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Lactobacillus kefiranofaciens and Lactobacillus satsumensis isolated from

Brazilian kefir grains produce alpha-glucans that are potentially suitable
for food applications

Article  in  LWT- Food Science and Technology · May 2016

DOI: 10.1016/j.lwt.2016.05.010

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 LWT - Food Science and Technology 72 (2016) 390e398

Contents lists available at ScienceDirect

LWT - Food Science and Technology

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

Lactobacillus kefiranofaciens and Lactobacillus satsumensis isolated

from Brazilian kefir grains produce alpha-glucans that are potentially
suitable for food applications
Igor Moura de Paiva a, *, Raphael da Silva Steinberg a, Ivana Silva Lula c,
Elaine Maria de Souza-Fagundes d, Thiago de Oliveira Mendes e,
Maria Jose


Valenzuela Bell e, Jacques Robert Nicoli b, Alvaro Cantini Nunes a,
b
Elisabeth Neumann
a
Department of General Biology, Federal University of Minas Gerais, Brazil
b
Department of Microbiology, Federal University of Minas Gerais, Brazil
c
Department of Chemistry, Federal University of Minas Gerais, Brazil
d
Department of Pharmacology, Federal University of Minas Gerais, Brazil
e
Department of Physics, Federal University of Juiz de Fora, Brazil

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

Article history: Lactic acid bacteria produce exopolysaccharides, which have broad application in the food industry,
Received 12 December 2015 especially as thickeners, stabilizers, and gelling agents. Additionally, some biopolymers have been
Received in revised form regarded as health promoters due to their role as prebiotics and their immunomodulatory properties.
12 April 2016
This study aimed to characterize three exopolysaccharides produced by novel Lactobacillus strains from
Accepted 2 May 2016
Brazilian milk or sugar water kefir and to determine their biocompatibility. Lactobacillus kefiranofaciens
Available online 3 May 2016
1P3 and Lactobacillus satsumensis 10P and 10P2 were grown in the presence of sucrose, and their exo-
polysaccharides were purified and structurally elucidated. Glucose was the only sugar found in the
Keywords:
Glucan
polysaccharide chains, so most of the glucose residues were linked by a-(1,6) glycosidic bonds (~90%) and
Lactobacillus the estimated molecular weight was 800 kDa. According to the NIR spectra, a-glucan 1P3, 10P, and 10P2
Kefir were all different compounds. The in vitro cytotoxicity of the polymers was low or nonexistent, especially
Biocompatibility in nontumor cells. The polymers had no significant effects on the expression of proinflammatory and
Food additive regulatory cytokines in the intestine of mice. In contrast, the intestinal IgAþ B cells were significantly
higher. Taken together, this study suggests that these exopolysaccharides might be useful as food ad-
ditives due to their safety features to the host.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Exopolysaccharides (EPS) are extracellular polymers consisting
of hundreds or thousands of monosaccharide residues that can be
produced by plants, bacteria, yeasts, fungi, and algae and provide
protection against harsh environments to the producers. In most
cases, EPS protect against drying of the cell, osmotic stress,
phagocytosis, the presence of antibiotics or toxic components,
phage attacks and predation by protozoa. In single-cell organisms,
* Corresponding author. 2-065 Katz Group Centre for Pharmacy and Health
Research (University of Alberta), 87 Ave NW, Edmonton, AB, T6G 2E1, Canada.
E-mail address: igmpaiva@gmail.com (I.M. Paiva).
the EPS, which serve as one of the main components of microbial
biofilm, provide intercellular adhesion and adhesion between the
cells and surfaces. These high molecular weight polymers are
widely exploited industrially, especially in food, chemical and
pharmaceutical industries, due to their properties of emulsification,
stabilization, flocculation, control of crystallization, inhibition of
syneresis, encapsulation, film formation, and removal of heavy
metals. In industrial and medical fields, the main biopolymers used
are produced by plants (pectin, starch) and algae (alginate, carra-
geenan), hydrocolloids animal protein (gelatin, casein), and some
microbial polysaccharides (dextran, xantan, gellan, curdlan) (De
Vuyst & Deegest, 1999; Wang, Ahmed, Feng, Li, & Song, 2008).
Exopolysaccharides from lactic acid bacteria (LAB) have attrac-
ted special interest among the scientific community because they

