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Behavior of Listeria innocua during the manufacturing and pit-ripening of Formaggio di Fossa di Sogliano PDO cheese

Formaggio di Fossa di Sogliano is a traditional Italian Protected Designation of Origin (PDO) cheese ripened for a minimum of 5 months, with the feature of a ripening of at least 80 to at most 100 days in pits, digged into tuffaceous rocks according to medieval tradition of Italy. In this study, a challenge test using Listeria innocua as a surrogate of Listeria monocytogenes was performed, with the aim of increasing knowledge concerning the impact of the Fossa cheese process, and especially of the traditional ripening process of this PDO, on the behaviour of L. monocytogenes. Pasteurized milk was experimentally inoculated with 4.5 log CFU/mL cocktail by three L. innocua strains, and L. innocua and Mesophilic Lactic Acid Bacteria (LAB) counts as well as the evolution of temperatures, pH and aw values were monitored throughout the manufacturing and ripening processes. Throughout the ripening in maturation room a constant temperature of 8°C was observed reaching a temperature between 10 and 15.5°C during ripening into pit. In the final products data for LAB concentration, pH and aw values were roughly in accordance with literature, even if some differences were, probably due to variability of artisanal cheese productions. The numbers of L. innocua showed a slight decrease but remained stable until the end of ripening in maturation room, whereas a significant reduction of the microorganism was observed in the final product, at the end of the ripening into the pit. The findings give scientific evidence that the process of this PDO prevented the L. innocua growth, allowing us to speculate a similar behaviour of L. monocytogenes. Based on this study, the recommendation to extend as much as possible the ripening into pit (from 80 to 100 days) was provided to food business operators as a risk mitigation strategy to be implemented.


Introduction
Italy has a prominent position in the European dairy industry being the largest producer country of Protected Designation of Origin (PDO) cheeses. The main use for cow milk in Italy is for cheese making: milk destination is productions of PDO cheeses (44%), followed by other destinations (39%), fresh milk (9%) and UHT milk (9%) (CLAL, 2019). A total of 527,105 tonnes of PDO cheeses were produced in 2016 in Italy (CLAL, 2019). Formaggio di Fossa di Sogliano, henceforth Fossa cheese, is registered as PDO by the European Community (EC) regulations (EC, 2009). Fossa cheese is a semi-hard cheese, produced using whole pasteurized milk, either from cow or sheep or a mixture of both, with the peculiarity of cheese ripening for a minimum of 5 months, of which the first 60 days take place in a maturation room, following by a variable period from at least 80 to at most 100 days in ripening pits, digged into tuffaceous rocks, that are prepared and sealed according to tradition originated in the Middle Ages (Avellini et al., 1999;De Santi et al., 2010;Gobbetti et al., 1999). This peculiar ripening has important effects for hardness, moisture, flavour and for the development of some organoleptical properties that characterize this traditional cheese (Barbieri et al., 2011;De Santi et al., 2010;Fontana et al., 2010;Pirisi et al., 2011).
