International Journal of Environmental Research and Public Health

Review Improvement of Raw Hygiene through the Selection of Starter and Non-Starter Lactic Acid Bacteria: The Successful Case of PDO Siciliano Cheese

Raimondo Gaglio, Massimo Todaro and Luca Settanni *

Department of Agricultural, Food and Forestry Science, University of Palermo, Viale delle Scienze 4, 90128 Palermo, ; [email protected] (R.G.); [email protected] (M.T.) * Correspondence: [email protected]

Abstract: This review article focuses on the technological aspects and microbiological critical points of pressed-cooked processed from raw ewe’s milk without the inoculation of starter cultures, in particular “Pecorino” cheese typology produced in Italy. After showing the composition of the biofilms adhering to the surface of the traditional dairy equipment (mainly wooden vat used to collect milk) and the microbiological characteristics of PDO cheese manufactured throughout , this cheese is taken as a case study to develop a strategy to improve its hygienic and safety characteristics. Basically, the natural lactic acid bacterial populations of fresh and ripened   cheeses were characterized to select an autochthonous starter and non-starter cultures to stabilize the microbial community of PDO Pecorino Siciliano cheese. These bacteria were applied at a small scale Citation: Gaglio, R.; Todaro, M.; level to prove their in situ efficacy, and finally introduced within the consortium for protection and Settanni, L. Improvement of Raw promotion of this cheese to disseminate their performances to all dairy factories. The innovation in Milk Cheese Hygiene through the PDO Pecorino Siciliano cheese production was proven to be respectful of the traditional protocol, the Selection of Starter and Non-Starter final cheeses preserved their typicality, and the general cheese safety was improved. An overview of Lactic Acid Bacteria: The Successful Case of PDO Pecorino Siciliano the future research prospects is also reported. Cheese. Int. J. Environ. Res. Public Health 2021, 18, 1834. https:// Keywords: lactic acid bacteria; microbial variability; starter selection; traditional cheese; bacterial sta- doi.org/10.3390/ijerph18041834 bilization

Academic Editors: Miguel Elias, Maria João Fraqueza, Marta Laranjo and Luís Patarata 1. Introduction The origin of cheese is particularly ancient; it is dated back to 8000 years ago when this Received: 8 January 2021 dairy fermented product was already produced in the Middle-East [1]. Italy is characterized Accepted: 10 February 2021 by a long history of cheese production. In Sicily, which represents the largest island in the Published: 13 February 2021 Mediterranean sea, the production of cheese began with the Phoenician community [2], and the discovery of several archaeological finds indicated that the dairy activity was Publisher’s Note: MDPI stays neutral routinely conducted in the island in the Eneolithic age [3]. with regard to jurisdictional claims in Generally, all hard cheeses made from ewes’ milk produced in Italy are referred to as published maps and institutional affil- “Pecorino” cheeses. The term Pecorino is an adjective that comes from “pecora,” the Italian iations. translation of ewe. The wider tradition of raw ewe’s milk transformation is concentrated in the southern regions [4,5]. Indeed, Pecorino cheese is produced almost throughout Italy, with names strictly indicating the geographical origin like , Pecorino Siciliano, , and among the most known products [6]. All Copyright: © 2021 by the authors. these cheeses are considered typical products since they are transformed by the application Licensee MDPI, Basel, Switzerland. of traditional production processes in given geographical areas [7]. Most of them enjoy the This article is an open access article “protected designation of origin” (PDO) status, a recognition of quality conferred by the distributed under the terms and European Community to protect some agricultural products and foodstuffs produced in a conditions of the Creative Commons specific area and following their production protocols [8]. Attribution (CC BY) license (https:// PDO Pecorino Siciliano cheese is produced from raw ewe’s milk without the inocula- creativecommons.org/licenses/by/ tion of starter cultures in milk using traditional wooden equipment. In these conditions, 4.0/).

Int. J. Environ. Res. Public Health 2021, 18, 1834. https://doi.org/10.3390/ijerph18041834 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, x 2 of 16

PDO Pecorino Siciliano cheese is produced from raw ewe’s milk without the inocu- lation of starter cultures in milk using traditional wooden equipment. In these conditions, the microbiota responsible for the acidification of curd and maturation of cheese origi- Int. J. Environ.nates Res. Public from Health the2021 raw, 18 ,milk, 1834 the equipment, the animal , and, in general, the trans- 2 of 16 formation (dairy) environment [9,10]. The necessary to transform milk into cheese are lactic acid bacteria (LAB): Those acting during the first hours/days of productions are starterthe microbiota species, responsible which have for to the generate acidification a high of curd amount and maturation of lactic acid of cheese ba- originates sically through thefrom fermentation the raw milk, of the lactose, equipment, while the the animal species rennet, acting and, induring general, ripening, the transformation important to develop(dairy) the environment final organoleptic [9,10]. The no microorganismstes, are indicated necessary as non-starter to transform LAB milk [11]. into cheese Both groups of LABare lacticcontribute acid bacteria to contro (LAB):lling Those the acting development during the of first undesired hours/days microor- of productions are starter species, which have to generate a high amount of lactic acid basically through the ganisms, but their activities and the stressing physicochemical conditions of the mature fermentation of lactose, while the species acting during ripening, important to develop cheeses, mainly representedthe final organoleptic by low pH, notes, high are concentrations indicated as non-starter of organic LAB acids, [11]. low Both water groups of LAB activity, and moderatecontribute sodium to controlling chloride the conten developmentt [12], might of undesired not be microorganisms, enough to eliminate but their activities the presence of spoilageand the stressingand opportunistic physicochemical pathogen conditions microorganisms of the mature cheeses,from Pecorino mainly represented Si- by ciliano cheese [6]. low pH, high concentrations of organic acids, low water activity, and moderate sodium Todaro et al.chloride [6] isol contentated and [12 ],identified might not Citrobacter be enough freundii to eliminate and theStenotrophomonas presence of spoilage and maltophilia amongopportunistic the potential pathogen pathogenic microorganisms bacteria and from Pseudomonas Pecorino Siciliano putida cheese within [6]. the spoilage agents at consistentTodaro etlevels, al. [6 ]together isolated andwith identified several Citrobacternon-starter freundii LAB, andin someStenotrophomonas PDO mal- tophilia Pseudomonas putida Pecorino Siciliano cheeses.among This the paper potential review pathogenics the strategy bacteria planned, and and the workswithin per- the spoilage agents at consistent levels, together with several non-starter LAB, in some PDO Pecorino formed to improve the hygienic and safety aspects of PDO Pecorino Siciliano cheese Siciliano cheeses. This paper reviews the strategy planned, and the works performed to based on the isolation,improve characterization, the hygienic and and safety selection aspects of of starter PDO Pecorino and non-starter Siciliano cheese LAB, basedas on the well as their applicationisolation, at characterization,the laboratory, small and selection scale, and of starterlarge scale and non-starter level. LAB, as well as their application at the laboratory, small scale, and large scale level. 2. PDO Pecorino Siciliano Cheese Technology 2. PDO Pecorino Siciliano Cheese Technology PDO Pecorino Siciliano is a semi-hard cheese produced following a century-old PDO Pecorino Siciliano is a semi-hard cheese produced following a century-old protocol from raw ewe’s milk collected into wooden vats where it is transformed, adding protocol from raw ewe’s milk collected into wooden vats where it is transformed, adding animal rennet in aanimal paste, rennetdissolved in a paste,into warm dissolved water into before warm addition, water before without addition, LAB without starter LAB starter inoculums. The productioninoculums. Thearea production of this PDO area ofcheese this PDO is quite cheese wide, is quite because wide, because it is repre- it is represented sented by the entireby theSicily entire region Sicily (South region (SouthItaly) Italy)with withits 25,711 its 25,711 km km2. The2. The protocol protocol of of productionpro- was duction was regulatedregulated in 1955 in 1955 [13] [13 making] making PDO PDO Pecorino Pecorino Siciliano Siciliano cheesecheese probably probably one one of the oldest of the oldest EuropeanEuropean cheeses cheeses whose whose producti productionon has has been been regulated regulated [14]. [14]. PDO PDO Pecorino Pecorino Siciliano Siciliano cheese ischeese counted is counted among among the thepre pressed-cookedssed-cooked cheeses cheeses [[15],15], andand the the transformation transfor- process mation process is isgraphically graphically reported reported inin FigureFigure1. 1.

