THE POTENTIAL ROLE OF LACTIC ACID BACTERIOCINS AS AN ANTIFUNGAL AGENT Nanis G. Allam1, Mahmoud AbdEl-Mongy 2, Noha S. El-Nahrawy 1, Mohammed A. Hefnawy 3. 1
Microbiology Unit, Botany Department Faculty of Science, Tanta University. 2 Microbial Biotechnology Department, Genetic Engineering and Biotechnology Institute, El-Sadat City University. 3 Microbiology Unit, Botany Department, Faculty of Science, Menoufia University.
Corresponding: Nanis G. Allam, Medical Campus, Faculty of Science, Tanta University, El-Bahr Street, Tanta, Egypt Email:
[email protected],
[email protected] ABSTRACT The aim of the present study was to produce a novel antifungal substances from lactic acid bacteria to be used in biocontrol of pathogenic fungi and yeasts. Among investigated lactic acid bacteria, Enterococcus hirae ATCC 9790 and Enterococcus faecium Aus0004 were responsible for production of the most potent antifungal substances. Two produced substances revealed high thermal stability at 121°C, high activity after sodium dodecyl sulfate treatment, complete inactivation in the presence of trypsin and proteinase K enzymes and were active over a wide range of pH (3 to 7). The active substance produced by E.hirae had high potency against growth of some of pathogenic fungi and yeasts like C. albicans, Trichophyton mentagrophytes, Microsporum gypseum, Microsporum nanum, Microsporum canis, Aspergillus niger and Fusarium oxysporum. Also, active substance produced by E. faecium was active against Microsporum gypseum, Microsporum canis, Aspergillus niger and Fusarium oxysporum. The active protein substances of E. hirae and E. faecium c
ed f
g
g
ml of amino acids respectively and could be identified as bacteriocins. KEY WORDS: Lactic acid bacteria, bacteriocins, antifungal activity
INTRODUCTION Mycoses are diseases caused by fungi. Mycoses have become a growing problem in modern medical care with the increase in the population of patients with immuno-deficiency or undergoing immunosuppressive therapy. The diagnosis of these diseases can be problematic, drug resistance is of great concern and fewer drugs are available compared to bacterial or viral
diseases (Sanglard and White, 2006; Blanco and Garcia, 2008; Miceli and Lee, 2011). Fungal infections lead to various diseases that can be Local, super-ficial, allergic or systemic (Blanco and Garcia, 2008). Systemic infections are particularly serious and potentially lifethreatening (Richardson, 2005; Richardson and Lassflorl, 2008; Miceli et al., 2011). Candida albicans is an opportunistic fungal pathogen that can cause super- ficial and systemic infections in immunocompromised patients (Rossetia et al., 2015). Candida albicans infections (candidiasis) has attracted a great deal of attention due to their high morbidity and mortality rates and accompanying increase in medical costs (Bassetti et al., 2011). Skin and mucous membrane infections include cutaneous candidiasis, chronic mucocutaneous candidiasis, oral, oropharyngeal, esophageal and vulvovaginal candidiasis (Cheng et al., 2005; Egusa et al., 2005). Invasive candidiasis encompasses a wide variety of severe or invasive diseases that include candidaemia, disseminated candidiasis, deep organ involvement, endocarditis and meningitis, excluding more superficial or less severe diseases such as oropharyngeal and esophageal candidiasis (De rosa et al., 2009). Candidaemia indicates the presence of Candida species in the blood. After entering the blood stream, the yeast cells can infect all internal organs and may cause life-threatening septicemia (Soliman et al., 2015). Also dermatophytic fungi of genera Trichophyton, Epidermophyton and Microsporum infect human skin, hair and nails (Kumar et al., 2009). For many years, fungal infections were treated with polyene compounds (Nystatin and Amphotericin B), despite their high toxicity. In the early 90s, the development of first generation triazoles (fluconazole and itraconazole) changed the epidemiology of candidiasis by offering new options for prevention and treatment (Hope et al., 2008). However, the extensive use of these new antifungal agents has resulted in resistant strains, which now creates urgency for the development of new treatment strategies (Canuto and Rodero, 2002; Petrikkos and Skiada, 2007). In the search for new treatments, antimicrobial peptides have attracted considerable attention due to their various unique properties (Lee and Lee, 2015). Antimicrobial peptides are important components of the innate immune defense against a variety of microbial infections, and do not easily induce resistance compared to conventional antibiotics (Matsuzaki, 2009). Probiotic LAB strains, Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 were found to inhibit the growth of Candida albicans (Kohler et al., 2012). Lactobacillus fermentum CRL 251 produce peptides which were active against Aspergillus niger CH101, Penicillium sp. CH 102, Geotrichium citri-aurantii INTA1 and Penicillium digitatum INTA2 (Gerez et al., 2013). Lactobacillus brevis PS1 produce peptide which was active against Fusarium species (Mauch et al., 2010).
