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Citation: Alkalbani, N.S.; Osaili, T.M.;
Al-Nabulsi, A.A.; Obaid, R.S.;
Olaimat, A.N.; Liu, S.-Q.; Ayyash,
M.M. In Vitro Characterization and
Identification of Potential Probiotic
Yeasts Isolated from Fermented Dairy
and Non-Dairy Food Products. J.
Fungi 2022,8, 544. https://doi.org/
10.3390/jof8050544
Academic Editor: Laurent Dufossé
Received: 23 April 2022
Accepted: 19 May 2022
Published: 23 May 2022
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Fungi
Journal of
Article
In Vitro Characterization and Identification of Potential
Probiotic Yeasts Isolated from Fermented Dairy and Non-Dairy
Food Products
Nadia S. Alkalbani 1, Tareq M. Osaili 2,3 , Anas A. Al-Nabulsi 3, Reyad S. Obaid 2, Amin N. Olaimat 4,
Shao-Quan Liu 5and Mutamed M. Ayyash 1,*
1Department of Food Science, College of Agriculture and Veterinary Medicine, United Arab Emirates
University (UAEU), Al Ain P.O. Box 15551, United Arab Emirates; 950223010@uaeu.ac.ae
2Department Clinical Nutrition and Dietetics, University of Sharjah,
Sharjah P.O. Box 27272, United Arab Emirates; tosaili@sharjah.ac.ae (T.M.O.); robaid@sharjah.ac.ae (R.S.O.)
3Department of Nutrition and Food Technology, Jordan University of Science and Technology,
Irbid 21121, Jordan; anas_nabulsi@just.edu.jo
4Department of Clinical Nutrition and Dietetics, Faculty of Applied Medical Sciences, The Hashemite
University, P.O. Box 330127, Zarqa 13133, Jordan; aminolaimat@hu.edu.jo
5Department of Food Science and Technology, Faculty of Science, National University of
Singapore, Singapore 117542, Singapore; fstlsq@nus.edu.sg
*Correspondence: mutamed.ayyash@uaeu.ac.ae
Abstract:
This study is about the isolation of yeast from fermented dairy and non-dairy products
as well as the characterization of their survival in
in vitro
digestion conditions and tolerance to bile
salts. Promising strains were selected to further investigate their probiotic properties, including cell
surface properties (autoaggregation, hydrophobicity and coaggregation), physiological properties
(adhesion to the HT-29 cell line and cholesterol lowering), antimicrobial activities, bile salt hydrolysis,
exopolysaccharide (EPS) producing capability, heat resistance and resistance to six antibiotics. The
selected yeast isolates demonstrated remarkable survivability in an acidic environment. The reduction
caused by
in vitro
digestion conditions ranged from 0.7 to 2.1 Log
10
. Bile salt tolerance increased
with the extension in the incubation period, which ranged from 69.2% to 91.1% after 24 h. The
ability of the 12 selected isolates to remove cholesterol varied from 41.6% to 96.5%, and all yeast
strains exhibited a capability to hydrolyse screened bile salts. All the selected isolates exhibited heat
resistance, hydrophobicity, strong coaggregation, autoaggregation after 24 h, robust antimicrobial
activity and EPS production. The ability to adhere to the HT-29 cell line was within an average
of 6.3 Log
10
CFU/mL after 2 h. Based on ITS/5.8S ribosomal DNA sequencing, 12 yeast isolates
were identified as 1 strain for each Candida albicans and Saccharomyces cerevisiae and 10 strains for
Pichia kudriavzevii.
Keywords: autoaggregation; coaggregation; antimicrobial resistance; probiotics; yeast
1. Introduction
Probiotics are defined as ‘live microorganisms that, when administered in adequate
amounts, confer a health benefit on the host’ [
1
]. Probiotics contain various microorgan-
isms, including bacteria and yeasts [
2
]. Lactic acid bacteria (LAB) and Bifidobacteria are
the main sources of probiotic strains [
3
,
4
], which are widely used as supplements or in
food industries. In contrast, to date, only a probiotic yeast, Saccharomyces cerevisiae var.
boulardii, has gained the qualified presumption of safety (QPS) status from the European
Food Safety Authority as a probiotic supplement [
5
]. S. cerevisiae var. boulardii is used in
numerous countries to prevent and treat several gastrointestinal disorders [
6
]. However,
the scientific community is witnessing a significant increase in the number of scientific
studies on the isolation, characterization and identification of non-Saccharomyces yeasts
J. Fungi 2022,8, 544. https://doi.org/10.3390/jof8050544 https://www.mdpi.com/journal/jof
J. Fungi 2022,8, 544 2 of 19
(e.g., Pichia,Schizosaccharomyces,Kluyveromyces,Rhodotorula and Candida) and reporting
them as promising probiotics [7–10].
Yeasts are unicellular eukaryotic microorganisms commonly found in soli, air, water,
and food and are of animal and plant origin; they constitute <0.1% of microbiota in the
human gut [
11
,
12
]. The use of yeasts as probiotics has gained increasing attention within
the last few years, owing to their high contents of minerals, vitamin B, peptides, proteins
and several immunostimulant compounds, such as mannan oligosaccharides, proteases
and
β
-glucans [
9
,
13
,
14
]. Moreover, yeasts exhibit good resistance to industrial conditions,
such as high temperature and lyophilization [15–17].
Currently, yeasts have gained increasing interest in the field of food biotechnology,
including their roles in recombinant protein production, alcoholic fermentation and vi-
tamin biosynthesis [
9
,
18
]. Furthermore, in the production of bread, beer, table olives,
wine or kefir, yeasts are used as starters [
19
,
20
]. Pichia kudriavzevii and a combination
of
S. cerevisiae
var. boulardii and inulin are used to produce fermented cereal-based food
and symbiotic yogurt, respectively [
21
,
22
]. Yeasts are also associated with the maturation
of certain cheeses [
23
]. Although yeasts may be a contaminant present in various foods
(e.g., fruit juices, chocolate and yoghurt) that could cause food spoilage, many yeasts have
been found to exhibit antimicrobial activity against foodborne pathogens and/or spoilage
microorganisms [24,25].
The characterization of new probiotic candidates needs to follow the criteria estab-
lished by the United Nations/World Health Organization (FAO/WHO) in 2002. The most
important among these criteria is tolerance to the gastrointestinal tract (GIT) [
26
] con-
ditions (low pH, digestive enzymes, bile salts and alkaline pH), adhesion to epithelial
cells, bile salt hydrolysis (BSH), assimilation of cholesterol in the human intestine and
food, antimicrobial activities and antibiotic sensitivity [
1
]. Furthermore, probiotic candi-
dates should exhibit high-temperature tolerance for industrial purposes and the ability to
produce exopolysaccharides (EPS) [27].
The biofunctional market continuously requires the diversification and application of
novel products that provide new probiotic strains with specific functional properties [
28
].
Probiotic yeasts can provide functional properties that bacterial probiotics cannot. Thus,
isolation of new probiotic yeasts is always required to meet the demands of the functional
food and beverage market. The present study aimed (1) to isolate novel yeasts from dairy
and non-dairy fermented food products, (2) to characterize the potential probiotic attributes
of these newly isolated yeasts, including tolerance to the GIT conditions, cell surface and
adhesive properties (autoaggregation, hydrophobicity, coaggregation and HT-29 cell line
adhesion), antimicrobial activities, antibiotic sensitivities, heat tolerance, EPS production,
ability to remove cholesterol and BSH activity, and (3) to identify the best potential probiotic
yeasts using molecular techniques.
