applied sciences Article Microbiota “Fingerprint” of Greek Feta Cheese through Ripening Athina Tzora 1,* , Aikaterini Nelli 1, Chrysoula Voidarou 1 , George Fthenakis 2, Georgios Rozos 3, Georgios Theodorides 4 , Eleftherios Bonos 1 and Ioannis Skoufos 1 1 Laboratory of Animal Health, Food Hygiene and Quality, Department of Agriculture, University of Ioannina, 47132 Arta, Greece; [email protected] (A.N.); [email protected] (C.V.); [email protected] (E.B.); [email protected] (I.S.) 2 Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece; [email protected] 3 Laboratory of Microbiology, Biotechnology & Hygiene, Department of Agricultural Development, Democritus University of Thrace, 68200 Orestiada, Greece; [email protected] 4 Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; [email protected] * Correspondence: [email protected]; Tel.: +30-26810-50202 Abstract: Feta is a Greek protected designation of origin (PDO) brined curd white cheese made from small ruminants’ milk. In the present research, Greek Feta cheese bacterial diversity was evaluated via matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Analysis of 23 cheese samples, produced in different regions of the country, was performed in two ripening times (three or six months post-production). The identified microbiota were primarily constituted of lactic acid bacteria. A total of 13 different genera were obtained. The dominant species in both ripening times were Lactobacillus plantarum (100.0% and 87.0%, at three or six months Lactobacillus brevis Lactobacillus paracasei post-production, respectively), (56.5% and 73.9%), (56.5% and 39.1%), Lactobacillus rhamnosus (13.0% and 17.4%), Lactobacillus paraplantarum (4.3% and 26.1%), Lactobacillus curvatus (8.7% and 8.7%). Other species included Enterococcus faecalis (47.8% and 43.5%), Citation: Tzora, A.; Nelli, A.; Voidarou, C.; Fthenakis, G.; Rozos, G.; Enterococcus faecium (34.8% and 17.4%), Enterococcus durans (13.0% and 17.4%), Enterococcus malodoratus Theodorides, G.; Bonos, E.; Skoufos, I. (4.3% and 4.3%), and Streptococcus salivarius subsp. thermophilus (21.7% and 30.4%). The increased Microbiota “Fingerprint” of Greek ripening time was found to be correlated to decreased total solids (r = 0.616; p = 0.002), protein Feta Cheese through Ripening. Appl. (r = 0.683; p < 0.001), and PH (r = 0.780; p < 0.001). The results of this study contribute to a better Sci. 2021, 11, 5631. https://doi.org/ understanding of the core microbiota of Feta cheese. 10.3390/app11125631 Keywords: Feta cheese; microbiota; safety; ripening; MALDI-TOF MS Academic Editor: Alessandra Lucchese Received: 26 May 2021 1. Introduction Accepted: 16 June 2021 Published: 18 June 2021 Feta cheese is a white brined cheese produced traditionally in Greece, made of either 100% sheep milk or a mixture of 0–30% goat milk and 70–100% sheep milk. Its flavor Publisher’s Note: MDPI stays neutral is distinguished by its natural taste and texture as well as its saltiness and slight acidity. with regard to jurisdictional claims in Since 2008, Feta cheese belongs to the Protected Designation of Origin Products (PDO) published maps and institutional affil- group [1], ensuring the strict quality and safety specifications imposed by the European iations. Union. It is by far the most consumed Greek cheese, representing 90% of the total national cheese production. Four basic ingredients are needed to produce Feta cheese: milk, rennet, microorganisms, and salt. These ingredients follow the specific process of gel formation, whey expulsion, acid production, and salt addition. Then, a period of ripening takes place [2]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI- This article is an open access article TOF MS) has been recently utilized as a tool to characterize microorganisms fast and distributed under the terms and accurately, while it has been used as a tool to identify bacteria, yeasts, mycobacteria, and conditions of the Creative Commons molds [3]. The microbial identification through mass spectrometry is achieved by analyzing Attribution (CC BY) license (https:// the expression of their intrinsic proteins. This mass spectral pattern of protein expression creativecommons.org/licenses/by/ is compared with reference patterns in a database and can be distinguished by a large 4.0/). spectrum of proteins in order to classify closely related organisms at the species level [4]. Appl. Sci. 2021, 11, 5631. https://doi.org/10.3390/app11125631 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 5631 2 of 14 This has as a result to generate a characteristic mass and intensity distribution of the mainly ribosomal proteins, representing