG
was also evaluated using modified MRS broth at
different conditions such as carbon source
(sucrose and lactose), concentration (10 and 30
g/L), nitrogen source and concentration (yeast
extract and ammonium sulphate, 5 and 15 g L-1),
temperatures (20 and 40 °C) and agitation (0 and
150 rpm) [23], an experimental design of
Plackett Burman was used, some
exopolysaccharides obtained exhibited higher
antioxidant and antibiofilm activity against E.
coli, S. aureus and P. aeruginosa with values
between 23 and 77%. Under optimal conditions
the production of Lactiplantibacillus plantarum
BAL-29-ITTG, was 619.66 ± 83.21 mg L-1. In
this work, the culture medium corresponding to
the optimal conditions was selected and used
[23], Lactiplantibacillus plantarum BAL-29-
ITTG was grown in a stirred tank fermenter and
the extracellular EPS was characterized by
viscometry to obtain its molecular weight, by
thermal analysis to determine its thermal
transition such as glass transition temperatures
(Tg) or melting point (Tm) and by FTIR-ATR and
NMR to obtain its main monosaccharides
composition.
2. Methodology
2.1 Biologic material and strain recollection
L. plantarum BAL-29-ITTG was obtained from
the Research Laboratory, Instituto Tecnológico
de Tuxtla Gutiérrez, Chiapas, México. The strain
was maintained in glycerol (30% v/v) at -18°C, it
was reactivated by two successive soup MRS (De
Man, Rogosa y Sharpe), using a (10 % v/v) of
inoculum. The vessels or tubes were incubated
for 12 h at 37 °C and stirred at 110 rpm using a
LumistellMR IRO 65 stirrer with controlled
temperature. The Applikon® Biotechnology
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model ez2-control bioreactor containing MRS
broth was then inoculated and the effect of
aeration (0 vvm and 1 vvm) and agitation (150
rpm and 250 rpm) was evaluated. After 24 h of
incubation. The cultures were centrifuged at
4500 rpm for 30 minutes at 4 °C, two volumes of
cold absolute ethanol were added to the obtained
free cells soup, these were shaken vigorously and
then incubated for 12 h at 4 °C. The precipitated
EPS were dissolved using ultrapure water and
dialyzed for two days using Spectra/Por 1,
MWCO 6-8 kD membranes, water was changed
each 8 h, EPS were freeze-dried at 0.860 bar a -
40 °C using a LABCONCO FreeZone4.5 freeze
dryer.
2.2 EPS characterization
Molecular weights were calculated by
viscometry using a manual glass Ubbelohde
viscometer, the temperature was maintained at 20
°C using an oil bath. EPS solutions were
prepared at five different concentrations, from 1
to 5 mg mL-1, using distilled water as solvent,
once the temperature was constant, the flow
times were recorded and the relative viscosity
(rel) was calculated using the equation 1,
specific viscosity (sp) was calculated using the
equation 2, reduced viscosity (red) was
calculated using the equation 3, inherent
viscosity (inh) was calculated using the equation
4 and, the Mark-Houwink Sakurada (equation 5)
was used to calculate the viscosity average
molecular weight (Mv)
𝜼𝒓𝒆𝒍 = 𝒕 𝒕𝒐
⁄ (1)
𝜼𝒔𝒑 = 𝜼𝒓𝒆𝒍 − 𝟏 (2)
𝜼𝒓𝒆𝒅 = 𝜼𝒔𝒑 𝑪
⁄ (3)
𝜼𝒊𝒏𝒉 =𝑳𝒏(𝜼𝒓𝒆𝒍) 𝑪
⁄ (4)
𝜼 = 𝑲 • 𝑴𝜶 (5)
where t is the time of flow of each sample, t0 is
the time of flow of the solvent, C is the
concentration (g mL-1), is the intrinsic viscosity
at zero concentration, and are empirically
determined constants.
Attenuated Total Reflectance-Fourier Transform
Infrared Spectroscopy (FTIR-ATR) was
recorded from 650 to 4000 cm-1 with 16 scans
using a Fourier Transform Perkin Elmer
(Spectrum 400, Walthem, MA, USA) in
Attenuated Total Reflection mode (ATR) using a
diamond/ZnSe crystal with single reflection.
