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Biofilm formation by agave epiphytic lactic acid bacteria fed with agave
fructans

Article in World Journal of Microbiology and Biotechnology · September 2023

DOI: 10.1007/s11274-023-03749-3

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World Journal of Microbiology and Biotechnology (2023) 39:299
https://doi.org/10.1007/s11274-023-03749-3

RESEARCH

Biofilm formation by agave epiphytic lactic acid bacteria fed with

agave fructans
Nayeli Martha-Lucero1 · Gustavo Viniegra-González1 · Luis González-Olivares2 · Alma Cruz-Guerrero1

Received: 29 June 2023 / Accepted: 31 August 2023

© The Author(s), under exclusive licence to Springer Nature B.V. 2023

Abstract
The aim of this work was to find out if biofilms can be made by lactic acid bacteria (LAB) isolated from agave plants
using agave fructans as sole carbohydrate substrates or if it was necessary to use fructose as a breakdown product of
such polymers. This is part of a research project geared to develop industrial lactic acid production from agave fructans,
an abundant raw material in Mexico’s agave plantations. Present results showed that nine strains of LAB isolated from
Agave salmiana and belonging to genus Lacticaseibacillus and Enterococcus produced exopolysaccharides directly from
agave fructans to a greater extent than with fructose. The best polysaccharide productions in planktonic cultures were
Lacticaseibacillus paracasei strains DG2, DG3, DG4 and DG8. Furthermore, all nine LAB strains produced biofilms
on polystyrene microplates, much better with agave fructans than with fructose. In most strains, biofilm formation was
favored at pH from 6.0 to 6.5, except for strains DG7 and DG9 where pH 5.5 was optimal. Biofilm formation required
between 3 and 5 days of incubation in all Lacticaseibacillus paracasei strains, whereas Enterococcus faecium required a
little less of 3 days. Present results support the straight use of agave fructans to develop LAB biofilms using agave epi-
phytic bacteria. This finding simplifies upstream processing of agave fructans to be used for future lactic acid fermentation
in LAB biofilm reactors.

Keywords Agave fructans · Biofilm · Enterococcus · Lactic acid bacteria · Lacticaseibacillus

Introduction residuals with 16% of agave fructans. Such biopolymers are

This work is part of a research project looking for an alter-
native and cost-effective substrate for industrial lactic acid
production to be used as a building block of bioplastics, such
as polylactic acid, since Mexico is a net importer of grains
and has no surplus of sugar cane. Agave fructans seem to
be a suitable alternative for such a purpose because tequila
industry consumes 2.6 million tons of Agave tequilana
heads (Consejo Regulador del Tequila 2023) comprising
only 54% of total agave biomass (Íñiguez-Covarrubias
et al. 2001) leaving, unused, 2.2 million tons of biomass
Alma Cruz-Guerrero
aec@xanum.uam.mx
1
Departamento de Biotecnología, Universidad Autónoma
Metropolitana, Iztapalapa, San Rafael Atlixco 186, Ciudad de
México, México
2
Universidad Autonoma del Estado de Hidalgo, Área
académica de química, Mineral de la Reforma, Hgo., México
known to be a good substrate for lactic acid fermentation
(Martha-Lucero 2018; Pineda-Tapia et al. 2022). Therefore,
agave fructans homolactic fermentation in biofilm reactors
appears to be a viable alternative for future development of
bioplastic industry in Mexico.
Agave fructans are branched polysaccharides made of
fructose linked by β (2 − 1) and β (2–6) glycosidic bonds
and external and internal glucose units and having a degree
of polymerization lower than thirty. They have high solu-
bility in water and low thermal stability as compared to
inulin (García-Villalba et al. 2022; Huezcas-Garrido et al.
2022; Crispín-Isidro et al. 2015; Espinosa-Andrews and
Rodríguez-Rodríguez 2018) and can be fermented by Lac-
tobacillus casei (Martha-Lucero 2018) and Lactobacillus
acidophilus (Ramírez-Pérez et al. 2022). Therefore, it is
important to find new lactic acid platforms that meet indus-
trial needs, such as, biofilm reactors (Germec et al. 2020;
Precedence Research 2022) where spent biomass is reused
and easily separated from lactic acid.