2. Materials and Methods
2.1. Sample Collection
A total of 105 samples of various fermented dairy and non-dairy food products
sources free of any food preservatives were collected from different local markets in the
United Arab Emirates (UAE). The samples were placed in an icebox and transported
to the food microbiology lab of the UAEU for the isolation and characterization of the
potential probiotic yeast strains. Unless otherwise stated, all chemicals were purchased
from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Isolation of Yeasts
The food samples were serially diluted with 1% peptone water (Neogen, Lansing,
MI, USA). The pour-plate technique was employed using Yeast Extract–Peptone–Dextrose
(YPD) agar (Himedia Laboratories Pvt. Ltd., Nashik, India), and the plates were aerobically
incubated at 25
◦
C for 5 days (Binder C 170, Tuttlingen, Germany). Three copies of each
colony isolates were subcultured in the YPD broth; subsequently, the stocks were prepared
J. Fungi 2022,8, 544 3 of 19
using glycerol (50% v/v) and then stored at
−
80
◦
C. The potential probiotic characteristics
of the yeast isolates were evaluated after two successive activations at 25 ◦C.
2.3. Acid Tolerance: Preliminary Probiotic Investigation
Acid tolerance of the yeast isolates was evaluated at pH 2.5. A suspension of the
tested yeast isolates was prepared in YPD broth and incubated at 25
◦
C for 24 h. The
suspension was centrifuged at 5000
×
gfor 10 min, washed with phosphate-buffered saline
(PBS)
(0.1 M, pH 7)
and resuspended in 3 mL YPD broth with the pH adjusted to 2.5 using
1 M HCl. Subsequently, the suspension was distributed in 24-well plates and incubated at
25
◦
C for 24 h. A 1 mL solution of the resuspended yeasts pellets in a YEP broth without
pH adjustment (pH 6.7) was considered a control. The growth levels of yeast strains were
measured at OD600.
2.4. Tolerance to In Vitro Digestion Conditions
In vitro
digestion tolerance was evaluated using the method described by Brod-
korb et al. [
29
]. The
in vitro
gastrointestinal INFOGEST 2.0 protocol was applied to the
yeast strains. A 2 mL aliquot of the yeast pellet suspension was subjected to
in vitro
diges-
tion, including the oral (amylase 75 U/mL, salivary fluid SSF pH 7.0, 0.3 M CaCl
2
, 2 min,
37
◦
C), gastric (pepsin 2000 U/mL, RGE 60 U/mL, gastric juice SGF pH 3.0, 0.3 M CaCl
2
,
120 min, 37
◦
C) and intestinal (pancreatin 100 U/mL, bile 10 mmol/L, duodenal juice SIF
pH 7.0, 0.3 M CaCl
2
, 120 min, 37
◦
C) phases. Continuous shaking at 120 rpm was applied
during the
in vitro
digestion process. Serial dilution was performed to directly measure
the yeast count before and after the in vitro digestion.
2.5. Bile Salt Tolerance
The bile salt tolerance of the selected yeast isolates was tested according to AlKa-
lbani et al. [
30
]. The selected yeasts were tested against 0.3% oxgall, 0.1% cholic acid and
0.1% taurocholic acid, individually, during 0, 6 and 24 h of incubation at 37
◦
C. The growth
levels of yeast strains were recorded at OD600.
2.6. Cholesterol Removal
According to Alameri et al. [
31
], the capability of the selected yeast isolates to remove
cholesterol was measured using o-phthalaldehyde at 550 nm. The cholesterol removal (%)
was expressed as follows:
Cholesterol removal (%)=100 −residual cholestrol at each incubation interval
100 ×100
2.7. Bile Salt Hydrolysis (BSH) Activity
The BSH activities were determined by measuring the amount of amino acids released
from conjugated bile salts by yeast strains according to the method described by AlKa-
lbani et al. [
30
]. The BSH activities were assayed against 6 mM sodium glycocholate, 6 mM
sodium taurocholate or 6 mM conjugated bile salt mixture (glycocholic, glycochenodeoxy-
cholic, taurocholic, taurochenodeoxycholic and taurodeoxycholic acids).
2.8. Autoaggregation
Autoaggregation assay of the activated cultures was performed according to the
method described in [
32
], and absorbance was measured at 600 nm at the time intervals of 0,
3, 6 and 24 h. The autoaggregation percentage was calculated using the
following equation:
Auto −aggregation(%)=1−At
A0×100 (1)
where ‘A
t
’ denotes the absorbance at the time ‘t’, and ‘A
00
denotes the absorbance at the
time ‘00.
J. Fungi 2022,8, 544 4 of 19
2.9. Hydrophobicity
Hydrophobicity was evaluated against three different hydrocarbons, n-hexadecane,
xylene and octane, according to the method described by Fadda et al. [
14
]. The final
absorbance was measured at 600 nm. The hydrophobicity percentage was expressed
as follows:
Hydrophobicity(%)=A−A0
A×100
where ‘A’ denotes the initial absorbance at 600 nm, and ‘A00denotes the final absorbance.
2.10. Coaggregation
The coaggregation experiment was conducted according to the method described by
Andrade et al. [
33
] at 37
◦
C during incubation for 4, 6 and 24 h against four pathogens:
Escherichia coli 0157:H7 1934, Staphylococcus aureus ATCC 25923, Salmonella Typhimurium
02–8423
and Listeria monocytogenes DSM 20649. The coaggregation percentage was calcu-
lated using the following equation:
Co −aggregation(%)=A0−At
A0×100
where ‘A
t
’ denotes the absorbance at the time ‘t’, and ‘A
00
denotes the absorbance at the
time ‘00.
2.11. Antimicrobial Activity
The cell-free supernatant of the activated selected yeast isolates was used to determine
the antibacterial activity against four foodborne pathogens: L. monocytogenes,Salmonella
Typhimurium 02-8423, E. coli O157:H7 and S. aureus. The antimicrobial test was conducted
according to the method described by Hossain et al. [34].
2.12. Antibiotic Susceptibility
The resistance of the selected yeast isolates to antibiotics (2-
µ
g clindamycin (CLI),
10-
µ
g ampicillin (AMP), 25-
µ
g trimethoprim-sulfamethoxazole (SXT), 10-
µ
g penicillin
(PEN), 30-
µ
g vancomycin (VA) and 15-
µ
g erythromycin (E) (Oxoid; Hampshire, UK)) was
evaluated using the YPD agar. This methodology was adapted from Tarique et al. [
35
]. The
interpretative zones of resistant (R), moderately susceptible (MS) and susceptible (S) were
defined according to the method described in [36].
2.13. Adhesion to the HT-29 Cell Line
To evaluate the adhesion ability of selected yeasts, the activated isolates were washed
twice with Dulbecco’s phosphate-buffered saline. The adhesion property was tested ac-
cording to the method described by Hong et al. [
37
] and measured in percentage using the
following equation:
Adhesion ability(%)=At
A0×100
where Atdenotes the number of the adhered cells (log CFU/mL) after incubation, and A0
denotes the initial cell number (log CFU/mL).
2.14. EPS Production
The ability of the selected yeast isolates to produce EPS (
−
ve/+ve) was measured
according to the method described by Angmo et al. [
38
], where yeasts cultured overnight
were streaked onto the surface of plates containing ruthenium red milk agar (10% w/vskim
milk powder, 1% w/vsucrose, 0.08-g/L ruthenium red, 1.5% w/vagar).
J. Fungi 2022,8, 544 5 of 19
2.15. Heat Resistance
Heat resistance of the selected yeast isolates was measured according to the method
described by Teles Santos et al. [
39
] at 60
◦
C for 5 min. Serial dilution was performed to
directly measure the yeast count before and after heat treatment.