In our study we reported characterization and identification of antifungal proteins produced by two strains of LAB Enterococcus hirae ATCC 9790 and Enterococcus faecium Aus0004, that showed high activity against C. albicans. MATERIALS AND METHODS THE USED MICRO-ORGANISMS A) Different probiotic bacteria (eighteen species) were isolated from traditional Egyptian foods. B) Candida albicans was kindly provided by Microbiology unit, Bacteriology laboratory, Faculty of Science, Tanta University.
c) Trichophyton mentagrophytes (RCMB 09225), Trichophyton rubrum (RCMB 09274), Epidermophyton floccosum (RCMB 099341), Microsporum gypseum (RCMB 091285), Microsporum
nanum
(RCMB
091276),
Microsporum
audouinii
(RCMB
091242),
Microsporum canis (RCMB 091219), Aspergillus niger (RCMB 02563), Cryptococcus neoformans (RCMB 05098), and Fusarium oxysporum (RCMB 080352) were used to study the activity of the antifungal substances. These fungi were purchased from the Regional Center for Mycology and Biotechnology, Antifungal Activity Unit, El Azhar University. SCREENING OF ANTIFUNGAL ACTIVITY OF INVESTIGATED LACTIC ACID BACTERIA Overnight cultures of the tested bacteria were prepared in De man, Rogosa and Sharpe (MRS) broth at 37 oC and then centrifuged at 8000 rpm for 15 min at 4 oC and the supernatants were filter sterilized using (Millipore/0.22 µm). Overnight culture of Candida albicans was prepared with 5×105 cfu ml-1 in 100 µl potassium-sodium-phosphate buffer, (pH 7) was spread onto nutrient agar plates. The supernatants of investigated bacteria (100 µl) were loaded into well made in the inoculated plates. The plates were incubated overnight at 37oC. Then the diameters of inhibition zones were measured according to Galvez et al. (1986).
IDENTIFICATION OF SELECTED ANTIFUNGAL PRODUCING BACTERIA BY 16S rRNA GENE SEQUENCE The most effective antifungal producing bacteria, were identified by 16S rRNA gene sequence. Genomic DNA was extracted as described by Carozzi et al. (1991), and the 16S rRNA gene was amplified by PCR using 16S rRNA primers (forward primers; AGA GTT TGA TCC TGG
CTC AG and reverse primers; GGT TAC CTT GTT ACG ACT T according to Rehman et al. (2007). Polymerase chain reaction was performed for 5 min by initial denaturation at 95 °C, followed by 35 cycles for each denaturation at 95°C for 30 sec, annealing at 65 °C for 1 min, extending at 72 °C for 1.30 min, and a final extension at 72 °C for 10 min. The PCR product of 0.5 kb was extracted from the gel using Fermentas gel extraction kit (Thermo), and the amplified products were electrophoresed on 1% agarose gel. Sequencing was carried out by the Genetic Ana y i Sy te
de Gene JET™ PCR Coulter Inc. Fullerton, CA, USA. The 16S
rRNA gene sequences were compared with known sequences in the Gene Bank database to identify the most homologous sequence.
