Probiotics and Antimicrobial Proteins

https://doi.org/10.1007/s12602-018-9471-2

Evaluation of the Probiotic Potential of Saccharomyces cerevisiae

Strain (C41) Isolated from Tibicos by In Vitro Studies
Haydee Eliza Romero-Luna 1 & Humberto Hernández-Sánchez 2 & Rosa María Ribas-Aparicio 3 &
Patricia Isidra Cauich-Sánchez 4 & Gloria Dávila-Ortiz 1

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Artisanal fermented beverages have been associated with beneficial effects for a long time. In Mexico, there are a wide variety of
artisanal fermented beverages such as Tepache, where the fermentation is initiated by the addition of grains of a polysaccharide-
containing biofilm matrix formed by a symbiotic association of bacteria and yeasts known as BTibicos.^ These microorganisms can
be responsible for the beneficial effect associated with fermented beverages inoculated with Tibicos. The probiotic potential of
microorganisms has been widely studied, mainly in lactic acid bacteria, while despite the wide distribution of yeasts, these have not
been so studied. Therefore, the aim of this study was to evaluate in vitro the probiotic potential properties of a yeast isolated from
Tibicos. For this, the yeast was identified by molecular techniques as Saccharomyces cerevisiae, which showed a good resistance to
pH 2.0, bile salts and in vitro digestion. The results also showed a good ability to form cellular aggregates as a result of having a
hydrophobic surface. In addition, it can be considered as safe since it does not show hemolytic activity and is sensitive to nystatin.
Additionally, the yeast presented an excellent antioxidant capacity to reduce the DPPH radical. The S. cerevisiae strain C41 isolated
from Tibicos was successfully compared by means of in vitro tests with the only recognized probiotic yeast, S. boulardii. These
findings point Saccharomyces cerevisiae C41 as a potentially probiotic yeast; nevertheless, it is necessary to consider further in vitro
and in vivo studies that establish the benefits that this yeast could provide.

Keywords Probiotic yeast . Saccharomyces cerevisiae . Tibicos . Fermented beverages

Introduction Tibicos are a symbiotic culture of bacteria and yeasts embed-

In Mexico, there are a wide variety of traditional fermented
beverages, among which, tepache stands out. This beverage is
obtained from the pineapple peel and can be fermented sponta-
neously or by the addition of Tibicos as an inoculum.
* Gloria Dávila-Ortiz
gdavilao@yahoo.com
1
Laboratorio de Proteínas Vegetales, Departamento de Ingeniería
Bioquímica, Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, Mexico City, Mexico
2
Laboratorio de Biotecnología de Alimentos, Escuela Nacional de
Ciencias Biológicas, Instituto Politécnico Nacional, Mexico
City, Mexico
3
Laboratorio de Producción y Control de Biológicos, Departamento
de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, Mexico City, Mexico
4
Laboratorio de Bacteriología Médica, Departamento de
Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, Mexico City, Mexico
ded in a polysaccharide biofilm matrix, generally dextrans, pro-
duced by bacteria [1]. The word BTibicos^ is used in Latin
America to refer to water kefir grains; moreover, it is known by
other names depending on the geographic location, such as
BGinger beer plants,^, BTibi grains,^ BCalifornia bees,^
BAfrican bees,^ Bale nuts,^ Bbalm of Gilead,^ BJapanese beer
seeds,^ and Bsugary kefir grains^ [2, 3]. Lactobacillus and
Leuconostoc are the main genera of bacteria reported in Tibicos,
as well as Candida, Rhodotorula, Brettanomyces, and
Saccharomyces yeasts [4, 5].
Yeasts are part of the predominant microorganisms in
fermented food, which is a safe historical human use, and are
of great importance in the gastrointestinal tract although it is
found in small numbers [6, 7]. Saccharomyces boulardii is the
only yeast recognized as probiotic, and it is used to normalize the
intestinal microbiota in patients with gut disorders [8]. This spe-
cies is genetically very close but metabolically different from
Saccharomyces cerevisiae [9]. S. cerevisiae presented probiotic
characteristics such as antagonistic interactions against patho-
gens, in addition to surviving through the intestinal tract [10].

