# Antifungal activity of potential probiotic Limosilactobacillus fermentum strains and their role against toxigenic aflatoxin-producing aspergilli

### LAB strains and culture conditions

Fresh traditional fermented yogurt samples were collected from different regions of Iran. Samples were transported on ice to the Food Biotechnology Laboratory of the Iranian Agricultural and Biotechnology Institute (ABRII), Tabriz, Iran. To isolate LAB from the sample, inoculate 50 mL of De Man Rogosa and Sharpe Broth (MRS, Merck, Darmstadt, Germany) with 25 g/L NaCl and incubate at 37 °C for 48 h. After the incubation period, all cultures were drawn separately on MRS agar plates and incubated anaerobically for 48 h at 37 °C in an anaerobic vessel equipped with an anaerobic gas generation kit. Colonies of various morphologies were isolated from cultured MRS agar plates. After morphological and biochemical analysis (Gram staining, catalase activity, cell morphology, and sporulation), Gram-positive and catalase-negative bacilli and cocci were identified as his LAB isolates.

### acid and bile resistance

To test acid tolerance, MRS broth containing pepsin enzyme (3 mg/mL) was applied as medium. The pH of the broth was changed to 2.5 with 1.0N HCl and the broth (pH 7.0) served as a control. Additionally, the cultures were inoculated for 3 hours and the optical density (OD) at 600 nm was measured. Resistance to low pH was calculated with the following formula:

$${text{viability}}left(%right):,left[ {{text{OD }}left( {text{after treatment}} right)/{text{OD }}left( {text{before treatment}} right)} right] , times { 1}00% .$$

Strains with >80% viability were selected for further study.twenty oneIsolates were tested for their ability to grow in the presence of 0.3% w/v bile salts. Samples were inoculated for 4 hours at 37 °C and the OD620 samples were evaluated to confirm cell viability. Oxgal resistivity was calculated using the following formula:twenty one:

$${text{viability}}left(%right):,left[ {{text{OD }}left( {text{After treatment}} right)/{text{OD }}left( {text{Before treatment}} right)} right] , times { 1}00% .$$

### Inhibition of aflatoxin-producing Aspergillus by LAB isolates

Inhibition of aflatoxin production was assessed using a modified overlay method Aspergillus flavus When Aspergillus nigertwenty two by two selected LAB isolates. To accomplish this, selected LABs were streaked onto MRS agar plates and incubated for 48 hours at 37 °C in an anaerobic vessel. Next, soft potato dextrose agar (PDA, 75 wt%) was prepared and the prescribed inoculum size (9.5 × 10Four spores/mL) determined by counting with a Neubauer hemocytometer. Plates were then incubated aerobically at 30° C. for 7 days.Each LAB isolate was tested twice for each Aspergillus strain, using one control for each. They were looking for a discrete zone of inhibition surrounding the bacterial streak called the “Positive Impact Zone”.

### antibacterial activity

Agar well diffusion experiments were used to determine the antibacterial properties of the isolates. For this purpose, Mueller-Hinton agar was used as the medium and several different indicator bacteria were used, including: Escherichia coli (PTCC 1276), salmonella enterica (PTCC-1709), Listeria monocytogenes (PTCC 1163), Staphylococcus aureus (PTCC 1764), Bacillus cereus (PTCC 1539), Streptococcus inie (PTCC 1887), Shigella flexneri (ATCC 9199), Klebsiella pneumoniae (ATCC 43816), and Yersinia enterocolitica (ATCC 23715) was used. A sterile, pasteurized pipette was utilized to create wells in the medium.

Finally, 100 mL of isolation supernatant was poured into each well and the plates were incubated at 37 °C for 24 hours.After the incubation period, the diameters of the measured inhibition halo zones were examined statisticallytwenty three.

### antibiotic susceptibility

The disc diffusion method was used to test the susceptibility of isolates to a number of high-dose and therapeutically important antibiotics (see Table 1). Isolates were swabbed with solidified MRS medium, antibiotic discs were placed on the medium and incubated overnight at 37 °C. Finally, the size of the suppression zone around the disc was measured with a digital caliper.twenty four.