Figure 1. Main steps of protected designation of origin (PDO) Pecorino Siciliano cheese production. Adapted from Settanni et al. [16].

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Figure 1. Main steps of protected designation of origin (PDO) Pecorino Siciliano cheese production. Adapted from Settanni et al. [16].

In particular, the milk is preheated at a temperature of 38 °C, even though in the practice of the dairy factories, a temperature of 35–37 °C is often registered. The curd is Int. J. Environ. Res. Public Health 2021, 18, 1834 3 of 16 then broken by a wooden stick beater (called “rotula” in the local dialect) until reaching pieces of rice grain dimensions, and the curd is pressed into rattan baskets in order to facilitate the draining of the whey. These containers also provide the final shape to the ◦ cheeses, which are cookedIn particular, under the hot milk (about is preheated 75 °C) deproteinized at a temperature whey, of 38 namelyC, even “scotta though in the ◦ whey” (residualpractice from of the dairy cheese factories, production a temperature [17]) for of about 35–37 3–4C ish oftenjust after registered. molding. The curd is then broken by a wooden stick beater (called “rotula” in the local dialect) until reaching Curdling and cooking are performed into the same wooden vat, called “tina” in the local pieces of rice grain dimensions, and the curd is pressed into rattan baskets in order to dialect, where rawfacilitate milk thewas draining collected. of theThe whey. traditional These containerswooden equipment also provide used the finalfor PDO shape to the Pecorino Sicilianocheeses, cheese which production are cooked is undermainly hot made (about of 75 chestnut◦C) deproteinized or Douglas whey, fir, namelygenus “scotta Pseudotsuga, woodwhey” [18], (residual even though from Ricotta recent cheese attemp productionts to introduce [17]) for other about 3–4local h justwood after ty- molding. pologies of the CurdlingSicilian forestry and cooking resources are performed showed intointeresting the same results wooden during vat, calledraw ewes’ “tina” in the milk cheese productionlocal dialect, [19,20]. where raw milk was collected. The traditional wooden equipment used for PDO Pecorino Siciliano cheese production is mainly made of chestnut or Douglas fir, 3. Role of the Woodengenus Pseudotsuga Vat during, wood PDO [18 Pecorino], even though Siciliano recent Cheese attempts Production to introduce other local wood typologies of the Sicilian forestry resources showed interesting results during raw ewes’ The Europeanmilk Regulation cheese production (EC) no. [19 ,201935/2004]. refers to the materials in contact with foods [21]. Basically, any material must not transfer its constituents to foods. Even though the measures regarding3. Role of theplastics, Wooden active Vat duringand intelligent PDO Pecorino materials, Siciliano epoxy Cheese derivatives, Production re- generated cellulose,The and European ceramics Regulation have been (EC) harmonized no. 1935/2004 and refers adopted to the at materials the European in contact with level, there are nofoods specific [21]. Basically, indications any for material wood must as a not food transfer contact its constituentsmaterial. Thus, to foods. member Even though countries legislatethe measuresat different regarding levels plastics,[22]. E.g., active in andFrance intelligent and Italy materials, (mainly epoxy in the derivatives, Sicily regen- region), woodenerated equipment cellulose, is andused ceramics for dairy have purposes been harmonized thanks to and the adopted EC no. at 2074/2005, the European level, there are no specific indications for wood as a food contact material. Thus, member coun- which allows derogation from the EC no. 852/2004 for foods with traditional characteris- tries legislate at different levels [22]. E.g., in France and Italy (mainly in the Sicily region), tics “as regardswooden the type equipment of materials is used of which for dairy the purposes instruments thanks and to the ECequipment no. 2074/2005, used which specifically for allowsthe preparation, derogation frompackaging, the EC no.and 852/2004 wrapping for foodsof these with products traditional are characteristics made” “as [23]. regards the type of materials of which the instruments and the equipment used specifically Traditionalfor Sicilian the preparation, cheeses are packaging, in contact and with wrapping wood of during these products the entire are made”production [23]. process, from milkingTraditional until ripening. Sicilian cheesesFigure are2 reports in contact the with main wood steps during of production the entire production of PDO Pecorino process,Siciliano from cheese, milking highlighting until ripening. the Figure constant2 reports contact the main with steps wooden of production equip- of PDO ment. Pecorino Siciliano cheese, highlighting the constant contact with wooden equipment.

Figure 2.FigureContact 2. ofContact raw milk of raw and milk ripened and cheeses ripened with cheeses wooden with equipment wooden duringequipment the production during the of production PDO Pecorino Sicilianoof cheeses. PDO Pecorino Siciliano cheeses.

Among the woodenAmong theequipment wooden equipmentgenerally generally used to used produce to produce the thetypical typical Sicilian Sicilian cheeses, cheeses, the vatthe used vat usedto collect to collect the thebulk bulk milk milk for for curdling assumes assumes the the most most important important microbiologi- cal role because it acts as a reservoir of starter LAB necessary to acidify the curd [18,24–26]. microbiological role because it acts as a reservoir of starter LAB necessary to acidify the The wooden vat is also important to provide the non-starter LAB responsible for the desir- curd [18,24–26].able The changes wooden occurring vat is duringalso important cheese ripening to provide [27–29 ].the Both non-starter starter and LAB non-starter re- LAB sponsible for theare desirable transferred changes from the occurring wooden vat during to the cheese milk during ripening the first [27–29]. minutes Both of contact starter [18 ,24,30] and non-starterthanks LAB toare the transferred biofilms formed from on the its internalwooden surface vat to [19 the,31]. milk A given during biofilm the is anfirst aggregate minutes of contactof microorganisms [18,24,30] thanks embedded to the biofilms into their formed exopolysaccharides on its internal (EPS) surface adhering [19,31]. to a solid surface [32,33]. The ultrastructure of wood surfaces is porous (Figure3a), facilitating the absorption and trapping of bacteria, which may develop a complex biofilm (Figure3b).