Some S. cerevisiae strains are also marketed as dietary supple-
ments because of their high nutrient and mineral content [11].
These characteristics indicate that S. cerevisiae is a good
candidate to be used as probiotic. And the Tibicos could be a
promising source of new microbial strains that can be used as
probiotics. So, the aim of this study was to isolate and identify
a yeast from Tibicos, considered as starters of traditional
fermented beverages, and evaluate its probiotic potential.
Materials and Methods
Yeast Isolation and Culture Conditions
The strain was isolated from a sample of Tibicos, which was
obtained from a market in Mexico City. One gram of Tibicos
was placed in a sterile plastic bag with 10 mL of saline solu-
tion (0.9%) and manually milled with a glass pestle until it
disintegrated. The sample was streaked on malt extract agar
(Dibico, Mexico). After 48 h at 28 °C, the yeast colony was
isolated and preserved.
S. boulardii was isolated from the commercial probiotic
product Floratil (Biocodex, France).
The yeast isolates were preserved in malt extract broth with
glycerol (20%) and maintained at − 20 °C.
Yeast Strain Identification
Initially, the isolated yeast was characterized according to
macro and microscopic features with an optical microscope
(Nikon 80i eclipse, Japan). The assimilation of carbon com-
pounds was evaluated by API 20C AUX (bioMériux, France).
On the other hand, the identification was carried out by mass
spectrometric and ITS-5.8S rDNA sequence analyses.
Identification by Mass Spectrometric Analysis
A pure 24–48-h culture of yeast grown in malt extract agar at
28 °C was used, from which a portion of a colony was taken
and then placed directly on a disposable target slide, using a
toothpick. Immediately afterward, the sample was lysed with
0.5 μL of formic acid (25%, v/v), which was allowed to dry at
room temperature; then, 1 μL of the matrix solution (3.1% (w/v)
α-cyano-4-hydroxycinnamic acid) was added and allowed to
dry at room temperature. The prepared sample was analyzed
with Vitek® MS-Plus mass spectrometer (bioMériux, Marcy-
l’Etoile, France) in linear positive-ion mode, across the mass-
to-charge ratio range of 2000 to 20,000 Da. The sample was
irradiated with 50 laser shots per second at 50 Hz. Prior to the
analysis of the sample, the equipment performed the calibration
using Escherichia coli ATCC 8739. The results were obtained
and displayed by Myla v2.4 middleware.
Probiotics & Antimicro. Prot.
Molecular Identification
The yeast strain was also identified by the amplification of 5.8
rRNA gene and the two ribosomal internal transcribed spacers
(ITS). For the DNA isolation, the yeast was cultured in 15 mL
of malt extract broth at 28 °C during 24 h for the DNA isolation.
For the amplification of 5.8S-ITS rRNA region of the yeast
DNA, the primers ITS1 (5′-TCC GTA GGT GAA CCT GCG
G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′)
[12] were used. The polymerase-chain reaction (PCR) was
performed with a 50 μL of the reaction mixture, which
contained 0.25 mM of each dNTP, 15 pmol of each primer,
1 U of Taq DNA polymerase (Invitrogen, USA), 1× of buffer
Taq Mg-free and 0.9 mM of MgCl2, and 100 ng of isolated
DNA. On the other hand, the amplification conditions of PCR
consisted of an initial 4-min denaturation at 95 °C, 35 cycles
that consisted of 30 s of denaturation at 94 °C, 1-min anneal-
ing at 56 °C and 1-min extension at 72 °C, and 7 min for final
extension at 72 °C, in a Veriti thermal cycler (Applied
Biosystems, USA). The PCR product was analyzed by elec-
trophoresis with 0.8% (w/v) agarose in 0.5× TBE (Tris-borate-
EDTA buffer). The PCR product was then purified using a
GeneJet PCR purification kit (ThermoScientific, USA). The
sequencing was carried out in the Molecular Biology
Laboratory of the Institute of Cell Physiology at the
Universidad Nacional Autónoma de México (UNAM) using
the same primers. Finally, the obtained sequences from isolat-
ed yeast were searched with the Basic Local Search Tool
(BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Resistance to pH 2 and Bile Salts
The survival to gastrointestinal digestion was determined ac-
cording to Castro-Rodríguez et al. [13] with some modifica-
tions. The yeast was cultured in malt extract broth for 24 h and
then harvested by centrifugation (3500×g, 10 min), washed
and resuspended with saline solution (0.9% w/v). Five millili-
ters of cellular suspension (107 CFU/mL) was added to a
45 mL of saline solution (0.9%) adjusted to pH 2 (with 6 N
HCl) and incubated 1 h at 37 °C. The cellular suspension was
also inoculated in NaCl solution (0.9% w/v) with bile salts
(Oxgall, 0.5% w/v) incubated 3 h at 37 °C.
Resistance to Simulated Gastrointestinal Digestion
The survival through gastric and intestinal simulated juices
was evaluated. Five milliliters of yeast suspension was added
to 45 mL of an artificial gastric fluid (3 mg/mL pepsin (Sigma,
USA) in saline solution at pH 2.0) and incubated 1 h at 37 °C.
Over time, an aliquot was taken and diluted in phosphate-
buffered saline (PBS) to evaluate viability by plate count,
and 5 mL was taken and added to 45 mL of an artificial
intestine juice (0.5% Oxgall (w/v); 1.9 mg/mL pancreatin,
Probiotics & Antimicro. Prot.
pH 6.5). An aliquot was taken after 30, 90 min, and 3 h to
evaluate the viability by plate count.
The viability was expressed as a percentage of viable cells
according to Ogunremi et al. [14].
CFU ml−1 ðFinalÞ
Survivalð%Þ ¼  100
CFU ml−1 ðInitialÞ
Hydrophobicity
The cell surface hydrophobicity was evaluated with chloro-
form and n-hexadecane. Three milliliters of yeast suspension
was in contact with 0.75 mL of chloroform or hexadecane; the
mixture was then vortexed for 2 min and left to rest for 1 h at
37 °C. Subsequently, the absorbance of the aqueous phase was
measured at 600 nm, and the results expressed as a percentage
of hydrophobicity according to the following equation:
ðAi −A f Þ
%H ¼  100
Ai
Ai is the OD600 of the cell pellet; Af is OD600 of aqueous
phase at the end of the experiment [13].
Autoaggregation Ability
The autoaggregation was evaluated according to Gil-
Rodríguez et al. [15], where fresh yeast culture in malt extract
broth was harvested by centrifugation (3000×g, 10 min),
washed, and resuspended with saline solution (0.9%). One
milliliter of the suspension was placed in a disposable cuvette.
The absorbance was measured at 0, 2, and 24 h in a spectro-
photometer (Multiskan GO Microplate, Thermo Scientific,
USA) at a wavelength of 600 nm without shaking the cell
suspension. The autoaggregation was calculated as follows:
 
