LFBT #723604, VOL 26, ISS 4

Characterization of Cell Wall

Extracts from Saccharomyces
cerevisiae with Immunological
Activity
Blanca Aguilar, Josu Sols, Juan Manuel Viveros,
Zaira Lpez, and Peter Knauth
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Characterization of Cell Wall Extracts from Saccharomyces cerevisiae with
Immunological Activity
Blanca Aguilar, Josu Sols, Juan Manuel Viveros, Zaira Lpez,
and Peter Knauth

Food Biotechnology, 26:113, 2012

Copyright Taylor & Francis Group, LLC
ISSN: 0890-5436 print / 1532-4249 online
DOI: 10.1080/08905436.2012.723604

Characterization of Cell Wall

Extracts from Saccharomyces
cerevisiae with Immunological
Activity
Blanca Aguilar1, Josu Sols1, Juan Manuel Viveros1,
Zaira Lpez2, and Peter Knauth2

Laboratorio de Microbiologa Industrial, Centro Universitario de Ciencias Exactas

e Ingenieras, Universidad de Guadalajara (UdG), Guadalajara, Jalisco, Mexico
2
Cell Biology Laboratory, Centro Universitario de la Cinega, UdG, Ocotln, Jalisco,
Mexico
The yeast cell wall (YCW) is composed mainly of -glucans, mannoproteins, and
chitin. -Glucans are well known immunomodulators, recognized by Toll-like receptor-4
(TLR-4) and inducing the nuclear factor-kappaB (NF-B) signaling pathway, which
is implicated in the synthesis of pro-inflammatory mediators such as interleukin-1beta (IL-1), tumor necrosis factor-alpha (TNF-), and nitric oxide (NO). Here
we evaluated the immunomodulating effect of the YCW extracts from three different strains of Saccharomyces cerevisiae (AR5, MG, and L013) obtained from Tequila
and bread-making processes by challenging intraperitoneal macrophages from BALB/c
mice with YCW extract and/or lipopolysaccharide (LPS) endotoxin from Escherichia
coli. Only the extract from AR5, which had a high glucan-to-mannan ratio, exhibited
an antagonistic effect upon LPS stimulation: The IL-1 and TNF- concentrations in
plasma increased slightly by 24% and 4%, respectively, and the NO level increased moderately by 200% compared with the control. When the YCW extracts had less glucan,
as found for MG and L013, the LPS stimulus had more of a synergistic effect on the
plasma IL-1 concentration, which increased about 175%, and on the NO level, which
rose 330470%. These results indicated that under certain conditions, YCW extracts
have an immune-attenuating effect; that is, the mice liberated much less IL-1 or nearly
equal amounts of TNF- or NO compared with the LPS stimulus alone.
Key Words: Saccharomyces cerevisiae; cell wall; beta-glucan; immunomodulator;
BALB/c mouse; macrophage

Address correspondence to Blanca Aguilar, Laboratorio de Microbiologa Industrial,

Centro Universitario de Ciencias Exactas e Ingenieras, Universidad de Guadalajara
(UdG), Boulevard Marcelino Garca Barragn 1421, 44420 Guadalajara, Jalisco,
Mexico; E-mail: agublanca@gmail.com

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B. Aguilar et al.