Differential Scanning Calorimetry (DSC) was
carried out using a Modilated DSC, TA
Instruments model Q2000. Samples were cooled
to -30 °C, an isothermal was maintained for 5
min; afterward the temperature was modulated to
+/−1 °C enery 60 s; and then, a heat ramp of 10
°C min-l up to 180 °C under nitrogen atmosphere
was applied. Two cycles were recorded, the
results showthe second one. Glass transition
temperatures (Tg) were calculated by using the
Universal Analysis 2000 software from TA
Instruments. Decomposition temperatures (Td)
were measured by thermogravimetric analysis
(TGA) using a TA Instrument, Discovery model,
Mew Castle. DE, USA. A heat ramp of 20 °C
mil-1 was used from 20 up to 600 °C with a
nitrogen flow of 50 mL min-1.
Nuclear magnetic resonance (NMR) data were
obtained at 25 or 70 °C using a Bruker AVANCE
III HD NMR spectrometer operating at 400.13 or
100.62 MHz for 1H and 13C respectively. 20 mg
of each sample were dissolved using 0.5 mL of
deuterium oxide (D2O) 99% from Merk, Toluca
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México, after that, samples were lyophilized and
dissolved again using 0.5 mL of D2O as solvent.
The main monosaccharides of this EPS was
selected by comparison between experimental
data from NMR and the database from the
software online CASPER [24], for this, the main
chemical shifts obtained by 1H, 13C, COSY,
TOCSY and HSQC NMR spectroscopy were
loaded and, once the software CASPER
generated the most probable monosaccharides
moieties, the ones with the lower error and
agreement with the data from FTIR were
selected.
3. Results and discussion
3.1 Molecular weight
Five different concentrations of EPS were
prepared from 1 to 5 mg mL-1 using distillated
water as solvent, the times of flow were
measured at 20 °C using a Cannon-Ubbelohde
viscometer. Once the intrinsic viscosity was
determined by Huggins and Kramer equations
(Figure 1), the viscosity average molecular
weight was calculated using the Mark-Houwink
equation, constant from dextran, = 0.0443 mL
g-1 and α = 0.043, were used. In Table 1 the
times of flow and different viscosities are shown.
The intrinsic viscosity calculated was 12.19,
therefore the Mn calculated was 471.8 kDa, this
value was in the same order of magnitude as an
EPS isolated from Limosilactobacillus reuteri
C66 (370 kDa) [25]. The low intrinsic viscosity
and high molecular weight suggest a compact
structure in solution.
Figure 1. Plot of: (•) reduced viscosity (Huggins) and (◊) inherent viscosity (Kramer).
Table 1. Data to obtain the intrinsic viscosity of the EPS
Concentration
(g/mL)
Time of flow*
(s)
rel
sp
red
(mL/g)
inh
0.00000
121.0
-
-
-
-
0.00142
123.0
1.0165
0.0165
11.6401
11.5449
0.00213
124.0
1.0248
0.0248
11.6237
11.4820
0.00320
125.3
1.0358
0.0358
11.1915
10.9957
0.00400
126.3
1.0441
0.0441
11.0193
10.7833
0.00500
127.3
1.0523
0.0528
10.4683
10.2036
*Average of three measurements
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3.2 Composition
3.2.1 FTIR-ATR analysis
FTIR-ATR spectrum of EPS (Figure 2) showed
de characteristic absorption bands, the
corresponding to the OH was centered around
3266 cm-1, the stretching vibration of the
methylene groups (-CH2) was observed at 2925
cm-1, the vibration of carboxylic groups (C=O) at
1644 cm-1 characteristic of acetyl group N-linked
or stretching vibration of mannose [26] which
corresponds with the observed by NMR and
suggested by CARPER, a band with low
intensity at 1531 cm-1, suggest N-acetylated
residues [27], the N-H vibration above 3200 cm-
1 was not observed, however, a strong band at
1219 cm-1 was attributed to acetyl groups [28]
and, pyranose ring [29]. The ether groups (C-O-
C) were assigned at 1030 and 1049 cm-1. In the
anomeric region, the band at 879 cm-1 could be
due to -glycosidic linkage between monomers
[30], the band at 860 cm-1 could be attributed to
-D-glucopyranose, the corresponding to -D-
glucopyranose was not observed [31], the three
bands at 913, 879 and 778 cm-1 corresponding to
-D-glucose [28], the two bands at 913 and 778
cm-1 could be due to -polyglucosanes with (1:6-
linkages) [31]. The band at 811 cm-1 could be
due to manose [28]. These bands indicate that the
EPS contained mainly glycosides linkages in
its structure, this data also is agreement with data
obtained from NMR (Table 2).