2.16. Molecular Identification of the Selected Yeast Isolates
A total of 12 yeasts were selected and subjected to PCR amplification of the ITS/5.8S ri-
bosomal DNA. DNA extraction and purification were performed using DNeasy UltraClean
Microbial Kit (Qiagen, Carlsbad, CA, USA) and PCR Kit (BIONEER, Daejeon, Korea) ac-
cording to the manufacturer’s protocols. PCR analysis was conducted as detailed in [
40
,
41
]
and according to Amorim et al. [
7
] using primers ITS1 (5
0
-TCCGTAGGTGAACCTGCGG-3
0
)
and ITS4 (5
0
-TCCTCCGCTTATTGATATGC-3
0
). Sequencing was performed at the Macrogen
sequencing facilities (Macrogen-Korea, Seoul, Korea). Yeast identification was achieved by
comparing the obtained sequences with those available from the NCBI database using the
BLAST algorithm. The accession numbers of the selected yeast isolates were obtained by
GenBank
®
. The neighbour-joining method was employed to determine the closest yeast
species using the MEGA software version 11 [42,43].
2.17. Statistical Analysis
To determine whether the variations between yeast isolates had a significant influence
on quantitative parameters, one-way ANOVA and Tukey’s test were conducted to examine
the differences between the mean values at p < 0.05. All tests were conducted at least
three times.
3. Results and Discussion
A total of 105 colonies with different morphological properties were isolated on YPD
agar from different food products sold in the local market. The selected yeast isolates were
purified and preserved at −80 ◦C in 50% glycerol containing YPD broth.
3.1. Preliminary Acid Tolerance
The acid tolerance percentages of 105 isolates at pH 2.5 during 24 h of incubation
at 37
◦
C are presented in Table S1 and summarized in Figure 1(boxplot). The yeasts
isolates exhibited various levels of survivability at low pH (0.0% to 100%). A total of
45 yeast
isolates that demonstrated noticeable acid tolerance were selected to investigate
their tolerance to in vitro digestion conditions and bile salt.
The beneficial aspects of probiotics can be exploited if they exhibit resistance to an
acidic environment. Thus, acid tolerance is a pivotal factor that allows the candidate
probiotic to pass through the gastrointestinal tract (GIT) in a vital and adequate amount
and to be used in the food industry. In this study, a low acidic medium pH of 2.5 at
37 ◦C
was used as a preliminary indicator for potential probiotic features that could be held
in our isolates. Generally, adjustment of yeast cell walls and activation of the cell wall
integrity and general stress response pathways are the main strategies that enable the
selected probiotic yeasts to resist a strong inorganic acid [44,45].
In the present study, high survivability in an acidic medium is preferred. The strains
were basically isolated from low-pH environments such as fermented dairy and non-dairy
products, where they cohabited with the lactic and/or acetic acid produced by bacteria.
In this context, the results of Santos et al. [
46
] and Moreira et al. [
47
] are consistent with
ours. ¸Sanlidere Alo˘glu et al. [
48
] tested the different yeast species they collected at pH 2.5
according to our acid tolerance conditions.
J. Fungi 2022,8, 544 6 of 19
J. Fungi 2022, 8, x FOR PEER REVIEW 6 of 19
Figure 1. Boxplot summarizing the survival rate (%) of the 105 yeast isolates under pH 2.5 for 2 h at
37 °C. Bullets represent outliners.
The beneficial aspects of probiotics can be exploited if they exhibit resistance to an
acidic environment. Thus, acid tolerance is a pivotal factor that allows the candidate pro-
biotic to pass through the gastrointestinal tract (GIT) in a vital and adequate amount and
to be used in the food industry. In this study, a low acidic medium pH of 2.5 at 37 °C was
used as a preliminary indicator for potential probiotic features that could be held in our
isolates. Generally, adjustment of yeast cell walls and activation of the cell wall integrity
and general stress response pathways are the main strategies that enable the selected pro-
biotic yeasts to resist a strong inorganic acid [44,45].
In the present study, high survivability in an acidic medium is preferred. The strains
were basically isolated from low-pH environments such as fermented dairy and non-dairy
products, where they cohabited with the lactic and/or acetic acid produced by bacteria. In
this context, the results of Santos et al. [46] and Moreira et al. [47] are consistent with ours.
Şanlidere Aloğlu et al. [48] tested the different yeast species they collected at pH 2.5 ac-
cording to our acid tolerance conditions.
3.2. Tolerance to In Vitro Digestion Conditions and Bile Salts
Table 1 presents the survival rates of potential yeast probiotics before and after being
subjected to in vitro digestion with simulated fluids and bile stress against oxgall, cholic
acid and taurocholic acid at different concentrations. The growth of all yeast isolates de-
creased (p < 0.05) under in vitro digestion conditions. The yeasts’ count reduction after in
vitro digestion ranged from ~0.7 to 2.1 Logs. In general, isolates O63, SH45, SH40, O12,
O26, SH46 and SH55 exhibited the highest resistance to in vitro digestion conditions. On
the other hand, the yeast isolates demonstrated remarkable resistance to oxgall compared
Figure 1.
Boxplot summarizing the survival rate (%) of the 105 yeast isolates under pH 2.5 for 2 h at
37 ◦C. Bullets represent outliners.
3.2. Tolerance to In Vitro Digestion Conditions and Bile Salts
Table 1presents the survival rates of potential yeast probiotics before and after being
subjected to
in vitro
digestion with simulated fluids and bile stress against oxgall, cholic
acid and taurocholic acid at different concentrations. The growth of all yeast isolates
decreased (p< 0.05) under
in vitro
digestion conditions. The yeasts’ count reduction after
in vitro
digestion ranged from ~0.7 to 2.1 Logs. In general, isolates O63, SH45, SH40, O12,
O26, SH46 and SH55 exhibited the highest resistance to
in vitro
digestion conditions. On
the other hand, the yeast isolates demonstrated remarkable resistance to oxgall compared
with cholic and taurocholic acids. The bile salt tolerance of the yeast isolates increased
with the extension in the incubation period, which ranged from 43.8% to 87.9%, 17.4%
to 85.7% and 68.4% to 86.7% after 6 h and from 48.9% to 90.5%, 26.5% to 89.5% and
69.2% to 91.1% after
24 h
. Overall, isolates SH104, SH105, SH 96, G1, SH46, O12 and O24,
among others, exhibited high bile resistance. Twelve isolates with high survivability in
in vitro
digestion conditions were selected according to their varying isolated sources for
subsequent investigations. These isolates were G1, O12, O13, O18, O21, O26, O36, O63,
O66, SH40, SH45 and SH55.
J. Fungi 2022,8, 544 7 of 19
Table 1. In vitro
digestion conditions and bile salt tolerances for 45 potential probiotic yeast isolates.