INTRODUCTION
Saccharomyces cerevisiae is one of the most widely studied yeasts due not only
to its traditional use in the bakery, brewing, and wine industries but also
because it is an important model organism in modern cell biology research
(Petranovic et al., 2010). In Mexico, fermentation of Agave must by S. cerevisiae
has a long tradition, from pre-Hispanic pulque to present-day (additionally
distilled) Tequila and mezcal. For Tequila production, at the end of the fermentation process, the S. cerevisiae population reached under optimal conditions
(5080 g/l of sugars, 30 C, aeration, and an additional nitrogen source) up
to 2 108 colony forming units (CFU)/mL and under suboptimal conditions
(140 g/L sugars), still about 1.2 108 CFU/mL. In 2006, about 243 million liters
of Tequila were produced (Lappe-Oliveras et al., 2008), but it must be considered that each liter of Tequila generates about 710 L of vinasses, that is,
the wastewater from ethanol distillation. These vinasses are highly recalcitrant, have a low pH (3.5), a high chemical and biochemical oxygen demand
(COD: 50 g/l; BOD: 25 g/l), 5 g/l total suspended solids, and 20 g/l total dissolved solids. Because they are highly colored, they prevent oxygenation of
rivers by blocking the light necessary for photosynthesis (iguez-Covarrubas
and Peraza-Luna, 2007). Despite the efforts carried out to date by the Tequila
industry to address the problem of vinasses, no significant progress in their
sustainable usage could be achieved. Therefore, it is of great interest to generate a biotechnological process to i) treat the polluting vinasses and/or ii) use the
residual yeasts to obtain bioactive compounds, which would change the status
of the vinasses from waste to that of recyclable and valuable commodity.
The cell wall of S. cerevisiae represents about 30% of the cells dry
weight and is made up of 15% of proteins and 85% of polysaccharides;
these are composed of 8090% glucose, 1020% mannose, and 12% of
N-acetylglucosamine. About 80% of the glucose residues are linked with each
other via -1,3-glycosidic bonds forming the -1,3-glucan chains with a degree
of polymerization of about 1,500 parts/chain. These chains are branched
by other -1,3-glucan chains, by -1,6-glucan chains, and by -1,4 polymers
of N-acetylglucosamine (chitin). Additionally, mannoproteins are linked with
-1,6-glucan by glycosyl-phosphatidylinositol (GPI) anchor or with -1,3-glucan
by alkali-labile bonds (Klis et al., 2006; Lesage and Bussey, 2006). While
glucans and chitin afford mechanical rigidity to the cell wall, mannoproteins
provide a negative charge at a slightly acidic pH in a hydrophilic environment
(Caridi, 2006).
-Glucans have been known for many years as the most effective
immunomodulators based on polysaccharides; they have very low toxicity
(lentinan in mouse: median lethal dose (LD50 ) > 1,600 mg/kg), are relatively
resistant to gastric acid, and are a good source of YCW (yeast cell walls)
(Novak and Vetvicka, 2009; Rop et al., 2009). Immunomodulating effects rely

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Cell Wall extract from S. cerevisiae with Immunological Activity