AT
Autoaggregation ð%Þ ¼ 1−  100
A0
AT is the absorbance at 600 nm at each time and A0 is the
absorbance at 600 nm at 0 time.
Hemolytic Activity
The hemolytic activity was assessed according to Argyri et al.
[16]. The yeast strain was inoculated in blood agar (5% sheep
blood) by streaking and incubated at 37 °C for 48 h. The
results were expressed according to the type of hemolysis,
i.e., α-hemolysis (green halos around the growth), β-
hemolysis (clear halos around the growth), or γ-hemolysis
(without halos).
Antimycotic Resistance
Fluconazole (25 μg), ketoconazole (10 μg), and nystatin (100 U)
were the antimycotics evaluated. The yeast was inoculated by
lawn on the surface of malt extract agar with sterile swabs; then,
paper discs impregnated with the specific concentrations of the
antimycotics were placed on the agar surface inoculated with
yeast. The antimycotics were dissolved in DMSO. Finally, the
diameter of the inhibition halos formed was measured after 24 h.
Antioxidant Activity
The antioxidant activity was evaluated according to the method
described by Gil-Rodríguez et al. [15] with some modifications.
Fresh yeast culture was harvested by centrifugation
(3000×g, 10 min), washed, and resuspended in saline solution
(0.9% NaCl). The yeast suspension (800 μL) was transferred
into a new tube, 1 mL of DPPH solution (0.2 mM in methanol)
was added, vortexed, and incubated at room temperature safe
from light. After 30 min, the tube was centrifuged (3000×g,
10 min) and 200 μL of the supernatant was transferred into
96-well plates to measure the absorbance at 517 nm. The
reduction of DPPH was expressed as a percentage calculated
according to the following equation:
 
