FOODBORNE PATHOGENS AND DISEASE

Volume 16, Number 9, 2019

ª Mary Ann Liebert, Inc.
DOI: 10.1089/fpd.2018.2606

Equilibrium Isotherm Approach to Measure the Capability

of Yeast Cell Wall to Adsorb Clostridium perfringens

Elisa Santovito,1 Donato Greco,1 Vito D’Ascanio,1 Virginie Marquis,2 Ruth Raspoet,2
Antonio F. Logrieco,1 and Giuseppina Avantaggiato1

Abstract
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Yeast cell wall (YCW) products are currently used as substitutes to antibiotic growth promoters, to improve
animal performances, and to reduce the incidence of infectious diseases in livestock. They are claimed to bind
enteropathogens, thus interfering with their colonization in the intestinal mucosa. Although the anti-infectious
activity of YCW products on Gram-positive pathogens like Clostridium perfringens has been reported in vivo,
in vitro evidences on the adsorption of C. perfringens by YCW fractions are not yet available. Preliminary
results showed that purified YCW products exert antimicrobial activity toward C. perfringens. Using the
adsorption isotherm approach, we measured the ability of YCW products in adsorbing C. perfringens, thus
reducing its viability. Dosages of YCW products >1 mg/mL adsorbed 4 Log colony-forming unit (CFU)/mL of
C. perfringens in buffered solution. The maximum adsorption of the bacterium was reached in 3 h, whereas only
one product of four YCW products retained the adsorption up to 6 h. The analysis of equilibrium isotherms and
adsorption kinetics revealed that all products adsorb C. perfringens in a dose- and time-dependent manner, with
high affinity and capacity, sequestering up to 4 Log CFU/mg of product. The determination of adsorption
parameters allows to differentiate among adsorbents and select the most efficient product. This approach
discriminated among YCW products more efficiently than the antimicrobial assay. In conclusion, this study
suggests that the ability of YCW products in reducing C. perfringens viability can be the result of an adsorption
mechanism.

Keywords: yeast cell wall, antimicrobials, Clostridium perfringens, equilibrium adsorption isotherm, adsorp-
tion parameters, growth promoters, necrotic enteritis, feed additives

Introduction Granum, 2002). Different toxinotypes induce a wide variety

of diseases throughout the animal kingdom, such as gas

C lostridium perfringens is a Gram-positive spore-

forming obligate anaerobic pathogen widely distributed
throughout the environment. It forms spores allowing sur-
vival at normal cooking temperatures and germination during
slow cooling or storage at ambient temperature (McClane
et al., 2013).
The etiology induced by C. perfringens typically manifests
as conditions where numerous cells of the pathogen colonize
the lumen of the small intestine and produce toxins that are
absorbed into the circulation, inducing damages to other
organs such as brain, lungs, and kidneys (Collins et al., 1989).
C. perfringens strains can produce *17 toxins that are re-
sponsible for tissue death, hemolysis, vasoconstriction, in-
creased vascular permeability, and shock (Brynestad and
gangrene, enterotoxaemia, food poisoning, and necrotic en-
teritis (NE) (Uzal et al., 2014).
In the past years, the over usage of antibiotics as growth
promoters (AGPs) in livestock has been considered respon-
sible for contributing to creating resistance among bacteria,
including the C. perfringens population (Hermans and Mor-
gan, 2007; Riaz et al., 2017). As a consequence, rising con-
cerns over the residual effect of antimicrobials on human
health and the transfer of antimicrobial resistance to human
pathogens led (EU, 2003) to the ban by EU of the marketing
and use of antibiotics (other than coccidiostats and histo-
monostats) as growth promoters from January 2006 (Her-
mans and Morgan, 2007; Riaz et al., 2017). The AGPs’ ban
created the necessity to look for compounds that can

1
National Research Council, Institute of Sciences of Food Production (CNR-ISPA), Bari, Italy.
2
Phileo-Lesaffre Animal Care, Marcq-en-Baroeul, France.