on increasing phagocytosis by granulocytes, monocytes, macrophages, and dendritic cells. Macrophages comprise part of the innate immune system that
utilizes pattern recognition receptors (PRR) to bind certain highly conserved
molecules present on the hostile microorganism, the so-called pathogenassociated molecular patterns (PAMP). -Glucan is recognized as a PAMP by
PRR, in concreto by Toll-like receptor-2 (TLR-2), by dectin 1 (also known as
CD11b/CD18 or Mac-1), CR3, and probably by others (Brown, 2006; Chen
and Seviour, 2007; Goodridge et al., 2009; Netea et al., 2004). The signaling pathways for gene expression modulated by -glucans have not been
fully elucidated to date, but generally, the pro-inflammatory nuclear factor kappaB (NF-B) pathway is induced, leading to a release of cytokines
tumor necrosis factor-alpha (TNF-), interleukin-1beta (IL-1), IL-2, IL-6, and
IL-12, causing phagocytosis and respiratory burst (Brown, 2006; Netea et al.,
2004). Additionally, interferon-gamma (IFN-) is liberated, which activates
macrophages and induces inducible nitric oxide synthase (iNOS) to produce
nitric oxide (NO), which in turn dilates the veins, causing a drop in blood pressure (Novak and Vetvicka, 2008). Other pro-inflammatory reactions include upregulation of cell adhesion molecules such as ICAM-1 or E-selectin (Morgensen,
2009), and arachidonic acid or the granulocyte-macrophage colony-stimulating
factor (GM-CSF) is liberated (Tsoni and Brown, 2008). Paradoxically, production of IL-10, an anti-inflammatory cytokine, is also stimulated (Tsoni and
Brown, 2008).
It is supposed that this induced immunostimulation explains the protective
effect of -glucans on experimental infections with different microorganisms in
several animal studies (Novak and Vetvicka, 2008). However, these biological
effects depend on the structure of the -glucans (solubility, branching degree,
molecular weight, charge) (Mantovani et al., 2008), and this in turn depends
on the source for -glucans as well as on the isolation process (Novak and
Vetvicka, 2009). Therefore, it is important to evaluate the immunostimulating
capacity of -glucans that are isolated in a simple and inexpensive procedure
from yeasts employed in the fermentation process of the Tequila production
with the aim of providing this waste (i.e., no longer used yeasts) with an
easily accessible value. This approach is different from other studies, as we
examine the immunomodulating properties of YCW components from cells not
especially grown for this purpose. Moreover, these components are even not
specially purified, as the isolation process should be simple, and therefore it
was unclear if sufficient immunostimulating effects can be achieved. We conducted this evaluation using two different strains from the Tequila industry
(and comparing these with one strain from the bakery industry); we allowed
these to grow under fermentation-like conditions, isolated the -glucans, and
tested the immunomodulating effect by determining the inflammatory mediators IL-1, TNF-, and NO produced by peritoneal macrophages of BALB/c
mice with and without additional stimulus by lipopolysaccharide (LPS).

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B. Aguilar et al.

MATERIALS AND METHODS

Strains
The Saccharomyces cerevisiae strains AR5 and MG, originally isolated from
two different Tequila distilleries in Jalisco (Mexico), were obtained from the
strain collection of the CIATEJ Research Centre (Guadalajara, Mexico) (Flores 120
et al., 2005). The third strain, L013, is a commercial strain commonly utilized
in the bakery industry (Ancel, France). All strains were kept on Potato dextrose
agar (PDA) plates and maintained under refrigeration for conservation. The
yeasts were lyophilized for long-term storage.

Yeast Cultivation

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To produce yeast biomass, 1-L Erlenmeyer flasks containing 350 ml

yeast-peptone-dextrose (YPD) medium (20 g/L yeast extract, Bioxon, Mexico;
20 g/L peptone, Bioxon, Mexico; and 30 g/L dextrose, pH 5.5) were inoculated
with 2 106 CFU/mL and grown at 30 C (not shaken). Growth of the yeasts
was monitored by measuring the Abs600 . In order to obtain sufficient cell wall 130
extract, this procedure was repeated five times for each strain.

Preparation of Cell Wall Extracts and Quantification of -Glucans

and Mannans
The yeasts were collected during the early stationary phase (Abs600 3.5),
considering that the yeasts were not yet under limiting nutritional conditions
(except for oxygen). The samples (50 mL) were harvested by centrifugation
(5 min, 4,500 g at 4 C), washed twice with ice-cold water, and the pellet
was resuspended in 1.0 mL ice-cold TE (10 mM Tris, 1 mM EDTA, pH 7.5).
To this we added 0.5 g 0.45-m glass beads and the cells were broken up
using a cell disrupter (Mini-Beadbeater; Biospec Products) with 20-s pulses
during 2060 min. In order to avoid overheating of the samples, they were
continuously cooled with ice-cold water. Cell lysis was confirmed microscopically. Afterwards, the extract was separated from the beads and washed twice
with ice-cold TE; the final crude cell wall extract was lyophilized, weighed, and
resuspended in 1 mL TE and stored at 20 C.
To analyze cell wall composition, a 100-L aliquot of each cell wall extract
(five different batches for each strain) was hydrolyzed with 72% H2 SO4 according to the method described by Dallies et al. (1998). The glucose and mannose
released were separated using HPLC (Varian ProStar 210) with a Metacarb
Ca Plus isothermal column at 80 C and a mobile water phase with a 0.6mL/min flow and quantified utilizing a refraction index detector (Varian model
356-LC).