http://dx.doi.org/10.1016/j.lwt.2016.05.010
0023-6438/© 2016 Elsevier Ltd. All rights reserved.
 I.M. Paiva et al. / LWT - Food Science and Technology 72 (2016) 390e398
are produced by food-grade microorganisms, which are generally
recognized as safe (GRAS), and are therefore harmless to humans.
Furthermore, LAB potentially secrete a broad range of EPS of great
versatility for food and pharmaceutical purposes (Badel, Bernardi,
& Michaud, 2011; Botelho et al., 2014). EPS have been used for
the production of yogurt, cheese and other dairy products to
improve the rheology and texture, as well as for conferring bene-
ficial effects on human health, including immunomodulatory and
^ te
anticarcinogenic activities (Co
, Skory, Unser, & Rich, 2013; Das &
Goyal, 2014; Dilna et al., 2015; Hidalgo-Cantabrana et al., 2012).
Kefir is a traditional beverage produced from the fermentation
of milk with kefir grains, and more recently, it has been produced
by the fermentation of a sucrose solution with the same grains. The
grains consist of a complex conglomeration of bacteria and yeasts
that remain firmly embedded in an EPS matrix (Garrote, Abraham,
& Antoni, 2001). The most reported EPS of the kefir polymeric
matrix is kefiran, a heteropolysaccharide produced by LAB from
milk kefir grains. This polysaccharide is mainly produced by
Lactobacillus kefiranofaciens, and many studies have reported its
biological and technological properties (Furuno & Nakanishi, 2012;
Medrano, Racedo, Rolny, Abraham, & Pe
rez, 2011; Piermaria,
Pinotti, Garcia, & Abraham, 2009; Uchida et al., 2010). On the
other hand, homopolysaccharides composed of repeated D-glucose
residues, especially the a-(1,6) glucans produced by Lactobacillus
hilgardii and Lactobacillus satsumensis, are the primary polymers
among the sugar water kefir grains (Co ^te