A517 ðsampleÞ
Reduction of DPPH ð%Þ ¼ 1−  100
A517 ðblankÞ
Results were classified according to Gil-Rodríguez et al.
[15]: low activity (20–30%), good activity (30–40%), very
good activity (40–50%), and excellent activity (> 50%).
Extracellular Enzyme Production
To test the production of protease, lipase, and esterase, mod-
ified agars were elaborated. The surface of modified agars was
inoculated with 5 mL of standardized yeast suspension of 24 h
old broth culture (107 UFC/mL). The uninoculated plate
served as control.
Protease activity: Skim milk agar (skim milk 10 g, glucose
1 g, peptone 5 g, yeast extract 2.5 g, agar 15 g, distilled water
1 L) was used to assess the production of protease. The for-
mation of clear zones around the colonies after incubation at
30 °C for 48 h is considered as a positive result [17].
Lipase activity: Tributyrin agar (tributyrin 10 g, peptone
5 g, yeast extract 3 g, agar 15 g, distilled water 1 L) was used
to assess the production of lipase. The presence of clear halos
around the colonies after incubation at 30 °C for 48 h is con-
sidered as a positive result [17].
Esterase activity: To determine the productions of esterase,
an agar which contained tween 80 (10 g), NaCl (5 g), CaCl·
2H2O (0.1 g), peptone (10 g), agar (20 g), and distilled water
(1 L) was elaborated. The formation of opaque halo was con-
sidered as a positive result [18].
 Probiotics & Antimicro. Prot.

Results Hydrophobicity

Yeast Isolation and Identification
The yeast strain was isolated from Tibicos and was character-
ized macro- and microscopically. The yeast presented a pearl
color colony, circular shape, umbonate elevation and entire
margin. It presents vegetative reproduction by multilateral
budding; hence, its cellular shape is round. According to
API 20C AUX gallery, the yeast strain was positive for S.
cerevisiae, due to its ability to fully ferment D-glucose, D-ga-
lactose, D-maltose, and D-sucrose substrates, while D-xylose,
D-rafinose, and methyl-alpha-D-glucoside were fermented in a
lesser extent.
Subsequently, the identification was carried out by mass
spectrometry, where the genus and species of yeast were
obtained with a high percentage of confidence (99.9%).
The same result was obtained by the molecular identifica-
tion, which confirms the isolate species. It was based on
100% of the identity of 5.8S-ITS amplicon sequence (ac-
cession number of GeneBank KY816912.1) compared to
the database sequences in the BLAST analysis. The yeast
was identified as Saccharomyces cerevisiae by both
methods.
Resistance to Acidic, Bile Salt Condition
and Simulated Gastrointestinal Digestion
In this work, the survival of S. cerevisiae C41 was evaluated in
acidic conditions, in contact with bile salts, and in conditions
that simulate the GI tract (Table 1). No change in viability was
observed within the 60 min of exposure to pH 2.0, and
180 min of exposure to bile salts for S. cerevisiae C41, show-
ing no significant difference with respect to S. boulardii, and
therefore the same behavior in acidic and bile conditions.
However, at the end of simulated digestion, S. cerevisiae
C41 showed a reduction in the survival, while S. boulardii
remained viable during the process of simulated digestion.
In this study, S. cerevisiae C41 showed higher percentages of
hydrophobicity than S. boulardii. Both yeasts showed a great-
er affinity to chloroform (84.36 and 37.72%, for S. cerevisiae
and S. boulardii, respectively). While the values for n-
hexadecane were 66.15% for S. cerevisiae C41 and 24.06%
for S. boulardii.
Autoaggregation Ability
The autoaggregation ability of yeasts increases over time. S.
cerevisiae C41 obtained a favorable and rapid autoaggrega-
tion, showing percentages of 52.95% at 2 h and 96.9% at
24 h, similar to those obtained with S. boulardii (55.15 and
96.7% for 2 and 24 h, respectively).
Hemolytic Activity
S. cerevisiae C41 did not present the formation of a halo
around the colonies. Therefore, this yeast did not show hemo-
lytic activity, so, the safety of this yeast is not a concern.
Antimycotic Resistance
The antimycotic resistant results are shown in Table 2. In this
case, the highest inhibition halos were observed with nystatin
in both yeasts. Only S. boulardii shows resistance to
fluconazole.
Antioxidant Activity
S. cerevisiae C41 presented a DPPH reduction percentage of
63.03%, which according to Gil-Rodríguez et al. [15] shows
an excellent antioxidant activity. While the probiotic S.
boulardii exhibited a very good antioxidant activity with a
DPPH reduction percentage of 43.60%.