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Cell Wall extract from S. cerevisiae with Immunological Activity

Animals
Eight-week-old male BALB/c mice (weight, 2025 g) were selected and
housed under standard environmental conditions (10 mice per cage) at room 155
temperature in a 12:12-h light-dark cycle and were fed ad libitum. The animals were managed according to guidelines for the use and care of laboratory
animals (Mexican Official Norm NOM-062-ZOO-1999), which was published in
the Mexican Official Gazette of the Federation on December 6, 1999.

Stimulation of Peritoneal Macrophages

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For each strain, the cell wall extracts from five different batches were
brought together to possess a sufficient and representative sample. These, as
well as the LPS from Escherichia coli 055:B5 (Sigma Chemical Co., St. Louis,
Mo., USA), were diluted in saline solution and administered into the peritoneum (i.p.) of each mouse at a final volume of 200 L. The following groups, 165
each with five mice, were analyzed:
1.

CT, control treatment, healthy mice without any treatment;

2.

CV, control vehicle, mice injected i.p. with 200 L saline solution;

3.

LPS, mice injected i.p. with 2.5 mg lyophilized LPS per kg of body weight;

4.

AR5, MG, or L013, mice injected i.p. with 2.5 mg lyophilized YCW (from 170
the respective strain) per kg of body weight; and

5.

AR5, MG, or L013+LPS, mice injected i.p. with 2.5 mg lyophilized YCW
(from the respective strain) per kg of body weight and 30 min later
additionally with 2.5 mg LPS per kg of body weight.

One and a half hours after the last session, the animals were anaesthetized 175
with diethyl ether and sacrificed. Blood was taken by cardiac puncture; the
plasma was immediately obtained by centrifugation (5 min, 2,500 g) and stored
at 20 C until subsequent determination of IL-1, TNF-, and NO.

IL-1 and TNF- Concentrations in Plasma

Plasma IL-1 and TNF- concentrations were measured using the respec- 180
tive High Sensitivity Colorimetric Sandwich ELISA kit (R&D Systems,
Minneapolis, Minn., USA). The sample was added into the specific antibodyprecoated microtiter plates and the unbound material was washed away.
Alkaline phosphatase-labeled antibodies, which also recognize the analyte,
were added and unbound antibodies were washed away. First NADPH as sub- 185
strate and then an amplifier solution were added to form a dark red stain,
whose absorbance was measured at 490 nm.

B. Aguilar et al.

Nitric Oxide (NO) Plasma Concentration

The NO that was generated and liberated into the plasma was measured as
a stable form of nitrite by using the Griess reagent (Green et al. 1982). Briefly,
100 L of plasma (sample) were mixed with 100 L Griess reagent (1:1 (v/v)
0.1% N-[1-naphthyl])ethylenediamine dihydrochloride in H2 O:1% sulphanilamide in 2 N HCl), incubated for 10 min at room temperature, and absorbance
at 540 nm was determined with a spectrophotometer.

Statistical Analysis

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Values are expressed as means (from five samples) standard deviation

(SD). Differences between groups were determined by one-way analysis of variance (ANOVA) and subjected post-hoc to Bonferroni multiple comparison tests.
A value of p < 0.05 was considered to indicate statistical significance.