et al., 2013; Waldherr,
Doll, Meissner, & Vogel, 2010). The biosynthesis of these homo-
polysaccharides is carried out from sucrose through the extracel-
lular activity of glucosyltransferases (GTF) or glucansucrases
(Leemhuis et al., 2013; Synytsya & Novak, 2014).
Recently, glucans have gained attention because of their func-
tional properties and potential use in the dairy industry (Korakli &
Vogel, 2006; Pachekrepapol, Horne, & Lucey, 2015; Vannucci et al.,
2013). In general, the applicability of these polymers relies on their
monosaccharide composition, type of linkages, degree of branching
and molecular weight. Thus, the aim of this study was to investigate
the structure of the exopolysaccharides produced by
L. kefiranofaciens and L. satsumensis strains isolated from Brazilian
kefir grains to show their cytotoxicity, immunomodulation capac-
ity, and biocompatibility as a food additive.
2. Materials and methods
2.1. Bacterial strains and cell lines
In a previous study (Zaniratti et al., 2015), we identified 52
species of cultured Lactobacillus from Brazilian sugar water and
milk kefir grains. The strains were maintained at 80  C with MRS
medium and 20% glycerol. They were grown in an anaerobic
chamber (Thermo Scientific, Marietta, USA) containing 85% N2, 5%
CO2 and 10% H2 at 37  C for 48 h.
The Jurkat (human immortalized line of T lymphocyte), HL-60
(wild type human promyelocytic leukemia) and THP-1 (acute
monocytic leukemia) cell lines were kindly donated by Dr. Gustavo
P. Amarante-Mendes from the University of Sa ~o Paulo (Brazil),
whereas the MCF-7 and MDA-MB 231 human breast cell lines were
a gift from Dr. Alfredo Goes (Federal University of Minas Gerais,
Brazil). The HCT-116 (human colorectal carcinoma) cells were
purchased from ATCC. African green monkey kidney (Vero) cells
were donated by Dr. Erna Kroon (Federal University of Minas Ger-
ais, Brazil). The cells were cultured in RPMI 1640 or DMEM media
(Gibco BRL, Grand Island, USA) supplemented by 100 U/ml peni-
cillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 10% fetal
bovine serum. All cultures were maintained in a humidified incu-
bator at 37  C and 5% CO2.
391
2.2. Screening of EPS-producing Lactobacillus strains
First, Lactobacillus isolates were seeded in Petri dishes contain-
ing modified MRS medium by replacing 20 g L1 glucose with
80 g L1 sucrose or 50 g L1 raffinose (Waldherr et al., 2010). EPS
producers were identified in triplicate by their slimy colony
appearance.
In addition, a molecular approach for screening putative gtf
genes was performed. Briefly, DNA from the EPS-producing isolates
was extracted from overnight cultures using a Wizard Genomic
DNA purification kit (Promega). PCR conditions, including the use
of the DegFor and DegRev primers, were based on those described
by Kralj, Geel-Schutten, Maarel, and Dijkhuizen (2003). Amplicons
were checked with agarose gel electrophoresis, and when neces-
sary, they were sequenced using the Genetic Analyzer ABI 3130
(Applied Biosystems). The nucleotide sequences were translated
into amino acid sequences using ORF Finder and were analyzed
through BLAST. Some strains were also distinguished by using the
primer (GTG)5, and their amplification products were compared in
polyacrylamide gels.
2.3. Production and purification of exopolysaccharides
The EPS production by the selected strains was conducted in
Erlenmeyer flasks containing 200 ml modified MRS broth containing
sucrose at 37  C for 48 h. The initial cell density was approximately
9.4 billion CFU mL1. Afterward, the cells were removed by centri-
fugation. Crude EPS solutions were mixed with two volumes of cold
ethanol and stored at 20  C overnight. The precipitates were
collected by centrifugation at 9000g for 30 min. For gravimetric
quantification purpose, the pellets were air dried at 37  C for 96 h,
and total weights were measured, whereas for the other analysis,
further purification was carried out. The polysaccharide pellets were
dissolved in ultrapure water, followed by three more steps of pre-
cipitation in ethanol and resuspension in water before ultracentri-
fugation (10 kDa cutoff; Amicon, Bedford, USA) and freeze-drying. In
addition, free monosaccharides and total protein were determined
by ion-exchange chromatography (described in section 2.4) and
Bradford’s method (Sigma, St. Louis, USA), respectively.
2.4. Monosaccharide composition
The polysaccharide chains were chemically hydrolyzed by
adding 3 mL 1.75 M perchloric acid to the sample and incubating at
80  C for 2 h and 16 h. For neutralization, 5M potassium hydroxide
was used, and salt precipitates were removed by centrifugation.
The supernatants, containing sugar monomers, were analyzed by
high performance liquid chromatography (HPLC) using an ion-
exchange column packed with polystyrene resin (Supelcogel C-
610H) and a refractive index detector (RID-10A). The mobile phase