Figure 2. FTIR-ATR spectrum of the EPS and amplification of the anomeric region.
3.2.2 Thermal characterization
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Thermo-gravimetric analysis showed four main
steps of weight loss (Figure 3), the first one at
temperature below 150 °C indicated moisture
[32], but at lower temperature, around 43 °C, loss
weight could be attributed to residual ethanol
from the extraction method [33]. A rapid
degradation started at 220 °C, this showed a
maximum around 265 °C, followed by another
with a maximum at 340 °C, after that, the
degradation continued gradually, the final
residue at 600 °C was 35.8%, it is known that
EPS can have ionizable functional groups [2]
such as phosphate or sulphate [4] then, this high
percentage can be due to inorganic material.
Similar values have been observed, for example,
in EPS from L. Plantarum CNPC003 [34]. On the
other hand, by modulated differential scanning
calorimetry, two glass transition temperatures
were observed (Figure 4) the first one, at low
temperature (~7-8 °C), could be due to
branching, whereas the second one, at high
temperature (~157 °C) was attributed to the mail
chain [33], these branched structure could
explain its high molecular weight calculated by
viscometry.
Figure 3. Thermo-Gravimetric Analysis and Differential Thermal Analysis (a) and, Differential Scanning Calorimetry of the
obtained EPS (b), lower value corresponding to the Tg of branched structure and higher one corresponding to Tg of the main
chain.
3.2.3 Nuclear Magnetic Resonance
To decrease the signals from hydroxyl groups,
sample was first dissolved using D2O to
exchange hydrogens atoms bonded to
heteroatoms and then. It was lyophilized, the
sample was dissolved again using the same
solvent and NMR spectra were recorded. For 1H
NMR the spectra were recorded at 25 or 70 °C,
the last one because many NMR spectra available
have been recorded at this temperature and, the
chemical shift from DHO moves to higher field
[35] allowing a better observation of the
anomeric region, reported chemical shifts are
relative to the signal of HDO which appeared at
4.71 ppm at 25 °C but, it appeared at 4.30 ppm at
70 °C, although, the values of chemical shifts
from EPS were similar at both temperatures.
Water suppression was applied to observe the
anomeric region between 4.4 and 5.6 ppm, the
hydrogens H2-H6 were observed between 3.2
and 4.4 ppm (Figure 4), similar patterns have
been observed for other EPS [17], finally weak
signals were observed between 0.8 and 2.4 ppm
which can be assigned to aliphatic moieties.
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Figure 4 1H NMR spectra with water suppression at a) 25 °C, b) 70 °C and c) overlapping spectra at 25 °C (dark line) and 70
°C (light line)
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13C NMR spectrum was recorded (Figure 5), in
the anomeric region of the 13C spectrum, between
98 and 106 ppm, eight signals were observed, this
result indicated that, this EPS is a
heteropolysaccharide with a complex structure.
For an EPS from Limosilactobacillus fermentum
D12, seven anomeric carbons were observed,
these were assigned to galactofuranose and
glucopyranose residues [36]. To the correct
assignation, 2D 13C-1H HSQC NMR spectrum
was recorded at 70 °C (Figure 6), in this
spectrum, eight correlations in the anomeric
region were also observed (Figure 6a).
Figure 5. 13C NMR spectrum of EPS.
Correlation spectroscopy H-H (COSY) was
recorded at 70 °C (Figure 7a). No correlation
was observed between the region of sugar rings
and the signals below 2.5 ppm, taking into
consideration the observed in the FTIR spectrum,
these last signals could be due to methyl from N-
acetyl groups. On the other hand, in the anomeric
region, only seven correlations from H1-H2
could be observed (Figure 7b), the hydrogen of
the anomeric carbon at 4.49 ppm showed the
highest intensity, therefore, the complete spin
system, H2-H3, H3-H4, H4-H5, H5-H6 and H5-
H6’ could be assigned by COSY. Chemical shifts
are shown in Table 2.