RESULTS

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Yeast Cell Wall Composition

The two strains, AR5 and MG, from Saccharomyces cerevisiae, isolated from
the Tequila industry, did not show more similarity among each other regarding
cell wall composition on comparison with the bakery strain L013. The -glucanto-mannan ratio for AR5 was 1:0.79, meaning that the cell wall contained more
glucan than mannan; for both of the remaining strains, this was vice versa: MG
had a ratio of 1:1.76 and L013, one of 1:2.56 (Fig. 1).
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g polysaccharide/mg cell wall

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Glucan
Mannan

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40
30
20
10
0
AR5

MG

L013

Strain
Figure 1: Cell wall composition with regard to the polysaccharides -glucan ( ) and mannan

( ), of the three studied strains from S. cerevisiae: AR5 and MG (both isolated from the Tequila
industry) and L013 (from the bakery industry). Bars indicate the standard deviation (SD) of five
experiments.

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Cell Wall extract from S. cerevisiae with Immunological Activity

IL-1 Release from Stimulated Intraperitoneal Macrophages

The immunomodulatory effect of YCW components can be estimated
by injecting them i.p. into mice to stimulate their macrophages, which in 210
turn liberate cytokines. As representative indicators, the plasma concentrations of IL-1, TNF-, and NO were determined and compared with the
immunostimulating effect of LPS from Gram-negative bacteria Escherichia
coli.
Mice immunostimulated by YCW (AR5, MG, or L013) alone did not show 215
any more significant difference in plasma IL-1 levels (2127 ng/L) than the
control group (CV, 19 ng/L). In contrast, injection of LPS increased IL-1
plasma levels significantly (71 ng/L). Plasma IL-1 levels were also significantly higher when the mice were stimulated by LPS and YCW from MG or
L013 (52 ng/L). The sole exception was the YCW from AR5, which exhibited 220
a much higher -glucan amount in the cell wall than the other strains: this
extract lowered the plasma IL-1 concentration, induced by the LPS stimulus,
back to normal values (24 ng/L) (Fig. 2).

TNF- Release from Stimulated Intraperitoneal Macrophages

When the mice were stimulated only by YCW components, TNF- plasma 225
levels increased significantly (710738 ng/L) for AR5 and MG and not significantly (658 ng/L) for L013, compared with the control (CV, 601 ng/L). Also the
stimulus by LPS led to an increase in TNF- levels but not a significant one

IL-1 plasma concentration [ng/l]

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10
PS
L0
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L0
13
+L
PS

+L

LP

5+

AR

AR

LP
S

Treatment
Figure 2: Plasma levels of IL-1, produced by peritoneal macrophages from mice after injection

of endotoxin (LPS) or pooled yeast cell wall (YCW) from AR5, MG, or L013. Stimulation first with
pooled YCW and afterwards with endotoxin is indicated as LPS + AR5, LPS + MG, and LPS +
L013, respectively. Control w/o treatment (CT) and control with vector (CV). Bars indicate the
standard deviation (SD) of five experiments.

B. Aguilar et al.

TNF- plasma concentration [ng/l]

(638 ng/L). In combination, YCW from AR5 and MG exhibited inhibitory effects
on the TNF- liberation induced by an LPS stimulus (574624 ng/L), while 230
L013 YCW exhibited a contrary, even stimulating, effect (752 ng/L) (Fig. 3).

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PS

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+L

L0

13
L0

PS

G
M

+L
G

S
AR
AR
5
5+
LP
S

LP

Treatment
Figure 3: TNF- plasma levels produced by peritoneal macrophages from mice after injection

of endotoxin (LPS) or pooled yeast cell wall (YCW) from AR5, MG, or L013. Stimulation first
with pooled YCW and followed by endotoxin is indicated as LPS + AR5, LPS + MG, and LPS
+ L013, respectively. Control w/o treatment (CT) and control with vector (CV). Bars indicate
the standard deviation (SD) of five experiments.

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NO plasma concentration [M]

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50

L
L0 013
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+L
PS

S
AR
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AR
5+
LP
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M
G
M
G
+L
PS

LP

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V

0
C

Treatment
Figure 4: Plasmatic nitric oxide (NO) concentration generated by peritoneal macrophages
from mice after the following different stimuli: Control w/o treatment (CT); control with vector
(CV); injection of endotoxin (LPS), or pooled yeast cell wall (YCW) from strain AR5, MG, or L013,
as well as injection first of pooled YCW and afterwards of LPS (LPS + AR5, LPS + MG, and LPS +
L013, respectively). Bars indicate the standard deviation (SD) of five experiments.