630
 CLOSTRIDIUM PERFRINGENS ADSORPTION BY YEAST DERIVATIVES
effectively replace them to control NE (Riaz et al., 2017), as
it has been a common perception that NE is a re-emerging
disease (Hermans and Morgan, 2007).
The role of animal feed in the production of safe food is
recognized worldwide, for its impact on animal health and
food security. Therefore, feed additives constitute an im-
portant group of preharvest interventions that can help in
controlling foodborne pathogens at farm level (Van Im-
merseel et al., 2002; Hermans and Morgan, 2007; Riaz et al.,
2017).
Some substances are being used or researched as feed
supplements for their antimicrobial effect, like prebiotics
(inulin, fructo-oligosaccharides, mannan-oligosaccharides,
among others), probiotics (yeasts, lactic acid bacteria, among
others), and other anti-infectious substances (organic acids,
botanical or herbal extracts, flavors and etheric oils, among
others) (Santovito et al., 2018). Yeast cell wall (YCW)
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products are used worldwide as alternatives to AGPs for the
promotion of health and performance in livestock, based on
their capacity to bind enteropathogenic bacteria (Ganner
et al., 2013), and for their immunomodulatory activity (Ko-
gan and Kocher, 2007; Volman et al., 2008). YCW fractions
are suggested as anti-adhesive agents and are thus proposed
to prevent attachment of pathogenic bacteria by interfering
with the adhesion of bacterial fimbriae or afimbrial adhesins
with the surface lectins of the intestinal cells of the host,
hence interfering with the colonization in the intestinal mu-
cosa (Herrera et al., 2000; Ganner et al., 2010; Trevisi et al.,
2012).
Using in vitro agglutination and microscopy observation,
different authors showed the capacity of YCW components to
bind pathogenic bacteria such as Escherichia coli and Sal-
monella spp. (Herrera et al., 2000; Ganner et al., 2013).
These studies also demonstrated the ability of YCW products
to bind enterotoxigenic bacteria, thus inhibiting their adhe-
sion on the brush border of intestinal villi (Trevisi et al.,
2012). Ganner et al. (2010) demonstrated that a purified
YCW can differentially bind Gram-negative pathogens, such
as some E. coli strains and Salmonella spp., but do not bind
Gram-positive pathogens as probiotic bacteria of the genera
lactobacilli and bifidobacteria, and C. perfringens. However,
in a recent study, using electron microscopy analysis, Posa-
das et al. (2017) showed that C. perfringens physically ad-
heres to live yeast cells. On the whole, all these in vitro results
suggest that YCW products may exert a selective binding
effect against some pathogenic bacteria without having ad-
verse effect on other bacteria. However, because of differences
Table 1. Chemical Composition of Yeast Cell Wall Products and Endogenous Microbial Contamination
Nitrogen Proteins Glucans b-Glucans Mannans Lipids/lipoproteins
(% DM) (% DM) (% DM) (% DM) (% DM) (% DM)
YCW1 2.9 18.2 30.3 27.6 26.8
YCW2 2.9 17.8 23.4 22.2 22.7
YCW3 2.8 17.5 22.2 19.1 13
YCW4 3.8 23.8 28.7 23.7 22.3
Data of YCW products composition are given as percentage on dry matter (% DM). Total viable aerobe count data are given as
mean – standard deviation of three independent experiments.
CFU, colony-forming unit; TVC, total viable aerobe count according to ISO 4833-1 (2013); YCW, yeast cell wall.
631
in composition, production process, and origin, generalizations
concerning the pathogens adsorption ability of YCW products
are inconsistent, and more research is needed to confirm the
effectiveness of YCW products to capture strict anaerobic
pathogens like C. perfringens. YCW products are underrep-
resented in the literature as anti-C. perfringens agents. Limited