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LAB biofilms are made of exopolysaccharides (EPS) that
allow the bacteria to adhere to different surfaces and play an
essential role by acting as a superglue that keeps them stable
and attached to each other. Additionally, EPS have the func-
tion of protecting cell integrity in different environments
(Zayed et al. 2022). For example, Leuconostoc spp. biofilms
made from sucrose found in sugar industry (Castilla-Mar-
roquín et al. 2020; De la Cruz-Noriega et al. 2022). It has
been found that Lactobacillus crispate excretes enzymes
related to biofilm formation (Li et al. 2020) and it is worth
noticing that LAB biofilms reactors have been found with
some advantages over planktonic cultures, such as, reduced
nutritional requirements (Akoğlu 2020; Ho et al. 1997a,
b), resistance to osmotic stress (Ercan and Demirci 2015),
continuous separation of the product (Edel et al. 2019) with
concomitant lower product inhibition (Wang et al. 2018),
higher productivity (Bastarrachea et al. 2022), high local
cell density (Ho et al. 1997b, c) and improved cell viabil-
ity with longer fermentation times (Gao et al. 2022). Such
advantages of LAB biofilms have been studied by Demirci
and Pometto (1995) in a repeated-batch fermentation of
Lactobacillus casei subsp. rhamnosus ATCC 11,443.
If agave extracts were to be used as raw material for LAB
biofilm fermentations it would be good to know if it is nec-
essary to breakdown agave fructans into fructose or if agave
fructans, by themselves, are good substrates for biofilm for-
mation. Thus, in this work biofilm formation was examined
with such alternative substrates by measuring the produc-
tion of bacterial polysaccharides and assessing biofilm for-
mation on polystyrene microplates.
Materials and methods
Glucose, fructose, and dextran were purchased from Sigma-
Aldrich (St. Louis, MO., USA). Agave fructans, free from
monosaccharides, were donated by Dr. Rosa Camacho
from Center for Research and Assistance in Technology
and Design of the State of Jalisco, A.C. (CIATEJ, Guadala-
jara) and came from Nutriagaves Group, S.A. de C.V. Yeast
extract, peptone, and meat extract were purchased from BD
Bioxon (Mexico State, Mexico). Sodium acetate, sorbitan
monooleate, ammonium citrate, magnesium sulfate, man-
ganese sulfate, potassium phosphate, sodium phosphate,
sodium chloride, and potassium chloride were purchased
from J.T. Baker TM (Phillipsburg, N.J., USA). Crystal vio-
let was purchased from Merck (Darmstadt, Germany).
Microorganisms
Nine LAB homolactic strains were selected for this study.
They were isolated from samples found in Agave salmiana
and identified by Gallardo (2023) with their correspond-
ing accession numbers (Table 1). They were screened to
be homolactic, fructan and glucose, fermenters. All strains
were stored at -20 °C in MRS: glycerol media.
Culture media
The MRS medium was used as a base, with the carbon
source (20 g/L) to be, either fructose or agave fructans. The
nitrogen source was kept constant, using a mixture of yeast
extract (5 g/L), meat extract (5 g/L) and peptone (10 g/L).
Also, it contained the following salts: sodium acetate
(5 g/L), sorbitan monooleate (1 mL/L), ammonium citrate
(2 g/L), magnesium sulfate (0.2 g/L), manganese (0.05 g/L).
The C: N ratio was five.