Q1

Cell Wall extract from S. cerevisiae with Immunological Activity

Nitric Oxide Release from Stimulated Intraperitoneal

Macrophages
Immunostimulation of mice with LPS significantly increased the plasma
NO level (105 M) compared with that of the control (CV, 46 M). YCW injected 235
alone had even a stronger stimulatory effect, which was more pronounced
for the high -glucan strain AR5 (266 M) than for MG or L013 (154176
M). However, in combination (i.e., YCW and LPS), the effects were opposite:
employing YCW from AR5, the plasma NO concentration decreased significantly (140 M), with YCW from the intermediate strain MG, NO release did 240
not change significantly (197 M), while with high-mannan YCW from L013,
NO release increased even further (263 M).

DISCUSSION
Ratio of -Glucan to Mannan
The -glucan-to-mannan ratio for the three strains (AR5, MG, and
L013) was between 1:0.79 and 1:2.56. Similar results utilizing the same
medium, and that which also was not shaken in order to simulate fermentation
conditions, were found previously by our group: the AR5 strain had a -glucanto-mannan ratio of 1:0.84; another strain (denominated SLR) from the Tequila
industry had a 1:1.10 ratio, and a control strain (denominated LP) from the
bakery industry had a ratio of 1:1.15 (Aguilar-Uscanga et al., 2007); the latter
was rather different compared with the bakery strain L013. Employing Agave
must as growth medium (as in Tequila production), the -glucan-to-mannan
ratio was 1:0.72 for AR5, 1:1.15 for SLR, and 1:1.28 for LP, that is, fairly similar
to the ratio found utilizing the YPD medium. The principal difference between
both media was that the total amount of polysaccharides was, with Agave must,
about 2540% lower than with YPD (Aguilar-Uscanga et al., 2007). Thus, we
employed YPD in this study as growth medium in order to obtain more cell wall
components for the subsequent studies, considering that -glucan-to-mannan
proportions are quite similar.

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Cytokine Release from Stimulated Intraperitoneal Macrophages

The mechanism of how -glucan induces immunomodulatory effects
remains unclear. Several reports claim a generally enhanced host defense
(Chen and Seviour, 2007; Novak and Vetvicka, 2009) but also suppression
of early pro-inflammatory cytokines, such as IL-1, IL-6, and TNF- (Bedirli 265
et al., 2007; Medeiros et al., 2006; Soltys and Quinn, 1999), has been reported.
The latter would be an important mechanism for preventing an excessive
immunoreaction, thus preventing septic shock (Bedirli et al., 2007).

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Here we can demonstrate that the immunomodulating response on YCW