CHARACTERIZATION OF THE PRODUCED ANTIFUNGAL SUBSTANCES EFFECT OF ENZYMES ON ACTIVITY OF ANTIFUNGAL SUBSTANCES The stability of the antifungal substances was tested after treatment with proteinase K, trypsin, and α-amylase (Sigma-Aldrich). The enzymes were added to 1 ml of neutralized supernatants to a final concentration of 2 mg ml _1 in all cases, and 100 µl was inoculated into assayed plates according to Cocolin et al. (2007). EFFECT OF pH ON ACTIVITY OF ANTIFUNGAL SUBSTANCES The effect of pH on the antifungal activity of the produced substances was tested by adjusting each of the supernatants to various pH values ranging from 3:10 using sterile 6 N NaOH or 5 N HCl, and incubated for 20 min at 37 oC according to Oh et al. (2000). The antifungal spectrum of activity was carried out by measuring the zones of inhibition against Candida albicans. EFFECT OF HEAT AND TIME ON ACTIVITY OF ANTIFUNGAL SUBSTANCES The effect of heat and time on the antifungal activity was studied by incubating the bacterial free supernatants at 70 oC, 100 oC and 121 oC (autoclaving) for 15min, 20 min for autoclaving and 30 min according to Zamfir et al. (1999). EFFECT OF SURFACTANTS ON ACTIVITY OF ANTIFUNGAL SUBSTANCES The effect of various surfactants, including Tween-80, SDS, urea (1.0% each) on the supernatants were tested according to Todorov et al. (1999). EFFECT OF EDTA AND UV ON ACTIVITY OF ANTIFUNGAL SUBSTANCES 1% of EDTA (ethylene diamine tetra acetic acid) added to the investigated supernatants and incubated at 37 oC for 5 hr (Todorov et al., 1999). Also the supernatants were subjected to ultra violet rays for 5 hr. Then the antifungal activity was determined by well agar diffusion method. Untreated supernatants were used as controls. Three replicas were performed for each test.
STUDY THE POTENCY OF ANTIFUNGAL SUBSTANCES AGAINST VARIOUS PATHOGENIC FUNGI The antifungal activity of the cell-free supernatants of E. hirae and E. faecium were determined against different pathogenic fungi by well agar diffusion method. The plates were inoculated by different pathogenic fungi (5×105 cfu ml-1) on the surface of Sabouraud dextrose agar media and 100 µl of cell free supernatants inoculated into the wells .The plates incubated at 25 oC for 5-7 days for all organisms except Aspergillus niger the plates were incubated at 28 oC for 48 hr. The antifungal activity indicated by measuring the diameter of inhibition zone. Three replica plates were prepared for each fungus. EXTRACTION, PURIFICATION AND PROTEIN AMINO ACIDS CONTENT OF THE ANTIFUNGAL SUBSTANCES Two liters of MRS cultures of the two organisms were prepared and the supernatants were collected by centrifugation and adjusted to pH 6.5, the supernatants were precipitated by 80% saturated ammonium sulphate at 4 oC (Sambrook et al., 1989). The supernatant ammonium sulphate mixtures were kept for 24 hr at 4 oC before separating by centrifugation .The precipitates were dialyzed in a dialysis bag to remove the excess of sulphate. The precipitates were dissolved in small volume of iso-propanol in 25 mM ammonium acetate buffer pH 6.5 (20% v/v) and loaded into equilibrated column which previously soaked with sephadex G-150. The antifungal substances were eluted with 40 % iso-propanol in 25 mM ammonium acetate buffer pH 6.5. The fractions were collected and their activities were determined according to Galvez et al. (1986).The active fractions were analyzed to detect amino acids content. Principal of amino acid analyzer: Hydrochloric acid was used to hydrolyze amino acids of the active fractions according to Pellet and Young (1980). Condition of amino acid analyzer for hydrolysate program: Column: Hydrolysate column SYKAM (S 4300) (4.6 ᵡ 150 mm) and its temperature 57°C. Sample: 100μ
Buffer system: Sodium acetate buffer A (pH 3.45), buffer B (pH 10.85), regeneration solution and sample dilution buffer (pH 2.2). Flow rate: 0.25 ml/min for Ninhydrin pump 0.45 ml/min for quaternary pump Detection: Ninhydrin is used f r the detecti n f a in acid at λ440 for proline and 570 nm for the other amino acids through oxidative decarboxylation reaction to the amino acid with Ninhydrin t give ruhe ann’
ur e a c
und detected by the a
f
ectr
h t
eter at
the above mentioned wave lengths. RESULTS SCREENING OF THE INVESTIGATED BACTERIA FOR PRODUCTION OF ANTIFUNGAL SUBSTANCE Among eighteen isolated lactic acid bacteria, isolate No.4 and No.17 showed high antifungal activities against Candida albicans with large inhibition zones (25 and 25.6 mm respectively) as presented in photo1. a a
b
Photo 1: The two potent organisms against Candida albicans a) isolate No.4, b) isolate No.17. IDENTIFICATION OF SELECTED ANTIFUNGAL PRODUCING BACTERIA BY 16S rRNA GENE SEQUENCE The selected bacteria were identified to strain level using 16S rRNA gene sequence. The sequence was compared to that in Gene Bank and the phylogenetic tree was constructed. The obtained sequence proved that the isolate No. 4 showed 96% identity to Enterococcus hirae ATCC 9790 and the isolate No. 17 showed 96% identity to Enterococcus faecium Aus0004 strain (Fig:1).