was 50 mM H2SO4 with a flow rate of 0.6 mL min1. Glucose,
fructose, arabinose and glycerol were used as the standards
(Maeda, Zhu, Suzuki, Suzuki, & Kitamura, 2004).
2.5. Molecular weight determination
Gel permeation chromatography (GPC) with a refractive index
detector (RID-10A) was used to determine the EPS molecular
weight. The samples were injected into the HPLC column (Shodex
KW-803). Elution was carried out by 0.1 M NaNO3 at a flow rate of
1 mL min1. Commercial dextrans with a range from 36 to 500 kDa
were used as the standards.
392 I.M. Paiva et al. / LWT - Food Science and Technology 72 (2016) 390e398
2.6. Spectroscopic analysis
Nuclear magnetic resonance (NMR) spectra were recorded using
a Bruker DRX-400 AVANCE operating at 400 MHz and equipped
with a 5 mm dual probe using direct detection. Deuterated water
(D2O, Cambridge isotopic 99.9%) was used as the solvent at 70  C
(343 K). One-dimensional 13C and 1H NMR spectra were obtained
with water suppression when necessary.
A Fourier-Transform spectrometer (FT) (model MPA FT-NIR)
from Bruker, equipped with a Te-InGaAs detector, quartz optics
and OPUS 6.5 software (Ettlingen, Germany), was used to analyze
near-infrared (NIR) spectra of each EPS. The analyses were per-
formed in reflectance mode, from 12,000 to 4000 cm1 with a
resolution of 4 cm1 and with 100 scans being accumulated for
sample. Each sample was placed in a glass vial and placed directly
into the equipment for spectra acquisition.
2.7. In vitro EPS cytotoxicity
Cells were seeded in 96-well plates at different densities for 24 h
(Jurkat: 100,000 cells well1; HL60: 50,000 cells well1; MCF-7,
HCT-116, MDA-MB-231 and Vero: 10,000 cells well1). Then, the
EPS extracts were dissolved in PBS and added into the wells. The
cells were treated with 100 mg/ml EPS for 48 h. Cell viability was
measured by MTT assay (Mosmann, 1983). Briefly, after treatment,
20 mL MTT solution (5 mg mL1) was added, and cells were incu-
bated at 37  C for 1 h. The medium was replaced by 200 mL of 0.04 M
HCl solution in isopropanol, and the absorbance was read at
595 nm. The values of the control wells (i.e., PBS treatment) were
considered to represent 100% cell viability. The assays consisted of
two independent experiments performed in triplicate.
2.8. In vivo EPS immunomodulation
Outbred NIH Swiss mice (aged 3 weeks, weight 14e17 g) were
housed in plastic cages (ALE.MIL.01.03, Alesco, Monte Mor, Brazil),
Fig. 1. Slimy colonies of Lactobacillus kefiranofaciens 1P3 (A), L. satsumensis 10P (B) and L. satsumensis 10P2 (C) in medium containing sucrose. No ropy colonies were observed in the
presence of glucose (control), as shown for L. satsumensis 10P2 (D).
which were maintained at controlled temperature under a 12 h
light/dark cycle, and given unrestricted access to commercial food
(Nuvilab CR1, Nuvital Nutriente, Curitiba, Brazil) and sterilized
filtered water. The mice were handled in accordance with inter-
national regulations for animal welfare (COBEA 2006). All pro-
cedures were approved by the Ethics Committee in Animal
Experimentation of the Federal University of Minas Gerais (protocol
number 96/2011). The experiment was performed twice, and each
test and control group consisted of five mice. Treated animals daily
received EPS extract (100 mg/kg body weight) dissolved in PBS by
intragastric route daily for seven consecutive days. Control animals
received only PBS during the same period. After treatment, the test
and control mice were anesthetized and sacrificed by cervical
dislocation.
2.8.1. IgA producing cells in intestinal lamina propria
The ileum tissue samples were taken and processed for paraffin
inclusion, and 4-mm sections were stained with hematoxylin-eosin
and analyzed by a pathologist. The IgA producing (IgAþ) cells were
identified by direct immunofluorescence test. For this purpose,
histological slices were prepared as described by Vinderola et al.
(2005) with slight modifications. First, they were deparaffinized
by xylol, rehydrated in a graded series of ethanol, incubated in a
wet chamber with bovine albumin serum (BSA) for 30 min and
washed in PBS solution. Air-dried histological samples were incu-
bated with 1:100-diluted anti-mouse IgA FITC conjugate (Sigma-
eAldrich) in PBS-BSA solution for 30 min at 37  C. After being
washed 3 times in PBS, the samples were examined using a fluo-
rescent light microscope. The results represent the number of IgAþ
cells in enough of the microscopic field (magnification 10) to
cover the entire ileum of each animal.
2.8.2. Cytokine gene expression in intestine
A fragment (1 cm) was collected from the jejunum, duodenum
and ileum of each animal. Tissue samples were ground together
using an electric-power homogenizer, total RNA was isolated, and
 I.M. Paiva et al. / LWT - Food Science and Technology 72 (2016) 390e398 393