components depends highly on their glucan-to-mannan ratio; the more glucan,
the better the effects for potentially preventing septic shock. This can best
be observed for the plasma IL-1 concentration: injection of any YCW alone
into the mice did not alter IL-1 levels while LPS led to an increase in the
level by 250%. Only mice pretreated with the high glucan YCW (AR5) did not
react on the additional LPS; their IL-1 levels increased only slightly by 20%.
However, when the mice were pretreated with YCW from MG or L013, the IL1 level continued to increase significantly by 160%, although less so than when
treated with LPS alone. A similar effect was reported by Bedirli et al. (2007):
rats increased IL-1 plasma levels nine-fold within 5 h after a sepsis-inducing
Cecal ligation and puncture (CLP) procedure, but when rats were treated with
2 mg/kg -glucan the IL-1 level increased only four-fold.
With TNF- as an early pro-inflammatory marker, the signs were less
clear: administration of YCW to the mice had already increased plasma TNF-
levels slightly (by 1023%), which was more than by LPS alone (7%). A stimulus by both led to a decrease in TNF- levels to normal values with YCW
from AR5 and MG (antagonistic effect), but with that from L013 (low glucan
content), it increased slightly by 14% (synergistic effect). Olson et al. (1996)
found that macrophages in vitro released TNF- in a -glucan concentrationdependent manner, with a peak at about 200 g/mL, which agrees with our
observations in vivo. Soltys and Quinn (1999) measured in vitro TNF- production of monocytes isolated from BALB/c mice: a stimulus with 10 g/mL
-glucan, which would be equivalent to 60 mg/kg in mouse, according to
Raghav et al. (2007), increased the TNF- concentration two-fold within 4 h, in
contrast to a six-fold increase induced by 10 g/mL LPS. When the mice were
pretreated with 1 mg/kg -glucan, the isolated monocytes liberated only threefold more TNF- upon the LPS stimulus compared with the control. These
results coincide with ours: that -glucan alone induces liberation of TNF-
and that pre-treatment with -glucan had a slight antagonistic effect on the
LPS-induced TNF- release.
Either LPS (via TLR-4 and MD-2; Miyake, 2004) or -glucan (via TLR-2;
Netea et al., 2004) stimulate the NF-B signalling pathway, leading to liberation of TNF-, IL-1, IL-6, and IL-12, which finally activates iNOS of, for
example, macrophages. Accordingly, in our experiment, the high glucan YCW
(AR5) raised NO production by 480% compared with that of the control, while
YCW from MG or L013 had a much lower effect (an increase of 235280%). The
LPS stimulus alone also increased the plasma NO concentration, but only by
130%. On injecting both into the mice, high-glucan YCW again exhibited the
greatest (protective) effect: NO production continued to increase by 200%, yet
this was comparable with that of the LPS stimulus alone. The remaining YCW
exerted a contrary effect: they led to an increase in plasma NO concentration
of 330% (MG) and of 480% (L013), respectively. This effect can be explained

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Cell Wall extract from S. cerevisiae with Immunological Activity

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by the concerted action of TNF- and IL-1; if either the IL-1 or the TNF concentration increased due to a stimulus, NO production also increased,
or both concentrations required being kept low in order to maintain the NO
concentration in blood low also.
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CONCLUSION
The immunomodulating effects induced by -glucans are not simply a general immunostimulation. Depending on their concentration, they also possess
immuno-attenuating properties. Especially, the latter effect could potentially
be used to prevent postsurgical septic shock because, after pretreatment with
YCW-derived -glucan, fewer pro-inflammatory cytokines are released, consequently, less NO is liberated. Hence, vascular permeability does not increase
and transmigration of neutrophils is hindered, which in turn increases the survival rate by inhibiting septic shock reactions (Akramiene et al., 2007; Bedirli
et al., 2007; Medeiros et al., 2006).
The aim of this work is to find usage for residual yeasts from alcohol fermentation process, which are produced annually in high amounts, by using
them as source for obtaining YCW components. To be profitable for industries,
the purification step should be simple and not expensive. Immunomodulating
properties of YCW extracts obtained under these conditions have not been
evaluated before. Here we could demonstrate that the effectiveness of these
extracts depends highly on their glucan-to-mannan ratio, which cannot be
predicted easily, because this is determined mainly by the yeast strain itself
and less by the growing conditions: While the YCW extract from one strain
(AR5) was quite effective, that from the other strain (MG) was not, although
both were S. cerevisiae and both were isolated from a similar fermentation process (Agave must) used in the same region (Jalisco State, Mexico).
This is especially important for artisan distilleries, which frequently use
spontaneous inoculation rather than specific inoculation with known strains
(Lappe-Oliveras et al., 2008).

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ACKNOWLEDGMENTS
We thank PROMEP and COECYTJAL-UdG for the support offered for carrying
out this work.

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