Maldi-tof mass spectrometry for the identification and detection of antimicrobial activity of lactic acid bacteria isolated from local cheeses

Research shows that LAB, especially in milk and milk products, are widely available in nature and used as starter cultures for fermentation in dairy industries. On order to shorten the fermentation process and to reduce the risk of fermentation failure, it is necessary to consciously select and use a starting culture (Leroy & De Vuyst, 2004). Despite the limited commercial availability of new starter cultures, LAB can replace chemical additives and help to provide attractive and diverse food products (Pidcock et al., 2002). LAB have recently gained growing interest due to their proteolytic activities and use as protective cultures (Tulini et al., 2016).


Introduction
When preparing food products, any hazards and risks that may affect human health must be considered.Consumers prefer natural and nutritious food products with no chemical additives.Use of lactic acid bacteria (LAB) is a natural and preferable preservation method.
Research shows that LAB, especially in milk and milk products, are widely available in nature and used as starter cultures for fermentation in dairy industries.On order to shorten the fermentation process and to reduce the risk of fermentation failure, it is necessary to consciously select and use a starting culture (Leroy & De Vuyst, 2004).Despite the limited commercial availability of new starter cultures, LAB can replace chemical additives and help to provide attractive and diverse food products (Pidcock et al., 2002).LAB have recently gained growing interest due to their proteolytic activities and use as protective cultures (Tulini et al., 2016).Macaluso et al. (2016) obtained 699 LAB strains isolated from traditional Sicilian cheese and raw milk.Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis bacteria were used to investigate antimicrobial activity.A total of 223 strains were found to inhibit the growth of L. monocytogenes.Ot has been reported that bacteriocin-producing cultures in dairy products improve the quality and safety of those products in a more practical and cost-effective way.El-Ghaish et al. (2017) state that bacteriocins and/or bacteriocin producing LAB should be used as food preservatives to improve the safety of food products (i.e.; cheese and yoghurt).To determine these lactic acid bacteria, a lot of research has been done.Nacef et al. (2017) identified the culturable population of LAB from a French cheese Maroilles made either with raw or pasteurized milk using MALDO-TDF mass spectrometry.All strains unambiguously identified.MALDO-TDF MS identification allowed identification of three genera belonging to LAB including Lactobacillus, Enterococcus and Leuconostoc.Meghoufel et al. (2017) MALDO-TDF MS procedure used to identify 36 lactic acid bacteria isolated from Jben cheese.Kačániová et al. (2017) identified LAB from cow cottage cheese using the MALDO-TDF MS.Respectively, the results showed the domination of the Enterococcus and Lactobacillus genus presents in these cheeses.Although there are various kinds of traditional cheeses in Turkey, LAB that can be used as possible starter cultures isolated from these cheeses have not yet been adequately identified.
The aim of this syudy is to identify potential starter cultures and bacteriocins in order to improve the production quality of various regional cheeses in Turkey.For this purpose, LAB that are used in traditional cheese production were isolated from natural products and identified using biochemical tests and MALDO-TDF MS (Matrix-Assisted Laser Desorption/İonization) method to investigate their antimicrobial properties.

Materials
A total of 21 non-commercial cheese samples were collected from eight villages in Turkey (The various locations are indicated with codes in Table 1).The codes indicated in Table 1 were used to show which isolates where obtained and in which cheese.The cheeses were produced by the local people of the villages using traditional methods.Samples were stored at 4 °C in sterile sample containers and brought to the Food Microbiology Laboratory of the Department of Food Engineering of Sakarya University

Lactic acid bacteria isolation from cheese samples
For the isolation of lactic acid bacteria (LAD), cheese samples (10 g) were homogenized with 90 mL sterilized buffered peptone water.Afterwards, serial dilutions (10 −1 to 10 −6 ) were performed and 0.1 mL of each dilution was platted onto de Man, Rogosa and Sharp (MRS) (Merck, Germany) agar, M17 (Merck, Germany) agar and Kanamycin Esculin Azide Agar (KAA) (Merck, Germany) plates.The M17 and MRS plates were incubated at 30 °C for 48 h and the KAA plates were incubated at 37 °C for 24 to 48 h.The plates were also incubated both in anaerobic and aerobic environments.Anaerocult A (Merck) was used to isolate bacteria developing in the anaerobic environment.150 individual morphologically distinct isolates/colonies were randomly-picked from MRS agar, M17 agar and KAA plates.They were sub-cultured and purified three times in an MRS medium.For the selection of isolates, small, white or pale rectified and smooth-edged colonies were selected for enterococci.Cream-colored, smooth-edged columns were selected for lactobacilli while white, smooth-edged and bright colonies were selected for lactococci.Osolates inoculated into MRS Broth or M17 Broth were incubated for 48 h.Stocks were prepared from 800 μl of LAB cultured in MRS broth or M17 broth.200 μl of sterile liquid glycerol (800 μL active isolate + 200 μL glycerol) was mixed in a 1 mL Eppendorf tube and stored at -80 °C (Harrigan & McCance, 1990).Prior to each analysis, the isolates were activated in MRS, M17 and KAA.