Fermentation conditions
Table 1 Description of genus and species of LAB used in the experi-
ments, as well as the place where they were isolated The LAB were preserved in MRS: glycerol (1:1) frozen
Code Bacteria Accession Isolation media. All bacterial strains were reactivated in glucose
number MRS broth by incubating for 24 h at 37 °C. Subsequently,
DG1 Lacticaseibacillus paracasei OM967267 Agave sap one milliliter was added to 9 mL of culture medium with
DG2 Lacticaseibacillus paracasei OM802845 Agave sap fructose or agave fructans, and incubated for 24 h at 37 °C.
DG3 Lacticaseibacillus paracasei OM967268 Agave sap
For each fermentation run, an inoculum of 8 mg/mL was
DG4 Lacticaseibacillus paracasei OM967269 Agave sap
previously activated and added up to volume of 10 mL.
DG5 Lacticaseibacillus paracasei OM802846 Agave sap
Incubation was for 72 h at 37 °C and growth was measured
DG6 Lacticaseibacillus paracasei OM967270 Agave sap
DG7 Lacticaseibacillus paracasei OM967271 Agave sap by turbidity in a spectrophotometer (Shimadzu UV-1800-
DG8 Lacticaseibacillus paracasei OM967272 Agave 120 V, Japan) at 600 nm. The OD is related to the equation
leaves y = 0.2424x + 0.0193, where, y: absorbance and x: biomass.
DG9 Enterococcus faecium OM80284 Agave
metzala
a
Metzal is defined as material scraped from the agave head (Peralta-
García et al. 2020)

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Carbohydrates determination Table 2 Yield (Yx/s) at the end of the fermentation for agave fructans
(FOS) and fructose (F). Statistical analysis was done by carbon source
(column)
Total sugar concentrations were measured according to LAB Yx/s (g biomass/ Yx/s (g biomass/ Yield
Dubois et al. (1956) following some modifications sug- g substrate) Fruc- g substrate) Fruc- ratio
gested by Martínez et al. (2000). Previously, a phenol- tans (FOS) tose (F) (FOS/F)
sulfuric solution was prepared, dissolving 10 mg of phenol DG1 0.769 ± 0.055 b 0.756 ± 0.009 b 0.98
together with 10 mL of concentrated sulfuric acid. Samples DG2 0.971 ± 0.008 a 0.676 ± 0.006 c 0.70
made of 1 mL of fermentation supernatant were blended DG3 0.787 ± 0.012 b 0.641 ± 0.005 d 0.81
with 2 mL of phenol-sulfuric acid and let to rest for 15 min DG4 0.570 ± 0.007 c 0.783 ± 0.004 b 1.37
on freezing water. Later, test tubes were placed in a water DG5 0.633 ± 0.012 c 0.916 ± 0.016 a 1.45
bath at 92 °C for 8 min. Finally, they were cooled in a water DG6 0.268 ± 0.005 e 0.331 ± 0.013 e 1.24
DG7 0.351 ± 0.007 d 0.347 ± 0.004 e 0.99
bath at 25 °C for 30 min and the absorbances were measured
DG8 0.829 ± 0.005 b 0.670 ± 0.007 cd 0.81
at 480 nm. A standard glucose curve (0- 0.1 mg/mL) was
DG9 0.271 ± 0.001 e 0.267 ± 0.002 f 0.99
used as a reference.
A positive linear correlation between carbon sources were
FOS = 0.6435 F + 0.136; R 2 = 0.867. Outliers: DG4 and DG5.
Yield parameters (Yx/s)

Fermentation runs were analyzed by triplicate. Mass bal-
ance analysis was used to estimate biomass yield, Yx/s, as
shown in Table 2.
EPS determination
EPS determination was performed according to the method
proposed by Carrero-Puentes et al. (2021). Briefly, fermen-
tation broth was centrifuged at 2,800 g for 5 min. Then 3 mL
of the supernatant were mixed with 0.5 mL of 80% trichlo-
roacetic acid (TCA), vortexed for 10 s and centrifuged at
11,200 g for 30 min at 4 °C. Afterwards, 1 mL of the super-
natant was recovered, and 1 mL of 96% ethanol was added.
Such mixture was let to rest for 20 min at 4 °C. After the
resting time, absorbance was measured at 720 nm and the
EPS concentration was assayed with the help of a standard
dextran curve (0–1 mg/mL).
EPS balance analysis was used to estimate product yield,
Yp/s, as shown in Fig. 1.
Fructosidase activity assay
Fructosidase activity was determined in the culture super-
natant by monitoring the increase in reducing sugars in an
agave fructans solution as reported by Cruz-Guerrero et al.
(2006). One unit of fructosidase (UF) was defined as the
amount of enzyme that liberates 1 µmol of reducing sugars
per minute at 37 °C and pH 7.0.
Auto-aggregation
Auto-aggregation was followed according to Maldonado et
al. (2012) using biomass recovered from the 24 h fermenta-
tion runs. The precipitate was washed and suspended with
PBS (phosphate buffer solution 1 M, pH 7.4) and incubated
for 2 h at 37 °C, without shaking. Finally, the absorbance
at 600 nm was measured. Auto-aggregation was estimated
according to the following equation:
Auto − aggregation (%) = |(A0 − A) / A0| × 100