in vivo reports have highlighted the beneficial effects of yeasts
in maintaining performance and health while minimizing
mortality and morbidity in broilers challenged with C. per-
fringens in an antibiotic-free production situation (Van Im-
merseel et al., 2004; Thanissery et al., 2010).
The objective of this study was to investigate the adsorp-
tion of the bacterium C. perfringens by purified YCW
products and to measure the effect of the adsorption on the
viability of the pathogen. For this purpose, we used the
equilibrium isotherm approach to study the adsorption ca-
pacity of YCW products toward C. perfringens, and the effect
of YCW product concentration, incubation time, and C.
perfringens concentration on the adsorption.
Materials and Methods
YCW products
Phileo LeSaffre Animal Care kindly provided the four
purified YCW products used in this study. The products
contained cell wall fragments derived from Saccharomyces
cerevisiae. Table 1 gives the chemical composition of YCW
products as provided by the supplier. YCW products were
analyzed for microbial contamination by aerobes and an-
aerobes, according to ISO methods (ISO4832, 2006;
ISO4833-1, 2013).
Bacterial strains and culture conditions
C. perfringens strains and their origins are given in
Table 2. All strains were grown at 37C under anaerobic
conditions in thioglycollate broth (TG; Biolife, Italy). The
inoculum density was standardized by photometric mea-
surement at 600 nm wavelength using a Ultrospec 3100 Pro
Spectrophotometer (Amersham, United Kingdom). The
enumeration of C. perfringens was performed by serial 10-
fold dilutions in physiological saline and pour-plating on
tryptose sulfite cycloserine (TSC) agar (Biolife) according to
ISO methods (2004). Strains were maintained in TG broth
supplied with 20% glycerol and stored at -80C. Culture
media and all the solutions were stored in anaerobic condi-
tions in a glove box (Plas-Lab, MI) to prevent oxidation.
Particle
size Yeast TVC
(nm) origin (Log[CFU/mg])
13.6 300 Bakery 0.30 – 0.01
20.5 50 Bakery 0.09 – 0.19
5.7 60 Bakery 0.48 – 0.01
Not available 50 Brewery 0.58 – 0.18
 632
Table 2. List of Clostridium perfringens Strains
and Their Source
Strain name Origin Supplier
ATCC13124T Reference LGC Standards, Milan, Italy
material
CP56 Poultry U-Ghent, Ghent, Belgium
AD211b Poultry ADRIA Développement,
Quimper, France
AD1600 Water ADRIA Développement,
Quimper, France
Antimicrobial activity assay
Minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) were determined by the
broth dilution method (Andrews, 2001). YCW products were
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tested in twofold dilution series in the range of 0.5–
10 mg/mL. Growth controls and blanks were obtained using
sterile broth in place of culture and YCW product suspen-
sions, respectively. Tubes were incubated at 37C for 18 h in
anaerobic conditions. The MIC is the concentration of the
higher dilution tube in which the absence of bacterial growth
occurred, with no visible turbidity with respect to the relevant
blank. For MBC determination, 1 mL of the suspension was
taken out, duly diluted, and used to determine the viable
residual cells (colony-forming unit [CFU]/mL) by plate
counting. The MBC is the lowest concentration of YCW
products that reduces the viability of the initial bacterial in-
oculum by ‡99.9%.
Preliminary adsorption assay
Preliminary adsorption experiments were performed using
the type strain ATCC13124 as reference. In sterile disposable
culture tubes with sealable cap, 1 mL of a C. perfringens
culture suspension containing 1.2 – 0.2 · 105 CFU was added
to 9 mL of Dulbecco’s 1 · phosphate-buffered saline (PBS;
pH 7.2), containing different amounts of YCW products to