Microbiological and biochemical characterization of lactic acid bacteria isolates
Morphological, physiological and biochemical characteristics were used to identify LAB. 150 pure bacterial isolates were tested for cell morphology, gram reaction and catalase production.Dnly 100 of them were Gram positive and catalase-negative.Growth tests were performed at different NaCl concentrations (4, 6.5%), temperatures (10, 15 and 45 °C) and pH values (9.2, 9.6).Mobility characteristics and gas production were tested (Harrigan & McCance, 1990;Halkman, 2005).

Gram staining and catalase test and mobility test
Gram staining and catalase test were performed in accordance with the methods described by Temiz (2000).Mobility test was performed in accordance with the method described by Halkman (2005).

Development tests of isolates at different temperatures, salt concentrations and pH
Sterile 5 mL M17 and MRS Broth media with 1% isolate were incubated at 10 °C, 15 °C and 45 °C for 48 h.Turbidity was taken into account when evaluating the data.M17, MRS and KAA mediums containing 4% and 6.5% NaCl were used to determine the salt tolerance of bacterial isolates.The isolates were inoculated on an agar medium and their growth was examined after incubation at 37 °C for 48 h.
3.0 mL M17 and MRS Broth media with pH 9.2 and 9.6 were inoculated with the isolates.1% isolate was added to the MRS and M17 Broth media.They were incubated at 30 °C for 7 d.Sterile filtered NaDH and HCl were used to adjust the pH of the media (Holt et al., 1994).

Gas production from glucose test of isolates
Tubes containing 10 mL MRS and M17 Broth media were prepared.The Durham tubes was placed in reverse position and sterilized in autoclave.0.1 mL was taken from the isolates and inoculated with the tubes, and the samples were incubated at 30 °C for 7 d.(Randazzo et al., 2004).

Identification of bacteria with MALDI-TOF MS biotyper
After identifying the isolates using biochemical methods, pure cultures were also identified using MALDO-TDF MS (Matrix Supported Laser Desorption/Oonization Flight Time Mass Spectrometry, Bruker, Germany) method.The samples were automatically analyzed using a MALDO-TDF mass spectrometry (Bruker, Germany) running Flexcontrol 3.4 software.The mass spectrometry was calibrated using the Bruker's bacterial test standard (Bruker Daltonics) in accordance with the method described by Özcan et al. (2016).

Determination of antimicrobial activity of LAB using Kirby-Bauer Disk diffusion method
100 LAB isolates were inoculated into MRS Broth.The cell concentration of the isolates was adjusted using a densitometry McFarland (Biosan) at a level of 0.5-0.6McFarland (10 7 cfu/ml).20 mL of sterile MRS Broth supplemented with 1% isolate were incubated at 30 °C for 48 h.After incubation, the isolates were centrifuged at 4 °C and 10,000 rpm for 45 min.Supernatants were sterilized using sterile membrane filters with a pore diameter of 0.22 μm.(Campos et al., 2008;Uludağ, 2015).Similarly, test microorganismsI: L. monocytogenes ATCC 7644, Staph.aureus ATCC 25923, E. coli D157I: H7, C. sakazakii ATCC 29544, B. cereus and Salmonella Typhimurium ATCC 140828 were inoculated into TSA and incubated at 37 °C for 24 h.Cell concentration was set to 10 7 cfu/mL.The developing test microorganisms was spread on a trypticase soy agar (TSA) plate.15 μL of supernatants were added onto the discs.The petri dishes were incubated for 24 h at the temperature appropriate for each indicator pathogen.The following day, the zones of inhibition around the discs in each plate were recorded in mm (Campos et al., 2008).