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A given biofilm is an aggregate of microorganisms embedded into their exopolysaccha- Int.Int. J. Environ. J. Environ.rides Res. Res.(EPS) Public Public Health Healthadhering 20212021, 18, 18, x , to 1834 a solid surface [32,33]. The ultrastructure of wood surfaces 4is of4 of16 16

porous (Figure 3a), facilitating the absorption and trapping of bacteria, which may de- velop a complex biofilm (Figure 3b). A given biofilm is an aggregate of microorganisms embedded into their exopolysaccha- rides (EPS) adhering to a solid surface [32,33]. The ultrastructure of wood surfaces is porous (Figure 3a), facilitating the absorption and trapping of bacteria, which may de- velop a complex biofilm (Figure 3b).

(a) (b) (a) (b) FigureFigure 3. Scanning3. Scanning electron electron microscopy microscopy observations observ of woodenations vat of surface. wooden (a) Before vat surface. microbial (a activation;) Before (microbialb) after contact with scotta whey. Figure 3. Scanning electron microscopy observations of wooden vat surface. (a) Before microbial activation; (b) after contactactivation; with (b) afterscotta contact whey. with scotta whey. In order to allow the development of the desired LAB onto the internal surfaces of In order to allowthe Inthe wooden order development to vats allow (early the bacterial developmentof the biofilmdesired of formation, the LAB desired onto Figure LAB the4 ),onto internal the the new internal vats, surfaces just surfaces after of their of the wooden vats (earlytheconstruction, wooden bacterial vats undergo biofilm(early bacterial the formation, first biofilm treatment Figureformation, with 4), hot Figurethe water new 4), (80the vats, ◦newC) just for vats, approximately after just aftertheir their 30 construction, undergoconstruction,consecutive the first daysundergo treatment (Figure the4 a).first wi This treatmentth treatmenthot water wi isth aimed hot(80 water °C) at removing for(80 °C)approximately thefor tanninapproximately components. 30 30 consecutiveSubsequently, days the (Figure internal 4a). surfaces This treatm of theent vats is aimed are vigorously at removing brushed the tannin with coarse compo- salt consecutive days (Figurenents.(Figure Subsequently, 44a).b) and This filled thetreatm in internal withent hot surfacesis water aimed once of the at again vatsremoving toare remove vigorously the the tannin salt. brushed After compo- with this surfacecoarse nents. Subsequently,saltpreparation the(Figure internal 4b) protocol and surfaces filled is applied,in withof the hot the vats water biofilms are once vigorously formed again throughto remove brushed the the daily salt. with contact After coarse this with sur- hot salt (Figure 4b) andface (approximatelyfilled preparation in with protocol 70 hot◦C) water scotta is applied, wheyonce (Figuretheagain biofilms4 toc) forremove formed almost the seventhrough salt. consecutive the After daily this contact days sur- [19 with]. face preparation protocolhot (approximately is applied, 70 °C)the scotta biofilms whey formed (Figure 4c) through for almost the seven daily consecutive contact dayswith [19]. hot (approximately 70 °C) scotta whey (Figure 4c) for almost seven consecutive days [19].

(a) (b) (c)

FigureFigure 4. 4.BacterialBacterial activation activation of of wooden wooden vat vat surfaces surfaces with with dairy dairy lactic lactic acid acid bacteria bacteria (LAB): (LAB): ( (aa)) Tan- Tannin ninremoval removal with with hot hot water water for for 30 30 days; days; (b )(b coarse) coarse salt salt brushing; brushing; (c )(c Scotta) Scotta whey whey contact contact for for 7 days.7 days.

(a) TheThe wooden wooden vat vat biofilms biofilms( associatedb associated) with with the the production production of of PDO PDO(c) Pecorino Pecorino Siciliano Siciliano cheesescheeses are are often often the the same same used used for for the the production production of of PDO PDO Vastedda Vastedda della della valle valle del del Belìce Belìce Figure 4. Bacterial activationcheese,cheese, which of which wooden was was thoroughly thoroughlyvat surfaces investigated investigated with dairy by by somelactic some authors acid authors bacteria [18,34]. [18,34 (LAB):To]. this To this (purpose,a) Tan- purpose, it is it nin removal with hotimportant wateris important for to 30 note todays; note that (b that most) coarse most of LAB ofsalt LAB brushing;were were identified identified (c) Scotta as asLactobacillus wheyLactobacillus contact, but, but forsince since 7 days. 2020 2020 the the nomenclaturenomenclature of of lactobacilli lactobacilli underwent underwent a aconsistent consistent revision revision by by Zheng Zheng et et al. al. [35], [35 ],and and the the bacteriabacteria previously previously grouped grouped into the genusgenusLactobacillus Lactobacillusare are currently currently split split into into 23 different23 dif- The wooden vat biofilms associated with the production of PDO Pecorino Siciliano cheeses are often the same used for the production of PDO Vastedda della valle del Belìce

cheese, which was thoroughly investigated by some authors [18,34]. To this purpose, it is important to note that most of LAB were identified as Lactobacillus, but since 2020 the nomenclature of lactobacilli underwent a consistent revision by Zheng et al. [35], and the bacteria previously grouped into the genus Lactobacillus are currently split into 23 dif-

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ferentgenera. genera. However, However, in orderin order to better to better understand understand theresults the results of the of research the research of Scatassa of Scatassaet al. [et18 al.,34 ],[18,34], as well as aswell those as those of the of other the other research research objectives objectives of this of review this review article, arti- LAB cle,whose LAB whose nomenclature nomenclature was revised, was revised, will be will reported be reported with thewith current the current names names and theand old thenames old names between between brackets brackets at the at first thecitation. first citation. The collection The collection of the of wooden the wooden vat biofilms vat biofilmsoccurred occurred by the by brushing the brushing technique technique reported re byported Didienne by Didienne et al. [30 ].et Briefly,al. [30]. the Briefly, vat surfaces the vatwere surfaces sampled, were sampled, positioning positioning UV-treated UV-t paperreated squares paper halfway squares onhalfway the bottom on the and bottom up the andside up ofthe the side vat of (Figure the vat5 ).(Figure 5).

(a) (b)

FigureFigure 5. Wooden5. Wooden vat vat biofilm biofilm collection: collection: (a) ( Brushing;a) Brushing; (b) ( collectionb) collection by by gauze. gauze.