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Figure 6. 1H-13C HSQC spectroscopy a) anomeric region and b) saccharide ring region.
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Figure 7. Correlation spectroscopy 1H-1H (COSY) of the EPS and b) H1-H2 correlation observed.
To observe the total spin systems in the
saccharide rings, total correlation spectroscopy
(TOCSY) was carried out but, due to the higher
number of signals, only two spin systems, from
two sugar residues could be totally assigned and,
for one more, only five correlations could be
observed (Table 2), after this assignation, the 13C
chemical shifts were obtained by the HSQC
NMR spectrum and the data were compared with
the database from the online software CASPER
(http://www.casper.organ.su.se/casper/determin
e.php) For each experimental data set, different
saccharides were suggested but, only those with
similar 1H and 13C chemical shifts values and
lower error were selected.
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Table 2. Selection of monosaccharides based on comparison between the experimental and database NMR chemical shifts
1H(13C).
1
2
3
4
5
6
Residue*
Error
4.49 (103.4)
3.30 (73.6)
3.45 (70.3)
3.63 (75.5)
3.82 (67.2)
3.94, 4.18 (78.7)
Exp**
4.53 (103.7)
3.34 (74.0)
3.51 (76.7)
3.47 (70.6)
3.62 (75.8)
3.88, 4.20 (69.5)
→6)--D-Glc-(1→
0.33
4.54 (103.2)
3.39 (73.9)
3.66 (75.1)
3.64 (79.7)
3.65 (75.8)
3.82, 3.99 (61.1)
→4)--D-Glc-(1→
0.33
5.24 (101.0)
4.09 (78.7)
3.90 (71.0)
3.78 (73.4)
3.50 (76.3)
3.62 (75.4)
Exp**
5.24 (101.9)
4.02(71.7)
3.95(71.7)
3.85 (75.0)
3.75 (73.2)
3.80, 3.88 (62.0)
→4)--D-Man-(1→
0.33
5.10 (98.9)
4.04 (70.7)
3.86 (78.5)
3.64 (67.5)
3.76 (73.4)
-
Exp**
4.90 (100.6)
3.99 (70.8)
3.83 (71.7)
3.74 (67.6)
3.82 (72.0)
3.77, 3,85 (61.8)
→6)--D-Man-(1→
0.00
5.07 (102.9)
4.18 (70.6)
3.95 (79.0)
3.77 (67.1)
3.78 (74.3)
3.77, 3,85 (61.8)
→3)--D-Man-(1→
1.00
**Saccharide residue suggested by comparison between experimental data and database from the CASPER sofware, the
selected ones were chosen based on the lower error.
*The experimental assignation was based on 1H-13C (HSQC), 1H-1H (COSY) and 1H-1H (TOCSY) NMR
Figure 8. TOCSY from ESP: a) total correlation of anomeric hydrogen at 4.50 ppm and b) correlation H2 up to H4 of residue
with anomeric hydrogen at 5.25 ppm.
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For the hydrogen of the anomeric carbon at 4.49
ppm, the total correlation could be assigned
(Figure 8a), this corresponds to the observed by
COSY. According to the predicted chemical
shifts from CAPER, the experimental signals
could correspond to →6)--D-Glc-(1→ or →4)-
-D-Glc-(1→ . It is important to note that these
results are consistent with those obtained by
FTIR where stretching vibration from mannose
was observed as well as vibrations from pyranose
ring, -glycosidic linkages and -D-
glucopyranose.
For anomeric hydrogens at 5.24, H1-H2
correlation was determined by COSY, the
correlation of H2 with H3 up to H6 could be
assigned (Table 2), the anomeric carbon at 101.0
ppm and its hydrogen at 5.24, could be assigned
to →4)--D-Man-(1→ residue. This corresponds
again with what was observed in the FTIR-ATR
spectrum.