A
B
Figure 1: A) Phylogenetic tree for Enterococcus hirae ATCC 9790. B) Phylogenetic tree for Enterococcus faecium Aus0004. CHARACTERIZATION OF THE PRODUCED ANTIFUNGAL SUBSTANCES EFFECT OF ENZYMES ON ACTIVITY OF ANTIFUNGAL SUBSTANCES Table 1 and photo 2 h wed that α-amylase enzyme had neglected effect on the activity of two antifungal substances. While, Proteinase k and Trypsin (proteolytic enzymes) resulted in loss of activities. Table 1: Effect of enzymes on activity of antifungal substances. Mean of IZ Diameter (mm) ± SD Enzyme
Enterococcus hirae Enterococcus faecium
Control
25 ± 1
24.6 ± 0.58
Proteinase k
-Ve
-Ve
Trypsin
-Ve
-Ve
α-amylase
22.6 ± 0.58
17.3 ± 0.58
Mean of inhibition zone diameter in mm ± standard deviation. –Ve result: no inhibition zone. a
b
Photo 2: Effect of enzymes on activity of antifungal substances. a) Enterococcus hirae, b) Enterococcus faecium. (C: control, am: α-amylase, T: trypsin and Pro: proteinase k enzymes).
EFFECT OF pH ON ACTIVITY OF ANTIFUNGAL SUBSTANCES The data presented in Table 2 showed that no antifungal activity was recorded at pH values from 8 to 10 against Candida albicans. On the other hand the highest activities of the
antifungal substances produced by Enterococcus hirae and Enterococcus faecium were obtained at pH 3 as presented in photo3. Table 2: Effect of pH on activity of antifungal substances. Mean of IZ Diameter (mm) ± SD PH
Enterococcus hirae Enterococcus faecium
Control (4.3)
25 ± 1
25.3 ± 0.58
3
27.3 ± 1.15
27.6 ± 0.58
4
25.6 ± 0.58
26.6 ± 0.58
5
20.6 ± 0.58
17.3 ±0.58
6
19.3 ± 0.58
16.3 ± 2.08
7
14.3 ± 0.58
13.6 ± 2.08
8
-Ve
-Ve
9
-Ve
-Ve
01
-Ve
-Ve
Mean of inhibition zone diameter in mm ± standard deviation. –Ve result: no inhibition zone.
a
c
b
d
Photo 3: Effect of pH on activity of antifungal substances. a,b) Enterococcus hirae and c,d) Enterococcus faecium. (C: control). EFFECT OF HEAT AND TIME ON ACTIVITY OF ANTIFUNGAL SUBSTANCES When supernatants of the two bacteria subjected to heat treatment the activity was maintained as presented in Table 3 and photo 4, only neglected effect appeared with increasing time of exposure at 70 °C. Table3: Effect of heat and time on activity of antifungal substances. Mean of IZ Diameter (mm) ± SD Heat and time
Enterococcus hirae Enterococcus faecium
Control
25 ±1
25.6 ± 0.58
70 °C for 15 min
24.6 ± 0.58
25.3 ± 0.58
70 °C for 30 min
24.3 ± 0.58
25.2 ± 0.058
Autoclaving
25.3 ± 0.58
25.7 ± 0
100 °C for 15 min
25.6 ± 0.58
25.3 ± 0.58
100 °C for 30 min
26.6 ± 0.58
27.6 ± 0.058
Mean of inhibition zone diameter in mm ± standard deviation.