Dairy production follows several stages, from raw milk production at the farm and further processing either at a dairy company or at the farm itself, and food safety hazards may enter at various stages along this dairy supply chain (van Asselt et al., 2017). Based on the Rapid Alert System for Food and Feed (RASFF) database on food safety hazards in dairy products for 2009 to 2014, a total of 243 notifications were retrieved, of which 203 were microbiological contaminations, and most of these involved cheese products. Listeria monocytogenes is involved in 52% of these notifications for microbiological contamination (van Asselt et al., 2017), and from 2015 to hitherto, other one hundred alerts for L. monocytogenes, categorized as serious risk, were notified by EU Countries in the product category milk and milk products. In the context of food safety, food business operators (FBO) are responsible for ensuring that the requirements of food law are met within the food business under their control (Regulation EC No 178/2002), and Regulation EC 2073/2005 sets out microbiological criteria for foodstuffs, including food safety criteria for Listeria monocytogenes in Ready-To-Eat (RTE) foods at specific time points along the food chain and as a function of the type of food. In fact, RTE foods are a broad and diverse food category, some of which support the growth of L. monocytogenes and others that do not or even result in microbial inactivation in specific storage and shelf life conditions (EFSA, 2018); the critical limit for this food safety criteria that must be satisfied depends on the evidence that the considered RTE food is able or not to support L. monocytogenes growth: products with pH≤4,4 or a w ≤0,92, or products with pH≤5,0 and a w ≤0,94, or products with a shelf-life of less than five days shall be automatically considered to belong to this category, but, other categories of products can also belong to this category, subject to scientific justification (Regulation EC No 2073/2005. When pasteurized milk is used for cheese making, the contamination by L. monocytogenes can be due to unsatisfactory pasteurization or contamination after heat treatment (Lomonaco et al., 2009) and in the specific case of soft-ripened cheese made of pasteurized milk, the factors influencing the risk of illness by L. monocytogenes are cross-contamination or re-contamination during manufacturing (linked to the hygiene of the processing environment) and the evolution of L. monocytogenes population during the ripening.
Few studies were performed on Fossa cheese, and they were mostly focused on characterization of the cheese composition and of the bacterial or fungal microflora in the cheese and in its ripening environment.
This study is the first challenge test performed on Fossa cheese, where a postpasteurisation contamination of milk by Listeria innocua was simulated, and followed the traditional manufacturing and ripening of this Italian PDO cheese in real tuffaceous pit, filled up and opened only after the pit ripening as the PDO specification. In this scenario, L. innocua was choosen as surrogate of L. monocytogenes given the use of L. monocytogenes as inoculum was avoided for the consequent risk of cheese environment contamination. Three L. innocua strains (one type strain: ATCC ® 3390 ™ and two wild strains belonging to IZSLER's collection: Li 48775 isolated from milk and Li 39673/2 isolated from butter) were used to contaminate the pasteurized milk. The bacterial cultures were prepared following the protocol described by Dalzini et al. Briefly,each strain, in Brain Heart Infusion (BHI) (Oxoid Italia, Milan, Italy) supplemented with 20% glycerol, was transferred (2% inoculum) into BHI, incubated at 37°C for 24 h in aerobic conditions and grown for two successive cultures. The cultures were centrifuged at 4°C at 4000 g (Jouan centrifuge CR422, Winchester, VA, USA) and the supernatant fluids were immediately drawn off and discarded. The pellet was washed with sterile physiological solution (H 2 O with 0.9% NaCl), centrifuged and resuspended in sterile physiological solution. Before use, the individual strains were combined in equal volumes in order to obtain a multistrain cocktail. Once warmed to 38°C, 150 L of milk were inoculated with 1% v:v of multi-strain cocktail of L. innocua to reach approximately 4.5 log CFU/mL in milk. The number of viable cells of the suspension was verified in milk before cheesemaking by 10-fold dilution and direct plating on Listeria selective agar (Oxford Formulation) (Oxoid Italia, Milan, Italy) plates incubated at 37°C for 48 h. according to production specification (MIPAAF, 2009 diacetylactis, Leuconostoc mesenteroides, and cheese rennet for the milk coagulation (Micromilk 20g/50 L of milk). The coagulum was cut for two successive times in order to crumble it into small cubes of about 0.5-1 cm, transferred into round moulds (20 cm diameter and 10 height) and the lets stand at room temperatures for 2 hours, turning the moulds upside down for a total of 3 times.

Formaggio di Fossa di Sogliano PDO process
A total of 18 cheeses of about 800 g each, were manufactured in this study. All the cheeses were transported to a dairy plant at Sogliano di Rubicone, Emilia-Romagna Region, Italy. Cheeses were stored at room temperature (22 ± 2°C) for 18 h, and then, dry-salted by spreading the salt over the cheese, and again put into the moulds for 8-9 hours. After washing, cheeses were put again into the moulds for 48 h at room temperature (22 ± 2°C). The cheese wheels were removed from the moulds, moved and placed in appropriate ripening iron steels grills in the ripening room. During these 60 days of ripening, all the cheese wheels were turned every 8 hours in the first week, and then once a day up to 30 days, and once every 3-4 days up to 60 days of ripening.