For the anomeric hydrogen at 5.10 ppm, only the
correlation of H2 up to H5 could be assigned
(Table 2). In this case, the structures suggested
using CASPER correspond to 1-3 and 1-6 linked
-D-Mannose. These main components are like
EPS produced by Streptococcus thermophilus
and Lactobacillus bulgaricus although with
different linkage [26].
For hydrogens of the other anomeric carbons,
because of the high number of signals, only some
correlations could be done, results are shown in
Table 3. The components were selected only by
comparison between C-H chemical shifts of the
anomeric carbon, the H1-H2 correlation from
sugar rings and the values obtained by CASPER.
In this case, NAc residues were suggested, these
results are according to the observed by COSY
and FTIR. For some experimental data, different
sugars were equally likely.
Table 3. Suggested monosaccharides residues by CASPER based on the chemical shifts of the anomeric carbon, C1-H1 and
H1-H2 correlation.
13C
HSQC
1 H
COSY
H1-H2
Residue*
103.5
4.50
4.53
4.71
4.50-3.31
4.53-3.50
4.71-3.35
→6)--Glu-(1→
→4)--Gal-(1→
→6)--Gal-(1→
→6)--Glu
102.5
5.04
5.13
5.04-4.04
5.13-NObs
→6) α-D-Mani (1→
101.0
5.25
5.25-4.09
→4)-α-D-Man-(1→
→4)-α-D-GlcNAc-(1→
100.0
4.89
4.88-3.98
→6)--D-Man-(1→
→6)-α-D-GlcNAc-(1→
→6)-α-D-Gal-(1→
→4)-α-D-Gali-(1→
98.98
5.04
5.08
5.04-4.04
5.08-4.01
→6)-α-D-GlcNAc-(1→
→3)-α-D-Gal (1→
98.95
5.04
5.08
5.04-4.04
5.08-4.01
6)-α-D-GlcNAc-(1→
→3)-α-D-Gal (1→
98.92
5.04
5.08
5.04-4.04
5.08-4.01
6)-α-D-GlcNAc-(1→
→3)-α-D-Gal (1→
98.88
5.04
5.04-4.20
6)-α-D-GalNAc-(1→
4)-α-D-GalNAc-(1→
*Residue was selected by minor error between the experimental chemical schiffs and the calculated ones using CASPER,
anomeric carbon and H1 and H2 were the main criteria.
13 ISSN: 2594-1925
Revista de Ciencias Tecnológicas (RECIT). Volumen 8 (3): e405.
4. Conclusions
Because the EPS produced by Lactiplantibacillus
plantarum BAL-29-ITTG under the optimal
reported culture conditions have shown
antioxidant and antibiofilm activity against E.
coli, S. Aureus and P. aeruginosa, the need for its
chemical and thermal characterization arises;
although a compete characterization is very
difficult due to its complex structure, according
to the data obtained in this work, the studied EPS
is a branched heteropolysaccharides with a
higher molecular weight and, compacted in
aqueous solutions; It had a degradation
temperature like others produced EPS but, it had
a higher content of inorganic material such as
mineral salts. According to the comparative
between database from the software CASPER
and the experimental NMR and FTIR data, this
EPS from BAL-29-ITTG, is essentially
composed of glucopyranose 1-4 and 1-6 linked,
mannopyranose, 1-3, 1-4 y 1-6 linked and some
N-acetylated moieties. The sugars that make up
these EPS are linked mainly by β and α-
glycosidic bonds. These same sugars linked by
these bonds coincide with those found in the EPS
produced by other strains of Lactiplantibacillus
plantarum isolated from other sources. All the
above indicates that BAL-29-ITTG produces
extracellular EPS with promising
physicochemical and biotechnological
characteristics.
5. Acknowledgment
Authors thank Tecnológico Nacional de México
for funding project 16426.23-P; Iván de Jesús
Zapata González and José Román Torres Lubián
by their support with NMR spectra acquisition
and, Rony Obed Suchiapa Díaz thanks Conahcyt
for the scholarship.
6. Authorship acknowledgment
Rony Obed Suchiapa Diaz: Methodology, formal
analysis, research, Lucia María Cristina Ventura
Carrasco: Validation, resources, writing:
proofreading, supervision, project administration
and acquisition of funds; Alejandro Ramírez-
Jiménez: Validation, formal analysis, research,
writing-original draft, supervision.