Table 1 Survival of simulated

gastrointestinal digestion and Time (min) % viability (S. cerevisiae C41) % viability (S. boulardii)
under acidic and bile conditions
Acidic condition (pH 2.0) 30 100 ± 0.00a 100 ± 0.00a
60 100 ± 0.00a 100 ± 0.00a
Bile condition (Oxgall) 180 100 ± 0.00a 100 ± 0.00a
Gastric juice 30 100 ± 0.00a 100 ± 0.00a
60 91.81 ± 10.17a 100 ± 0.00a
Intestinal juice 30 83.46 ± 9.24a 100 ± 0.00a
90 83.46 ± 9.24a 100 ± 0.00a
180 78.95 ± 2.87b 100 ± 0.00a

Mean ± SD. Means with the same letter are not significantly different (P < 0.05)
Probiotics & Antimicro. Prot.
Table 2 The diameter of inhibition halos formed by antimycotics (mm)
Antimycotic S. cerevisiae C41 S. boulardii
Fluconazole 21.5 ± 0.5a 0.0 ± 0.0b
Ketoconazole 20.0 ± 2.0a 18.5 ± 0.5a
Nystatin 25.0 ± 2.0a 24.0 ± 1.0a
Mean ± SD. Means with the same letter are not significantly different (P
< 0.05)
Extracellular Enzyme Production
S. cerevisiae C41 did not exhibit the production of the extra-
cellular enzymes tested, being in the same case Saccharo-
myces boulardii. The results were negative for esterase, lipase,
and protease for both yeasts.
Discussion
The identity of yeast isolated from Tibicos as S. cerevisiae
provides a greater opportunity to present probiotic character-
istics, because although it is already generally recognized as
safe (GRAS), besides, the yeast considered as probiotic be-
longs to Saccharomyces genera [19].
The first condition that a microorganism, which is going to be
used as probiotic, must satisfy is the survival in acidic and biliary
conditions, as well as the presence of other compounds such as
digestive enzymes, which are stress factors found in the gastro-
intestinal tract [20]. In this regard, yeasts are capable of with-
standing a wide range of pH, which is a favorable characteristic
since yeasts will find acidic pH conditions (2.5 to 3.5) in the
stomach, to later find an increase in gut pH (6.5–7.5), in addition
to bile and digestive enzymes. These factors may affect the
viability of probiotics, and in this way prevent them from caus-
ing the associated beneficial effects [21]. The results obtained in
this study show that S. cerevisiae C41 is able to survive with
these factors, indicating that the yeast has mechanisms that allow
it to adapt to stress conditions found in the gastrointestinal tract
(acid pH, bile acids, and digestive enzymes), and thus be able to
reach and stay viable to the implantation site and generate the
beneficial effect. These results are in agreement with previous
studies where tolerance to acidic conditions and bile salts in
yeasts isolated from foods is shown [22].
After passing through previously mentioned stress condi-
tions, S cerevisiae C41 must be able to adhere to epithelial
cells. Hydrophobicity and autoaggregation are properties as-
sociated with the adhesion. The hydrophobicity was assessed
measuring the interaction with chloroform and n-hexadecane,
which is related with the adhesion of the yeast to the intestinal
epithelium [23], due to the adhesion of microbial cells are
given by passive forces, electrostatic interactions, lipoteichoic
acids, lectins, and hydrophobic forces [24]. The technique
employed in this study has been used to measure the hydro-
phobicity on the surface of the cell of probiotics and is based
on the microbial affinity to an acid monopolar solvent
(chloroform) and a non-polar solvent (n-hexadecane) [25].
The results obtained in this work demonstrate that S.
cerevisiae C41 is more hydrophobic than S. boulardii. This
could indicate the presence of proteinaceous material on the
cell surface of the yeast, which could interact by hydrophobic
forces to achieve adhesion to the intestinal epithelium [26,
27]. Meanwhile, autoaggregation is the ability of probiotics
to form cell aggregates; this property is also mediated by cell
surface and has been associated with adherence to epithelial
cells [28], since when the cell aggregates are formed the ad-
hesion to the intestine increases, and allow the colonization in
