Identication and biotechnological characterization of Lactic Acid Bacteria Isolated from White Cheese Samples

In this study, the isolation of lactic acid bacteria were carried out from approximately one hundred white cheese samples collected from different regions of Turkey. Subsequently, phenotypic and genotypic characterization of the isolates were performed. Finally, biotechnological enzyme and bacteriocin production potentials of the isolates were determined. As a result of the analysis, a total of fourty one bacteria were isolated and seventeen of them were found to be different species. The isolates generally grew at 4–6 pH values, 0–8% NaCl and 30–40 o C. Genomic ngerprint proles of the isolates were determined by using BOX-PCR. According to 16S rRNA sequence results, test strains belong to Lentilactobacillus keri, Levilactobacillus brevis, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Pediococcus lolii, Staphylococcus haemolyticus, Lysnibacillus sinduriensis, P. parvulus, Lactiplantibacillus paraplantarum, Staphylococcus hominis, Lactiplantibacillus plantarum, Enterococcus faecium, Micrococcus yunnanensis, Microbacterium paraoxydans and Micrococcus aloeverae species. Since the isolate coded MA56 is 96.41% similar to Lentilactobacillus buchneri, it is thought to be a new species. Also MA19, MA25, MA43 and MA47 were determined to have multi-enzyme production potential. MA43 was found to be the only isolate producing bacteriocin.


Introduction
Cheese is produced by coagulating the milk with the effect of a suitable proteolytic enzyme or organic acids. and processing, ltering, suppressing and maturing in various ways (Carafa et  Besides the health aspect, LABs can also be the source of new species with enzymatic activities for biotecnological process (Mathur et al. 2020).
Microbial enzymes are more preferred than other enzymes because they have high catalytic activity and e ciency. Major enzymes used in biotechnological processes include amylases, proteases, and lipases. LAB with amylase, lipase xylanases and protease activities have been reported in previous studies. Considering these properties, interest in lactic acid bacteria is increasing day by day and there are many LAB species with technological potential yet to be discovered. In the light of all this information, in this study, isolation, identi cation and molecular characterization of lactic acid bacteria from cheese samples collected from different regions (Erzurum, Van, Konya, Karaman and Kars) of Turkey were carried out. Later, biotechnologically important enzyme and bacteriocin production potentials of the isolates were determined.

Material And Methods
Sampling and lactic acid bacteria isolation A total of one hundred Cow cheese samples taken from markets in different regions of Turkey were brought to the laboratory under aseptic conditions and kept at + 4°C until they were studied. 225 ml of sterile physiological water (0.9% NaCl) was added to 25g cheese sample and homogenization process was carried out and dilution series (10 0 -10 − 7 ) were prepared (Luiz et al. 2017). The dilution liquids were spread on MRS and M17 Agar media and incubated at 35°C for 48 hours. After the incubation, isolates were stored at -86 o C in a stock medium containing 15% glycerol (Kirmaci et al. 2016).

Phenotypic Characterization
To determine phenotypic characterization, test strains were grown in MRS and M17 medium at different temperatures 15°C to 50°C (with 5°C intervals) for up to 72 h. The pH ranges were analyzed in MRS and M17 at pH 3.0-11.0 (1 pH unit intervals), Tolerance of NaCl was determined using MRS and M17 supplemented with 0-12 % NaCl (at intervals of 1.0 %) for 72 h. All experiment were tested in triplicate and growth was measured at OD600 nm ). Gram-staining of test strains was performed according to method of Gerhardt et al (Gerhardt et al. 1981). Catalase activity was performed by the production of bubbles of a drop of 3 % H 2 O 2 (v/v). Oxidase reagent (Sigma) was used for testing oxidase activity (Adiguzel et al. 2020). Biochemical characterization of isolates were conducted using API 50CHL test (Anekella and Perez-Diaz 2020).

Determination of Bacteriocin Production Potentials
After determination of isolates species, The presence of related bacteriocin genes were investigated. For this purpose, PCR analyzes were performed using primers speci c to each bacteriocin gene (Chiorean et al. 2018). The 16S PCR cycle given above was performed by changing anneling temperatures of bacteriocin primers.