References
[1] S. L. Flitsch, “Enzymatic Carbohydrate
Synthesis”, en Comprehensive Chirality,
Elsevier, 2012, pp. 454-464. doi: 10.1016/B978-
0-08-095167-6.00727-8.
[2] A. I. Netrusov, E. V. Liyaskina, I. V.
Kurgaeva, A. U. Liyaskina, G. Yang, y V. V.
Revin, “Exopolysaccharides Producing Bacteria:
A Review”, Microorganisms, vol. 11, n.o 6, p.
1541, jun. 2023, doi:
10.3390/microorganisms11061541.
[3] S. A. M. Moghannem, M. M. S. Farag, A. M.
Shehab, y M. S. Azab, “Exopolysaccharide
production from Bacillus velezensis KY471306
using statistical experimental design”, Brazilian
Journal of Microbiology, vol. 49, n.o 3, pp. 452-
462, jul. 2018, doi: 10.1016/j.bjm.2017.05.012.
[4] S. R. Dave, K. H. Upadhyay, A. M. Vaishnav,
y D. R. Tipre, “Exopolysaccharides from marine
bacteria: production, recovery and applications”,
Environmental Sustainability, vol. 3, n.o 2, pp.
139-154, jun. 2020, doi: 10.1007/s42398-020-
00101-5.
[5] L. A. Silva, J. H. P. Lopes Neto, y H. R.
Cardarelli, “Exopolysaccharides produced by
Lactobacillus plantarum: technological
properties, biological activity, and potential
application in the food industry”, Ann Microbiol,
vol. 69, n.o 4, pp. 321-328, abr. 2019, doi:
10.1007/s13213-019-01456-9.
[6] E. Korcz y L. Varga, “Exopolysaccharides
from lactic acid bacteria: Techno-functional
application in the food industry”, Trends in Food
14 ISSN: 2594-1925
Revista de Ciencias Tecnológicas (RECIT). Volumen 8 (3): e405.
Science & Technology, vol. 110, pp. 375-384,
abr. 2021, doi: 10.1016/j.tifs.2021.02.014.
[7] A. T. Adesulu-Dahunsi, A. I. Sanni, K.
Jeyaram, J. O. Ojediran, A. O. Ogunsakin, y K.
Banwo, “Extracellular polysaccharide from
Weissella confusa OF126: Production,
optimization, and characterization”,
International Journal of Biological
Macromolecules, vol. 111, pp. 514-525, may
2018, doi: 10.1016/j.ijbiomac.2018.01.060.
[8] Nguyen, Phu-Tho, T.-T. Nguyen, D.-C. Bui,
P.-T. Hong, Q.-K. Hoang, y H.-T. Nguyen,
“Exopolysaccharide production by lactic acid
bacteria: the manipulation of environmental
stresses for industrial applications”, AIMS
Microbiology, vol. 6, n.o 4, pp. 451-469, 2020,
doi: 10.3934/microbiol.2020027.
[9] A. K. Abdalla et al., “Exopolysaccharides as
Antimicrobial Agents: Mechanism and Spectrum
of Activity”, Front. Microbiol., vol. 12, p.
664395, may 2021, doi:
10.3389/fmicb.2021.664395.
[10] T. Lin, C. Chen, B. Chen, J. Shaw, y Y.
Chen, “Optimal economic productivity of
exopolysaccharides from lactic acid bacteria with
production possibility curves”, Food Science &
Nutrition, vol. 7, n.o 7, pp. 2336-2344, jul. 2019,
doi: 10.1002/fsn3.1079.
[11] Z. Liu et al., “Characterization and
bioactivities of the exopolysaccharide from a
probiotic strain of Lactobacillus plantarum
WLPL04”, Journal of Dairy Science, vol. 100,
n.o 9, pp. 6895-6905, sep. 2017, doi:
10.3168/jds.2016-11944.
[12] R. D. Ayivi et al., “Lactic Acid Bacteria:
Food Safety and Human Health Applications”,
Dairy, vol. 1, n.o 3, pp. 202-232, oct. 2020, doi:
10.3390/dairy1030015.