After the first ripening in room, cheeses were wrapped in natural cotton bags and differentiated with laces for the second and traditional ripening into pit. The pit was prepared with a floor of wooden boards and walls lined with layers of straw, and it was filled up with the cheeses of this study and also with other 40,000 Kg of cheeses. The experimental cheeses were put, within the pit, at three different points of the pit, respectively at the bottom, in the middle and at the opening of the pit.
Finally, the pit was hermetically sealed from the end of January to the end of April, and after 93 days, it was opened: overall, Fossa cheeses were ripened for a total of 153 days. The 18 experimental Fossa cheeses, items of the challenge test, were transported to the IZSLER laboratory. Figures 1 and 2 show, respectively, the flowchart reporting the main steps of the experimental study's design and some pictures of the cheese ripening.

Sampling, microbiological and physicochemical analyses
Three replicate samples of 25 g were collected during cheese making (milk and curd) and during the ripening at 7, 30, 60 and 153 days (cheese) and prepared according to ISO 6887 1 (ISO, 1999) and ISO 6887 5 (ISO 2010) for the analyses. The microbiological analyses were performed on milk, curd and cheeses: for curd and cheeses, samples were first transferred separately into plastic one-chamber filter stomacher bags (NEOMED, Milan, Italy) and then homogenized for 3 min in a Stomacher 400 blender (Seward Medical, London, UK) with 225 mL of 0.1% w/v of Buffered Peptone Water (BPW) (containing 1% v/v of Tween  80 and 0.85% w/v of NaCl, pH 7.0 ± 0.2).

Article
To verify the absence of natural contamination, milk was analyzed at time 0 for the presence of L. monocytogenes according to ISO 11290-1 (ISO, 2004). The enumeration of L. innocua was performed by 10-fold dilution and plating (0.1 mL) on Listeria selective agar (Oxford Formulation) (Oxoid Italia, Milan, Italy) plates incubated at 37°C for 48 h. Mesophilic Lactic Acid Bacteria (LAB) were enumerated according to ISO 15214 (ISO, 1998). For each culture medium, the quality has been evaluated in accordance with ISO 11133-1 (ISO, 2009) and ISO 11133-2 (ISO, 2011). All the microbiological analysis were performed according to ISO 7218 (ISO, 2007).
For the physiochemical analyses, pH was measured on 10 g of samples using an HI 223 Calibration check TM Microprocessor pH meter (Hanna Instrument, USA) equipped with a Gel-Glass electrode (Hamilton, Switzerland); water activity (a w ) was measured at 25°C with the a w recorder AquaLab, series 3, Model TE (Decagon Devices, Inc., Pullman, USA) in accordance with ISO/FDIS 21807 (ISO, 2004) and the time/temperature profiles during cheese making and ripening were monitored directly into the milk, curd and cheeses using electronic data loggers (Thermo Button 22T, Astori tecnica s.n.c. Brescia, Italia).

Data analysis
Bacterial counts were converted to log CFU/g or mL. The individual means and standard deviations of microbiological and physicochemical results were determined on the basis of the average of three replicate. The difference of results during the manufacturing process and in relation to the different positions in the pit, were evaluated by variance analysis (ANOVA) by Fisher test. Significance was evaluated when the pvalue was lower than 0.05 (P<0.05) in this case a Tukey's HSD (Honest Significant Difference) test was performed to reveal significant changes using R v 3.4.0 software (R Development Core Team, 2008).

Results and Discussion
During each manufacturing and ripening step for the production of Formaggio di Fossa di Sogliano PDO cheeses, temperature profiles are shown in Figure 3 whereas the evolution of L. innocua and LAB counts as well as pH and a w values are reported in Table 1. In the first hours of the cheese making, temperature decreased from 38°C to 21°C until the beginning of the ripening stage and again decreased to 8°C, remaining constant throughout the ripening in maturation room (Figure 3).