Isolate
Tolerance to GIT Bile Salt Tolerances (%)
6 h 24 h
Before After Log Reduction 0.3 CA 1.0 TA 1.0 OX 0.3 CA 1.0 TA 1.0 OX
G.1 7.3 ±0.01 5.5 ±0.03 1.8 54.2 36.9 74.1 68.3 81.4 89.1
G.2 7.5 ±0.09 5.4 ±0.02 2.1 53.7 36.1 74.8 66.7 73.5 88.1
G.3 7.6 ±0.24 6.2 ±0.12 1.4 71.6 52.5 70.7 84.3 82.1 83.3
G.6 7.4 ±0.09 6.3 ±0.11 1.1 73.8 61.6 77.2 78.9 81.1 87.5
G.7 7.4 ±0.13 6.1 ±0.12 1.3 66.4 68.6 79.7 80.8 83.4 88.0
G.8 7.5 ±0.11 6.1 ±0.06 1.4 71.0 81.7 79.6 83.8 85.6 88.3
G.9 7.5 ±0.07 6.2 ±0.10 1.3 67.8 76.8 80.0 79.9 81.5 84.5
G.10 7.5 ±0.02 6.2 ±0.02 1.4 70.5 64.7 80.8 83.0 80.7 87.6
O.12 7.3 ±0.06 6.3 ±0.04 0.9 80.5 78.9 81.0 87.4 84.1 87.4
O.13 7.5 ±0.06 6.2 ±0.03 1.3 72.8 68.2 79.2 80.5 81.1 87.1
O.18 7.5 ±0.01 6.3 ±0.02 1.2 67.0 80.0 84.8 79.9 81.4 88.7
O.19 7.4 ±0.04 6.1 ±0.03 1.3 69.0 45.9 85.8 78.2 58.0 86.2
O.20 7.5 ±0.02 6.3 ±0.01 1.2 79.8 42.4 80.6 86.1 81.5 87.1
O.21 7.6 ±0.19 6.4 ±0.20 1.3 82.2 59.2 86.3 86.9 80.7 89.1
O.22 7.5 ±0.04 6.3 ±0.05 1.2 82.9 76.7 84.2 87.5 79.4 87.9
O.23 7.4 ±0.08 6.2 ±0.03 1.2 73.7 67.8 83.8 79.8 75.6 88.0
O.24 7.5 ±0.03 6.5 ±0.09 1.0 84.5 70.3 86.7 87.9 83.7 91.1
O.26 7.4 ±0.06 6.2 ±0.09 1.2 80.3 69.5 82.0 83.4 77.9 88.2
O.30 7.5 ±0.01 6.3 ±0.10 1.2 67.0 61.6 79.7 78.7 75.4 86.0
O.33 7.4 ±0.03 6.4 ±0.06 0.9 73.9 62.7 80.5 83.1 69.9 83.9
O.36 7.4 ±0.05 6.2 ±0.01 1.3 84.3 79.2 84.3 87.7 85.7 89.8
SH.40 7.4 ±0.08 6.6 ±0.09 0.9 73.7 63.4 81.3 81.4 77.9 86.9
SH.45 7.1 ±0.02 6.1 ±0.04 1.0 65.1 62.7 84.0 84.6 74.4 86.1
SH.46 7.2 ±0.10 6.3 ±0.11 0.9 70.5 63.4 82.2 85.0 76.6 90.7
SH.55 7.0 ±0.24 6.0 ±0.16 1.0 73.2 68.2 72.7 86.0 80.9 84.4
O.63 7.1 ±0.12 6.4 ±0.08 0.7 64.2 64.8 81.4 66.9 65.9 86.4
O.65 7.2 ±0.00 6.3 ±0.04 1.0 68.0 62.7 81.4 77.1 75.1 86.7
G.69 7.3 ±0.06 6.3 ±0.06 1.0 43.8 17.4 68.6 48.9 26.5 69.8
O.66 7.2 ±0.15 5.4 ±0.07 1.8 53.1 51.7 69.2 57.5 66.0 69.9
G.75 7.4 ±0.20 5.9 ±0.11 1.5 52.5 57.0 69.4 57.5 61.3 70.3
G.71 7.5 ±0.13 6.2 ±0.16 1.3 76.4 70.4 72.7 86.6 82.7 83.3
G.77 7.2 ±0.17 5.8 ±0.12 1.4 78.5 75.7 76.6 87.2 87.3 85.6
G.78 7.1 ±0.06 6.1 ±0.04 1.0 81.7 72.7 77.3 88.1 85.5 86.1
G.80 7.2 ±0.21 5.9 ±0.10 1.3 67.2 78.0 76.5 80.3 84.3 86.4
G.82 7.4 ±0.09 5.9 ±0.06 1.5 78.1 70.1 75.5 85.9 81.1 84.8
G.84 7.2 ±0.08 5.9 ±0.04 1.3 78.9 70.7 72.0 87.8 80.8 86.0
J. Fungi 2022,8, 544 8 of 19
Table 1. Cont.
Isolate
Tolerance to GIT Bile Salt Tolerances (%)
6 h 24 h
Before After Log Reduction 0.3 CA 1.0 TA 1.0 OX 0.3 CA 1.0 TA 1.0 OX
SH.96 7.4 ±0.17 5.8 ±0.14 1.6 86.8 84.8 75.7 89.7 89.2 90.1
SH.97 7.4 ±0.08 6.2 ±0.04 1.3 85.9 84.8 78.1 90.2 89.3 89.8
SH.98 7.2 ±0.23 5.7 ±0.09 1.5 87.3 82.4 71.9 89.5 88.8 81.9
SH.99 7.3 ±0.32 6.3 ±0.27 1.0 84.9 77.3 68.4 89.6 89.0 69.2
SH.100 7.4 ±0.13 5.9 ±0.18 1.5 84.9 83.0 81.1 89.8 89.1 90.3
SH.102 7.3 ±0.13 5.6 ±0.07 1.7 83.9 85.7 73.3 89.4 88.7 89.4
SH.103 7.1 ±0.19 5.5 ±0.17 1.6 87.9 81.7 79.9 90.8 89.5 90.3
SH.104 7.4 ±0.30 6.2 ±0.22 1.2 86.6 79.8 80.0 90.5 89.4 90.6
SH.105 7.4 ±0.14 6.1 ±0.08 1.3 86.3 70.5 74.8 89.4 87.8 89.4
Values are expressed as mean
±
standard deviation of triplicates. CA, cholic acid; OX, oxgall; TA, taurocholic acid.
GIT, stimulated gastrointestinal tract by INFOGEST.
A probiotic candidate must exhibit high survivability in stressful conditions that it
will inevitably face inside the human gastrointestinal tract (GIT) to exert its functionality.
At the start of the digestion process, the potential probiotics should demonstrate tolerance
to the amylase present in the oral cavity. After ingestion, the potential probiotics must
resist several harsh conditions in the stomach, e.g., presence of low pH, gastric fluid
and pepsin [
49
]. Next, the probiotic cells must exhibit resistance to the small intestine
conditions, such as the presence of pancreatin, bile salts and alkaline stress [
28
]. Moreover,
tolerance to mild heat shock is necessary for the survivability of probiotic strains. The
probiotic candidate has to retain its viability and functionality at the internal temperature
of the human body (37
◦
C) because 28–30
◦
C is mostly the optimal temperature for yeast
growth [50].
Consequently, the potential probiotic should exhibit low reduction in viability after
being subjected to
in vitro
digestion [
51
]. Generally, the yeast probiotic tolerance mecha-
nism to the GIT conditions depends on the species/strain. Bile salts possess antimicrobial
activity that could suppress any microorganism, including yeasts. Thus, for microorgan-
isms to be classified as probiotics, they need to resist bile salts. The bile salt resistance of
S. cerevisiae could be attributed to an increase in its lipid content after being exposed to bile
salts and low pH. These lipids contents probably act as a protective agent against bile salt
stress [52,53].
In light of our results, the resistances of all isolates to the GIT conditions and bile salts
are remarkably different depending on the species/strain specificity. Other works yielded
promising findings for P. kudriavzevii [
54
] and S. boulardii var. boulardii strains [
55
], which
tolerated simulated GIT juices, isolated from fermented cereal foods and commercial food
supplements. In agreement with our findings, Chen et al. [
56
], Menezes et al. [
57
] and
Amorim et al. [
7
] proved the capability of different yeast strains isolated from a variety of
food sources to tolerate bile salt.
3.3. Cholesterol Removal and Bile Salt Hydrolysis (BSH)
Table 2presents the cholesterol removal and BSH activities of 12 yeast strains. All
12 yeast
strains were capable of effectively removing cholesterol from YPD media. Ta-
ble 2demonstrates that the cholesterol removal ability significantly differed among the
yeast strains, which varied from 41.6% to 96.5%. Strains O21, O26, SH55 and O13 exhib-
ited a higher ability to remove cholesterol compared with the other investigated yeast
strains. Regarding BSH, all yeast strains exhibited the capability to hydrolyse screened
bile salts forming free cholic acid. This capability ranged from 3.48 to 4.62, 3.40 to 4.01 and
J. Fungi 2022,8, 544 9 of 19
3.56 to 4.77 U/mg
for sodium glycocholate, sodium taurocholate and mixture of bile salts,
respectively. Strains O12, O26 and O66 demonstrated higher BSH activities than the other
investigated yeast strains (Table 2).