Partial purification of bacteriocin
The supernatants obtained previously from lactic acid bacteria contain lactic acid and other organic acids, hydrogen peroxide, bacteriocin and bacteriocin-like substances.Some operations were carried out to observe only the antimicrobial effect of the bacteriocin and bacteriocin-like substances.
First, sterile NaDH and HCl were used to adjust the pH of the supernatant to 7.0 in order to prevent the antimicrobial effect of the organic acids.Afterwards, ammonium sulfate was slowly added to precipitate the proteins into the supernatant.Ot was mixed with vortex until it was dissolved.The final concentration ratio was found to be 40%.The supernatants were stored overnight at +4 °C and then the samples were centrifuged at +4 °C and 10,000 rpm for 45 min.After centrifugation, the upper phase was poured.The remaining precipitate was dissolved in 4 mL of sterile 0.05 M potassium phosphate buffer (pH 7.0).The suspended precipitate mixture was used as a partial bacteriocin extract (Uludağ, 2015).Using the same method, antimicrobial tests were performed in three parallel directions on the partially purified bacteriocin extracts.Zone diameters were measured in mm, and means and standard deviations were calculated.

Statistical analysis
All data were expressed as mean ± standard deviation.Data were analyzed using one-way analysis of variance ANDVA in the Statistical Package for Social Sciences (Version 19.0; SPSS Onc.) at a significance level of P < 0.05.

Identification of lactic acid bacteria
LAB were biochemically classified according to Bergey's Manual of Systematic Bacteriology published in 1984.Table 2 shows the results of the biochemical tests carried out for the identification of the isolates.Osolate from Bolu Gerede cheese could not be obtained.On this regard, there is no information in this section.
Biochemical identification methods fail to differentiate strains down to the species level.Ot is, therefore, better to identify strains at the genus level when those methods are used (Dimitonova et al., 2008).Based on the results, homofermentative cocci isolates that grew at 45 °C, 6.5% NaCl and pH 9.6 were identified as Enterococcus (44%) (Kim & Lee, 2013).Cocci-shaped isolates that grew at 10 °C, but not at 45 °C, 6.5% NaCl and pH 9.6 were identified as Lactococcus (Axelsson, 2004).46% of isolates that were rod-shaped and did not produce CD 2 from glucose were identified as Lactobacillus while 10% of those that produced gas from glucose were identified as heterofermentative Leunoconostoc (10%) (Sezer, 2007).All isolates in the table are Gram (+) and catalase (-).Glucose-free gas formation was not observed in the majority of isolates, indicating that they were homofermentative strains.All identified isolates were stationary.
The combination of molecular and biochemical identification methods allows a more accurate identification (Tabasco et al., 2007).Therefore, these results should be confirmed at the molecular level as well.Table 2 also shows the identification results at the species level by the MALDO-TDF method.LAB colonies isolated from non-commercial cheeses were identified using the recently developed MALDO-TDF MS method, which allows the comparison of resulting profiles with the reference spectrum and rapid classification of isolates.E. durans (6), E. faecalis (18), E. faecium (24), E. italicus (2), Lb. brevis (1), Lb. paracasei (2), Lb. plantarum (1), Lactococcus lactis (3), Leuconostoc lactis (1), Leu.mesenteroides (11), and Streptococcus parauberis (2) were identified using this method.29 isolates were eliminated because they were identified as yeast and non-lactic acid bacteria.
The results of the MALDO-TDF MS and biochemical identification methods were compared.55 strains were identified as the same strain at the genus level while 16 strains were identified as different strains at the genus level.Moreover, the identification at the molecular level revealed that although some strains had the same characteristics, they were different bacteria.On parallel with the literature, this result also indicated that the biochemical identification methods are insufficient in differentiating species.Fguiri et al. (2015) identified Lc. lactis, Lb. pentosus, Lb. plantarum, Lb. brevis and Pediococcus pentosaceus using biochemical methods, however, they identified E. faecium using molecular methods.Ot has been determined that molecular analysis is the most reliable method for identification.
On general, identification of LAB from cheeses relies on methods based on phenotypic features and/or on physiological and biochemical criteria.However, these complex and time-consuming methods can underestimate the microbiological diversity of a food ecosystem (Ercolini et al., 2001).On contrast, the nucleic acid-based molecular approaches have proven to be powerful tools for the identification of microbial diversity in food samples (Coeuret et al., 2003).The MALDO-TDF MS identification is a rapid, cost-effective, robust and reliable method (Pavlovic et al., 2013).Ot is, therefore, considered to be an attractive alternative to biochemical and even molecular biological methods (Dec et al., 2014;Vithanage et al., 2014).Dušková et al. (2012) reported that the MALDO-TDF MS method has a higher success rate (93%) than the polymerase chain reaction (PCR) method (77%) in identifying Lactobacillus species at the species level.On some cases, the MALDO-TDF MS method allows the identification of bacteria at subspecies level (Carbonnelle et al., 2011).Ot can be concluded from these results that the MALDO-TDF MS method is affordable, sustainable and robust.
On this study E. faecium and E. faecalis were dominant in all isolates obtained from cheeses according to MALDO-TDF MS method (Figure 1).
Enterococcus bacteria were also detected in 13 cheese varieties.Similarly, studies have reported that enterococci are dominant in many cheeses such as Cebreiro, Kefalotyri, Manchego, Picante in Beira Baixa, Semicotto Caprino, White, Teleme Tomas, Brydza (Öner et al., 2004;Jurkovič et al., 2006;Tuncer, 2009;Yoğurtçu, 2011).Enterococci show very good adaptation to the environment (Tenreiro et al., 2012), which may explain the dominance of cheeses that contain Enterococcus bacteria.E. faecium and E. faecalis are two probiotic species (Erginkaya et al., 2007).Therefore, the probiotic properties of these strains should be further researched and their use as commercial starter cultures should be extensively investigated.