TheThe biofilms biofilms were were subjected subjected to to plate plate counts, andand thethe colonies colonies showing showing different different mor- morphologicalphological appearances appearances were were isolated, isolated, purified, purified, and and identified identified by 16S by rRNA 16S rRNA gene sequenc-gene sequencinging as belonging as belonging to several to several LAB generaLAB genera [18,34 [18,34].]. In particular, In particular, the species the species identified identi- were: fiedEnterococcus were: Enterococcus durans, duransEnterococcus, Enterococcus faecium faecium, Enterococcus, Enterococcus faecalis faecalis, Enterococcus, Enterococcus hirae, hiraeStrepto-, Streptococcuscoccus lutetiensis lutetiensis, Streptococcus, Streptococcus gallolyticus gallolyticussubsp. subsp.macedonicus macedonicus, Streptococcus, Streptococcus thermophilus ther- , mophilusPediococcus, Pediococcus acidilactici acidilactici, Lactococcus, Lactococcus lactis, Levilactobacillus lactis, Levilactobacillus brevis (formerly brevis (formerlyLactobacillus Lacto- brevis), bacillusLimosilactobacillus brevis), Limosilactobacillus fermentum (formerly fermentumLactobacillus (formerly fermentum Lactobacillus), Lacticaseibacillus fermentum), Lactica- casei (for- seibacillusmerly Lactobacillus casei (formerly casei Lactobacillus), Lactobacillus casei delbrueckii), Lactobacillus, and Lacticaseibacillus delbrueckii, and rhamnosus Lacticaseibacillus(formerly rhamnosusLactobacillus (formerly rhamnosus Lactobacillus). rhamnosus). In lightIn light of ofthe the Commission Commission Regulation Regulation (EC) (EC) No 2073/20052073/2005 [[36],36], ScatassaScatassa et et al. al. [18 [18]] also alsoapplied applied the the microbiological microbiological criteria criteria for for foodstuffs foodstuffs to to the the wooden wooden vats vats used used to to transform trans- formmilk milk into into cheese. cheese. To To this this purpose, purpose, the the presence presence of ofListeria Listeria monocytogenes monocytogenesand andSalmonella Salmo- nellaspp. spp. was was investigated investigated as as food food safety safety criteria, criteria, while while that that of ofEscherichia Escherichia coli coliand and coagulase- coag- ulase-positivepositive staphylococci staphylococci as process as process hygiene hygiene criteria. criteria. The The investigation investigation results results showed showed that Ls. thatmonocytogenes Ls. monocytogenesand Salmonella and Salmonellaspp. were spp. not were detected, not detected, coagulase-positive coagulase-positive staphylococci staphy- were lococcibelow were the below detection the limit,detection while limit,Es. coli whilewas Es. found coli was only found in one only vat outin one of the vat five outobjects of the of fivestudy. objects Some of study. authors Some stated authors that the stated absence that of the these absence pathogenic of these species pathogenic onto the species surface of ontothe the wooden surface vats of the is due wooden to their va inabilityts is due to to adhere their inability or to survive to adhere in the or microbial to survive biofilms in the for a combination of stress conditions such as the production of organic acids, the competition microbial biofilms for a combination of stress conditions such as the production of or- for nutrients, and the generation of antimicrobial compounds, mainly bacteriocins, by LAB ganic acids, the competition for nutrients, and the generation of antimicrobial com- and also to the high temperatures applied for cheese cooking [26,37]. To this purpose, pounds, mainly bacteriocins, by LAB and also to the high temperatures applied for Scatassa et al. [18,34] tested all isolates through the general assays applied to evaluate the cheese cooking [26,37]. To this purpose, Scatassa et al. [18,34] tested all isolates through bacteriocin-like inhibitory substance production [38], finding that a consistent percentage the general assays applied to evaluate the bacteriocin-like inhibitory substance produc- of vat LAB inhibited some indicator strains, including Ls. monocytogenes (strain ATCC tion [38], finding that a consistent percentage of vat LAB inhibited some indicator strains, 19114). These results provided evidence that the wooden vat biofilms contribute actively to including Ls. monocytogenes (strain ATCC 19114). These results provided evidence that the microbial safety of the traditional cheeses. the wooden vat biofilms contribute actively to the microbial safety of the traditional cheeses.

4. Microbial Ecology of PDO Pecorino Siciliano Cheese

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4. Microbial Ecology of PDO Pecorino Siciliano Cheese Even though PDO Pecorino Siciliano cheese has been produced for centuries, the first documentation regarding the microbial ecology of similar Pecorino Siciliano cheeses was published in 2006 by Vernile et al. [39]. Those authors characterized only the levels of LAB in cheese produced in different seasons, showing that after three months of ripening, mesophilic rods and cocci were approximately at the same level, which was estimated at about 107 cfu/g. Two years later, the same authors [40] reported the genotypic characteri- zation of those LAB showing that the species associated with the ripening of PDO Pecorino Siciliano cheese were E. faecium, E. faecalis, Lacticaseibacillus paracasei (formerly Lactobacillus paracasei), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum), Lactiplantibacillus pentosus (formerly Lactobacillus pentosus), Lt. rhamnosus, Latilactobacillus curvatus (formerly Lactobacillus curvatus) and leuconostocs. Todaro et al. [6] evaluated the microbiological char- acteristics of PDO Pecorino Siciliano cheeses of different weights subjected to two different salting technologies (dry salting and a combined dry-brine salting). That work showed that LAB and the undesired spoilage and potentially pathogenic bacteria (pseudomon- ads and members of Enterobacteriaceae family) were almost at the same levels (105 cfu/g) after five months of ripening. LAB populations included E. durans, E. faecium, E. faecalis, Lactococcus garvieae, L. brevis, P. acidilactici, Pediococcus pentosaceus, S. gallolyticus subsp. macedonicus, and Streptococcus infantarius. Randazzo et al. [41] revealed the presence of Lc. lactis, S. thermophilus, Streptococcus bovis, E. faecalis, and Leuconostoc mesenteroides in artisanal Pecorino Siciliano cheeses through the application of a combined culture-dependent and– independent approach. Caggia et al. [42] specifically performed the isolation of non-starter LAB from Pecorino Siciliano cheeses in order to collect a group of LAB to be screened for probiotic features identifying several Lt. rhamnosus and Lt. paracasei. The species associated with the wooden equipment and those identified in PDO Pecorino Siciliano cheeses included mesophilic (Lc. lactis) and thermophilic (S. thermophilus) starter LAB. In general, the thermophilic species are associated with the cheese core [43], where the temperature reached during cooking is maintained for a longer time than at the under rind [44]. However, the majority of the species identified from the wooden vats and ripened PDO Pecorino Siciliano cheeses were allotted into the non-starter LAB group. Among these, in the last years, S. gallolyticus subsp. macedonicus and Lc. garvieae represent adjunct cultures inoculated during different Italian cheese productions [45,46]. Enterococci are responsible for several typicality notes of cheese [47] but may carry antibiotic resistance genes and virulence factors [48]. These bacteria, besides being naturally present in raw milk [49,50] and in the wooden vat biofilms [18,34], have also been isolated from the animal rennet pastes used in traditional Sicilian cheese production [51], indicating that the presence of enterococci in the final cheeses might have different origins. Once their harmlessness is ascertained [48], enterococci can be selected as secondary adjunct cultures also for their contribution to extending cheese shelf life due to the bacteriocin production [47,52]. With the aim of investigating the antibiotic resistance distribution among Pecorino Siciliano cheese enterococci, Russo et al. [53] isolated several strains belonging to the species E. faecium. The authors detected a high resistance of the strains towards rifampicin, erythromycin, and ampicillin and also found that several strains exhibited multidrug-resistant phenotypes highlighting concerns regarding the spread of antibiotic resistance by dairy enterococci. Todaro et al. [6] focused the attention on the isolation and identification of the unde- sirable bacteria of PDO Pecorino Siciliano cheeses, finding out that this community was composed of Pseudomonas putida, Pseudomonas vronovensis, C. freundii, Enterobacter spp., Es. coli, Klebsiella oxytoca, Serratia grimesii, and Sn. maltophilia. Although the presence of the potentially pathogenic bacteria Enterobacter spp., K. oxytoca, C. freundii, and Es. coli was documented for Italian raw milk Pecorino cheese typology during the first month of ripening [54], the surprising finding of the work of Todaro et al. [6] was that these species were still viable in cheeses ripened for five months. Members of Enterobacteriaceae family at high levels have also been reported for raw ewes’ milk cheeses produced in Greece and Portugal [55,56] and their main implication with human health is basically related to the Int. J. Environ. Res. Public Health 2021, 18, 1834 7 of 16