gain final concentrations at 0.5, 1.0, 2.5, 5.0, and 10.0 mg/mL.
Test tubes were manipulated under anaerobic conditions in a
glove box. Sealed tubes were vortexed and incubated at 37C
with a constant shake at 250 rpm to promote YCW–cells
interaction. Controls were prepared without the addition of
YCW (YCW = 0 mg/mL). After 3 h of incubation, major ag-
gregates were allowed to settle for 15 min, and 1 mL of the
suspension was gently taken out, duly diluted, and used to
determine the viable residual cells (CFU/mL) by plate
counting. We chose not to centrifuge the tubes to avoid the
sedimentation of partly adsorbed bacteria that might still be
viable. Sediments were resuspended 1:10 in PBS, and the
presence of viable cells was verified by plate counting as
described previously. Blanks were prepared per YCW con-
centration, using PBS buffer in place of the bacterial sus-
pension. All adsorption experiments were performed in
triplicate. Cell count reduction values expressed in percent
were plotted as a function of YCW dosage.
Adsorption experiments
Adsorption experiments were performed at constant tem-
perature (37C), pH (pH 7.2), and bacterial concentration.
SANTOVITO ET AL.
One milliliter of a C. perfringens culture suspension con-
taining 1.2 – 0.2 · 105 CFU was added to 9 mL of Dulbecco’s
1 · PBS buffer containing a fixed amount of YCW. Unless
otherwise mentioned, in all experiments the test tubes were
prepared in anaerobic conditions in a glove box. Sealed tubes
were vortexed and incubated at 37C with a constant shake at
250 rpm. At the selected incubation time, aggregates were
allowed to settle for 15 min, pH was verified, and 1 mL of the
liquid was gently taken out. The CFU/mL count of non-
adsorbed cells in the upper liquid was determined by plate
counting. Controls were prepared without the addition of
YCW (YCW = 0 mg/mL). Blanks were prepared per YCW
concentration, using PBS buffer in place of the bacterial
suspension. All adsorption experiments were performed in
triplicate. The following equation [Eq. (1)] was used to cal-
culate the amount of adsorbed bacterial cells (A):
(C0  C)
A¼
C0
where C0 is the bacterial concentration (Log[CFU/mL]) in
controls with no YCW products; C, CFU count (Log[C-
FU/mL]) found in the suspension in the test tubes with the
YCW. The calculated quantity was expressed in percent and
used as an indicator of the cell count reduction. Nonspecific
adhesion of bacteria to the test tubes was <1% of the quantity
added in all experiments. Furthermore, any reduction in
bacterial concentration of the suspensions was assumed to be
caused by bacterial adsorption by YCW products.
With the purpose of investigating the effect of contact time
on the adsorption, C. perfringens (1.2 – 0.2 · 104 CFU/mL)
was incubated in PBS containing 1 mg/mL of each YCW, for
1–6 h.
To determine the adsorption capacity of YCW products,
adsorption isotherms were carried out at constant temperature
and pH, testing a fixed amount of YCW products, with dif-
ferent C. perfringens concentrations. Equilibrium isotherms
were carried out using optimal contact time (3 h) and opti-
mal YCW product dosage (0.1% w/v, corresponding to
1 mg/mL), as determined by preliminary experiments. One
milliliter of bacterial suspension at increasing concentrations
from 103 to 107 CFU/mL was added to 9 mL of PBS con-
taining weighted amounts of YCW products in sterile test
tubes. Tubes were gently shaken at 250 rpm to attain the
equilibrium. After 3 h, aggregates were allowed to settle for
15 min, suspension aliquots were carefully collected from the
top of the solution, and the residual CFU were determined by
plate counting.
The amount of adsorbed cell per unit mass of YCW at
equilibrium (Qeq) was calculated using the following equation:


