Molecular detection, phylogenetic analysis, and antibacterial performance of lactic acid bacteria isolated from traditional cheeses, North‐West Iran

Abstract Lactic acid bacteria (LAB) are candidate probiotic bacteria that can provide health benefits when delivered via functional foods. The purpose of this study was to isolate and characterize LAB from traditional cheeses consumed in north‐west regions of Iran. A number of sixty traditional cheeses samples were collected and initially screened as LAB using biochemical and molecular methods. A fragment of 1,540 bp in size of 16s rRNA gene was amplified from 70 bacterial isolates. Restriction fragment length polymorphism (RFLP) was employed to differentiate LAB isolates. LAB isolates generated three different RFLP patterns using HinfI restriction enzyme. Phylogenetic analysis revealed that LAB isolates belonged to three genera including Enterococcus, Lactobacillus, and Lactococcus. Most of the isolated LAB strains belonged to Enterococcus spp. The antimicrobial performance of eight LAB isolates with different RFLP patterns ranged from 6.72 to 14.00 mm. It was concluded that molecular characterization of LAB strains in traditional cheeses will enhance our understanding of traditional food microbiota and will help to find bacterial strains with probiotic potential with great benefit both in health and industry.

there is a global interest to isolate LAB from food commodities for application in functional food and dietary supplement (Chiang & Pan, 2012). LAB isolated from different food sources drawn a lot of attention in combating food-associated pathogens and spoilers, biodegradation of chemical contaminants, and development of unique food with special interests for the consumers (Koohestani, Moradi, Tajik, & Badali, 2018;Lim, Yeu, Hong, & Kang, 2018). LAB secrete diverse bioactive metabolites such as organic acids, short-chain fatty acids, carbohydrates, antimicrobial peptides, enzymes, vitamins, cofactors, immune-signaling compounds, and complex agents, and most of them, for example, organic acids, hydrogen peroxide, bacteriocins, etc. exhibit different antibacterial activity in individual and synergistic forms on foodborne pathogens .
The bactericidal effects of such agents on a wide range of pathogens such as Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus have been studied (De Vuyst & Leroy, 2007;Hassanzadazar, Ehsani, & Mardani, 2014).
To date, various food sources, for example, human and animal milk, cheese, meat products, etc. have been investigated for isolation and identification of new LAB strains with novel functional properties (Al-Gamal et al., 2019;Awaisheh & Ibrahim, 2009;Hassanzadazar et al., 2014). This has resulted in the acquisition of new strains. However, researchers are still trying to isolate and identify new isolates with unique properties from different sources. Koopeh, Shal, and Shoor are three popular traditional cheese varieties consumed in the North-West of Iran. Koopeh cheese is one of the most popular fermented dairy food not only in Iran but also in some parts of Turkey and Iraq (Amirbozorgi, Samadlouie, & Shahidi, 2016).
It is a semisoft type cheese made mainly from raw ewe's milk and less commonly from cow milk or a mixture of both kinds of milk without using any starter in clay jugs (Hassanzadazar et al., 2014). Shoor is a salty cheese that traditionally is produced from boiled buttermilk with a reasonable amount of salt, and Shal cheese is another popular milk product in the west of Iran which is freshly prepared in large casts.
In the present study, we attempted to isolate and discriminate LAB from three traditional cheese varieties consumed in West Azerbaijan province, Iran, using biochemical and molecular methods.
The phylogenetic tree of the isolated LABs was generated based on the partial sequences of the 23s rRNA gene. Additionally, the antibacterial performance of LAB isolates was investigated on L. monocytogenes.

| Cheese collection
Sixty traditional cheese samples (~200 g) composed of Koopeh (n = 20), Shal (n = 20), and Shoor (n = 20) were collected from markets from three different geographical locations in West Azerbaijan province, Iran. The samples were aseptically transferred to the Faculty of Veterinary Medicine, Urmia University, under cold and aseptic conditions.

| Bacteria isolation and biochemical characterization
First, an amount of 25 g of each cheese sample was added to 225 ml 0.1% w/v peptone water (Merck, Darmstadt, Germany) and homogenized using a stomacher (Seward Medical Ltd., London, U.K) at 280 rpm for three min. The cheese suspension was diluted in 2% w/v sodium citrate (Merck) and cultured on two De Man, Rogosa and Sharpe (MRS) agar (Quelab, Montréal, Canada) plates and incubated under anaerobic (anaerobic jar) and aerobic conditions for 1-2 days at 37°C. The 3-4 different single colonies were randomly selected from each cultured plate. The selected colonies were Gram stained, examined microscopically, and catalase test was also performed. Gram-positive and catalase-negative bacilli/ cocci were chosen and stored in cryotube containing 15% (v/v) glycerol (Merck) at −20°C for further molecular and antibacterial characterization.