[13] L. Yu et al., “Purification, characterization
and probiotic proliferation effect of
exopolysaccharides produced by
Lactiplantibacillus plantarum HDC-01 isolated
from sauerkraut”, Front. Microbiol., vol. 14, p.
1210302, jun. 2023, doi:
10.3389/fmicb.2023.1210302.
[14] J. Wang et al., “Optimization of
Exopolysaccharide Produced by Lactobacillus
plantarum R301 and Its Antioxidant and Anti-
Inflammatory Activities”, Foods, vol. 12, n.o 13,
p. 2481, jun. 2023, doi: 10.3390/foods12132481.
[15] J. Xiong, D. Liu, y Y. Huang,
“Exopolysaccharides from Lactiplantibacillus
plantarum: isolation, purification, structure–
function relationship, and application”, Eur Food
Res Technol, vol. 249, n.o 6, pp. 1431-1448, jun.
2023, doi: 10.1007/s00217-023-04237-6.
[16] D. Meghwal, K. K. Meena, R. Singhal, L.
Gupta, y N. L. Panwar, “Exopolysaccharides
producing lactic acid bacteria from goat milk:
Probiotic potential, challenges, and opportunities
for the food industry”, ap, vol. 12, n.o 2, dic.
2023, doi: 10.54085/ap.2023.12.2.22.
[17] M. Ayyash et al., “Exopolysaccharide
produced by the potential probiotic Lactococcus
garvieae C47: Structural characteristics,
rheological properties, bioactivities and impact
on fermented camel milk”, Food Chemistry, vol.
333, p. 127418, dic. 2020, doi:
10.1016/j.foodchem.2020.127418.
[18] Q. Xu, M.-M. Wang, X. Li, Y.-R. Ding, X.-
Y. Wei, y T. Zhou, “Antioxidant and anti-
inflammatory activities and action mechanisms
of exopolysaccharides from Lactiplantibacillus
plantarum Z-1”, Food Bioscience, vol. 62, p.
105247, dic. 2024, doi:
10.1016/j.fbio.2024.105247.
[19] T. Bouzaiene et al., “Exopolysaccharides
from Lactiplantibacillus plantarum C7 Exhibited
Antibacterial, Antioxidant, Anti-Enzymatic, and
Prebiotic Activities”, Fermentation, vol. 10, n.o
7, p. 339, jun. 2024, doi:
10.3390/fermentation10070339.
[20] M. Kowsalya et al., “Extraction and
characterization of exopolysaccharides from
Lactiplantibacillus plantarum strain PRK7 and
PRK 11, and evaluation of their antioxidant,
emulsion, and antibiofilm activities”,
International Journal of Biological
Macromolecules, vol. 242, p. 124842, jul. 2023,
doi: 10.1016/j.ijbiomac.2023.124842.
15 ISSN: 2594-1925
Revista de Ciencias Tecnológicas (RECIT). Volumen 8 (3): e405.
[21] Y. Jiang y Z. Yang, “A functional and
genetic overview of exopolysaccharides
produced by Lactobacillus plantarum”, Journal
of Functional Foods, vol. 47, pp. 229-240, ago.
2018, doi: 10.1016/j.jff.2018.05.060.
[22] J. I. Ramírez-Pérez et al., “Effect of linear
and branched fructans on growth and probiotic
characteristics of seven Lactobacillus spp.
isolated from an autochthonous beverage from
Chiapas, Mexico”, Arch Microbiol, vol. 204, n.o
7, p. 364, jul. 2022, doi: 10.1007/s00203-022-
02984-w.
[23] L. M. C. Ventura Canseco, R. O. Suchiapa
Díaz, M. C. Luján Hidalgo, y M. Abud Archila,
“Producción y bioactividades de los
exopolisacáridos de Lactiplantibacillus
plantarum BAL-29-ITTG utilizando un diseño
experimental Plackett-Burman”, Rev.
Mesoamericana de Investigación, vol. 3, n.o 3,
2023, doi: 10.31644/RMI.V3N3.2023.A03.
[24] K. M. Dorst y G. Widmalm, “NMR
chemical shift prediction and structural
elucidation of linker-containing oligo- and
polysaccharides using the computer program
CASPER”, Carbohydrate Research, vol. 533, p.