Fig. 1 Correlation analysis

between biomass yield (Yx/s)
with respect to EPS yield (Yp/x)
considering the two carbon
sources, agave fructans (FOS)
and fructose (F). The points (●)
show the behavior close to the
line of the DG1 to DG7 strains;
in addition, there are two atypi-
cal ones that are DG9 (○) and
DG8 is not because is an outlier
(1.24,45). Linear equation: FOS
= -1.0513 F + 2.1181; R² =
0.7254

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 299 Page 4 of 10
Fig. 2 Biomass concentration at the end of the fermentation at different
carbon sources. Black bars represent fructose and white bars represent
fructans. Capital letters with open bars show the significant difference
between strains in agave fructans and lower-case letters with closed
bars represent fructose (p < 0.01)
where, A0 and A are the absorbances before and after
incubation, respectively. Strains were classed with, high
(71–100%), medium (36–70%), and low (0–35%) auto-
aggregation features.
Biofilm formation
Biofilms formation was evaluated in polystyrene micro-
plates, as follows, 10 mL samples of culture medium were
inoculated with 0.5 mL of BAL having 8 mg/mL of bio-
mass. From such mixture, a 200 µL sample was added to
each well. Subsequently, they were fermented for 72 h at
37 °C.
Different conditions for the biofilm formation were eval-
uated with (20 g/L) fructose or agave fructans as carbon
sources. The effect of pH was assessed using initial value,
5.5, 6, and 6.5. Fermentation times were 3 and 5 days.
Biofilm quantification
Biofilm quantification in polystyrene microplates was per-
formed according to Lebeer et al. (2007). Biofilms were
washed thrice with PBS. Later, 200 µL of 0.1% crystal
violet solution were added and let to stand for 30 min. The
excess dye was washed out with 200 µL of water. Biofilms
were air-dried for 30 min, the crystal violet attached to the
adhered LAB was extracted with 200 µL of ethanol-ace-
tone (80:20) and its optical density (OD) was measured at
565 nm. The culture medium without inoculum was used as
a control.
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World Journal of Microbiology and Biotechnology
Fig. 3 EPS production at the end of the fermentation for different car-
bon sources. Black bars represent fructose and white bars represent
fructans. Capital letters show the significant difference between strains
in agave fructans, and lower-case letters represent fructose (p < 0.01)
Statistical analyses
Statistical analyses were performed with Statgraphics Plus
5.1 (New Jersey, USA). Analysis of variance (α = 0.01) and
Tukey tests were performed to find significant differences
between the experiments. The data were expressed as means
of three replicates and standard error of each mean value.
Results
Planktonic growth
Figure 2 shows that all LAB isolates listed in Table 1, were
able to grow as planktonic cultures on fructose or agave
fructans. Table 2 shows the average biomass yield values
(Yx/s) in fructose and agave fructans. Statistical errors were
lower than 8% of mean values therefore there were not a
statistical differences on this parameter showing all strains
grew well in both substrates.
EPS production
Figure 3 shows EPS production per gram of biomass (Yp/x)
of planktonic cultures inoculated with LAB, either with
agave fructans Yp/x (FOS) or fructose Yp/x (F) as carbon
source. The ratio of those coefficients, Yp/x (FOS)/ Yp/x (F),
indicated the relative efficiency to produce EPS comparing
fructans to fructose in planktonic cultures. When such ratio
was plotted vs. the ratio of biomass yield, Yx/s (FOS)/ Yx/s
(F), a linear decreasing correlation was found, except for
two outliers Lacticaseibacillus paracasei OM967272 and
Enterococcus faecium OM80284 (DG8 and DG9) as shown
World Journal of Microbiology and Biotechnology Page 5 of 10 299