| Extraction of bacterial genomic DNA
Genomic DNA from previously cultivated LAB in MRS broth (Quelab) for 12-18 hr at 37°C was used. The DNA was extracted using DNA extraction kit (SinaClon, Tehran, Iran) according to the kit's manufacturer instruction. The quality and amount of extracted DNA were evaluated by Nano-Drop 2000c (Termo Scientific, Massachusetts, USA). Then, extracted DNA samples were kept at −20°C for subsequent application.

| Purification and restriction endonuclease digestion of PCR products
All amplified PCR products were subjected to purification using Combo GP Kit (GeneALL, Seoul, Korea) following the kit's manufacturer instruction. For restriction endonuclease digestion of purified PCR products, HinfI endonuclease (Jena Biosciense, Germany) was used. Digestion reaction was performed in 15 µl reaction volume, containing 1 µl FastDigest HinfI enzyme, 1.5 µl universal buffer, 4.5 µl dH 2 O, and 8 µl of the purified PCR product. The reaction mixture was incubated at 37°C for 5 min. The digested PCR products were electrophoresed, stained with Safe Stain, and visualized using a UV transilluminator.

| DNA sequencing and phylogenetic analysis
Purified PCR products of eight bacterial isolates with different restriction fragment length polymorphism (RFLP) patterns were selected for further nucleotide sequencing. Purified PCR products from the gel were sequenced (Bioneer Company, Daejeon, Korea). The obtained nucleotide sequences of 16S rRNA were BLAST searched in GenBank (National Centre for Biotechnology Information, Rockville Pike, Bethesda, USA) using the advanced BLAST similarity search option and compared to the 16S rRNA sequences of Lactococcus, Lactobacillus, and Enterococcus subsp. from GenBank (Table 1).
Nucleotide sequences were aligned and compared to other nucleotide sequences retrieved from GenBank using Clustal W, and the phylogenetic tree was generated using the neighbor-joining method in MEGA software (version 6.0; Biodesign Institute, Tempe, USA) (Kumar, Stecher, Li, Knyaz, & Tamura, 2018

| Antibacterial activity of LAB isolates
To determine the antibacterial performance of 8 LAB isolates with different RFLP patterns, spot-on-the-lawn method was applied (Koohestani et al., 2018). For this, bacterial suspension was prepared by inoculation of each isolate in MRS broth under anaerobic or aerobic conditions at 37 ± 1°C and then 10 µl of each bacterial suspension was carefully dropped on a plate containing MRS agar.
monocytogenes ATCC 19,115, by visible-ultraviolet spectrophotometer at 600 nm, was poured on the palate and inoculated at 37 ± 1°C for 24 hr. Zone of inhibition (ZOI), as a clear area around each spotted bacterial suspension, was checked by a caliper in triplicate. Lb.
casei 431 was used as a control strain with antibacterial activity.

PCR and RFLP
A DNA fragment of 1,540 bp in size was amplified for all isolates from 70 bacterial isolates (Figure 1a). RFLP analysis of the PCR products revealed three different digestion patterns (patterns I-III) ( Figure 1b). Of 70 isolates, amplified fragments of 16s rRNA from 63 (90%) isolates showed RFLP pattern I. RFLP patterns II and III each with two and five isolates were all the RFLP patterns identified using HinfI endonuclease enzyme.

| Phylogenetic analysis
Phylogenetic tree constructed based on neighbor-joining analysis of 16S rRNA clustered eight examined isolates in four different clusters ( Figure 2 were the most prevalent LAB which were identified in the traditional cheeses in the present study.

| Antibacterial activity
The ZOI (Figure 3) of selected LAB isolates were shown in Table 2.
Results showed that antimicrobial performances of all investigated isolates with ZOI were ranged from 6.72 to 14.00 mm, while Lb.  crobiota of traditional dairy products will help to improve and standardize these products and thus enhance consumer acceptability in broader regions (Yu et al., 2015).