V C0  Ceq
Qeq ¼ ,
m
where C0 is the Log CFU in the control tubes without YCW
(Log[CFU/mL]); Ceq, residual Log CFU in the experimental
tubes with YCW at equilibrium (Log[CFU/mL]); V, volume
of solution (mL); and m, mass of YCW (mg).
Adsorption isotherms were obtained by plotting the num-
ber of CFU adsorbed per unit of mass (mg) of YCW (Qeq)
against the residual concentration of the bacterium in the
suspension (Ceq), under equilibrium conditions [Qeq = f(Ceq)].
 CLOSTRIDIUM PERFRINGENS ADSORPTION BY YEAST DERIVATIVES
The effectiveness of YCW products in adsorbing C. per-
fringens was proven on the strains given in Table 2. Cell
suspensions in PBS containing 1 mg/mL of each YCW were
incubated for 3 h. The initial bacterial concentration was
1.2 – 0.2 · 104 CFU/mL in all cases. The presence of viable
cells in the sediments was determined as described previously.
Results
Effect of YCW products on C. perfringens viability
Similar to overall YCW composition, the four YCW
products used in this study contained polysaccharides (19–
28% of b-glucans, 22–30% of glucans, and 13–27% of
mannans), and 18–24% of proteins. YCW products did not
differ substantially in chemical composition. Particle size
was 50 nm for YCW2 and YCW4, and 60 nm for YCW3,
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whereas the size of YCW1 particles was 300 nm (Table 1).
As all YCW products were not sterilized before they were
used for the study, in order not to compromise their structure,
they were analyzed for endogenous microbial contamination.
In all cases, the total viable aerobe count was negligible,
being <0.5 LogCFU/mg of product (Table 1). Furthermore,
neither C. perfringens isolates nor anaerobic bacteria were
found. All YCW products showed antimicrobial activity. The
MIC values were 10 mg/mL for YCW1, and 5 mg/mL for
YCW2, YCW3, and YCW4. The MBC value was 10 mg/mL
for all YCW products. To verify that the antimicrobial ac-
tivity of YCW products was the result of an adsorption pro-
cess, we studied the reduction in the viable cell count in the
presence of YCW products in a buffered solution. The use
of PBS buffer minimized the bacterial replication, and kept
C. perfringens concentration constant, as confirmed by plate
FIG. 1. Effect of YCW concentration on Clostridium perfringens adsorption. Adsorption isotherms were obtained in PBS
at constant temperature (37C), pH (7.2), C. perfringens concentration (4.07 – 0.06 Log[CFU/mL]) and contact time (3 h),
and varying YCW concentration (1–10 mg/mL). Values are mean of three replicates, and error bars indicate standard
deviation. CFU, colony-forming unit; PBS, phosphate-buffered saline; YCW, yeast cell wall.
633
counting of the control tests (YCW = 0 mg/mL) at the end of
the incubation period. Therefore, the reduction of bacterial
viability measured by the adsorption tests can be ascribed to
the effect of YCW products on C. perfringens.
In the preliminary adsorption assay, the number of viable
cells decreased significantly when YCW products concen-
tration increased. No viable cell was found in the upper liquid
fraction using YCW product dosage ‡1 mg/mL (Fig. 1). At
the dosage of 0.5 mg/mL, the residual C. perfringens count
was 0.19 – 0.03 Log(CFU/mL) for YCW1, 0.54 – 0.02
Log(CFU/mL) for YCW2, 0.35 – 0.02 Log(CFU/mL) for
YCW3, and 0.22 – 0.01 Log(CFU/mL) for YCW4. Sedi-
mentation of YCW product–bacteria aggregates was fast and
well visible. Sediments in the test tubes did not contain viable
cells at any of the YCW product concentrations. Blanks did
not show any bacterial growth after 24 of incubation. These
results suggest that the optimal YCW dosage to study C.
perfringens adsorption is 1 mg/mL. Indeed, it is not useful to
increase the dosage of YCW products beyond 1 mg/mL to
reduce the viability of the pathogen.
Adsorption of C. perfringens by YCW products
The optimal experimental conditions for bacterial ad-
sorption (contact time and C. perfringens concentration)
were determined to measure the extent of C. perfringens
adsorption by YCW products.
The effect of contact time for the adsorption of bacterial
cells by YCW products was studied for 6 h (Fig. 2). Adsorption
kinetics showed that all YCW products sequestered most in-
oculated cells within the initial 3 h. At this time, maximum
adsorption values expressed as percentage and logarithm of
adsorbed CFU per mg of product were as follows: 95% – 2%
 634 SANTOVITO ET AL.
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FIG. 2. Effect of contact time on adsorption rate of Clostridium perfringens by YCW products. Adsorption experiments
were performed in PBS at constant temperature (37C), pH (7.2), YCW concentration (1 mg/mL), C. perfringens con-
centration (4.06 – 0.15 Log[CFU/mL]), and varying contact time (1–6 h). Values are mean of three replicates, and error bars
indicate standard deviation. CFU, colony-forming unit; YCW, yeast cell wall.