in Fig. 1. Statistical errors, similar to yield estimates, were

lower than 8% of mean values. This supports a decreasing
propensity to produce EPS in agave fructans when cell bio-
synthesis is more efficient. Namely, EPS synthesis could be
an adaptative response to metabolic stress when fructan uti-
lization is difficult. We do not have clues on why there are
such outliers, but they could be the subject of further inquiry
on the regulatory networks of fructan hydrolysis and poly-
saccharide synthesis.

Fructosidase activity

In the medium containing agave fructans as carbon source

(Fig. 4) the production of fructosidase by strains L. para-
casei OM 967,268 (DG3) and L. paracasei OM 967,272
(DG8) were higher, while the strain E. faecium OM80284
(DG9) showed a smaller growth and as a consequence pro-
duced less enzyme. Moreover, in the culture media with
fructose as carbon there was no fructosidase production for
any strain.
Auto-aggregation
As can be seen in Fig. 5, all the LAB strains have a high
self-aggregation capacity above 70%, that is, they have
the capacity for cell-cell adhesion between bacteria of the
same strain. Auto-aggregation is often among the first steps
in forming biofilms and all present strains seem have quite
similar auto-aggregation properties.
Effect of carbon sources on biofilm formation
Figure 6a and b shows, the formation of biofilms over
a polystyrene surface, using agave fructans or fructose.
Here there is a quite remarkable difference. All the wells
with fructose showed little biofilm formation, whereas with
agave fructans, biofilm was quite evident. Such differences
Fig. 4 Fructosidase production by 24 h cultures of LAB strains with
agave fructans as a carbon source
show the importance and advantage of using agave fruc-
tans as carbon source instead of fructose to develop LAB
biofilms on plastic surfaces. Best strains with agave fruc-
tans were L. paracasei: OM802845 (DG2), OM 967,268
(DG3), OM967269 (DG4), and OM967272 (DG8), without
showing a significant difference (p < 0.01), followed by L.
paracasei OM802846 (DG5), which did not show a sig-
nificant difference (p < 0.01) with respect to DG3 and later
followed by L. paracasei OM967270 (DG6), L. paracasei
OM967271 (DG7) and E. faecium OM80284 (DG9) where
there is no significant difference between them (p < 0.01).
Only strain DG8 produced a significant amount of biofilm
on polystyrene using fructose. It is worth recalling that
such strain is an outlier in the linear correlation between
Yp/x and Yx/s and produced little EPS in planktonic culture
supporting important regulatory differences of this strain
with respect to the rest of them (Fig. 1). This experiment
is conclusive evidence that agave fructans, are quite good
substrates for biofilm formation of LAB homolactic strains

Fig. 5 Auto-aggregation of LAB

isolated from agave. The capacity
of the LAB to form aggregates
is considered: Low: 0 to 35%.
Medium: from 36 to 70%. High:
from 71 to 100%. Lowercase
letters show significant difference
between strains (p < 0.01)

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Fig. 6 Effect of different carbon

sources on biofilm formation on
polystyrene microplates. In 6 A,
which shows the OD at 575 nm,
the ability to form biofilms can be
found: low: 0 to 0.499; medium:
0.5 to 1.499; high: >1.5. In 6B,
biofilms form on polystyrene
stained with crystal violet. Black
bars represent fructose and white
bars represent fructans. In each
well, there is a fermentation; in
the horizontal lines are the strains
from DG1 to DG9, and in the
last line in the three wells, there
is a negative control where the
culture media without bacteria
was placed