Antimicrobial activities of LAB
The capacity of substances to inhibit microbial growth is referred to as antimicrobial activity.On the study, antimicrobial activity tests were performed on 100 LAB isolated from cheeses.As can be seen in Table 3, antimicrobial activity was observed in 10 isolates.Accordingly, about 10% of LAB showed antimicrobial activity against 6 pathogens, indicating that most lactic acid bacterial isolates do not exhibit antimicrobial activity.The antimicrobial activity of substances from different isolates of the same strain against pathogens appears to be different from each other, which may be due to different metabolites produced by the sub-species of the isolate.
Many studies have reported that LAB obtained from cheeses show antimicrobial activity against pathogens such as L. monocytogenes, B. cereus and E. coli (Bilgin 2008;Abanoz 2014;Uludağ 2015).Similarly, Lb. plantarum isolate (OS2) from Bryndza showed very good antimicrobial activity against L. monocytogenes ATCC 7644, E. coli D157I:H7, C. sakazakii ATCC 2954 and B. cereus, and the diameter of zones of inhibition ranged from 9 mm to 15 mm.E. faecium isolate (O71) from Bryndza also showed antimicrobial activity (15 mm) against C. sakazakii ATCC 29544.The isolated LAB successfully inhibited these pathogenic strains.Therefore, addition of these isolated LAB to commercial food products can provide effective protection against infections caused by those pathogens.None of the isolates showed antimicrobial activity against Staph.aureus ATCC 25923.Similarly, Samantır (2014) reported that LAB strains isolated from swede did not show any antimicrobial activity against Staph.aureus ATCC 10832.

Determination of antimicrobial activity of bacteriocins
On this study, only two of the partial bacteriocins obtained from isolates (F72, OA2) showed effective antimicrobial activity against pathogens, indicating that the antimicrobial activity of LAB results from organic acids and low pH values.Dur results agree well with those reported by Sezer (2007) who reported that only 35 of the 601 isolates retained the effect after neutralization.
The partial bacteriocin produced by E. faecium obtained from herb cheese formed a zone of inhibition (9 mm in diameter) against E. coli D157I: H7 and the partial bacteriocin produced by E. faecalis isolated from Bryndza formed a zone of inhibition (11 mm in diameter) against S. Typhimurium ATCC 14028.Dnly bacteriocins (enterosin) obtained from enterococci protected antimicrobial effects.This may be due to the fact that bacteriocin produced from E. faecium and E. faecalis strains has better physico-chemical properties and biological activities than other bacteriocins (Foulquié Moreno et al., 2003).Ot has been concluded that bacteriocin produced from E. faecium can be used as biological preservatives to extend the shelf life of dairy products.

Conclusion
Consumers' demand for unprocessed food products and reduced use of chemical preservatives in the food industry have led to an increase in the number of studies on the potential use of LAB as biopreservatives against pathogenic bacteria.Traditional cheese products could serve as good and alternative sources of LAB for consumers.On this study, traditional cheeses were found to contain many different antimicrobial LAB and different promising bacteriocins that can be used as food additives.LAB were found to have components that inhibit pathogen development.Therefore, they can replace the chemical additives in the food industry and contribute to the production of diverse food products.We believe that the use of bacteriocin-producing cultures will improve the quality and safety of food products in an efficient and cost-effective way.Therefore, bacteriocins and/or bacteriocin producing LAB should be added as food preservatives to food products (i.e.; cheese and yoghurt) in order to improve food safety.The results also show that the MALDO-TDF MS is a more accurate, rapid and cost-effective method than biochemical tests for identification of LAB.

Figure 1 .
Figure 1.Percent distribution of lactic acid bacteria isolated from all cheeses according to MALDO-TDF MS method.

Table 1 .
Provinces, codes and names of the cheeses used in the research.

Table 2 .
Biochemical and MALDO-TDF identification results of LAB isolated from cheese samples.