LAB counts increased significantly (P<0.05) in the first hours reaching 6.93 ± 0.16 log CFU/g in the curd (Table 1), and produced a mild gradual but significant decrease in pH values (P<0.05) from 6.66 to 5.00 at day 60 due to the production of organic acids. Once into the pit, the observed temperatures increased from 10 to 15°C with changes accordingly to the position of the cheeses into the pit (Figure 3) and in case of cheeses placed in the opening of the pit, the observed temperatures, moving up and down until the end of pit ripening, were probably due to the greater effect of the external environment and climate. With respect to the published data, Authors reported temperatures throughout the ripening time ranging from 25 to 16°C depending on the moment of the ripening (usually temperatures were higher in the beginning of the ripening and began to drop slowly almost at the end of the ageing into the pit) and with variations depending on the season of the ripening. Our findings were lower to studies of Avellini et al., 204;De Santi et al., 2010;Mascaro et al., 2010but in line with Toppino et al., 1992. The final products, cheeses placed in the bottom of the pit and ripened for 93 days in pit, were characterized by a LAB concentration of 6.86 ± 0.39 log CFU/g, a pH value of 5.06 ± 0.06 and a w of 0.916 ± 0.002 showing no differences in relation to the position of the cheeses into the pit (Table 1). Data we observed for LAB concentration in the final products were in accordance with those reported in literature studies: Barbieri et al. (2011) showed a LAB concentration of about 6 log counts in 150 day-old cheese ripened 90 days in a pit, Fontana et al. (2010) reported a level of 6.92±0.76 log CFU/g on Fossa cheese surface, as well as a wide variability from 5.8 to 7.8 log count was observed by Gobetti et al. (1999) in Fossa cheese at the end of the ripening in pit.
Our results were in agreement with literature also for the pH values, varying between 5.00 and 5.75 (Toppino et al., 1992;Gobbetti et al., 1999;Mascaro et al., 2010), while the final a w values were slightly higher than those observed by Gobetti et al. (1999) but similar to Mascaro et al. (2010). This variability could be due to the fact that artisanal production of cheese is not completely standardized, and therefore, it creates variability in intrinsic product characteristic, mainly in case of semi hard cheese that are fermented products with a variable and dynamic microbial community that is still biological active during further storage (Lahou et al., 2017).
The success of this challenge test is related to the fact that L. innocua behaviour should be evaluated during the whole 5 months of manufacturing process: given this behaviour is unknown both during the cheese manufacturing and the long ripening time into pit, a high concentration of the inoculum was chosen, in agreement NACMCF (2010), to prevent undetectable or not significant findings. In addition, the absence of L. monocytogenes in milk used for the experiment was verified. The L. innocua concentration remained essentially unchanged from day 0 to day 32 of the ripening in maturation room (from 4.51±0.02 to 4.36 ± 0.20 log CFU/g) and only at the end of the ripening at 60 days showed a significant decrease (P<0.05) to 3.89±0.26 log CFU/g (Table 1); the final concentration of L. innocua was 2.92 ± 0.20 log CFU/g, 2.49 ± 0.83 log CFU/g and 2.32 ± 0.73 log CFU/g after the ripening respectively in the bottom, middle and in opening of the pit, showing significant reductions (P<0.05) from a minimum of 1.33 to a maximum of 2.95 log CFU/g among the tested replicates. This study is the first microbiological challenge test performed on Fossa cheese and it gives scientific evidence that the process of this PDO prevented the L. innocua growth. Factors affecting L. monocytogenes growth, and in general of Listeria spp., mainly include the product characteristics (pH, a w , concentration of antimicrobials, food preservatives), storage temperature and time. The microstructure of the food matrix can also affect the growth by imposing physical constraints on microorganisms, by limiting the diffusion of essential nutrients and oxygen or preventing the diffusion of metabolic products (Aspridou et al, 2014;EFSA, 2018). Additional factors that can affect L. monocytogenes growth is the presence of competitive microflora as well as the socalled Jameson effect, which is expressed as L. monocytogenes growth cessation when lactic acid bacteria reach a critical population density markedly higher than L. monocytogenes (Lardeux et al., 