b
a
d
c
Photo 4 : Effect of heat and time ( 1: 70 °C for 15 min, 2: 70 °C for 30 min, 3: autoclaving (121 °C for 20 min), 4: 100 °C for 15 min, 5: 100 °C for 30 min, C : control ) on activity of antifungal substances. a,b) Enterococcus hirae and c,d) Enterococcus faecium. EFFECT OF SURFACTANTS ON ACTIVITY OF ANTIFUNGAL SUBSTANCES All the investigated surfactants had no effect on the activity of the antifungal substances. SDS increased the antifungal activity of Enterococcus hirae and Enterococcus faecium by giving large inhibition zones their diameters were 26.6, 27 mm as illustrated in Table 4 and photo 5. Table 4: Effect of surfactants on activity of antifungal substances. Mean of IZ Diameter (mm) ± SD Surfactants Enterococcus hirae Enterococcus faecium Control
25 ± 0
24.6 ± 0.58
SDS
26.6 ± 2.89
27 ± 1
Urea
22.8 ± 1.5
21.2 ± 0.58
Tween-80
15.3 ± 0.58
16 ± 1.73
Mean of inhibition zone diameter in mm ± standard deviation.
a
b
Photo 5: Effect of surfactants on activity of antifungal substances. a) Enterococcus hirae and b) Enterococcus faecium. EFFECT OF EDTA AND UV ON ACTIVITY OF ANTIFUNGAL SUBSTANCES The data presented in Table 5 showed that EDTA increased the antifungal activity of the two Enterococcal strains also UV showed slight decrease in activity as presented in photo 6. Table 5: Effect of EDTA and UV on activity of antifungal substances. Mean of IZ Diameter (mm) ± SD Effect of
Enterococcus hirae Enterococcus faecium
Control
25.3 ± 0.58
25 ± 1
UV
22.6 ± 0.58
20 ± 1
EDTA
26.6 ± 0.58
26.6 ± 0.58
Mean of inhibition zone diameter in mm ± standard deviation. a
b
Photo 6: Effect of EDTA and UV on activity of antifungal substances. a) Enterococcus hirae and b) Enterococcus faecium.
STUDY THE POTENCY OF ANTIFUNGAL SUBSTANCES AGAINST VARIOUS PATHOGENIC FUNGI Enterococcus hirae had inhibitory effect against the growth of Trichophyton mentagrophytes, Microsporum gypseum, Microsporum nanum, Microsporum canis, Aspergillus niger and Fusarium oxysporum. Also Enterococcus faecium had inhibitory effect against the growth of Microsporum gypseum, Microsporum canis, Aspergillus niger and Fusarium oxysporum as shown in Table 6.
Table 6: Study the effect of bacterial cell free supernatants of E. hirae and E. faecium on various pathogenic fungi. Sample
Mean of IZ Diameter (mm) ± SD Enterococcus hirae
Enterococcus faecium
Trichophyton mentagrophytes
17.6 ± 0.72
-Ve
Trichophyton rubrum
-Ve
-Ve
Epidermophyton floccosum
-Ve
-Ve
Microsporum gypseum
20.3 ± 0.72
18.2 ± 0.58
Microsporum nanum
21.2 ± 0.58
-Ve
Microsporum audouinii
-Ve
-Ve
Microsporum canis
23.4 ± 1.5
20.6 ± 0.63
Aspergillus niger
22.8 ± 1.5
19.2 ± 1.5
Cryptococcus neoformans
-Ve
-Ve
Fusarium oxysporum
22.4 ± 0.58
19.2 ± 0.63
Tested fungi
Mean of inhibition zone diameter in mm ± standard deviation. –Ve result: no inhibition zone.
EXTRACTION, PURIFICATION AND PROTEIN AMINO ACIDS CONTENT OF THE ANTIFUNGAL SUBSTANCES Active fractions of Enterococcus hirae and Enterococcus faecium were extracted by precipitation with 80% saturated ammonium sulphate, then the active fractions were separated and fractionated by gel filtration (Sephadex G-150 column). g
The active fraction of Enterococcus hirae was containing while that of Enterococcus faecium was containing
g
of amino acids of amino acids as
presented in Table 7.The bacteriocins of the two Enterococcal species contained glutamic as the highest concentration of the produced amino acids. Table 7: Protein amino acids content of Enterococcus hirae and Enterococcus faecium.