Table 3 Effect of different initial pH values on biofilm formation using biofilm formation was favored at pH 6 and 6.5, except
agave fructans as a carbon source. A statistical analysis of the interac- for strain L. paracasei OM967271 (DG7) and E. faecium
tion between strains and pH values was performed
OM80284 (DG9). it was pH 5.5. Particularly, the strains
LAB pH 5.5 (OD) pH 6 (OD) pH 6.5 (OD)
d c of L. paracasei: OM967267 (DG1), OM967268 (DG3),
DG1 2.677 ± 0.18 3.062 ± 0.12 1.879 ± 0.09 e
DG2 4.004 ± 0.13 b
4.344 ± 0.27 a
4.592 ± 0.04 a
OM802846 (DG5), OM967270 (DG6) and OM967272
DG3 4.188 ± 0.32 b
4.745 ± 0.10 a
4.233 ± 0.38 a
(DG8) were better at pH 6; while strains of L. paracasei:
DG4 4.061 ± 0.27 b
3.692 ± 0.21 b
4.322 ± 0.23 a OM802845 (DG2) and OM967269 (DG4) were highest at
DG5 2.994 ± 0.18 c
4.007 ± 0.18 b
3.604 ± 0.13 b pH 6.5.
c b
DG6 2.946 ± 0.11 4.148 ± 0.07 2.097 ± 0.15 d
DG7 4.854 ± 0.09 a
3.355 ± 0.16 c
2.206 ± 0.40 d Effect of fermentation time on biofilm formation
c a
DG8 3.399 ± 0.27 4.660 ± 0.23 4.647 ± 0.08 a
DG9 4.235 ± 0.20 a
3.648 ± 0.27 b
2.546 ± 0.18 d Biofilm formation in the presence of agave fructans was
performed at different fermentation times showing a linear
previously screened to be able to grow in those agave poly- increase with time with similar rate of biofilm formation for
mers as a major carbon source. all strains, except for a slower rate by E. faecium OM80284
(DG9). As can be seen in Table 4, the biofilm formation is
Effect of pH on biofilm formation higher at 5 days than at 3 days in all strains except strain E.
faecium OM80284 (DG9). In the Fig. 7 shows the behav-
The biofilm formation in the presence of agave fructans ior of biofilm formation, finding that there is a relationship
was analyzed by adjusting the initial pH to 5.5, 6, and 6.5; between all strains L. paracasei (DG1 to DG8), obtaining
showing that the behavior of biofilm formation is affected in a different behavior in E. faecium OM80284 (DG9). The
diverse ways with respect to pH (Table 3). In most strains average rate of biofilm formation (µ) is 4.18 days− 1 for all

13
World Journal of Microbiology and Biotechnology Page 7 of 10 299

Table 4 Effect of different LAB 3 days (OD) 5 days (OD) Rate of

fermentation times on biofilm biofilm
formation using agave fructans as formation
a carbon source. According to the (days − 1)
OD, the ability to form biofilms
DG1 1.879 ± 0.092c 7.509 ± 0.219C 3.75
can be found as follows: low: 0
to 0.499; medium: 0.5 to 1.499; DG2 4.592 ± 0.043a 8.993 ± 0.114AB 4.39
high: >1.5. Capital letters show DG3 4.233 ± 0.380ab 8.786 ± 0.579 AB 4.39
the significant difference between DG4 4.322 ± 0.229ab 9.829 ± 0.100A 4.91
the strains at 3 days, and lower- DG5 3.604 ± 0.131b 7.365 ± 0.178C 3.68
case letters indicate a significant
DG6 2.097 ± 0.153c 8.399 ± 0.140B 4.12
difference at 5 days (p < 0.01)
DG7 2.206 ± 0.401c 6.813 ± 0.048C 3.41
DG8 4.647 ± 0.076a 9.607 ± 0.178A 4.8
DG9 2.546 ± 0.178c 1.285 ± 0.095D 0.84