production of biogenic amines [57]. A rapid decline of K. oxytoca detected in curd was reported during the ripening of Serra ewe’s cheese [58], and this phenomenon is important to reduce the risk of hemorrhagic colitis caused by this bacterium [59]. Even though C. freundii is considered as a low virulence , it could be responsible for several infections, including pneumonia, diarrhea, and septicemia [60]. Escherichia coli is naturally present in the intestine of warm-blooded animals and represents an indicator of fecal contamination, but it is also implicated in gastrointestinal diseases [61]. Regarding spoilage bacteria, Pseudomonas spp. are generally isolated from raw ewes’ milk cheeses with a limited ripening time [62]. Generally, pseudomonads are present at high cell densities when pH is high [63]; thus, a rapid pH drop is necessary to hamper their development. Fresh Pecorino Siciliano “primosale” cheeses, namely “primosale”, were specifically investigated at a retail sale by Giammanco et al. [64] for the presence of Staphylococcus aureus and Es. coli finding out that a consistent percentage of the samples analyzed were positive for these two species. However, the study evidenced that those results depended on post-production contamination, in particular, the retail sale conditions may have played a key role in the development of St. aureus and Es. coli. Furthermore, Cardamone et al. [65] inoculated the four main dairy pathogenic species (Es. coli O157, Ls. monocytogenes, Salmonella Enteritidis, and St. aureus) at the production stage of Pecorino Siciliano cheese and evaluated their behavior during ripening. The results of the investi- gation indicated that PDO Pecorino Siciliano cheese production conditions determined a consistent decrease in the growth of all four bacteria inoculated. PDO Pecorino Siciliano cheese was also specifically investigated for the presence of biogenic amines with the aim of correlating these compounds with cheese microbiol- ogy [66]. Indeed, biogenic amines are toxic compounds generated through the microbial decarboxylation of amino acids [67]. Only histamine was detected at a high concentration in PDO Pecorino Siciliano cheese. Guarcello et al. [68] selected some cheese LAB that did not carry amino acid decarboxylase genes and found that some of them even showed a biogenic amine degrading ability determining the selection of LAB starters useful to prevent the accumulation of these toxic compounds in experimental cheeses. In light of the several microorganisms implicated in human pathogenicity and shelf- life issues isolated from ripened Pecorino cheeses, the selection and application of starter strains are of paramount importance to obtain safe and stable cheeses.

5. Improvement of PDO Pecorino Siciliano Cheese Hygiene through Selected LAB Addition Although the wooden vats used to produce PDO Pecorino Siciliano cheeses were proven to be safe systems for milk transformation, the presence of consistent numbers of spoilage/pathogenic bacteria in the final cheeses determined an alert on the entire production system of this PDO cheese. The sole wooden vat LAB biofilms are unable to sanitize the microbiological conditions of raw ewes’ milk, which is often very close to the European limit (<500,000 cfu/mL) for the “good microbiological quality” of raw ewes’ milk to be processed into cheese when it is not thermally treated [69]. Thus, a revision of the production protocol of PDO Pecorino Siciliano cheese based on the addition of selected autochthonous starter and non-starter LAB was suggested to improve the hygienic conditions of the final cheeses. According to the classification of Mucchetti and Neviani [70], based on the treatment of milk and the type of selected starters added, the Italian cheeses can be produced from pasteurized milk inoculated with selected starters, pasteurized milk inoculated with nat- ural starters, thermal treated milk inoculated with natural starters, raw milk inoculated with selected starters, raw milk inoculated with natural starters, and raw milk without starter addition. PDO Pecorino Siciliano cheese belongs to the last cheese typology, which deserves major microbiological attention due to the survival of the raw milk bacteria during cheese production [71], even though vat LAB exerts antagonistic measures [26,37]. From this perspective, PDO Pecorino Siciliano cheese represented a case study to convert the Int. J. Environ. Res. Public Health 2021, 18, x 8 of 16

measures [26,37]. From this perspective, PDO Pecorino Siciliano cheese represented a case study to convert the transformation process from raw milk without starters to a Int. J. Environ.production Res. Public Health performed2021, 18, 1834 with raw milk added with natural starters. 8 of 16 Settanni et al. [16] isolated potential starter LAB from acidified PDO Pecorino Sicil- iano curds, while non-starter LAB was collected from ripened cheeses [6]. Both LAB groups were selectedtransformation for their main process dairy from traits: raw milk Starter without LAB starters determined to a production the rapid performed acidi- with fication of UHT milkraw and milk showed added with rapid natural auto starters.lysis, while non-starter LAB was chosen for Settanni et al. [16] isolated potential starter LAB from acidified PDO Pecorino Siciliano their opposite behavior since a low acidification capacity and slow autolysis are com- curds, while non-starter LAB was collected from ripened cheeses [6]. Both LAB groups patible with long wereripening selected times for their [50]. main Sixty-two dairy traits: curd Starter LAB LABisolates determined were collected the rapid acidificationand tested for acidificationof UHT and milk autolysis and showed kinetics rapid, but autolysis, only two while strains, non-starter bothLAB identified was chosen as Lc. for their lactis subsp. lactis, oppositeresulted behavior in rapid since acidifiers a low and acidification highly autolytic. capacity and The slow secondary autolysis adjunct are compatible culture was composedwith long of three ripening strains times (one [50]. Sixty-twostrain per curd Lc. LAB garvieae isolates, E. were faecalis collected and andS. gal- tested for lolyticus subsp. macedonicusacidification), andwhich autolysis were kinetics, the weakest but only acid twoifiers strains, and both showed identified the as slowestLc. lactis subsp. lactis, resulted in rapid acidifiers and highly autolytic. The secondary adjunct culture was autolytic activity. These five cultures, individually or in starter/non-starter LAB combi- composed of three strains (one strain per Lc. garvieae, E. faecalis and S. gallolyticus subsp. nation, were appliedmacedonicus to produce), which experimental were the weakest cheeses acidifiers in the and dairy showed factory the slowest whose autolytic cheeses activity. showed the highestThese levels five of cultures, spoilage individually and potentially or in starter/non-starter pathogenic bacteria LAB combination, by Todaro wereet al. applied [6]. The bulk milkto was produce collected experimental into the cheeses wooden in the vat dairy daily factory used whose by the cheeses cheese-maker showed the in highest order to enrich thelevels milk of with spoilage the andvat potentiallybiofilms. The pathogenic entire bacteriamilk volume by Todaro was et divided al. [6]. The into bulk milk seven aliquots andwas transferred collected into into the plastic wooden vats. vat daily One used vat by was the cheese-makernot inoculated in orderwith to any enrich the milk with the vat biofilms. The entire milk volume was divided into seven aliquots and exogenous LAB culturetransferred and into represented plastic vats. the One control vat was nottrial, inoculated while six with vats any exogenousreceived LAB culture inoculums accordingand representedto the experimental the controltrial, plan while (Figure six vats 6). received The cheeses LAB inoculums were produced according to the applying the traditionalexperimental PDO planPecorino (Figure Siciliano6). The cheeses cheese-making were produced protocol, applying and the the traditional ripen- PDO ing lasted five months.Pecorino Siciliano cheese-making protocol, and the ripening lasted five months.