real-time RT-qPCR was performed as described by Steinberg et al. Table 1

(2014). Gene-specific primers for cytokines TGF-b, INF-g, TNF-a, Screening of exopolysaccharide (EPS)-producing lactobacilli isolates from Brazilian
kefir grains.
IL-4, IL-5, IL-6, IL-10, IL-12p40, and IL-17 were used, and the
expression levels of the cytokine genes were calculated using two Lactobacillus strain EPS production Lactobacillus strain EPS production
reference genes (b-actin and GAPDH). Sugar water kefir grains L. perolens 11P3 e
L. casei 8P e L. perolens 13P e
2.9. Statistical analysis L. casei 8P2 e L. perolens 14P2 e
L. casei 8P3 e L. perolens 16P3 e
L. casei 9P2 þ L. perolens 17P2 þ
Values are presented as the mean and standard deviation or as L. casei 13P2 e L. perolens 19P þ
the of three measurements. When necessary, the difference among L. casei 13P3 e L. satsumensis 10Pa þ
groups was checked by KruskaleWallis analysis using SPSS soft- L. casei 14P e L. satsumensis 10P2a þ
L. casei 17P e L. satsumensis 18P þ
ware. Data were considered significantly different when p < 0.05.
L. casei 20U e Milk kefir grains
L. casei 20U1 e L. casei 22P2 e
3. Results L. casei 20U2 e L. casei 25P e
L. casei 15U e L. kefiranofaciens 1P þ
3.1. Screening of EPS-producing strains L. casei 15U1 e L. kefiranofaciens 1P3a þ
L. casei 15U2 þ L. kefiranofaciens 2P2 e
L. casei 16U þ L. kefiranofaciens 6P e
Sucrose is known to be a substrate for both glucosyltransferases L. casei 17U e L. kefiranofaciens 8U þ
(GTF) and fructosyltransferases (FTF), whereas only fructan- L. casei 18U e L. kefiri 24P3I e
producing enzymes have the ability to use raffinose as an initi- L. diolivorans 1Z e L. kefiri 3P2 e
ator for polymerization. No ropy colony was visualized in the media L. mali 19U þ L. kefiri 3P3 þ
L. mali 21U1 e L. kefiri 4P2I e
containing glucose or raffinose. In the presence of sucrose, how- L. mali 21U2 e L. kefiri 4P3I e
ever, 16 EPS producers were identified (Fig. 1). Five Lactobacillus L. mali 22U e L. kefiri 4P3II e
species were observed from the sugar water kefir (Table 1), L. parafarraginis 12P þ L. kefiri 3U2 þ
including L. satsumensis, Lactobacillus perolens, Lactobacillus casei, L. parafarraginis 16P e L. mali 23P e
L. perolens 11P þ L. satsumensis 23P3 e
Lactobacillus mali, and Lactobacillus parafarraginis. Two species
L. perolens 11P2 e L. satsumensis 2P3 e
were also screened from the milk kefir: L. kefiranofaciens and
 Means no slimy colonies on the MRS agar containing sucrose, lactose or raffinose
Lactobacillus kefiri.
instead of glucose.
After this first screening, PCR amplicons from the catalytic þ Means slimy colonies only on the MRS containing sucrose.
domain of the glucosyltransferases were resolved in agarose gel, a
Strains further selected for EPS structural and biological characterization.
and three strains were selected for nucleotide sequencing:
L. satsumensis 10P, 10P2, and L. kefiranofaciens 1P3. Their amplifi-
cation products were the only ones that presented just one band retention times of these EPS were similar to each other (5.218;
migrating at 660 bp. Amplicons from the other selected strains had 5.223; and 5.236 min, respectively). Concerning the Bradford assay
more than one band or a single band with different migration of the EPS extracts, less than 21 mg/ml protein was measured.
behavior (data not shown). Inaccurate flanking nucleotide se- The 1H NMR and 13C NMR spectra of the three EPS revealed that
quences were removed, and the lengths of these amplicons were they present similar glycosidic bonds (Fig. 3). The a-(1,6) bonds
approximately 600 bp. A BLAST search revealed few analogies with were identified by a high hydrogen signal at 4.9 ppm, and the a-
known sequences from enzymes belonging to the glycosyl hydro- (1,3) bonds were identified by a lower intensity, anomeric signal at
lase family 70 (GH70). The glucansucrase that was phylogenetically approximately 5.3 ppm (Das & Goyal, 2014). According to the
closest was that produced by L. hilgardii TMW1828 (75% identity,
80% similarity), which had also been isolated from water kefir
grains (Waldherr et al., 2010).
The (GTG)5-PCR fingerprinting among the selected strains
showed that the band pattern of the L. kefiranofaciens 1P3,
L. satsumensis 10P and 10P2 were slightly different (Fig. 2), indi-
cating they might be different strains.

