# Microbiota \"Fingerprint\" of Greek Feta Cheese through Ripening
## 2 Materials and Methods
### Microbiological Analysis  
Cheese sampling was carried out by cutting sectors from the core of the blocked shaped cheeses, at a depth of approximately 5 cm, under sterile conditions. From the core of each cheese block, two samples (of 20 g each) were collected. The samples were transferred to stomacher bags, mixed with 180 mL sterilized buffered peptone water solution (LAB M, Bury, Lancashire, UK) and homogenized, used a stomacher (Bag Mixer R 400, Interscience, ST Nom, Saint-Nom-la-Breteche, France) for 60 s at room temperature. For each sample, appropriate decimal in buffer peptone water dilutions (10\({}^{-1}\) to 10\({}^{-6}\)) were prepared and 0.1 mL of each dilution was spread, in duplicate on agar plates. For total mesophilic bacteria, surface coating was performed on PCA agar and incubated at 30 \({}^{\circ}\)C for 72 h. Samples were analyzed also for mesophilic and thermophilic lactococci on M-17 agar (Merck, Darmstadt, Germany) anaerobically at 30 and 37 \({}^{\circ}\)C for 48 h, respectively, and lactobacilli on de Man, Rogosa and Sharp (MRS) (Merck, Darmstadt, Germany) agar, at 30, 37, and 42 \({}^{\circ}\)C anaerobically. Kanamycin aesculin azide (KAA) agar (Merck, Darmstadt, Germany) for enterococci at 37 \({}^{\circ}\)C for 24 h, incubated both in aerobic and anaerobic environments [16]. The samples were also tested for microorganisms which affect the safety of the cheeses according to relevant legislation [17; 18; 19; 20; 21]. For this purpose, analysis performed on blood agar base (Merck, Darmstadt, Germany) and MacConkey agar plates (Merck, Darmstadt, Germany) for the detection of foodborne pathogens while cheese samples were also tested for the presence of the pathogenic bacteria _Salmonella_ spp. and _L. monocytogenes_ according to ISO 6579:2002 and ISO 11290-1, 2017, respectively [22; 23]. For the bacterial enumeration, Miles and Misra plate method (surface drop) was performed and 10 \(\upmu\)L of each dilution was also inoculated to the appropriate culture media and conditions [24].  
Identification of Bacteria with MALDI-TOF MS Biotypere  
Pure cultures were identified using Microflex LT MALDI-TOF MS (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry/Bruker Daltonics, Germany). Five single colonies were taken directly from the nutrient agar plate and the sample preparation was performed according to the instructions of Bruker Daltonics and analyzed running Flexcontrol 3.4 software. Each sample was spotted in triplicate. For each sample, the MS signals were acquired in positive linear, according to the manufacture's instruction. The obtained experimental MALDI-TOF mass spectrum profiles were then compared tothe reference spectra and the matching between them was expressed by a Biotyper Log (score). The Log (score) considers the number of matching peaks, the total number of peaks, the peak weight representing species specificity, and a correlation factor related to the matching peak intensity. All samples were measured on the mass spectrometer (Bruker Daltonics, Germany) using the method \"MBT_AutoX\" and the peaks were evaluated with the processing method \"MBT_Process\" using flexControl (version 3.4, Bruker Daltonics, Germany). A calibration took place by using the Bacterial Test Standard (BTS) from Bruker. All spectra were compared with reference spectra of the BDAL database (version 8.0) and main spectrum profiles (MSPs) created [25].  
Dendrograms of identified strains were plotted with the BioTyper MSP Dendrogram Creation Standard Method (version 1.4) using the default settings: the distance measure set as correlation; the linkage set as average; the score oriented dendrogram was selected, the score threshold value for single organism was 300 and the score threshold value for related organisms was zero (0).