Detection of Antibacterial Activity
For detection of antimicrobial activity, disc diffusion assay was used. Pathogenic bacteria were spread on the surfaces of Mueller Hinton agar media. Overnight cultures, on MRS medium, of the strains to be tested were centrifuged and cell free supernatant was loaded on discs. The plates were incubated at 37°C for 24h. The antagonistic effects of the test strains were determined by measuring the zone diameters (Mezaini et al. 2009

Results And Discussion
Isolation of LAB In the study, a total of 41 bacteria isolated from cheese samples obtained from Erzurum Kars, Karaman, Konya and Van provinces for the isolation of lactic acid bacteria and stock cultures were prepared. Since seventeen of the isolated bacteria belonged to different species, the study was continued with these isolates.   ). According to this information, MA56 might be a novel species belonging to the genus Lentilactobacillus. It is thought that MA56 will be added to the literature as a novel species as a result of the whole genome sequence analysis in future studies. Detailed sequence results of the isolates and related species are given in Table 2. Also, 16S rRNA gene based phylogenetic tree is shown in Fig. 1. Previous studies have reported that rep-PCR is an easy method that can be used to classify bacteria. Mohammed et al. used BOX-PCR analysis to characterize lactic acid bacteria isolated from traditional milk samples in their study (Mohammed et al. 2009). We also performed genomic ngerprint analysis of isolates using BOX-PCR in this study. While 12 polymorphic bands were observed in some of the test strains, it was observed that there was 1 band in some isolates Fig. 2. It was observed that the BOX-PCR was not su cient to clasify the all LAB.

Determination of Biotechnological Enzyme Production Potentials of Isolates
The potential of isolates to produce amylase, lipase, protease and xylanase enzymes, which are biotechnologically important, were determined. As a result of analysis one strains have xylanase and lipase activity, ve strains with amylase activity, and ten strains with protease activity were observed (Table 3). Also petri images of some isolates are given in Fig. 3. Matthews et al. investigated the enzyme production potential of lactic acid bacteria and determined that especially Lactobacillus and Pediococcus species are important producers of lipase, cellulase and xylanase enzymes. In another study, Konkit and Kim examined that Lactoccocus chungangensis produces amylase, proteinase, and lipase enzymes (Konkit and Kim 2016).  There are many studies in the literature in which enzymes obtained from lactic acid bacteria are used in biotechnological processes. For example, The xylanase enzyme puri ed from Pedioccus acidilactici was applied in clari cation of fruit juices (Adiguzel et al. 2019). In another study, it was reported that the protease enzyme obtained from Lactobacillus plantarum had an antimicrobial effect on pathogenic microorganisms (Lin and Pan 2019). So, in this study, isolates coded MA19, MA25, MA43 and MA47 were determined to have multi-enzyme production potential (Table 3) and these isolates are attractive for biotechnological processes, because they have more than one enzyme activity.

Determination of Bacteriocin Production Potential
Whether the isolates produced bacteriocin was determined using speci c bacteriocin primers with PCR. As a result of the PCR analysis, it was determined that only the MA43 coded isolate produced bacteriocin. The gel image of plantaricin belonging to MA43 and Lactobacillus plantarum ATCC 8014 are shown in Fig. 4.
After it was determined that MA43 has bacteriosin gene, its effect against pathogenic bacteria was investigated. It has been determined that MA43 has a highest antagonistic effect against Escherichia coli O157:H7 ATCC 43888 and Pseudomonas aeruginosa ATCC 9027. Previous studies have also reported that Lactobacillus plantarum has an antimicrobial effect against various pathogens (Layus et al. 2020;Wang et al. 2017). In this respect, MA43 has an antagonistic effect against foodborne pathogens shows that it can use food preservatives.

Conclusions
With this study, it was determined that the white cheese samples have a very wide micro ora. 16S rRNA sequence similarity to the closest species of the MA56 was determined as 96.41%. This isolate is highly likely to be a novel species of lactic acid bacteria. MA43 not only has amylase, lipase and protease activity but also produces bacteriosin makes it unique for biotechnological processes. In addition, this study leads to new studies for MA56 and MA43.

Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.
Neighbour joining phylogenetic tree based on 16S rRNA gene sequences of test strains and related type species. Yersinia enterocolitica ATCC 9610 was used as out-group. Bootstrap values based on 1000 replications are listed as percentages at branching points. The accession numbers are given in parentheses. The scale bar represented 0.5% divergence.