Table 2.
Cholesterol removal (%) and bile salt hydrolase (BSH) activities (specific activity, U/mg) of
12 potential probiotic yeasts.
Isolate CR (%)
BSH
Na-SG SA Na-TA SA Bile salt
mixture SA
G1 47.98 ±7.55 ab 1.79 ±0.05 abc 3.70 1.83 ±0.07 bc 3.79 1.72 ±0.05 a3.56
O12 50.16 ±8.68 ab 1.80 ±0.07 bc 3.68 1.72 ±0.07 ab 3.52 1.84 ±0.07 bc 3.77
O13 71.96 ±5.20 d2.13 ±0.10 e4.46 1.88 ±0.04 c3.93 1.73 ±0.07 a3.62
O18 62.31 ±2.35 cd 1.90 ±0.06 d3.85 1.72 ±0.04 ab 3.49 2.11 ±0.08 d4.27
O21 95.02 ±1.43 e1.87 ±0.03 cd 4.01 1.70 ±0.06 a3.65 2.22 ±0.05 e4.77
O26 91.59 ±2.47 e2.17 ±0.03 ef 4.55 1.91 ±0.02 c4.01 2.26 ±0.04 e4.73
O36 53.58 ±1.08 bc 1.89 ±0.02 d3.95 1.82 ±0.05 abc 3.81 1.92 ±0.05 c4.02
O63 47.98 ±1.95 ab 1.74 ±0.04 ab 3.57 1.71 ±0.05 ab 3.50 1.89 ±0.04 bc 3.87
O66 65.42 ±2.80 cd 1.94 ±0.04 d4.04 1.88 ±0.02 c3.90 2.04 ±0.05 d4.25
SH40 39.56 ±2.86 a1.76 ±0.02 ab 3.48 1.71 ±0.02 ab 3.40 1.81 ±0.02 ab 3.59
SH45 59.81 ±1.87 bc 1.71 ±0.05 a3.48 1.71 ±0.02 ab 3.48 1.90 ±0.09 bc 3.86
SH55 91.90 ±2.35 e2.23 ±0.03 f4.62 1.83 ±0.03 bc 3.79 1.86 ±0.07 bc 3.84
Values are expressed as mean
±
standard deviation of triplicates. Na-SG, sodium glycocholate (6 mM); Na-TA,
sodium taurocholate (6 mM); bile salt mixture (6 mM; glycocholic acid, glycochenodeoxycholic acid, taurocholic
acid, taurochenodeoxycholic acid, taurodeoxycholic acid); SA, specific activity (U/mg).
a–f
Means in same column
with different lowercase letters differed significantly (p< 0.05). SA, specific activities (U/mg).
Cholesterol removal is one of the desirable features of probiotics. In the current
study, the investigated isolates exhibited cholesterol reduction capability and BSH activities.
Cholesterol assimilation by a probiotic microorganism has been attributed to four main
mechanisms, namely, attachment to the cell wall, reduction of cholesterol to coprostanol,
incorporation of the cholesterol in the cell wall and disruption of the cholesterol micelles
by BSH [
58
,
59
]. Our findings on the cholesterol-lowering ability of the isolated yeasts are
superior to those reported in [48,60–62].
Probiotics possess BSH activities to act as bile salt detoxifiers and promote competition
in the microbial communities within the small intestine [
63
,
64
]. The ability of probiotic
strains to resist the toxicity of conjugated bile salts present in the duodenum is associated
with their BSH activity. In agreement with our results, Fadda et al. [
14
] and ¸Sanlidere
Alo˘glu et al. [
48
] reported several yeast strains isolated from foods exhibiting BSH activity.
3.4. Autoaggregation and Hydrophobicity
Table 3presents the autoaggregation (%) during 24 h of incubation at 37
◦
C and
hydrophobicity (%) against hexadecane, xylene and octane. The 12 yeast isolates exhibited
a significant percentage of autoaggregation ranging from 37.6% to 66%, 44.5% to 84.0%
and 50.7% to 85.8% during 3, 6 and 24 h of incubation, respectively. In general, the
autoaggregation percentages increased with the increase in the incubation period. After
24 h, isolates SH45, O36, O26, O66, O23, O28 and O21 showed a higher autoaggregation
ability than the other screened isolates. Table 3demonstrates that the hydrophobicity of
the 12 isolates to hexadecane and octane was higher than to xylene. The hydrophobicity
percentages ranged from 23% to 50.4%, 28.2% to 46.5% and 4.3% to 42.5% for hexane,
J. Fungi 2022,8, 544 10 of 19
octane and xylene, respectively (Table 3). Isolates SH40, O36, O40, O36, O12, O21 and O26
presented higher hydrophobicity than the other evaluated isolates.
Table 3. Autoaggregation (%) and hydrophobicity (%) of 12 potential probiotic yeast isolates.
Isolate Autoaggregation (%) Hydrophobicity (%)
3 h 6 h 24 h n-Hexane Octane Xylene
G1 42.3 ±0.28 b56.7 ±1.13 b69.8 ±1.57 b36.8 ±3.04 bcde 42.31 ±1.85 fg 6.51 ±2.21 a
O12 58.9 ±0.55 cd 73.6 ±0.60 c80.7 ±0.32 c32.6 ±5.71 abcd 36.7 ±5.24 cde 25.16 ±2.55 bcde
O13 60.7 ±0.44 de 75.8 ±1.14 c83.2 ±0.75 de 30.1 ±1.15 ab 40.65 ±0.86 efg 13.08 ±7.56 ab
O18 64.1 ±0.51 fg 78.4 ±0.46 c82.8 ±1.00 d31.5 ±1.95 abc 43.46 ±3.02 g24.86 ±4.20 bcde
O21 65.1 ±0.21 gh 77.0 ±2.41 c83.7 ±0.13 de 41.9 ±1.45 de 35.21 ±1.07 bcd 20.73 ±2.72 abcd
O26 65.6 ±0.35 gh 77.5 ±0.75 c84.4 ±1.11 def 37.6 ±2.76 bcde 34.46 ±1.47 abcd 37.72 ±3.31 e
O36 59.2 ±2.49 cd 75.0 ±2.64 c84.8 ±1.01 ef 42.9 ±1.11 e42.27 ±2.68 fg 15.62 ±2.98 abc
O63 37.7 ±0.75 a47.0 ±2.53 a51.0 ±0.28 a30.7 ±2.36 ab 30.67 ±1.27 a23.71 ±4.37 bcde
O66 62.6 ±0.34 ef 77.8 ±0.22 c82.9 ±1.15 de 24.9 ±1.12 a31.84 ±3.67 ab 18.03 ±1.78 abc
SH40 42.8 ±1.38 b57.2 ±0.49 b83.7 ±0.05 de 41.2 ±3.61 cde 44.98 ±1.57 g29.55 ±8.17 cde
SH45 66.6 ±0.31 h75.3 ±4.86 c86.1 ±0.55 f33.6 ±1.84 abcde 38.51 ±3.84 def 21.19 ±3.46 abcd
SH55 58.5 ±0.06 c71.3 ±0.51 c80.3 ±1.43 c28.3 ±1.72 ab 32.85 ±1.14 abc 33.11 ±9.87 de
Values are expressed as mean
±
standard deviation of triplicates.
a–h
Means in same column with different
lowercase letters differed significantly (p< 0.05).