3.2. Chemical characterization

Although the growth conditions for the EPS production had not
been optimized, the strains 1P3, 10P, and 10P2 showed similar
behavior. According to the gravimetric estimation, they produced
an average of 12.65 ± 1.76 mg mL1 biopolymer after 48 h.
Partial and total hydrolysis confirmed the presence of only
glucose in the polysaccharide chains of the EPS. The acid exposure
for 2 h was conducted to identify other possible free sugars, such as
fructans, as some monosaccharides could be degraded when
exposed to acidic conditions for a prolonged time (Bounaix et al.,
2009). However, only glucose and oligosaccharides were found
after that. Chromatograms from the hydrolysates obtained after
16 h had a peak corresponding to glucose, and those peaks corre-
sponding to partial hydrolysis products were not detected Fig. 2. (GTG)5-PCR fingerprinting between Lactobacillus kefiranofaciens 1P3 (B),
anymore. GPC analysis revealed that the molecular weights of EPS L. satsumensis 10P (C) and L. satsumensis 10P2 (D). Their amplicons were separated in a
1P3, 10P, and 10P2 were approximately 800 kDa because the polyacrylamide gel. Lane A corresponds to the 100 bp DNA ladder.
394 I.M. Paiva et al. / LWT - Food Science and Technology 72 (2016) 390e398

integration of these signals, the polymers have 7.88± 0.92% a-(1,3)
linkage, which indicates the percentage of branching. The 13C NMR
spectra showed six signals related to the carbon atoms. In the
anomeric region, a signal was detected at 98.4 ppm corresponding
to C-1, which is typically indicative of a-(1,6) glucans. Those signals
with chemical shifts at 70e75 ppm were due to C-2, C-3, C-4, and C-
5. The last carbon signal at 66 ppm corresponded to C-6 (Ahmed,
Siddiqui, Arman, & Ahmed, 2012; Zhao, Kan, Li, & Chen, 2005).
At least five bands were identified from the NIR spectra of the
EPSs (Fig. 4a). Bands between 4200 and 4900 cm1 indicated hy-
droxyl stretching and deformation. Macedo, Laporte, and Lacroix
(2002) used primarily those bands, as well as the one migrating

13
Fig. 3. Four hundred megahertz C and 1H NMR spectroscopy of exopolysaccharides produced by Lactobacillus kefiranofaciens 1P3 (A), L. satsumensis 10P (B), and L. satsumensis
10P2 (C).
 I.M. Paiva et al. / LWT - Food Science and Technology 72 (2016) 390e398 395

Fig. 4. Near-infrared (NIR) spectra of the exopolysaccharides (A). Orthogonal projection of the latent structures with discriminant analysis (B) using NIR spectra from polymers
produced by L. kefiranofaciens 1P3 (O), L. satsumensis 10P (D), and L. satsumensis 10P2 (þ).

at 5800 cm1, to quantify exopolysaccharides through statistical Table 2

analysis. Although the discrimination of the glucans was not Exopolysaccharide (EPS) in vitro cytotoxicity against tumoral and nontumoral cell
lines.Table 2
possible based on their apparent band pattern, the multivariate
analysis of the spectra demonstrated that the three EPSs are Cell lines Proliferation inhibition (%)a
significantly different among themselves (Fig. 4b). A model of the EPS 1P3 EPS 10P EPS 10P2
orthogonal projection of the latent structures with discriminant
JURKAT 8.49 ± 3.43 0 0
analysis (OPLS-DA) showed a difference between the a-glucan 1P3 HL-60 15.05 ± 9.73 3.36 ± 10.23 0
and the others on the left-hand side. As the horizontal direction is HCT-116 20.67 ± 5.36 28.41 ± 11.95 28.70 ± 3.81
the direction of greatest variance, the model suggests that the a- THP-1 14.96 ± 5.97 4.35 ± 1.51 9.67 ± 1.56
glucan 1P3 is significantly different from the EPS 10P and 10P2, MCF-7 4.80 ± 2.01 0 10.64 ± 4.74
MDA-MB-231 17.14 ± 6.70 22.50 ± 1.65 28.32 ± 5.05
with the latter two sharing greater similarity with each other. VERO 0 0 0
a
Cells were treated for 48 h with a polymer concentration of 100 mg/ml. Data are
3.3. Antitumoral and cytotoxic activity expressed as the mean ± standard deviation of the percentage of cell proliferation
inhibition compared to control (cells treated with PBS), which were considered to
The a-(1,3) branched a-(1,6) glucans produced by the strains show 100% cell viability. The data represent two independent experiments per-
formed in triplicate.
1P3, 10P and 10P2 presented weak antitumor activity in all cell lines
tested in this study (Table 2). They showed approximately 30% in-
hibition of colorectal carcinoma HCT-116 and breast cancer MDA- produced EPS. Based on a molecular screening, three of the EPSs
MB 231. The other cancer cell lines were weakly or unaffected by produced - L. satsumensis 10P and 10P2 from the sugar water kefir
the extracts, proliferating as well as the PBS control. Furthermore, and L. kefiranofaciens 1P3 from the milk kefir - were further
the extracts of these strains did not inhibit the viability of the investigated. Contrary to most lactic acid bacteria species, which
nontumoral Vero cells at all, demonstrating no cytotoxicity among typically show low yields of polysaccharide production, these
all tested groups.