this work is the use Lactobacillus paracasei DSM23505

Fig. 7 Behavior of the strains over time (0, 3 and 5 days). The triangles
(▲) represent DG1 to DG8 with a correlation R² = 0.8955 with a lin-
ear equation: y = 1.6404x − 0.4211; The circles (●) represent DG9 an
atypical behavior
strains of L. paracasei. In E. faecium OM80284 (DG9) the
µ = 0.84 days− 1 (Table 4).
Discussions
The main result of this work is that epiphytic homolactic
agave LAB strains, previously screened by Gallardo (2023)
for their ability to ferment agave fructans, produce much
higher amounts of biofilms on polystyrene microplates
when fed with agave fructans, as compared to fructose.
Such results support the use of agave extracts as alternative
resource for industrial lactic acid production instead of the
previous use of glucose in USA (Vink and Davies 2015) or
sucrose in Thailand (Groot and Borén 2010) and seems to
eliminate the need to break down agave fructans into fruc-
tose as a substrate for biofilm reactors. As a precedent of
produced natural inulinase and hydrolyze inulin from chic-
ory flour (Petrova et al. 2015) and the strains of Lactoba-
cillus paracasei, KCTC13090 and KCTC13169 fermented
the inulin of extract of Jerusalem artichoke tuber (Choi et
al. 2012). But to the best of our knowledge this is the first
report using agave fructans, as only carbohydrate source,
fermented by agave epiphytic LAB strains. These results
seem to be competitive with respect to other reports: using
a glucose and inulin mix with Lacticaseibacillus rhamnosus
GG (Lebeer et al. 2007); sucrose using L. rhamnosus GG
(Jiang et al. 2018), Leuconostoc lactis (Awad and Ahmaed
2018) and Lactobacillus sakei MN1 (Nácher-Vázquez et al.
2017); fructose with Lactobacillus reuteri biofilms of the
L2/6 and B2/1 (Schwab et al. 2007; Guan et al. 2020); raffi-
nose and galactose using Lacticaseibacillus rhamnosus GG
ATCC 53,103, Lactobacillus acidophilus ATCC 4356, Lac-
ticaseibacillus casei ATCC 393, Lacticaseibacillus casei
subsp. paracasei ATCC BAA-52, and Lactiplantibacillus
plantarum ATCC 8014 (Suissa et al. 2022).
The comparison of EPS synthesis using agave fructans
and fructose in planktonic cultures did not show statistical
differences between themselves. However, the comparison
of the ratios of biomass product yield (Yp/x) in agave fruc-
tans over fructose, as compared to the corresponding ratios
of biomass substrate yields (Yx/s) showed a negative linear
regression supporting the notion that EPS could be an adap-
tive response to metabolic stress. In Nguyen et al. (2020)
report that EPS synthesis is related to environmental and
nutritional stress, since LAB can alter their cell surface by
producing more polysaccharides. Increased EPS produc-
tion results in thicker, firmer cell walls that help them retain
water and maintain their integrity. Regarding nutritional
stress, with respect to carbon sources such as sucrose or
fructans, it can be explained because the unlimited supply of
basic sugar components and the high availability of energy
promote the production of EPS. Since in Van Hijum et al.
(2006) it has been found that LAB have enzymes such as
fructosidases that have a double function that is hydrolysis