The adherence of microorganisms to epithelial cells in the human intestine can be de-
duced by their cell surface properties, represented by testing the autoaggregation capability
and hydrophobic properties of probiotic candidates [
65
]. A higher aggregation capacity
provides high cell intensity involving the adhesion mechanism, whereas a robust hydropho-
bic property facilitates the attachment between the microbe and epithelial cells [
28
]. In
the present study, the yeast strains exhibited significant percentages of autoaggregation
and hydrophobicity to the investigated hydrocarbons. However, there were remarkable
distinctions among the screened isolates, which may be attributed to the difference in
the hydrophilic and hydrophobic regions in the cell wall of the microbial isolates [
66
]. In
addition, Verstrepen and Klis [
67
] reported that the differential expression of the adhesin
genes in the yeast allows them to rapidly adjust their adhesive properties to a specific
environment. It is noteworthy that the size of the yeasts cell are 10 times larger than that
of bacteria [
12
]. Therefore, an individual yeast cell requires a larger area to adhere to the
human intestinal cell surface [68].
In this work, the increasing trend of autoaggregation throughout 24 h is consistent with
the findings of Bonatsou et al. [
32
], whereas both the autoaggregation and hydrophobicity
results are superior to those reported by Zullo and Ciafardini [
62
]. The drawback of
the latter study [
62
] was that the hydrophobicity of yeasts was examined against one
hydrocarbon (hexadecane). Moreover, the autoaggregation capacity of the yeasts was
tested for only 4 h.
3.5. Coaggregation and Antimicrobial Activity
The coaggregation percentages of 12 yeast strains in the presence of E. coli O157:H7,
Salmonella Typhimurium, L. monocytogenes and S. aureus at 3, 6 and
24 h
of incubation
at 37
◦
C and antimicrobial activities against the same four pathogens are presented in
Table 4. The coaggregation capability increased (p< 0.05) during the incubation period of 3
to
24 h
at 37
◦
C, particularly with Salmonella Typhimurium. However, from another view,
the yeast isolates had the highest coaggregation percentages with L. monocytogenes than the
other three pathogens during the incubation period. Overall, isolates O12, O21, O26, O66
and SH45 had a higher coaggregation percentage than the other investigated strains. The
antimicrobial activity presented in Table 4ranges from 0.1 to >2.0 mm zone. Interestingly,
all yeast strains exhibited substantial inhibition activities against all four pathogens, except
the G1, O26 and O13 isolates.
J. Fungi 2022,8, 544 11 of 19
Table 4. Coaggregation (%) and antimicrobial activity of 12 potential probiotic yeast isolates against 4 foodborne pathogens.
Isolate S. Typhimurium E. coli O157:H7 S. aureus L. monocytogenes
3 h 6 h 24 h A.M 3 h 6 h 24 h A.M 3 h 6 h 24 h A.M 3 h 6 h 24 h A.M
G1 12.2 ±
1.53 b23.9 ±
0.46 a42.7 ±
1.79 a+++ 12.8 ±
0.55 h16.5 ±
0.97 a38.3 ±
0.62 a+++ 18.0 ±
0.82 f26.8 ±
0.97 f48.3 ±
0.98 e+23.8 ±
0.65 a33.7 ±
0.12 a52.1 ±
0.20 a+++
O12 17.3 ±
0.01 c46.7 ±
0.54 cd 59.7 ±
1.18 cd +++ 46.1 ±
1.04 a51.3 ±
0.07 f64.2 ±
0.08 ij +++ 23.0 ±
0.62 d48.7 ±
0.33 a62.4 ±
1.76 a+++ 38.5 ±
0.45 d45.9 ±
1.15 b61.9 ±
0.83 c+++
O13 25.8 ±
0.61 e52.9 ±
1.33 ef 65.3 ±
0.15 e+38.9 ±
0.26 cd 46.2 ±
0.09 def 62.0 ±
0.85 gh +++ 26.8 ±
0.78 c37.4 ±
0.46 cd 53.8 ±
0.46 cd +28.9 ±
0.21 b40.1 ±
0.59 ab 57.2 ±
0.14 b+++
O18 35.3 ±
0.93 g58.6 ±
1.55 g65.4 ±
2.67 e+++ 37.2 ±
1.04 d47.4 ±
2.45 def 59.9 ±
0.86 ef +++ 21.9 ±
0.08 de 31.9 ±
1.16 e48.3 ±
1.06 e+++ 49.9 ±
1.08 f60.2 ±
0.95 d68.9 ±
2.07 d+++
O21 21.5 ±
0.37 d50.1 ±
0.42 de 62.2 ±
0.59 de +++ 43.5 ±
1.05 ab 51.7 ±
0.83 f65.4 ±
0.45 j+++ 19.5 ±
0.78 ef 37.6 ±
0.10 cd 49.8 ±
1.50 de +++ 47.1 ±
0.26 e57.6 ±
2.25 cd 69.6 ±
1.19 d+++
O26 21.9 ±
0.84 d50.8 ±
1.08 de 62.6 ±
1.03 de +++ 41.1 ±
0.73 bc 50.3 ±
0.38 f63.1 ±
0.11 hi +++ 21.0 ±
0.70 def 46.8 ±
1.94 ab 60.1 ±
0.91 ab +45.7 ±
1.09 e52.9 ±
0.30 c66.8 ±
0.62 d+++
O36 12.0 ±
0.95 b43.4 ±
1.25 bc 57.4 ±
0.06 bcd +++ 22.6 ±
0.54 f35.0 ±
0.42 c52.1 ±
1.21 c+++ 14.1 ±
0.12 g27.3 ±
0.49 f48.5 ±
1.28 e+++ 48.0 ±
0.10 ef 55.2 ±
3.36 cd 68.5 ±
1.12 d+++
O63 36.0 ±
2.60 gh 42.6 ±
0.16 bc 54.2 ±
0.36 b+++ 17.2 ±
1.15 g26.9 ±
1.09 b44.4 ±
0.72 b+++ 32.7 ±
0.68 b37.6 ±
1.66 cd 52.7 ±
0.86 cde +++ 33.0 ±
0.45 c40.0 ±
0.93 ab 53.4 ±
3.19 ab +++
O66 31.6 ±
0.63 f56.0 ±
0.39 fg 65.0 ±
1.23 e+++ 32.4 ±
0.88 e42.9 ±
3.03 de 58.5 ±
1.68 e+++ 40.6 ±
1.41 a48.0 ±
0.52 a62.1 ±
0.82 a+++ 52.5 ±
0.30 g60.1 ±
2.06 d70.0 ±
0.42 d+++
SH40 37.9 ±
0.00 h55.8 ±
1.41 fg 67.3 ±
2.02 e+++ 33.5 ±
0.24 e41.9 ±
1.92 d54.6 ±
1.45 d+++ 23.1 ±
0.56 d35.3 ±
0.68 de 33.3 ±
1.00 f+++ 55.5 ±
0.71 h59.7 ±
2.94 d69.3 ±
2.19 d+++
SH45 9.30 ±
0.31 a40.2 ±
0.04 b55.4 ±
1.49 bc +++ 30.4 ±
0.49 e44.3 ±
1.41 de 60.4 ±
0.97 fg +++ 34.0 ±
1.06 b43.2 ±
0.23 b58.6 ±
1.71 ab +++ 47.0 ±
0.20 e59.2 ±
1.07 cd 69.8 ±
2.24 d+++
SH55 18.8 ±
0.36 c48.1 ±
1.93 d58.3 ±
0.04 bcd +++ 39.4 ±
0.80 cd 47.9 ±
0.66 ef 61.6 ±
0.07 fgh +++ 34.6 ±
1.61 b39.2 ±
1.07 c56.4 ±
0.39 bc +++ 29.0 ±
0.97 b37.5 ±
0.42 a57.5 ±
4.19 b+++
Values are expressed as mean ±standard error of triplicates. A.M: antimicrobial activity. a–j Means in same column with different lowercase letters differed significantly (p< 0.05). (+)
inhibition zone 0.1 to 1.0 mm; (+++) inhibition zone > 2.1 mm.