Wooden vat

CC EC1 EC2 EC3 EC4 EC5 EC6 Plastic vats Plastic

Lc. lactis CAG4 Lc. lactis CAG37 Lc. lactis CAG4 Lc. lactis CAG4 Lc. lactis CAG37 Lc. lactis CAG4 Lc. lactis CAG37 Lc. garvieae PSL67 Lc. garvieae PSL67 Lc. lactis CAG37 E. faecalis PSL71 E. faecalis PSL71 Lc. garvieae PSL67 S. macedonicus PSL72 S. macedonicus PSL72 E. faecalis PSL71 S. macedonicus PSL72 FigureFigure 6. Experimental 6. Experimental design of design PDO Pecorino of PDO Siciliano Pecorino cheese Siciliano production cheese performed production at the performed factory-scale at level. the Adaptedfac- fromtory-scale Settanni et al.level. [16]. Adapted from Settanni et al. [16].

The experimental trials were first followed for their acidification kinetics by monitor- The experimental trials were first followed for their acidification kinetics by moni- ing pH and microbiological levels. Both starter strains determined a similar pH decrease toring pH and microbiologicalin the six inoculated levels. trials, Both and starter their pHstrains drop determined was faster than a similar that registered pH de- for the crease in the six inoculatedcontrol trial. trials, After and 7 days their of acidification,pH drop was the faster control than cheeses that showed registered higher for levels of the control trial. AfterPseudomonads 7 days ofspp. acidification, and members the of Enterobacteriaceaecontrol cheesesfamily showed than higher cheeses levels produced of with Pseudomonads spp.the and starter/non-starter members of Enterobacteriaceae LAB inoculums. family At the endthan of cheeses the ripening produced period, with prolonged the starter/non-starterfor five LAB months inoculums. to perform At thethe direct end comparisonof the ripening with theperiod, cheeses prolonged analyzed byfor Todaro et al. [6], both Pseudomonads spp. and members of Enterobacteriaceae family were below five months to performthe detection the direct limit comparison in all inoculated with cheeses, the cheeses while their analyzed levels wereby Todaro 3.7 cfu/g et al. (log10) in [6], both Pseudomonads spp. and members of Enterobacteriaceae family were below the detection limit in all inoculated cheeses, while their levels were 3.7 cfu/g (log10) in con- trol cheeses. The species Lc. lactis subsp. lactis successfully reduced the presence of En-

Int. J. Environ. Res. Public Health 2021, 18, x 9 of 16

Int. J. Environ. Res. Public Health 2021, 18, 1834 9 of 16

terobacteriaceae family members also when added as starter culture during the production of raw ewes’ milk Portuguese Serra de Estrela cheese [72] and different traditional Ser- control cheeses. The species Lc. lactis subsp. lactis successfully reduced the presence of bian cheeses [73]. The addition of selected Lc. lactis subsp. lactis isolated from PDO Peco- Enterobacteriaceae family members also when added as starter culture during the production rino Siciliano curds in milk with microbiological levels close to the limits of the CE Reg- of raw ewes’ milk Portuguese Serra de Estrela cheese [72] and different traditional Serbian ulation 853 [69] ameliorated the hygienic conditions of the final cheeses. Furthermore, the cheeses [73]. The addition of selected Lc. lactis subsp. lactis isolated from PDO Pecorino Sicilianosensory curdsanalysis in milk showed with microbiologicalthat the cheeses levels prod closeuced to with the limits the starter/non-starter of the CE Regulation LAB in- 853oculums [69] ameliorated retained a the typical hygienic sens conditionsory profile. of the Thus, final the cheeses. performances Furthermore, of thethe sensoryselected LAB analysisneeded showedto be tested that the at cheesesa large produced scale level with considering the starter/non-starter other dairy LAB factories inoculums within the retainedconsortium a typical for the sensory protection profile. and Thus, prom the performancesotion of PDO of Pecorino the selected Siciliano LAB needed cheese. to be tested at a large scale level considering other dairy factories within the consortium for the protection6. Microbial and Stabilization promotion of PDOof PDO Pecorino Pecorino Siciliano Siciliano cheese. Cheese Production

6. MicrobialThe approach Stabilization of Settanni of PDO et Pecorino al. [16] was Siciliano followed Cheese by Production Guarcello et al. [74] to extend the results of the selection of the mixed starter (Lc. lactis subsp. lactis)/non-starter (E. fae- The approach of Settanni et al. [16] was followed by Guarcello et al. [74] to extend the resultscalis, Lc. of thegarviae selection, S. gallolyticus of the mixed subsp. starter macedonicus (Lc. lactis subsp.) LABlactis inoculum)/non-starter to six (E. dairy faecalis factories, Lc.located garviae in, S. different gallolyticus areassubsp. of macedonicusSicily (Figure) LAB 7) inoculum within five to six provinces dairy factories (Agrigento, located inCatania, differentEnna, Palermo, areas of Sicilyand Trapani), (Figure7) withinall being five part provinces of the (Agrigento, consortium Catania, for PDO Enna, Pecorino Palermo, Siciliano andcheese Trapani), protection all being and part promotion. of the consortium The application for PDO Pecorino of the selected Siciliano LAB cheese was protection performed in andan attempt promotion. to stabilize The application the microbiological of the selected characteristics LAB was performed of PDO in an Pecorino attempt toSiciliano stabilizecheese, thepreserving microbiological its typicality, characteristics throughout of PDO the Pecorino regional Siciliano territory cheese, where preserving the disciplinary its typicality,allows the throughout production. the regional territory where the disciplinary allows the production.

FigureFigure 7. 7.Location Location of of the the dairy dairy factories factories producing producing PDO PDO Pecorino Pecorino Siciliano Siciliano cheese. cheese.