13
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and biosynthesis. This allows using a part of the fructans as
a nutrient and another for the synthesis of EPS. In example,
LAB strains are prone to produce more EPS when biomass
synthesis requires more metabolic work.
It has been found that LAB can grow and have meta-
bolic activities with various carbon sources such as lactose,
glucose, fructans, sucrose, raffinose, galactose, fructans,
among others. The reason may be that they have adapted
to the carbon sources available in the environment in which
they inhabit (Saeed and Salam 2013). Therefore, the LAB
found in dairy products will have a metabolic machinery is
designed to use carbohydrates such as glucose, lactose, and
galactose, instead of using agave fructans as main carbo-
hydrate because are not part of their natural environment.
On the other hand, the LAB isolated from agave that have
adapted to an environment where the predominant source is
agave fructans produce other enzymes to survive and can use
this carbohydrate (Plaza-Vinuesa et al. 2023). García-Gam-
boa et al. (2018) analyzed enzymes produced by L. casei
and L. paracasei, suggesting a metabolic pathway where
LAB strains hydrolyze fructans into fructose and sucrose,
however it is necessary to analyze whether LAB produce
fructan β fructosidase. Likewise, it can be considered that
the strains of L. paracasei isolated from leaves, analyzed in
this work have better yields with fructose and agave fruc-
tans because they are more abundant carbohydrates in that
part of the plant. With respect to Enterococcus, it has been
reported that they have different pathways for carbohydrate
metabolism, allowing them to use a wide variety of sugars,
which allows them to grow on agave fructans, but not form
biofilms (Ayala-Monter et al. 2018; Montañez-Soto et al.
2011).
The fact that the LAB strains evaluated have the ability
to produce fructosidase in the presence of agave fructans
was due probably to the source from which they were iso-
lated having a high content of oligofructans, allowing the
microorganisms to adapt to this medium. In Petrov et al.
(2017) report that L. paracasei DSM 23,505 is able to pro-
duce β-fructosidase.
Agave fructans production is already developed in
Mexico at industrial scale (Ávila-Fernández et al. 2009).
Therefore, it is quite possible to adapt current lactic acid
fermentation technology to such industries if biofilm tech-
nology is developed as suggested by Iram et al. (2023) but
adapted to local homolactic LAB strains and agave fructans
as those studied in this work.
Another interesting application of this work is to sup-
port the use of agave fructans as prebiotic ingredients in
human and animal nutrition (García-Villalba et al. 2022)
because intestinal biofilm formation helps to stabilize the
presence of probiotic organisms in the foregut mucosa (Du
et al. 2022; Elsadek et al. 2023). For example, this type of
13
World Journal of Microbiology and Biotechnology
fructans is involved in the activation of the immune system
through interactions with probiotics such as Lactobacillus
casei and Bifidobacterium lactis (Moreno-Vilet et al. 2014).
Also, some of those agave LAB strains could become future
candidates as probiotic organisms since most of present
commercial strains have been developed from dairy prod-
ucts (Rezaei et al. 2021).
Furthermore, the remarkable difference between plank-
tonic and surface exopolysaccharide synthesis found in
present experiments opens the question of basic studies on
the epigenetic networks related to biofilm physiology. Of
special interest is the decrease of nutritional requirements of
biofilms already shown by Ho et al. (1997b, c) with quite dif-
ferent LAB strains using glucose as carbohydrate substrate.
Also, it seems important to study the nature and function of
glycosidase enzyme systems with abilities to break down
agave fructans and participate in the biosynthesis of biofilm
exopolysaccharides as those studied by (Van Hijum et al.
2006). Future work could confirm the resistance of LAB to
extreme pH (Emanuel et al. 2010), product inhibition (Ho
et al. 1997b, c) and biofilm kinetics and stability (Demirci
and Pometto 1995). It is worth noticing that all the strains
analyzed in here, have been isolated as epiphytic organisms
of Agave salmiana and have been characterized to belong
to genus Enterococcus and Lacticaseibacillus with proper
accession numbers in bioinformatic public data bases. Some
of them could be used as future proprietary strains in future
industrial bioindustries.
Acknowledgements This work was supported by the Biotechnology
Department of Universidad Autonoma Metropolitana at Iztapalapa.
NAML received a graduate fellowship from CONAHCYT.
Author contributions MLN performed material preparation and anal-
ysis. Quality control of the data and analyses was performed by CGA,
VGG, and GOL. The first draft of the manuscript was written by MLN
and CGA. All authors read, revised, and approved the final manuscript.
Funding MLN has received research support to study for a doctorate
from CONACYT (CVU 853186).
Data Availability The datasets generated during and/or analyzed dur-
ing the current study are available from the corresponding author on
reasonable request.
Declarations
Competing interests The authors declare no competing interests.
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