J. Fungi 2022,8, 544 12 of 19
The capability of the probiotics to coaggregate with the foodborne pathogens and their
potential to displace these pathogens are critical for protection against enteric infections [
69
].
Yeast probiotics prevent the pathogens from adhering to the intestinal epithelial cells by
adhering to them instead and then cocurating their binding sites [
33
]. Generally, probiotics
adapt a coaggregation behaviour to form a competitive microenvironment surrounding
the pathogen [
70
]. The suggested coaggregation mechanism between yeasts and bacterial
pathogens has been proposed by Millsap et al. [
71
], who stated that particular bacterial
pathogens have binding molecules on their surfaces that allow them to bind to mannose
residues on the yeast cell surface. In addition to mannans, glucans and chitin, which are
the main components of the yeast cell wall, all may be associated with yeast coaggregation
with pathogenic bacteria [
18
]. Several studies have also confirmed particular pathogenic
bacteria bound to S. boulardii,Debaryomyces hansenii and Yarrowia lipolytica [
72
–
74
]. Our
strains exhibited an intermediate coaggregation ability. However, the higher coaggregation
results for all four investigated pathogens are superior to those for Kluyveromyces lactis and
Torulaspora delbrueckii toward the same four pathogens [33].
The antimicrobial activity of probiotics is an essential characteristic represented by
antimicrobial compound production, completing exclusion of the pathogens and promo-
tion of the intestinal barrier function [
75
]. Several mechanisms have been postulated for
antagonistic yeasts against pathogenic bacteria, including (1) competition for nutrients and
space between yeast probiotic and microbial pathogens; (2) pH changes in the environment
due to the metabolic activity of the yeasts, leading to stressful conditions for the pathogens;
(3) production of high-concentration ethanol; and (4) release of antibacterial substances
and secretion of antimicrobial compounds, such as mycocins or killer toxins [
18
,
76
–
78
]. In
this work, P. kudriavzevii represents the majority of the tested isolates, and it belongs to
the Pichia genus, which was deeply reviewed as a producer of killer toxins that can inhibit
particular pathogens by Belda et al. [79].
Our antimicrobial activity results are in contrast to those of Amorim et al. [
7
] because
no antimicrobial activity was exhibited by their tested yeast isolates (Candida lusitaniae and
Meyerozyma caribbica). However, the results obtained by Hossain et al. [
34
] coincide with the
current study. Furthermore, the results of the current study indicated that the differences
in the antimicrobial activity among the yeast isolates might be attributed to species and
strain specificity.
3.6. Antibiotic Susceptibility and Attachment to the HT-29 Cell Line
The antibiotic resistance of 12 yeast strains against 6 antibiotics is presented in Table 5.
All yeast strains were sensitive or moderately sensitive to all the investigated antibiotics,
except strains G1, O12, O13 and O26. Table 5demonstrates that the yeast strains were
more susceptible to erythromycin and clindamycin. Regarding the HT-29 cell line adhesion,
the range of the yeasts’ adhesion to the HT-29 cell line was 5.97–6.99 Log
10
CFU/mL
(Table 5). Generally, isolates G1, O12, O13 and SH45 had the highest ability for HT-29 cell
line attachment.
The antibiotic resistance of probiotics is deemed a safety concern because there is a
chance of an antimicrobial resistance gene horizontally transmitting to the pathogens [
28
].
Therefore, potential probiotics with antibiotic sensitivity are desirable. In our work, eight
strains were found to be susceptible or moderately susceptible to various commercial antibi-
otics. Our results are almost in line with those of Amorim et al. [
7
] and Hossain et al. [
34
],
who isolated yeast species from pineapple and soya paste, respectively. The minor dispari-
ties between our study and others can be attributed to strain and species variations.
The capability to adhere to the intestinal epithelium is one of the primary criteria
for probiotic candidate selection. This capability is considered a pre-condition to exclude
enteropathogenic bacteria or promote host immunomodulation [
80
,
81
]. Expressed proteins
located on the surface of the cell walls are associated with microbial adhesion to intestinal
epithelial cells [
68
,
82
]. Generally, the results obtained from the present work showed
J. Fungi 2022,8, 544 13 of 19
suitable attachment to the HT-29 cell line. Several studies verified the adhesion abilities of
different yeast strains isolated from food sources using the HT-29 cell line [37,60,83].
Table 5. Antibiotic resistance to 6 different antibiotics and attachment to HT-29 cells.
Isolate Antibiotic Resistance Attachment to HT-29 Cells
CLI AMP SXT PEN VAN ERY Log10 CFU
G1 MS MS MS R MS S 6.66 ±0.06 e
O12 MS S MS MS R S 6.82 ±0.17 e
O13 MS MS MS R R MS 6.65 ±0.06 e
O18 S S S S S S 6.27 ±0.06 bcd
O21 MS MS MS S MS MS 6.00 ±0.06 a
O26 S R R MS S S 6.23 ±0.26 bcd
O36 MS MS MS MS MS MS 6.15 ±0.04 abc
O63 S MS MS MS S S 6.16 ±0.19 abc
O66 MS MS MS MS MS MS 6.37 ±0.04 cd
SH40 MS MS S S MS MS 6.36 ±0.17 cd
SH45 MS S S S S MS 6.41 ±0.02 d
SH55 MS S S MS S S 6.06 ±0.03 ab
Values are expressed as mean
±
standard deviation of triplicates.
a
CLI, clindamycin (2
µ
g); AMP, ampicillin
(10
µ
g); SXT, trimethoprim-sulfamethoxazole (25
µ
g); PEN, penicillin (10
µ
g); VAN, vancomycin (30
µ
g); ERY,
erythromycin (15
µ
g); R, resistant; MS, moderately susceptible; S, susceptible.
a–e
Means in same column with
different lowercase letters differed significantly (p< 0.05).
3.7. EPS Production and Heat Resistance
Interestingly, all 12 isolates showed the potential to produce EPS, as presented in
Table 6.
Table 6.
Exopolysaccharide (EPS) production and heat resistance (Log
10
CFU/mL) of 12 potential
probiotic yeast isolates.
Isolate EPS Production Heat Resistance (Log10 CFU/mL)
Before After
G1 + 6.6 ±0.01 a4.4 ±0.02 a
O12 + 7.5 ±0.13 efg 5.2 ±0.17 c
O13 + 7.7 ±0.03 g5.3 ±0.00 cd
O18 + 7.3 ±0.05 bcd 5.6 ±0.06 f
O21 + 7.3 ±0.02 bcd 5.5 ±0.02 ef
O26 + 7.3 ±0.07 bcd 5.3 ±0.07 cd
O36 + 7.5 ±0.00 def 5.4 ±0.03 cde
O63 + 7.2 ±0.06 bcd 5.3 ±0.17 cde
O66 + 7.3 ±0.04 cde 4.7 ±0.10 b
SH40 + 7.1 ±0.02 b5.4 ±0.02 def
SH45 + 7.6 ±0.07 fg 5.3 ±0.13 cd
SH55 + 7.2 ±0.03 bc 4.6 ±0.24 ab
Values are expressed as mean
±
standard deviation of triplicates.
a–g
Means in same column with different
lowercase letters differed significantly (p< 0.05). “+” denoted to ability to produce EPS.