2015). During the pit ripening, cheeses maintained intrinsic variables (a w 0.92 and pH around 5.1) values that may be hostile for the bacterial survival as L. innocua. The combination of these hurdles, low pH and a w and high LAB population for long times (over 93 ripening days) probably played a key role in the behaviour of L. innocua. In addition, for the extrinsic variable temperature that could let the bacteria growth, similarly to findings observed by Valero et al. (2014) for a traditional semihard aromatic cheese typically aged for three to six months in which the impact of temperature on L. monocytogenes behavior was investigated during storage at 4, 12 and 22°C, the observed temperatures into pit (increasing from 10 to 15°C) could have produced the significant reduction of L. innocua. However, as observed by Lahou and Uyttendaele (2017) from several challenge tests in different dairy products, L. monocytogenes growth is significantly higher in pasteurized than raw milk cheeses, given more bacterial competition is expected to be present in raw milk cheeses, which may be an explanation of the lower growth potential of L. monocytogenes in these cheeses (Izquierdo et al., 2009;Mellefont et al., 2008;Schvartzman et al., 2011). In this challenge test, an high amount of L. innocua inoculum in a pasteurized milk with no or very low competitors was performed, and therefore, a non-specific competition for nutrients by the microrganism and the indigenous microflora present in the matrix could be considered, but only the competition with starters cultures. A limitation of our challenge test, in terms of extending our findings to a similar behaviour of L. monocytogenes during the process of this PDO, could be identify in the use of L. innocua. L. innocua is described as an attractive surrogate for L. monocytogenes due to its close genetic relationship (Glasser et al., 2001) and in literature it has been used in a variety of systems designed to control the growth, eliminate the presence, or reduce the transmission of this foodborne pathogen in foods. However, O'Bryan et al. (2006) observed that L. innocua surrogate strains may not accurately reflect the behavior of L. monocytogenes in all test conditions/foods, including thermal and other stresses responses. This suggestion should be taken into account considering that the growth of L. monocytogenes is among the most important factors affecting the risk of human listeriosis associated with consumption of RTE foods and that its growth was pointed out to be a function of the type of food and storage conditions (EFSA, 2018). In this context, the pit ripening seems the key point for the control of L. innocua and L. monocytogenes in this PDO cheese, and the risks to human health could be reduced to an acceptable level by both the containing of the Listeria contamination during cheese making and an appropriate ripening period into the pit. Based on the results of this challenge test, the recommendation to food business operator for risk mitigation to extend as much as possible the ripening into pit according to the Fossa Cheese specification (from 80 to 100 days) was provided, and a further L. innocua decay trend during shelf life at refrigerated temperatures is expected. Obviously, the maintenance of Good Hygiene Practice at primary production and during cheese making is suggested: avoidance of Listeria contamination in milk remains of high priority for the dairy industry.

Conclusions
The present challenge test is the first to investigate the behavior of L. innocua, as surrogate of L. monocytogenes, in the Formaggio di Fossa di Sogliano cheese process, and mostly in traditional ripening process of this PDO. This study can be considered an useful example of the European food safety approach, with the integration of self-control by food business operator, food companies and the monitoring and auditing by Veterinary Competent Authority. The application of microbial criteria is one of the essential activities to ensure that ready to eat foods presented a low risk to public health, and even in these experimental unrealistic conditions, the numbers of L. innocua showed a significant reduction in the final product, at the end of the ripening into the pit. These findings give scientific evidence that the process of this PDO prevented the L. innocua growth, allowing us to speculate a similar behaviour of L. monocytogenes. The recommendation of extending the ripening time alignes the need to control of L. monocytogenes in a more proactive way by implementing an effective food safety management system.