(3.86 – 0.01 Log[CFU/mg YCW2]), 93% – 1% (3.80 – 0.01
Log[CFU/mg YCW4]), 91% – 1% (3.70 – 0.01 Log[CFU/mg
YCW3]), and 87% – 2% (3.56 – 0.01 Log[CFU/mg YCW1]).
The YCW2 was selected as the most effective in adsorbing
C. perfringens. From the kinetic experiments, the plot of CFU
adsorption versus time showed that the rate of adsorption by
YCW2 is fast at the initial stages of the contact and then
becomes slower near equilibrium. Half the total C. perfringens
adsorption occurred in <1 h, and maximum adsorption was
reached in 3 h. Of interest, no further changes in the adsorption

FIG. 3. Effect of Clostridium perfringens concentration on the adsorption by YCW products. Equilibrium adsorption
isotherms were obtained keeping constant temperature (37C), pH (7.2), contact time (3 h), YCW dosage (1 mg/mL), and
varying C. perfringens concentration (3–7 Log[CFU/mL]). Values are mean of three replicates, and error bars indicate
standard deviation. CFU, colony-forming unit; YCW, yeast cell wall.
 CLOSTRIDIUM PERFRINGENS ADSORPTION BY YEAST DERIVATIVES
were observed beyond 3 h up to 6 h for YCW2. On the con-
trary, all other YCW products yielded lower adsorption values
at longer times (4–6 h). At 6 h of contact time, adsorption values
for YCW products decreased, being 64% – 1% (2.60 – 0.01
Log[CFU/mg]) for YCW1, 54% – 1% (2.18 – 0.01 Log[CFU/
mg]) for YCW4, and 18% – 1% (0.71 – 0.01 Log[CFU/mg)])
for YCW3. These results suggest that YCW2 retained adsorbed
cells over time, whereas YCW3 released most adsorbed cells
that were still culturable. A rapid adsorption of C. perfringens,
an establishment of equilibrium in a short time, and a retaining
of the adsorbed fraction imply the efficacy of YCW2 in ad-
sorbing C. perfringens.
The equilibrium isotherms for C. perfringens adsorption
by YCW products are given in Figure 3. Isotherms were
obtained at a constant temperature, pH, YCW dosage, and
equilibrium adsorption time, whereas C. perfringens con-
centration varied in a range of five orders of magnitude. All
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YCW products yielded similar adsorption isotherms. A de-
crease in the quantity of adsorbed bacteria was observed for
all YCW products when the bacterial concentration was in-
creased. C. perfringens adsorption values varied from 34% to
100%. Experimental values for maximum adsorption ca-
pacity were 4.43 – 0.16 Log(CFU/mg YCW2), 3.96 – 0.02
Log(CFU/mg YCW1), 3.72 – 0.03 Log(CFU/mg YCW4),
and 3.60 – 0.05 Log(CFU/mg YCW3). For all isotherms, a
fall in slope occurred after the first inflection.
Finally, all YCW products were further tested to determine
the capability of binding C. perfringens strains other than the
type strain. As given in Figure 4, YCW products showed
some differences in the adsorption of bacterial strains. In par-
ticular, YCW2 and YCW4 adsorbed almost 100% of CP56,
FIG. 4. Effect of YCW on the viability of different
Clostridium perfringens strains. The strains Ad1600, CP56,
Ad211b, and ATCC13124 were incubated at the concen-
tration of *4 Log(CFU/mL)in PBS, at constant temperature
(37C), pH (7.2), and contact time (3 h), with 1 mg/mL of
each YCW. Values are mean of three replicates, and error
bars indicate standard deviation. CFU, colony-forming unit;
YCW, yeast cell wall.
635
Ad211b, and ATCC13124 strains, and adsorbed Ad1600 strain
by *70% and 50%, respectively. YCW3 adsorbed 70–80% of
Ad211b and ATCC13124 strains, and *40% of Ad1600 and
CP56. YCW1 adsorbed almost 100% of Ad211b strain, and
<55% of the other strains.
Discussion
YCW fractions and derived products have been proposed
to bind enteropathogenic bacteria, and their use as feed
supplements has been suggested for the prophylaxis and the
control of gut pathogens in livestock (Ganan et al., 2012).
The overall effect of YCW products is a reduction of the
viability of specific bacterial pathogens and an increase in the
number of beneficial bacteria, with a consequent improve-
ment of animal’s performance and immunity (Santovito et al.,
2018). Despite the commercial success of YCW products, the
mechanisms at the base of YCW products–pathogen interac-
tion are not completely clear. The three-dimensional structure
of mannose within the YCW is presumed to be relevant for
pathogen immobilization (Ganner and Schatzmayr, 2012), and
also receptor interaction, hydrophobic and other nonspecific
interactions might be involved in the adhesion process (Ofek
et al., 2003).
The inhibition mechanism exerted by YCW products on
pathogens seems to be limited to some specific Gram-
negative enteropathogens (Salmonella and E. coli). However,
recent in vivo studies reported the efficacy of YCW products
in improving the immune system response in starter broilers
under C. perfringens challenge (Fowler et al., 2015), as well
as an improvement in performance, and a reduction in mor-
tality and gut lesions associated with NE (M’Sadeq et al.,
2015). C. perfringens has been found to bind to the wall of
live yeast cells (Posadas et al., 2017), although no informa-
tion was provided on the extent of adsorption or on the via-
bility of the target bacterium after adsorption. We designed
an isotherm adsorption study to assess the ability of YCW
products to bind C. perfringens and reduce the viability of
adsorbed cells. Unlike the agglutination method, which is
commonly used to qualitatively investigate the binding capacity