3.4. Immunomodulatory capacity

The a-glucans provision for seven days increased IgA þ cells in

the ileum of mice. The IgA producing area in the lamina propria was
significantly higher (p < 0.05) in the EPS-treated mice compared to
the control mice (Fig. 5). As shown in Fig. 6, the a-glucan treatment
reduced the expression of the IL-10 and TGF-b, but the difference
was statistically significant only in the TGF-b expression among the
mice that received the EPS 1P3. The treatment with EPS 10P or 10P2
also showed a slight increase in expression of the INF-g, TNF-a, IL6,
and IL-17. The expression levels of the IL5 and IL12p40 genes were
almost the same in the experimental groups and the control.

4. Discussion

The search for new sources of biocompatible exopoly-

saccharides has been encouraged because of their potential use in
the food and pharmaceutical industries. Herein, we have demon-
strated that kefir grains are an interesting source of EPS-producing
microorganisms. We demonstrated, even with no growth optimi-
zation, that 16 of the 52 Lactobacillus isolates from Brazilian kefir
Fig. 5. Exopolysaccharide (EPS) immunomodulatory activity in mice intestinal lamina
propria. The percentage of the IgA-producing area of the saline-treated mice (control)
and mice treated with a-glucans produced by L. kefiranofaciens 1P3, L. satsumensis 10P
and L. satsumensis 10P2 for seven days. *indicates significant differences (p < 0.05)
compared with control animals.
396 I.M. Paiva et al. / LWT - Food Science and Technology 72 (2016) 390e398
Fig. 6. Effect of a-glucans on the expression levels of IL-10 (A), TGF-b (B), TNF-a (C), and IFN-g (D) in the small intestine. The cytokine mRNA was measured by RT-qPCR using saline-
treated mice as the control. Mice treated with polymers produced by L. kefiranofaciens 1P3 and L. satsumensis 10P and 10P2 for seven days. * indicates statistically significant
difference (p < 0.05).
selected strains produced a large amount of polymer. Although this
was not the focus of this study, all strains tested herein produced,
under similar culture conditions, an amount of EPS four to five
times higher than L. hilgardii TMW1828, a LAB previously isolated
from kefir grains and described as an efficient EPS producer
(Waldherr et al., 2010).
Polysaccharide structures were elucidated, and essentially, the
glucans were found to vary in their type and degree of branching,
length of branch chains, spatial arrangement and molecular weight.
We characterized three dextran-like polysaccharides as a-(1,3)
branched a-(1,6) glucans, containing approximately 8% branch
linkage and with an estimated molecular weight of 800 kDa.
Despite the inaccuracies incurred from the failure to use higher
molecular weight standards, the three EPS presented smaller mo-
lecular weight than similar biopolymers produced by lactic acid
bacteria (Ahmed et al., 2012; Bounaix et al., 2009; Das & Goyal,
2014; Wang, Zhao, Tian, Yang, & Yang, 2015). Despite the similar-
ities among our biopolymers, the discrimination analysis from the
NIR spectra demonstrated that they are different compounds.
The production of an a-(1,3) glucan by L. satsumensis was pre-
viously described by Co^ te