108937, nov. 2023, doi:
10.1016/j.carres.2023.108937.
[25] A. K. M. H. Kober et al.,
“Exopolysaccharides from camel milk-derived
Limosilactobacillus reuteri C66: Structural
characterization, bioactive and rheological
properties for food applications”, Food
Chemistry: X, vol. 25, p. 102164, ene. 2025, doi:
10.1016/j.fochx.2025.102164.
[26] A. A. Al-Nabulsi et al., “Characterization
and bioactive properties of exopolysaccharides
produced by Streptococcus thermophilus and
Lactobacillus bulgaricus isolated from labaneh”,
LWT, vol. 167, p. 113817, sep. 2022, doi:
10.1016/j.lwt.2022.113817.
[27] T. Hong, J.-Y. Yin, S.-P. Nie, y M.-Y. Xie,
“Applications of infrared spectroscopy in
polysaccharide structural analysis: Progress,
challenge and perspective”, Food Chemistry: X,
vol. 12, p. 100168, dic. 2021, doi:
10.1016/j.fochx.2021.100168.
[28] M. Mathlouthi y J. L. Koenig, “Vibrational
Spectra of Carbohydrates”, en Advances in
Carbohydrate Chemistry and Biochemistry, vol.
44, Elsevier, 1987, pp. 7-89. doi: 10.1016/S0065-
2318(08)60077-3.
[29] X. Wang, C. Shao, L. Liu, X. Guo, Y. Xu, y
X. Lü, “Optimization, partial characterization
and antioxidant activity of an exopolysaccharide
from Lactobacillus plantarum KX041”,
International Journal of Biological
Macromolecules, vol. 103, pp. 1173-1184, oct.
2017, doi: 10.1016/j.ijbiomac.2017.05.118.
[30] O. Braissant et al., “Characteristics and
turnover of exopolymeric substances in a
hypersaline microbial mat: EPS turnover in a
hypersaline microbial mat”, FEMS Microbiology
Ecology, vol. 67, n.o 2, pp. 293-307, feb. 2009,
doi: 10.1111/j.1574-6941.2008.00614.x.
[31] S. A. Barker, E. J. Bourne, M. Stacey, y D.
H. Whiffen, “Infra-red spectra of carbohydrates.
Part I. Some derivatives of D-glucopyranose”, J.
Chem. Soc., p. 171, 1954, doi:
10.1039/jr9540000171.
[32] J. Wang, X. Zhao, Y. Yang, A. Zhao, y Z.
Yang, “Characterization and bioactivities of an
exopolysaccharide produced by Lactobacillus
plantarum YW32”, International Journal of
Biological Macromolecules, vol. 74, pp. 119-
126, mar. 2015, doi:
10.1016/j.ijbiomac.2014.12.006.
[33] F. Zamora, M. C. González, M. T. Dueñas,
A. Irastorza, S. Velasco, y I. Ibarburu,
“Thermodegradation and thermal transitions of
an exopolysaccharide produced by Pediococcus
damnosus 2.6”, Journal of Macromolecular
Science, Part B, vol. 41, n.o 3, pp. 473-486, jun.
2002, doi: 10.1081/MB-120004348.
[34] V. B. Bomfim et al., “Partial
characterization and antioxidant activity of
exopolysaccharides produced by Lactobacillus
plantarum CNPC003”, LWT, vol. 127, p. 109349,
jun. 2020, doi: 10.1016/j.lwt.2020.109349.
[35] H. E. Gottlieb, V. Kotlyar, y A. Nudelman,
“NMR Chemical Shifts of Common Laboratory
Solvents as Trace Impurities”, J. Org. Chem.,
16 ISSN: 2594-1925
Revista de Ciencias Tecnológicas (RECIT). Volumen 8 (3): e405.
vol. 62, n.o 21, pp. 7512-7515, oct. 1997, doi:
10.1021/jo971176v.
[36] N. Čuljak et al., “Limosilactobacillus
fermentum strains MC1 and D12: Functional
properties and exopolysaccharides
characterization”, International Journal of
Biological Macromolecules, vol. 273, p. 133215,
jul. 2024, doi: 10.1016/j.ijbiomac.2024.133215.
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