the gastrointestinal tract [29]. The high percentage of
autoaggregation obtained in this work is promising since this
feature could favor adhesion and therefore the beneficial effect
that could be exercised by S. cerevisiae C41. Nonetheless,
hydrophobicity and autoaggregation are characteristics that
vary from organism to organism and from strain to strain,
and these variations may be due to differences in the expres-
sion of cell surface proteins as well as environmental condi-
tions that may affect cell surface expression [30].
Safety is another main characteristic of probiotic microor-
ganisms. Since a probiotic must lack factors that may affect the
host, such as hemolysis, which is a factor of virulence, with
which the availability of iron is facilitated causing anemia in
the host [31]. So, the results show that this virulence factor did
not represent a risk in the use of the S. cerevisiae C41.
As for antimycotics, nystatin is the most used for candidiasis
infections. However, this antimycotic affects the viability in its
totality of probiotic yeast, but a reabsorbable antifungal agent as
fluconazole can be used [32]. Another study had indicated that
if fluconazole is ingested between 4 and 6 h after ingestion of
probiotic yeast, this is not affected by the antifungal [33].
Probiotics can have other beneficial characteristics which
are specific to each strain. One of the most important and
desired characteristics is the antioxidant activity, since previ-
ous studies have indicated that diseases such as arthritis, car-
diovascular disease, and some tumors are the result of damage
caused by reactive oxygen species (ROS) [34–36], and mini-
mizing the damage caused by free radicals could prevent these
conditions [37, 38], so the interest in studying natural antiox-
idants has increased. Probiotics have cell components with
antioxidant activity, which can complement the antioxidants
obtained in the diet and the endogenous antioxidants in the
body [39, 40]. Numerous authors have mentioned that the
antioxidant activity of yeasts is associated to the high content
of (1 → 3)-β-D glucan in the cell wall and other cellular com-
ponents like superoxide dismutase, catalase, etc. [41–43].
On the other hand, the ability of a probiotic to produce extra-
cellular enzymes such as lipase, esterase, and protease, provides
health benefits by improving nutrient utilization in the intestine

[43]. These enzymes are also involved in the sensory properties
of fermented foods because they break down complex com-
pounds in food during fermentation [44, 45]. In this case, S.
cerevisiae did not present the production of the enzymes tested;
however, it could produce other desirable enzymes.
Conclusions
Most of the microorganisms recognized as probiotics are bac-
teria, and only one of them is a yeast, which belongs to the
genus Saccharomyces. In this study, the probiotic potential of
a yeast belonging to the same genus and widely used for food
production was evaluated. The yeast identified as Saccharo-
myces cerevisiae was isolated from water kefir grains known
as Tibicos, an artisanal starter with which some traditional
fermented beverages are made in Latin America. The yeast
studied showed probiotic characteristics similar to those shown
by Saccharomyces boulardii. This points to Tibicos and tradi-
tional fermented beverages as a source of useful yeasts, and the
Saccharomyces cerevisiae C41 as a yeast with potentially pro-
biotic characteristics.
Funding This research was supported by the National Council for
Science and Technology from Mexico (260107).
Compliance with Ethical Standards
Conflict of Interests The authors declare that they have no conflict of
interest.
Ethical Approval This study does not contain any studies with human
participants or animals performed by any of the authors.
Informed Consent For this type of study, formal consent is not required,
because it does not contain studies with human participants.
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