of yeast products (Pérez-Sotelo et al., 2005), the isotherm ad-
sorption method proposed herein allowed determining the
maximum adsorption capacity for the target pathogen and the
selection of the most efficient adsorbent material.
Quantifying the adsorbed cells and moreover, the non-
adsorbed ones, is of primary importance in assessing the
antimicrobial potential of an adsorbent product, because
nonadsorbed cells are still capable of replicating in the host
organism, thus starting an infectious process. Indeed, we
measured the nonadsorbed cells, which were still viable and
culturable after incubation in the presence of YCW products.
The results of MIC and MBC analysis showed that YCW
fractions (50–300 nm) reduce the viability of C. perfringens
and adsorb the pathogen. C. perfringens is rod-shaped bac-
terium measuring 4–8 lm in length and 0.8–1.5 lm in di-
ameter (Murray et al., 2015). Given the particle size of YCW
products, it can be assumed that YCW products adsorb onto
the bacterial surface and probably block vital metabolism of
the bacterium.
The equilibrium isotherm approach revealed that all YCW
products adsorbed C. perfringens in a dosage- and time-
dependent manner, and with high affinity and capacity.
 636
A decrease in the quantity of adsorbed bacteria was observed
for all YCW products when the bacterial concentration was
increased. The relationship between the CFU count in the
suspension at equilibrium and the number of adsorbed cells
could not be described by the typical adsorption isotherm
equations (such as the Langmuir or the Freundlich models)
(Freundlich, 1907; Langmuir, 1918). For all products, the
isotherm adsorption curves resemble the shape of the class
‘‘H’’ (high affinity) isotherms (subgroup mx), as defined by
Giles et al. (1960). The ‘‘H’’ curve is a special case of the
Langmuir type ‘‘L’’ curve. Three regions can be recognized
in these isotherms: the region of complete adsorption; the
region of gradual rise; and the region with a downward trend
resulting in lower adsorption capacity. In the first part of the
isotherm, C. perfringens had such high affinity with YCW
products that it was completely adsorbed until maximum
adsorption was reached. The initial part of the isotherm was
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therefore vertical, running parallel to the y-axis. After that, a
change in the slope occurred in the curves and the adsorp-
tion continued to increase until maximum adsorption was
reached. The fall in slope is probably because of the associ-
ation of the solutes (bacterial cells) in solution; with an in-
crease in concentration, the solute–solute attraction begins
to increase more rapidly than the adsorbent–solute attrac-
tion. Indeed, the solutes yielding H-type isotherms are often
large units, as in this study being bacterial cells (Giles et al.,
1960).
Adsorption kinetics showed that all YCW products se-
questered most inoculated cells within the initial 3 h. C.
perfringens adsorption by YCW products was fast at the
initial stages of the contact with the bacterial cells, and half of
the total adsorption occurred in <1 h. Only one product re-
tained the adsorbed cells up to 6 h, whereas all other YCW
products yielded lower adsorption values at 4–6 h. The less
effective products released most adsorbed cells, which were
still culturable. On the other side, adsorbed cells in the sed-
iment of the test tubes were not culturable, thus suggesting
that C. perfringens adsorption by YCW products determined
a reduction in metabolic activity, as hypothesized by other
authors ( Jiang et al., 2007; Ganner et al., 2013).
Conclusions
The findings of this study show that the in vitro efficacy of
YCW products in reducing the number of culturable cells of
C. perfringens can be the result of an adsorption process.
All YCW products tested in this work adsorbed C. per-
fringens with high affinity and capacity within the initial 3 h.
The most efficient product was YCW2, which retained the
adsorbed cells up to 6 h. Differences on the adsorption ob-
served among strains of the pathogen need to be further
elucidated.
In our hypothesis, YCW fragments (50–300 nm) may ad-
here to the surface of the bacterial cell, with the consequence
of interfering with its primary functions (such as replication)
and reducing viability. The standard plating method used in
this study measures only the subset of bacteria in suspension
that are viable and culturable, although no information is
available on the presence of viable bacteria that are not cul-
turable. The improvement of the use of YCW—as AGPs or as
treatment—involves a better understanding of the genetic
and the biochemistry underlying the adhesion phenomenon,
SANTOVITO ET AL.
which could eventually lead to its superexpression in spe-
cially engineered or selected yeast strains.
Further research is required to elucidate at the molecular
level, the mechanisms triggered by YCW products’ adhesion
to bacteria, especially the effects of adsorption on toxin
production by enterotoxigenic bacteria.
Acknowledgments
The authors thank Prof. Filip Van Immerseel (U-Ghent,
Belgium) and ADRIA Développement (Quimper, France) for
providing the strains used in this study.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Elisa Santovito, PhD
National Research Council
Institute of Sciences of Food Production (CNR-ISPA)
Via Amendola 122/O
Bari 70126
Italy
E-mail: elisa.santovito@ispa.cnr.it