### cell surface hydrophobicity

As previously mentioned, we tested the ability of the isolates to adhere to xylene and toluenetwenty fourThe test is run 3 times and is represented by the following formula:

$${text{Hydrophobic }}left( % right) , = , left( {{1 } – {text{ A}}_{{1}} /{text{A } }_{0} } right) , times { 1}00,$$

here0 is the absorbance at 600 nm before adding xylene and toluene, and A1 Absorbance after addition of xylene and toluene for 4 hours.

### Self-aggregation assay

The ability of the isolates to adhere to xylene and toluene was examined as previously reported. The auto-aggregate percentage was measured using the following formula:

$${text{auto summation }}left( % right) , = { 1 } – , left( {{text{A}}_{{text{t}}} /{ text{A}}_{0} } right) , times { 1}00,$$

here0 is the absorbance at time t = 0 and at is the absorbance at time t.

### Aggregation assay

Isolates were tabulated based on the method used Staphylococcus aureus When B. Cereustwenty fiveThe coaggregation rate was calculated based on the following formula:

$${text{A}}_{0} {-}{text{ A}}_{{text{t}}} /{text{A}}_{{text{t}} } times { 1}00.$$

### biofilm formation

The ability of isolates to generate biofilms was assessed using the method of Gomez et al. 26, with some changes. Add 500 mL of the overnight isolate (107-Ten8 CFU/mL). For this purpose, cultures were incubated anaerobically at 37 °C for 48 h. After this, the wells were gently washed three times with 5 mL of sterile distilled water. Adherent bacteria were then fixed using 3 mL of methanol for 15 minutes, after which the plates were emptied and dried at room temperature. Then 3 mL of 2% (v/v) crystal violet solution was poured into the wells and left at room temperature for 5 min. Finally, lyse the dye from the adherent cells using 2 mL of 33% (v/v) glacial acetic acid and measure the His OD of each well in a plate reader (Bio-Rad, Hercules, CA, USA).26.

### molecular identification

#### Genomic DNA extraction

Total genomic DNA was isolated from isolates using techniques developed in our laboratory. A single His colony of each isolate was inserted into a 0.2 PCR tube and 20 µL of lysis buffer was added to the tube for this purpose. The tube was gently vortexed until the contents were homogenous and left at room temperature for 1 hour. They were then incubated in a thermal cycler PTC 200 (MJC Research, Waltham, USA) at 85 °C for 10 min. After this, the tube was filled with 150 L deionized water and centrifuged at 8000×.g 5 minutes. Finally, the top layer containing genomic DNA was removed from the tube and poured into a new tube.Genomic DNA was kept in the refrigerator until needed27.

#### Amplification of 16S-rRNA gene by polymerase chain reaction (PCR)

Genomic DNA samples from isolates were amplified on a PTC 200 thermal cycler using LAB-specific universal primers.twenty threeThe following temperature profile was used for amplification. First, DNA denaturation at 95 °C for 5 min, followed by 32 cycles of denaturation at 94 °C for 60 s, annealing at 59 °C for 60 s, extension at 72 °C for 60 s, and a final extension step72. °C for 5 minutes. PCR products were separated using electrophoresis on 0.8% (w/v) agarose gels and stained with ethidium bromide.

#### 16S-rRNA gene sequencing

A PCR product for the 16S rRNA gene (1544 bp) was generated using the previously described primer set. Korea-based Macrogene Corporation sequenced the PCR products. The sequences were then examined using his BLAST program at the National Center for Biotechnology Information. (http://www.ncbi.nlm.nih.gov/BLAST).

### statistical analysis

Analysis of variance (ANOVA) and Duncan’s test were used to determine significant differences (P ≤ 0.05) between parameters for each isolate. Additionally, Excel 2013 (Microsoft Corporation) and SPSS (IBM SPSS Statistics 20) were used for formal statistical analysis.