TheThe production production of theof cheesesthe cheeses in each in factory each wasfactory performed was performed from the bulk from milk the after bulk its milk contact with the wooden vat surface and then transferred into two plastic vats (Figure8). The after its contact with the wooden vat surface and then transferred into two plastic vats control cheeses were processed without LAB inoculums, while the experimental cheeses were (Figure 8). The control cheeses were processed without LAB inoculums, while the ex- inoculated with the mixed starter/non-starter LAB culture. Both cheese productions followed PDOperimental Pecorino cheeses Siciliano were cheese inoculated protocol, and with the the final mixed cheeses starter/non-starter were ripened for five LAB months culture. as Both forcheese previous productions investigations. followed PDO Pecorino Siciliano cheese protocol, and the final cheeses were ripened for five months as for previous investigations.

Int.Int. J. Environ. J. Environ. Res. Res. Public Public Health Health 20212021, ,1818,, x 1834 10 of 1610 of 16

FigureFigure 8. Working plan plan for for PDO PDO Pecorino Pecorino Siciliano Siciliano cheese chee productionse production applied applied to test the to starter/non-test the start- er/non-starterstarter LAB inoculums LAB inoculums at a large at scale a large level. scale level.

In general,general, the the level level of LABof LAB cocci cocci in the in wooden the wooden vat biofilms vat biofilms was at higher was levelsat higher than levels thanthose those of LAB of rods. LAB Regarding rods. Regarding the bulk ,the bulk they milks, showed they similar showed levels forsimilar LAB coccilevels and for LAB total mesophilic counts, thus, LAB dominated the microbial community of all raw milks cocci and total mesophilic counts, thus, LAB dominated the microbial community of all processed into cheese. The cheese-making parameters were particularly variable among the raw milks processed into cheese. The cheese-making parameters were particularly vari- six dairy factories, but all curds of the trials with selected LAB addition displayed a faster ablepH drop among than the control six dairy curds. factories, The levels but of LABall curds in ripened of the inoculated trials with cheeses selected were LAB higher addition displayedthan those a registered faster pH in drop control than cheeses. control Cheese curds. microbiotas The levels were of LAB analyzed, in ripened applying inoculated a cheesesculture-dependent were higher approach than exclusively.those registered The authors in control also performed cheeses. aCheese polymorphic microbiotas profile were analyzed,recognition applying (by randomly a culture-dependent amplified polymorphic appr DNAoach analysis)exclusively. of the The bacterial authors isolates also per- formedfrom the a agar polymorphic plates at the profile highest recognition cell suspension (by dilutions randomly showing amplified the dominance polymorphic of the DNA analysis)inoculated of LAB the overbacterial the vat isolates LAB and from those the indigenous agar plates of at milk. the Regardinghighest cell the suspension undesired dilu- tionsbacterial showing groups, the coagulase-positive dominance of the staphylococci inoculated wereLAB statisticallyover the vat different LAB and among those the indige- nousfactories, of milk. members Regarding of the Enterobacteriaceae the undesired familybacterial were groups, at low levels,coagulase- whilepositive pseudomonads staphylococ- were below the detection limit. ci were statistically different among the factories, members of the Enterobacteriaceae fam- The microbial stabilization of PDO Pecorino Siciliano cheese production through ilythe were addition at low of thelevels, selected while starter/non-starter pseudomonads LABwere was below better the investigated detection limit. by a culture- independentThe microbial approach stabilization based on MiSeq of PDO Illumina Pecori technologyno Siciliano by Gaglio cheese et al.production [75]. Cheese through themicrobiota addition analysis of the showed selected that starter/non-starter all control cheeses wereLAB dominatedwas better by investigated streptococci andby a cul- ture-independentlactobacilli, while theapproach cheeses processedbased on addingMiSeq theIllu selectedmina technology LAB were characterizedby Gaglio et byal. [75]. Cheesedominant microbiota levels of streptococci,analysis showed lactobacilli, that all and control lactococci cheeses (Figure were9). dominated In particular, by in strepto- cocciaddition and to lactobacilli, the LAB species while inoculated,the cheeses this processed approach adding also revealed the selected the presence LAB were of Lt. charac- terizedcasei (formerly by dominantLactobacillus levels zeae of) instreptococci, all cheeses, L.lactobacilli, brevis and Ln.and mesenteroides lactococci only(Figure in some 9). In par- ticular,factories in in addition both control to the and LAB inoculated species cheeses. inoculated, The selected this approach LAB culture also determinedrevealed the a pres- enceslight of increase Lt. casei in (formerly the levelsof Lactobacillus enterococci. zeae) in all cheeses, L. brevis and Ln. mesenteroides only in some factories in both control and inoculated cheeses. The selected LAB culture determined a slight increase in the levels of enterococci.

Int. J. Environ. Res. Public Health 2021, 18, x 11 of 16

variable among the six dairy factories, but all curds of the trials with selected LAB addition displayed a faster pH drop than control curds. The levels of LAB in ripened inoculated cheeses were higher than those registered in control cheeses. Cheese microbiotas were analyzed, applying a culture-dependent approach exclusively. The authors also performed a polymorphic profile recognition (by randomly amplified polymorphic DNA analysis) of the bacterial isolates from the agar plates at the highest cell suspension dilutions showing the dominance of the inoculated LAB over the vat LAB and those indigenous of milk. Regarding the undesired bacterial groups, coagulase-positive staphylococci were statistically different among the factories, members of the Enterobacteriaceae family were at low levels, while pseudomonads were below the detection limit. The microbial stabilization of PDO Pecorino Siciliano cheese production through the addition of the selected starter/non-starter LAB was better investigated by a culture-independent approach based on MiSeq Illumina technology by Gaglio et al. [75]. Cheese microbiota analysis showed that all control cheeses were dominated by streptococci and lactobacilli, while the cheeses processed adding the selected LAB were characterized by dominant levels of streptococci, lactobacilli, and lactococci (Figure 9). In particular, in addition to the LAB species inoculated, this approach also revealed the presence of Lt. casei (formerly Lactobacillus zeae) in all cheeses, L. brevis and Ln. Int. J. Environ. Res. Public Healthmesenteroides2021, 18, 1834 only in some factories in both control and inoculated 11 cheeses. of 16 The selected LAB culture determined a slight increase in the levels of enterococci.