et al. (2013). On the other hand, dextran
production by a strain of L. kefiranofaciens has not been described,
yet the microorganism in question is often associated with the
production of kefiran (Ahmed, Wang, Anjum, Ahmadc, & Khan,
2013; Maeda et al., 2004; Wang et al., 2008). Thus, the present
study showed that L. kefiranofaciens, as well as L. kefiri, are capable
of producing homopolysaccharides via glucansucrase in the pres-
ence of sucrose. This finding is important because it indicates how
some homemade milk kefir grains adapt when the milk medium is
replaced by a sucrose solution, as commonly observed in Brazil.
According to previous studies of the antitumoral activity of plant
extracts and other chemical substances that were conducted by
Lessa et al. (2011) and Michel et al. (2015), none of the polymers
studied herein presented significant antiproliferative activity,
confirming that the cancer cell growth-inhibition capacity among
a-glucans is usually low. Some papers have demonstrated that the
anticancer property of those substances could increase after sul-
fation, sulfonylation or carboxymethylation (Wiater, Paduch,
Pleszczyn, & Szczodrak, 2011; Zhang & Cheung, 2002; Zhang,
Zhang, Zhou, Chen, & Zeng, 2000). In contrast, b-glucans are
often reported as having high cytotoxicity against tumors
(Vannucci et al., 2013). Interestingly, the three a-glucans had no
cytotoxicity against the noncancerous Vero cells; this result is in
accordance with results showing a biocompatible glucan from
Lactobacillus plantarum DM5 (Das & Goyal, 2014).
No interference was observed in the cytokine expression by a-
glucans 1P3, 10P and 10P2 in the small intestine of mice after seven
days of oral administration. The gene expression of the IL-10 and
TGF-b was slightly decreased, but no significant increase was
observed in the expression of the pro-inflammatory cytokines.
Moreover, histological study showed that this weak effect in cyto-
kine response did not compromise the ileum integrity (data not
shown). These results are important because some dextrans, such
as low molecular weight dextran sodium sulfate, induce serious
colon damage in vivo (Laroui et al., 2012).
Concerning immunomodulatory capacity, a significant increase
of IgA-producing cells in the ileum of mice was observed after
seven days of EPS treatment through gavage. Similarly, the oral
 I.M. Paiva et al. / LWT - Food Science and Technology 72 (2016) 390e398
administration of a glucogalactan from L. kefiranofaciens enhanced
the IgA production in mouse intestine (Medrano et al., 2011;
Vinderola, Perdigo
n, Duarte, Farnworth, & Matar, 2006). IgA is the
most abundant antibody isotype in mucous secretions and works
together with innate nonspecific defense mechanisms to block
microbial adhesion to epithelial cells without causing tissue-
damaging inflammation (Cerutti, Chen, & Chorny, 2011). The role
of a-(1,4) glucans in the induction of the innate immune response is
already known through some publications (Bittencourt et al., 2006;
Kakutani et al., 2007; Nair, Melnick, Ramachandran, Escalon, &
Ramachandran, 2006). Zhao et al. (2005) reported that a-(1,6)
glucan also has immunostimulatory activity. However, a-glucans
are nondigestible ingredients and might affect the growth of some
bacteria in the gut, which would cause changes in the immune
response (Olano-Martins, Mountzouris, Gibson, & Rastall, 2000).
Thus, the effect of the present exopolysaccharides on a germ-free
mouse model in different doses and for different periods must be
conducted to investigate further the immune effect of the EPS in the
gastrointestinal tract.
5. Conclusion
We characterized three a-glucans produced by Lactobacillus spp.
isolated from Brazilian kefir grains. Although further technological
and biological characterization remains, in vitro and in vivo studies
demonstrate that these polymers are biocompatible and indicate
the potential of the polymers for use as a food and pharmaceutical
ingredient. In addition, we demonstrate that L. kefiranofaciens
produces glucan instead of kefiran when sucrose is present, which
might be useful for improving the rheological characteristics of
kefir. Finally, our results encourage further investigations about the
immunomodulatory properties of a-glucans, especially regarding
IgA intestinal production.
Acknowledgments
The authors would like to acknowledge the Fundaça ~o de
Amparo  a Pesquisa de Minas Gerais (FAPEMIG, APQ-02353-10),
Conselho Nacional de Desenvolvimento Científico e Tecnolo
gico
(CNPq, 559370/2010-5), and Pro
-Reitoria de Pesquisa/Universidade
Federal de Minas Gerais (PRPq/UFMG, ADRC-01/2013) for the
financial support of this research and scholarships.
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