a “molecular fingerprint”. Some advantages stemming from this technology include the increase in the reporting of some relatively rare species over traditional methods, the easy pretreatment of samples, the quick identification results, and the lower costs. At the moment, MALDI-TOF MS profiling is accepted as a univer- sally valid identification method and is used in food microbial ecology studies, including those on dairy products [5]. A recent study used MALDI-TOF MS to identify non-starter lactic acid bacteria (NSLAB) isolates from four artisanal kinds of cheeses, namely Kefalo- tyri, Anthotyro, Xynotyri, and Touloumotyri, synthesized from raw goat and sheep milk. The identified lactic acid bacteria (LAB) species included Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus paracasei, Leuconostoc mesenteroides, Entero- coccus faecium, Lactococcus lactis, and Pediococcus pentosaceus [6]. Albesharat et al. assessed the LAB strains in breast milk, feces of mothers and babies, and local foods (fermented) using genotypic tools such as 16S RNA gene sequencing, random amplified polymorphic DNA (RAPD) and MALDI-TOF [7]. Feta PDO is defined by its distinctive organoleptic features. Various microbial popula- tions coexist, operate, and interact, providing through their metabolism, the development of taste, texture, aroma, safety, and shelf-life of the cheese [8]. These microbiota dynamics and changes control both organoleptic and biosafety characteristics, thus defining the quality of the product [9]. The microflora of cheeses differ through time and are based on the cheese type, as well as the environmental, processing, and aging conditions. Feta cheese microbiota are originated from the milk microbiota of indigenous sheep and goat breeds, the starter lactic acid bacteria and the secondary microflora [6]. The starter lactic acid bacte- ria (SLAB), are inoculated starter cultures, involved in acid production during manufacture and contribute to the ripening process. Secondary microorganisms are not responsible for acid production during manufacture, but generally play an important role during ripening as these bacteria contribute to the lipolysis and proteolysis of cheese [10]. The secondary microflora consist of non-starter lactic acid bacteria (NSLAB) which grow internally and are often unique to specific cheese varieties. Specifically, the microbial ecology of cheese is based on the complex interaction among SLAB and NSLAB [11]. The screening of microbiota which affects Feta’s unique organoleptic properties and re- flects product quality, authenticity, and safety is critical, as this is the Greek cheese with the higher consumption worldwide. Cheese microbiota diversity and overall safety is affected by production regions, traditional techniques, and ripening time. This diversity can also de- termine the sensorial appeal and nutritional value for the consumer. The microbial profile of Feta PDO has been reported in a very limited number of scientific papers. Rantsiou et al. studied the microbiota in four Feta cheese samples from pasteurized milk obtained from retail shops by polymerase chain reaction–denaturing gradient gel electrophoresis (PCR- DGGE) and identified LAB and yeast species [12]. Additionally, Bozoudi et al. studied the NSLAB, by molecular methods such as random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and pulsed-field gel electrophoresis (PFGE) on fresh and ma- ture artisanal Feta cheese samples at three mountainous areas of Greece [13]. Furthermore, Spyrelli et al. used 16s rRNA gene sequencing to examine the effect of container material on fresh and mature Feta cheese bacterial diversity [14]. The main goal of our work was to define the standard core microbiota of Feta PDO cheeses through the identification of the bacteria by the culture-dependent technique MALDI-TOF MS Biotyper. Moreover, this study aimed to identify and report the effect of the ripening period before consumption, which is often a period of three to six months (i.e., 90 to 180 days) post-production, on the microbial diversity and chemical composition of Feta PDO cheese. Appl. Sci. 2021, 11, 5631 3 of 14 2. Materials and Methods 2.1. Sample Collection A total of 23 Feta cheese samples were collected from various production sites (dairy establishments) in different geographical regions in Greece. All Feta cheese samples were prepared in accordance with the requirements of a Protected Destination of Origin (PDO) cheese [15]. All the samples were placed in sterile sample containers and transported to the laboratory for microbiological analysis. The cheeses were analyzed in two different ripening periods, three and