Control Cheeses Experimental Cheeses

100%100

90%90

80%80

70%70 (%) 60%60

50%50 abundance

40%40

Relative Relative 30%30

20%20

10%10

0%0 FactoryPC1 1 FactoryPC2 2 FactoryPC3 3 FactoryPC4 4 FactoryPC5 5 FactoryPC6 6 FactoryPS1 1 FactoryPS2 2 FactoryPS3 3 FactoryPS4 4 FactoryPS5 5 FactoryPS6 6

ActinobacteriaMicrococcaceae (Micrococcaceae) BacteroidetesChryseobacterium (Chryseobacterium) Staphylococcus;s__aureusSt. aureus StaphylococcaceaeStaphylococcaceae Other LABLAB EnterococcaceaeEnterococcus (Enterococcus) Lactobacillus;s__brevisL. brevis Lactobacillus Lactobacillus;s__zeaeL. zeae Pediococcus LeuconostocaceaeLeuconostoc mesenteroides (Leuconostoc;s__mesenteroides) Lactococcus;s__Lactococcus Lactococcus;s__garvieaeLc. garviae Streptococcus Streptococcus;s__luteciaeSt. luteciae Enterobacteriaceae;g__;s__Enterobacteriaceae Chromohalobacter;s__Chromohalobacter Moraxellaceae Figure 9.Figure Relative 9. Relative abundances abundances (%) (%)of bacteria of bacteria identified identified by MiSeqMiSeq Illumina Illumina in controlin control and experimentaland experimental PDO Pecorino PDO Pecorino SicilianoSiciliano cheeses. cheeses. Adapted Adapted from from Gaglio Gaglio et al. et al.[70] [70.].

The presence of Staphylococcus spp. in control and experimental cheeses of three factoriesThe presence indicated of that Staphylococcus even after the addition spp. in of selectedcontrol LAB, and someexperimental cheeses may cheeses be char- of three factoriesacterized indicated by the presence that even of pathogenic after the species addition and that of selected milk hygiene LAB is, important some cheeses for the may be characterizedsafety of the by final the cheeses. presence Other of minor pathogenic groups including speciesChryseobacterium and that milk, Chromohalobacter, hygiene is important for theMicrococcaceae, safety ofand theMoraxellaceae final cheeses.were alsoOther detected. minor groups including Chryseobacterium, Chromohalobacter,The final cheeses Micrococcaceae were analyzed, forand the Moraxellaceae chemical composition. were also The detected. addition of the selected LAB did not affect cheese fatty acids (FA), mainly represented by saturated fatty acidsThe likefinal palmitic cheeses and were myristic analy acids.zed Thefor ratiothe chemical between saturated composition. FA and The unsaturated addition of the selectedFA, in LAB particular did not oleic affect acid, cheese for control fatty andacids selected (FA), LABmainly added represented cheeses were by saturated almost fatty acidssuperimposable like palmitic (2.31and andmyristic 2.29, respectively). acids. The ratio Regarding between the volatile saturated organic FA compound and unsaturated composition of the cheeses, their profiles were composed of alkanes, aldehydes, terpenes, esters, acids, ketones, and benzene. The cheeses inoculated with the selected LAB were different from those of the control cheeses only for butanoic acid, but the factories where the cheeses were produced impacted differently the presence of hexane and hexanoic acid ethyl ester that was generated at higher concentrations from the control cheeses [75]. The addition of the starter/non-starter LAB addition impacted the final pH of the ripened cheeses that was 0.1 points lower than the average value registered for the control cheeses. Specifically, the final pH of control cheeses was 5.76, while that of LAB added cheeses was 5.66. This finding could be of relevance in the practice of producing cheeses from raw milk. Even though 0.1 points pH does not strongly impact the acidity perception by consumers, a lower pH of the curd due to the addition of the starter culture rather than non-starter LAB exerts a stressing effect on the undesired microorganisms present in raw milk retarding or inhibiting their development. Some differences regarding cheese yield, water activity (aw), ash content, and salt percentage were registered among factories. In particular, cheese yield % and aw was higher for the control cheeses, while ash % on dry matter and salt % were higher in the cheeses added with the selected LAB [74]. Int. J. Environ. Res. Public Health 2021, 18, 1834 12 of 16

The same cheeses were evaluated by 10 expert panelists who scored 21 descriptive attributes, including aroma, taste, surface structure, and texture [74]. The sensory tests evidenced how the addition of LAB influenced color, eye formation, uniformity of structure, and unpleasant taste. The panelists detected fewer eyes in the cheeses produced by adding the selected LAB while a higher presence of unpleasant taste was scored for the control cheeses. Furthermore, a triangle test indicated the highest preference for the cheeses produced with the addition of the LAB inoculums. Although the selected LAB determined a lowering of pH, they did not generate an overproduction of organic acids excluding that they negatively impacted the overall assessment of the cheeses.

7. Conclusions and Future Perspectives Future Perspectives Cheese is one of the foods that are undergoing a negative impact among consumers due to their fat content and low presence of active compounds. However, very recently, cheeses are being rediscovered as foods with functional properties, in particular, when ingredients with bioactive compounds are added. From this perspective, the current research is focusing on the incorporation of several ingredients, mainly by-products rich in polyphenols and other antioxidant molecules [76]. Our research group is preparing works on the incorporation of grape pomace wastes and aromatic herbs in Pecorino Siciliano cheeses for which LAB resistant to plant polyphenols have been selected ad hoc [77–79]. Furthermore, Pecorino Siciliano cheese was evaluated as a promising matrix to deliver probiotic cultures [80], opening new routes for production and commercialization of this traditional cheese.

8. Conclusions The quality of cheese productions carried out without the addition of starter cultures is quite unpredictable. In order to stabilize the characteristics of these cheeses and to preserve the typical organoleptic notes avoiding the flattening of the sensory profile of the final products, the application of commercial starter cultures is not advisable [81]. The development of starter and secondary adjunct (non-starter) cultures from autochthonous LAB has been reported as a winning strategy to maintain cheese typicality. Autochthonous LAB are those isolated from the production environment, associated to the local raw materials, and adapted to the traditional process technology [7]. The studies conducted on PDO Pecorino Siciliano cheese showed that the wooden vats used for milk collection and curdling are a primary source of useful LAB, starter cultures selected from autochthonous populations safeguard the typicality of the cheese with little modifications of the traditional manufacture technology, their application in combination with non-starter LAB determines their dominance over milk LAB and controls the development of spoilage and potential pathogenic species. These investigations also indicated that the final cheese safety does not depend exclusively on raw milk, but it is rather a consequence of several technological and microbiological factors occurring during production. Specifically, the addition of the selected mixed culture composed of Lc. lactis subsp. lactis, E. faecalis, Lc. garviae, and S. gallolyticus subsp. macedonicus at a large scale level among several dairy factories gathered into PDO Pecorino Siciliano cheese consortium was able to determine rapid curd acidification and to activate mechanisms of antagonism for the control of undesired microorganisms. This strategy provided useful evidence to preserve the identity of typical cheeses and to improve food security. However, the cooking step undoubtedly represents a key factor for the final safety of raw milk cheeses. From this perspective, the possible interaction among the cooking process, starter selection, and reduction of pathogenic and spoilage bacteria still needs to be investigated for PDO Pecorino Siciliano cheese.

Author Contributions: Conceptualization, L.S.; data curation, R.G.; writing—original draft prepa- ration, R.G., M.T., L.S.; writing—review and editing, L.S.; project administration, M.T.; All authors have read and agreed to the published version of the manuscript. Int. J. Environ. Res. Public Health 2021, 18, 1834 13 of 16

Funding: This work was supported by AGER 2 Project, grant no. 2017-1144. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data included in this study are available upon request by contacting the corresponding author. Conflicts of Interest: The authors declare that they have no conflict of interest.

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