The EPS production of the yeast isolates was inferred by creating a white ropy mucus
on ruthenium red skim milk agar plates. Numerous microorganisms, including yeasts, can
produce EPSs, which may vary in their monomer composition, molecular weight and type
and degree of branching [
84
]. Therefore, EPSs differ in their functions and applications,
J. Fungi 2022,8, 544 14 of 19
which are most related to adhering to, protecting and retaining compounds [
85
]. The
research group [
86
] had reported EPS production and isolation by yeast, K. marxianus and
P. kudriavzevii, which were isolated from dairy products. On the other hand, Fekri et al. [
87
]
revealed that their p yeast strains isolated from traditional sourdough, K. marxianus,
K. lactis
and K. aestuarii, produced a higher amount of EPS compared with those of isolated yeasts
in the same research [87].
The heat resistance of 12 yeast isolates is presented in Table 6. The growth of all isolates
reduced (p< 0.05) after they were treated at 60
◦
C for 5 min. The decrease in yeast growth
ranged from 1.7 to 2.6 Log
10
CFU/mL. Isolates O18, O21, O63 and SH40 presented higher
heat resistance compared with other isolates.
Heat resistance is a fundamental challenge faced by probiotics when used in the food
industry. In the present study, all yeast isolates demonstrated good tolerance to heat. One of
the suggested mechanisms for the yeasts to resist extreme heat is the production of trehalose,
a sugar produced by a wide variety of microorganisms. The intracellular accumulated
trehalose is involved in promoting thermotolerance of the yeasts [
88
]. Several studies have
evaluated the heat resistance of yeast probiotics using a method that mainly focuses on
testing at only 37
◦
C, which is the internal temperature of the human body [
9
,
89
,
90
]. The
drawback of this method is that it only evaluates the use of probiotics as a supplement, not
its use in the food industry, which requires higher temperature. In the studies conducted
by Hu et al. [
91
] and Hossain et al. [
34
], the heat resistance of S. cerevisiae and S. cerevisiae
var. boulardii was tested up to 42
◦
C and 48
◦
C for 30 min and 72 h, respectively. The
isolates in both studies [
34
,
91
] exhibited a significant reduction in growth rate after heat
treatment compared with our isolates. The trend of the heat resistance of S. cerevisiae has
been reported by Kalyuzhin [92].
3.8. Molecular Identification of Selected Yeast Isolates
A total of 12 potential yeast probiotics were identified using ITS/5.8S ribosomal DNA
sequences. Each isolate’s name and accession number obtained from GenBank are pre-
sented in Table 7. Molecular phylogeny analysis was conducted, and a phylogenetic tree
constructed to identify yeasts to a species level based on the 1ITS/5.8S ribosomal DNA
sequences from evolutionary distances using the neighbour-joining method. The phyloge-
netic tree of the 12 isolates is presented in Figure 2. The genotyping of S. cerevisiae, one of
the yeast species included in the current paper, has been widely discussed [
93
,
94
]. One of
the most reliable methods used to amplify the genomic sequences is PCR amplification of
inter-delta sequences, where delta elements create the LTR flanking retrotransposons TY1
and TY2 in S. cerevisiae [
41
]. Therefore, in order to distinguish the S. cerevisiae strain, the
use of inter-delta sequencing is recommended.
Table 7. Identification of yeast isolates using ITS/5.8S ribosomal DNA and their accession numbers
obtained from GenBank.
Isolate Microorganism Accession No Source
G1 Candida sp. OK441052 Gamed (traditional fermented dairy product)
O12 Pichia kudriavzevii OK441055 Jordanian Olive
O13 Pichia kudriavzevii OK441056 Jordanian Olive
O18 Pichia kudriavzevii OK441057 Jordanian Olive in oil
O21 Pichia kudriavzevii OK441060 Jordanian Olive in oil
O26 Pichia kudriavzevii OK441064 Moroccan green olives
O36 Pichia kudriavzevii OK441067 Jordanian green olives
O63 Pichia sp. OK441068 Jordanian green olives
O66 Saccharomyces
cerevisiae OK441070 Jordanian green olives
SH40 Pichia kudriavzevii OK441071
Shanklish (traditional fermented dairy product)
SH45 Pichia kudriavzevii OK441072
Shanklish (traditional fermented dairy product)
SH55 Pichia kudriavzevii OK441073
Shanklish (traditional fermented dairy product)
J. Fungi 2022,8, 544 15 of 19
J. Fungi 2022, 8, x FOR PEER REVIEW 14 of 19
3.8. Molecular Identification of Selected Yeast Isolates
A total of 12 potential yeast probiotics were identified using ITS/5.8S ribosomal DNA
sequences. Each isolate’s name and accession number obtained from GenBank are pre-
sented in Table 7. Molecular phylogeny analysis was conducted, and a phylogenetic tree
constructed to identify yeasts to a species level based on the 1ITS/5.8S ribosomal DNA
sequences from evolutionary distances using the neighbour-joining method. The phylo-
genetic tree of the 12 isolates is presented in Figure 2. The genotyping of S. cerevisiae, one
of the yeast species included in the current paper, has been widely discussed [93,94]. One
of the most reliable methods used to amplify the genomic sequences is PCR amplification
of inter-delta sequences, where delta elements create the LTR flanking retrotransposons
TY1 and TY2 in S. cerevisiae [41]. Therefore, in order to distinguish the S. cerevisiae strain,
the use of inter-delta sequencing is recommended.
Figure 2. Neighbour-joining phylogenetic tree based on ITS/5.8S ribosomal DNA. The numbers in parentheses are acces-
sion numbers of the identified sequences from the GenBank. The filled circles are the reference strains from NCBI.
Figure 2.
Neighbour-joining phylogenetic tree based on ITS/5.8S ribosomal DNA. The numbers in
parentheses are accession numbers of the identified sequences from the GenBank. The filled circles
are the reference strains from NCBI.
4. Conclusions
Selected yeast strains from fermented dairy and non-dairy products demonstrated
probiotic characteristics. The probiotic yeasts exhibited an excellent survival rate after the
in vitro
digestion, with a 0.7 Log reduction for the highest
in vitro
digestion resistance.
The yeast isolates were able to hydrolyse bile salts and significantly reduce cholesterol.
The susceptibility of these strains to the tested antibiotics did not present any concerns.
The autoaggregation of 12 isolates ranged from 50.7% to 85.8% during 24 h of incubation.
All those isolates exhibited a higher percentage of hydrophobicity to hexadecane and
octane compared with xylene. Generally, the increase in coaggregation percentages during
incubation time from 3 h to 24 h was remarkable (p< 0.05). The isolates showed significant
inhibition activities against the four screened pathogens except G1, O26, and O13 isolates.
Overall, the 12 isolates had moderate ability to attach to the HT-29 cell line. The reduction
in the growth of 12 isolates after heat treatment ranged from 1.7 to 2.6 LoG
10
CFU/mL.
All the yeast isolates can produce exopolysaccharides (EPS), and isolates SH40 (Pichia
kudriavzevii OK441071), SH55 (P. kudriavzevii OK441073), O63 (Picha sp. OK441068) and
O66 (S. cerevisiae OK441070) have promising probiotic traits, which necessitate further
characterization for their use in the food industry.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/jof8050544/s1, Table S1: Acid tolerance at pH 2.5 during 24 h of
incubation at 37 ◦C for 105 potential probiotic yeast isolates.
J. Fungi 2022,8, 544 16 of 19
Author Contributions:
N.S.A., writing—original draft, investigation, data curation, formal analysis;
T.M.O., A.N.O., A.A.A.-N., S.-Q.L. and R.S.O., writing—review and editing; M.M.A., conceptualiza-
tion, writing—original draft, funding, supervising, writing—review and editing, supervision. All
authors have read and agreed to the published version of the manuscript.
Funding:
This research and APC were funded by the United Arab Emirates University (UAEU),
Al-Ain, United Arab Emirates.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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