Name of amino acid
E. hirae
E. faecium
Aspartic
5076
7868.5
Threonine
2554.2
3261.25
Serine
2575.8
4411.5
Glutamic
12877.35
18480
Proline
11439.45
15665.5
Glycine
3219.15
4632.25
Alanine
1975.2
2707.75
Systine
96.75
40.25
Valine
5607.9
8947
Methionine
851.85
1047
Isoleucine
4459.05
6593
Leucine
4973.55
7445.5
Tyrosine
1808.4
2763.25
Phenylalanine
3879.45
5909.75
Histiodine
1744.2
2758
Lysine
2695.65
4246.75
Ammonium
3223.95
5579.5
Arginine
1242.45
2000.75
Total
58861.2
88692.25
DISCUSSION In an era of increased incidence of fungal infections in immunocompromised patients (Ostrosky, 2002; Venkatesan et al., 2005) and acquiring resistance t ‘fr nt ine’ antifunga therapies (Prasad and Kapoor, 2005), there is a growing need to discover new antifungal therapies. Antimicrobial and antifungal peptides were used as the first line of defense between the host organism and its surrounding environment, because these peptides were able to inhibit a wide spectrum of infectious microbes without significant toxicity to the host (Shekh and Roy, 2012). The genus Enterococcus belongs to a group of important lactic acid bacteria (LAB) that participate and contribute towards different fermentation processes. The present study revealed that Enterococcus hirae ATCC 9790 and Enterococcus faecium Aus0004 had high potency against growth of Candida albicans as they produce extracellularly antifungal substances. The activity was completely disappeared after treatment with proteolytic enzymes that indicated proteinaceous nature of the antifungal substances. In the same connection, Ahmadova et al. (2013) found that the active compound produced by Enterococcus faecium AQ71 was completely destroyed by proteolytic enzymes so it was classified as bacteriocin. The current study revealed that the highest antifungal activities of the produced substances by E. hirae and E. faecium were obtained at pH 3 and the still active from pH 4 to 7. No antifungal activities were recorded at pH values from 8 to 10 against Candida albicans. In the same connection, Rehaiem et al. (2009) and Hadji Sfaxi et al. (2011) reported that Enterococcal bacteriocins were stable over a wide range of pH. The antifungal activities of E. hirae and E. faecium were increased upon thermal treatment after heating at 100 °C for 30 min and after autoclaving for 20 min at 121 °C. Similar results have been reported for bacteriocin of Enterococcus faecium AQ71 (Ahmadova et al., 2013). Generally bacteriocins are heat stable peptides with high level of stability after heating and exposure for autoclaving as reported by Rehaiem et al. (2009 and Renye and Van Hekken (2009) for Enterococcal bacteriocins.
In this study the antifungal substances were not sensitive to treatment with SDS, Tween-80, Urea, EDTA and ultraviolet rays such stability evidencing a good potential of these substances to survive in the gastrointestinal tract. The stability of antifungal activity in the presence of SDS was already reported for some E. faecium strains (Rehaiem et al., 2009; Hadji-Sfaxi et al., 2011). In the same connection, Todorov and Dicks (2005b) revealed that SDS, Tween 20, Tween 80 and EDTA had no effect on the activity of bacteriocin produced by E. faecium. It could be concluded from previous evidence that the antifungal substances produced by E. hirae and E. faecium are bacteriocins. The antifungal activity of investigated bacteriocins revealed strong potency against the growth of some pathogenic fungi. Bacteriocin of E. hirae had inhibitory effect on the growth of Trichophyton mentagrophytes, Microsporum gypseum, Microsporum nanum, Microsporum canis, Aspergillus niger and Fusarium oxysporum. Also, bacteriocin of E. faecium inhibited the growth of Aspergillus niger, Fusarium oxysporum, Microsporum canis and Microsporum gypseum. Similar results for other LAB were observed such as Lactobacillus plantarum IMAU10014 was active against Penicillium roqueforti, Aspergillus niger (Wang et al., 2012) and Lactobacillus brevis was active against Microsporum canis, Microsporum gypseum and Epidermophyton floccosum (Guo et al., 2012). The active proteins of bacteriocins were purified in two steps involving ammonium sulphate precipitation and gel filtration (Sephadex G-150 column). Then the amino acid content of produced proteins of E. hirae and E. faecium strains were
g
and 88692.25 mg ml
respectively. This study showed that the bacteriocins of the two Enterococcal strains had high activity against C. albicans and other various pathogenic fungi so these active bacteriocins could be used as an alternative antifungal agents and could be considered as important tool for the biological control of the pathogenic fungi.
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ated fr
‘Rayeb’ a traditi na Tuni ian dairy beverage
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