| DISCUSS IONS
Enterococcus subsp. was the most prevalent LAB genus identi- fied in the examined traditional cheese in the present study. The genus Enterococcus comprises many species, in which only a few have been studied to be used as probiotics, for example, Ent. faecalis, Ent. faecium, Ent. lactis, and more recently Ent. hirae (Adnan, Patel, & Hadi, 2017) and Ent. durans . These strains have the ability to produce a variety of antimicrobial compounds, such as organic acids and bacteriocins (Hassanzadazar et al., 2014). In a study by Silva et al. (2015), about 50% of identified LAB isolated from Brazilian water buffalo mozzarella cheese belonged to Enterococcus subsp. It was also reported that four strains out of six identified LAB isolates from traditionally fermented Xinjiang cheese were Ent. hirae (Azat et al., 2016). Ghahremani, Mardani, and Rezapour (2015) reported that 81% of LAB isolated from traditional cheese in Khorramabad city of Iran belonged to Enterococcus subsp. All these studies demonstrated that the majority of LAB isolated from different traditional cheese from various regions have belonged to Enterococcus genus. In Russia, seven Lactobacillus and Bifidobacterium species were identified in the traditional fermented dairy products sampled from different regions (Yu et al., 2015). All these mentioned reports and other works from different countries demonstrating that traditional dairy products have complex compositions of LAB species (Campagnollo et al., 2018;Parsaeimehr, Khazaei, Jebellijavan, & Staji, 2019).
Two strains, Lb. farciminis and Lb. paracasei, were identified in the examined cheese samples. Lb. farciminis is a probiotic strain commercialized by Dupont-Danisco for animal nutrition as a feed additive (Tareb, Bernardeau, Amiel, & Vernoux, 2017). It was shown that Lb.
farciminis as a feed additive would help to improve ruminal fermentation digestibility without any adverse effect on the pH of rumen and enhance ruminant productivity through manipulation of the rumen microbial ecosystem (Elghandour et al., 2019). Lb. paracasei and Lac. lactis were the next LAB strains which were identified in the examined cheese in the present study.
Antibacterial potential of LAB is one of the most promising specifications that influence the application of LAB in the food industry F I G U R E 2 The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.49584526 was reported. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. This analysis involved 27 nucleotide sequences. Codon positions included were 1st + 2nd+3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 616 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018) F I G U R E 3 An example of antibacterial zone of inhibition of one selected LAB isolate based on spot-on-the-lawn method (Moradi, Mardani, & Tajik, 2019). Antibacterial agents such as bacteriocins, organic acids, enzymes, alcohols, and low molecular mass substances are among the most studied antimicrobial agents responsible for the antimicrobial action of LAB .
As reported in this study (Table 2), this activity is species-and strain-dependent. For example, isolates with RFLP pattern of II and III that belong mainly to Lactococcus subsp. and Lactobacillus subsp. revealed strong antibacterial activity than isolates with RFLP pattern I that consist of Enterococcus subsp. Similar results were reported on antibacterial activity of LAB isolated from raw camel milk (Rahmeh et al., 2019), Genestoso cheese (González et al., 2007) and Brazilian artisanal cheeses (Margalho et al., 2020). Organic acids production, as the main antibacterial agents, by LAB is mainly a genus-specific phenomenon and partially specie-specific. In this regard, lactic acid production by LAB strains is significantly higher than acetic acid production (Thierry et al., 2015). There is a relationship between organic acid production, type of culture media, and antibacterial activity of LAB. For example, LAB at fish broth and MRS produce succinic and acetic acids at the highest and lowest contents (Özcelik, Kuley, & Özogul, 2016). However, the EPS derived from LAB represent moderate to strong antibacterial activity on some foodborne microorganisms (i.e., pathogens and spoilage groups), depending on bacterial species and EPS source and characterization .

| CON CLUS IONS
In conclusion, traditional cheeses examined in the present study were identified as sources of LAB especially Enterococcus subsp.
It was revealed that PCR-RFLP combined with 16s rRNA gene sequencing will be a valuable tool for the identification and molecular characterization of LAB in the food commodities especially dairy products. It seems that the observed antibacterial activity appears to be strain-dependent.

ACK N OWLED G M ENTS
The authors would like to thank the Dean of Research of the Ferdowsi University of Mashhad for funding this project and the Faculty of Veterinary Medicine, Urmia University for the technical support.
The technical assistance of Ms. E. Divsalar and Dr. P. Khademi is greatly appreciated.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L S TATEM ENTS
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are available upon request.